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

Revision as of 07:16, 16 October 2016

SCAU

Design

1. Vector for genes stacking

The biosynthesis of astaxanthin from geranylgeranyl-PP requires four genes, PSY, CrtI, BKT and BHY. When endosperm specifically expresses PSY and CrtI genes, together with the expression of rice endogenous β-LCY gene, the β-carotene (pro-vitamin A) will be synthesized to produce the famous Golden Rice. BHY (β-carotene hydroxylase) catalyzes β-carotene to zeaxanthin, and BKT (β-carotene ketolase) catalyzes zeaxanthin to form the end product astaxanthin. For suitable expression in rice, the all four genes were codon-optimized and the pea RUBISCO chloroplast transit peptide was fused on three non-plant-origin genes (CrtI, BKT and BHY), respectively. Therefore, we utilized a multigene vector system to assemble and transformed these four genes into rice to study the metabolic synthesis of astaxanthin in endosperm.

To assemble these four genes of astaxanthin biosynthetic pathway in rice endosperm, a modified multigene vector system, TransGene Stacking II (TGSII), was used. 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. Finally, after completing four-gene assembly, the marker-free element, containing a HPT selective 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. By this way, multiple genes and a maker-free element can be easily stacked into a TAC-based binary acceptor vector (Figure 3.) (Liu et al., PNAS, 1999, 96: 6535-6540; Lin et al., PNAS, 2003, 100: 5962-5967; Zhu et al., unpublished).You can read more details by click here! part and protocol

Figure 3. Physic map of the multigene vector 380MF-BBPC for biosynthesizing astaxanthin and marker-free deletion.

2. Experimental design

Firstly, the nucleic acid sequences of four genes have been codon optimized and directly synthesized for stable expression in rice. Then, these genes were subcloned into endosperm-specific gene cassettes of two donors. Secondly, these genes and a marker-free element were assembled into a TAC-based binary vector by using a transgene stacking II system. Finally, the obtained marker-free multigene vector was transferred into Agrobacterium tumefaciens strain EHA105 for rice callus transformation. The transgenic plants were identified by analyses of PCR, RT-PCR, qRT-PCR and HPLC. The schematic diagram of our project was shown in Figure 4.

Figure 4. The schematic diagram of Astaxanthin Rice project.

3. Marker free

Selective markers are applied in the process of positive individuals screening, while markers are considered to be useless after screening. However, resistant gene-based selective markers, such as HPT (hygromycin), attracted much more attention due to its biologically potential danger. One is that drug resistance of pathogenic microbes would be obtained through gene drift. Another is products coded by these selective genes might be a new types of allergen in food made from transgenic plants. Thus, it is strongly necessary to remove the selective markers after transgenic screening.

In this part, we used Cre/loxp site-specific recombination method to delete the selective marker (Figure 5). To delete the selective resistance gene in transgenic rice, a marker-free element was used to assemble into four-gene multigene vector. This marker-free element was placed between two loxP sites, and consists of a HPT (hygromycin) resistance gene expression cassette and a Cre gene expression cassette controlled by anther-specific promoter. When Cre gene was expressed in transgenic rice anther, the Cre enzyme deleted the marker-free element between two loxP sites.

Figure 5. The schematic diagram of the marker-free process. PV4 is an anther-specific promoter that drives Cre gene expression in anther.

References

【1】Varda Mann, Mark Harker, Iris Pecker, and Joseph Hirschberg. Metabolic engineering of astaxanthin production in tobacco flowers. Nature Biotechnology . 18, 888-892 (2002)

【2】Salim Al-Babili, Peter Beyer. Golden Rice–five years on the road–five years to go? Trends in Plant Science. 10, 12, 565-573 (2005)

【3】Jacqueline A Paine, Catherine A Shipton, Sunandha Chaggar, Rhian M Howells, Mike J Kennedy, Gareth Vernon, Susan Y Wright, Edward Hinchliffe, Jessica L Adams, Aron L Silverstone, Rachel Drake. Improving the nutritional value of Golden Rice through increased pro-vitamin A content. Nature Biotechnology. 23, 4, 482-487 (2005)

【4】Cong-Ping Tan, Fang-Qing Zhao, Zhong-Liang Su, Cheng-Wei Liang, Song Qin. Expression of β-carotene hydroxylase gene (crtR-B) from the green alga Haematococcus pluvialis in chloroplasts of Chlamydomonas reinhardtii. J Appl Phycol . 19, 347–355 (2007)

【5】Giovanni Giuliano. Plant carotenoids: genomics meets multi-gene engineering. Plant Biology. 19, 111–117 (2014)

【6】Yook JS, Okamoto M, Rakwal R, Shibato J, Lee MC1, Matsui T, Chang H, Cho JY, Soya H. Astaxanthin Supplementation Enhances Adult Hippocampal Neurogenesis and Spatial Memory in Mice. Molecular nutrition & food research. 60 , 589-599 (2016).

【7】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).

【8】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).

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

@@ Line 103: / Line 103: @@
 Design
 </div>
-	<div class="h2_font_size">1.	Vector for genes stacking </div>
+	<div class="h2_font_size">1. Vector for genes stacking </div>
      <div class="p_font_size" style="text-indent:0em">The biosynthesis of astaxanthin from geranylgeranyl-PP requires four genes, <font style="font-style:italic">PSY, CrtI, BKT</font> and <font style="font-style:italic">BHY</font>. When endosperm specifically expresses <font style="font-style:italic">PSY</font> and <font style="font-style:italic">CrtI</font> genes, together with the expression of rice endogenous <font style="font-style:italic">β-LCY</font> gene, the β-carotene (pro-vitamin A) will be synthesized to produce the famous Golden Rice.  <font style="font-style:italic">BHY</font> (β-carotene hydroxylase) catalyzes β-carotene to zeaxanthin, and <font style="font-style:italic">BKT</font> (β-carotene ketolase) catalyzes zeaxanthin to form the end product astaxanthin. For suitable expression in rice, the all four genes were codon-optimized and the pea RUBISCO chloroplast transit peptide was fused on three non-plant-origin genes <font style="font-style:italic">(CrtI, BKT</font> and <font style="font-style:italic">BHY)</font>, respectively. Therefore, we utilized a multigene vector system to assemble and transformed these four genes into rice to study the metabolic synthesis of astaxanthin in endosperm.
 </div>
@@ Line 109: / Line 109: @@
 <div class="p_font_size">
-To assemble these four genes of astaxanthin biosynthetic pathway in rice endosperm, a modified multigene vector system, TransGene Stacking II (TGSII), was used. 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. Finally, after completing four-gene assembly, the marker-free element, containing a HPT selective 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. By this way, multiple genes and a maker-free element can be easily stacking into a TAC-based binary acceptor vector (Figure 3) (Liu et al., PNAS, 1999, 96: 6535-6540； Lin et al., PNAS, 2003, 100: 5962-5967；Zhu et al., unpublished).You can read more details by click here!   <a href="https://2016.igem.org/Team:SCAU-China/Basic_Part">part</a> and <a href="https://2016.igem.org/Team:SCAU-China/Protocol"> protocol </a>
+To assemble these four genes of astaxanthin biosynthetic pathway in rice endosperm, a modified multigene vector system, TransGene Stacking II (TGSII), was used. 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. Finally, after completing four-gene assembly, the marker-free element, containing a HPT selective 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. By this way, multiple genes and a maker-free element can be easily stacked into a TAC-based binary acceptor vector (Figure 3.) (Liu et al., PNAS, 1999, 96: 6535-6540; Lin et al., PNAS, 2003, 100: 5962-5967; Zhu et al., unpublished).You can read more details by click here!   <a href="https://2016.igem.org/Team:SCAU-China/Basic_Part">part</a> and <a href="https://2016.igem.org/Team:SCAU-China/Protocol"> protocol </a>
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 <div class="col s1">
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 <div class="p_font_size"><small> <font style="font-weight:bold">Figure 3.</font> &nbsp;&nbsp;Physic map of the multigene vector 380MF-BBPC for biosynthesizing astaxanthin and marker-free deletion.</small>
 </div>
-<div class="h2_font_size">2.Experimental design</div>
+<div class="h2_font_size">2. Experimental design</div>
 <div class="p_font_size">Firstly, the nucleic acid sequences of four genes have been codon optimized and directly synthesized for stable expression in rice. Then, these genes were subcloned into endosperm-specific gene cassettes of two donors. Secondly, these genes and a marker-free element were assembled into a TAC-based binary vector by using a transgene stacking II system. Finally, the obtained marker-free multigene vector was transferred into <em>Agrobacterium tumefaciens</em> strain EHA105 for rice callus transformation. The transgenic plants were identified by analyses of PCR, RT-PCR, qRT-PCR and HPLC. The schematic diagram of our project was shown in Figure 4.
 </div>
@@ Line 136: / Line 136: @@
 </div>
 <br>
-<div  class="h2_font_size">3.Marker free</div>
+<div  class="h2_font_size">3. Marker free</div>
 <div class="p_font_size">
-Selective markers are applied in the process of positive individuals screening, while markers are considered to be useless after screening. However, resistant gene-based selective markers, such as HPT (hygromycin), attracted much more attention due to its biologically potential danger. One is that drug resistance of pathogenic microbes would be obtained through gene drift. Another is products codes by these selective genes would be a new types of allergen in food made from transgenic plant. Thus, it is strongly necessary to remove the selective markers after transgenic screening.
+Selective markers are applied in the process of positive individuals screening, while markers are considered to be useless after screening. However, resistant gene-based selective markers, such as HPT (hygromycin), attracted much more attention due to its biologically potential danger. One is that drug resistance of pathogenic microbes would be obtained through gene drift. Another is products coded by these selective genes might be a new types of allergen in food made from transgenic plants. Thus, it is strongly necessary to remove the selective markers after transgenic screening.
 </div>
 <div class="p_font_size">