橡胶树半胱氨酸蛋白酶基因HbCP2的克隆与表达特性分析

Molecular Cloning and Expression Analysis of a Cysteine Proteinase Gene( HbCP2) from Hevea brasiliensis

根据EST和基因组序列设计引物,应用RT-PCR技术从橡胶树的根组织中分离到1条长1056bp的cDNA,该序列包含了1 023 bp的开放读码框(ORF),17 bp的5’UTR和16 bp的3’UTR;ORF预测编码340个氨基酸,理论分子量为37.52 kD,等电点为5.25,属于液泡定位的分泌型蛋白;蛋白含有1个Inhibitor_I29和1个Peptidase_C1A结构域,隶属于木瓜蛋白酶C1家族;蛋白与蓖麻、杨树中同源蛋白的相似性均在80%以上,将基因命名为HbCP2。表达分析显示,在所分析的根、树皮、木质部、芽、叶片、叶柄和乳管等组织中,HbCP2仅在根中被检测到,表现出明显的组织特异性。

Based on the EST and genome sequences,a 1 056 bp cDNA was isolated from the root tissue of Hevea brasiliensis with specific primers via RT-PCR.The sequence contains a 1 023 bp long open reading frame （ORF）,17 bp 5′UTR and 16 bp 3′UTR.The ORF was predicted to encode 340 amino acids with a theoretical mo-lecular weight （Mw）of 37.52 kD and isolectric point （pI）of 5 .25;The sequence was predicted to be a secretory protein located to vacuoles;the protein contains one Inhibitor_I29 domain and one Peptidase_C1 A domain and can be categoried as the papain C1 family;the protein shares a similarity of more than 80%with the homologues in cas-tor bean （Ricinus communis）,poplar （Populus trichocarpa）,and then this gene was designated as HbCP2.Semi-quantitative RT-PCR analysis showed that HbCP2 exhibits a root-specific expression pattern in examed tissues,i.e. root,bark,xylem,bud,leaf,petiole and laticifer.