甘薯IbPSY和IbSGR基因的克隆与功能分析
发布时间：2019-06-10 19:04
【摘要】：甘薯是我国重要的粮食作物,其块根颜色多样。紫肉甘薯块根富含花色素苷,而橙肉甘薯块根则富含类胡萝卜素。β-胡萝卜素作为类胡萝卜素的一种,对人体健康有着重要的作用,在人体维生素A的合成途径中,β-胡萝卜素能够作为其合成的前体物质,当机体缺乏维生素A时,适量补充β-胡萝卜素能缓解由维生素A缺乏引起的病症。此外β-胡萝卜素还是一种很好的抗氧化剂,能有效的抑制机体中自由基的产生。本研究从橙肉甘薯品系渝薯11-10-97新品系中克隆了八氢番茄红素合成酶基因IbPSY,该基因是类胡萝卜素合成途径中的第一个关键酶基因。IbPSY基因编码区全长为1320bp,编码的蛋白质含有439个氨基酸。IbPSY含有一个47个氨基酸的质体转运肽,将Ib PSY蛋白与绿色荧光蛋白GFP融合,进行亚细胞定位分析,结果表明IbPSY蛋白定位于叶绿体中,这一结果与其他物种中PSY蛋白的定位情况一致,并且符合类胡萝卜素的合成场所就是质体这一事实。利用产类胡萝卜素的缺陷型工程菌进行功能验证实验,Ib PSY能够弥补细菌β-胡萝卜素合成途径中的crtB的作用,使缺陷型工程菌能合成β-胡萝卜素。这表明,本研究所得到的IbPSY具有八氢番茄红素合成酶的功能。在拟南芥中超量表达IbPSY能显著提高拟南芥叶片中类胡萝卜素的含量。这再一次证实了本研究所获得的IbPSY就是编码八氢番茄红素合成酶的基因。本研究从甘薯中克隆得到了编码滞绿蛋白的基因IbSGR,IbSGR编码区全长801bp,编码的蛋白质含有266个氨基酸,Ib SGR含有一个52个氨基酸的质体转运肽。亚细胞定位结果显示Ib SGR蛋白定位于叶绿体中,采用qPCR技术分析IbSGR在甘薯叶和茎中的相对表达量,结果表明IbSGR在成熟叶片中的表达量最高,其次是幼嫩的叶片和老茎,在幼嫩的茎中表达量最低。在叶片衰老的过程中,SGR能帮助植物快速降解叶绿素,所以这一表达模式与SGR的功能是相符的。超量表达IbSGR的拟南芥,其叶片中的叶绿素含量与野生型相比有显著的下降。这表明本研究所得到的IbSGR确实是甘薯中编码滞绿蛋白的基因。利用BiFC技术证明了IbSGR与Ib PSY能够发生相互作用,将IbSGR基因转入产β-胡萝卜素的工程菌中,工程菌的菌体颜色由橘黄色变成了白色,且HPLC并未检测到工程菌中有β-胡萝卜素的积累。超量表达IbSGR的拟南芥叶片中类胡萝卜素的含量与野生型相比有明显的下降。这说明IbSGR能与Ib PSY相互作用并能抑制IbPSY的功能,调控植物体类胡萝卜素的积累。本研究确证了克隆得到的IbPSY和IbSGR的确就是甘薯的PSY基因和SGR基因,并且初步证明了IbSGR能与Ib PSY相互作用,进而调控植株中类胡萝卜素的积累。
[Abstract]:Sweet potato is an important food crop in China, and its root tuber has a variety of colors. The root of purple sweet potato is rich in anthocyanins, while the root of orange sweet potato is rich in carotenoids. 尾-carotene, as a kind of carotenoids, plays an important role in human health. 尾-carotene can be used as the precursor of its synthesis. when the body lacks vitamin A, proper supplementation of 尾-carotene can alleviate the symptoms caused by vitamin A deficiency. In addition, 尾-carotene is also a good antioxidant, which can effectively inhibit the production of free radicals in the body. In this study, the octahydrolycopene synthase gene IbPSY, the first key enzyme gene in carotenoid synthesis pathway, was cloned from a new orange sweet potato strain Yushu 1110X97. The coding region of IbPSY gene is 1320bp. the coding region of IbPSY gene is 1320bp. The encoded protein contained 439 amino acids. IbPSY contained a 47 amino acid Plastid transporter peptide. IbPSY protein was fused with green fluorescent protein GFP and subcellular localization analysis was carried out. the results showed that IbPSY protein was located in chloroplast. This result is consistent with the localization of PSY protein in other species and is consistent with the fact that the site of carotenoid synthesis is plastids. The functional verification experiment of carotenoid-producing engineering bacteria, Ib PSY can make up for the role of crtB in the pathway of 尾-carotene synthesis, and enable defective engineering bacteria to synthesize 尾-carotene. This shows that the IbPSY obtained in this study has the function of octahydrolycopene synthase. Overexpression of IbPSY in Arabidopsis thaliana could significantly increase the content of carotenoids in leaves of Arabidopsis thaliana. This confirms once again that the IbPSY obtained in this study is the gene encoding octahydrolycopene synthase. In this study, the full length of 801bp coding gene IbSGR,IbSGR coding green protein was cloned from sweet potato. The encoded protein contains 266amino acids and IbSGR contains a 52 amino acid Plastid transporter peptide. The results of subcellular localization showed that IbSGR protein was located in chloroplast. The relative expression of IbSGR in sweet potato leaves and stems was analyzed by qPCR. The results showed that the expression of IbSGR was the highest in mature leaves, followed by young leaves and old stems. The expression level was the lowest in young stems. During leaf senescence, SGR can help plants degrade chlorophyll rapidly, so this expression pattern is consistent with the function of SGR. The chlorophyll content in leaves of Arabidopsis thaliana overexpressing IbSGR was significantly lower than that of wild type. This suggests that the IbSGR obtained in this study is indeed a gene encoding green protein in sweet potato. BiFC technique was used to prove that IbSGR and Ib PSY could interact with each other. When IbSGR gene was transferred into 尾-carotene producing engineering bacteria, the color of engineering bacteria changed from orange to white. The accumulation of 尾-carotene in engineering bacteria was not detected by HPLC. The content of carotenoids in Arabidopsis thaliana leaves overexpressing IbSGR was significantly lower than that in wild type. This suggests that IbSGR can interact with IbPSY and inhibit the function of IbPSY and regulate the accumulation of carotenoids in plants. In this study, it was confirmed that the cloned IbPSY and IbSGR were indeed PSY genes and SGR genes of sweet potato, and it was preliminarily proved that IbSGR could interact with IbPSY and regulate the accumulation of carotenoids in plants.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S531
本文编号：2496667
[Abstract]:Sweet potato is an important food crop in China, and its root tuber has a variety of colors. The root of purple sweet potato is rich in anthocyanins, while the root of orange sweet potato is rich in carotenoids. 尾-carotene, as a kind of carotenoids, plays an important role in human health. 尾-carotene can be used as the precursor of its synthesis. when the body lacks vitamin A, proper supplementation of 尾-carotene can alleviate the symptoms caused by vitamin A deficiency. In addition, 尾-carotene is also a good antioxidant, which can effectively inhibit the production of free radicals in the body. In this study, the octahydrolycopene synthase gene IbPSY, the first key enzyme gene in carotenoid synthesis pathway, was cloned from a new orange sweet potato strain Yushu 1110X97. The coding region of IbPSY gene is 1320bp. the coding region of IbPSY gene is 1320bp. The encoded protein contained 439 amino acids. IbPSY contained a 47 amino acid Plastid transporter peptide. IbPSY protein was fused with green fluorescent protein GFP and subcellular localization analysis was carried out. the results showed that IbPSY protein was located in chloroplast. This result is consistent with the localization of PSY protein in other species and is consistent with the fact that the site of carotenoid synthesis is plastids. The functional verification experiment of carotenoid-producing engineering bacteria, Ib PSY can make up for the role of crtB in the pathway of 尾-carotene synthesis, and enable defective engineering bacteria to synthesize 尾-carotene. This shows that the IbPSY obtained in this study has the function of octahydrolycopene synthase. Overexpression of IbPSY in Arabidopsis thaliana could significantly increase the content of carotenoids in leaves of Arabidopsis thaliana. This confirms once again that the IbPSY obtained in this study is the gene encoding octahydrolycopene synthase. In this study, the full length of 801bp coding gene IbSGR,IbSGR coding green protein was cloned from sweet potato. The encoded protein contains 266amino acids and IbSGR contains a 52 amino acid Plastid transporter peptide. The results of subcellular localization showed that IbSGR protein was located in chloroplast. The relative expression of IbSGR in sweet potato leaves and stems was analyzed by qPCR. The results showed that the expression of IbSGR was the highest in mature leaves, followed by young leaves and old stems. The expression level was the lowest in young stems. During leaf senescence, SGR can help plants degrade chlorophyll rapidly, so this expression pattern is consistent with the function of SGR. The chlorophyll content in leaves of Arabidopsis thaliana overexpressing IbSGR was significantly lower than that of wild type. This suggests that the IbSGR obtained in this study is indeed a gene encoding green protein in sweet potato. BiFC technique was used to prove that IbSGR and Ib PSY could interact with each other. When IbSGR gene was transferred into 尾-carotene producing engineering bacteria, the color of engineering bacteria changed from orange to white. The accumulation of 尾-carotene in engineering bacteria was not detected by HPLC. The content of carotenoids in Arabidopsis thaliana leaves overexpressing IbSGR was significantly lower than that in wild type. This suggests that IbSGR can interact with IbPSY and inhibit the function of IbPSY and regulate the accumulation of carotenoids in plants. In this study, it was confirmed that the cloned IbPSY and IbSGR were indeed PSY genes and SGR genes of sweet potato, and it was preliminarily proved that IbSGR could interact with IbPSY and regulate the accumulation of carotenoids in plants.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S531
【参考文献】
相关期刊论文 前10条
1 Yufan Fu;Lili Ma;Fei Qiu;Chunxian Yang;Chunxia Hu;Ying Zhao;Zhi Lin;Min Chen;Zhihua Liao;Xiaozhong Lan;;A Prenyltransferase Gene Confirmed to Be a Carotenogenic CRTE Gene from Sweetpotato[J];Journal of Genetics and Genomics;2014年11期
2 陈俊毅;朱晓宇;蒯本科;;绿色器官衰老进程中叶绿素降解代谢及其调控的研究进展[J];植物生理学报;2014年09期
3 ZHANG Jian-cheng;ZHOU Wen-jing;XU Qiang;TAO Neng-guo;YE Jun-li;GUO Fei;XU Juan;DENG Xiu-xin;;Two Lycopene β-Cyclases Genes from Sweet Orange (Citrus sinensis L. Osbeck) Encode Enzymes With Different Functional Efficiency During the Conversion of Lycopene-to-Provitamin A[J];Journal of Integrative Agriculture;2013年10期
4 霍培;季静;王罡;关春峰;;植物类胡萝卜素生物合成及功能[J];中国生物工程杂志;2011年11期
5 张建成;周文静;邓秀新;;超表达草生欧文氏菌crtB基因促进转基因番茄类胡萝卜素合成的研究[J];园艺学报;2010年03期
6 占爱瑶;由香玲;詹亚光;;植物萜类化合物的生物合成及应用[J];生物技术通讯;2010年01期
7 李莉;侯丽霞;施木田;吕鑫;何启伟;;番茄红素环化酶基因片段克隆及反义表达载体构建[J];山东农业科学;2009年01期
8 张建成;刘和;;植物类胡萝卜素生物合成及其调控与遗传操作[J];中国农学通报;2007年11期
9 黄彬城;季静;王罡;郑阳霞;;植物类胡萝卜素的研究进展[J];天津农业科学;2006年02期
10 赵小明;杜琨;王宪伟;;甘薯的营养价值及开发利用[J];安徽农业科学;2006年04期
，本文编号：2496667
本文链接：https://www.wllwen.com/kejilunwen/jiyingongcheng/2496667.html
最近更新
教材专著
