茄子雄性不育相关基因的克隆及功能分析

Eggplant is one of the important vegetable widely cultivated throughout the world. Eggplant has a strong heterosis, and it will bring significant economic efficiency and broad application prospects when the male sterile lines are used in hybrid seed production. However, up to now the studies on the molecular mechanism of male sterility eggplant are few. Based on the results of the previous studies, long-read 454 GS FLX high-throughput sequencing is performed to produce a substantial expressed sequence tags (ESTs) dataset from critical period of fertility reversal and transcriptome datas, as reference genome, gained by bioinformatic analysis including functional annotation, classification and metabolic pathway assignment. The gene expression profiling of two materials in the same developmental phase is obtained using short-read Solexa sequencing technology, and then, differential expressed ESTs are screened by comparing expression profiles and representative metabolic pathways of two materials.The full-length sequences of the putative relevant ESTs are obtained by RACE-PCR. Their functional analysis, using a series of experiments of overexpression and RNA interference (RNAi) strategies, is performed to elucidate their specific roles in the fertility reversal process of eggplant functional male sterility. So, this study will help to uncover molecular mechanism of eggplant functional male sterility and lay an important foundation for later research of gene interactions, regulated relation and breeding of new varieties.

茄子是世界各地广泛栽培的重要蔬菜之一，具有很强的杂种优势，将雄性不育系应用于茄子杂交制种，具有显著的经济效益和广阔的应用前景。然而截至目前对于茄子雄性不育育性调控的分子机理研究较少。本项目在前期研究基础上，通过长读长的454 GS FLX高通量测序得到茄子花药开裂关键时期的宏量ESTs，并对其进行数据注释、功能分类和代谢途径分析，以获得海量转录组信息；再通过基于短读长的Solexa高通量测序的两种类型茄子相同发育时期的表达谱比较，高通量筛选差异表达ESTs，然后针对可能在茄子功能性雄性不育育性转换过程中起重要作用的基因克隆其全长，并通过转基因超量表达和RNA干涉等进行功能验证。本项目的完成将有助于揭示茄子功能性雄性不育的分子机制，也为后续基因互作及调控关系的研究以及加速茄子新品种选育奠定重要的理论基础。

为了探索茄子功能性雄性不育产生的分子机理，本项目以两个茄子功能性雄性不育系和一个可育系为材料，提取开花当天花药RNA进行了高通量转录组测序，完成了9个样品的转录组分析，共获得57.80Gb Clean Data，各样品Clean Data均达到6.13Gb，Q30碱基百分比在94.12%及以上。分别将各样品的Clean Reads与指定的参考基因组进行序列比对，基于比对结果进行可变剪接预测分析、基因结构优化分析以及新基因的发掘，发掘新基因2,971个，其中2,192个得到功能注释。根据基因在不同样品中的表达量识别差异表达基因，对其进行功能注释和富集分析。对差异表达基因进行GO功能分析显示，共有16个差异基因。全部基因均归属于生物合成、分子功能和细胞组分3大分支的51个功能组，其中主要为与酶类相关的差异基因、细胞信号传导相关基因和与能量代谢相关基因。有41个差异表达基因集基因，其中上调基因26个，下调基因15个，在COG数据库中共有12个基因被注释9种功能。对茄子雄性不育花药进行蛋白质差异组学研究，从分离的43个蛋白点中成功鉴定4个极显著差异蛋白点，并对其中一个差异表达蛋白COI1的编码基因及JAZ1、MYC2 两个基因进行了同源克隆、生物信息学分析及实时荧光定量PCR表达分析，发现COI1与茄子花药开裂有直接关系；对茄子茉莉酸信号转导途径的LOX 、AOC、OPR3、COI1基因进行了克隆和定量表达分析，LOX、AOS、AOC、OPR3开花前低水平表达影响了不育系花药的开裂。以上研究为揭示茄子功能性雄性不育系花药不开裂的机理奠定了一定的基础，也为加速茄子雄性不育种质资源的创制和杂种优势育种的利用提供了可行的参考。