基于盐逆境下栽培和野生大豆CLCs同源基因的发掘和功能解析

So far, the study on physiological and molecular mechanisms of plant salt tolerance mostly focus on Na+ toxicity and adaptation, but anionic toxicity of plant salt injury is mainly caused by Cl-, and Cl- toxicity has been yet ignored by most researchers. The effect of Cl- stress on the cultivated soybean is greater than that of Na+ stress, while wild soybean shows a better resistance to Cl- stress. As a kind of anion channels or transporters, the chloride channel proteins (CLCs), which are closely related to the absorption, transportation and molecular regulation of Cl- in soybean plants, such as GmCLC1, are involved in regulation of Cl- homeostasis and chloride (salt) tolerance under salt stress. This project will focus on the physiological response difference of chloride (salt) injury on cultivated and wild soybeans, and whole soybean genome discovery of CLC homologous genes. By adopting multiple analysis tools of molecular biology, such as CLCs gene cloning, subcellular co-localization of CLCs protein with proteins encoded by organelles marker genes, yeast mutant complementary test, soybean plant genetic transformation (including transgenic hairy roots mediated by Agrobacterium rhizogenes, cotyledonary node tissue transformation mediated by Agrobacterium tumefaciens) and virus induced gene silence (VIGS), the key CLCs homologous genes and their special physiological functions related to the chloride (salt) toxicity difference between cultivated and wild soybeans will be revealed, and we hope to provide the important scientific theory basis for future utilization of the key CLCs homologous genes on chloride (salt) resistance improvement in soybean and other crops.

迄今对植物耐盐生理及分子机制的研究大都集中在Na+毒害和适应方面,但植物盐害的阴离子毒害主要由Cl-造成,一直为多数研究学者所忽视。栽培大豆的Cl-胁迫大于Na+,而盐生野大豆对Cl-表现出颇强的抗性。与大豆植株体内Cl-吸收、转运及分子调控密切相关的氯离子通道蛋白(CLCs)做为阴离子通道或转运蛋白,如GmCLC1,已表明参与了盐逆境下植株体内Cl-稳态和耐氯(盐)性调控。本项目重点关注栽培和野生大豆氯(盐)害的生理响应差异和CLCs同源基因的发掘,通过基因克隆、细胞器标记基因编码蛋白亚细胞共定位、酵母(突变体)互补、大豆转基因(发根农杆菌侵染、根癌农杆菌子叶节诱导)和病毒诱导的基因沉默（VIGS）技术等多种分子生物学手段,解析和揭示引起栽培和野生大豆耐氯(盐)性差异的关键CLCs同源基因及其生理功能,以期为今后用于改良大豆或其它作物的耐氯(盐)性提供重要的科学依据。

围绕NaCl胁迫下作物Cl-毒害机制这一重要科学问题，我们前期研究表明:野生大豆较栽培大豆耐Cl-/盐性强。因此，盐胁迫下栽培和野生大豆Cl-敏感性差异的分子机制非常值得研究。本研究从栽培和滩涂野生大豆材料中克隆出包括已报道GmCLC1的8个CLCs同源基因成员。通过定量PCR、大豆发根组合植株转化和酵母突变体互补等技术，筛选出可能参与栽培和野生大豆耐Cl-/盐性差异调控的CLC-c2、CLC-b1等关键成员，开展了基因RNAi与过表达大豆发根组合植株耐Cl-/盐性、膜片钳电生理以及转化拟南芥突变体等多种分析试验，研究了它们对大豆耐Cl-/盐性差异调控的影响机制。结果表明：大豆CLCs基因家族中，仅少数成员，如CLC-c2核苷酸和编码蛋白氨基酸序列在供试栽培和野生大豆间存在细微差异，但它们（还包括CLC-c1）受NaCl诱导上调的表达水平在野生大豆根中更明显。盐胁迫下，将大豆CLCs基因导入酵母突变体中，CLC-c2，CLC-d1和GmCLC1等表现互补和生长恢复效应；除过表达CLC-d1大豆发根组合植株盐害较对照加重外，过表达其它CLCs成员植株的生长有所改善，其中CLC-c2或CLC-b1较明显；除CLC-d1/2增强外，GmCLC1、CLC-b1/2、CLC-c1/2及CLC-g RNAi植株耐盐性降低。GsCLC-b1和GsCLC-c2基因编码蛋白都具有电压依赖性Cl-通道属性。与GmCLC1相比，GsCLC-c2基因编码蛋白的离子通道活性较强，且对Cl-选择性大于NO3-，该基因过表达大豆发根组合植株调控Cl−在体内吸收和降低向地上部转运及增强耐盐性的效应明显强于GmCLC1。转GsCLC-b1, GsCLC-c2等拟南芥突变体植株盐害明显减轻。这主要与维持盐胁迫下植株地上部较高相对含水量和抗氧化酶活性，以及较低Cl-、Na+含量及Cl-/NO3-及Na+/K+值有关。可初步表明：造成栽培和野生大豆耐Cl-/盐性差异与其CLCs家族少数成员间存在关联性，比如GsCLC-b1和GsCLC-c2，尤其是后者，可能是关键成员。这为今后深入研究栽培和野生大豆耐Cl-/盐性差异的分子调控网络机制和寻求提高大豆、棉花、水稻等作物耐Cl-/盐性的分子遗传改良或新种质培育途径，提供了重要科学依据。