蒺藜苜蓿MtbHLH148转录因子的克隆与转化及其功能分析
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引用本文:王菊萍,王 珍,张铁军,龙瑞才,杨青川,康俊梅.蒺藜苜蓿MtbHLH148转录因子的克隆与转化及其功能分析[J].西北植物学报,2019,39(6):963~973
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王菊萍,王 珍,张铁军,龙瑞才,杨青川,康俊梅* (中国农业科学院 北京畜牧兽医研究所北京 100193) 
基金项目:中国农业科学院科技创新工程(ASTIP IAS14);
中文摘要:bHLH(Basic helix loop helix, bHLH)转录因子家族是植物最大的转录因子家族之一,广泛参与植物生长发育和盐胁迫应答机制。该研究利用同源克隆的方法克隆蒺藜苜蓿(Medicago truncatula)的MtbHLH148基因,采用qRT PCR方法分析MtbHLH148基因在蒺藜苜蓿中的表达特性,构建超表达载体并通过农杆菌侵染法转化拟南芥(Arabidopsis thaliana),对转基因拟南芥的耐盐性相关功能进行分析研究。结果显示:(1)从蒺藜苜蓿中获得MtbHLH148基因,该基因cDNA全长1 343 bp,包含开放阅读框为603 bp,编码 201 个氨基酸,蛋白分子量22.7 kD,等电点为11.76;蛋白结构分析显示,该蛋白无跨膜结构域,无信号肽,为亲水性蛋白;含有精氨酸/赖氨酸残基的保守结构域和典型的bHLH结构域;二级结构以α 螺旋和无规则卷曲为主。(2)亚细胞定位表明,MtbHLH148蛋白定位在细胞核。(3)进化树分析表明,MtbHLH148与大豆(Glycine max)的亲缘性最近;启动子分析发现,该基因启动子区域含有光响应元件、MYB结合位点以及ABA应答元件ABRE,可能参与非生物胁迫。(4)qRT PCR分析发现,MtbHLH148基因在蒺藜苜蓿的茎中表达量最高,叶中表达量最低,且MtbHLH148基因受ABA(100 μmol/L)诱导并在盐胁迫(200 mmol/L NaCl)处理8 h内表达量上调,而在低温(4 ℃)处理时表达量明显下调。(5)成功构建超表达载体pCAMBIA3301 MtbHLH148并转化拟南芥获得16个抗性株系,经鉴定有12个过表达株系,其中表达量最高的转基因株系为OE8;对OE8株系耐盐性功能分析发现,转基因拟南芥植株的发芽率明显高于野生型,盐胁迫下转基因拟南芥的根长是野生型的1.5倍,表明其耐盐性得到了增强。研究表明,MtbHLH148基因可能在盐胁迫调节机制中具有一定的调控作用。
中文关键词:蒺藜苜蓿  转录因子  盐胁迫
 
Cloning and Analysis of a Basic Helix loop helix (bHLH) Transcription Factor MtbHLH148 from Medicago truncatula L.
Abstract:The bHLH transcription factor family, one of the largest transcription factor families in plants, has been reported to involve in plant growth and salt stress response. In this study, homologous cloning was used to acquire the MtbHLH148 gene from barrel clover (Medicago truncatula). qRT PCR was used to analyze the expression pattern of MtbHLH148 gene in barrel clover. The overexpression vector was constructed and transformed into Arabidopsis through agrobacterium infection, so as to analyze the related functions of salt tolerance of transgenic Arabidopsis. The result shows: (1) the full length of MtbHLH148 cDNA was 1 343 bp, Which contained 603 bp open reading frame and encoding 201 amino acids. The theoretical pI of MtbHLH148 protein was 11.76 and whoes molecular weight was 22.7 kD. Protein structure analysis showed that the protein had no transmembrane domain, no signal peptide and it was a hydrophilic protein. This gene contained highly conserved bHLH domains. As expected, the secondary structure was predominatly α helix and random curl. (2) Subcellular localization results showed that the proteins were locate in the nucleus. (3) Phylogenetic analysis revealed that MtbHLH148 was closely related to Glycine max Analysis of the cis regulatory element demonstrated that the promoters of MtbHLH148 contained light, hormone and stress response elements, suggesting their involvement in the biological processes. (4) qRT PCR analysis of the expression pattern of the MtbHLH148 in barrel clover showed that the highest level in stem and the lowest level in leaf. For treatment, the genes were induced by ABA (100 μmol/L), and were repressed by cold (4 ℃) but up regulated by NaCl (200 mmol/L) within the first 8 h. (5) The pCAMBIA3301 MtbHLH148 overexpression vector was successfully constructed and transformed into Arabidopsis. We obtained 16 resistant lines and identified 12 over expressed lines. Among these over expressed lines, the transgenic line OE8 had the highest expression level. Analysis of salt tolerance function of OE8 showed that a significantly higher germination rate was observed in spite of their indiscernible phenotypic difference from wild type. Statistical analysis showed that the root length of the transgenic seedlings over expressing MtbHLH148 was about 1.5 times of the non transgenic plants, suggesting enhanced salt tolerance. Based on these results, we infer that MtbHLH148 may play a regulatory role in plant response to salinity.
keywords:Medicago truncatula  transcription factors  salt tolerance
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