大豆GmCYS20基因在百脉根共生结瘤过程中的功能研究  

Function of the soybean GmCYS20 gene in symbiotic nodulation of Lotus japonicus

作　　者：柯丹霞[1,2] 彭昆鹏 张孟珂[1] 贾妍 KE Dan-xia;PENG Kun-peng;ZHANG Meng-ke;JIA Yan(College of Life Sciences, Xinyang Normal University, Xinyang 464000, China;Institute for Conservation and Utilization of Agro-bioresources in Dabie Mountains, Xinyang Normal University, Xinyang 464000, China)

机构地区：[1]信阳师范学院生命科学学院,河南信阳464000 [2]大别山农业生物资源保护与利用研究院,河南信阳464000

出　　处：《草业学报》2018年第9期132-141,共10页Acta Prataculturae Sinica

基　　金：国家自然科学基金项目(31400213);河南省科技攻关计划项目(182102110448);农业部油料作物生物学与遗传育种重点实验室开放课题基金(2017002);信阳师范学院青年骨干教师资助计划(2015)和信阳师范学院"南湖学者奖励计划"青年项目(2016)资助

摘　　要：半胱氨酸蛋白酶抑制剂(cystatin)参与豆科植物与根瘤菌共生结瘤过程。克隆了大豆的1个半胱氨酸蛋白酶抑制剂基因GmCYS20,基因编码区全长为291bp。氨基酸多序列比对分析发现,大豆GmCYS20蛋白与野生大豆cystatin蛋白相似性高达99%。构建GmCYS20基因的植物表达载体,通过发根农杆菌介导的百脉根发状根转化法获得转GmCYS20基因复合体植株,利用GUS染色和RT-PCR方法鉴定阳性发状根。复合体植株接种根瘤菌4周后,转GmCYS20基因植株结瘤数目明显高于空载体对照,结瘤指示基因的转录水平也呈上升趋势。进一步对接种10d复合体植株中根瘤菌的侵染表型进行统计发现,过量表达GmCYS20基因并不影响根瘤菌的侵染过程,而是在根瘤起始与发育阶段发挥正调控作用。结果表明:GmCYS20基因通过促进根瘤的起始与发育,从而参与调控共生结瘤过程。研究结果可为揭示豆科植物与根瘤菌的共生互作机制提供新的分子生物学证据。Cystatin plays an important role in the symbiotic relationship between leguminous plants and rhizobium.We cloned the soybean cystatin gene GmCYS20(291bp)for analysis in this study.The analysis of amino acid multiple sequence alignment with homologous proteins showed that the similarity between soybean GmCYS20and wild soybean(Glycine soja)CYS was up to99%.The plant overexpression vector was constructed and GmCYS20-overexpression composite plants were obtained in Lotus japonicas using the hairy root transformation method.Positive hairy roots were identified by GUS staining and RT-PCR assay.After inoculation with rhizobia for4weeks,overexpression of GmCYS20increased the number of nodules in composite Lotus japonicus and up regulated the expression levels of symbiotic related genes.Further analysis of rhizobial infection phenotypes after inoculation with rhizobia for10days showed that overexpression of GmCYS20gene did not affect the rhizobium infection process but played a positive role in initiation and development of nodules.These results showed that the GmCYS20gene was involved in the regulation of symbiotic nodulation by promoting nodule initiation and development in L.japonicus.The results provide new molecular biological avenues to reveal the mechanisms of the symbiotic interaction between leguminous plants and rhizobium.

关 键 词：大豆 半胱氨酸蛋白酶抑制剂 百脉根 共生结瘤 根瘤发育 

分 类 号：S565.1[农业科学—作物学]