盐胁迫下甜菜Na+吸收机制及其分子基础研究

Natrophilic plants are able to absorb larger quantities of Na+ by roots from soil and compartmentalize Na+ into vacuole in leaves in order to use Na+ for a beneficial osmotic adjustment to adapt to saline environment. However, the precise mechanisms underlying Na+ uptake and transport in the root of natrophilic plants remain elusive. It has been shown that high-affinity K+ transporter (HKT) plays critical roles in Na+ uptake by the cortical and endodermal cells in the roots of some crops, such as rice (Oryza sativa), wheat (Triticum aestivum) and barley (Hordeum vulgare). All these salt-exclusion glycophytic crops, however, might not be the best choice to understand how ion transporters involved in Na+ uptake and transport work in salt tolerance in crop plants due to their weaker abilities for Na+ uptake in roots and Na+ compartmentalization in leaves. In this project, therefore, sugar beet (Beta vulgaris L.), an important sugar crop that can absorb and accumulate vast amount of Na+, will be used to understand the function of Na+ uptake mediated by HKT transporter in salt tolerance. The BvHKT gene encoding the high-affinity K+ transporter will be cloned and characterized; the transcript abundance of the BvHKT gene under salt treatment will be quantitatively analyzed by using real-time RT-PCR; the BvHKT gene in sugar beet will be silenced by RNA interference (RNAi) method；22Na+ influx, Na+ net uptake rate, and sugar content in transgenic plants will be tested. Results of this project should provide the theoretical basis and gene resources involved in salt tolerance for the genetic improvement of crops in the saline regions.

嗜盐作物能从土壤中吸收大量Na+并将其转运至地上部叶片、区域化于液泡中作为一种有益的渗透调节剂来适应盐渍生境，但Na+在其根部的吸收与转运机制尚不清楚。已有研究表明高亲和性K+转运蛋白(HKT)在植物根系表皮和皮层细胞Na+吸收中起重要作用，但所用材料(如水稻、小麦和大麦)均为叶片中Na+区域化能力很弱的拒盐植物，且缺乏足够发达的根，吸收Na+的能力十分有限，并不是研究Na+吸收机制的理想材料。本项目拟以大量吸收并积累Na+的糖料作物甜菜为材料，克隆其高亲和性K+转运蛋白基因BvHKT；利用real-time RT-PCR技术对BvHKT基因在盐处理下的转录丰度进行定量分析；采用RNA干扰技术沉默BvHKT基因表达，分析转基因植株根部22Na+内流、Na+净吸收速率和糖含量等变化。以解析HKT类转运蛋白介导的Na+吸收在甜菜耐盐性中的作用，为农作物抗盐性遗传改良提供理论依据及抗逆基因资源。

本项目取得了以下主要研究进展和成果：(1) 基于农学(如干重)和生理学(如Na+、K+、Ca2+、可溶性糖和脯氨酸)指标，采用隶属函数法综合评价筛选出耐盐性较强的甜菜品种“甘糖7号”。(2) 发现50 mmol/L NaCl能够促进甜菜幼苗的生长并减缓由渗透胁迫造成的不利影响。渗透胁迫下添加NaCl使甜菜体内、特别是地上部积累较多的Na+，即使不添加NaCl其体内也能维持Na+含量的稳定。表明Na+在甜菜适应渗透胁迫中起着重要作用。(3) 发现外界高浓度K+(10和50 mmol/L)能通过增强K+吸收及限制Na+吸收，使甜菜维持较低的Na+/K+比，从而增强其对盐分的耐受能力。在NaCl条件下， 5和10 mmol/L TEA+(K+通道抑制剂)均对甜菜根Na+和K+净吸收速率没有显著性影响。然而，添加3和6 mmol/L Cs+(K+内向整流通道抑制剂)，使甜菜根K+净吸收速率明显降低，但对Na+净吸收速率的影响不明显。添加5和10 mmol/L Ba2+(K+通道和转运蛋白抑制剂)不但降低K+吸收速率，而且降低Na+吸收速率。表明甜菜根Na+吸收对Ba2+敏感，而对TEA+和Cs+不敏感；K+吸收对Cs+和Ba2+敏感，而对TEA+不敏感。由此可见，AKT1介导甜菜根K+吸收，而HKT1介导Na+吸收。(4) 鉴定甜菜K+内向整流通道基因BvAKT1，其cDNA全长3250 bp，包含2604 bp的开放阅读框，71 bp的5’非翻译区，575 bp的3’非翻译区及34 bp的poly(A)尾巴。此cDNA编码867个氨基酸的多肽，推测分子量为97.82 kDa，等电点为6.57。系统进化分析表明，甜菜BvAKT1与其它植物AKT1的同源性在60%以上。(5) 发现不同浓度(5-150 mmol/L)NaCl处理下，甜菜根中的BvAKT1转录丰度显著高于叶；150 mmol/L NaCl处理下该基因表达量明显高于100 mmol/L。表明高盐胁迫可提高BvAKT1的转录水平。另外，50 mmol/L NaCl处理6 h后，根中的BvAKT1转录丰度呈现降低趋势。不同浓度(0-10 mmol/L)KCl处理下，BvAKT1转录丰度并未发生变化，由此推测BvAKT1受转录后调控。(6) 构建甜菜BvAKT1基因的RNA干扰载体，为深入研究该基因的功能奠定了基础。