水稻基腐病细菌毒素的遗传特性和产毒相关的分子标记

[目的]水稻基腐病(Erwinia chrysanthemi pv.zeae)是水稻上重要的细菌病害之一,本论文对该病菌的毒素遗传特性和产毒相关的分子标记进行了研究.[方法]通过化学诱变方法,筛选基腐细菌去质粒的突变体Ech7-mu1;应用RAPD技术,筛选产毒素相关的分子标记.[结果]毒素活性测定结果表明,野生菌Ech7和去质粒菌株Ech7-mu1都能产生毒素.从260条随机引物中,筛选出引物K10,该引物能从不产生毒素的突变株Ech7-4中扩增出大小为2139bp的DNA特异片段,但不能扩增野生菌Ech7,将该片段克隆,测序分析,设计特异引物,在突变体Ech7-4中获得了与毒素产生相关的SCAR分子标记(标记符合率为100%).该基因片段有5个ORFs,其中2个ORFs分别编码NADH-黄素还原酶和N-乙酰转移酶,另外2个不完整的ORFs编码的蛋白分别与Pseudomonas aerginosa(ZP00136947)和Yersinia Pestis(ZP01177873)的抗菌素代谢转运蛋白通透酶(DMT)具有66%和46%的同源率.[结论]水稻基腐细菌毒素的生物合成是由染色体基因编码,与质粒无关.不产生毒素的突变菌株基因突变的位点位于SCAR标记DNA的3'末端.

Genetic and molecular characters of toxin producting Erwinia chrysanthemi pv. Zeae

OBJECTIVE: Bacterial foot rot, caused by Erwinia chrysanthemi pv. zeae, is one of the most important diseases in rice. Genetic and molecular characters of toxin producting for Erwinia chrysanthemi pv. zeae were conducted in this paper. METHODS: A plasmid-deficient strain, Ech7-mul, was obtained by chemical mutation,and the relative specific molecular mark with toxin was screened from Random Amplified Polymorphic DNA (RAPD) by PCR. RESULTS: The wild strain Ech7 and the plasmid-deficient strain Ech7-mul could both produce toxin.We screened 260 random primers in PCR, and found that a specific fragment (2139bp) could be amplified with K10 primer from theminus-toxin strain Ech7-4 DNA, but could not from the wild strain Ech7 DNA. The amplified fragment DNA was cloned and sequenced, and specific primers were designed to amplify it. The 2139bp fragment DNA could be a specific molecular mark with 100% SCAR identity between wild strain and the toxin mutant strain. Sequence analysis showed that there were five open reading frame (ORF), two of them were NADH-flavin reductase and N-acetyltransferase,respectively. Another ORF, located in the end region of 2139bp fragment, had 66% and 46% homologies with permeases of the drug/metabolite transporter (DMT) from Pseudomonas aerginosa (ZP00136947) and Yersinia Pestis (ZP01177873). CONCLUSION: Toxin biosynthesis in E. chrysanthemi pv. zeae might be coded by chromosome, but not by the bacterial plasmid.The position of gene mutation in the mutant Ech7-4 might be at the 3' region of toxin-relation SCAR DNA fragment.