马铃薯锌转运蛋白基因StZIP4调控植株锌吸收转运功能研究

Human Zn deficiency has become a widespread public health problem. While, increasing Zn concentrations in potato tuber has positive meaning in alleviating Zn deficiency in human. In our early study, we found foliar zinc fertilizers can significantly increase Zn concentrations in potato tuber, and a Zn transporter gene StZIP4 which was highly induced by Zn deficiency has been found in potato leaf based on the high-throughput sequencing, combined with the existing study, it was indicated StZIP4 play an important role in Zn transport from leaf to underground part. To verify this, in this project, firstly, we planted to analysis the function of StZIP4 in transporting Zn through yeast heterologous complementary method, and investigate the expression pattern and localization by real-time fluorescent quantitative PCR and subcellular localization technology; Secondly, Zn efficiency of different potato varieties would be evaluated, and the StZIP4 express level and sequence would be researched in different potato varieties with different zinc efficiency; Finally, molecular method such as constitutive expression and gene silencing of StZIP4 will carried out to explore the StZIP4 function in zinc uptake and transport. This project will supplement new data of molecular biology for potato zinc uptake and transport, offer new gene for cultivating high Zn efficient potato varieties, and have positive significance for alleviating human Zn deficiency.

人体缺锌已成为普遍的公共健康问题。提高马铃薯块茎锌含量对于缓解人体缺锌具有积极意义。我们前期发现叶面喷施硫酸锌溶液能显著提高薯块锌含量，通过转录组测序在叶片中发现一个锌转运蛋白基因StZIP4，其表达量受缺锌条件诱导，结合前人研究，推测StZIP4在锌从叶片向地下部运转过程中起到重要作用。为了验证这一推测，本研究首先利用酵母异源功能互补的方法研究StZIP4运转锌的功能，利用实时荧光定量PCR及亚细胞定位技术研究其组织表达特异性和表达部位；再次，通过不同马铃薯品种锌效率评价，研究StZIP4在不同锌效率马铃薯品种中的表达情况；最后，应用过量表达和基因沉默等分子生物学手段，探讨StZIP4转运蛋白基因在锌吸收转运过程中的作用，进一步揭示马铃薯锌吸收运转分子机制。本研究为解析马铃薯锌吸收运转分子生物学补充新的数据，为培育马铃薯锌高效品种提供新的基因储备，对于缓解人体缺锌具有积极意义。

锌对人体健康具有非常重要的作用。我们前期在马铃薯中发现一个锌转运蛋白基因StZIP4，其表达量受缺锌条件诱导，推测StZIP4在锌运转过程中起到重要作用。在本项目中，利用生物信息学手段鉴定出马铃薯锌转运蛋白StZIP家族共12个成员，根据它们在染色体上的分布情况进行命名，在项目申请时的StZIP4被更名为StZIP12，获得了StZIP12的全长序列；克隆并分离了马铃薯StZIP12基因，该基因全长为1820 bp，编码407个氨基酸;利用锌吸收缺陷型酵母zrt1 /zrt2 Δ双突变体研究StZIP12转运锌的功能，证实了StZIP12具有体外转运锌的功能；利用亚细胞定位技术，进行StZIP12的亚细胞定位分析，发现StZIP12蛋白主要定位于拟南芥原生质体的细胞膜上；分别构建StZIP12的过量表达和RNAi沉默载体，利用转基因技术开展StZIP12在马铃薯中的遗传转化，及后期的功能验证分析，发现过量表达StZIP12增加了转基因薯块的锌含量，初步证实了StZIP12在马铃薯Zn吸收转运中发挥了重要的功能。本研究为马铃薯锌吸收运转分子生物学补充新的数据，为培育马铃薯锌高效品种提供新的基因储备。