田菁茎瘤固氮根瘤菌ORS571类T3SS效应蛋白的预测、原核表达及相关功能分析

摘要

Rhizobium type Ⅲ secretion system (T3SS) effector proteins play important roles in nodulation,nitrogen fixation and decision of host range.In order to express and analysis the type Ⅲ-like secretion system effector proteins of Azorhizobium caulinodans ORS571,the whole proteome of ORS571 was analyzed by bioinformatics software BPBAac and Effective T3 and related literature reports.Seven candidate effector proteins of ORS571 were obtained,and they were AZC_0308,AZC_0649,AZC_0667,AZC_2699,AZC_2928,AZC_3165 and AZC_4087,respectively.Specific amplification primers with restriction enzyme cutting sites were designed and synthesized,and whole gene sequences of the 7 effector proteins were obtained by PCR.Than the 7 gene fragments were cloned into the prokaryotic expression vector pMAL-c4x,respectively.Through optimizing the expression conditions,a large amount of soluble fusion proteins were obtained.Amylose Resin affinity chromatography was used to purify fusion proteins,and the results indicated that purification effect was better.Detection of fusion proteins expression were completed by Western blot using anti-maltose binding protein (MBP) monoclonal antibody (horse radish peroxidase conjugated),and the results implied that these 7 fusion proteins expressed correctly and the effect of protein expression was better.AZC_2928 gene deletion mutant was constructed and infected wheat (Triticum aestivum) XiaoYan 22,and the results showed that AZC_2928 gene had inhibitory effect on the growth of wheat.In conclusion,this research lays a foundation for the application of the Azorhizobium caulinodans ORS571 type m-like secretion system effector proteins in symbiotic nitrogen fixation of non-leguminous plants.%根瘤菌Ⅲ型分泌系统(typeⅢsecretion system,T3SS)效应蛋白在固氮结瘤及决定根瘤菌的宿主范围等方面具有重要作用.为了对田菁(Sesbaniac annab ina)茎瘤固氮根瘤菌(Azorhizobium caulinodans)ORS571的类T3SS效应蛋白进行预测、表达和相关功能分析,本研究利用生物信息学软件BPBAac和Effective T3对ORS571的全蛋白组进行分析,并结合相关文献报道获得了7个效应蛋白,分别是AZC0308、AZC_0649、AZC_0667、AZC_ 2699、AZC_ 2928、AZC_3165和AZC 4087;设计并合成具有相应酶切位点的特异性扩增引物,利用PCR获得了以上7个效应蛋白的全基因序列;以pMAL-c4x作为原核表达载体,成功构建了7种重组表达载体,通过优化诱导表达条件获得了可溶性高的融合蛋白;随后利用Amylose Resin亲和层析对表达的融合蛋白进行纯化,获得了较高纯度的融合蛋白;以辣根过氧化物酶标记抗麦芽糖结合蛋白(maltose binding protein,MBP)单克隆抗体作为一抗进行Western blot检测,结果表明,7种效应蛋白高效正确表达.构建AZC 2928基因缺失的ORS571突变体并侵染小麦(Triticumaestivum)小偃22,结果表明,AZC 2928基因对小麦的生长具有抑制作用.研究结果为今后从茎瘤固氮根瘤菌ORS571类T3SS效应蛋白方面研究根瘤菌与非豆科植物的共生固氮提供实验基础.