Na+提高甜菜抗旱性的作用机制研究

Sugar beet (Beta vulgaris L.) is considered as a natrophilic crop with the stronger resistance to adverse environment. Researches on beneficial effects of Na+ in sugar beet were limited mainly the growth and salt tolerance, however, our recent studies showed that Na+ also plays very important roles in the response of sugar beet plants to drought stress. Tonoplast Na+/H+ antiporter (NHX) proved to be involved in the compartmentation of Na+ into vacuoles from cytosol. However, the precise mechanisms underlying this transporter in drought tolerance of sugar beet remained unclear. This project, based on our previous studies, will analyze the intracellular micro-distribution of Na+ in both root and shoot of two sugar beet cultivars with differing drought tolerance under osmotic stress, to investigate relationship between Na+ compartmentation and drought tolerance. BvNHX gene encoding tonoplast Na+/H+ antiporter will be cloned and characterized in sugar beet; the transcript abundance of this gene will be quantitatively analyzed by using real-time PCR in two cultivars exposed to osmotic stress; BvNHX gene in drought-resistance cultivar will be silenced by RNA interference method, and this gene will also overexpressed in drought-sensitive one, then cellular micro-distribution of Na+ and the changes of drought tolerance will be tested in both BvNHX-silenced plants and BvNHX-overexpressed plants, respectively. Results of this project may reveal the mechanisms underlying Na+ in adaptation of sugar beet to drought, and also should provide the theoretical basis for elucidating adaptative mechanisms of natrophilic crops to adverse environment. Overall, this project has the important theory significance and the practice value for the genetic improvement of crops and protection of ecological environment in arid and semi-arid regions in North China.

甜菜是一种抗逆性较强的嗜钠作物，以往有关Na+对甜菜有益作用的研究多集中在生长和耐盐性上，而我们最近发现Na+在甜菜响应干旱胁迫过程中也发挥着重要作用；液泡膜Na+/H+逆向转运蛋白(NHX)是介导液泡Na+区域化的重要功能蛋白，但其在甜菜抗旱性中的作用机理尚不清楚。本项目拟在前期工作基础上，通过系统分析渗透胁迫下2个抗旱性不同的甜菜品种根和叶细胞中Na+微区分布，探明甜菜Na+区域化与抗旱性的关系；克隆甜菜NHX蛋白基因BvNHX，采用Real-time PCR技术分析该基因在渗透胁迫下的转录丰度，通过在抗旱性强的品种中沉默BvNHX基因的表达、在抗旱性弱的品种中超表达该基因，分析沉默植株和超表达植株Na+微区分布及抗旱性的变化。以期揭示Na+在甜菜适应干旱中的作用机理，为阐明嗜钠作物逆境适应机制奠定理论基础。对我国北方地区农作物的抗逆性遗传改良和生态环境建设具有重要理论意义和实践价值。

干旱和盐胁迫是制约全球作物生长和产量的主要非生物因素。本项目以糖料作物甜菜为材料，探究了Na+在其抗逆性中的作用机制及其分子基础。取得了如下主要研究进展和成果：（1）发现干旱胁迫下甜菜叶中的Na+浓度随着胁迫强度的增强而呈增加的趋势；而K+浓度则始终维持在恒定的水平。（2）克隆和鉴定甜菜液泡膜Na+/H+逆向转运蛋白BvNHX基因，其cDNA全长2284 bp，包括1653 bp的开放阅读框，19 bp的5’非翻译区，612 bp的3’非翻译区及25 bp的poly（A）尾巴；该基因编码552个氨基酸，推测分子量为61.3 kDa，等电点为6.609。系统进化分析表明，BvNHX与盐爪爪KfNHX和盐穗木HcNHX进化关系较近，而与水稻OsNHX和拟南芥AtNHX1关系较远。（3）发现BvNHX在甜菜根和叶中均有所表达，但叶中的表达量明显高于根；随着NaCl浓度的增加，叶中的BvNHX转录丰度逐渐呈增加的趋势，在50 mmol/L NaCl下达到最大。（4）构建CaMV 35S启动子驱动的含BvNHX基因片段反向重复序列的RNAi植物表达载体pARB，通过冻融法将其导入根癌农杆菌GV3101，并转入甜菜幼苗获得BvNHX-RNAi转化株系。半定量RT-PCR分析表明，该干扰载体能特异性地导致转化植株BvNHX基因表达的沉默。（5）构建BvNHX过量表达载体pCAMBIA1302-BvNHX，采用实生苗侵染法将其导入豆科牧草红豆草。在100和200 mmol/L NaCl下，转基因红豆草植株叶鲜重较野生型植株分别增加了27%和44%，K+浓度则分别提高了22%和16%，表明过量表达BvNHX能够增强转基因红豆草的耐盐性。（6）发现盐胁迫下四倍体甜菜品种体内积累较少的Na+和更多的可溶性糖以及维持较低的Na+/K+比和较强的K+、Na+选择性吸收能力，其生物量和耐盐性明显高于二倍体品种。（7）发现过量表达旱生植物霸王ZxNHX和ZxVP1-1基因能够提高甜菜离子区域化能力，使其地上部积累更多的Na+、Ca2+、脯氨酸、果糖和葡萄糖，贮藏根中积累更多的蔗糖，从而增强甜菜植株的抗旱性。这些研究成果对我国北方干旱半干旱地区农作物的抗逆性遗传改良以及生态环境建设具有重要的理论意义和实践价值。