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园艺学报 ›› 2015, Vol. 42 ›› Issue (1): 47-55.doi: 10.16420/j.issn.0513-353x.2014-0782

• 蔬菜 • 上一篇    下一篇

番茄LemiR828的克隆表达及其靶基因的鉴定

刘伟伟,于丽丽,方媛,周莹,李洋,周波   

  1. 东北林业大学生命科学学院,哈尔滨 150040
  • 出版日期:2015-01-25 发布日期:2015-01-25
  • 基金资助:

    东北林业大学国家基础科学人才培养基金——科研训练及科研能力提高项目子项目(201220A5);国家自然科学基金项目(30800082和31272200);中央高校基本科研业务费专项资金项目(DL11CA03,2572014EA03-02)

Clone and Expression Analysis of LemiR828 and Identification of Its Target Gene in Tomato

LIU Wei-Wei, YU Li-Li, FANG Yuan, ZHOU Ying, LI Yang, ZHOU Bo   

  1. College of Life Science,Northeast Forestry University,Harbin 150040,China
  • Online:2015-01-25 Published:2015-01-25

摘要: 以番茄(Lycopersicum esculentum)LA1996(Aft基因型,紫色花青素积累品系)cDNA为模板,对LemiR828前体基因和预测到的靶基因LeTAS4(Trans-Acting SiRNA Gene 4)进行克隆,采用RT-PCR技术检测其在番茄果实发育中的差异表达,并用RLM 5′RACE技术验证LemiR828对预测靶基因LeTAS4 mRNA的剪切作用及靶作用位点。结果表明,LemiR828的前体序列含有完整的茎环结构;在果实发育成熟的过程中,LemiR828及LeTAS4在番茄中的表达量存在差异。在绿色果阶段的表达量较低且LemiR828对LeTAS4的负调控关系不明显;而在成熟果阶段,LemiR828及LeTAS4的表达量均升高,且存在明显的负调控关系。LemiR828能够靶作用于LeTAS4的转录本上调控其降解,其剪切位点为LemiR828序列5′端的第10位碱基。因此,在LA1996番茄果实发育过程中,LemiR828和LeTAS4参与果实发育的调控,并且LemiR828负调控其靶基因LeTAS4的表达。

关键词: 番茄, LemiR828, LeTAS4, 花青素合成

Abstract: In order to understand the character of LemiR828 in tomato(Lycopersicum esculentum)LA1996(with anthocyanin fruit,Aft),the precursor sequence and potential target gene of LemiR828 were obtained by PCR using cDNA as template. The expression of LemiR828 and its target gene in tomato fruits were also evaluated by real-time quantitative PCR. LemiR828-guided cleavage and the target site identified on putative Trans-Acting SiRNA Gene 4(LeTAS4)mRNA were validated using RLM 5′RACE. The precursor sequence of LemiR828 was shown to contain a complete hairpin structure. Moreover,the expression of LemiR828 and its target gene LeTAS4 in tomato fruits differed in developmental stages. During green fruit stages,the transcriptional level of LemiR828 and LeTAS4 was low and did not seem to be related to negative regulation;however,in mature fruit stages,their expression was high,and LemiR828 negatively regulated the expression of LeTAS4. The mature LemiR828 targeted a sequence of high complementarity in LeTAS4 mRNA and sheared the 10th position in LeTAS4 mRNA from LemiR828 5? end.Thus,during specific developmental stages in Aft tomato fruits,LemiR828 and LeTAS4 were involved in the regulation of fruit development and the expression of LeTAS4 was negatively regulated by LemiR828,probably regulated anthocyanin synthesis at particular stages.

Key words: tomato, LemiR828, LeTAS4, anthocyanin biosynthesis

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