菊花Pht1家族质膜磷酸盐转运蛋白基因CmPTs克隆和功能解析

Chrysanthemum is one of the ten traditional important ornamental flowers in China and one of the four most popular cut flowers all over the world , it is of high ornamental and economic value. Phosphorus (P) is essential for chrysanthemum growth, production and flower quality, however, most of commercial chrysanthemum cultivars are defective in Pi utilization. Soil salinity, water pollution resulted from over-fertilization of Pi and run off of phosphate rock resources all challenged the chrysanthemum producing. In this proposal, we planed to clone plasam membrane localized phosphate transporter of Pht1 gene family from chrysanthemum. Bio-information analysis, spatial and temporal expression profiles and promotor cloning and cis-elements analysis will be conducted to disclose the genes' expression pattern. We will determine the regulation of post-transcription through site-directed mutagenesis as well as subcellular localization observation and provide yeast evidence specifically for CmPT's function in a heterologous expression system. The function of Pht1 family in vivo will be determined via generation of over-expression and antisense transgenic plants. Consequently, the proposal will shade light on functional characterization of chrysanthemum PM pht1 gene family with regard to its nutrients uptake and translocation,which is very important to understand the mechanism involved in Pi uptake and will lay foundation for chrysanthemum germplasm enhancement.

菊花是中国十大传统名花和世界四大切花之一，有很高的观赏和经济价值。磷是影响菊花生长发育、产量与品质的最重要营养元素之一。主栽切花菊品种磷利用率低下，生产上过度施用磷肥导致的土壤次生盐渍化、水体环境污染，以及磷矿资源的日益枯竭等均对切花菊生产提出了严峻挑战。为此，本申请项目拟从菊花中同源克隆Pht1家族质膜磷酸盐转运蛋白编码基因CmPTs，通过生物信息学分析、基因时空表达特性、启动子克隆与顺式作用元件分析阐述其转录水平调控特性；对其蛋白修饰位点进行定点突变，通过酵母异源表达体系及亚细胞定位变化观察初步阐述其功能；利用超表达和反义转基因技术获得CmPTs转基因菊花，阐述不同基因家族成员对磷吸收与转运调控的机制。本申请项目的实施可丰富菊花磷高效利用分子机理，并为培育磷高效利用菊花的生物技术育种提供基础。

菊花为世界四大切花之一。磷缺乏会严重影响切花的质量和观赏品质。切花菊设施周年生产中，普遍存在磷肥过度施用、利用率低和损失严重等，并导致水系统富营养化及土壤次生盐渍化。为此，本研究从磷高效吸收菊花 ‘南农银山’中克隆了CmPht1家族2个基因，命名为CmPht1;1和CmPht1;2（以下简称CmPT1和CmPT2），ORF分别为1596 bp、1602 bp，编码532和534个氨基酸。CmPT1,CmPT2与已知的高亲和磷转运蛋白同源性为71.8～78.6%，73.94%～81.01%。CmPT1、CmPT2均包含12个跨膜区，以及Pht1家族磷转运蛋白保守特征序列GGDYPLSATIMSE。CmPT1在根中高表达，而CmPT2在茎中表达量较高，两者在根中受低磷明显诱导。CmPT1和CmPT2可回补酵母突变体磷吸收功能，为与质子相偶联的运输蛋白，其Km值分别为35.2 µM、19.3 µM，最大吸收速率分别为594 pmol Pi（mg yeast cells•min）-1、170pmol Pi (mg yeast cells•min)-1，说明CmPT1、CmPT2为高亲和磷酸盐转运蛋白。对CmPT2蛋白关键修饰氨基酸位点进行了点突变，发现G75A，G80A，G84A，G222A，T239A，Y242A，N422A，T424A，G449A位点对CmPT2的磷酸盐转运功能十分重要。CmPT2及其突变子均定位在细胞膜上。CmPT1超表达菊花的株高较对照提高38.7%和35.5%，根系体积增加53.1%和66.7%，干物质重增加37.1%和48.6%，植株磷含量增加1.39倍和1.72倍。低磷条件下，CmPT2超表达转基因菊花根长较野生型增加50.0%和20.4%，根干重增加2.71和2.50倍，侧根数增加8.3%和26.4%；株高增加28.1%和31.2%，地上部干重增加1.16倍和1.47倍。正常磷浓度条件下，CmPT2超表达株系的根长、根干重、侧根数、株高及地上部干重较野生型差异不显著。花期叶片中的总磷含量分别增加19.8%和8.0%，花中的总磷分别增加60.6%和24.6%，而根中低于对照，表明CmPT2在菊花磷吸收和转运中发挥作用。培养研究生4名，发表SCI论文9篇，中文核心期刊3篇。获华耐园艺科技奖和江苏省农业技术推广奖二等奖各1项。