大豆持绿基因Gmsgk的精细定位、分子克隆和功能验证

Plant remains greenness of leaves during senescence is known as stay-green.The stay-green character can continue to plant photosynthesis and increase plant adversity resistance. Study on stay-green gene is an element task to inquire into stay-green mechanisms in plant. A novel soybean stay-green mutant, M5136, was selected from excellent cultivar Kefeng14 by radiation with 60Co-ray and had a genetic stability in stay-green character after years. Genetic analysis showed that one recessive gene was controlling the stay-green character in M5136 by identify a separate group of (Kefeng14×M5136)F2, named Gmsgk. Based on the previous research, Gmsgk will be fine mapped using a larger F2 segregation population from a cross (Kefeng14×Zhonghuang 35) in this research. The candidate gene sequences of Gmsgk will be calculated, cloned and analyzed by the method of bioinformatics with soybean DNA sequence in GenBank databases. The candidate gene will be transfer to the soybean and Arabidopsis by agrobacterium-mediated method to verify the expression function of Gmsgk. The research results will lay the foundations for the development and utilization of stay-green gene, and has also the certain theory value for research the plant senescence mechanism and breeding the crop variety of high photosynthetic efficiency.

植物衰老期间叶片依然保持绿色的现象称为持绿，持绿能够延续植物的光合作用和增加抗逆性，研究持绿基因是探讨植物持绿机理的基础工作。我们利用60Co-γ射线辐照，从大豆优良品种科丰14诱变后代中获得1个持绿突变体M5136，经多年鉴定持绿性遗传稳定。(M5136×科丰14)F2分离群体的遗传分析表明，M5136的持绿性由1对隐性基因控制，暂命名为Gmsgk。本项目利用M5136与非持绿大豆中黄35杂交，构建大规模的F2分离群体，开展Gmsgk基因的精细定位；在此基础上，根据已知大豆基因组序列，结合生物信息学方法，确定并克隆Gmsgk候选基因，分析其结构，通过遗传转化验证候选基因的功能。研究结果不仅为持绿基因Gmsgk的开发和利用奠定基础，而且对研究植物的衰老延缓机制和高光效作物的遗传改良具有一定的理论价值。

本项目以自主发现的大豆持绿突变体“M5136”为材料，持绿性由1对隐性基因Gmsgk控制，通过对Gmsgk的精细定位、克隆、遗传转化功能验证等研究，为该基因的利用提供依据。获得的主要结果如下：.（1）利用重组自交系（“M5136”ד L74-826”）F5:6的分离群体结合BSA法对Gmsgk进行分子标记，定位于大豆D1a连锁群上，紧密连锁的SSR分子标记关系为：Sat_345-290bp - Satt468-210bp - Satt436-190bp - Gmsgk - Satt283-170bp - Satt370-290bp - Sat_160-220bp。其中，Satt436-190bp 和Satt283-170bp与Gmsgk的遗传距离分别为12.9cM和15.1cM。这两个标记也可作为Gmsgk分子设计的标记。同时，获得了一项国家发明专利“一种辅助鉴定具有绿子叶性状的方法”（专利号：ZL201210330293.0）；.（2）以大豆叶绿素降解的相关基因GmSGR1和GmSGR2序列为参考，采用RT-PCR的方法，克隆 “M5136”和野生型“科丰14”中GmSGR1和GmSGR2的基因序列。通过比对，发现“M5136”中Gmsgk为GmSGR2的突变型，GmSGR2序列长度为815bp，Gmsgk长度为814bp，在编码区182bp处出现一个T碱基的缺失，导致随后的第6个碱基处读码为终止密码子，引起蛋白质合成受阻，基因功能丧失，表现持绿性状。同时，构建GmSGR1和 GmSGR2与荧光蛋白基因的融合基因表达载体遗传转化烟草，借助荧光蛋白的细胞定位法，将GmSGR1和GmSGR2定位在叶绿体中，由于Gmsgk为GmSGR2的突变型，故Gmsgk定位在叶绿体中；.（3）由于Gmsgk为隐性基因，利用RNAi干扰载体遗传转化大豆法，对Gmsgk的进行功能验证。T0代种子完全或部分呈现绿色，T1叶片成熟期持绿，T1种子为绿色，表明GmSGR2对于调控大豆持绿性起关键性作用，缺失GmSGR2则叶绿素在成熟期无法正常降解，而Gmsgk为GmSGR2的缺陷型，证明Gmsgk确为持绿基因； .（4）在研究持绿基因Gmsgk的同时，对持绿突变体进行了利用研究，选育出3个大豆新品种。“北农106”通过北京市农作物品种审定，使用权转让给北京市一家种子企业；北农107和北农1