3个紫花苜蓿乙烯应答因子基因的克隆与分析

【目的】克隆3个紫花苜蓿乙烯应答因子基因，分析其在不同条件下的表达特性；构建植物表达载体并转化烟草，对转基因烟草的耐盐性进行初步鉴定。【方法】根据已获得的cDNA序列设计引物，扩增MsERF5、MsERF8和MsERF11的DNA序列，并分析基因结构；利用半定量RT-PCR技术分析其组织表达特异性和胁迫条件下的表达特性；通过农杆菌介导的方法转化烟草，鉴定盐胁迫条件下转基因烟草的表型和生理生化特性，初步验证其功能。【结果】MsERF5、MsERF8和MsERF11都不包含内含子。MsERF5在根、叶和花蕾中的表达量高于茎和花；MsERF8在根和叶中的表达量高于茎、花蕾和花；MsERF11在叶中的表达量最高；3个基因都能被多种非生物胁迫（盐、干旱、铝）和激素（脱落酸、赤霉素、乙烯利、水杨酸和茉莉酸甲酯）诱导，但表达模式不同。在盐浓度为200 mmol?L-1的筛选培养基上只有导入目的基因的愈伤组织能产生不定芽，250 mmol?L-1 NaCl处理再生植株10 d，转基因植株和野生型植株叶片的电导率和可溶性糖含量均呈现上升趋势，但野生型植株叶片的电导率显著高于转基因植株，可溶性糖含量则显著低于转基因植株（P＜0.05）；转基因植株和野生型植株叶片的叶绿素含量均呈现下降趋势，野生型植株叶片叶绿素含量显著低于转基因植株（P＜0.05）；盐胁迫条件下，转化不同基因的再生植株的电导率、叶绿素和可溶性糖含量没有显著差异（P＞0.05）。【结论】3个紫花苜蓿乙烯应答因子基因都不包含内含子，其表达具有组织特异性，都能被多种非生物胁迫和激素诱导，能够使烟草愈伤组织在含盐培养基上形成不定芽并最终产生转基因植株，且转基因植株的耐盐性高于野生型植株。

Cloning and Analysis of Three Ethylene Response Factor Genes in Alfalfa (Medicago sativa L.)

【Objective】 Cloning and characterization of three ethylene response factor genes in alfalfa to provide a foundation for further study of their function.【Method】Three ethylene response factor genes,MsERF5,MsERF8,and MsERF11 were isolated from alfalfa by PCR,and their expression patterns under different treatments and in different tissues were analyzed using semi-quantitative RT-PCR.The expression vectors of these three genes,was constructed,and then were introduced into tobacco by Agrobacterium mediated transformation system and the salt-tolerance of trangenic plants was analyzed.【Result】 MsERF5,MsERF8,and MsERF11 had no intron.The expression patterns of these three genes were significantly different in different tissues,the expression level of MsERF5 in roots,leaves and flower buds was higher than in other tissues.The expression of MsERF8 was higher in roots and leaves,and the expression of MsERF11 in leaves was higher than in other tissues,the expression of MsERF5,MsERF8,and MsERF11 were both induced by salt,drought,Al3+ and diverse hormones.The expression vectors of these three genes were constructed,and they were introduced into tobacco by Agrobacterium mediated transformation system.The calluses transformed MsERF5,MsERF8,and MsERF11 were changed into adventitious bud differ from the non-transformed calluses.The chlorophyll and soluble sugars contents of the transgenic plants were significantly higher than wild-type plants.The electrolyte leakage was significantly lower than wild-type plants.【Conclusion】MsERF5,MsERF8,and MsERF11 had no intron,the expression patterns of these three genes were significantly different in different tissues,and both of them could be induced by abiotic stresses and diverse hormones treatments.All of them could change the calluses into adventitious buds on the saline medium and produce transgenic plants,and the salt-tolerance of transgenic plants was higher than wild-type plants.