致病疫霉坏死基因PcF/SCR.1的克隆及功能分析  

Cloning and Function Analysis of Necrosis- inducing Gene PcF/SCR.1in Phytophthora infestans

作　　者：赵冬梅[1] 徐进[1] 杨志辉[1] 朱杰华[1] 朱丽丹[1] 

机构地区：[1]河北农业大学植物保护学院,保定071000

出　　处：《农业生物技术学报》2014年第6期744-752,共9页Journal of Agricultural Biotechnology

基　　金：现代农业产业技术体系建设专项资金资助(No.CARS-10-P12)

摘　　要：PcF(Phytophthora cactorum-fragaria)蛋白是一类只存在于疫霉属(Phytophthora)中的重要致病因子。致病疫霉(Phytophthora infestans)基因组学研究预测出了20个PcF/SCR(small cysteine-rich)-like基因,但其对寄主植物的致坏死功能尚未被证实。本研究选取其中编码蛋白被预测具有4个二硫键的基因PcF/SCR.1(GenBank:XM_002902885),对其进行基因克隆和致坏死功能分析。利用RT-PCR获得336 nt的全长cDNA,将其连入表达载体pBI121后,转化农杆菌(Agrobacterium tumefaciens)LBA4404,注射接种本生烟(Nicotiana benthamiana),观察叶片症状。在注射接种后5 d时叶片开始出现黄化,7 d时叶片出现严重皱缩和坏死症状,初步表明该基因具有致坏死功能。序列比对分析表明,PcF/SCR.1基因编码的蛋白具有与PcF蛋白相类似的3个二硫键(C89和C99,C68和C100,C73和C104)和1个仅存在于PcF/SCR.1的潜在二硫键(C88和C110),推测其在维持蛋白结构功能方面发挥重要作用。为深入了解PcF/SCR.1基因的致坏死功能,利用试剂盒定点突变该基因编码蛋白的第99、100、104和110位上的半胱氨酸,构建缺失二硫键突变体的重组质粒,并在本生烟中进行瞬时表达。仅突变110位半胱氨酸,或者同时突变99位和110位两个位点的半胱氨酸时,PcF/SCR.1基因的致坏死功能并没有受到影响;当半胱氨酸的突变个数增加到3个(C99/100/110A)或者4个(C99/100/104/110A)时,接种烟草叶片没有出现坏死症状,表明二硫键在该基因的致坏死功能中具有重要作用。为进一步了解PcF/SCR.1基因在致病疫霉侵染马铃薯(Solanum tuberosum)过程中的表达模式,利用荧光定量RT-PCR分析其在不同侵染时期表达量的变化,结果显示,该基因在整个侵染过程中均有不同程度的上调,而且在接种48 h时表达量达到最高值,说明该基因可作为毒素因子在病菌侵染马铃薯叶片的整个过程中都起作用,而且主要作用可能是在侵染后期(48 h)通过分�Phytophthora cactorum-fragaria （PcF）proteins, distributing in Phytophthora only, are considered to be important virulence factors. There are 20 PcF/SCR（small cysteine- rich）- like genes which have beenfound by genomics research in Phytophthora infestans, and no necrosis-inducing function confirmation forplants yet. In this study, one of them was selected to testify its necrosis-inducing function, which was predicted to have 4 disulfide bonds in the mature protein, hereby named PcF/SCR.1 （GenBank： X_M002902885）. The full-length cDNA of PcF/SCR.1 was obtained by RT-PCR with the length of 336 nt and then inserted into binary expression vector pBI121 to produce recombinant plasmid pBI121-PcF/SCR.1. Therecombinant plasmid was transformed into Agrobacterium tumefaciens LBA4404 and analyzed necrosis- inducing function by heterologous expression in Nicotiana benthamiana. The results showed that PcF/SCR. 1 caused leaf yellow at 5 days post induction （dpi） and shrinkage and necrotic symptoms at 7 dpi, indicating that the gene was able to induce plant necrosis. Furthermore, sequence alignments among PcF/SCR.1 and PcFprotein showed that there were 3 disulfide bonds （between C89 and C99, C68 and C100, C73 and C104）, similar to that of PcF proteins, and 1 additional potential disulfide bonds （between C88 and Cll0） existedonly in PcF/SCR. 1. All those disulfide bonds were predicted to important for maintaining protein structures and function. In order to furtherly understand the necrosis-inducing function of PcF/SCR.1, site directedmutagenesis that replaced 4 cysteines with alanine at amino acid sites at 99, 100, 104 and 110 of PcF/SCR. 1 was used to destroy each disulfide bridge. The resulted plasmids then heterologously expressed in N.benthamiana. The agro-infiltration assay results showed that a cysteine point mutation at aa 110, or double mutations at aa 99 and aa 110 had no effects on the necrosis-inducing function of PcF/SCR.1. However, 3cysteine mutations （C99/100/110） or 4 （C99/100/104/11

关 键 词：PcF(Phytophthora cactorum—fragaria)SCR(small cysteine—rich)-like基因 致病疫霉 致坏死功能 

分 类 号：S435.32[农业科学—农业昆虫与害虫防治]