大豆混合盐碱胁迫应答基因GmDUF247-1的克隆及功能分析

Cloning and Functional Characterization of the GmDUF247-1 Gene in Soybean Response to Saline-alkaline Stress

我国盐碱地分布广、面积大,是重要的后备耕地资源、粮食增产的“潜在粮仓”。挖掘负调控大豆耐混合盐碱性的基因,通过基因敲除创制耐混合盐碱大豆新品种,是合理开发利用盐碱地,提高我国大豆产量的有效途径之一。课题组前期筛选到1个混合盐碱胁迫下调表达的基因Glyma.02g271000(GmDUF247-1)。其编码的GmDUF247-1蛋白包含1个DUF247结构域和1个跨膜结构域,利用烟草叶片瞬时表达发现GmDUF247-1-GFP融合蛋白定位在细胞膜上。荧光定量PCR显示,GmDUF247-1基因在大豆根中表达量最高,在混合盐碱处理下显著下调。为研究GmDUF247-1在大豆混合盐碱胁迫下的功能,利用大豆毛状根系统过表达GmDUF247-1基因,发现混合盐碱胁迫处理后,GmDUF247-1过表达大豆毛状根复合植株叶片萎蔫程度明显高于空载体对照,存活率、根长和株高显著低于对照。对GmDUF247-1基因在大豆自然群体中的单倍型分析发现,其启动子区有8个SNPs和4个InDels,可能导致与逆境应答和生长发育相关的转录因子识别元件序列发生改变;CDS区存在3种单倍型,GmDUF247-1^(H1)基因型受到了明显的人工选择。本研究初步明确了GmDUF247-1基因负调控大豆混合耐盐碱性,为系统研究GmDUF247-1基因功能和育种利用奠定了研究基础。

Saline-alkaline lands,which are found with large area in China,are considered as potential exploitable land resource inagriculture.Breeding of new soybean cultivars showing salt-alkaline tolerance in these regions is a way to improve the soybean production in China.Therefore,identification of the negative regulatory genes to saline-alkaline stress is of interest,and editing of them in elite varieties is able to create new soybean cultivars with salt-alkaline tolerance.Previously,we found that the gene Glyma.02g271000(named as GmDUF247-1)was down-regulated under mixed salt-alkaline stress condition.GmDUF247-1 was annotated with one DUF247(Domain of Unknown Function 247)domain and one transmembrane domain,and the GmDUF247-1-GFP fusion protein was observed on the cell membrane when transiently expressing in tobacco leaves.GmDUF247-1 was detected with the highest transcripts in roots,and its expression was down-regulated under mixed saline-alkaline treatment.Through generating the hairy root composite plants,the transgenic plants overexpressing GmDUF247-1 showed more wilted leaves,lower survival rates,shorter roots and shoots if compared with these lines expressing the empty vector.Based on the haplotype analysis of GmDUF247-1 in soybean population,eight SNPs and four InDels in the promoter region were detected,implying alterations potentially in the binding of growth-and stress-related transcription.Three haplotypes were identified based on the coding sequence variations,and the haplotype GmDUF247-1^(H1) was detected with artificial selection.In summary,we provided the preliminary results on negative regulation of GmDUF247-1 under mixed salt-alkaline stress treatment,which will facilitate to systematically characterize its function and elucidate its potential in breeding new soybean cultivars with superior tolerance to mixed salt-alkaline stress.