白花前胡CAL基因的克隆和原核表达分析  

Gene Cloning and Prokaryotic Expression Analysis for CAL Gene from Peucedanum praeruptorum Dunn

作　　者：管凤雅 吴君贤 姚金辰 查良平 储姗姗 谢晋[2] GUAN Feng-ya;WU Jun-xian;YAO Jin-chen;ZHA Liang-ping;CHU Shan-shan;XIE Jin(School of Pharmacy,Anhui University of Chinese Medicine,Hefei 230012,China;School of Pharmacy,Anhui Medical University,Hefei 230032,China)

机构地区：[1]安徽中医药大学药学院,安徽合肥230012 [2]安徽医科大学药学院,安徽合肥230032

出　　处：《中国现代中药》2023年第8期1723-1729,共7页Modern Chinese Medicine

基　　金：国家自然科学基金项目(81803675);安徽省自然科学基金项目(2208085QH269);安徽高校省级质量工程项目(2021jyxm0678)。

摘　　要：目的:克隆得到白花前胡花发育关键基因CAULIFLOWER(CAL)并对其进行原核表达和基因表达分析。方法:根据白花前胡转录组数据中的基因序列信息,通过逆转录聚合酶链式反应(RT-PCR)从白花前胡中克隆CAL基因,命名为PpCAL1和PpCAL2。构建原核表达载体pET-32a-PpCAL1和pET-32a-PpCAL2,并转化至大肠埃希菌Transetta(DE3)进行诱导表达。运用实时荧光定量PCR(qRT-PCR)分析抽薹开花前后白花前胡不同组织的基因相对表达量。结果:PpCAL1和PpCAL2的开放阅读框(ORF)长度分别为645、738 bp,分别编码214、245个氨基酸。结构功能域分析表明,PpCAL1和PpCAL2蛋白都具有MADS_MEF2_like和K-box保守结构域。原核表达结果显示,异丙基-β-D-硫代半乳糖苷可成功诱导目的蛋白表达。系统进化树分析显示,白花前胡PpCAL1与胡萝卜DcCAL聚为一支,白花前胡PpCAL2与橡胶树HbCAL聚为一支。基因表达结果显示,PpCAL1和PpCAL2在不同组织中均在开花后表达量上调。结论:克隆并分析白花前胡PpCAL1和PpCAL2基因,为进一步研究白花前胡开花基因提供参考。Objective:In this study,the key genes in flower development in Peucedanum praeruptorum Dunn were cloned.Meanwhile,prokaryotic expression and gene expression analysis were carried out.Method:According to the gene sequence information from the P.praeruptorum transcriptome data,genes encoding CAULIFLOWER(CAL)were cloned from P.praeruptorum by reverse transcription-polymerase chain reaction(RT-PCR),named PpCAL1 and PpCAL2,respectively.Prokaryotic expression vectors pET-32a-PpCAL1 and pET-32a-PpCAL2 were transformed into Escherichia coli Transetta(DE3)for induction.A real-time quantitative PCR(qRT-PCR)analysis was performed to profile the expression in different tissue of both non-bolting and bolting plants.Result:The open reading frame(ORF)lengths of PpCAL1 and PpCAL2 were 645 and 738 bp,encoding 214 and 245 amino acids.Structural functional domain analysis showed that PpCAL1 and PpCAL2 contained conserved MADS_MEF2_like and K-box domains.Prokaryotic expression results showed that IPTG induction could successfully induce the expression of the target protein.Phylogenetic tree analysis showed that PpCAL1 and DcCAL were clustered into a branch,and PpCAL2 and HbCAL were clustered into a branch.The expression levels of PpCAL1 and PpCAL2 in the different tissue of bolting plants were higher than those in non-bolting plants.Conclusion:The PpCAL1 and PpCAL2 genes of P.praeruptorum have been cloned and analyzed for the first time,and this study provides a reference for further research on the flowering genes of P.praeruptorum.

关 键 词：白花前胡 CAL基因 抽薹开花 基因克隆 原核表达 

分 类 号：R282.12[医药卫生—中药学]