小麦TaTDR-Like基因的克隆及表达分析  

Cloning and expression analysis of TaTDR-Like genes in wheat

作　　者：都晶晶 夏玉 任扬 李燕红 李政 张改生[1,3] 王军卫 马守才[1] 宋瑜龙[1,3] 齐倩[1] 牛娜 DU Jing-jing;XIAYu;REN Yang;LI Yan-hong;LI Zheng;ZHANG Gai-sheng;WANG Jun-wei;MAShou-cai;SONG Yu-long;QI Qian;NIU Na(College of Agronomy,Northwest A&F University/National Yangling Agricultural Biotechnology Breeding Center/Yangling Branch of National Wheat Improvement Center/Engineering Research Center of Wheat Breeding of Ministry of Education,Yangling,Shaanxi 712100,China;College of Life Science,Henan University/State Key Laboratory of Crop Adversity Adaptation and Improvement,Kaifeng,Henan 475001,China;Key Laboratory of Crop Heterosis Research and Utilization in Shaanxi Province,Yangling,Shaanxi 712100,China)

机构地区：[1]西北农林科技大学农学院/国家杨凌农业生物技术育种中心/国家小麦改良中心杨凌分中心/小麦育种教育部工程研究中心,陕西杨凌712100 [2]河南大学生命科学学院/作物逆境适应与改良国家重点实验室,河南开封475001 [3]陕西省作物杂种优势研究与利用重点实验室,陕西杨凌712100

出　　处：《南方农业学报》2021年第9期2329-2338,共10页Journal of Southern Agriculture

基　　金：陕西省重点研发计划一般项目(2019NY-003);陕西省重点实验室后补助项目(2018SZS-22);陕西省博士后基金项目(2016BSHED113);西北农林科技大学大学生创新创业训练项目(X202010712224)。

摘　　要：【目的】克隆小麦绒毡层退化基因(Tapetum degeneration retardation,TaTDR-Like),研究其组织表达及不同育性材料间花药不同发育时期的时空表达模式,为深入揭示小麦花药异常发育的分子机制打下理论基础。【方法】以普通小麦品种西农1376(MF-XN1376)为材料,通过PCR扩增克隆得到TaTDR-Like基因的3个同源拷贝,利用生物信息学分析TaTDR-Like蛋白特性及Ta TDR-Like基因启动子顺式作用元件,利用实时荧光定量PCR检测TaTDR-Like基因的组织表达情况及在可育、不育材料花药不同时期中的时空表达模式,利用酵母双杂交技术进行自激活检测,并构建植物融合表达载体35S-TaTDRLD-EGFP,通过农杆菌介导转入烟草叶片表皮细胞,观察TaTDRL-D蛋白亚细胞定位情况。【结果】成功克隆小麦Ta TDR-Like基因,发现该基因存在3个同源拷贝即TaTDRL-A、TaTDRL-B和TaTDRL-D,分别编码550、553、557个氨基酸,其蛋白分子量分别为58.50、58.90和59.21 kD,等电点(pI)分别为4.77、4.66和4.63。TaTDR-Like蛋白均在C端有1个bHLH保守结构域,其中TaTDRL-A与TaTDRL-B的保守结构域相似度达100%,与TaTDRL-D的保守结构域相似度为96%,TaTDRL-D与OsTDR、AtAMS保守结构域相似度分别为98%和88%;系统发育进化树表明TaTDR-Like蛋白与大麦(KAE8787147.1)、二穗短柄草TDR(XP_(0)14756384)、水稻TDR(Os02g0120500)和拟南芥AMS(AT2G16910.1)的进化关系较密切;启动子分析表明TaTDR-Like基因启动子处含有较多光响应元件、非生物应激反应、激素响应元件等顺式作用元件;组织特异性表达分析显示TaTDRL-D在花药中的表达量最高,而TaTDRL-A和TaTDRL-B在幼穗表达量较高;对花药发育不同时期表达分析显示TaTDRL-D在花药发育单核早期表达量最高,之后逐渐降低,单核晚期到三核期不育材料(CMS-XN1376)表达量均高于可育材料(MF-XN1376);自激活检测结果表明TaTDRL-D具有转录激活活性;亚细胞定位显示TaTDRL-D蛋白定位于【Objective】Wheat tapetum degeneration retardation-like(TaTDR-Like)genes were cloned and their expression patterns were analyzed between different tissues and different developmental stages of anthers in order to gain insight into the molecular mechanisms underlying abnormal wheat pollen development.【Method】In this study,orthologues of rice(Oryza sativa L.)gene TDR(Os02 t0120500)were cloned from the common wheat cultivar,MF-XN1376.Bioinformatics was used to analyze the characteristics of the proteins and cis-acting elements of the gene promoters.Moreover,real-time fluorescence quantitative PCR(q RT-PCR) was used to assess the tissue-specific expression of TaTDR-Like genes in different tissues(anther,leaf,ovary,stem and spike)and expression pattern of TaTDRL-D genes in anthers at different developmental stages.The subcellular localization of TaTDRL-D protein was observed,and yeast two-hybrid technology was used to detect its self-activation.The plant fusion expression vector 35 S-TaTDRLD-EGFP was constructed and transferred into tobacco leaf epidermal cells through agrobacterium-mediated transfer.【Result】It was found that there were three homologous copies,namely Ta TDRL-A,TaTDRL-B and TaTDRL-D,encoding 550,553 and 557 amino acids(aa),corresponding to molecular weights of 58.50,58.90 and 59.21 kD and isoelectric points(pI)of 4.77,4.66 and 4.63,respectively.TaTDR-Like proteins all have a bHLH conserved domain at the C-terminal.Ta TDRL-A and Ta TDRL-B had 100% and 96% similarity with TaTDRL-D,respectively,while TaTDRL-D showed 98% and 88% similarity with OsTDR and AtAMS,respectively.A phylogenetic tree analysis showed that Ta TDR-Like proteins were closely related to rice TDR(Os02 g0120500),Brachypodium distachyum TDR(XP014756384),barley(KAE8787147.1)and Arabidopsis AMS(AT2 G16910.1).Promoter analysis results showed that TaTDRL-A,TaTDRL-B and TaTDRL-D promoters contain many cis-acting elements,such as those involved in the response to light conditions,abiotic stress and phytohormones,which may participate

关 键 词：小麦 TaTDR-Like基因 克隆 转录因子 表达分析 

分 类 号：S512.1[农业科学—作物学]