作 者 ：郭 鹏,张万筠,马红玉,姜 健*

期 刊 ：西北植物学报 2014年 7期 页码：1371~1376

关键词：miRNAs；Sly-MIR167；克隆；冷胁迫；番茄；

Keywords:miRNAs, Sly-MIR167, clone, cold stress, tomato,

摘 要 ：以番茄为材料,采用Northern杂交技术,分析番茄MIR167(Sly-MIR167)在低温胁迫下的表达模式,以明确Sly-MIR167在冷胁迫下的分子调控机制,为基因工程在改良番茄品种中的实际应用提供依据。结果显示:(1)25 ℃下Sly-MIR167在番茄根 、茎、 花瓣、果实、叶片都有表达,4 ℃低温胁迫下的表达量均增加,表明Sly-MIR167表达受低温诱导。(2)采用农杆菌侵染构建表达载体并转化番茄获得转基因植株,冷胁迫实验结果显示:转基因植株在冷胁迫处理的生长状况明显优于对照;另外,冷胁迫下2个转基因株系(T₂-5和T₂-19)的最大光化学效率、叶绿素含量下降幅度明显低于野生型;脯氨酸含量高于野生型;MDA含量低于野生型,表明Sly-MIR167能够提高番茄对冷胁迫的耐受性。(3)通过miRU在线软件预测Sly-MIR167的靶基因为NF-YA1、NF-YA2,利用RT-PCR技术分析其表达下调,证明它们被MIR167负调控。

Abstract:In this paper,Northern hybridization was used to analyze the Sly-MIR167 expression pattern of tomato under cold stress.The result shows that:(1)Sly-MIR167 constitutively expressed in roots,stems,petals,fruits and leaves of tomato at 25 ℃.However,the expression trends become more greatly at 4 ℃.The result indicated that Sly-MIR167 was induced by low temperature.(2)The 35S∶MIR167 expression vector was constructed and transferred into tomato by Agrobacterium infection.The cold stress experimental results show that the transgenic lines have better growth index than that of control.Moreover,the chlorophyll content and F_v/F_m of two transgenic lines(T₂-5 and T₂-19) decreased significantly lower than that of wild type;more proline content and less MDA content in the transgenic lines than that in the wild type.Sly-MIR167 can obviously improve the cold tolerance of tomato.(3)The expression assay of target gene NF-YA1,NF-YA2 by RT-PCR demonstrated the down-regulation caused by Sly-MIR167.It indicated that NF-YA1 and NF-YA2 were negative regulated.In a word,the research contributes to provide more application prospects in transgenic tomato.