一个与油菜黄化相关的未知功能基因克隆及表达

Cloning and analysis of the cDNA of a gene encoding an unknown protein in Brassica napus

以构建的油菜幼叶黄化突变体Cr3529消减文库中一个未知功能的差异表达基因片段为基础,应用RACE-PCR技术,扩增并克隆到两个cDNA全序列,分别命名为BnCr4和Bn-Cr4-1.测序结果显示,BnCr4编码区序列大小为1395 bp,编码465个氨基酸,而BnCr4-1编码区序列长1311 bp,编码437个氨基酸.BLAST结果表明它们与拟南芥中的一个未知功能基因的cDNA同源性分别为87%和80%.功能预测显示它们含有与蛋白质的修饰作用有关的多个活性位点,如磷酸化、糖基化、酰胺化以及磷酸泛酰巯基乙胺结合位点,可能是一种新的与cAMP介导的蛋白质磷酸化与去磷酸化作用有关的蛋白.Northen杂交结果显示该基因在Cr3529子叶期和幼叶期的表达高于野生型油菜,显示该基因的表达与突变性状紧密相关.最后,原核表达了BnCr4,得到了与预计分子量相同的融合蛋白.

Two full-length cDNA sequences were amplified and cloned by RT-PCR and RACE-PCR according to a differentially expressed fragment of the backward subtraction library of young leaves in chlorophyll-reduced mutant Cr3529 of Brassica napus, named BnCr4 and BnCr4-1 respectively. They encoded 465 and 437 amino acids respectively. Results of BLAST in GentBank showed they shared 87 % and 85 % identity to the cDNAof an unknown function gene（accession number： At5g19540）in Arabidopsis thaliana, respectively. Prediction of protein function showed they contain multiple types of functional sites. Northern blots showed that the expression of the unknown gene presented more strongly in cotyledon and young leaves of chlorophyll- reduced mutant than that of wild type. The open reading frame of BnCr4 was cloned into expression vector pET-32a（ ＋ ）, and then transformed into host bacterium, BL21 （DE3）. The host cells were induced by IPTG. The molecular weight of the expressed protein in E. coli was identified with the deduced protein.