苜蓿雄性不育相关基因的克隆及分子机理研究

Male sterile lines that have genetic stability and excellent traits is important tool for alfalfa heterosis .Molecular mechanism of fertility genes is the foundation of sterile lines application. It has also important guiding significance for the MS in production.At present,male stetile lines Ms-4 had not been applied .And molecular mechanism of fertility genes had not been studied in deepth.We cann't improve and create good sterile lines by genetic technology. In this study, suppression subtractive hybridization (SSH) will be used to build alfalfa male sterility and fertility cDNA of differentially expressed genes in the same genetic background library on the basis of the preliminary studies. This method will overcome the conventional molecular markers can't display differences in gene defects. It will screen specific genes of alfalfa male sterility and determine functional sterile genes. The project research results can not only reveal the molecular mechanism of alfalfa male sterility ,but also for the future to carry out genetic engineering to create a good foundation, and also has important theoretical significance in the production of sterile line as soon as possible.

稳定遗传的、性状优良的雄性不育系是苜蓿利用杂种优势的重要工具。育性基因分子机理是不育系应用的基础，对于不育系在生产上的应用也具有重要指导意义。目前，我国发现的苜蓿雄性不育系MS-4还没有在生产上应用，其不育基因分子机理不清，无法利用基因工程改良或创造新的不育系。因此，本研究拟在前期研究的基础上，克服常规分子标记无法有选择地显示差异基因的缺陷，采用抑制性消减杂交法(SSH)构建同一遗传背景下苜蓿雄性不育与可育差异表达基因的cDNA文库，筛选克隆苜蓿雄性不育特异基因，确定不育功能基因。本项目研究成果不仅可以揭示苜蓿雄性不育的分子机理，还可为今后开展基因工程创造苜蓿雄性不育系的研究奠定良好基础，对不育系尽快在生产上应用也具有重要理论指导意义。

本项目研究了苜蓿雄性不育系MS-4和可育系差异基因功能，数量和表达，通过分别构建不育条件下和可育条件下基因差异表达消减cDNA文库，共获得功能已知的EST序列190条，其中与MADS-box基因家族有关的基因有3个。苜蓿雄性不育恢复基因诱导表达的相关基因涉及基础代谢、胁迫应答、蛋白活性、转运、翻译、花发育、信号转导等。经GO功能分类，推定出6个相关基因进行荧光定量PCR，结果表明：6个基因在苜蓿雄性不育系花蕾不同发育时期中出现差异表达，推测这6个基因可能与苜蓿雄性育性相关，差异基因的获得为苜蓿雄性育性恢复基因的深入研究提供了依据；并获得了苜蓿雄性不育系MS-4 RNA研究的有效取样方法，解决远距离携带液氮取材困难的问题。苜蓿雄性不育系MS-4花蕾和小花冰盒保存6h是基地取样短途运输的允许时间。

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