Na+依赖型共转运体GhBASS5调控棉花耐盐性的机理研究

The uptake activity of sodium and specific substrate for the sodium-dependent symporters were dependent on the sodium-gradient across the membrane. Under salt stress, the regulation of sodium-dependent symporters would not only effect the pathway that the cotransporting substrate supplied, but also function in the the uptake and distribution of Na+, and hence have an important effect on the plant salt tolerance. By comparing two upland cotton cultivars with significantly difference in the salt tolerance, GhBASS5 was considered as a negative effecting gene in cotton salt tolerance, which might encode a plastidial sodium:keto acid symporter. The expression level of GhBASS5 in salt tolerance cotton was significantly lower than salt sensitive cultivar no matter under control or stress condition. We speculated that the down-regulation of GhBASS5 would weaken the effect of salt stress on the plastid function, and promote cotton survival under salt stress. Silencing GhBASS5 in cotton by VIGS technique improved cotton salt tolerance ability significantly, and reduced the sodium content in the leaves. Next, we will confirm the sodium:keto acid cotransporting ability for GhBASS5 by the application of patch clamp technique and mutant functional complementation experiment. And identify the tissue expression and subcellular localization characteristics of GhBASS5 through studying GhBASS5 promoter and fluorescent labeling experiment. Furthermore, we will mensurate the physiological and biochemical character of both GhBASS5-silencing and over-expressing materials under salt stress, and confirm the regulation of the Na+ transporter genes and plasmid functional genes in GhBASS5-silencing cotton in respond to salt stress by qRT-PCR. Based on the above results, the negative regulation mechanism of GhBASS5 in cotton salt tolerance will be concluded.

Na依赖型共转运体依赖Na梯度共转运Na和特异底物，盐胁迫下该型转运体的调控不仅影响其转运底物参与的代谢通路，还影响植物对Na的运输，因而对植物的耐盐性有重要影响。本研究通过对比两个耐盐性差异的棉花材料，发现一个棉花耐盐负调控基因GhBASS5，其可能编码一个质体Na依赖型酮酸共转运体。耐盐材料GhBASS5的表达水平显著低于敏盐材料，推测降低其表达将减弱盐胁迫对质体功能的影响，有利于棉花耐盐。VIGS沉默GhBASS5后棉花叶部Na含量降低，耐盐性提高。后续将应用膜片钳技术和突变体互补实验鉴定GhBASS5的钠依赖底物共转功能；通过启动子研究和荧光标记实验确定GhBASS5的组织表达特点及亚细胞定位；测定GhBASS5沉默和超表达材料的耐盐生理生化指标，定量分析GhBASS5沉默后棉花Na转运体基因和质体功能基因的耐盐应答。综合以上结果，阐明GhBASS5负调控棉花耐盐性的分子机理。

调控Na+的吸收和运输是植物耐盐的主要策略之一。Na依赖型胆汁酸共转运体BASS（Bile acid sodium symporters）依赖Na离子共运输底物，但对其在植物耐盐应答过程中功能还未知。本研究通过对比两个耐盐差异棉花根系转录组，发现GhBASS5的表达调控与棉花的耐盐性呈负相关。GhBASS5在棉花根系中高水平表达，其在敏盐棉花材料根系中的表达水平显著高于耐盐材料，并在盐胁迫后显著下调应答。GhBASS5编码了一个质体膜定位的Na+转运蛋白，并在内皮层和木质部特异表达。沉默下调敏盐材料中GhBASS5的表达能显著降低木质部茎流中Na+含量，并增强根系的Na+外排量，使得地上部Na+积累水平显著低于对照，因而可以显著提高棉花的耐盐性。而超表达GhBASS5则造成细胞内Na+含量升高，木质部茎流中Na+含量升高，并导致根系Na+外排量降低，地上部Na+过度积累，进而致使超表达GhBASS5植物非常敏盐。此外，超表达GhBASS5会损害质体的耐盐应答，如光合调控，渗透物合成，过氧化物清除等功能。基于实验结果，本研究发现GhBASS5主要定位于棉花根部内皮层和木质部，介导棉花对Na+的吸收和长距离运输，高水平的GhBASS5表达会导致棉花敏盐。本研究为下一步应用GhBASS5的耐盐应答机制改良棉花耐盐性奠定了基础。