水稻基因OsGLP1-2的克隆、表达及功能分析  

Cloning,expression and functional analysis of rice gene Os GLP1-2

作　　者：李明玉 石杨 李斌[1] 王若霖 肖强 陈龙[1] 陈稷[2] 杨其亮 黄进 LI Ming-yu;SHI Yang;LI Bin;WANG Ruo-lin;XIAO Qiang;CHEN Long;CHEN Ji;YANG Qi-liang;HUANG Jin(College of Ecology and Environment,Chengdu University of Technology,Chengdu 610059,China;College of Agronomy,Sichuan Ag-ricultural University,Chengdu 611130,China;Guangzhou Forevergen Medical Laboratory,Guangzhou 510700,China)

机构地区：[1]成都理工大学生态环境学院,成都610059 [2]四川农业大学农学院,成都611130 [3]广州永诺医学检验所有限公司,广州510700

出　　处：《西南农业学报》2023年第2期217-223,共7页Southwest China Journal of Agricultural Sciences

基　　金：国家自然科学基金项目(31870383)。

摘　　要：【目的】克隆水稻镉(Cadmium, Cd)响应基因OsGLP1-2,并对其进行生物信息学及Cd胁迫处理的表达模式分析,为探索OsGLP1-2基因在水稻中应对重金属胁迫的分子机制提供基础。【方法】以水稻为材料,利用PCR克隆OsGLP1-2基因,并利用生物信息学方法对其顺式作用元件、二级结构和疏水性等进行分析,用实时荧光定量PCR(qRT-PCR)检测其在100μmol/L Cd处理下,水稻茎部和根部中的表达特征,再利用转基因酵母进行Cd耐受性分析。【结果】水稻OsGLP1-2基因编码区(CDS)序列长度为651 bp,编码216个氨基酸,其编码蛋白的相对分子量为22.48 kDa,理论等电点(PI)为7.16,为稳定性的中性疏水性蛋白,含有Cupin结构域,属于Cupin家族,该家族在植物防御等方面发挥着重要作用。同源家族进化树表明该基因是单独的一支,但与大麦HvGER5a的亲缘关系最为接近,表明OsGLP1-2可能具有HvGER5a类似的功能。二级结构分析表明该蛋白含有10个β折叠、8个α螺旋和17个无规卷曲结构。并且OsGLP1-2具有光、干旱等环境诱导相关的调节元件以及生长素、赤霉素和茉莉酸甲酯等激素相关调节元件,表明目的基因可能参与水稻对非生物胁迫的响应。100μmol/L Cd处理下,在6 h时地上部分目的基因的表达水平约为对照组3倍,且地下部分也为对照组的3倍,即OsGLP1-2基因能对Cd产生响应。斑点实验表明该基因在酵母中无明显表型。【结论】成功克隆了水稻OsGLP1-2基因,且该基因对Cd有一定的响应,可为水稻应对重金属胁迫的反应机制提供参考依据。【Objective】In the present study,the rice cadmium-responsive gene Os GLP1-2 was cloned,and the bioinformatics and expression pattern analysis of Cd stress treatment were performed to provide a basis for exploring the molecular mechanism of Os GLP1-2 gene in response to heavy metal stress in rice.【Method】The Os GLP1-2 gene was cloned,and the characteristics of the gene and protein were analyzed by bioinformatics.Real-time fluorescent quantitative PCR(qRT-PCR)was used to analyze the expression pattern of Os GLP1-2 under Cd stress in rice stems and roots,respectively.At last,Cd tolerance analysis was performed using transgenic yeast.【Result】The cloned rice Os GLP1-2 gene coding region(CDS)sequence length was 651 bp,encoding 216 amino acids,the relative molecular weight of the encoded protein was 22.48 k Da,and the theoretical isoelectric point(PI)was 7.16,which was the stable and neutral hydrophobic protein,containing the Cupin domain and belonging to the Cupin family.The Cupin family played an important role in plant defense.The homologous family phyloge-netic tree showed that this gene was a single branch,but it was the closest relative to Hv GER5a in barley.It indicated that Os GLP1-2 may have similar functions as Hv GER5a.The results of the secondary structure showed that the protein contained 10βsheets,8αhelices and 17 random coil structures.And Os GLP1-2 had regulatory elements related to environmental induction such as light and drought,as well as hor-mone-related regulatory elements such as auxin,gibberellin and methyl jasmonate,indicating that the target gene may participate in the re-sponse of rice to abiotic stress.Under the treatment of 100μmol/L Cd,the expression level of the target gene in the above-ground part was about 3 times that of the control group at 6 hours,and the expression level of the underground part was also 3 times that of the control group,indicating that Os GLP1-2 gene could respond to Cd.Spot experiments showed that the gene had no obvious phenotype in yeast.【Conclusio

关 键 词：镉 水稻 OsGLP1-2 表达分析 

分 类 号：S511[农业科学—作物学]