水稻OsPUT1基因的鉴定和功能初步验证

Identification and Preliminary Functional Verification of OsPUT1 Gene

为探究氨基酸转运蛋白(AAT)的功能和可能的分子机制,首先,通过建立隐马尔可夫新模型对水稻AAT家族成员进行鉴定,构建系统进化树分析水稻与拟南芥AAT成员之间的进化关系;其次,利用生物信息学手段对水稻多胺转运蛋白基因1(OsPUT1)的启动子、蛋白结构和功能结构域进行了分析;再次,构建了OsPUT1-GFP载体在水稻原生质体中表达以确定其亚细胞定位,利用荧光定量PCR检测OsPUT1基因在不同组织以及不同逆境胁迫下的表达模式;最后,采用水稻OsPUT1基因过表达(OE)、日本晴(Nip)和RNA干扰(RNAi)株系初步研究了基因的功能。结果显示:在水稻中含有96个OsAAT蛋白家族成员,分为13个亚家族;PUT亚家族含有6个OsPUT成员,其中OsPUT1和AtPUT3遗传距离最近且分布在同一个分支;OsPUT1基因启动子中含有涉及生长发育、光调节、植物激素和胁迫响应的顺式作用元件;OsPUT1蛋白含有多胺转运蛋白结构域PotE,亚细胞定位试验表明其位于细胞膜上;在叶片中OsPUT1基因表达量相对较高,在花中表达量相对较低;基因表达受JA、甘露醇和ABA抑制;低温胁迫下,基因表达先下降后恢复正常;在SA、亚精胺和百草枯处理下,基因表达先上升后降低;在氯化钠处理下,基因表达量先上升,在1 h达到最高,之后下降,在12 h达到最低,在24 h恢复到正常水平;OE株系显著降低了对0.2μmol/L百草枯的抗性,而RNAi株系则显著增强了对0.2μmol/L百草枯的抗性。综上说明,OsPUT1蛋白可能具有运输多胺和参与胁迫响应的功能。

To explore the function and possible molecular mechanisms of amino acid transporters(AATs),firstly,a new hidden markov model was established to identify the members of the rice AAT family,and a phylogenetic tree was constructed to analyze the evolutionary relationship of AAT members between rice and Arabidopsis;secondly,the promoter,protein structure,and functional domain of rice polyamine transporter gene 1(OsPUT1)were analyzed using bioinformatics methods;thirdly,the subcellular localization of OsPUT1 was determined by constructing the OsPUT1-GFP vector and expressing it in rice protoplasts;fourthly,the expression patterns of OsPUT1 gene in different tissues and under different stress conditions were detected using qRT-PCR;finally,the function of OsPUT1 gene was preliminarily studied using the OE,Nip and RNAi lines.The results showed that there were 96 OsAATs divided into 13 subfamilies in rice;the PUT subfamily contained 6 OsPUTs,among which OsPUT1 and AtPUT3 had the closest genetic distance and were distributed in the same branch.The promoter of OsPUT1 gene contained cis-acting elements related to growth and development,light regulation,plant hormones,and stress response;OsPUT1 protein contained the polyamine transporter domain PotE,and subcellular localization experiment indicated that it was located on the cell membrane;the expression of OsPUT1 gene was relatively high in leaves,while was low in flowers;gene expression was inhibited by JA,mannitol,and ABA,while decreased and then returned to normal under low temperature stress,yet increased and then decreased under SA,Spm and paraquat treatment,however,under sodium chloride treatment,the gene expression level first increased,reached its peak at 1 h,then decreased,reached its lowest at 12 h,and returned to normal levels at 24 h;OE lines significantly reduced resistance to paraquat,while RNAi lines significantly increased resistance to paraquat.Overall,the OsPUT1 protein might have the function of transporting PA and participating in stress response.