百脉根Rboh基因家族在共生固氮中的功能研究
Function of Rboh gene family in Lotus japonicas during symbiotic nitrogen fixation

为探索植物呼吸爆发氧化酶（respiratory burst oxidase homologue,Rboh）基因家族在共生固氮中的功能和作用机制，利用分子生物学、细胞生物学和遗传学等方法，围绕百脉根Rboh基因家族中在根瘤中特异性表达的基因LjRbohA（Lj5g3v1497840）以及在根瘤中高表达的基因LjRbohB（Lj6g3v1549190）和LjRbohC（Lj5g3v1497820），分别对其蛋白的亚细胞定位、基因的表达模式、基因的启动子活性及蛋白的功能进行了研究。结果显示：LjRbohA、LjRbohB和LjRbohC蛋白都定位在植物的细胞膜上；LjRbohA、LjRbohB和LjRbohC基因在接种根瘤菌后表达上调；LjRobhA和LjRobhB基因在根瘤、维管束和侧根原基表达；利用CRISPR Cas9技术对LjRbohA、LjRbohB和LjRbohC 3个基因分别进行敲除导致结瘤数目减少。这些研究结果表明，LjRbohA、LjRbohB和LjRbohC这 3个基因可能都参与调控百脉根与根瘤菌的共生结瘤过程。
genes including RbohA(Lj5g3v1497840),RbohB(Lj6g3v1549190) and RbohC (Lj5g3v1497820) were found to be primarily expressed in nodules based on the transcriptions of Rboh genes in symbiosis in Lotus japonicus. Their function involved in symbiosis were further studied in Lotus japonicus.The subcellular location of proteins,the expression pattern of Rboh genes,the proteins function and the promoter activity of Rboh genes in Lotus japonicas were studied with methods of molecular biology,cell biology and genetics. The results showed that LjRbohA,LjRbohB and LjRbohC proteins were localized at the plasma membrane.The transcript of LjRbohA,LjRbohB and LjRbohC accumulated abundantly in roots and nodules. The activity of LjRbohA and LjRbohB promoters were detected at vascular tissue,nodule,and the lateral root primordia.The numbers of nodules were reduced in LjRbohB,LjRbohA LjRbohC  and LjRbohA LjRbohB LjRbohC knockout transgenic roots compared with control transgenic roots. It is indicated that these three Rbohs might have an overlapping function in regulating nitrogen fixation symbiosis in L. japonicus.