嗜盐作物甜菜体内Na+长距离运输机制及其分子基础研究

Sugar beet (Beta vulgaris L.) is a typically natrophilic crop that can promptly transport larger quantities of Na+ absorbed by root to shoot and compartmentalize Na+ into vacuoles in leaves, as a cheap osmotic adjustment in order to adapt to saline environment. However, the precise molecular mechanisms underlying the long-distance transport of Na+ from root to shoot in sugar beet remain unclear. It has been shown that plasma membrane Na+/H+ antiporter (SOS1) might play important roles in the root xylem loading of Na+ and spatial distribution of Na+ in higher plants. In view of this, the present project will use two varieties of sugar beet with different salinity tolerance as materials to clone and identify the BvSOS1 gene encoding plasma membrane Na+/H+ antiporter. The transcript abundance of the BvSOS1 gene in different tissues of two varieties exposed to salt treatments will be analyzed quantitatively by using qRT-PCR methods. The BvSOS1 gene in salt-tolerant variety will be edited by CRISPR/Cas9 technology, and this gene will be overexpressed in salt-sensitive one, then Na+ concentrations of various tissues and xylem sap, and changes of sugar contents and salt tolerance will be determined in both BvSOS1-mutated plants and BvSOS1-overexpressed plants, respectively. The objective of this project is to clarify the functions of BvSOS1 in long-distance transport and spatial distribution of Na+, and the influences of it on salt tolerance in natrophilic crop. Overall, results of this project should provide theoretical basis and gene resouece involved in salt resistance for the genetic improvement of crops in north region of China.

嗜盐作物甜菜能够将根系吸收的大量Na+及时有效地转运至地上部叶片并区域化在液泡中,将其作为一种廉价的渗透调节剂以适应盐渍生境，但Na+从根系向地上部叶片长距离运输的分子机制尚不清楚。研究表明质膜Na+/H+逆向转运蛋白(SOS1)可能在植物根系木质部Na+装载及其空间分布中起重要作用。鉴于此，本项目拟以两个不同耐盐性甜菜品种为材料，克隆其质膜Na+/H+逆向转运蛋白基因BvSOS1；采用qRT-PCR技术对BvSOS1基因在盐处理下的转录丰度进行定量分析；利用CRISPR/Cas9技术定点编辑耐盐性较强品种的BvSOS1、在耐盐性较弱品种中过量表达该基因，分析BvSOS1基因突变植株和过量表达植株各组织及木质部汁液Na+浓度、糖含量和耐盐性的变化。以期阐明BvSOS1在嗜盐作物体内Na+长距离运输和空间分布中的作用及对耐盐性的影响，为我国北方地区农作物抗逆性遗传改良提供理论依据和基因资源。

盐胁迫是制约全球农作物生长和产量的主要环境因素之一。本项目以嗜盐作物甜菜为材料，探究了质膜Na+/H+逆向转运蛋白BvSOS1、BvCBLs、BvCIPKs和BvSnRK2s等基因在甜菜响应盐胁迫中的作用机制，取得了如下主要研究进展和成果：(1)克隆和鉴定甜菜质膜Na+/H+逆向转运蛋白基因BvSOS1，其CDS全长3489 bp，编码1162个氨基酸，等电点(pI)6.34，分子量128.5，含有12个跨膜区，定位在第6条染色体。(2)发现BvSOS1在甜菜地上部和根中均有所表达，但地上部的表达水平显著高于根部；随着盐处理时间延长，地上部和根中的表达量均呈增加趋势。(3)构建了BvSOS1过量表达载体，得到重组质粒pEarleyGate 100-BvSOS1，采用花序浸染法遗传转化拟南芥，最终获得23株转基因株系。(4)发现过表达BvSOS1使转基因植株种子发芽率、幼苗根长、鲜重、叶绿素和脯氨酸含量显著增加，抗氧化酶相关基因AtSOD1、AtCAT1、AtPOD1及脯氨酸合成相关基因AtP5CS1表达水平高于野生型植株。可见BvSOS1通过调节抗氧化酶活性和渗透调节能力，增强转基因植株的耐盐性。(5)鉴定出7个BvCBLs，其含有保守的EF-hand基序，而BvCBL1、-2和-5的N-末端含有豆蔻酰化位点，BvCBL4和-6的N端包含一个跨膜结构域。在盐处理下，BvCBLs在地上部和根部的表达量均有显著增加，而在山梨醇处理下其在根部的表达量显著诱导。(6)鉴定出20个BvCIPKs基因，根据系统发育分析和基因结构及保守基序分布将其分为A-E五个亚簇，C端含有高度保守的NAF/FISL和PPI结构域。50和100 mM NaCl显著诱导甜菜地上部和根中BvCIPKs表达。(7)鉴定到6个BvSnRK2s基因，其CDS在1008 bp~1095 bp；pI从4.85~5.73不等，GRAVY均为负值，表明BvSnRK2s为亲水蛋白。根据是否受ABA诱导，将SnRK2分为3簇Ⅰ、Ⅱ和Ⅲ。发现BvSnRK2s在甜菜叶和根中均有所表达。在盐处理下地上部和根中的表达量上调，而干旱处理下在地上部的表达量相对较高。可见，BvSnRK2s在响应逆境胁迫中具有重要作用。以上研究成果对我国北方干旱半干旱地区农作物抗逆性遗传改良和生态环境保护与建设具有重要的理论意义和实践价值。