甘薯IbPSY和IbSGR基因的克隆與功能分析
發(fā)布時(shí)間：2019-06-10 19:04
【摘要】：甘薯是我國(guó)重要的糧食作物,其塊根顏色多樣。紫肉甘薯塊根富含花色素苷,而橙肉甘薯塊根則富含類胡蘿卜素。β-胡蘿卜素作為類胡蘿卜素的一種,對(duì)人體健康有著重要的作用,在人體維生素A的合成途徑中,β-胡蘿卜素能夠作為其合成的前體物質(zhì),當(dāng)機(jī)體缺乏維生素A時(shí),適量補(bǔ)充β-胡蘿卜素能緩解由維生素A缺乏引起的病癥。此外β-胡蘿卜素還是一種很好的抗氧化劑,能有效的抑制機(jī)體中自由基的產(chǎn)生。本研究從橙肉甘薯品系渝薯11-10-97新品系中克隆了八氫番茄紅素合成酶基因IbPSY,該基因是類胡蘿卜素合成途徑中的第一個(gè)關(guān)鍵酶基因。IbPSY基因編碼區(qū)全長(zhǎng)為1320bp,編碼的蛋白質(zhì)含有439個(gè)氨基酸。IbPSY含有一個(gè)47個(gè)氨基酸的質(zhì)體轉(zhuǎn)運(yùn)肽,將Ib PSY蛋白與綠色熒光蛋白GFP融合,進(jìn)行亞細(xì)胞定位分析,結(jié)果表明IbPSY蛋白定位于葉綠體中,這一結(jié)果與其他物種中PSY蛋白的定位情況一致,并且符合類胡蘿卜素的合成場(chǎng)所就是質(zhì)體這一事實(shí)。利用產(chǎn)類胡蘿卜素的缺陷型工程菌進(jìn)行功能驗(yàn)證實(shí)驗(yàn),Ib PSY能夠彌補(bǔ)細(xì)菌β-胡蘿卜素合成途徑中的crtB的作用,使缺陷型工程菌能合成β-胡蘿卜素。這表明,本研究所得到的IbPSY具有八氫番茄紅素合成酶的功能。在擬南芥中超量表達(dá)IbPSY能顯著提高擬南芥葉片中類胡蘿卜素的含量。這再一次證實(shí)了本研究所獲得的IbPSY就是編碼八氫番茄紅素合成酶的基因。本研究從甘薯中克隆得到了編碼滯綠蛋白的基因IbSGR,IbSGR編碼區(qū)全長(zhǎng)801bp,編碼的蛋白質(zhì)含有266個(gè)氨基酸,Ib SGR含有一個(gè)52個(gè)氨基酸的質(zhì)體轉(zhuǎn)運(yùn)肽。亞細(xì)胞定位結(jié)果顯示Ib SGR蛋白定位于葉綠體中,采用qPCR技術(shù)分析IbSGR在甘薯葉和莖中的相對(duì)表達(dá)量,結(jié)果表明IbSGR在成熟葉片中的表達(dá)量最高,其次是幼嫩的葉片和老莖,在幼嫩的莖中表達(dá)量最低。在葉片衰老的過(guò)程中,SGR能幫助植物快速降解葉綠素,所以這一表達(dá)模式與SGR的功能是相符的。超量表達(dá)IbSGR的擬南芥,其葉片中的葉綠素含量與野生型相比有顯著的下降。這表明本研究所得到的IbSGR確實(shí)是甘薯中編碼滯綠蛋白的基因。利用BiFC技術(shù)證明了IbSGR與Ib PSY能夠發(fā)生相互作用,將IbSGR基因轉(zhuǎn)入產(chǎn)β-胡蘿卜素的工程菌中,工程菌的菌體顏色由橘黃色變成了白色,且HPLC并未檢測(cè)到工程菌中有β-胡蘿卜素的積累。超量表達(dá)IbSGR的擬南芥葉片中類胡蘿卜素的含量與野生型相比有明顯的下降。這說(shuō)明IbSGR能與Ib PSY相互作用并能抑制IbPSY的功能,調(diào)控植物體類胡蘿卜素的積累。本研究確證了克隆得到的IbPSY和IbSGR的確就是甘薯的PSY基因和SGR基因,并且初步證明了IbSGR能與Ib PSY相互作用,進(jìn)而調(diào)控植株中類胡蘿卜素的積累。
[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.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S531
本文編號(hào)：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.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S531
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