一个玉米Pht1家族磷转运蛋白基因克隆和功能分析

Cloning and Characterization of a Phosphate Transporter Gene of Pht1 Family in Maize

从耐低磷玉米自交系178中分离和鉴定高亲和力磷转运蛋白质基因,为开展磷高效分子育种奠定理论基础。本研究以水稻和拟南芥中鉴定的磷转运蛋白基因为基础,运用生物信息学方法,对玉米进行全基因组预测及系统进化分析;并运用克隆、实时荧光定量PCR和亚细胞定位方法对其家族成员进行深入研究。结果表明,从玉米自交系B73全基因组序列中筛选出37个磷转运蛋白候选基因,并被聚类为五大家族。从耐低磷材料中扩增了一个属于Pht1家族成员ZmPht1;9的全长cDNA,该基因编码区长1620bp,编码539个氨基酸,含有典型的MFS超家族蛋白的保守结构域和12个跨膜结构;荧光定量PCR分析表明,在低磷胁迫下,该基因的相对表达量显著增加,且叶片中的表达量高于根系,同时在基因型之间也存在差异;原生质体转化的亚细胞定位结果显示,ZmPht1;9表达蛋白主要分布于细胞膜上。ZmPht1;9编码位于细胞膜上的高亲和力磷转运蛋白,对调节磷素的动态平衡起重要作用。

To provide usefully theoretical information for improvement of phosphorus utilization, a high-affinity PHT gene from low-P tolerant maize inbred line 178 was isolated in this study. The PHT families of maize at whole genome-wide level were predicted based on the gene sequences of PHT orthologs in rice and Arabidopsis using bioinformatics approaches , and their phylogcnetic relationship was also been analyzed based on amino acid sequence. The methods of clone, quantitative real-time PCR and subcellular localization were performed for further research of PHT family member. A total of 37 maize PHT genes were predicted from whole genome sequences of maize inbred line B73, which can be classed into five groups based on their characters of functional domains. A complete eDNA of ZmPhtl ;9, belongs to Phtl family and encodes 539 putative amino acids with the complete coding region of 1 620bp in length, was further cloned from 178＇ s cDNA template. The protein of ZmPhtl ;9 contains 12 transmembrane domains and has a typical MFS conservation structure domain, which shows highly conserved within plants. The relative expression levels of ZmPhtl ;9 detected by quantitative real-time PCR exhibited up-regulation under low phosphorus stress and was higher in the leaves than in the roots, and expression patterns were shown differently between two extreme maize lines with distinct tolerance to phosphorus deficiency. Subcellular localization analysis revealed that the ZmPhtl;9 was expressed in cytoplasm membrane via protoplast-mediated. The results suggested that ZmPhtl ;9 was a high-affinity PHT gene and was possibly involved in maintaining phosphate dynamic homeostasis under phosphate deficiency.