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目的:获取青杄的PSAK全长cDNA序列,对其进行生物信息学分析,并鉴定其在青杄各组织中相对表达量。方法:以多年生青杄(Picea wilsonii)的cDNA文库为模板,通过RACE PCR的方法获取PSAK的末端序列,经过与EST序列拼接得到PSAK基因的cDNA全长序列。通过特异引物将其克隆在pEASY-T1载体上,基于其氨基酸序列,利用DNAMAN、ExPASy、SWISS-MODEL等工具对其进行生物信息学分析,进行了半定量RT-PCR与RT-qPCR实验来检测其在mRNA水平的组织表达情况。结果:青杄PSAK基因编码140个氨基酸,蛋白分子量为14.23kDa,理论等电点为10.29。蛋白的C端有较为保守的结构域,与拟南芥、烟草等物种具有较高的相似性。PwPSAK主要在青杄的针叶与茎中进行表达。结论:成功获取了PwPSAK的单克隆并进行了生物信息学分析,为青杄中PSAK后续的功能研究提供理论依据。
OBJECTIVE: To obtain the full-length cDNA of PSAK from barley and analyze its bioinformatics and to identify the relative expression of PSAK in various tissues of barley. Methods: The cDNA library of Picea wilsonii was used as a template to obtain the terminal sequence of PSAK by RACE PCR. The complete cDNA sequence of PSAK was obtained by splicing with EST sequence. The PCR products were cloned into pEASY-T1 vector by using specific primers. Bioinformatics analysis was carried out by using DNAMAN, ExPASy and SWISS-MODEL based on their amino acid sequences and semi-quantitative RT-PCR and RT-qPCR assays Its tissue expression at the mRNA level. Results: The barley PSAK gene encodes a protein of 140 amino acids with a molecular weight of 14.23 kDa and a theoretical isoelectric point of 10.29. Protein C-terminal more conserved domain, and Arabidopsis, tobacco and other species have a high similarity. PwPSAK is mainly expressed in the conifers and stems of the barley. Conclusion: The monoclonal PwPSAK was successfully obtained and bioinformatics analysis was carried out, providing a theoretical basis for the subsequent functional study of PSAK in barley.