苜蓿中华根瘤菌042B nodD基因的克隆、序列分析及其表达

CLONING, SEQUENCING AND EXPRESSION OF SINORHIZOBIUM MELILOTI STRAIN 042B GENE nod D

苜蓿中华根瘤菌（ Ｓｉｎｏｒｈｉｚｏｂｉｕｍ ｍｅｌｉｏｔｉ） ０４２Ｂ 是一株能在苜蓿和大豆上结瘤的菌株。将０４２Ｂ 的ｎｏｄＤ 基因克隆到载体ｐＢＢＲ１ ＭＣＳ５ ，并在豌豆根瘤菌（ Ｒｈｉｚｏｂｉｕ ｍ ｌｅｇｕｍｉｎｏｓａｒｕ ｍ ｂｖ ．ｖｉｃｉａｅ）ＬＲＲ５０４５ 系统中进行功能分析，发现０４２Ｂ 的ＮｏｄＤ 蛋白能与大豆的类黄酮化合物ｇｅｎｉｓｔｅｉｎ 结合，也能与苜蓿的类黄酮化合物ｌｕｔｅｏｌｉｎ 反应。表明０４２Ｂ ｎｏｄＤ 基因很可能是其能够在两类寄主植物上结瘤的寄主专一性决定因子。将ｎｏｄＤ 基因片段分别克隆到表达载体ｐＴｈｉｏＨｉｓ Ａ、Ｂ和Ｃ，得到了３ 个重组质粒ｐＸＤＡ、ｐＸＤＢ和ｐＸＤＣ。通过序列分析发现，ｐＸＤＣ 中的ｎｏｄＤ 基因与ｐＴｈｉｏＨｉｓ Ｃ 中的ｔｒｘＡ 基因阅读框吻合。将大肠杆菌（ Ｅ．ｃｏｌｉ）Ｔｏｐ１０（ｐＸＤＣ） 经ＩＰＴＧ 诱导后用ＳＤＳＰＡＧＥ 分析，发现融合蛋白表达成功，其分子量恰为ＴｒｘＡ 与ＮｏｄＤ 之和。利用Ｗｅｓｔｅｒｎ 印迹法证明Ｅ．ｃｏｌｉ Ｔｏｐ１０（ｐＸＤＣ） 所表达的蛋白质是由目的基因编码的。

S.meliloti strain 042B is a rhizobium strain which can form nodules both on alfalfa and on soybean. In this study, nod D gene of 042B was cloned into pBBR1MCS 5.By functional analysis in the system of R.leguminosarum bv. viciae LPR5045(pSym -,pMP154),it was found that the NodD has responses both to luteolin and to genistein. This result showed that the 042B nod D gene is probably a specific nodulation determinant which determined its capability of nodulation on both alfalfa and soybean. \ \ The nod D fragment was then cloned into the expressional vector pThioHis A,B and C, and three recombinant plasmids pXDA,pXDB and pXDC were constructed. The plasmid pXDC was identified to be in the same open reading frame with the trxA gene of pThioHis C throuth sequencing analysis. Inducing IPTG and analyzing with SDS PAGE,it was found that the fusion protein expressed from E.coli Top10(pXDC) with the molecular weight of TrXA and NodD together. The Western bolt result demonstrated that the expression result is the target gene nod D product.