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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 was isolated from the calyx of Cocos sinensis using RT-PCR and RACE techniques, using Citrus sinensis (L.) Olinda as the material, named CsCysP (GenBank Accession No. KJ093387). The full-length cDNA of this gene is 1 485 bp in length and has an open reading frame (ORF) of 1 083 bp, suggesting that it encodes a polypeptide of 360 amino acid residues. Its genome sequence and cDNA showed three introns. It was found that CsCysP belongs to the papain-like (papain-C1A) family of cysteine proteases and has 73% -83% similarity with homologous proteins of Arabidopsis, soybean, tobacco, poplar and the like. Subcellular localization results show that CsCysP protein is located on the cell wall. The results of qRT-PCR showed that the expression of CsCysP in the leaves and flowers of the calyx and the fruit of mature fruits was significantly higher than that of the roots, stems and leaves of seedlings. The expression of CsCysP was induced by abscisic acid, high salt and PEG6000, and its expression was inhibited by hypothermia, ethylene, brassinolide, salicylic acid and methyl jasmonic acid. Five transgenic lines expressing citrus overexpression CsCysP were obtained by genetic engineering.