茶树磷转运蛋白基因CsPT4的克隆、亚细胞定位及表达分析

摘要

茶园中磷肥的利用效率取决于茶树体内与磷元素吸收、转运及生理利用等相关蛋白的协同调控,而磷转运蛋白(Phosphate transporter proteins,Phts)在此过程中起着关键的调控作用.本研究以茶树品种龙井长叶(Camellia sinensis cv.Longjing-changye)为试验材料,采用同源克隆的方法首次克隆获得茶树磷转运蛋白编码基因CsPht1:4(CsPT4)的全长cDNA.该基因全长1642 bp,开放阅读框(ORF)1620 bp(GenBank登录号:KY132100),编码539个氨基酸.生物信息学分析显示,CsPT4基因编码蛋白分子量为59.12 kD,理论等电点(pI)为8.51;具有典型的Pht1家族特性:"6-亲水-6"跨膜结构.亚细胞定位结果显示,该蛋白分布于质膜上,与Softberry软件预测结果一致.荧光定量PCR表明:CsPT4在正常生长的茶树根、茎、嫩叶、老叶中均有表达,在老叶中的表达量较高,在根部表达量最低.低磷处理,根和叶中CsPT4上调表达水平均先上升后下降;根部CsPT4表达量48 h内各个时间点均高于叶部.缺磷处理,根和叶中CsPT4上调表达水平均升高;根部和叶部分别在72 h和48 h达最大值.本研究为茶树响应低磷的分子机制提供了参考.%Phosphate transporter proteins (Phts) play important roles in plant phosphorus (P) absorption and transportation. Furthermore, Phts affect usage efficiency of the tea garden fertilizer. A full-length phosphate transporter complementary DNA (cDNA) CsPht1:4 (also named CsPT4) was cloned from tea plant (Camellia sinensis cv. Longjingchangye) by rapid amplification of cDNA ends (RACE) techniques. CsPT4 had an open reading frame of 1620 bp (GenBank accession No. KY132100) and encoded a 539 amino acid polypeptide. Bioinformatic analyses showed that CsPT4 had a molecular weight of 59.12 kD and a theoretical isoelectric point of 8.51. The protein secondary structure was a "6+Hydrophilic+6" configuration,which was consistent with the typical structure of Phts. Subcellular localization assay showed that the CsPT4 protein localized in plasma membrane, which was consistent with the predicted results of Softberry. The expression pattern of CsPT4 gene was tissue-specific. Its transcript abundance in old leaves was much higher than that in tender leaves, stems and roots. The lowest expression of CsPT4 gene was identified in roots. Quantitative real-time PCR showed that the gene expression trends in root and leaves were different under low-P and P-deficiency treatments. Under low-P treatment, its induced level was first increased and then decreased, with higher expression in roots than leaves. While under P-deficiency treatment, the induced expression of CsPT4 gene kept stable, with its peak in roots and leaves at 72 h and 48 h, respectively. The results of this study provided a reference for the study of the molecular mechanism of tea adaptation to low P.