本文選題：超矮生基因scp-1 + 油菜素內(nèi)酯��； 參考：《西北農(nóng)林科技大學(xué)》2017年碩士論文

【摘要】：株高是影響黃瓜生產(chǎn)的重要農(nóng)藝性狀,目前對(duì)黃瓜株高調(diào)控機(jī)制的研究報(bào)道較少。油菜素甾醇類化合物(BRs)是調(diào)控植物株高的主要激素之一,對(duì)BR矮生突變體的研究有助于揭示黃瓜株高調(diào)控的分子機(jī)制。課題組前期以一個(gè)超矮生黃瓜突變體C257為材料,已將其矮生突變基因scp-1定位在6.6cM區(qū)間,結(jié)合MutMap分析,在其定位區(qū)間內(nèi)發(fā)現(xiàn)有8個(gè)基因的14個(gè)SNP發(fā)生了非同義突變。本研究在上述工作基礎(chǔ)上,克隆了scp-1候選基因,并對(duì)scp-1進(jìn)行功能初步分析。此外,黃瓜的下胚軸是黃瓜植株的第一節(jié)間,下胚軸及其以上節(jié)間共同決定了黃瓜的株高,下胚軸的長(zhǎng)度也與壯苗和田間定植密切相關(guān)。本試驗(yàn)以CCMC的葉片和下胚軸為材料,構(gòu)建了黃瓜酵母雙雜交cDNA文庫(kù),并篩選黃瓜長(zhǎng)下胚軸突變體候選基因CsLH1的互作蛋白。主要結(jié)果如下:1、依據(jù)突變體C257的表型和暗形態(tài)建成,表明C257為BR相關(guān)突變體。外源油菜素內(nèi)酯(BL)噴施處理能夠恢復(fù)C257的部分表型,說明C257為BR合成缺陷突變體�？寺×薈257和CCMC的scp-1候選基因CsCYP85A,序列比對(duì)結(jié)果表明,在C257的CsCYP85A基因編碼區(qū)470bp位置,發(fā)生了G到A的堿基突變,導(dǎo)致該基因編碼的蛋白被截短。利用該位點(diǎn)設(shè)計(jì)CsCYP85A-dCAPS標(biāo)記,在C257與GY14的F2小群體中進(jìn)行遺傳連鎖分析,結(jié)果表明,CsCYP85A-dCAPS標(biāo)記與scp-1共分離;在412份黃瓜種質(zhì)中對(duì)C257的CsCYP85A突變位點(diǎn)進(jìn)行位點(diǎn)多樣性分析,結(jié)果表明此突變位點(diǎn)是唯一的。以上結(jié)果表明CsCYP85A是scp-1最可能的候選基因。通過生物信息學(xué)分析發(fā)現(xiàn),在黃瓜基因組有3個(gè)CsCYP85A基因,依次命名為CsCYP85A1、CsCYP85A2和CsCYP85A3,其中CsCYP85A1在突變體和野生型植株的雄花、雌花、莖、葉、根均表達(dá),而CsCYP85A2和CsCYP85A3的表達(dá)量難以檢測(cè)。CsCYP85A1在野生型植株中的表達(dá)受BL反饋調(diào)節(jié)抑制,而在突變體中則不受影響。2、以華北型密刺黃瓜CCMC植株幼苗期葉片和下胚軸為材料,構(gòu)建酵母雙雜交cDNA文庫(kù),經(jīng)檢測(cè)庫(kù)容為9.45×105 cfu,文庫(kù)滴度為9.72×10~7 cfu/ml,文庫(kù)插入片段主要分布在250~2000bp之間,重組克隆陽性率≥80%。3、構(gòu)建黃瓜長(zhǎng)下胚軸突變體候選基因CsLH1的誘餌載體,通過酵母雙雜交點(diǎn)對(duì)點(diǎn)驗(yàn)證,初步證明CsLH1與CsHY5互作。利用構(gòu)建的酵母雙雜交cDNA文庫(kù),初步篩選到一個(gè)與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，

本文編號(hào)：1900585