黄瓜果实多刺基因ns的克隆及功能分析

Fruit spine is one of important quality trait in cucumber, its density is a major contents for breeding program. One recessive gene, ns, confers numerous spines on the fruit of cucumber. There was no research of the molecular mechanism for regulation and expression about the ns gene until to now. The fruit spines of cucumber are multicellular epidermal trichomes. The development of trichome was regulated by MYB-bHLH-WD40 complex in plant. We detected a WD40 protein gene in the preliminary chromosomal mapping region of the ns gene. This discovery laid good foundations for gene cloning of the ns and making clarification the molecular mechanism for the formation of numerous spines. Taking this as the breakthrough point, the proposal aimed at cloning the ns gene based on the whole genome sequence of 9930 and the re-sequencing for 115 core germplasms. And re-sequencing and RNA sequence of parental lines, genome-wide association study (GWAS) for the numerous spines trait will also be conducted. Functional analysis will be done for the ns candidate genes through trans-gene technology and using sequence comparison, gene homology, over-expression analysis, and RNA-seq technology. It’s the first time to carry out the study of the ns gene in cucumber at the molecular level. This study aimed at mining the key gene conferring numerous spines on the fruit in cucumber. The results of the present study have significant impact on exploring the molecular mechanism of the development of fruit spines in cucumber, and it can provide academic guidance and practical molecular markers to the MAS breeding program.

黄瓜果刺密度作为重要外观性状是品质育种的重点之一。黄瓜果实多刺性状由隐性单基因ns控制，其在分子水平如何进行调控和表达的研究处于空白。黄瓜果刺是多细胞无腺体的表皮毛，植物表皮毛的发育涉及MYB-bHLH-WD40复合体的调控机制。项目组在ns基因定位区段发现了与植物表皮毛发育相关的WD40蛋白基因，为克隆ns以及揭示其调控表达的分子机理奠定了良好基础。本项目以此为切入点，在充分挖掘9930全基因组序列信息和完成115份核心种质重测序的基础上，结合对亲本材料的重测序、转录组测序及GWAS技术，旨在克隆控制果实多刺的ns基因，并通过序列比较、同源性分析、功能互补验证、转基因功能验证、RNA-seq等手段，明确ns基因调控和表达的分子基础。本项目拟通过高效技术路线挖掘调控果刺发育的关键基因，研究结果不仅可以揭示果实多刺发育的内在分子机制，而且为MAS技术创新不同果刺类型的种质材料提供理论依据。

黄瓜果刺密度作为重要外观性状是品质育种的重点之一。黄瓜果实多刺性状由隐性单基因ns控制，其在分子水平如何进行调控和表达的研究处于空白。本项目在充分挖掘9930全基因组序列信息和完成115份核心种质重测序的基础上，结合对亲本材料的重测序、转录组测序及GWAS技术，利用F2大群体结合高密度遗传图谱，将ns基因定位在黄瓜2号染色体nsIndel55和nsIndel39之间，32.4kb物理区间内，该区域内包含5个候选基因Csa2M264060、Csa2M264560、Csa2M264570、Csa2M264580、Csa2M264590。测序分析候选基因Csa2M264590发现在母本NCG122中相对于父本NCG121该基因的内含子上存在多处单碱基或小片段的插入、缺失和替换。qRT-PCR结果表明Csa2G264590基因在黄瓜果实上特异表达，且在两亲本中的表达量存在极显著的差异。结合转录组测序技术，确定了果实多刺候选基因Csa2G264590为本研究目的基因，完成了该基因的克隆。亚细胞定位结果表明ns蛋白主要定位于细胞膜。通过过表达、原位杂交、蛋白互作、生长素测定等技术方法，完成了目的基因的功能分析。获得了高准确率且简便实用的Indel标记和SNP标记，为创新不同果刺类型的黄瓜种质材料提供了技术支撑。