黄瓜超矮生基因scp-1功能初步分析及酵母双杂交cDNA文库的构建

发布时间：2018-05-17 07:59

  本文选题：超矮生基因scp-1 + 油菜素内酯　； 参考：《西北农林科技大学》2017年硕士论文

【摘要】：株高是影响黄瓜生产的重要农艺性状,目前对黄瓜株高调控机制的研究报道较少。油菜素甾醇类化合物(BRs)是调控植物株高的主要激素之一,对BR矮生突变体的研究有助于揭示黄瓜株高调控的分子机制。课题组前期以一个超矮生黄瓜突变体C257为材料,已将其矮生突变基因scp-1定位在6.6cM区间,结合MutMap分析,在其定位区间内发现有8个基因的14个SNP发生了非同义突变。本研究在上述工作基础上,克隆了scp-1候选基因,并对scp-1进行功能初步分析。此外,黄瓜的下胚轴是黄瓜植株的第一节间,下胚轴及其以上节间共同决定了黄瓜的株高,下胚轴的长度也与壮苗和田间定植密切相关。本试验以CCMC的叶片和下胚轴为材料,构建了黄瓜酵母双杂交cDNA文库,并筛选黄瓜长下胚轴突变体候选基因CsLH1的互作蛋白。主要结果如下:1、依据突变体C257的表型和暗形态建成,表明C257为BR相关突变体。外源油菜素内酯(BL)喷施处理能够恢复C257的部分表型,说明C257为BR合成缺陷突变体。克隆了C257和CCMC的scp-1候选基因CsCYP85A,序列比对结果表明,在C257的CsCYP85A基因编码区470bp位置,发生了G到A的碱基突变,导致该基因编码的蛋白被截短。利用该位点设计CsCYP85A-dCAPS标记,在C257与GY14的F2小群体中进行遗传连锁分析,结果表明,CsCYP85A-dCAPS标记与scp-1共分离;在412份黄瓜种质中对C257的CsCYP85A突变位点进行位点多样性分析,结果表明此突变位点是唯一的。以上结果表明CsCYP85A是scp-1最可能的候选基因。通过生物信息学分析发现,在黄瓜基因组有3个CsCYP85A基因,依次命名为CsCYP85A1、CsCYP85A2和CsCYP85A3,其中CsCYP85A1在突变体和野生型植株的雄花、雌花、茎、叶、根均表达,而CsCYP85A2和CsCYP85A3的表达量难以检测。CsCYP85A1在野生型植株中的表达受BL反馈调节抑制,而在突变体中则不受影响。2、以华北型密刺黄瓜CCMC植株幼苗期叶片和下胚轴为材料,构建酵母双杂交cDNA文库,经检测库容为9.45×105 cfu,文库滴度为9.72×10~7 cfu/ml,文库插入片段主要分布在250~2000bp之间,重组克隆阳性率≥80%。3、构建黄瓜长下胚轴突变体候选基因CsLH1的诱饵载体,通过酵母双杂交点对点验证,初步证明CsLH1与CsHY5互作。利用构建的酵母双杂交cDNA文库,初步筛选到一个与CsLH1互作的蛋白GATA transcription factor 24-like。
[Abstract]:Plant height is an important agronomic trait affecting cucumber production. Brassinosterol is one of the main hormones regulating plant height. The study of Br dwarf mutants is helpful to reveal the molecular mechanism of plant height regulation in cucumber. In the early stage of the study, a super dwarf cucumber mutant C257 was used as the material, and its dwarf mutant gene scp-1 was located in the 6.6cM region. Combined with MutMap analysis, 14 SNP with 8 genes were found to have non-synonymous mutations. Based on the above work, candidate genes of scp-1 were cloned and the function of scp-1 was preliminarily analyzed. In addition, the Hypocotyl of cucumber is the first internode of cucumber plant. Hypocotyl and its above internodes jointly determine the plant height of cucumber, and the length of Hypocotyl is closely related to strong seedling and field planting. In this study, a two-hybrid cDNA library of cucumber yeast was constructed from the leaves and hypocotyls of CCMC, and the interaction proteins of the candidate gene CsLH1 of cucumber long Hypocotyl mutant were screened. The main results are as follows: 1. According to the phenotype and dark morphogenesis of mutant C257, C257 is a Br related mutant. Exogenous Brassinolide (BLL) spraying could restore some phenotypes of C257, which indicated that C257 was a mutant of Br synthesis defect. The scp-1 candidate gene CsCYP85A of C257 and CCMC was cloned. The sequence alignment results showed that there was a base mutation from G to A in the CsCYP85A coding region of C257, which resulted in the truncation of the protein encoded by the gene. Using this CsCYP85A-dCAPS marker, genetic linkage analysis was carried out in F2 population of C257 and GY14. The results showed that CsCYP85A-dCAPS marker was coisolated with scp-1, and C257 CsCYP85A mutation locus was analyzed in 412 cucumber germplasms. The results showed that the mutation site was unique. These results suggest that CsCYP85A is the most likely candidate gene for scp-1. According to bioinformatics analysis, there were three CsCYP85A genes in cucumber genome, named CsCYP85A2CsCYP85A2 and CsCYP85A3, in which CsCYP85A1 was expressed in male flower, female flower, stem, leaf and root of mutant and wild-type plant. The expression of CsCYP85A1 in wild type plants was inhibited by BL feedback regulation, but not in mutants. The leaves and Hypocotyl of CCMC plants of North China type were used as materials. The yeast two-hybrid cDNA library was constructed. The library capacity was 9.45 脳 105cfu. the titer of the library was 9.72 脳 100.7cfu-ml. the inserted fragment of the library was mainly distributed between 250~2000bp and the positive rate of recombinant clone was 鈮，

本文编号：1900585