白花丹参谷氧还蛋白SmGRX2基因的克隆及其对丹参的转化  

Cloning,molecular characterization and expression of GRX gene of Salvia miltiorrhiza Bge.f.alba and subsequent transformation into Salvia miltiorrhiza

作　　者：赵法兴[1] 李艳玲 郝岗平 ZHAO Fa-xing;LI Yan-ling;HAO Gang-ping(Jiangxi College of Traditional Chinese Medicine,Fuzhou Jiangxi 344000,China;Department of Biological Science,Shandong First Medical University,Taian Shangdong 271000,China)

机构地区：[1]江西中医药高等专科学校,江西抚州344000 [2]山东第一医科大学生命科学学院,山东泰安271016

出　　处：《时珍国医国药》2022年第3期725-729,共5页Lishizhen Medicine and Materia Medica Research

基　　金：国家自然科学基金(81173489);山东省中医药科技计划项目(2020M064)。

摘　　要：目的获取白花丹参GRX基因的全长cDNA序列(命名为SmGRX2),并分析其序列特征和不同外源胁迫下的表达特征,构建其表达载体并转化到白花丹参。方法利用已有白花丹参cDNA全长文库,随机测序获得SmGRX2全长序列。BLAST进行序列比对,Motif Scan软件分析氨基酸序列的功能区段,real-time RT-PCR技术分析基因表达特征;将SmGRX2连接到含有双增强子的高效植物双元表达载体pGreen0029-GFP上,获得含融合DHA基因的植物双元表达载体pGreen0029-SmGRX2-DHA,采用电击法将含SmGRX2的植物表达载体转入根癌农杆菌LBA4404中,用叶片浸染法转化白花丹参。结果获得ORF为372bp的SmGRX2全长cDNA序列,编码氨基酸123个,氨基酸序列分析发现其具有典型的GRX prosite patters CCMC,NCBI登录号为KJ009322。Real-timePCR分析其在根部中表达量最高。SA和MeJA处理均能较强的诱导该基因的表达。对再生植株用PCR检测证实了SmGRX2基因的导入,pGreen0029-SmGRX2-DHA的转化率为24.4%。结论首次获得白花丹参SmGRX2全长基因序列,分析其可能参与对抗病等生物胁迫的响应;成功获得过表达SmGRX2转基因植株,为SmGRX2基因的进一步功能分析奠定了基础。Objective To clone the full-length cDNA of a thioltransferase gene glutaredoxin(GRX)in Salvia miltiorrhiza.f.al-ba and investigate its sequence characteristics and analyze its expression in different organs and under different stresses.To con-struction of SmGRX2 high efficiency expression vector and subsequent transformation into Salvia miltiorrhiza.Methods SmGRX2 gene was obtained by sequencing cDNA library constructed by us.BLAST was used for alignment,and Motif Scan was used to a-nalysis the protein characterization.Real-time RT-PCR was used to detect the gene expression level.SmGRX2 was subcloned into the plant binary vector pGreen0029-GFP and pGreen0029-SmGRX2-DHA was produced.pGreen0029-SmGRX2-DHA was transformed into Agrobacterium tumefaciens LBA4404,and then the leaves of Salvia miltiorrhiza bge.f.alba were inocu-lated in LBA4404 with pGreen0029-SmGRX2-DHA.Results The full-length cDNA of SmGRX2 was 372bp that putatively encoded a polypeptide of 123 amino acids.The deduced amino acid sequence of SmGRX2 shared high homology with other known GRX,and GRX prosite patters CCMC were found in this amino acid sequence.Real-time PCR analysis showed the highest ex-pression in root.The SmGRX2 expression could be induced under the treatments of SA and MeJA.Genetic transformation was confirmed by PCR,and the transformation efficiency rate was 24.4%.Conclusion A novel SmGRX2 gene was cloned from Salvia miltiorrhiza Bge.f.alba.Its functional areas CC[M/L][C/S]are highly conservative,and is possibly associated with pathogen stress resistance of Salvia miltiorrhiza Bge.f.alba.Transgenic Salvia miltiorrhiza bge.f.alba plants of SmGRX2 overexpreesion was obtained,which facilitates further investigation of SmGRX2 function in transgenetic wide plants.

关 键 词：白花丹参 SmGRX2基因克隆 表达分析 表达载体构建 遗传转化 

分 类 号：R2-03[医药卫生—中医学]