异子蓬SabHLH169基因的克隆及抗旱功能分析

Cloning of Basic Helix-loop-helix(bHLH)Transcription Factor Gene SabHLH169 in Suaeda aralocaspica and Analysis of Its Resistances to Drought Stress

碱性螺旋-环-螺旋(basic belix-loop-helix,bHLH)转录因子是真核生物中最大的转录因子家族之一,其成员广泛参与植物生长、发育和胁迫响应。前期在异子蓬(Suaeda aralocaspica)光合作用关键酶PEPC-1的研究中筛选到一个可能与SaPEPC-1启动子相互作用的bHLH转录因子SabHLH169,探明该基因功能将为后期异子蓬bHLH基因的深入研究奠定基础。通过克隆获得该基因,采用生物信息学、实时荧光定量PCR等方法,初步探究SabHLH169基因的耐旱功能。结果显示,SabHLH169包含2 100 bp核苷酸,编码699个氨基酸,其蛋白质C末端具有典型的bHLH结构域,原核表达和Western blot分析表明,SabHLH169重组蛋白能够正确表达,其分子量大小与预测结果一致。过表达SabHLH169的拟南芥转基因株系在萌发期具有更高的耐旱性;干旱胁迫下,转基因拟南芥中3个干旱胁迫相关基因AtRD22、AtRD29A和AtDREB2A以及3个光合作用关键酶基因AtLhcb2、AtRubicso和AtPEPC的表达水平显著升高,且AtPEPC酶活性被显著增强。由此推测,SabHLH169基因可能在干旱胁迫响应中具有潜在功能。

The basic helix-loop-helix(bHLH)transcription factor(TF)is one of the largest TF families in eukaryotes,its members are widely involved in plant growth,development and response to stress.In our previous work of studying photosynthesis key enzyme PEPC-1 in Suaeda aralocaspica,a bHLH TF(SabHLH169)potentially interacting with the SaPEPC-1 promoter was screened.In the present study,the biological characteristics and preliminary function of this gene was explored,which may provide evidence for further studies of bHLH related genes in S.aralocaspica.The gene was obtained by homogous cloning,and the drought-resistant properties of SabHLH169 gene were characterized by bioinformatics and qRT-PCR analyses.The results showed that the open reading frame(ORF)of SabHLH169 contained 2100 bp nucleotides,encoding an protein containing 699 amino acids.The protein had a typical bHLH structural domain at the C-terminus.The prokaryotic expression and Western blot of the recombinant protein showed that the protein was expressed correctly,and the size was consistent with the predicted result.Arabidopsis thaliana transgenic lines of SabHLH169 presented higher drought tolerance at germination stage.Under drought stress,three drought-responsive genes(AtRD22,AtRD29A and AtDREB2A)and three photosynthesis key enzyme genes(AtLhcb2,AtRubicso and AtPEPC)were up-regulated in transgenic A.thaliana.Meanwhile,the PEPC enzyme activity was significantly enhanced.It is speculated that SabHLH169 gene may be involved in response to drought stress.