小麦木瓜类半胱氨酸蛋白酶基因TaRD21的克隆及赤霉病菌诱导下的表达分析

木瓜类半胱氨酸蛋白酶（PLCPs）作为一类重要的蛋白水解酶，在植物生长发育以及胁迫应答过程中都发挥着重要作用。本研究从抗、感赤霉病小麦品种差异表达基因谱中获得1个注释为RD21 Cysteine proteases的EST（表达序列标签），以此序列检索小麦最新基因组数据库并设计引物，从小麦中克隆到3个基因，分别命名为TaRD21-2A、TaRD21-2B和TaRD21-2D，属于PLCPs RD21家族。序列分析表明，3个基因的开放阅读框长度分别为1 410、1 428和1 419 bp，分别编码469、475和472个氨基酸。序列比对发现，3个基因的序列相似性为89.3％，所编码蛋白的氨基酸序列相似性为95.6％。系统进化分析表明，TaRD21-2A、TaRD21-2B和TaRD21-2D蛋白的同源性较高，且与乌拉尔图小麦TuRD21A蛋白聚为一类。qRT-PCR分析表明，3个TaRD21基因均受水杨酸(SA)、乙烯利(ETH)以及赤霉病菌诱导表达;感病品种中,TaRD21-2A对SA和赤霉病菌的响应更迅速，且表达量较高；抗病品种中,TaRD21-2B和TaRD21-2D基因对ETH的响应更迅速。

Cloning of Papain-Like Cysteine Protease Gene TaRD21 and Its Expression Analysis to Fusarium graminearum in Wheat

Papain-like cysteine proteases(PLCPs) belong to proteolytic enzymes, which play important roles in plant growth and stress responses. Based on the analysis of the gene expression profiling from resistant and susceptible wheat varieties induced by Fusarium graminearum,an EST with RD21 Cysteine proteases was obtained. Three genes were cloned from wheat by searching the latest wheat genome database and designing primers, designated as TaRD21-2A,TaRD21-2B and TaRD21-2D based on their chromosomal positions, belong to RD21 family of papain-like cysteine proteases. The open reading frames(ORFs) of the three genes are 1 410, 1 428 and 1 419 bp, and the length of the deduced proteins are 469, 475 and 472 amino acid residues, respectively. Sequence alignment of the three genes showed the similarity was 89.3% and the encoded amino acids was 95.6%. Phylogenetic analysis showed that TaRD21-2A, TaRD21-2B and TaRD21-2D proteins had high homology, clustered with TuRD21A protein of Triticum urartu. qRT-PCR assay revealed that the three TaRD21 genes canbe induced by defense related signal molecules, such as salicylic acid(SA) and ethrel(ETH), as welll as F.graminearum. However, TaRD21-2A was more responsive to SA and F.graminearum, and the expression level was higher in susceptible variety.TaRD21-2B and TaRD21-2D genes were more responsive to ETH with the higher expresssion level in resisstant variety.