陆地棉钾转运体基因GhHAK5启动子的克隆与功能分析  

Cloning and functional analysis of promoter of potassium transporter gene GhHAK5 in upland cotton(Gossypium hirsutum L.)

作　　者：晁毛妮[1] 胡海燕[1] 王润豪 陈煜[2] 付丽娜 刘庆庆 王清连[1] CHAO Mao-Ni;HU Hai-Yan;WANG Run-Hao;CHEN Yu;FU Li-Na;LIU Qing-Qing;WANG Qing-Lian(Henan Institute of Science and Technology/Henan Collaborative Innovation Center of Modern Biological Breeding,Xinxiang 453003,Henan,China;Cotton Research Center of Shandong Academy of Agricultural Sciences,Jinan 250100,Shandong,China)

机构地区：[1]河南科技学院/现代生物育种河南省协同创新中心,河南新乡453003 [2]山东棉花研究中心,山东济南250100

出　　处：《作物学报》2020年第1期40-51,共12页Acta Agronomica Sinica

基　　金：国家自然科学基金项目(31601347);河南省博士后科学基金项目(1902042);河南省科技攻关计划项目(192102110030);河南省高等学校重点科研计划项目(19A210013)资助~~

摘　　要：KUP/HAK/KT钾转运体基因的转录调控是植物响应低钾胁迫的一项重要机制。克隆和分析棉花钾转运体基因的启动子,不仅有助于了解其表达模式及调控机制,对于改良棉花的钾吸收特性也具有重要意义。陆地棉钾转运体基因GhHAK5是一个在根中特异性高表达的基因,其表达受低钾胁迫诱导,目前关于该基因启动子的功能还不清楚。本研究以陆地棉品种百棉1号为材料,通过PCR方法对GhHAK5上游2000bp启动子片段(pGhHAK5)进行克隆,并通过转化拟南芥、GUS组织定位和低钾诱导表达特性分析来研究其功能。结果表明, pGhHAK5除具有TATA-box和CAAT-box等基本顺式作用元件外,还含有多个响应于光、逆境胁迫、植物激素和生物钟等的顺式作用元件。pGhHAK5与雷蒙德氏棉pGrHAK5在重要调控元件的数量和位置分布上具有较高的一致性,均具有5个参与根特异性表达调控的元件(ATAAAAT)和1个参与低钾条件下转录调控的ARF转录因子结合位点(TGTCNN)。GUS组织化学染色结果显示,转基因拟南芥幼苗的叶脉和胚轴维管束组织染色较深,根系染色较浅;成熟期转基因拟南芥植株的根、叶脉和花萼维管束组织染色较深,茎和荚皮染色较浅,表明pGhHAK5驱动的GUS主要在拟南芥成熟的根和地上部维管束组织中表达。进一步低钾诱导表达特性分析表明, PGhHAK5驱动的GUS在拟南芥幼苗幼嫩根中的表达很弱,且其表达不受低钾胁迫诱导而增强,表明PGhHAK5可能是一个主要在成熟根中具有功能的低钾诱导型启动子。转录组分析和荧光定量PCR结果表明, GhHAK5主要在成熟的根中表达,且其表达受发育时期的影响,该结果与pGhHAK5驱动的GUS在拟南芥根中的表达结果一致。本研究结果有助于深入了解GhHAK5表达调控的分子机制,并为棉花钾吸收效率的提高及钾高效棉花品种的培育提供理论依据。Transcriptional regulation of KUP/HAK/KT potassium transporter gene is an important mechanism of plant response to low potassium stress. Cloning and analysis of promoter of potassium transporter gene in cotton is not only helpful to understand its expression pattern and regulation mechanism, but also important to improve the potassium absorption in cotton. Potassium transporter gene Gh HAK5 is a highly expressed in roots and induced by low potassium stress in upland cotton, but the function of its promoter is still unclear. In this study, the 2000 bp promoter fragment of GhHAK5 was cloned from upland cotton variety Baimian 1 by using PCR amplification, and its function was analyzed by GUS histochemical staining and induced expression analysis of GUS under low potassium in p Gh HAK5 transgenic Arabidopsis thaliana. In addition to TATA-box, CAAT-box and other basic cis-acting elements, pGhHAK5 also contained a number of cis-acting elements responsive to light, stress, phytohormone and circadian. p Gh HAK5 was highly consistent with p Gr HAK5 in the number and location of important regulatory elements, and had five root-specific expression regulatory elements(ATAAAAT) and an ARF transcription factor binding site(TGTCNN) involved in transcription regulation under low potassium conditions. GUS histochemical staining of transgenic Arabidopsis thaliana seedlings showed that the leaf veins and vascular tissue of hypocotyl were deeply stained, and the roots were shallowly stained. For mature Arabidopsis thaliana plants, enhanced GUS staining was observed in roots, leaf veins and the vascular tissue of calyx, and weakened GUS staining was observed in stem and pod skin, suggesting that pGhHAK5-driven GUS was mainly expressed in mature roots and vascular tissue of shoots. Induced expression analysis of GUS under low potassium in pGhHAK5 transgenic Arabidopsis thaliana showed that the expression of GUS driven by pGhHAK5 was weak in young roots of Arabidopsis thaliana seedlings, and its expression was not enhanced by low potas

关 键 词：启动子 维管束组织 钾转运体 低钾 陆地棉 

分 类 号：Q94[生物学—植物学]