拟南芥酪氨酸蛋白磷酸酶PTP135基因克隆及功能分析

Expression and Function Analysis on PTP135 Gene of Protein Tyrosine Phosphatase in Arabidopsis thaliana

拟南芥PTP135 T-DNA插入突变体ptp135具有与phyB突变体相似的特征,萌发时下胚轴伸长、子叶张开角度小、展开慢,后期表现为开花早、叶色浅、叶柄长、叶面积增大。采用PCR法从拟南芥叶片中克隆PTP135启动子基因,PTP135启动子连接到载体pCAMBIA1391,构建植物双元表达载体p CAMBIA1391-135p,构建的载体经过鉴定后,运用农杆菌介导的沾花法将PTP135启动子转化野生型拟南芥,用潮霉素筛选转化阳性株,对阳性株进行GUS组织化学染色,结果表明PTP135基因定位在维管束、柱头和花丝。采用PCR方法从拟南芥叶片中克隆PTP135基因,连接到载体pEZS-NL,构建瞬时表达载体pEZS-NL-135e,采用基因枪法转化洋葱表皮,结果表明PTP135编码的蛋白定位在细胞核和细胞膜。拟南芥野生型、ptp135和phyB,待即开花时取样,做光周期特异性基因CO、FT的RT-PCR表达分析,结果表明CO和FT基因在ptp135突变体中表达时间延长,表达量增加,证明PTP135可以在转录水平上调控CO和FT,PTP135参与植物开花调控途径,作用位点在CO上游。

The characteristics of ptp135 mutant of Arabidopsis thaliana was similar to phyB mutant.The young seeding had a higher hypocotyl,opening cotyledon with a smaller angle,and slower expansion.At the later stage,it showed early flowering,lighter leaf color.We cloned the PTP135 promoter gene from type A.thaliana leaves by PCR.The PTP135 promoter gene was connected to the vector pcambia1391,and the vector pcambia1391-135p was constructed.Then the vecter was transformed into wild type A.thaliana by Agrobacterium-mediated flower dipping method.The results showed that ptp135gene was located in vascular bundle,stigma and filament.We cloned the PTP135 gene from Arabidopsis thaliana leaves by PCR.The PTP135 gene was connected to the vector pEZS-NL,and the vector pEZS-NL-135e was constructed and transformed into onion epidermis by particle bombardment.The results showed that the protein of ptp135 was localized in the nucleus and cell membrane.The RT-PCR results showed that the expression time of CO and FT in ptp135 was longer and the expression level was more than in the wild.It was proved that PTP135 could regulate CO and FT at the transcriptional level.PTP135 worked in the flowering regulation pathway,and the action site was in the upstream of CO gene.