番木瓜环斑病毒弱毒株的构建及分析

The Construction and Analysis of the Attenuated Mutants for Cross Protection Against PRSV

弱毒株是利用交叉保护防治植物病毒病害的关键限制因子。通过对马铃薯Y病毒属病毒(强、弱毒株)的全长及部分氨基酸序列比对分析,筛选出在亲本强毒株PRSV-LM的P1和HC-Pro基因上的8个可能与致病性相关的氨基酸突变位点(I_(309)、K_(481)、K_(598)、R_(728)、F_(753)、L_(754)、N_(787)和D_(944))。采用定点突变和Gibson拼接法,分别成功构建了4种含有2个突变位点(L_(754)和N_(787))、4个突变位点(I_(309)、K_(481)、F_(753)和D_(944))、6个突变位点(I_(309)、K_(481)、F_(753)、L_(754)、N_(787)和D_(944))和8个突变位点(I_(309)、K_(481)、K_(598)、R_(728)、F_(753)、L_(754)、N_(787)和D_(944))的PRSV-LM全长c DNA侵染性克隆突变体:p Gprsvm^2、p Gprsvm4、p Gprsvm6和p Gprsvm8。经人工接种、病症观察和RT-PCR检测,4种突变体克隆均能系统性侵染非转基因番木瓜植株,除克隆p Gprsvm^2的病症表现与亲本强毒株PRSV-LM一致外,其他多位点突变的克隆p Gprsvm4、p Gprsvm6和p Gprsvm8在番木瓜植株上的病症严重程度出现依次减弱,而克隆p Gprsvm8的病症最弱,且延迟2周发病。本实验初步验证了6个氨基酸突变位点(I_(309)→S、K_(481)→Q、K_(598)→E、F_(753)→L、R_(728)→I和D_(944)→N)与PRSV-LM的病症表现及致病力相关,其中K_(598)→E和R_(728)→I可能是影响致病性的关键位点。

A nonpathogenic mild strain is a major limiting factor for controlling plant viruses by cross protection.Alignment of the full or part amino acid sequences of mild and severe strains of potyviruses, the sequences analyses of the gene P1 and HC-Pro revealed that eight point mutations of parental isolate PRSV-LM at I309, K481,K598, R728, F753, L754, N787 and D944were potentially associated with the attenuated symptom. Four full-length c DNA infectious clone mutants of PRSV-LM were successfully constructed by site-directed mutagenesis and Gibson Assembly cloning method which separately contained two mutations（at L754 and N787）, four mutations（at I309, K481,F753 and D944）, six mutations（at I309, K481, F753, L754, N787 and D944）, and eight mutations（at I309, K481, K598, R728, F753,L754, N787 and D944）, respectively named p Gprsvm-2, p Gprsvm4, p Gprsvm6 and p Gprsvm8. System infectivity developed on non-transgenic papaya after inoculation with that four clone mutants were monitored by reverse transcription-polymerase chain reaction and visual observation. Symptom severity on papaya of the p Gprsvm-2 was similar to the parental isolate PRSV-LM. With the exception of p Gprsvm-2, the symptoms induced by three other mutants（ p Gprsvm4, p Gprsvm6 and p Gprsvm8） compared with the parental PRSV-LM showed milder symptom severity. By contrast, p Gprsvm8 induced mildest symptom with two weeks delay. In the current study, we demonstrated that six amino acids change（at I309→S, K481→Q, K598→E, F753→L, R728→I and D944→N） was involved in symptom development and pathogenicity, two amino acids change（at K598→E and R728→I） are critical for the attenuated symptom.