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    <li><a href=" https://2016.igem.org/Team:SCAU-China/Notebook">Notebook</a></li>
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      <li><a href="#vecter" class="on">Introduction of astaxanthin</a></li>
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      <li><a href="#tissue">Bioreactor in rice endosperm</a></li>
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      <div class="text">Astaxanthin is a kind of naturally-occurring keto-carotenoids which is found in some microalgaes, shrimps and crabs. This compound is insoluble in water while soluble in most of organic solvent like pyridine, ethanol and benzene. In fact, it is merely in some specific species of algaes, bacteria and yeasts that astaxanthin can be synthesized. It is impossible for animals to synthesize astaxanthins on their own so that the accumulation of astaxanthin in the body of animal is the consequence of diet. In some organisms, astaxanthin takes on a colour of brown or blue as it forms into some types of pigment-protein complexes. For humans, astaxanthin is a powerful antioxidant with broad health implications. Thus, astaxanthin has been claimed a good commercial prospect for its value in medical and health care.<br><br>
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Currently, the industrial ways to produce astaxanthin are extract from microalgae Haematococcus pluvialis,Phaffia yeast,shrimp processing waste and chemical product.However these ways aren’t safety enough and the purification is difficult. While higher plants are supposed to be an efficient and safe bioreactor to produce astaxanthin,because it has advanced protein processing system to produce complex product, such as senior terpenoids. In nature, there are many precious products were terpenoid , such as carotenoids, microbial A, paclitaxel, etc. And in the low-level and annimal unable to synthesize some complex product.<br><br>
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    <li><a href=" https://2016.igem.org/Team:SCAU-China/Engagement">Engagement</a></li>
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Although higher plants such as Zea mays are capable to synthesize zeaxanthin, which is the metabolic precursor of astaxanthin, due to their lack ofβ- carotene ketolase, astaxanthin still can not be synthesized in these higher plants unless with the help of metabolism engineering.
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  According to the advantages listing below, we take rice (Orazy sativa) endosperm as the bioreactor for astaxanthin production.<br><br>
 
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  <p>Rice is a low-cost, high-productivity, high-safety and commonly used model plant</p>
 
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  <p>Rice is easy to plant on a large scale and has very high yield</p>
 
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  <p>Rice seed is an excellent biomass container</p>
 
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  <p>Genetic modification technology is quite mature in rice</p>
 
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  <p>As a special nutrition storage organs,rice seed is convenient to store, extract and purify</p>
 
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  <p>Astaxanthin accumulation at seed would not interrupt the growth of the  whole plant</p><br><br>
 
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  Genes responsible for carotenoid synthesis are inactive in rice endosperm. So it is impossibleo harvest astaxanthin from wild-type rice. Using multiple-gene metabolic engineering, we introduced the astaxanthin biosynthesis pathways which is specifically expressed in rice endosperm. Thus, possible negative effects of astaxanthin would not influence the growth of plants. In this way, rice endosperm serves as a special container for astaxanthin, which provides conveniences for storage and extraction.
 
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      <div class="text">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,BCP and BBCP) to study the metabolic of the synthesis of astaxanthin in the endosperm of rice.
 
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      <div class="text">The system of multiple genes vectors is composed of one acceptance vector which could transform artificial chromosome and two donor vectors(322-d1/322-d2) carrying genes. Using Cre/loxP recombination system, acceptance vector accept the gene from donor vectors alternatively to accomplish the construction of objective vectors(Lin et al., PNAS, 2003, 100: 5962-5967；Zhu et al., unpublished). Assembling the four genes(CrtI,PSY,BKT and BHY) and the specific promoters of endosperm by the principle of Gibson Assembly(Gibson, Methods Enzymol., 2011, 498: 349–361).  We got those vectors,(Ⅰ)pYL322d1-CrtⅠ,(Ⅱ)pYL322d2-PSY,(Ⅲ) pYL322d1-BKT and (Ⅳ)pYL322d2-BHY via assembling expression cassettes to donor vectors alternatively. Another time, using the assembly of Cre/LoxP system, transfer objective genes to acceptance vector according to the line of Ⅰ\Ⅱ\Ⅲ\Ⅳ to finish every steps. After the steps ofⅠ\Ⅱ, we got the binary expression vector 380-PC. The second step of Ⅲ, we got 380-BPC. After the Ⅳ, we got the 380-BBPC. Then we transformed the three vectors into Agrobacterium tumefaciens EHA105. We successfully got the rice which can produce astaxanthin in endosperm by Agrobacterium-mediated transformation of rice callus.<a href="https://2016.igem.org/Team:SCAU-China/PART">If you want to know more about it, please click here! </a>
 
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      <div class="text">We get the callus by callus induction of rice seed, after that we will transfer the carrier into callus by agrobacterium-mediated transformation. If we get positive callus, we should cultivate it to complete plant by plant tissue culture. Finally we will obtain the transgenic rice plants and harvest the rices.
 
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Design
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      <li><a href="#vecter">Introduction of astaxanthin</a></li>
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<div class="h2_font_size">1. Vector for genes stacking </div>
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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<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(Liu et al., PNAS, 1999, 96: 6535-6540; Lin et al., PNAS, 2003, 100: 5962-5967; Zhu et al., unpublished). By this way, multiple genes and a maker-free element can be easily stacked into a TAC-based binary acceptor vector (Figure 3) 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="text">In this part, we mainly introduce the work we used cre/loxp recombination，a site-specific recombinase technology to delete the selective marker.<a href="https://2016.igem.org/Team:SCAU-China/Safety#pcr">You can read more about this part here! </a>
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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>
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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 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.
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<div class="p_font_size"><small> <font style="font-weight:bold">Figure 4</font> &nbsp;&nbsp;The schematic diagram of Astaxanthin Rice project.</small>
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In this part, we used<em> Cre/loxP</em> 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<em> loxP</em> sites, and consists of a HPT (hygromycin) resistance gene expression cassette and a <font style="font-style:italic">Cre</font> gene expression cassette controlled by anther-specific promoter. When <font style="font-style:italic">Cre</font> gene was expressed in transgenic rice anther, the Cre enzyme deleted the marker-free element between two <em>loxP</em> sites.
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<small><font style="font-weight:bold">Figure 5</font> &nbsp;&nbsp;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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【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 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).
 +
</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).
 +
</div>
 +
<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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      <li><a href="#vecter">Introduction of astaxanthin</a></li>
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      <div class="text">【1】Varda Mann, Mark Harker, Iris Pecker, and Joseph Hirschberg. Metabolic engineering of astaxanthin production in tobacco flowers. Nature Biotechnology . 18, 888-892 (2002)<br><br>
+
−
  【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)<br><br>
+
−
  【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)<br><br>
+
−
  【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) <br><br>
+
−
  【5】Giovanni Giuliano. Plant carotenoids: genomics meets multi-gene engineering.  Plant Biology.  19, 111–117 (2014)<br><br>
+
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  【6】Yook Jang Soo, Okamoto Masahiro, Rakwal Randeep, Shibato Junko, Lee Min Chul, Matsui Takashi, Chang Hyukki,  Cho Joon Yong, Soya Hideaki. <br><br>
+
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  Astaxanthin supplementation enhances adult hippocampal neurogenesis and spatial memory inmice. Molecular nutrition & food research. 60 , 589-599(2016)
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Latest revision as of 11:50, 19 October 2016

SCAU

Design
1. Vector for genes stacking
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(Liu et al., PNAS, 1999, 96: 6535-6540; Lin et al., PNAS, 2003, 100: 5962-5967; Zhu et al., unpublished). By this way, multiple genes and a maker-free element can be easily stacked into a TAC-based binary acceptor vector (Figure 3) 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
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)