柑橘半胱氨酸蛋白酶基因CsCysP的分离、亚细胞定位及表达分析

Isolation,Subcellular Localization and Expression Analysis of a Citrus Cysteine Protease Gene,CsCysP

以‘奥林达’夏橙[Citrus sinensis(L.)‘Olinda’]为材料,采用RT-PCR结合RACE技术从果萼离层中分离到1个半胱氨酸蛋白酶类基因,命名为CsCysP(GenBank登录号:KJ093387)。该基因的cDNA全长为1 485 bp,开放阅读框(ORF)为1 083 bp,推测可编码360个氨基酸残基的多肽。其基因组序列与cDNA比对后显示有3个内含子。分析发现,CsCysP属于papain-like(木瓜蛋白酶,C1A)家族的半胱氨酸蛋白酶,与拟南芥、大豆、烟草、杨树等的同源蛋白有73%~83%的相似性。亚细胞定位结果显示CsCysP蛋白定位在细胞壁上。qRT-PCR结果表明CsCysP在老叶、成熟果实果萼离层和花中的表达量明显高于幼苗的根、茎、叶。CsCysP的表达被脱落酸、高盐和PEG6000诱导,低温、乙烯、芸薹素内酯、水杨酸和甲基茉莉酸可抑制其表达。利用基因工程手段获得了柑橘过表达CsCysP的5个转基因株系。

A cysteine protease gene,CsCysP(KJ093387),was cloned from the fruit abscission zone of sweet orange[Citrus sinensis(L.)‘Olinda'] using RT-PCR and RACE. The 1 485 bp full-length cDNA of CsCysP contains a 1 083 bp open reading frame(ORF)encoding a protein of 360 amino acid residues. Comparison between cDNA and genomic DNA sequences showed that the gene contains three introns. The phylogenetic analysis placed the gene into papain-like(C1A)group. BLASTp analysis showed that CsCysP protein shared 73%–83% amino acid identities with CPRs from Arabidoposis thaliana,soybean,tobacco,Populus trichocapa and other species. Transient expression of a 35S-CsCysP-GFP fusion gene in onion epidermal cells revealed that CsCysP protein was localized in cell wall. Quantitative Real-time PCR results showed that the expression of CsCysP was higher in senescent leaves,fruit abscission zone cells and flowers than in leaves,stems and roots of young seedlings. CsCysP was induced by salt,PEG6000 and ABA but repressed by low temperature,ethylene(ET),brassinolide(BR),methyl jasmonic acid(MeJA)and salicylic acid(SA). Five transgenic citrus lines overexpressing CsCysP were also obtained in this study and will be further characterized.