小型西瓜抗裂果基因挖掘及功能分析

The problem of fruit cracking is serious in mini-watermelon. In this study, F6 was developed from a cross between the homozygous fruit crack-resistant line "LQ06" and homozygous fruit crack-prone "XinA" mini-watermelon by single-seed descent(SSD). According to rind thinckness，firmness and degree of epicarps,F6 was divided into two sub-populations of crack-resistant and crack-prone in which the differential expression of proteins and genes correlated with crack-resistant will be identificated using two-dimensional electrophoresis combined with mass spectrometry. Candidate genes of crack-resistant will be found by real time PCR. The candidate genes shRNA silencing plasmids will be designed and constructed ，then introduced into Agrobacterium tumefaciens. The silencing plasmids and expressed vectors containing the candidate genes were transferred into fruit crack-resistant line, crack-prone line and the sub-populations mediated by Agobacterium tumefaciens，respectively. Then the phenotypes of the silencing and over-expressed candidate genes plants will be investigated. The genes correlated with crack-resistant will be acquirement. The roles of these genes may be clear in fruit cracking. These results may be helpful to clarify the molecular mechanism of watermelon fruit cracking occurred from fruit peel structure point of view, establish foundation on breeding new varieties of fruit crack-resistant watermelon through genetic improvement and solve the fruit cracking problem fundamentally.

针对小果型西瓜裂果严重问题，本研究以小果型抗裂果品系“LQ06”和易裂果品系“XinA”为亲本杂交，通过单粒传得到F6代，以果皮厚度、果皮硬度和外果皮木质化程度为标准，将F6代分为易裂和抗裂两个亚群，用蛋白质双向电泳结合质谱技术分析两亚群中的差异表达蛋白，挖掘出可能与抗裂果性能相关的蛋白与基因，利用real time PCR进行鉴定和验证找出真正的抗裂候选基因。根据抗裂候选基因的mRNA序列，设计并构建基于农杆菌表达系统的shRNA表达质粒，分别侵染易裂和抗裂品系及其杂交后代的两个亚群，同时以农杆菌系统为基础在易裂和抗裂品系中过表达这些候选基因，结合表型鉴定，获得抗裂果相关基因，明确这部分基因在裂果中的作用，从小果型西瓜果皮结构变化的角度阐明裂果发生的分子机理，为通过遗传改良选育抗裂果西瓜新品种，从根本上解决裂果问题奠定基础。

小型西瓜裂果现象严重限制了其产业发展。本文开展果实相关性状遗传、抗裂果QTL定位及不同裂果性材料果皮结构比较研究，以期为西瓜抗裂果育种奠定基础。以抗裂小型西瓜自交系“K2”和易裂“L1”为亲本研究果实相关性状的遗传规律，并分析了果实性状之间的相关性，结果显示果实切裂应度、果皮厚度、果皮硬度、果形指数、果实重量性状均符合正态分布，均可稳定遗传。果实切裂应度与果皮厚度呈极显著性负相关关系，与果皮硬度呈显著负相关关系；果皮厚度与果皮硬度呈显著性正相关关系；果实切裂应度与果皮厚度、果皮硬度相关性最高。运用全基因组重测序结合BSA法将果实裂果性状的主效控制基因初步定位在西瓜10号染色体的22,380,000bp-25,790,000bp区间内，区间大小为3.41Mb，区间内基因功能注释共找到197个候选基因，除去推测拟定基因外已注释基因有34个，关联区域内比对出500个SNP位点。抗裂果材料中果皮小细胞有逐渐变大过程，排列紧密，石细胞较小，分布密集，呈不连续团状，表皮层细胞整齐紧密；易裂果材料中果皮小细胞逐渐变大，排列分散，石细胞较大，分布不规则，呈团状分散，表皮层较薄，有明显破坏现象。本研究可为进一步开展裂果相关基因克隆，西瓜抗裂育种分子标记辅助选择及深入阐明西瓜裂果机理提供参考。以本项目第一标注发表了SCI论文2篇，国家级核心期刊论文3篇，培养硕士研究生3名。