Difference between revisions of "Team:SCAU-China/Protocol"

Latest revision as of 06:13, 18 October 2016

SCAU

Protocol

Vector construction protocol

To construct the multigene vector for astaxanthin biosynthesis, we used a modified multigene vector system, TransGene Stacking II (TGSII) (Zhu et al., unpublished). This novel system is based on our previous studies on the transformation-competent artificial chromosome (TAC) and multigene assembly system (Liu et al., PNAS, 1999, 96: 6535-6540; Lin et al., PNAS, 2003, 100: 5962-5967). This system consists of a transformation-competent artificial chromosome (TAC)-based binary acceptor vector (pYLTAC380GW), together with two donor vectors (pYL322-d1/ pYL322-d2). By using the Cre/loxP recombination system and two pairs of mutant loxP sites, multiple rounds of gene assembly cycles were carried out with alternative use of the donor vectors, and multiple genes were sequentially delivered into the TAC vector (Zhu et al., unpublished).

To sequentially assemble them, the four endosperm-specific promoters drove four genes (CrtI, PSY, BKT and BHY) expression cassettes were constructed into two donor vectors, respectively. And these obtained donors, (Ⅰ) d1-CrtI, (Ⅱ) d2-PSY, (Ⅲ) d1-BKT and (Ⅳ) d2-BHY, were sequentially assembled into the 380GW acceptor by four rounds of gene assembly cycles, according to Ⅰ\Ⅱ\Ⅲ\Ⅳ order. Finally, after completing four-gene assembly, the marker-free element, containing a HPT selectable resistance gene expression cassette and a Cre-induced gene expression cassette by an anther-specific promoter, was integrated into the four-gene acceptor by Gateway BP reaction. The obtained six-gene marker-free multigene vector, named 380MF-BBPC, was transferred into Agrobacterium tumefaciens strain EHA105 for further rice calli transformation.

Four genes assembly cycles:

Round Ⅰ: Cre/loxP reversible recombination of the d1-CrtI plasmid into the TAC-based acceptor plasmid pYLTAC380GW (i), followed by release of the d1 backbone by Cre-mediated irreversible recombination between one pair of mutant loxP sites (ii).The first CrtI gene expression cassette was assembled into the acceptor vector to form 380GW-C.

Round Ⅱ: Cre/loxP reversible recombination of the d2-PSY plasmid into the 380GW-C (iii), and release of the d2 backbone by Cre-mediated irreversible recombination between another pair of mutant loxP sites (iv). The second PSY gene expression cassette was assembled into the 380GW-C to form 380GW-PC.

Round Ⅲ: Similar to the first Round, the third BKT gene expression cassette from d1-BKT was assembled into 380GW-PC to obtain 380GW-BPC.

Round Ⅳ: Similar to the Round Ⅱ, the fourth BHY gene expression cassette from d2-BHY was assembled into380GW-BPC to obtain 380GW-BBPC.

Repeat above multigene assembly cycles, more genes can be easy to stack into the TAC binary vector.

Marker-free element integration

After four-round genes assembly, the Gateway BP reaction was used to integrate the marker-free element, containing a HPT selectable resistance gene expression cassette and a Cre-induced gene expression cassette driven by an anther-specific promoter, into the four-gene acceptor 380GW-BBPC. Finally, we obtained the marker-free multigene vector 380MF-BBPC for rice transformation.

Transformation of rice

1.Brief on the procedure:

Calli induction→ Subculture of calli → Co-culture of calli with Agrobacterium → Selection of resistant calli→ Differentiation of resistant calli → Rooting the seedling → Hardening the seedling →Transplanting the seedling.

2.Detailed steps

2.1 Calli induction

2.1.1 Medium Preparation

Component	Dosage
10×N6 macroelement solution	50 mL
1000×B5 microelement solution	0.5 mL
100×B5 vitamin solution	5 mL
100×Ferric salt solution	5 mL
0.5 mg/mL 2,4-D	3 mL
Casein enzymatic hydrolysate	150 mg
L-Proline	250 mg
L-Glutamine	250 mg
Sucrose	15 g
Add distilled water until the medium volume reach 500 mL
Blend with magnetic stirrer
Adjust medium pH to 5.8 with 1 mol/L NaOH
Phytagel	1.5 g

2.1.2 Experimental procedures

(1) Put 400 rice seeds into a 50 mL Erlenmeyer flask.

(2) Prepare 75% ethanol solution.

(3) Pour some 75% ethanol solution into the Erlenmeyer flask which contain the seeds. Keep shaking the Erlenmeyer flask in 1 minute at most.

(4) Decant the 75% ethanol from the Erlenmeyer flask.

(5) Add about 35 mL distilled water to the Erlenmeyer flask immediately and keep shaking for 40 seconds. Decant the water. Repeat this step for 5 times.

(6) Prepare 1.5% sodium hypochlorite solution.

(7) Pour some 1.5% sodium hypochlorite solution into the Erlenmeyer flask and seal the Erlenmeyer flask with flask sealing film. Shake the Erlenmeyer flask in a shaker for 30 minutes.

(8) Decant the 1.5% sodium hypochlorite solution.

(9) Add about 35 mL distilled water to the Erlenmeyer flask immediately and keep shaking for 40 seconds. Decant the water, Repeat this step for 5 times.

(10) Repeat step 6 to 9.

(11) Pick out seeds by a sterile tweezer and put seeds on a sterile filter paper.

(12) Place seeds in the laminar flow cabinet and dried by blowing for about 2 hours.

(13) Put sterile seeds on the surface of the calli initiation medium and seal plates with parafilm.

(14) Incubate seeds in the dark for about 14 days.

2.2 Subculture of calli

2.2.1 Medium preparation

Component	Dosage
10×N6macroelement solution	50 mL
1000×B5microelement solution	0.5 mL
100×B5vitamin solution	5 mL
100×Ferric salt solution	5 mL
0.5 mg/mL 2,4-D	3 mL
Casein enzymatic hydrolysate	150 mg
L-Proline	250 mg
L-Glutamine	250 mg
Sucrose	15 g
Add distilled water until the medium volume reach 500 mL
Blend with magnetic stirrer
Adjust medium pH to 5.8 with 1 mol/L NaOH
Phytagel	1.5 g（3 g/L）

2.2.2 Experimental procedures

(1) Place the calli initiation medium in the laminar flow cabinet.

(2) Use a sterile tweezer to divide calli from seeds and put calli on the subculture medium. Seal plates with parafilm.

(3) Incubate calli in the dark for 5 days.

2.3 Co-culture of calli with Agrobacterium

2.3.1 Medium preparation

(1)

Component	Dosage
10×MSmacroelement solution	25 mL
1000×B5microelement solution	0.25 mL
100×B5 vitamin solution	2.5 mL
100×Ferric salt solution	2.5 mL
0.5 mg/mL 2,4-D	1.5 mL
Casein enzymatic hydrolysate	125 g
Inositol	0.5 g
Sucrose	7.5 g
Glucose	7.5 g
Add distilled water until medium volume reach 250 mL
Blend with magnetic stirrer
Adjust medium pH to 5.2 with 1 mol/L NaOH
1000×Acetosyringone (Add before using the medium)	0.25 mL

(2)

Component	Dosage
10×MSmacroelement solution	25 mL
1000×B5microelement solution	0.25 mL
100×B5 vitamin solution	2.5 mL
100×Ferric salt solution	2.5 mL
0.5 mg/mL 2,4-D	1.5 mL
Casein enzymatic hydrolysate	125 g
Inositol	0.5 g
Sucrose	7.5 g
Glucose	7.5 g
Add distilled water until medium volume reach 250 mL
Blend with magnetic stirrer
Adjust medium pH to 5.3 with 1 mol/L NaOH
Agar	3.5 g
1000×Acetosyringone (Add before dividingthe medium into plates)	0.25 mL

2.3.2 Experimental procedures

(1) Place the subculture medium into the laminar flow cabinet.

(2) Pick out the calli and put them on a sterile filter paper. Dry the surface of calli by blowing.

(3) Add 0.25 mL Acetosyringone to co-culture liquid medium.

(4) Pour about 35 mL co-culture liquid medium into a 50 mL Erlenmeyer flask.

(5) Use a sterile stainless steel spoon to collect Agrobacterium transforment strain and suspend in the co-culture liquid medium. The co-culture liquid medium contain Agrobacterium also called Agrobacterium suspension.

(6) Adjust the OD600 of Agrobacterium suspension to 0.1.

(7) Seal the Erlenmeyer flask with flask sealing film. Shake the Erlenmeyer flask in a shaker in 27 ℃ for 30 minutes.

(8) After 30 minutes, take the Erlenmeyer flask out and put it in the laminar flow cabinet. Pick calli into the Agrobacterium suspension and gentle shake the Erlenmeyer flask for 20 minutes. Ensure that each calli contact with the Agrobacterium suspension directly.

(9) Decant the Agrobacterium suspension. Pick out calli and put them on a sterile filter paper.
Dry the surface of calli by blowing.

(10) At the same time, put a sterile filter paper on the surface of the co-culture medium. Dry the surface of the co-culture medium by blowing.

(11) When the surface of calli become dry, put them on the surface of the filter paper above the co-culture medium.

(12) Seal plates with parafilm. Incubate in the dark at 25 ℃ for 3 days.

2.4 First time selection of resistant calli

2.4.1 Medium preparation

Component	Dosage
10×N6macroelement solution	50 mL
1000×B5 microelement solution	0.5 mL
100×B5 vitamin solution	5 mL
100×Ferric salt solution	5 mL
0.5 mg/mL 2,4-D	3 mL
Casein enzymatic hydrolysate	150 mg
L-Proline	250 mg
L-Glutamine	250 mg
Sucrose	15 g
Add distilled water until the medium volume reach 500 mL
Blend with magnetic stirrer
Adjust medium pH to 5.8 with 1 mol/L NaOH
Phytagel	3 g
1000×Hygromycin B (Add before dividing the medium into plates)	0.5 mL
1000×Cefazolin sodium (Add before dividing the medium into plates)	0.5 mL
1000×Carbenicillin sodium (Add before dividing the medium into plates)	0.5 mL

2.4.2 Experimental procedures

(1) Place the co-culture medium in the laminar flow cabinet.

(2) Add 1 mL 1000×Cefazolin sodium and 1 mL 1000×Carbenicillin sodium to 1 L sterile distilled water. This solution is Agrobacterium eluent.

(3) Pick out calli and put them in a 50 mL Erlenmeyer flask.

(4) Add about 35 mL Agrobacterium eluent to the Erlenmeyer flask. Shake the Erlenmeyer flask for one minute. Decant the Agrobacterium eluent.

(5) Repeat step 4 for about 20 times. Ensure that most of the Agrobacterium on calli are eluted.

(6) Pick out calli and put them on a sterile filter paper. Dry the surface of calli by blowing.

(7) Put calli on the selecting medium.

(8) Seal plates with parafilm.

(9) Culture in the dark for 30 days.

2.5 Second time selection of resistant calli

2.5.1 Medium preparation

Component	Dosage
10×N6 macroelement solution	50 mL
1000×B5 microelement solution	0.5 mL
100×B5 vitamin solution	5 mL
100×Ferric salt solution	5 mL
0.5 mg/mL 2,4-D	3 mL
Casein enzymatic hydrolysate	150 mg
L-Proline	250 mg
L-Glutamine	250 mg
Sucrose	15 g
Add distilled water until the medium volume reach 500 mL
Blend with magnetic stirrer
Adjust medium pH to 5.8 with 1 mol/L NaOH
Phytagel	3 g
1000×Hygromycin B (Add before dividing the medium into plates)	0.5 mL
1000×Cefazolin sodium (Add before dividing the medium into plates)	0.5 mL
1000×Carbenicillin sodium (Add before dividing the medium into plates)	0.5 mL

2.5.2 Experimental procedures

(1) Place the first time selecting medium in the laminar flow cabinet.

(2) Pick out calli and put them on the second time selecting medium.

(3) Seal plates with parafilm.

(4) Culture in the dark for 30 days.

2.6 Differentiation of resistant calli

2.6.1 Medium preparation

Component	Dosage
10×N6 macroelement solution	50 mL
1000×MS microelement solution	0.5 mL
100×B5 vitamin solution	5 mL
100×Ferric salt solution	5 mL
0.5 mg/mL 6-B,A	3 mL
0.1 mg/mL NAA	5 mL
Sorbitil	9.1 g
Sucrose	10 g
Add distilled water until the medium volume reach 500 mL
Blend with magnetic stirrer
Adjust medium pH to 5.8 with 1 mol/L NaOH
Phytagel	2 g
1000×Hygromycin B (Add before dividing the medium into plates)	0.5 mL
1000×Cefazolin sodium (Add before dividing the medium into plates)	0.5 mL
1000×Carbenicillin sodium (Add before dividing the medium into plates)	0.5 mL

2.5.2 Experimental procedures

(1) Place the first time selecting medium in the laminar flow cabinet.

(2) Pick out calli and put them on the second time selecting medium.

(3) Seal plates with parafilm.

(4) Culture in the dark for 30 days.

2.6 Differentiation of resistant calli

2.6.1 Medium preparation

Component	Dosage
10×N6 macroelement solution	50 mL
1000×MS microelement solution	0.5 mL
100×B5 vitamin solution	5 mL
100×Ferric salt solution	5 mL
0.5 mg/mL 6-B,A	3 mL
0.1 mg/mL NAA	5 mL
Sorbitil	9.1 g
Sucrose	10 g
Add distilled water until the medium volume reach 500 mL
Blend with magnetic stirrer
Adjust medium pH to 5.8 with 1 mol/L NaOH
Phytagel	2 g
1000×Hygromycin B (Add before dividing the medium into plates)	0.5 mL
1000×Cefazolin sodium (Add before dividing the medium into plates)	0.5 mL
1000×Carbenicillin sodium (Add before dividing the medium into plates)	0.5 mL

2.6.2 Experimental procedures

(1) Place the second time selecting medium in the laminar flow cabinet.

(2) Pick out those yellow calli that generate after two times of selection. Put them on the differentiation medium.

(3) Seal plates with parafilm.

(4) Culture under the condition of 14 hours light and 10 hours dark a days, for about 25 days.

(5) After 25 days, green spot will appear on the surface of the resistant calli.

(6) Place the differentiation medium in the laminar flow cabinet.

(7) Choose calli which has green spot. Transfer them to a new differentiation medium.

(8) Seal cuture bottles.

(9) Culture under the condition of 14 hours light and 10 hours dark a days, for about 30 days.

2.7 Rooting the seedling

2.7.1 Medium preparation

Component	Dosage
10×N6 macroelement solution	50 mL
1000×MS microelement solution	0.5 mL
100×N6vitamin solution	5 mL
100×Ferric salt solution	5 mL
0.1 mg/mL NAA	5 mL
Sucrose	10 g
Add distilled water until the medium volume reach 500 mL
Blend with magnetic stirrer
Adjust medium pH to 5.8 with 1 mol/L NaOH
Phytagel	2 g
1000×Hygromycin B (Add before dividing the medium into plates)	0.5 mL
1000×Cefazolin sodium (Add before dividing the medium into plates)	0.5 mL
1000×Carbenicillin sodium (Add before dividing the medium into plates)	0.5 mL

2.7.2 Experimental procedures

(1) Place the differentiation medium in the laminar flow cabinet.

(2) Carefully pick the seedling out with a sterile tweezer. Transfer it to rooting medium.

(3) Seal culture bottles.

(4) Culture under the condition of 14 hours light and 10 hours dark a days, for about 20 days.

2.8 Hardening the seedling

2.8.1 Experimental procedures

(1) Choose seedling where are 8 cm high and grown strong roots.

(2) Open the lid of culture bottle. Add appropriate amount of distilled water.

(3) Culture in the greenhouse for 7 days.

2.9 Transplanting the seedling

2.9.1 Experimental procedures

(1) After hardening the seedling, take the seeding out and remove the medium on its roots with running water.

(2) Transplant the seedling to soil. Make sure that the seedling isn’t submergence or insolated.

Extraction and detection

1. The method of extracting the astaxanthin in rice endosperm

(1) Take 0.1 g rice seeds into mortar. After grinding it into powder on the ice, add 2 mL of methanol in mortar, and continue to grind for 5 minutes.

(2) Transfer the powder to a 2 mL centrifuge tube, spin at 10000 rpm for 10 min in the dark and low temperature condition to extract.

(3) Centrifuge at 8000rpm at 4 ℃ for 5 minutes and collect supernatant.

(4) Extract the sediment with methanol repeatedly, until the precipitation become white.

(5) Centrifuge again(8000rpm, 4 ℃, 5 min)and collect supernatant .

(6) Merge supernatant, concentrate supernatant into powder in the dark and low temperature conditions

(7) Add 600 µL methanol into dry astaxanthin extraction.

2. HPLC(High Performance Liquid Chromatography) determination of astaxanthin

After enrichment, put the samples into a 2 mL brown bottle through 0.22 µm syringe-driven filter, then detect the samples by HPLC. The mobile phase is methanol: water = 95:5. The flow rate is 1 mL/min. Column temperature is room temperature. The detection wavelength is 480 nm.

3. Standard curve for the determination of astaxanthin

Dissolve accurately 10 mg of standard sample of astaxanthin in 1 mL methylene chloride, and dilute with methanol to 100 mL in a brown volumetric flask and get the 0.1 mg/mL solution. Dilute the above-mentioned solution with methanol into 2 µg/mL, 4 µg/mL, 6 µg/mL, 8 µg/mL, 10 µg/mL. Take 20 µL sample to detect separately according to the conditions that mentioned above. With peak area as the ordinate, the concentration of the standard sample for horizontal, apply linear regression analysis, and get the regression equation.

4. Conclusion

The method of detecting the astaxanthin content in transgenic rice endosperm: grind transgenic rice seeds into powder on the ice, extract the sediment with methanol repeatedly, until the precipitation become white, concentrate the supernatant into powder, dissolved in methanol, use the high performance liquid chromatography (HPLC) to measure the content of astaxanthin.

References

【1】Y. J. Zhong, J. C. Huang, J. Liu, Y. Li, Y. Jiang, Z. F. Xu, G. Sandmann, F. Chen, Functional characterization of various algal carotenoid ketolases reveals that ketolating zeaxanthin efficiently is essential for high production of astaxanthin in transgenic Arabidopsis. Journal of Experimental Botany. 62, 10, 3659–3669 (2011)

【2】J. C. Huang, Y. J. Zhong, G. Sandmann, J. Liu, F. Chen, Cloning and selection of carotenoid ketolase genes for the engineering of high-yield astaxanthin in plants. Planta. 236, 691–699 (2012)

【3】J. C. Huang, Y. J. Zhong, J. Liu, G. Sandmann, F. Chen, Metabolic engineering of tomato for high-yield production of astaxanthin. Metabolic Engineering. 17, 59–67(2013)

【4】J. Breitenbach, C. Bai, S. M. Rivera, R. Canela, T. Capell, Paul Christou, C. f. Zhu, G. Sandmann, A novel carotenoid, 4-keto-a-carotene, as an unexpected by-product during genetic engineering of caroteno genesis in rice callus. Phytochemistry. 98, 85–91(2014)

【5】Liu Y-G, Shirano Y, Fukaki H, Yanai Y, Tasaka M, Tabata S, Shibata D. Complementation of plant mutants with large genomic DNA fragments by a transformation-competent artificial chromosome vector accelerates positional cloning. PNAS. 96, 6535–6540 (1999).

【6】Lin L, Liu Y-G, Xu X, L B. Efficient linking and transfer of multiple genes by a multigene assembly and transformation vector system. PNAS. 100: 5962-5967(2003).

【7】Zhu Q, Liu Y-G. A novel TransGene Stacking II system (TGSII) for plant multigene metabolic engineering. (in prepared and unpublished)

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 			<div class="h2_font_size">Vector construction protocol</div>
-			<div class="p_font_size" style="text-indent:0em">To construct the multigene vector for astaxanthin biosynthesis, we used a modified multigene vector system, TransGene Stacking II (TGSII) (Zhu et al., unpublished). This novel system is based on our previous studies on the transformation-competent artificial chromosome (TAC) for the larger DNA fragments transformation (Liu et al., PNAS, 1999, 96: 6535-6540) and the early version of the multigene assembly and transformation vector system (Lin et al., PNAS, 2003, 100: 5962-5967). This system consists of a transformation-competent artificial chromosome (TAC)-based binary acceptor vector (pYLTAC380GW), together with two donor vectors (pYL322-d1/ pYL322-d2). By using the Cre/loxP recombination system and two pairs of mutant loxP sites, multiple rounds of gene assembly cycles were carried out with alternative use of the donor vectors, and multiple genes were sequentially delivered into the TAC vector (Zhu et al., unpublished).</div>
+			<div class="p_font_size" style="text-indent:0em">To construct the multigene vector for astaxanthin biosynthesis, we used a modified multigene vector system, TransGene Stacking II (TGSII) (Zhu et al., unpublished). This novel system is based on our previous studies on the transformation-competent artificial chromosome (TAC) and multigene assembly system (Liu <em>et al.</em>, PNAS, 1999, 96: 6535-6540; Lin <em>et al.</em>, PNAS, 2003, 100: 5962-5967). This system consists of a transformation-competent artificial chromosome (TAC)-based binary acceptor vector (pYLTAC380GW), together with two donor vectors (pYL322-d1/ pYL322-d2). By using the <em>Cre/loxP</em> recombination system and two pairs of mutant <em>loxP</em> sites, multiple rounds of gene assembly cycles were carried out with alternative use of the donor vectors, and multiple genes were sequentially delivered into the TAC vector (Zhu et al., unpublished).</div>
-			<div class="p_font_size">To sequentially assemble them, the four endosperm-specific promoters drove four genes (CrtI, PSY, BKT and BHY) expression cassettes were constructed into two donor vectors, respectively. And these obtained donors, (I) d1-CrtI, (II) d2-PSY, (Ⅲ) d1-BKT and (Ⅳ) d2-BHY, were sequentially assembled into the 380GW acceptor by four rounds of gene assembly cycles, according to I\II\Ⅲ\Ⅳorder. Finally, after completing four-gene assembly, the marker-free element, containing a HPT selectable resistance gene expression cassette and a Cre-induced gene expression cassette by an anther-specific promoter, was integrated into the four-gene acceptor by Gateway BP reaction. The obtained six-gene marker-free multigene vector, named 380MF-BBPC, was transferred into Agrobacterium tumefaciens strain EHA105 for further rice callus transformation. </div>
+			<div class="p_font_size">To sequentially assemble them, the four endosperm-specific promoters drove four genes (<font style="font-style:italic">CrtI, PSY, BKT</font> and <font style="font-style:italic">BHY</font>) expression cassettes were constructed into two donor vectors, respectively. And these obtained donors, (Ⅰ) d1-CrtI, (Ⅱ) d2-PSY, (Ⅲ) d1-BKT and (Ⅳ) d2-BHY, were sequentially assembled into the 380GW acceptor by four rounds of gene assembly cycles, according to Ⅰ\Ⅱ\Ⅲ\Ⅳ order. Finally, after completing four-gene assembly, the marker-free element, containing a <em>HPT</em> selectable resistance gene expression cassette and a <em>Cre</em>-induced gene expression cassette by an anther-specific promoter, was integrated into the four-gene acceptor by Gateway BP reaction. The obtained six-gene marker-free multigene vector, named 380MF-BBPC, was transferred into <font style="font-style:italic">Agrobacterium tumefaciens</font> strain EHA105 for further rice calli transformation. </div>
 			<div class="h3_font_size">Four genes assembly cycles:</div>
-			<div class="p_font_size"><font style="font-weight:bold">Round I:</font> Cre/loxP reversible recombination of the d1-CrtI plasmid into the TAC-based acceptor plasmid pYLTAC380GW (i), followed by release of the d1 backbone by Cre-mediated irreversible recombination between one pair of mutant loxP sites (ii).The first CrtI gene expression cassette was assembled into the acceptor vector to form <font style="font-weight:bold">380GW-C</font>. </div>
+			<div class="p_font_size"><font style="font-weight:bold">Round Ⅰ:</font> <em>Cre/loxP</em> reversible recombination of the d1-CrtI plasmid into the TAC-based acceptor plasmid pYLTAC380GW (i), followed by release of the d1 backbone by Cre-mediated irreversible recombination between one pair of mutant <em>loxP</em> sites (ii).The first <font style="font-style:italic">CrtI</font> gene expression cassette was assembled into the acceptor vector to form <font style="font-weight:bold">380GW-C</font>. </div>
-			<div class="p_font_size"><font style="font-weight:bold">Round II:</font> Cre/loxP reversible recombination of the d2-PSY plasmid into the 380GW-C (iii), and release of the d2 backbone by Cre-mediated irreversible recombination between another pair of mutant loxP sites (iv). The second PSY gene expression cassette was assembled into the 380GW-C to form <font style="font-weight:bold">380GW-PC</font>.</div>
+			<div class="p_font_size"><font style="font-weight:bold">Round Ⅱ:</font> <em>Cre/loxP</em> reversible recombination of the d2-PSY plasmid into the 380GW-C (iii), and release of the d2 backbone by Cre-mediated irreversible recombination between another pair of mutant <em>loxP</em> sites (iv). The second <font style="font-style:italic">PSY</font> gene expression cassette was assembled into the 380GW-C to form <font style="font-weight:bold">380GW-PC</font>.</div>
-			<div class="p_font_size"><font style="font-weight:bold">Round III:</font> Similar to the first Round, the third BKT gene expression cassette from d1-BKT was assembled into 380GW-PC to obtain <font style="font-weight:bold">380GW-BPC</font>.</div>
+			<div class="p_font_size"><font style="font-weight:bold">Round Ⅲ:</font> Similar to the first Round, the third <font style="font-style:italic">BKT</font> gene expression cassette from d1-BKT was assembled into 380GW-PC to obtain <font style="font-weight:bold">380GW-BPC</font>.</div>
-			<div class="p_font_size"><font style="font-weight:bold">Round IV:</font> Similar to the Round II, the fourth BHY gene expression cassette from d2-BHY was assembled into380GW-BPC to obtain <font style="font-weight:bold">380GW-BBPC</font>.</div>
+			<div class="p_font_size"><font style="font-weight:bold">Round Ⅳ:</font> Similar to the Round Ⅱ, the fourth <font style="font-style:italic">BHY</font> gene expression cassette from d2-BHY was assembled into380GW-BPC to obtain <font style="font-weight:bold">380GW-BBPC</font>.</div>
 			<div class="p_font_size">Repeat above multigene assembly cycles, more genes can be easy to stack into the TAC binary vector.</div>
 			<div class="h3_font_size">Marker-free element integration</div>
-			<div class="p_font_size">After four-round genes assembly, the Gateway BP reaction was used to integrate the marker-free element, containing a HPT selectable resistance gene expression cassette and a Cre-induced gene expression cassette driven by an anther-specific promoter, into the four-gene acceptor <font style="font-weight:bold">380GW-BBPC</font>. Finally, we obtained the marker-free multigene vector <font style="font-weight:bold">380MF-BBPC</font> for rice transformation.</div>
+			<div class="p_font_size">After four-round genes assembly, the Gateway BP reaction was used to integrate the marker-free element, containing a <em>HPT</em> selectable resistance gene expression cassette and a <em>Cre</em>-induced gene expression cassette driven by an anther-specific promoter, into the four-gene acceptor <font style="font-weight:bold">380GW-BBPC</font>. Finally, we obtained the marker-free multigene vector <font style="font-weight:bold">380MF-BBPC</font> for rice transformation.</div>
 			<div class="h2_font_size">Transformation of rice</div>
 			<div class="h3_font_size">1.Brief on the procedure:</div>
-			<div class="p_font_size">Callus induction→ Subculture of callus → Co-culture of callus with Agrobacterium → Selection of resistant callus→ Differentiation of resistant callus → Rooting the seedling → Hardening the seedling →Transplanting the seedling.</div>
+			<div class="p_font_size">Calli induction→ Subculture of calli → Co-culture of calli with <font style="font-style:italic">Agrobacterium</font> → Selection of resistant calli→ Differentiation of resistant calli → Rooting the seedling → Hardening the seedling →Transplanting the seedling.</div>
 			<div class="h3_font_size">2.Detailed steps</div>
-			<div class="h4_font_size">2.1 Callus induction</div>
+			<div class="h4_font_size">2.1 Calli induction</div>
 			<div class="p_font_size"><strong>2.1.1 Medium Preparation</strong></div>
+<div align="center">
 			<table class="table">
 				<thead>
@@ Line 175: / Line 190: @@
 				</tbody>
 			</table>
+</div>
 			<div class="p_font_size"><strong>2.1.2 Experimental procedures</strong></div>
-			<div class="p_font_size">(1)Put 400 rice seeds into a 50 mL Erlenmeyer flask.</div>
+			<div class="p_font_size">(1) Put 400 rice seeds into a 50 mL Erlenmeyer flask.</div>
-			<div class="p_font_size">(2)Prepare 75% ethanol solution.</div>
+			<div class="p_font_size">(2) Prepare 75% ethanol solution.</div>
-			<div class="p_font_size">(3)Pour some 75% ethanol solution into the Erlenmeyer flask which contain the seeds. Keep shaking the Erlenmeyer flask in 1 minute at most.</div>
+			<div class="p_font_size">(3) Pour some 75% ethanol solution into the Erlenmeyer flask which contain the seeds. Keep shaking the Erlenmeyer flask in 1 minute at most.</div>
-			<div class="p_font_size">(4)Decant the 75% ethanol from the Erlenmeyer flask.</div>
+			<div class="p_font_size">(4) Decant the 75% ethanol from the Erlenmeyer flask.</div>
-			<div class="p_font_size">(5)Add about 35 mL distilled water to the Erlenmeyer flask immediately and keep shaking for 40 seconds. Decant the water. Repeat this step for 5 times.</div>
+			<div class="p_font_size">(5) Add about 35 mL distilled water to the Erlenmeyer flask immediately and keep shaking for 40 seconds. Decant the water. Repeat this step for 5 times.</div>
-			<div class="p_font_size">(6)Prepare 1.5% sodium hypochlorite solution.</div>
+			<div class="p_font_size">(6) Prepare 1.5% sodium hypochlorite solution.</div>
-			<div class="p_font_size">(7)Pour some 1.5% sodium hypochlorite solution into the Erlenmeyer flask and seal the Erlenmeyer flask with flask sealing film. Shake the Erlenmeyer flask in a shaker for 30 minutes.</div>
+			<div class="p_font_size">(7) Pour some 1.5% sodium hypochlorite solution into the Erlenmeyer flask and seal the Erlenmeyer flask with flask sealing film. Shake the Erlenmeyer flask in a shaker for 30 minutes.</div>
-			<div class="p_font_size">(8)Decant the 1.5% sodium hypochlorite solution.</div>
+			<div class="p_font_size">(8) Decant the 1.5% sodium hypochlorite solution.</div>
-			<div class="p_font_size">(9)Add about 35 mL distilled water to the Erlenmeyer flask immediately and keep shaking for 40 seconds. Decant the water, Repeat this step for 5 times.</div>
+			<div class="p_font_size">(9) Add about 35 mL distilled water to the Erlenmeyer flask immediately and keep shaking for 40 seconds. Decant the water, Repeat this step for 5 times.</div>
-			<div class="p_font_size">(10)Repeat step 6 to 9.</div>
+			<div class="p_font_size">(10) Repeat step 6 to 9.</div>
-			<div class="p_font_size">(11)Pick out seeds by a sterile tweezer and put seeds on a sterile filter paper.</div>
+			<div class="p_font_size">(11) Pick out seeds by a sterile tweezer and put seeds on a sterile filter paper.</div>
-			<div class="p_font_size">(12)Place seeds in the laminar flow cabinet and dried by blowing for about 2 hours.</div>
+			<div class="p_font_size">(12) Place seeds in the laminar flow cabinet and dried by blowing for about 2 hours.</div>
-			<div class="p_font_size">(13)Put sterile seeds on the surface of the callus initiation medium and seal plates with parafilm.</div>
+			<div class="p_font_size">(13) Put sterile seeds on the surface of the calli initiation medium and seal plates with parafilm.</div>
-			<div class="p_font_size">(14)Incubate seeds in the dark for about 14 days.</div>
+			<div class="p_font_size">(14) Incubate seeds in the dark for about 14 days.</div>
-			<div class="h4_font_size">2.2 Subculture of callus</div>
+			<div class="h4_font_size">2.2 Subculture of calli</div>
 			<div class="p_font_size"><strong>2.2.1 Medium preparation</strong></div>
+			<div align="center">
 			<table class="table">
 				<thead>
@@ Line 249: / Line 266: @@
 				</tbody>
 			</table>
+			</div>
 			<div class="p_font_size"><strong>2.2.2 Experimental procedures</strong></div>
-			<div class="p_font_size">(1)Place the callus initiation medium in the laminar flow cabinet.</div>
+			<div class="p_font_size">(1) Place the calli initiation medium in the laminar flow cabinet.</div>
-			<div class="p_font_size">(2)Use a sterile tweezer to divide callus from seeds and put callus on the subculture medium. Seal plates with parafilm.</div>
+			<div class="p_font_size">(2) Use a sterile tweezer to divide calli from seeds and put calli on the subculture medium. Seal plates with parafilm.</div>
-			<div class="p_font_size">(3)Incubate callus in the dark for 5 days.</div>
+			<div class="p_font_size">(3) Incubate calli in the dark for 5 days.</div>
-			<div class="h4_font_size">2.3 Co-culture of callus with Agrobacterium.</div>
+			<div class="h4_font_size">2.3 Co-culture of calli with <font style="font-style:italic">Agrobacterium</font></div>
 			<div class="p_font_size"><strong>2.3.1 Medium preparation</strong></div>
 			<div class="p_font_size">(1)</div>
+			<div align="center">
 			<table class="table">
 				<thead>
@@ Line 313: / Line 332: @@
 				</tbody>
 			</table>
+			</div>
 			<div class="p_font_size">(2)</div>
+			<div align="center">
 			<table class="table">
 				<thead>
@@ Line 375: / Line 396: @@
 				</tbody>
 			</table>
+			</div>
 			<div class="p_font_size"><strong>2.3.2 Experimental procedures</strong></div>
-			<div class="p_font_size">(1)Place the subculture medium into the laminar flow cabinet.</div>
+			<div class="p_font_size">(1) Place the subculture medium into the laminar flow cabinet.</div>
-			<div class="p_font_size">(2)Pick out the callus and put them on a sterile filter paper. Dry the surface of callus by blowing.</div>
+			<div class="p_font_size">(2) Pick out the calli and put them on a sterile filter paper. Dry the surface of calli by blowing.</div>
-			<div class="p_font_size">(3)Add 0.25 mL Acetosyringone to co-culture liquid medium.</div>
+			<div class="p_font_size">(3) Add 0.25 mL Acetosyringone to co-culture liquid medium.</div>
-			<div class="p_font_size">(4)Pour about 35 mL co-culture liquid medium into a 50 mL Erlenmeyer flask.</div>
+			<div class="p_font_size">(4) Pour about 35 mL co-culture liquid medium into a 50 mL Erlenmeyer flask.</div>
-			<div class="p_font_size">(5)Use a sterile stainless steel spoon to collect Agrobacterium transforment strain and suspend in the co-culture liquid medium. The co-culture liquid medium contain Agrobacterium also called Agrobacterium suspension.</div>
+			<div class="p_font_size">(5) Use a sterile stainless steel spoon to collect <font style="font-style:italic">Agrobacterium transforment</font> strain and suspend in the co-culture liquid medium. The co-culture liquid medium contain <font style="font-style:italic">Agrobacterium</font> also called <font style="font-style:italic">Agrobacterium</font> suspension.</div>
-			<div class="p_font_size">(6)Adjust the OD600 of Agrobacterium suspension to 0.1.</div>
+			<div class="p_font_size">(6) Adjust the OD600 of <font style="font-style:italic">Agrobacterium</font> suspension to 0.1.</div>
-			<div class="p_font_size">(7)Seal the Erlenmeyer flask with flask sealing film. Shake the Erlenmeyer flask in a shaker in 27 ℃ for 30 minutes.</div>
+			<div class="p_font_size">(7) Seal the Erlenmeyer flask with flask sealing film. Shake the Erlenmeyer flask in a shaker in 27 ℃ for 30 minutes.</div>
-			<div class="p_font_size">(8)After 30 minutes, take the Erlenmeyer flask out and put it in the laminar flow cabinet. Pick callus into the Agrobacterium suspension and gentle shake the Erlenmeyer flask for 20 minutes. Ensure that each callus contact with the Agrobacterium suspension directly.</div>
+			<div class="p_font_size">(8) After 30 minutes, take the Erlenmeyer flask out and put it in the laminar flow cabinet. Pick calli into the <font style="font-style:italic">Agrobacterium</font> suspension and gentle shake the Erlenmeyer flask for 20 minutes. Ensure that each calli contact with the <font style="font-style:italic">Agrobacterium</font> suspension directly.</div>
-			<div class="p_font_size">(9)Decant the Agrobacterium suspension. Pick out callus and put them on a sterile filter paper.<br>Dry the surface of callus by blowing.</div>
+			<div class="p_font_size">(9) Decant the <font style="font-style:italic">Agrobacterium</font> suspension. Pick out calli and put them on a sterile filter paper.<br>Dry the surface of calli by blowing.</div>
-			<div class="p_font_size">(10)At the same time, put a sterile filter paper on the surface of the co-culture medium. Dry the surface of the co-culture medium by blowing.</div>
+			<div class="p_font_size">(10) At the same time, put a sterile filter paper on the surface of the co-culture medium. Dry the surface of the co-culture medium by blowing.</div>
-			<div class="p_font_size">(11)When the surface of callus become dry, put them on the surface of the filter paper above the co-culture medium.</div>
+			<div class="p_font_size">(11) When the surface of calli become dry, put them on the surface of the filter paper above the co-culture medium.</div>
-			<div class="p_font_size">(12)Seal plates with parafilm. Incubate in the dark at 25 ℃ for 3 days.</div>
+			<div class="p_font_size">(12) Seal plates with parafilm. Incubate in the dark at 25 ℃ for 3 days.</div>
-			<div class="h4_font_size">2.4 First time selection of resistant callus</div>
+			<div class="h4_font_size">2.4 First time selection of resistant calli</div>
 			<div class="p_font_size"><strong>2.4.1 Medium preparation</strong></div>
+			<div align="center">
 			<table class="table">
 				<thead>
@@ Line 459: / Line 482: @@
 				</tbody>
 			</table>
+			</div>
 			<div class="p_font_size"><strong>2.4.2 Experimental procedures</strong></div>
-			<div class="p_font_size">(1)Place the co-culture medium in the laminar flow cabinet.</div>
+			<div class="p_font_size">(1) Place the co-culture medium in the laminar flow cabinet.</div>
-			<div class="p_font_size">(2)Add 1 mL 1000×Cefazolin sodium and 1 mL 1000×Carbenicillin sodium to 1 L sterile distilled water. This solution is Agrobacterium eluent.</div>
+			<div class="p_font_size">(2) Add 1 mL 1000×Cefazolin sodium and 1 mL 1000×Carbenicillin sodium to 1 L sterile distilled water. This solution is <font style="font-style:italic">Agrobacterium</font> eluent.</div>
-			<div class="p_font_size">(3)Pick out callus and put them in a 50 mL Erlenmeyer flask.</div>
+			<div class="p_font_size">(3) Pick out calli and put them in a 50 mL Erlenmeyer flask.</div>
-			<div class="p_font_size">(4)Add about 35 mL Agrobacterium eluent to the Erlenmeyer flask. Shake the Erlenmeyer flask for one minute. Decant the Agrobacterium eluent.</div>
+			<div class="p_font_size">(4) Add about 35 mL <font style="font-style:italic">Agrobacterium</font> eluent to the Erlenmeyer flask. Shake the Erlenmeyer flask for one minute. Decant the <font style="font-style:italic">Agrobacterium</font> eluent.</div>
-			<div class="p_font_size">(5)Repeat step 4 for about 20 times. Ensure that most of the Agrobacterium on callus are eluted.</div>
+			<div class="p_font_size">(5) Repeat step 4 for about 20 times. Ensure that most of the <font style="font-style:italic">Agrobacterium</font> on calli are eluted.</div>
-			<div class="p_font_size">(6)Pick out callus and put them on a sterile filter paper. Dry the surface of callus by blowing.</div>
+			<div class="p_font_size">(6) Pick out calli and put them on a sterile filter paper. Dry the surface of calli by blowing.</div>
-			<div class="p_font_size">(7)Put callus on the selecting medium.</div>
+			<div class="p_font_size">(7) Put calli on the selecting medium.</div>
-			<div class="p_font_size">(8)Seal plates with parafilm.</div>
+			<div class="p_font_size">(8) Seal plates with parafilm.</div>
-			<div class="p_font_size">(9)Culture in the dark for 30 days.</div>
+			<div class="p_font_size">(9) Culture in the dark for 30 days.</div>
-			<div class="h4_font_size">2.5 Second time selection of resistant callus</div>
+			<div class="h4_font_size">2.5 Second time selection of resistant calli</div>
 			<div class="p_font_size"><strong>2.5.1 Medium preparation</strong></div>
+			<div align="center">
 			<table class="table">
 				<thead>
@@ Line 540: / Line 565: @@
 				</tbody>
 			</table>
+			</div>
 			<div class="p_font_size"><strong>2.5.2 Experimental procedures</strong></div>
-			<div class="p_font_size">(1)Place the first time selecting medium in the laminar flow cabinet.</div>
+			<div class="p_font_size">(1) Place the first time selecting medium in the laminar flow cabinet.</div>
-			<div class="p_font_size">(2)Pick out callus and put them on the second time selecting medium.</div>
+			<div class="p_font_size">(2) Pick out calli and put them on the second time selecting medium.</div>
-			<div class="p_font_size">(3)Seal plates with parafilm.</div>
+			<div class="p_font_size">(3) Seal plates with parafilm.</div>
-			<div class="p_font_size">(4)Culture in the dark for 30 days.</div>
+			<div class="p_font_size">(4) Culture in the dark for 30 days.</div>
-			<div class="h4_font_size">2.6 Differentiation of resistant callus</div>
+			<div class="h4_font_size">2.6 Differentiation of resistant calli</div>
 			<div class="p_font_size"><strong>2.6.1 Medium preparation</strong></div>
+			<div align="center">
 			<table class="table">
 				<thead>
@@ Line 612: / Line 639: @@
 				</tbody>
 			</table>
+			</div>
 			<div class="p_font_size"><strong>2.5.2 Experimental procedures</strong></div>
-			<div class="p_font_size">(1)Place the first time selecting medium in the laminar flow cabinet.</div>
+			<div class="p_font_size">(1) Place the first time selecting medium in the laminar flow cabinet.</div>
-			<div class="p_font_size">(2)Pick out callus and put them on the second time selecting medium.</div>
+			<div class="p_font_size">(2) Pick out calli and put them on the second time selecting medium.</div>
-			<div class="p_font_size">(3)Seal plates with parafilm.</div>
+			<div class="p_font_size">(3) Seal plates with parafilm.</div>
-			<div class="p_font_size">(4)Culture in the dark for 30 days.</div>
+			<div class="p_font_size">(4) Culture in the dark for 30 days.</div>
-			<div class="h4_font_size">2.6 Differentiation of resistant callus</div>
+			<div class="h4_font_size">2.6 Differentiation of resistant calli</div>
 			<div class="p_font_size"><strong>2.6.1 Medium preparation</strong></div>
+			<div align="center">
 			<table class="table">
 				<thead>
@@ Line 684: / Line 713: @@
 				</tbody>
 			</table>
+			</div>
 			<div class="p_font_size"><strong>2.6.2 Experimental procedures</strong></div>
-			<div class="p_font_size">(1)Place the second time selecting medium in the laminar flow cabinet.</div>
+			<div class="p_font_size">(1) Place the second time selecting medium in the laminar flow cabinet.</div>
-			<div class="p_font_size">(2)Pick out those yellow callus that generate after two times of selection. Put them on the differentiation medium.</div>
+			<div class="p_font_size">(2) Pick out those yellow calli that generate after two times of selection. Put them on the differentiation medium.</div>
-			<div class="p_font_size">(3)Seal plates with parafilm.</div>
+			<div class="p_font_size">(3) Seal plates with parafilm.</div>
-			<div class="p_font_size">(4)Culture under the condition of 14 hours light and 10 hours dark a days, for about 25 days.</div>
+			<div class="p_font_size">(4) Culture under the condition of 14 hours light and 10 hours dark a days, for about 25 days.</div>
-			<div class="p_font_size">(5)After 25 days, green spot will appear on the surface of the resistant callus.</div>
+			<div class="p_font_size">(5) After 25 days, green spot will appear on the surface of the resistant calli.</div>
-			<div class="p_font_size">(6)Place the differentiation medium in the laminar flow cabinet.</div>
+			<div class="p_font_size">(6) Place the differentiation medium in the laminar flow cabinet.</div>
-			<div class="p_font_size">(7)Choose callus which has green spot. Transfer them to a new differentiation medium.</div>
+			<div class="p_font_size">(7) Choose calli which has green spot. Transfer them to a new differentiation medium.</div>
-			<div class="p_font_size">(8)Seal cuture bottles.</div>
+			<div class="p_font_size">(8) Seal cuture bottles.</div>
-			<div class="p_font_size">(9)Culture under the condition of 14 hours light and 10 hours dark a days, for about 30 days.</div>
+			<div class="p_font_size">(9) Culture under the condition of 14 hours light and 10 hours dark a days, for about 30 days.</div>
 			<div class="h4_font_size">2.7 Rooting the seedling</div>
 			<div class="p_font_size"><strong>2.7.1 Medium preparation</strong></div>
+			<div align="center">
 			<table class="table">
 				<thead>
@@ Line 752: / Line 783: @@
 					</tr>
 				</tbody>
 			</table>
+			</div>
 			<div class="p_font_size"><strong>2.7.2 Experimental procedures</strong></div>
-			<div class="p_font_size">(1)Place the differentiation medium in the laminar flow cabinet.</div>
+			<div class="p_font_size">(1) Place the differentiation medium in the laminar flow cabinet.</div>
-			<div class="p_font_size">(2)Carefully pick the seedling out with a sterile tweezer. Transfer it to rooting medium.</div>
+			<div class="p_font_size">(2) Carefully pick the seedling out with a sterile tweezer. Transfer it to rooting medium.</div>
-			<div class="p_font_size">(3)Seal culture bottles.</div>
+			<div class="p_font_size">(3) Seal culture bottles.</div>
-			<div class="p_font_size">(4)Culture under the condition of 14 hours light and 10 hours dark a days, for about 20 days.</div>
+			<div class="p_font_size">(4) Culture under the condition of 14 hours light and 10 hours dark a days, for about 20 days.</div>
 			<div class="h4_font_size">2.8 Hardening the seedling</div>
 			<div class="p_font_size"><strong>2.8.1 Experimental procedures</strong></div>
-			<div class="p_font_size">(1)Choose seedling where are 8 cm high and grown strong roots.</div>
+			<div class="p_font_size">(1) Choose seedling where are 8 cm high and grown strong roots.</div>
-			<div class="p_font_size">(2)Open the lid of culture bottle. Add appropriate amount of distilled water.</div>
+			<div class="p_font_size">(2) Open the lid of culture bottle. Add appropriate amount of distilled water.</div>
-			<div class="p_font_size">(3)Culture in the greenhouse for 7 days.</div>
+			<div class="p_font_size">(3) Culture in the greenhouse for 7 days.</div>
 			<div class="h4_font_size">2.9 Transplanting the seedling</div>
 			<div class="p_font_size"><strong>2.9.1 Experimental procedures</strong></div>
-			<div class="p_font_size">(1)After hardening the seedling, take the seeding out and remove the medium on its roots with running water.</div>
+			<div class="p_font_size">(1) After hardening the seedling, take the seeding out and remove the medium on its roots with running water.</div>
-			<div class="p_font_size">(2)Transplant the seedling to soil. Make sure that the seedling isn’t submergence or insolated.</div>
+			<div class="p_font_size">(2) Transplant the seedling to soil. Make sure that the seedling isn’t submergence or insolated.</div>
 			<div class="h2_font_size">Extraction and detection</div>
 			<div class="h3_font_size">1. The method of extracting the astaxanthin in rice endosperm</div>
-			<div class="p_font_size">1. Take 0.1 g rice seeds into mortar. After grinding it into powder on the ice, add 2 mL of methanol in mortar, and continue to grind for 5 minutes.</div>
+			<div class="p_font_size">(1) Take 0.1 g rice seeds into mortar. After grinding it into powder on the ice, add 2 mL of methanol in mortar, and continue to grind for 5 minutes.</div>
-			<div class="p_font_size">2. Transfer the powder to a 2 mL centrifuge tube, spin at 10000 rpm for 10 min in the dark and low temperature condition to extract </div>
+			<div class="p_font_size">(2) Transfer the powder to a 2 mL centrifuge tube, spin at 10000 rpm for 10 min in the dark and low temperature condition to extract. </div>
-			<div class="p_font_size">3. Centrifuge at 8000rpm at 4 ℃for 5 minutes and collect supernatant.</div>
+			<div class="p_font_size">(3) Centrifuge at 8000rpm at 4 ℃ for 5 minutes and collect supernatant.</div>
-			<div class="p_font_size">4. Extract the sediment with methanol repeatedly, until the precipitation become white.</div>
+			<div class="p_font_size">(4) Extract the sediment with methanol repeatedly, until the precipitation become white.</div>
-			<div class="p_font_size">5. Centrifuge again(8000rpm, 4 ℃, 5 min)and collect supernatant .</div>
+			<div class="p_font_size">(5) Centrifuge again(8000rpm, 4 ℃, 5 min)and collect supernatant .</div>
-			<div class="p_font_size">6. Merge supernatant, concentrate supernatant into powder in the dark and low temperature conditions</div>
+			<div class="p_font_size">(6) Merge supernatant, concentrate supernatant into powder in the dark and low temperature conditions</div>
-			<div class="p_font_size">7. Add 600 µL methanol into dry astaxanthin extraction.</div>
+			<div class="p_font_size">(7) Add 600 µL methanol into dry astaxanthin extraction.</div>
-			<div class="h3_font_size">2. HPLC(High Performance Liquid Chromatography)determination of astaxanthin</div>
+			<div class="h3_font_size">2. HPLC(High Performance Liquid Chromatography) determination of astaxanthin</div>
-			<div class="p_font_size">After enrichment, put the samples into a 2 mL brown bottle through 0.22µm syringe-driven filter, then detect the samples by HPLC. The mobile phase is methanol: water = 95:5. The flow rate is 1 mL/min. Column temperature is room temperature. The detection wavelength is 480 nm.</div>
+			<div class="p_font_size">After enrichment, put the samples into a 2 mL brown bottle through 0.22 µm syringe-driven filter, then detect the samples by HPLC. The mobile phase is methanol: water = 95:5. The flow rate is 1 mL/min. Column temperature is room temperature. The detection wavelength is 480 nm.</div>
 			<div class="h3_font_size">3. Standard curve for the determination of astaxanthin</div>
-			<div class="p_font_size">Dissolve accurately 10 mg of standard sample of astaxanthin in 1mL methylene chloride, and dilute with methanol to 100mL in a brown volumetric flask and get the 0.1 mg/mL solution. Dilute the above-mentioned solution with methanol into 2µg/mL, 4µg/mL, 6 µg/mL, 8 µg/mL, 10 µg/mL. Take 20µL sample to detect separately according to the conditions that mentioned above. With peak area as the ordinate, the concentration of the standard sample for horizontal, apply linear regression analysis, and get the regression equation.</div>
+			<div class="p_font_size">Dissolve accurately 10 mg of standard sample of astaxanthin in 1 mL methylene chloride, and dilute with methanol to 100 mL in a brown volumetric flask and get the 0.1 mg/mL solution. Dilute the above-mentioned solution with methanol into 2 µg/mL, 4 µg/mL, 6 µg/mL, 8 µg/mL, 10 µg/mL. Take 20 µL sample to detect separately according to the conditions that mentioned above. With peak area as the ordinate, the concentration of the standard sample for horizontal, apply linear regression analysis, and get the regression equation.</div>
-			<div class="h3_font_size">4.Conclusion</div>
+			<div class="h3_font_size">4. Conclusion</div>
 			<div class="p_font_size">The method of detecting the astaxanthin content in transgenic rice endosperm: grind transgenic rice seeds into powder on the ice, extract the sediment with methanol repeatedly, until the precipitation become white, concentrate the supernatant into powder, dissolved in methanol, use the high performance liquid chromatography (HPLC) to measure the content of astaxanthin.</div>
 			<div class="h3_font_size">References</div>
@@ Line 788: / Line 820: @@
 			<div class="p_font_size">【3】J. C. Huang, Y. J. Zhong, J. Liu, G. Sandmann, F. Chen, Metabolic engineering of tomato for high-yield production of astaxanthin. Metabolic Engineering. 17, 59–67(2013) </div>
 			<div class="p_font_size">【4】J. Breitenbach, C. Bai, S. M. Rivera, R. Canela, T. Capell, Paul Christou, C. f. Zhu, G. Sandmann, A novel carotenoid, 4-keto-a-carotene, as an unexpected by-product during genetic engineering of caroteno genesis in rice callus.  Phytochemistry. 98, 85–91(2014) </div>
-		</div>
+                        <div class="p_font_size">【5】Liu Y-G, Shirano Y, Fukaki H, Yanai Y, Tasaka M, Tabata S, Shibata D. Complementation of plant mutants with large genomic DNA fragments by a transformation-competent artificial chromosome vector accelerates positional cloning. PNAS. 96, 6535–6540 (1999).
-<div class="p_font_size">【5】Liu Y-G, Shirano Y, Fukaki H, Yanai Y, Tasaka M, Tabata S, Shibata D. Complementation of plant mutants with large genomic DNA fragments by a transformation-competent artificial chromosome vector accelerates positional cloning. PNAS. 96, 6535–6540 (1999).
 </div>
-		</div>
+                        <div class="p_font_size">【6】Lin L, Liu Y-G, Xu X, L B. Efficient linking and transfer of multiple genes by a multigene assembly and transformation vector system. PNAS. 100: 5962-5967(2003).
-<div class="p_font_size">【6】Lin L, Liu Y-G, Xu X, L B. Efficient linking and transfer of multiple genes by a multigene assembly and transformation vector system. PNAS. 100: 5962-5967(2003).
 </div>
-		</div>
+                        <div class="p_font_size">【7】Zhu Q, Liu Y-G. A novel TransGene Stacking II system (TGSII) for plant multigene metabolic engineering. (in prepared and unpublished)
-<div class="p_font_size">【7】Zhu Q, Liu Y-G. A novel TransGene Stacking II system (TGSII) for plant multigene metabolic engineering. (in prepared and unpublished)
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