西瓜性型遗传规律及性别决定相关基因的鉴定与功能分析

发布时间：2018-05-17 17:35

本文选题：西瓜 + 性别　；参考：《中国农业大学》2016年博士论文

【摘要】：葫芦科(Cucurbitaceae)瓜类作物因包含被子植物全部7种性型而成为植物性别决定机理的模式作物。与甜瓜(Cucumis melo)、黄瓜(Cucumis sativus)相比,西瓜(Citrullus lanatus)性型遗传规律和性别决定机理的研究相对落后。本论文利用西瓜雌雄异花同株、雄花完全花同株、全雌株等3种性别表型(性型)的4个材料构建了5组六世代群体,解析了西瓜性型遗传规律。在此基础上,利用相关遗传群体,定位、克隆并鉴定到2个主要性别决定基因——控制西瓜雄花完全花同株性型的a基因CitACS4和控制西瓜全雌株性型的gy基因ClWIP1,并分析了这2个基因的功能,旨在探索西瓜性别决定的分子机理,并为西瓜育种及良种繁育实践提供理论与技术基础。本论文取得的主要结果如下：利用雌雄异花同株性型材料XHB,全雌株性型材料XHBGM以及2个雄花完全花同株性型材料SL3H. AKKZW构建了5组六世代群体。通过春夏两个季节的性型调查,进行了西瓜性型的遗传规律分析。验证了雄花完全花同株性型由隐性单基因a控制,全雌株性型由隐性单基因gy控制。首次发现雄花雌花完全花同株性型由另一隐性基因tm控制,a基因对tm基因隐性上位。发现季节变化不影响单株性型特征但影响单株着生花器官的性别比率。利用西瓜全基因组信息同源克隆西瓜乙烯合成酶(ACS)基因CitACS4,该基因与甜瓜a基因CmACS-7同源性高。通过对西瓜基因组重测序材料CitACS4序列比对,发掘与雄花完全花同株性型紧密连锁的SNP位点C364W,并开发了dCAPs标记。遗传分离群体和自然群体性型连锁分析表明：该SNP位点与雄花完全花同株性型共分离。功能分析表明,CitACS4在雌花和完全花发育两性期的心皮原基特异表达,其功能是选择性败育雄蕊原基,与雄蕊原基的PCD进程相关。位点C364W破坏了CitACS4所编码蛋白结构稳定性,导致ACS酶活性显著降低、雄蕊原基败育进程受阻,使西瓜完全花发育。利用AKKZW和XHBGM的重测序信息,分析其基因组之间差异位点。设计标记对两个材料的BC1群体进行染色体步移,将全雌株性型定位在染色体2(Chr.2)与染色体3(Chr.3)上。通过生物信息学、PCR以及FISH杂交等手段,发现全雌株性型材料XHBGM的Chr.2和Chr.3之间发生了染色体易位。明确其易位断点位于Chr.2:33348296和Chr.3:4043234bp..4043236bpo Chr.2的易位断点位于C2H2型锌指蛋白转录因子基因Cla008537的外显子区域,与甜瓜g基因CmWIP1同源,将该基因命名为ClWIP1。功能分析表明,ClWIP1在西瓜雄花发育两性期心皮原基特异表达,其功能是选择性败育心皮原基,与心皮原基的PCD进程相关。染色体易位导致ClWIP1失活,心皮原基败育进程受阻,造成全雌株性型突变体出现。
[Abstract]:Cucurbitaceae (Cucurbitaceae) melons have become a model crop for sex determination because of the inclusion of all 7 sexual types of angiosperms. Compared with Cucumis meloides and Cucumis sativus (Cucumis sativus), the study of sex type inheritance and sex determination mechanism of Citrullus lanatus was relatively backward. In this paper, five groups of six-generation populations were constructed from the same plant of male and female, the same plant of male flower and the same plant of male flower, and the whole female plant, and the inheritance law of watermelon sex type was analyzed. On this basis, using relevant genetic populations, mapping, Two major sex-determining genes were cloned and identified: a gene CitACS4, which controls the male flower type of watermelon, and gy gene ClWIP1, which controls the female type of watermelon, and the function of the two genes was analyzed. The aim of this study was to explore the molecular mechanism of sex determination in watermelon and to provide theoretical and technical basis for watermelon breeding and breeding practice. The main results obtained in this paper are as follows: XHBs, XHBGM, SL3H, and SL3H, respectively. AKKZW constructed five groups of six generations. The genetic patterns of watermelon were investigated in spring and summer. It was verified that the homozygous type of male flower was controlled by recessive single gene a, while the whole female type was controlled by recessive single gene gy. For the first time, it was found that the female flowers of male flowers were completely homozygous by another recessive gene tm, which controlled the recessive epistasis of a gene to tm gene. It was found that seasonal variation did not affect the sexual characteristics of individual plants but affected the sex ratio of flowering organs per plant. CitACS4, a watermelon ethylene synthase gene, was cloned by using the whole genome information homology of watermelon. It has high homology with melon a gene CmACS-7. Based on the CitACS4 sequence alignment of watermelon genome, C364W, which is closely linked to the male flower, was identified and the dCAPs marker was developed. Genetic segregation population and natural population linkage analysis showed that the SNP locus was coisolated from male flower. Functional analysis showed that CitACS4 was specifically expressed in the carpel primordia at the stage of female flower and full flower development. The function of CitACS4 was selective abortion of the stamen primordium, which was related to the PCD process of the stamen primordium. The site C364W destroyed the structural stability of the protein encoded by CitACS4, and led to a significant decrease in the activity of ACS enzyme, and blocked the abortion process of the stamen primordium, which resulted in the complete flower development of watermelon. The differential loci of AKKZW and XHBGM were analyzed by using the resequenced information. Two BC1 populations with two materials were designed for chromosome walking, and the whole female sex type was located on chromosome 2 Chr. 2) and chromosome 3 Chr. 3). By means of bioinformatics and FISH hybridization, it was found that chromosomal translocation occurred between Chr.2 and Chr.3 in XHBGM. It was confirmed that the translocation breakpoints of Chr.2:33348296 and Chr.3:4043234bp..4043236bpo Chr.2 were located in the exon region of Cla008537 of C2H2 type zinc finger protein transcription factor gene, which was homologous to the CmWIP1 gene of muskmelon g gene, and the gene was named ClWIP1. The functional analysis showed that ClWIP1 was specifically expressed in the male flower of watermelon, and its function was to selectively abort the carpenter primordia, which was related to the PCD process of the carpenter primordia. Chromosomal translocation resulted in inactivation of ClWIP1 and obstruction of abortive process of carpenter primordia, resulting in the emergence of female mutant.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S651

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