苹果褪绿叶斑病毒互作寄主因子的筛选与鉴定

Apple latent viruses are economically important and common pathogens on commercial apple cultivars in China, which caused great harm to agricultural production and brought huge losses. Apple chlorotic leafspot virus (ACLSV) is one of the most serious latent viruses, the occurrence and distribution of which in China was investigated and detected by us. The results showed that the virus spread in all the 13 major apple producing provinces (23 regions). The positive rate of ACLSV is 69.9%. In order to make clear the interactive mechanism between the virus and host, full length cDNA libraries were constructed using tissue culturing seedlings of Malus sylvestris cv. R12740-7A as material, and three important functional proteins of ACLSV, CP, P216 and P50, were used as bait to screen proteins interacting with the bait proteins by yeast two-hybrid. And then the protein interaction was further verified by Co-Immunoprecipitation and Bimolecular fluorescence complementation. The expression of mRNA in the health and ACLSV-infected apple leaves was also examined by Real-Time PCR. The difference of mRNA expression were compared, and the effect on mRNA expression,which caused by ACLSV infection and inleraction of viral proteins with host proteins,was analysed. At last the role of the interaction between viral proteins and host proteins in the pathogenic process of ACLSV and the process of host response to virus infection was predicted by bioinformatics analysis．This study can provide reliable evidences for further clarifying the function of ACLSV proteins, understanding the pathogenic mechanism and molecular mechanisms of host resistance and susceptibility to ACLSV.

潜隐病毒在我国苹果主产区广泛发生，影响果实品质，降低果树产量，造成严重经济损失，ACLSV是为害最大的一类潜隐病毒。申请者对我国苹果主产区13个省（23个地区）的调查和检测发现，所有地区都有ACLSV发生，平均发病率达69.9%。为了揭示ACLSV与寄主的互作机制，该项目以ACLSV木本指示植物苏俄苹果(R12740-7A)组培苗为试材，构建全长cDNA文库，以ACLSV三个重要功能蛋白CP、P216和P50基因为诱饵，利用酵母双杂交技术筛选文库中能与其互作的寄主因子，利用免疫共沉淀技术和双分子荧光互补实验对蛋白互作进行进一步验证，利用荧光定量PCR等技术对互作基因在发病寄主和健康寄主中的表达差异情况进行检测。然后通过生物信息学分析，推测互作基因在ACLSV侵染过程中可能具有的生物学功能及蛋白互作的意义，从而为揭示ACLSV与寄主的互作机制提供理论依据。

苹果褪绿叶斑病毒（Apple chlorotic leaf spot virus，ACLSV）是苹果生产上为害最大的潜隐性病毒之一，国内外学者对ACLSV生物学特性进行了较为广泛的研究，但多从病毒本身出发，基本明确了该病毒的危害症状、血清学特性和分子变异等特征，病毒所编码蛋白的功能作用、病毒的致病机制及病原与寄主互作机制等还不是很明确，尤其与寄主互作研究存在欠缺。因此，进行了以下研究：.1、以苏俄苹果组培苗为材料，构建了以真核表达载体pGADT7为基础的苏俄苹果酵母双杂交cDNA文库。通过异硫氰酸胍法提取了组培苗的总RNA，分离纯化得到了mRNA，通过SMART法合成dscDNA，dscDNA经酶切与pGADT7文库质粒载体连接，电转大肠杆菌DH10B，经涂板检测，初始文库滴度为2.67×105 cfu/mL，库容量为4.0×106 cfu，随后将文库进行了百万扩增，扩增后文库滴度为1.0×108 cfu/mL，库容为1.5×1010 cfu，在文库中随机挑取46个克隆，PCR鉴定插入片段，1 kb以上的片段测得大于75%，且重组率为100%，说明文库质量较高，符合文库构建标准，为后续实验的展开奠定了基础。.2、以pGBKT7载体为基础，构建了ACLSV外壳蛋白（coat protein, CP）和运动蛋白（P50）酵母双杂交诱饵载体。通过RT-PCR获得ACLSV CP、P50功能蛋白完整基因序列，通过酶切连接构建到pGBKT7载体上，获得重组子pGBKT7-CP和pGBKT7-P50，测序验证序列准确性，转化到酵母菌Y2H gold中，进行毒性试验和自激活活性检测，证明了诱饵载体无毒性及自激活活性，可作为后续酵母双杂交诱饵载体。.3、以ACLSV CP和P50为诱饵蛋白，从苏俄苹果cDNA文库中筛选到131个互作靶基因，通过生物信息学分析，推测其中12个在与病毒互作过程中起重要作用，初步分析8个基因在苹果生长发育、病毒侵染应答等过程中的生物学功能。.4、获得了与ACLSV CP、P50均互作的Malus 15基因，明确其完整基因序列，通过酵母双杂交、pull down技术确定其与病毒功能基因之间的互作。.5、利用半定量RT-PCR技术初步明确染病和健康寄主Malus 15基因表达量的差异，分析在病毒致病过程中和病毒与寄主互作过程中可能发挥的作用。