發(fā)布時(shí)間：2018-04-06 01:18

  本文選題：甘藍(lán)型油菜　切入點(diǎn)：絨氈層　出處：《華中農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：油菜是人類最重要的食用植物油來源之一,雜種優(yōu)勢利用是提高其產(chǎn)量和品質(zhì)的有效策略,而雄性不育已經(jīng)成為油菜雜種優(yōu)勢利用的主要途徑。在甘藍(lán)型油菜近等基因系7365AB(Bnams3ms3ms4bms4b/Bna Ms3ms3ms4bms4b)中,不育材料7365A和可育材料7365B的差異僅在Bn Ms3位點(diǎn)上,本實(shí)驗(yàn)室前期研究表明在Bnms3突變體(7365A)中,一些與絨氈層降解相關(guān)的半胱氨酸蛋白酶基因的表達(dá)受到影響。絨氈層細(xì)胞的降解被認(rèn)為是細(xì)胞程序性死亡(PCD)的過程,而papain類半胱氨酸蛋白酶在絨氈層細(xì)胞的適時(shí)降解過程中起到關(guān)鍵控制作用。本研究中,我們以半胱氨酸蛋白酶基因?yàn)橥黄瓶?解析在甘藍(lán)型油菜中絨氈層細(xì)胞降解的分子機(jī)理。主要研究結(jié)果如下:1.獲得4個(gè)在材料7365AB中具有差異表達(dá)的半胱氨酸蛋白酶基因在擬南芥中,有31個(gè)papain類半胱氨酸蛋白酶基因。我們利用RT-PCR的方法分析與擬南芥同源的31個(gè)甘藍(lán)型油菜基因在材料7365AB中的表達(dá)情況,根據(jù)其表達(dá)模式的不同可將它們分為以下三類:a.27個(gè)基因在不育材料7365A和可育材料7365B中均有表達(dá);b.2個(gè)基因(Bn CP21和Bna CP13)在不育材料7365A中表達(dá),而在可育材料7365B中不表達(dá);c.2個(gè)基因(Bn CP1和Bn CP20)在不育材料7365A中不表達(dá),而在可育材料7365B中表達(dá)。2.提前表達(dá)擬南芥基因CP20和CP13表現(xiàn)雄性不育模式植物擬南芥和油菜同屬于十字花科,而且前人研究表明其同源基因的功能相似,由于一個(gè)擬南芥基因?qū)��?yīng)的甘藍(lán)型油菜基因存在多個(gè)拷貝,因此我們先研究這4個(gè)有差異表達(dá)對應(yīng)的擬南芥同源基因的功能。首先我們分別考查了擬南芥突變體cp1、cp13、cp20和cp21的花藥發(fā)育情況。與野生型相比,無論是營養(yǎng)生長還是生殖生長,其突變體都未表現(xiàn)出明顯的差異。為進(jìn)一步分析這4個(gè)基因在花藥發(fā)育中的功能,利用雙35S作為啟動(dòng)子構(gòu)建過表達(dá)載體轉(zhuǎn)化擬南芥,結(jié)果顯示其轉(zhuǎn)化株也沒有不育表型出現(xiàn);由于絨氈層PCD從四分體開始,而RT-PCR的結(jié)果表明這4個(gè)基因都是在花藥發(fā)育中后期才開始表達(dá)的,所以用一個(gè)從花粉母細(xì)胞時(shí)期開始表達(dá)的絨氈層特異啟動(dòng)子A9來啟動(dòng)它們的表達(dá)。結(jié)果證實(shí)在A9啟動(dòng)子的驅(qū)動(dòng)下,其中CP13和CP20的轉(zhuǎn)化株表現(xiàn)為雄性不育,因此,本研究對半胱氨酸蛋白酶基因的功能探索主要集中在Bn CP13和Bn CP20這兩個(gè)基因上。3.Bna C.CP20.1和Bna C.CP13.4基因的功能分析分別從不育材料7365A和可育材料7365B的花蕾中分離得到半胱氨酸蛋白酶基因Bna C.CP13.4和Bna C.CP20.1,對其氨基酸序列分析發(fā)現(xiàn),它們都含有典型的papain類半胱氨酸蛋白酶保守結(jié)構(gòu)域:EX3RX3FX2NX3IX3N、GCNGG motif、Gln殘基和三聯(lián)體(Cys-His-Asn)。對這兩個(gè)基因的表達(dá)模式進(jìn)行分析發(fā)現(xiàn):Bna C.CP20.1僅在可育材料花藥發(fā)育的單核期至花粉發(fā)育成熟期的絨氈層及小孢子中表達(dá),而Bna C.CP13.4僅在不育材料花藥發(fā)育的小孢子釋放期到小孢子成熟期的絨氈層中表達(dá)。同時(shí),我們檢驗(yàn)了Bna C.CP20.1和Bna C.CP13.4在其它材料中的表達(dá),如甘藍(lán)型油菜兩型系S45AB、芥菜型油菜hau CMS和D.berthautii CMS,結(jié)果顯示Bna C.CP20.1只在可育材料花藥發(fā)育中后期表達(dá),而Bna C.CP13.4只在不育材料花藥發(fā)育中后期表達(dá),與在7365AB中的表達(dá)模式是一致的。絨氈層特異啟動(dòng)子Bn A9啟動(dòng)Bna C.CP20.1和Bna C.CP13.4的表達(dá),其轉(zhuǎn)化株細(xì)胞學(xué)分析表明在四分體時(shí)期,不育材料絨氈層發(fā)育異常,大部分小孢子不能從四分體中釋放,最后絨氈層和小孢子一起降解導(dǎo)致雄性不育。隨后的苯胺藍(lán)染色證實(shí)小孢子不能正常分離的原因是由于四分體周圍的胼胝質(zhì)不能被及時(shí)地降解。透射電鏡觀察發(fā)現(xiàn)不育植株的外壁頂蓋結(jié)構(gòu)缺失,花粉外壁發(fā)育異常;TUNEL實(shí)驗(yàn)證實(shí)絨氈層PCD提前發(fā)生。這些結(jié)果表明,轉(zhuǎn)化不育株的敗育發(fā)生在四分體時(shí)期,由于絨氈層的提前降解,其分泌功能發(fā)生紊亂,不能及時(shí)地為小孢子的發(fā)育提供營養(yǎng)物質(zhì),從而導(dǎo)致不育。因此,在絨粘層的降解及花粉發(fā)育過程中,半胱氨酸蛋白酶基因的適時(shí)表達(dá)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.

【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S565.4

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