辣椒疫霉质外体疏水小蛋白SCR82编码基因的转录特征、异源表达和功能

Gene transcriptional pattern, prokaryotic expression and functional analysis of an apoplastic, hydrophobic and small effector SCR82 from Phytophthora capsici

摘要:

[目的]分析PcF/SCR质外体疏水小蛋白SCR82编码基因在辣椒疫霉生长发育和侵染寄主阶段的转录特征，克隆其cDNA和基因组全长序列，分析蛋白性状及其序列保守性，利用大肠杆菌表达并获得纯化蛋白，分析其生物学功能。[方法]提取辣椒疫霉菌丝、游动孢子囊、游动孢子、萌发休止孢和7个侵染时间点（1.5、3、6、12、24、36、72 h）的本氏烟根部总RNA，利用半定量RT-PCR分析scr82的转录表达水平；通过高保真PCR从萌发休止孢cDNA和基因组DNA中克隆出该基因全长序列；利用软件对SCR82进行生物信息学分析，挖掘其他物种中的同源序列，预测蛋白性状；将cDNA全长序列克隆到含6×His-SUMO序列的pET28a（+）中，在不同温度（22、37℃）和IPTG浓度（0.1、0.2、0.5、1.0 mmol/L）组合条件下利用大肠杆菌Rossette 2菌株表达该蛋白，利用Ni²⁺亲和层析柱纯化蛋白；将蛋白浸润本氏烟和拟南芥叶片，分析它是否引起植物细胞死亡，蛋白浸润12、24 h后利用RT-qPCR分析本氏烟中3个抗性相关基因（NbMC1、NbSOD、NbPOX）的表达量变化。[结果]scr82基因在辣椒疫霉萌发休止孢和侵染寄主阶段上调表达；该基因为辣椒疫霉中单拷贝基因，不含内含子，开放阅读框为249 bp；预测其编码82个氨基酸，包含长度为21个氨基酸的信号肽序列和7个半胱氨酸但不含有任何已知功能域，蛋白疏水性强，二级结构主要为α-螺旋和不规则卷曲，在疫霉菌中保守；含重组质粒的菌株在22℃经0.2 mmol/L的IPTG诱导过夜（~16 h）产生大小约24 kDa的融合蛋白，纯化获得30 mg/mL的可溶性蛋白；融合蛋白不引起本氏烟和拟南芥叶片的细胞死亡，但导致本氏烟抗性相关基因均上调表达。[结论]本研究获得了辣椒疫霉胞外疏水小蛋白SCR82的原核表达蛋白，该蛋白不引起植物细胞死亡，但触发植物的防卫反应。

Abstract:

[Objective] The aim was to analyze the gene transcriptional profiles, express the protein in Escherichia coli and analyze functions of the PcF/SCR effector SCR82 from Phytophthora capsici. [Methods] Gene transcripts during the developmental stages (mycelia, sporangia, zoospores and germinated cysts) and infection period (1.5, 3, 6, 12, 24, 36, 72 h post-inoculation) of Nicotiana benthamiana by P. capsici were checked by semi-quantitative RT-PCR. The cDNA full length was cloned into a modified pET28a(+) expression vector in a way that a 6×His-SUMO tag was fused with the protein N-terminal. Protein was induced using the E. coli isolate Rossette 2 at two temperatures (22℃ and 37℃) combined with different IPTG concentrations (0.1, 0.2, 0.5, 1 mmol/L). Protein was purified by Ni²⁺ column affinity chromatography and infiltrated into the leaves of N. benthamiana and Arabidopsis thaliana to test its ability of inducing plant cell death. Meanwhile, the expression changes of three resistance-related marker genes (NbMC1, NbSOD and NbPOX) of N. benthamiana was examined at 12 and 24 h post-infiltration of the recombinant protein. [Results] The expression of scr82 was upregulated during the cyst germination and host infection. The gene was single-copy without any introns and its open reading frame was 249-bp long. The deduced protein was of 82 amino acids consisting of a 21-aa signal peptide and 7 cysteines. It was predicted to be hydrophobic and contain no known domains. The secondary structure consisted of random coil and α-helix. Its homologues existed only in Phytophthora spp. After inducing by 0.2 mmol/L IPTG at 22℃ for about 16 h, the recombinant protein of about 24 kDa was observed on SDS-PAGE gels. About 30 mg/mL of purified protein was obtained. The recombinant did not trigger plant cell death, but caused up-regulated expression of NbMC1, NbSOD and NbPOX genes. [Conclusion] The recombinant could not induce plant cell death, but triggered plant defense reaction.