LING Na,YANG Zhaoxia,YANG Yalan,et al.Cloning of SmWRKY1 Transcription Factor Gene and Its Transformation in Arabidopsis thaliana[J].Northern Horticulture,2024,(4):81-89.[doi:10.11937/bfyy.20232986]
丹参转录因子SmWRKY1基因克隆及遗传转化拟南芥
- Title:
- Cloning of SmWRKY1 Transcription Factor Gene and Its Transformation in Arabidopsis thaliana
- 文章编号:
- 1001-0009(2024)04-0081-09
- Keywords:
- Savila miltiorkriza; SmWRKY; gene cloning; genetic transformation
- 分类号:
- Q 943.2
- 文献标志码:
- A
- 摘要:
- 以丹参为试材，提取总RNA和合成cDNA，采用PCR扩增获得丹参转录因子SmWRKY1基因序列，通过生物信息软件及在线工具检测基因编码蛋白质的理化性质及氨基酸多序列比对，构建过表达载体并使用花序侵染法转入拟南芥，研究了转SmWRKY1基因对拟南芥生长性状和生理指标的影响，以期初步探究丹参SmWRKY1转录调控因子的生物学功能。结果表明：SmWRKY1基因全长1 041 bp，编码346个氨基酸，NCBI数据库保守区BLAST分析显示具有典型的“WRKY”DNA结合保守结构域和C2H2型锌指结构。编码蛋白分子量为37 310.4 Da，等电点pI为9.77，GRAVY为-0.571，ProtParam分析SmWRKY1属于亲水性蛋白。构建过表达载体pCAMBIA-SmWRKY1，并使用花序侵染法转入拟南芥，经鉴定获得11株转[STBX]SmWRKY1[STBZ]基因的阳性苗，转基因植株可以使叶片和莲座直径显著变大，叶绿素和可溶性糖含量增加，MDA和脯氨酸含量减少，因此推测丹参[STBX]SmWRKY1[STBZ]可能是一个与植物基础防御有关的负调控因子，参与植物非生物胁迫反应。
- Abstract:
- Taking Salvia miltiorrhiza as the test material,total RNA and synthetic cDNA were extracted.The transcription factor SmWRKY1 gene sequence of Salvia miltiorrhiza was obtained by PCR amplification.The physicochemical properties of the gene encoding protein and amino acid multi sequence alignment were detected using bioinformatics software and online tools.An overexpression vector was constructed and transformed into Arabidopsis using floral infection method.The effect of SmWRKY1 gene transformation on the growth traits and physiological indicators of Arabidopsis was studied,in order to preliminarily explore the biological functions of SmWRKY1 transcription regulatory factors.The results showed that the SmWRKY1 gene had a total length of 1 041 bp and encodes 346 amino acids.BLAST analysis of the conserved region in the NCBI database showed a ‘WRKY’ DNA binding conserved domain and a C2H2 zinc finger structure.The molecular weight of the encoded protein was 37 310.4 Da，the Isoelectric point pI was 9.77，and the GRAVY was -0.571.ProtParam analysis showed that SmWRKY1 was a hydrophilic protein.Construction of overexpression vector pCAMBIA-SmWRKY1 and transfer to Arabidopsis using inflorescence infection method，11 positive seedlings with SmWRKY1 gene were identified.Transgenic plants could significantly increase leaf and rosette diameter，increased chlorophyll and soluble sugar content，and reduced MDA and proline content.Therefore，it was speculated that the SmWRKY1 factor of Salvia miltiorrhiza might be a negative regulatory factor related to plant basic defense，participating in plant abiotic stress response.
参考文献/References:
［1］李兴军,王江,柳红.丹参脂溶性成分结构优化研究进展［J］.有机化学,2023,43(2):471-490.［2］EULGEM T,RUSHTON P J,ROBATZEK S,et al.The WRKY superfamily of plant transcription factors［J］.Trends in Plant Science,2000,5(5):199-206.［3］刘万峰,蒲文宣,杨爱国,等.普通烟草NtWRKY53基因克隆、鉴定及表达分析［J］.烟草科技,2021,54(1):1-9.［4］杨敏,李庆萌,周陈平,等.番木瓜WRKY转录因子CpWRKY11的克隆和表达［J］.西北农林科技大学学报（自然科学版）,2023,51(5):119-130,138.［5］刘苗苗,臧连生,孙晓玲,等.茶树WRKY转录因子CsWRKY17的克隆与表达分析［J］.茶叶科学,2021,41(5):631-642.［6］刘佼佼,王学敏,马琳,等.紫花苜蓿MsWRKY42的分离、鉴定及其对非生物胁迫的响应［J］.中国农业科学,2020,53(17):3455-3466.［7］易小哲,王梦月,邬兰,等.艾活性成分生物合成调控相关的WRKY转录因子筛选及表达分析［J］.世界科学技术-中医药现代化,2022,24(5):1811-1824.［8］AKAGI A,FUKUSHIMA S,OKADA K,et al.WRKY45-dependent priming of diterpenoid phytoalexin biosynthesis in rice and the role of cytokinin in triggering the reaction［J］.Plant Molecular Biology,2014,86(1/2):171-183.［9］JIMMY J L，BABU S.Gene network mediated by WRKY13 to regulate resistance against sheath infecting fungi in rice (Oryza sativa L.)［J］.Plant Science,2019(28):269-282.［10］SPYROPOULOU E A,HARING M A,SCHUURINK R C.RNA sequencing on Solanum lycopersicum trichomes identifies transcription factors that activate terpene synthase promoters［J］.BMC Genomics,2014(15):402-417．［11］张雷,刘香英,孙洪蕊,等.南果梨PuLOX2S基因的鉴定及与 PuWRKY、PuMYB转录因子家族成员互作研究［J］.北方园艺,2022(15):16-24.［12］李明,曹慧,段高飞,等.葡萄VvLBD28基因的基本特征和表达分析［J］.北方园艺,2022(17):57-63.［13］徐金鹏,祁亚男,于延冲.盐、干旱胁迫对拟南芥WRKY71基因突变体种子萌发的影响［J］.山东农业科学,2020,52(3):34-37.［14］王双,杨瑞,白薇.水稻WRKY转录因子OsWRKY76 RNA干扰载体的构建及表达分析［J］.华北农学报,2019,34(6):9-13.［15］楚宗丽,张睿男,李亮杰,等.小麦WRKY转录因子的鉴定及其在胚性愈伤组织形成中的表达分析［J］.麦类作物学报,2021,41(12):1469-1478.［16］薛鑫,周心智,决登伟.龙眼DlWRKY52基因克隆及表达分析［J］.热带作物学报,2020,41(4):730-736.［17］刘开,余炳伟,李丹丹,等.茄子SmWRKY65基因克隆及其对青枯病的抗性分析［J］.广东农业科学,2021,48(3):42-52.［18］段俊枝,杨翠苹,王楠,等.WRKY转录因子在植物耐盐基因工程中的应用进展［J］.江苏农业科学,2023,51(5):71-80.［19］于恩,侯立贺,刘涛,等.药用植物中WRKY转录因子基因家族的研究进展［J］.基因组学与应用生物学,2021,40(9):3230-3236.［20］ALI M A,AZEEM F N,NWAZ M A,et al.Transcription factors WRKY11 and WRKY17 are involved in abiotic stress responses in Arabidopsis［J］.Journal of Plant Physiology,2018,226:12-21.
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备注/Memo
第一作者简介：凌娜（1986-），女，硕士，副教授，现主要从事植物遗传育种等研究工作。E-mail：lingnas1985@163.com.责任作者：孟凡荣(1973-)，女，博士，教授，现主要从事植物表观遗传学等研究工作。E-mail:mengfanrong73@yahoo.com.cn.基金项目：河南省科技攻关资助项目(182102110311)；2023年度河南省高等学校青年骨干教师培养计划资助项目（2023GGJS188）；2023年度河南省社会科学届联合会调研课题资助项目（SKL20231135）。收稿日期：2023-08-29