水稻白叶枯病菌噬菌体溶源/溶菌周期转换机制的研究

Lysogenic/lytic cycle switch in bacteriophage is important. However, it is still not clear about the timing and factors that can induce the cycle switch. Previous study within our group shows that the cycle switch of 10 strains of Xanthomonas oryzae pv. oryzae can be activated upon the addition of exogenous phages, but cannot be induced by using the traditional mitomycin C. Here, to elucidate the mechanism of lysogenic/lytic cycle switch in bacteriophage of X. oryzae pv. oryzae, we collected and purified 28 prophages released upon exogenous insult. Further transmission electron microscope technique and enzymic fingerprinting of phage genome were applied to verify the induced prophages. Then the whole genome sequence of the released lytic phages will be identified to support the lysogenic/lytic cycle switch. Moreover, qPCR will be applied to determine the gene expressions, which are predicted to be involved in the lysogenic/lytic cycle switch in bacteriophage of X. oryzae pv. oryzae. Furthermore, the mutant and complementary strains will be constructed and compared with the wild type to determine the characterizations, which are related to the cycle switch upon exogenous insults. Accordingly, the mechanism of lysogenic/lytic cycle switch will be elucidated. Taken together, understanding on mechanism for cycle switch in bacteriophage of X. oryzae pv. oryzae induced by exogenous insult will contribute greatly to its biological control.

噬菌体溶源/溶菌周期转换是自然界重要的生物学现象，但目前并不清楚何时何种条件下会发生周期转换。申请人所在课题组前期已发现传统的丝裂霉素C无诱导效果，而外源噬菌体刺激能诱导10株水稻白叶枯病菌噬菌体溶源/溶菌周期的转换，为进一步搞清楚外源刺激诱导溶源/溶菌周期转换的机制，本项目拟在前期已分离纯化获得28个外源刺激诱导白叶枯病菌溶源/溶菌周期转换所释放的前噬菌体，透射电镜观察结合基因组酶切分析排除外源噬菌体的基础上，首先通过测定水稻白叶枯病菌诱导释放的烈性噬菌体基因组，以进一步鉴定噬菌体溶源/溶菌周期的转换；其次qPCR测定白叶枯病菌编码前噬菌体基因的表达情况，以鉴定溶源/溶菌周期转换的相关基因；最后构建溶源/溶菌周期转换相关基因的突变体和回补体，测定和比较其与野生型的差异，以明确水稻白叶枯病菌噬菌体溶源/溶菌转换的机制，为通过调控溶源/溶菌周期转换来生态控制水稻白叶枯病提供理论依据。

噬菌体溶源/溶菌周期转换是自然界重要的生物学现象。为了明确水稻白叶枯病菌（Xanthomonas oryzae pv. oryzae，Xoo）基因组中的前噬菌体序列，本研究分析122株Xoo菌株利用PHASTER均预测到前噬菌体序列。为阐明水稻白叶枯病菌噬菌体溶源/溶菌周期转换诱导释放前噬菌体的现象，双平板试验发现噬菌体能诱导27/31株，5种常规诱导剂只诱导1-3株。分离纯化获得24个噬菌体，经透射电镜观察结合基因组酶切图谱与基因组序列鉴定发现这些噬菌体与烈性噬菌体X2序列相似度非常高，与宿主细菌序列相似度很小。但X2的特异基因仅PCR可检测，推测菌液中自然存在的噬菌体与非宿主噬菌体协同高效裂解细菌。.为阐明噬菌体的DNA是否可以整合到细菌基因组上，前期已鉴定噬菌体可以吸附在细胞表面，钾流变化与荧光染色鉴定噬菌体注射DNA至细菌细胞。分析细菌与噬菌体共培养物的序列，发现contig169和contig516与噬菌体基因组序列相似度较高。接着，利用PCR鉴定到40个可能的插入/整合位点。进一步构建基因敲除突变体证实了噬菌体可以利用转座酶、重组酶、整合酶等整合至细菌的基因组中。接着RNA-seq鉴定到噬菌体引起约67.9%转座酶编码基因差异表达；其中150个转座酶基因显著上调，4个下调。为阐明与水稻白叶枯病菌噬菌体高效裂解相关基因，利用电转化法构建了2746个随机插入突变体库，表型筛选到84个突变体。利用反向PCR鉴定发现插入位点大多位于转座酶、乙酰转移酶、糖基转移酶等功能基因上，其中转座酶家族占约27%。钾流和生长表型表明转座酶突变体不阻止噬菌体进入细胞内，且不影响菌体生长。利用单基因敲除构建了转座酶基因敲除突变体K4和K19，双平板鉴定到突变体表型消失，证实了转座酶起重要作用。.综上所述，本文解析了水稻白叶枯病菌C2的基因组精细图谱，鉴定了31株Xoo菌株在的噬菌体高效裂解现象；分析了非宿主噬菌体整合至细菌基因组是高效裂解的前提；阐明了转座酶编码基因在噬菌体高效裂解中起重要作用，为利用噬菌体防治水稻白叶枯病害有着十分重要的实践意义。