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

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<div class="h2_font_size">1. Vector for genes stacking </div>
 
<div class="h2_font_size">1. Vector for genes stacking </div>
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     <div class="p_font_size" style="text-indent:0em"><em>PSY</em> (phytoene synthase) catalyzes Geranylgeranyl-PP into Phytoene. The gene <em>CrtI</em> from Erwinia uredovora could finish the catalysis process from Phytoene to Lycopene. Therefore, when these two genes(<em>PSY</em> and <em>CrtI</em>) with specific promoters of endosperm ahead ,they will express CRTISO and β-LCY enzymes which synthesize β-Carotene to produce the famous Golden Rice. There are still two steps from β-Carotene to astaxanthin: <em>BHY</em> (β-carotenehy droxylase) catalyze β-Carotene to Zeaxanthin. And <em>BKT</em> catalyzes directly to synthesize the end product astaxnthin. The expression of  the endogenous gene <em>BHY</em> in rice is still unknown, accordingly it needs at least 3 genes (<em>PSY</em>+<em>CrtI</em> I+<em>BKT</em>, BPC) or 4 genes(<em>PSY</em>+<em>CrtI</em>+<em>BKT</em>+<em>BHY</em>, BBPC) to synthesize astaxanthin. For the combination of three genes, if the endogenous gene <em>BHY</em> of rice has little expression, maybe there will be just a little astaxanthin produced(even nothing!). But for the combination of four genes, it will create a complete metabolic pathway for astaxanthin production. Surely, it could produce astaxanthin. Thus we use the systems of Assembly of multiple genes and transformation, and the specific promoters of endosperm to construct three vectors(380-PC, BPC and BBPC) to study the metabolic of the synthesis of astaxanthin in the endosperm of rice. And the four genes we use were synthesized and codon optimization according to the preferred codons in rice.
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     <div class="p_font_size" style="text-indent:0em">The biosynthesis of astaxanthin from Geranylgeranyl-PP requires at four genes, PSY, CrtI, BKT and BHY. When endosperm-specifically expressed PSY and CrtI genes, together with the expression of rice endogenous β-LCY gene, the β-Carotene (pro-vitamin A) was synthesized to produce the famous Golden Rice. BHY (β-carotene hydroxylase) catalyze 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 transform these four genes into rice to study the metabolic synthesis of astaxanthin in endosperm.
 
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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. By this way, multiple genes can be easily stacking into a TAC-based binary acceptor vector. (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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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>
 
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<div class="p_font_size">Figure 3 Physic map of the multigene vector 380MF-BBPC for biosynthesizing astaxanthin and marker-free deletion.
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<div class="h2_font_size">2.Experimental design</div>
 
<div class="h2_font_size">2.Experimental design</div>
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<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 elementwere 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 3.
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<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 elementwere 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.
 
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<div class="p_font_size"><small> <font style="font-weight:bold">Figure. 3.  </font> The schematic diagram of Astaxanthin Rice project.</small>
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<div class="p_font_size"><small> <font style="font-weight:bold">Figure 4.  </font> The schematic diagram of Astaxanthin Rice project.</small>
 
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In this part, we used Cre/loxp site-specific recombination method to delete the selective marker (Figure 4). 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.
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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.
 
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<small><font style="font-weight:bold">Figure. 4.</font>  The schematic diagram of the marker-free process. PV4 is an anther-specific promoter that drives <em>Cre</em> gene expression in anther.</samll>
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<small><font style="font-weight:bold">Figure 5.</font>  The schematic diagram of the marker-free process. PV4 is an anther-specific promoter that drives <em>Cre</em> gene expression in anther.</samll>
 
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</div><div class="p_font_size">【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)  
 
</div><div class="p_font_size">【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)  
 
</div><div class="p_font_size">【5】Giovanni Giuliano. Plant carotenoids: genomics meets multi-gene engineering.  Plant Biology.  19, 111–117 (2014)
 
</div><div class="p_font_size">【5】Giovanni Giuliano. Plant carotenoids: genomics meets multi-gene engineering.  Plant Biology.  19, 111–117 (2014)
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</div><div class="p_font_size">【6】Yook Jang Soo, Okamoto Masahiro, Rakwal Randeep, Shibato Junko, Lee Min Chul, Matsui Takashi, Chang Hyukki, Cho Joon Yong, Soya Hideaki.  
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</div><div class="p_font_size">【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).
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</div><div class="p_font_size">【7】Astaxanthin?supplementation?enhances?adult?hippocampal neurogenesis?and?spatial?memory?inmice. Molecular nutrition & food research. 60 , 589-599(2016)
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</div><div class="p_font_size">【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).
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<div class="p_font_size">【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).
 +
</div>
 +
<div class="p_font_size">【9】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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Revision as of 13:46, 15 October 2016

SCAU

Design
1. Vector for genes stacking
The biosynthesis of astaxanthin from Geranylgeranyl-PP requires at four genes, PSY, CrtI, BKT and BHY. When endosperm-specifically expressed PSY and CrtI genes, together with the expression of rice endogenous β-LCY gene, the β-Carotene (pro-vitamin A) was synthesized to produce the famous Golden Rice. BHY (β-carotene hydroxylase) catalyze 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 transform 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 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!  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 elementwere 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 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.
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)