Team:SCAU-China/Protocol

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for astaxanthin biosynthesis, we used a modified multigene vector system, TransGene Stacking II (TGSII) (Zhu et al., unpublished).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alternative use of the donor vectors, and multiple genes were sequentially delivered into the TAC vector (Zhu et al., unpublished).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
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【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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