甘蓝型油菜转录因子NST3基因编辑和超表达载体构建

发布时间：2018-05-18 19:22

本文选题：甘蓝型油菜 + NST3　；参考：《西南大学》2017年硕士论文

【摘要】：油菜是世界上分布最广的油料作物之一,也是我国第一大油料作物。在我国,菌核病和油菜倒伏是限制油菜产量最主要的因素。木质素的含量的多少跟作物的抗病性、抗倒伏能力有关。植物细胞木质化提高细胞壁的强度,不仅可以增强植物抗病性,也可以增强抗倒伏能力。油菜生长后期是倒伏易发生的时期,也是菌核病的高发期,两者变现为交互作用。前期研究发现在油菜茎秆中,S型木质素单体含量越高,植物抗倒伏能力越强,并且对菌核病的抗性也越强。本研究论文主要包括首先对甘蓝型油菜不同器官的NST3基因的四个成员进行表达分析,在线对四个成员进行基因结构分析,并对其编码的蛋白进行生物信息学分析,构建NSR3家族的CRISPR-Cas9基因编辑系统,构建BnaA09g24190D成员的超表达载体。成功构建载体后,将其转化甘蓝型油菜,并得到阳性的转基因苗。提取油菜转基因苗DNA,设计特异引物扩片段及测序。本实验的主要结果如下:1、油菜的NST3基因有四个成员,分别是BnaA05g17950D、BnaA09g24190D、BnaC05g24890D和BnaC05g28390D。四个成员中,BnaA09g24190D、BnaC05g24890D的表达量均高于其他两个成员,在根、茎、花蕾花、角果和种子中BnaA09g24190D的表达量均比BnaC05g24890D高,在根中,BnaA09g24190D的表达量几乎是BnaC05g24890D的5倍。在茎中,BnaA09g24190D的表达量几乎是BnaC05g24890D的2.5倍。四个成员中,BnaA05g17950D、BnaC05g28390D的表达量较低,在子叶、叶和花蕾均不表达,在其他部位的表达量也较低。通过基因的表达量分析,可以推测,在油菜的这四个成员中,BnaA09g24190D、BnaC05g24890D在调节油菜次生细胞壁合成和调控木质素单体组分起主要作用,尤其是BnaA09g24190D。2、对BnaA05g17950D、BnaA09g24190D、BnaC05g24890D和BnaC05g28390D在线进行基因结构分析。BnaA05g17950D全长1819bp,CDS长927bp,该基因有4个外显子,3个内含子;BnaA09g24190D全长1776bp,CDS长1095bp,该基因有3个外显子,2个内含子;BnaC05g24890D全长1832bp,CDS长1116bp,3个外显子,2个内含子;BnaC05g28390D全长为2146bp,CDS长978bp,5个外显子,4个内含子。对BnaA05g17950D、BnaA09g24190D、BnaC05g24890D和BnaC05g28390D编码的蛋白质进行分析,分别含308,364,371,325个氨基酸,分子量在35.75~42.99KD之间,等电点均小于7,二级结构中,α-螺旋占24.45%~29.54%,β-转角占8.36%~8.77%,随机卷曲占40.62%~48.35%,延伸链占17.25%~21.23%。它们都具有NAM典型特征的保守域,根据它们的保守域能够预测出三级结构。3、本实验共得到22株阳性转基因苗,在转基因苗中,不同的基因成员的编辑情况也各不相同。在22株基因阳性植物中,只有2株的BnaA05g17950D得到编辑,编辑的效率为9.09%。BnaA09g24190D在11株油菜中都得到编辑,编辑的效率为50%。BnaC05g24890D在8株转基因油菜中都得到编辑,编辑的效率为36.4%。BnaC05g28390D在4株转基因油菜中得到编辑,编辑的效率为18.2%。第3、22株油菜转基因苗四个基因同时被编辑,第1、7株的BnaA09g24190D、BnaC05g24890D同时被编辑,第9株的油菜转基因苗的BnaA09g24190D、BnaC05g24890D、BnaC05g28390D都被编辑,第12株转基因油菜的BnaC05g24890D、BnaC05g28390D同时被编辑,第17株的转基因油菜的BnaA09g24190D、BnaC05g24890D同时被编辑。CRISPR-Cas9编辑得到结果可分为三类:靶位点及其附近碱基缺失、插入、既有缺失又有插入。4、在四个成员中,BnaA09g24190D在调节油菜次生细胞壁合成和调控木质素单体组分起最主要作用,因而本实验构建了BnaA09g24190D的超量表达载体,并转化油菜。本实验的后续工作,CRISPR-Cas9编辑和超表达的转基因苗的木质素单体含量变化情况、对抗菌核病的抗感情况和抗倒伏情况需要实验室的相关人员跟进。
[Abstract]:Rape is one of the most widely distributed oil crops in the world, and it is also the largest oil crop in China. In China, sclerotius disease and rape lodging are the most important factors to limit the yield of rape. The content of lignin is related to the resistance and lodging resistance of the crops. The resistance to disease can also enhance the ability to resist lodging. The late growth of rape is a period of prone to lodging and also a period of high incidence of Sclerotinia. The earlier studies have found that the higher the content of S lignin monomers, the stronger the lodging resistance of plants and the stronger the resistance to Sclerotinia. To include the analysis of four members of the NST3 gene of different organs of Brassica napus (Brassica napus), four members were analyzed on line, and the encoded proteins were analyzed by bioinformatics. The CRISPR-Cas9 gene editing system of the NSR3 family was constructed, and the overexpression vector of BnaA09g24190D members was constructed. The main results of this experiment are as follows: 1, the NST3 gene of rapeseed has four members, which are four members of BnaA05g17950D, BnaA09g24190D, BnaC05g24890D and BnaC05g28390D., BnaA09g24190D, Bna, respectively. The expression of C05g24890D is higher than that of the other two members. The expression of BnaA09g24190D in root, stem, bud flower, pod and seed is higher than that of BnaC05g24890D. In the root, the expression of BnaA09g24190D is almost 5 times as much as BnaC05g24890D. In the stem, the expression of BnaA09g24190D is almost 2.5 times that of BnaC05g24890D. Among four members, BnaA05g17950D, The expression of BnaC05g28390D is low, not expressed in cotyledon, leaves and buds, and is also low in other parts. Through the analysis of gene expression, it is possible to speculate that in these four members of rape, BnaA09g24190D and BnaC05g24890D play a major role in regulating the secondary cell wall of rape and regulating the lignin monomer components, especially Bn. AA09g24190D.2, BnaA05g17950D, BnaA09g24190D, BnaC05g24890D and BnaC05g28390D are on line gene structural analysis.BnaA05g17950D full length 1819bp, CDS long 927bp, the gene has 4 exons and 3 introns, BnaA09g24190D full length 1776bp, CDS long, this gene has 3 exons and 2 introns. P, 3 exons and 2 introns, the full length of BnaC05g28390D is 2146bp, CDS long 978bp, 5 exons and 4 introns. BnaA05g17950D, BnaA09g24190D, BnaC05g24890D and BnaC05g28390D encoded proteins are analyzed with 308364371325 amino acids, the molecular weight is between 35.75~42.99KD, and the isoelectric point is less than 7, and the two structure, alpha - The spiral accounts for 24.45%~29.54%, the beta angle is 8.36%~8.77%, the random curls account for 40.62%~48.35%, the extension chain accounts for the conservative domain with the typical NAM characteristics of the 17.25%~21.23%., and the three stage structure.3 can be predicted according to their conservative domain. This experiment results in a total of 22 positive transgenic seedlings, and the editing of different gene members in the transgenic seedlings is also the same. Of the 22 gene positive plants, only 2 of the BnaA05g17950D were edited, and the efficiency of the editing was 9.09%.BnaA09g24190D in 11 rapeseed. The efficiency of editors was edited in 8 transgenic rapeseed. The efficiency of the editing was that the efficiency of the editing was to be compiled in 4 transgenic rapeseed. Editing, the efficiency of editing is that four genes of 18.2%. 3,22 plant transgenic seedlings were edited simultaneously, the BnaA09g24190D of the 1,7 strain, BnaC05g24890D were edited simultaneously, the BnaA09g24190D, BnaC05g24890D, BnaC05g28390D of the ninth transgenic rape seedlings were edited, the BnaC05g24890D of twelfth transgenic rapeseed, BnaC05g28390D were edited at the same time, seventeenth The results of BnaA09g24190D and BnaC05g24890D edited.CRISPR-Cas9 in transgenic rapeseed can be divided into three categories: target site and its adjacent base deletion, insertion, both deletion and insertion of.4, and in the four members, BnaA09g24190D plays the most important role in regulating the secondary cell wall synthesis and regulating the lignin component of the rape. In this experiment, the overexpression vector of BnaA09g24190D was constructed and rapeseed was transformed. The follow-up work of this experiment, the changes of lignin content in the CRISPR-Cas9 edited and overexpressed transgenic seedlings, the resistance to the Sclerotinia and the resistance to lodging need to be followed up by the related personnel in the laboratory.
【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】：Q943.2;S565.4

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