半胱氨酸蛋白酶参与油菜绒毡层发育的分子调控机制

发布时间：2018-04-06 01:18

本文选题：甘蓝型油菜　切入点：绒毡层　出处：《华中农业大学》2016年博士论文

【摘要】：油菜是人类最重要的食用植物油来源之一,杂种优势利用是提高其产量和品质的有效策略,而雄性不育已经成为油菜杂种优势利用的主要途径。在甘蓝型油菜近等基因系7365AB(Bnams3ms3ms4bms4b/Bna Ms3ms3ms4bms4b)中,不育材料7365A和可育材料7365B的差异仅在Bn Ms3位点上,本实验室前期研究表明在Bnms3突变体(7365A)中,一些与绒毡层降解相关的半胱氨酸蛋白酶基因的表达受到影响。绒毡层细胞的降解被认为是细胞程序性死亡(PCD)的过程,而papain类半胱氨酸蛋白酶在绒毡层细胞的适时降解过程中起到关键控制作用。本研究中,我们以半胱氨酸蛋白酶基因为突破口,解析在甘蓝型油菜中绒毡层细胞降解的分子机理。主要研究结果如下:1.获得4个在材料7365AB中具有差异表达的半胱氨酸蛋白酶基因在拟南芥中,有31个papain类半胱氨酸蛋白酶基因。我们利用RT-PCR的方法分析与拟南芥同源的31个甘蓝型油菜基因在材料7365AB中的表达情况,根据其表达模式的不同可将它们分为以下三类:a.27个基因在不育材料7365A和可育材料7365B中均有表达;b.2个基因(Bn CP21和Bna CP13)在不育材料7365A中表达,而在可育材料7365B中不表达;c.2个基因(Bn CP1和Bn CP20)在不育材料7365A中不表达,而在可育材料7365B中表达。2.提前表达拟南芥基因CP20和CP13表现雄性不育模式植物拟南芥和油菜同属于十字花科,而且前人研究表明其同源基因的功能相似,由于一个拟南芥基因对应的甘蓝型油菜基因存在多个拷贝,因此我们先研究这4个有差异表达对应的拟南芥同源基因的功能。首先我们分别考查了拟南芥突变体cp1、cp13、cp20和cp21的花药发育情况。与野生型相比,无论是营养生长还是生殖生长,其突变体都未表现出明显的差异。为进一步分析这4个基因在花药发育中的功能,利用双35S作为启动子构建过表达载体转化拟南芥,结果显示其转化株也没有不育表型出现;由于绒毡层PCD从四分体开始,而RT-PCR的结果表明这4个基因都是在花药发育中后期才开始表达的,所以用一个从花粉母细胞时期开始表达的绒毡层特异启动子A9来启动它们的表达。结果证实在A9启动子的驱动下,其中CP13和CP20的转化株表现为雄性不育,因此,本研究对半胱氨酸蛋白酶基因的功能探索主要集中在Bn CP13和Bn CP20这两个基因上。3.Bna C.CP20.1和Bna C.CP13.4基因的功能分析分别从不育材料7365A和可育材料7365B的花蕾中分离得到半胱氨酸蛋白酶基因Bna C.CP13.4和Bna C.CP20.1,对其氨基酸序列分析发现,它们都含有典型的papain类半胱氨酸蛋白酶保守结构域:EX3RX3FX2NX3IX3N、GCNGG motif、Gln残基和三联体(Cys-His-Asn)。对这两个基因的表达模式进行分析发现:Bna C.CP20.1仅在可育材料花药发育的单核期至花粉发育成熟期的绒毡层及小孢子中表达,而Bna C.CP13.4仅在不育材料花药发育的小孢子释放期到小孢子成熟期的绒毡层中表达。同时,我们检验了Bna C.CP20.1和Bna C.CP13.4在其它材料中的表达,如甘蓝型油菜两型系S45AB、芥菜型油菜hau CMS和D.berthautii CMS,结果显示Bna C.CP20.1只在可育材料花药发育中后期表达,而Bna C.CP13.4只在不育材料花药发育中后期表达,与在7365AB中的表达模式是一致的。绒毡层特异启动子Bn A9启动Bna C.CP20.1和Bna C.CP13.4的表达,其转化株细胞学分析表明在四分体时期,不育材料绒毡层发育异常,大部分小孢子不能从四分体中释放,最后绒毡层和小孢子一起降解导致雄性不育。随后的苯胺蓝染色证实小孢子不能正常分离的原因是由于四分体周围的胼胝质不能被及时地降解。透射电镜观察发现不育植株的外壁顶盖结构缺失,花粉外壁发育异常;TUNEL实验证实绒毡层PCD提前发生。这些结果表明,转化不育株的败育发生在四分体时期,由于绒毡层的提前降解,其分泌功能发生紊乱,不能及时地为小孢子的发育提供营养物质,从而导致不育。因此,在绒粘层的降解及花粉发育过程中,半胱氨酸蛋白酶基因的适时表达是非常必要的。
[Abstract]:Rape is one of the most important sources of human edible vegetable oil, the heterosis utilization is the effective strategy to improve the yield and quality, and the male sterility has become the main way to utilize the Heterosis of Brassica napus. 7365AB gene in Brassica napus (Bnams3ms3ms4bms4b/Bna Ms3ms3ms4bms4b) in system, differences between sterile and fertile material 7365A material 7365B only in the Bn locus Ms3, preliminary studies show that the Bnms3 mutant (7365A), and the expression of some tapetum degradation related cysteine protease gene affected. The degradation of tapetal cells of cashmere is considered to be programmed cell death (PCD) process, and the papain class of cysteine protease in the degradation process of cashmere timely tapetal cells play a key role in the control. In this study, we use cysteine protease gene as the breakthrough point, analysis in Brassica napus tapetum cells The molecular mechanism of degradation. The main results are as follows: 1. and 4 in 7365AB with the differential expression of cysteine protease gene in Arabidopsis, 31 papain cysteine protease gene expression. We use the method of RT-PCR analysis and homology of 31 Arabidopsis genes in Brassica napus in 7365AB, according to the different the expression patterns can be divided into the following three categories: a.27 genes in sterile and fertile materials 7365A showed the expression of B.2 genes in material 7365B; (Bn CP21 and Bna CP13) expression in male sterile 7365A, and in May was not expressed in 7365B; C.2 genes (Bn CP1 and Bn CP20) was not expressed in sterile material 7365A, and in.2. early gene expression of CP20 and CP13 in Arabidopsis showed male sterile Arabidopsis and rapeseed belongs to Cruciferae and expression of fertility in 7365B and previous research The result shows that the homologous genes with similar functions, due to the presence of a Brassica napus gene of Arabidopsis gene corresponding to multiple copies, so we study these 4 differentially expressed Arabidopsis homolog of corresponding function. First we investigated the Arabidopsis mutant CP1, cp13, cp20 and CP21 in anther development. Compared with the the wild type, both vegetative or reproductive growth, the mutant showed no significant difference. To further analyze the function of these 4 genes in anther development, using double 35S as promoter and construct the expression vector into Arabidopsis, the results showed that the transformant is not sterile phenotype; the tapetum PCD from the four split, and the results of RT-PCR showed that the 4 genes are in anther development in the mid late stage of expression, so with a tapetum from pollen mother cell stage to express Specific promoter A9 to start their expression. The results show that under the control of A9 promoter, the CP13 and CP20 transformants showed male sterility, therefore, the study on cysteine protease gene function mainly explore the functional analysis of.3.Bna C.CP20.1 and Bna C.CP13.4 genes in the two genes Bn CP13 and Bn CP20 respectively from the male sterile 7365A and fertile material 7365B buds isolated cysteine protease gene Bna C.CP13.4 and Bna C.CP20.1, the amino acid sequence analysis showed that they all contain the papain cysteine protease domain: EX3RX3FX2NX3IX3N, GCNGG motif, Gln residues and three CIS (Cys-His-Asn). The expression pattern of these two genes: Bna C.CP20.1 analysis found only in the tapetum fertile anther development of mononuclear stage to pollen maturation and pollen in the table As Bna C.CP13.4, but only in the male sterile anther development of microspore release period to expression of tapetum microspore in mature stage. At the same time, we examined the expression of Bna C.CP20.1 and Bna C.CP13.4 in other materials, such as Brassica napus type two S45AB, Hau CMS and D.berthautii in Brassica juncea CMS, the results show that Bna C.CP20.1 only in the fertile anther development in late expression, and the expression of Bna C.CP13.4 in the late male sterile anther development, is consistent with the expression pattern in 7365AB. Tapetum specific promoter Bn A9 C.CP20.1 and Bna C.CP13.4 to start Bna, the transformants cytological analysis indicated that in four split period sterile materials, tapetum abnormalities, most microspores cannot release from four points in the body, the tapetum and microspore together result in male sterility. The degradation of aniline blue staining confirmed that the microspore is not followed The reason is not often separated due to around four callose split to degradation. Transmission electron microscopy showed the lack of outer cover structure of sterile plants, pollen dysplasia; TUNEL experiments confirmed that the tapetum PCD occur in advance. These results suggest that the transformation of male sterility occurred in the four period, because of cashmere the early tapetum degradation, the endocrine function disorder, not timely provide nutrients for microspore development, resulting in infertility. Therefore, in the process of degradation and pollen tapetal development, cysteine protease gene expression timely is very necessary.

【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S565.4

【参考文献】