固氮粪产碱菌ntrC-lacZ融合基因的构建及其在水稻根部的表达

将粪产碱菌（Alcaligenesfaecalis）A1501ntrC基因和lacZ基因正向克隆于广泛性转移载体pLA2917上，获得多拷贝ntrC质粒pLAC1和含ntrC－lacZ融合基因的重组质粒pLAC2，采用双亲结合方法，将上述两个重组质粒导入A1501中，获得含ntrC－lacZ融合基因的结合子A15C2和ntrC多拷贝结合子A15C1。采用X－gal原位显色技术、显微切片、扫描电镜观察及ntrC部分缺失突变株研究粪产碱菌ntrC在根部的表达及功能，结果表明粪产碱菌A1501在水稻根部有较强的定殖能力，并能侵进入水稻根内定殖。在水稻主侧根伸长区及根内皮层薄壁细胞及侧根分生区ntrC－lacZ融合基因表达活性明显高于别的部位。高铵条件下多拷贝ntrC结合子根表定殖能力大于野生型，而ntrC突变株则低于野生型。表明ntrC基因参与固氮菌根表结合的过程。

THE CONSTRUCTION OF ALCALIGENES FAECALIS ntrC-IacZ FUSION GENE AND ITS EXPRESSION DURING ASSOCIATION WITH RICE ROOTS

A broad host range vector pLA2917 containing ntrC gene or ntrC-lacZ fusion were constructed, namely pLACI and PLAC2. The plasmids pLACI and pLAC2 were introduced into A. faecalis wild type strain A1501 by conjugation,subsequently to abtain A15C1 and A15C2. The expression and regulation of ntrC gene of A. faecalis associated with rice roots was investigated under the condition of the associative nitrogen fixation using X-Gal decoration method, micrograph and ntrC partially deletion mutant The blue precipitation was strongly existed in parenchyma cells as well as in the lateral root primordial. It showed that ntrC gene could express at much higher level in these sites. In the presence of ammonia the number of multi-copy ntrC conjugatants colonized on surface of rice roots is higher than that of wild type A1501, and the colonization of ntrC mutant is weakest among these three strains. This provided an evidence that ntrC gene might be involved in procedure of colouization of A. faecalis to rice roots.