叶绿素合成酶基因在甘蓝型油菜及拟南芥种子生育酚合成过程中的作用研究

发布时间：2018-06-03 05:47

本文选题：甘蓝型油菜 + 拟南芥　；参考：《华中农业大学》2017年硕士论文

【摘要】：维生素E,包括生育酚和生育三烯酚,是一种重要的脂溶性有机物,它具有很强的抗氧化性,在动植生命中有着不可替代的重要地位。人类对维生素E的需求量巨大,但其中90%左右靠人工合成。天然维生素E主要来源于植物叶片和油料作物种子,人工合成的维生素E在生物活性上与天然维生素E相差甚远。因此提高绿色植物特别是油料作物种子中维生素E的含量就显得尤为重要。油菜是世界上最重要的油料作物之一,油菜种子富含维生素E且种植面积大,因此油菜维生素E育种日渐成为研究的热点之一,越来越受到育种工作者的关注。GWAS分析表明,油菜BnA9CHLSYN位点对种子中生育酚含量有着很强的效应。因此猜测油菜种子中生育酚合成的“瓶颈”在于叶绿素合成酶对生育酚合成的前体物质PDP进行“回收”,重新合成叶绿素,导致PDP浓度过低。想要打破这一“瓶颈”,就有必要研究叶绿素合成酶(Chlorophyll synthetase,CHLSYN)与生育酚合成之间的关系。本研究借助拟南芥这一载体研究种子中CHLSYN的表达量与种子中生育酚合成之间的关系,主要研究结果如下:1.不同甘蓝型油菜的BnA9CHLSYN位点对种子中生育酚含量的效应不同,据此将这些材料分为两组,H组和L组(分别对应BnA9CHLSYN位点对种子中生育酚含量呈正向效应和负向效应),H组材料种子中BnA9CHLSYN的表达水平低于L组材料种子中该基因的表达水平,生育酚含量相反。2.野生型背景下,采用特异性启动子干扰拟南芥种子中CHLSYN的表达之后,种子中生育酚含量提高,最多可提高33.69%,种子特异性超表达CHLSYN,最多可使拟南芥种子中生育酚含量降低44.73%,即拟南芥种子中CHLSYN的表达水平与生育酚含量呈负相关关系。拟南芥vte5纯合突变体背景下也得到相同的结论。3.采用相同种子特异性启动子干扰拟南芥种子中CHLSYN的同时超表达AtHPT,种子中生育酚含量最多能提高125.38%,而在种子中特异性超表达AtHPT最多可使生育酚含量提高123.56%,两者未达到显著差异。4.更换超表达AtHPT的启动子,采用不同种子特异性启动子干扰拟南芥种子中CHLSYN的同时超表达AtHPT,种子中生育酚含量最多能提高161.71%,而使用新的启动子超表达AtHPT之后,最多只能使生育酚含量提高95.99%。
[Abstract]:Vitamin E, including tocopherol and reproductive trienol, is an important lipophilic organic substance, which has strong antioxidant activity and plays an irreplaceable role in plant life. Human demand for vitamin E is enormous, but about 90% of it is artificially synthesized. The natural vitamin E mainly comes from plant leaves and oil crop seeds. The synthetic vitamin E is far from natural vitamin E in biological activity. Therefore, it is very important to increase the content of vitamin E in green plants, especially oil crop seeds. Rape is one of the most important oil crops in the world. Rape seeds are rich in vitamin E and have large planting area. The BnA9CHLSYN site of rape has a strong effect on the content of tocopherol in seeds. Therefore, it is speculated that the bottleneck of tocopherol synthesis in rapeseed seeds is that chlorophyll synthetase "recovers" the precursor of tocopherol synthesis, PDP, and resynthesizes chlorophyll, which leads to the low concentration of PDP. In order to break this bottleneck, it is necessary to study the relationship between chlorophyll synthetase Chlorophyll synthetic enzyme CHLSYN and tocopherol synthesis. In this study, Arabidopsis thaliana was used to study the relationship between the expression of CHLSYN and the synthesis of tocopherol in seeds. The main results were as follows: 1. The effect of BnA9CHLSYN locus on the content of tocopherol in different Brassica napus was different. These materials were divided into two groups: group H and group L (the positive and negative effects of BnA9CHLSYN locus on the content of tocopherol in seeds). The expression level of BnA9CHLSYN in H group was lower than that in L group. The content of tocopherol is opposite. Under the wild type background, the content of tocopherol in Arabidopsis thaliana seeds increased after the specific promoter was used to interfere with the expression of CHLSYN in Arabidopsis thaliana seeds. At most, the content of tocopherol in Arabidopsis thaliana seeds was decreased by 44.73%. The expression level of CHLSYN in Arabidopsis thaliana seeds was negatively correlated with the content of tocopherol. The same conclusion was obtained under the background of Arabidopsis vte5 homozygous mutants. The content of tocopherol in Arabidopsis thaliana seeds could be increased by using the same seed specific promoter to interfere with the simultaneous overexpression of AtHPT.The content of tocopherol in seeds could be increased by 125.38%, but the content of tocopherol in seeds could be increased by 123.56 at most, but there was no significant difference between the two. 4. When the promoter of AtHPT was replaced with different seed specific promoters which interfered with CHLSYN in Arabidopsis thaliana seeds, the content of tocopherol in seeds increased at most 161.71%, but after the new promoter was used to express AtHPT in Arabidopsis thaliana seeds, the content of tocopherol in seeds was increased by 161.71%. At most, the content of tocopherol increased 95.99%.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S565.4

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