野油菜黄单胞菌NK-01编码生物合成多糖基因的克隆

采用甲基磺酸乙酯诱变野油菜黄单胞菌ＮＫ－０１得到多糖合成缺陷的不产粘突变株。经ＳａｌⅠ部分酶切得到的野生型ＮＫ－０１染色体ＤＮＡ片段，连接到广泛寄主载体ｐＲＫ２９３上，在Ｅ．ｃｏｌｉ中建立完整的ＮＫ－０１基因库。通过使一株不产多糖突变株在协助质粒ｐＲＫ２０１３存在下，分别与含基因库的Ｅ．ｃｏｌｉ菌落群体进行三亲结合转移筛选具有卡那霉素抗性的产粘接合后体，对接合后体进行的重组质粒的酶切分析表明，

CLONING OF GENES ESSENTIAL FOR XANTHAN POLYSACCHARIDE BIOSYNTHESIS FROM XANTHOMONAS CAMPESTRIS NK-01

Xanthomonas campestris NK-01 produces amounts of xanthan polysaccharide. Nonmucoid mutants, defective in synthesis of xanthan polysaccharide, were isolate after treatment with ethylmethanesulfonate. To isolate genes essential for xanthan polysaccharide synthesis complete libraries of DNA fragments from wild-type Xanthomonas campestris NK-01 were constructed in E. coli HB101. The pooled clone bank was conjugated en masse from E. coli into a nonmucoid mutant. Kanamycin-resistant exconjugants were then screened for the ability to form mucoid colonies. Analysis plasmids form muciod exconjugants indicated that overlapping segments of DNA had been cloned. These plasmids were tested for complementation of nine additional nonmucoid mutants. A 13. 4 kb region of DNA was determined to contain at least some genes essential for xanthan polysacchdride synthesis.