大豆铁结合蛋白基因cDNA的克隆、序列分析和表达载体的构建

The cloning and sequence analysis of cDNA fragment of soybean ferritin gene and construction of its expression vector for plant transformation

根据已报道的植物铁结合蛋白基因的保守序列设计引物 ,用RT PCR方法 ,成功地从大豆总RNA中扩增出一大小为0 771kb的cDNA片断。将该片段克隆到pUCm T载体上 ,测序和序列的同源性分析表明该片断与Proudhon等 (1996)报道的大豆铁结合蛋白基因的氨基酸序列同源性达 95 %。利用pUCm -T和pBI12 1载体上共同的XbaⅠ和SacⅠ双酶切位点 ,构建了克隆片段的植物表达载体pSFKna。该载体含CaMV3 5S启动子、NOS终止子和NPT Ⅱ基因 ,在遗传转化中可用基因枪导入受体 ,并通过卡那霉素抗性筛选转化子。

Special primers were designed based on the sequence information of plant ferritin genes reported in the literature and were used for reverse transcription polymerase chain reaction(RT PCR)with the total RNA of Glycine max cv Xidou No 3 as PCR template in an attempt to clone its ferritin gene A single cDNA fragment of 771 bp was successfully obtained from the RT PCRs and was successfully cloned into vector pUCm T in this study The sequence homology analysis of the cloned fragment showed that the deduced amino acids of the fragment had ninety five percent homology to that of the soybean ferritin gene reported by Proudhon et al (1996) The recombinant pUCm T with the cDNA fragment and that of pBI121 vector were digested by double restriction enzymes XbaI and SacI The digested solutions were electrophoresed on agrose gel and the target fragments were recovered from the gel Through ligating the recovered target fragments by T4 DNA ligase,the cDNA was linked to pBI121 vector with CaMV35S as promoter The double enzyme digestion and PCR test on the recombinant pBI121 vector were carried out to confirm that soybean cDNA was cloned correctly into pBI121 vector The confirmed construct,named as pSFKna,contains CaMV35S promoter,NOS terminator and NPT Ⅱ gene can be used in gene gun delivered genetic transformation using kanamycin resistance as selecting marker