甘蓝型油菜半胱氨酸蛋白酶cDNA的克隆及组织特异性表达分析  

Full-length cDNA Cloning and Tissue Specified Expression Analysis of a Cysteine Protease in Brassica napus L.

作　　者：张红岩[1] 薛华[1] 马欣荣[2] 赵云[1] 王茂林[1] 

机构地区：[1]四川大学生命科学学院,成都610064 [2]中国科学院成都生物研究所,成都610041

出　　处：《应用与环境生物学报》2008年第2期172-176,共5页Chinese Journal of Applied and Environmental Biology

基　　金：国家“863”项目(2001AA241104);四川省“十一五”油菜育种攻关项目(200607001)资助~~

摘　　要：采用SSH(Suppression subtractive hybridization)和Race-PCR(Rapid-amplification of cDNA ends)技术克隆得到来自甘蓝型油菜(Brassica napus L.)矮化突变体‘NDF-1’的一个差异表达基因编码区的全长cDNA序列,命名为Bncp5.该基因全长1224bp,含有1080bp的完整开放阅读框,编码359个氨基酸.该基因编码区与甘蓝中一个与衰老相关的半胱氨酸蛋白酶基因(登录号AF454960)同源性达97%,氨基酸序列同源性达到100%.Bncp5编码的蛋白相对分子质量为39.5×103,等电点为5.57.对其活性位点的分析表明其具备真核生物半胱氨酸蛋白酶的活性中心,且有很高的功能区段保守性,推测为一跨膜蛋白.相对定量PCR的检测结果表明,Bncp5在野生型和矮化突变体的根、茎、子叶和真叶中都有表达,但是表达量存在显著性差异.在野生型高秆油菜中,根的表达量最低,在真叶的表达量最高;在矮化突变体中,根、茎中的表达明显高于野生型,而在子叶和真叶中的表达量与野生型油菜的表达量基本一致.A full-length cDNA encoding a differential expression gene in dwarf mutant ＇ NDF-1 ＇ of Brassica napus was cloned by suppression subtractive hybridization and rapid-amplification of cDNA ends, which was named as Bncp5. The sequencing and analysis showed that the length of Bncp5 was 1 224 bp which contained 1 080 bp open reading frame （ORF） encoding 359 amino acid residues. The amino acid sequence of Bncp5 had 100% homology with a senescent protein of B. olercea, and was highly homologous to some other plant species. This protein was presumed as a transmembrane protein with a molecular mass of 39.5 × 10^3 and isoelectric point of 5.57. The sequence analysis indicated that Bncp5 had one highly conserved active center which was consistent with other cysteine proteases in eukaryote. The results showed that Bncp5 expressed in all the tissues of both the dwarf mutant and its wild-type parent by semi-quantitative RT-PCR. However, the semi-quantitative RT-PCR demonstrated that the expression of Bncp5 was discrepant in different tissues. As for the mutant, the expression of Bncp5 was the highest in stem, but lowest in cotyledon. For the wild-type, the expression of Bncp5 was the highest in leaf, but lowest in root. The expression of Bncp5 in root, cotyledon and stem was quite different between the mutant and wild type, but it was the same in leaf. Fig 3, Tab 1, Ref 27

关 键 词：甘蓝型油菜 矮化突变体 半胱氨酸蛋白酶 相对定量PCR 衰老 

分 类 号：S565.4[农业科学—作物学]