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| − | + | <div class="h1_font_size">1. Vector for genes stacking </div> | |
| − | + | <div class="p_font_size">PSY (phytoene synthase) catalyzes Geranylgeranyl-PP into Phytoene. The gene CrtI from Erwinia uredovora could finish the catalysis process from Phytoene to Lycopene. Therefore, when these two genes(PSY and CrtI) with specific promoters of endosperm ahead ,they will express CRTISO and β-LCY enzymes which synthesizeβ-Carotene to produce the famous Golden Rice. There are still two steps from β-Carotene to astaxanthin: BHY (β-carotenehy droxylase) catalyze β-Carotene to Zeaxanthin. And BKT catalyzes directly to synthesize the end product astaxnthin. The expression of the endogenous gene BHY in rice is still unknown, accordingly it needs at least 3 genes (PSY+Crt I+BKT，BPC) or 4 genes(PSY+Crt I+BKT+BHY, BBPC) to synthesize astaxanthin. For the combination of three genes, if the endogenous gene BHY 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. | |
| − | + | </div> | |
| − | + | <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. 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 (Liu et al., PNAS, 1999, 96: 6535-6540； Lin et al., PNAS, 2003, 100: 5962-5967；Zhu et al., unpublished)You can read more details by click here! part (转到Part栏目）and protocol (转到Notebook 栏目下的protocol栏目） | |
| − | + | <div class="p_font_size"> | |
| − | + | <div class="h1_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 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 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. | |
| − | + | </div> | |
| − | + | <div class="p_font_size">Figure 3 The schematic diagram of Astaxanthin Rice project. | |
| − | + | </div> | |
| − | + | <h2>Diagram of our project design: | |
| − | </div> | + | </h2> |
| − | + | <div class="p_font_size"> | |
| − | + | (1)The sequence of the four transgenes are accessed from Genbank and codon optimized for expression in Orazy sativa. | |
| − | + | </div><div class="p_font_size">(2)Genes together with expressive elements are integrated into one plasmid utilizing E.coli; | |
| − | + | </div><div class="p_font_size">(3)Construct competent for transformation is transferred into Agrobacterium; | |
| − | + | </div><div class="p_font_size">(4)Multiple genes are delivered into rice genome. And then, culture the transgenic plant and harvest astaxanthin rice! | |
| − | + | </div> | |
| + | <div class="h1_font_size">3.Marker free</div> | ||
| + | <div class="p_font_size"> | ||
| + | In this part, we mainly introduce the work we used cre/loxp recombination，a site-specific recombinase technology to delete the selective marker. | ||
| + | One of the reason why transgenetic plant thought to be dangerous is that various selective markers are applied in the process of positive individuals screening. Selective markers utilize in screening to distinct the transgenetic individuals from negative ones while markers are considered to be useless after screening. Both resistant gene and reporter gene, such as hygromycin phosphotransferase and green fluorescent protein, can be used as selective markers. However, resistant gene-based selective markers attracted much more attention due to its biologically potential danger. Questions about resistant gene-based selective markers’ safety can divide into two parts. 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 transgenetic plant. Thus, it is necessary to remove the selective markers after screening. | ||
| + | </div> | ||
| + | <div class="p_font_size"> | ||
| + | Concerning the potential dangers of selective markers, we design a Cre-loxP based side-direct recombinant system to knock out hygromycin phosphotransferase gene in pollen utilizing Cre recombinase under the regulation of pollen-specific promoter Pv4. After performing selfing, we obtain the marker-free homozygous transgenetic organisms. Though plant had been used in the past iGEM projects, we utilize Orazy Sativa as a chassis and propose a marker-deletion system for the first time in iGEM. | ||
| + | </div> | ||
| + | <div class="p_font_size">DNA recombination mediated by Cre recombinase has become an important tool to generate marker-free transgentic plants because it is time-specific, tissue-specific and site-specific. | ||
| + | </div> | ||
| + | <div class="p_font_size"> | ||
| + | The Cre/loxP system of bacterio-phage P1 is a site-specific recombination system that consists of two components:the recombinase(Cre)and its recognition sites(loxP).Cre mediates recombination events and causes the excision of the DNA segment between two directly adjacent loxP sites. Besides，The result of recombination depends on the orientation of the loxP sites. For two lox sites on the same chromosome arm, inverted loxP sites will cause an inversion of the intervening DNA, while a direct repeat of loxP sites will cause a deletion event.If loxP sites are on different chromosomes, it is possible fortranslocation events to be catalysed by Cre thus inducing the recombination. | ||
| + | </div> | ||
| + | <div class="p_font_size"> | ||
| + | Figure 4 The schematic diagram of the marker-free element. PV4 is an anther-specific promoter that drives Cre gene expression in anther. | ||
| + | </div> | ||
| + | <div class="p_font_size"> | ||
| + | If we want to assemble an tissue-specific promoter ahead of the Cre gene, the process of delection could be regulated when we designed a direct repeat of loxP sites with the intervening marker gene. As a result, we can delete the gene wherever it is located on. By using tissue-specific promoter, we have got the marker-free pollens. | ||
| + | </div> | ||
| + | <div class="p_font_size"> | ||
| + | We found Pv4, a rice anther specific promoter. When our transgentic plants grow up, the Cre gene will be expressed which should be drived by Pv4, thus only in the anther of stamens. Hence,we got the marker-free pollens. Through selfing , we can get the homozygote of marker-free transgentic plants. | ||
| + | </div> | ||
| + | <div class="h1_font_size">references</div> | ||
| + | <div class="p_font_size"> | ||
| + | 【1】Varda Mann, Mark Harker, Iris Pecker, and Joseph Hirschberg. Metabolic engineering of astaxanthin production in tobacco flowers. Nature Biotechnology . 18, 888-892 (2002) | ||
| + | </div><div class="p_font_size">【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) | ||
| + | </div><div class="p_font_size">【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) | ||
| + | </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">【6】Yook Jang Soo, Okamoto Masahiro, Rakwal Randeep, Shibato Junko, Lee Min Chul, Matsui Takashi, Chang Hyukki, Cho Joon Yong, Soya Hideaki. | ||
| + | </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) | ||
| + | </div> | ||
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Revision as of 14:40, 13 October 2016
1. Vector for genes stacking
PSY (phytoene synthase) catalyzes Geranylgeranyl-PP into Phytoene. The gene CrtI from Erwinia uredovora could finish the catalysis process from Phytoene to Lycopene. Therefore, when these two genes(PSY and CrtI) with specific promoters of endosperm ahead ,they will express CRTISO and β-LCY enzymes which synthesizeβ-Carotene to produce the famous Golden Rice. There are still two steps from β-Carotene to astaxanthin: BHY (β-carotenehy droxylase) catalyze β-Carotene to Zeaxanthin. And BKT catalyzes directly to synthesize the end product astaxnthin. The expression of the endogenous gene BHY in rice is still unknown, accordingly it needs at least 3 genes (PSY+Crt I+BKT，BPC) or 4 genes(PSY+Crt I+BKT+BHY, BBPC) to synthesize astaxanthin. For the combination of three genes, if the endogenous gene BHY 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.
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! part (转到Part栏目）and protocol (转到Notebook 栏目下的protocol栏目）
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 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.
Figure 3 The schematic diagram of Astaxanthin Rice project.
Diagram of our project design:
(1)The sequence of the four transgenes are accessed from Genbank and codon optimized for expression in Orazy sativa.
(2)Genes together with expressive elements are integrated into one plasmid utilizing E.coli;
(3)Construct competent for transformation is transferred into Agrobacterium;
(4)Multiple genes are delivered into rice genome. And then, culture the transgenic plant and harvest astaxanthin rice!
3.Marker free
In this part, we mainly introduce the work we used cre/loxp recombination，a site-specific recombinase technology to delete the selective marker.
One of the reason why transgenetic plant thought to be dangerous is that various selective markers are applied in the process of positive individuals screening. Selective markers utilize in screening to distinct the transgenetic individuals from negative ones while markers are considered to be useless after screening. Both resistant gene and reporter gene, such as hygromycin phosphotransferase and green fluorescent protein, can be used as selective markers. However, resistant gene-based selective markers attracted much more attention due to its biologically potential danger. Questions about resistant gene-based selective markers’ safety can divide into two parts. 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 transgenetic plant. Thus, it is necessary to remove the selective markers after screening.
Concerning the potential dangers of selective markers, we design a Cre-loxP based side-direct recombinant system to knock out hygromycin phosphotransferase gene in pollen utilizing Cre recombinase under the regulation of pollen-specific promoter Pv4. After performing selfing, we obtain the marker-free homozygous transgenetic organisms. Though plant had been used in the past iGEM projects, we utilize Orazy Sativa as a chassis and propose a marker-deletion system for the first time in iGEM.
DNA recombination mediated by Cre recombinase has become an important tool to generate marker-free transgentic plants because it is time-specific, tissue-specific and site-specific.
The Cre/loxP system of bacterio-phage P1 is a site-specific recombination system that consists of two components:the recombinase(Cre)and its recognition sites(loxP).Cre mediates recombination events and causes the excision of the DNA segment between two directly adjacent loxP sites. Besides，The result of recombination depends on the orientation of the loxP sites. For two lox sites on the same chromosome arm, inverted loxP sites will cause an inversion of the intervening DNA, while a direct repeat of loxP sites will cause a deletion event.If loxP sites are on different chromosomes, it is possible fortranslocation events to be catalysed by Cre thus inducing the recombination.
Figure 4 The schematic diagram of the marker-free element. PV4 is an anther-specific promoter that drives Cre gene expression in anther.
If we want to assemble an tissue-specific promoter ahead of the Cre gene, the process of delection could be regulated when we designed a direct repeat of loxP sites with the intervening marker gene. As a result, we can delete the gene wherever it is located on. By using tissue-specific promoter, we have got the marker-free pollens.
We found Pv4, a rice anther specific promoter. When our transgentic plants grow up, the Cre gene will be expressed which should be drived by Pv4, thus only in the anther of stamens. Hence,we got the marker-free pollens. Through selfing , we can get the homozygote of marker-free transgentic plants.
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)
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