巴西橡胶树胶乳亮氨酸氨肽酶基因克隆及表达

以巴西橡胶树(Hevea Brasiliensis)无性系热研7-33-97的胶乳为材料,根据已报道的植物亮氨酸氨肽酶基因的保守序列设计简并引物,应用RACE技术克隆同源基因(HbLAP1).用生物信息学软件(DNA-MAN,DNAStar,ScanProsite,ClustalW)对HbLAP1进行生物信息学分析,通过Northern-blot分析HbLAP1的表达.结果表明,HbLAP1的cDNA全长1 985 bp,包括1个1 581 bp的阅读框,编码526个氨基酸的蛋白质,一个105 bp的5'非编码区和一个299bp的3'非编码区(括16个腺苷酸尾巴);HbLAP1是一种可溶性的细胞质氨肽酶结构域,割胶显著下调舶HbLAP1的表达.HbLAP1可能是乳管细胞茉莉酸信号途径的负调控因子.

Cloning and Expression of Leucine Aminopeptidase Gene in Hevea brasiliensis

Leucine aminopeptidases (LAPs) catalyze the cleavage of N-terminal amino acids from peptides and proteins. Although various physiological roles of LAPs have been demonstrated, little is known about the mechanism by which LAPs act. Degenerated primer was designed based on the conserved sequence of the reported leucine aminopeptidase genes in plants by searching the Genbank databases. Total RNA was isolated from the latex of Hevea brasiliensis. A full-length cDNA of leucine aminopeptidase gene (HbLAP1) was cloned by the rapid amplification of cDNA ends (RACE) technique. The putative protein, HbLAP1, was analyzed with the aid of several bioinformatics softwares, such as DNAMAN, DNAStar, ScanProsite, and ClustalW, and its gene expression pattern was revealed by Northern blot. Results showed that the full-length cDNA was 1 985 bp in length, containing a 1 581 bp ORF encoding a putative protein of 526 amino acid residues, flanked by a 105 bp 5'-UTR and a 299 bp 3'-UTR including a poly(A) tail of 16 bp. Bioinformatics analysis showed that the putative HbLAP1 was a soluble protein, localized in cytoplasm, and belonged to M17 peptidase family. The expression of HbLAP1 was remarkably down-regulated by tapping, suggesting that HbLAP1 be a negative regulator of jasmonate signaling pathway in laticifer cells.