CAJ | 학술논문

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 SiPRP (Sesamum indicum proline-rich protein)。根据该基因 ORF 两翼序列设计引物在芝麻 cDNA 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, SiPRP 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。
지마청고병시영향아국남방지마산량급품질적중요병해。해병속세균병해,유청고뢰이씨균인기。본연구이용수궤인물대지마수청고뢰이씨균유도전후진행기인차이표체분석,발현일개유도명현하조적기인(편단)。장해편단극륭、측서후,재지마기인조수거고중비대기서렬,득도일개 ORF 완정적전장기인。서렬분석표명해기인무내함자, ORF 장도위1458 bp,편마486안기산,기 N 단부함포안산급부함포안산적중복서렬,속우부함포안산단백(proline-rich protein, PRP),장기명명위 SiPRP (Sesamum indicum proline-rich protein)。근거해기인 ORF 량익서렬설계인물재지마 cDNA 중극륭도해기인,측서결과여예측서렬일치。Blast 분석표명해기인편마단백여기타식물중발현적PRP 단백동원성흔저,차기부함포안산적중복서렬재기타식물중야미발현,추측위일개신형부함포안산단백。진일보설계기인전화적정량급반정량 PCR 인물진행기유도표체분석,재차증명해기인수병균유도후하조표체。여기타식물중발현적대다수 PRP 단백정위우세포벽불동, SiPRP 주요정위우세포막,소량가이분비도세포외。연초표피세포순시표체현시해단백정위재세포막상적특수결구,추측해단백재지마화청고뢰이씨균적호작중발휘중요작용。
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 regu-lated 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 (Sesamum indicum 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 pre-dicted 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 de-signed 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.