苜蓿中华根瘤菌042B nodD基因的克隆、序列分析及其表达

苜蓿中华根瘤菌０４２Ｂ是一株能在苜蓿和大豆上结瘤的菌株。将０４２Ｂ的ｎｏｄＳＤ基因克隆到时载体ｐＢＢＲ１ＭＣＳ－５，并在豌豆根瘤菌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，也怀苜蓿原类黄酮化合物ｌｕｔｅｏｌｉｎ反应。

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

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.