芝麻中一个富含脯氨酸新基因的克隆与特征分析

芝麻青枯病是影响我国南方芝麻产量及品质的重要病害。该病属细菌病害,由青枯雷尔氏菌引起。本研究利用随机引物对芝麻受青枯雷尔氏菌诱导前后进行基因差异表达分析,发现一个诱导明显下调的基因(片段)。将该片段克隆、测序后,在芝麻基因组数据库中比对其序列,得到一个ORF完整的全长基因。序列分析表明该基因无内含子,ORF长度为1458 bp,编码486氨基酸,其N端富含脯氨酸及富含脯氨酸的重复序列,属于富含脯氨酸蛋白(proline-rich protein,PRP),将其命名为Si PRP(Sesamum indicum proline-rich protein)。根据该基因ORF两翼序列设计引物在芝麻c DNA中克隆到该基因,测序结果与预测序列一致。Blast分析表明该基因编码蛋白与其他植物中发现的PRP蛋白同源性很低,且其富含脯氨酸的重复序列在其他植物中也未发现,推测为一个新型富含脯氨酸蛋白。进一步设计基因专化的定量及半定量PCR引物进行其诱导表达分析,再次证明该基因受病菌诱导后下调表达。与其他植物中发现的大多数PRP蛋白定位于细胞壁不同,Si PRP主要定位于细胞膜,少量可以分泌到细胞外。烟草表皮细胞瞬时表达显示该蛋白定位在细胞膜上的特殊结构,推测该蛋白在芝麻和青枯雷尔氏菌的互作中发挥重要作用。

Cloning and Characterization of a Novel Gene Encoding Proline-Rich Protein in Sesame

Bacterial wilt of sesame is a major threat in sesame production in south China, resulting seriously in yield and quality losses. The disease is caused by bacterial pathogen Ralstonia solanacearum. This study profiled the gene expression of sesame inoculated with Ralstonia solanacearum by using fifty random primers. A gene (fragment) was found to be drastically down regulated by the pathogen. The gene fragment was cloned and sequenced. Using the sequence as queries, the sesame genome database (http://www.ncbi.nlm.nih.gov/) was searched and the corresponding DNA sequence containing a complete ORF was obtained. The full-length of the gene shows that its encoding region is 1458 bp, encoding a putative protein of 486 amino acids. The protein is rich in proline on its N-terminus, and has several repeat sequences (motifs) rich in proline, suggesting that it belongs to proline-rich protein (PRP) family. The protein was named as SiPRP (Sesaumu indicunm Proline-rich Protein). The encoding region of SiPRP was further amplified in sesame cDNAs, sequencing analysis demonstrated that it has the same sequence with the predicted one. Blast analysis revealed that the protein has the lower homology with other plant PRPs, and has new types of proline-rich motifs, suggesting that SiPRP is a new member in PRP family. Semi quantitative RT-PCR and qPCR with newly designed gene-specific primers verified that SiPRP expression was drastically down regulated upon pathogen infection. Previous studies showed that most plant PRPs were located on plant cell wall, however, transient expression in onion epidermal cells showed that SiPRP-YFP fusion protein was located on cell membrane, with a bit secreted outside the cell. Transient expression in tobacco cells revealed that SiPRP protein might be located on special structures of the membrane. SiPRP protein identified in this study may play pivotal roles in Ralstonia solanacearum-sesame interactions.