Sl-miR482在番茄果实中的表达分析及STTM沉默载体的构建

doi:10.13560/j.cnki.biotech.bull.1985.2019-0566

摘要/Abstract

摘要： 研究番茄Sl-miR482在果实成熟阶段特殊的表达模式,对其功能进行生物信息学分析并构建STTM沉默载体,为深入阐明Sl-miR482在番茄果实成熟软化中的作用机制奠定基础。采用定量PCR进行基因表达检测;利用启动子分析软件和靶基因预测网站进行生物信息学分析;利用PCR扩增及酶切、连接反应进行STTM载体构建,并转化农杆菌。Sl-miR482在开花期表达量逐渐上升,到未成熟绿期表达量达到最高峰,随后逐渐下降。进一步生物信息学分析结果表明,Sl-miR482启动子中存在乙烯应答元件等。靶基因预测网站结果显示,番茄果胶裂解酶Sl-PL13为Sl-miR482的靶基因;基因表达分析结果表明,Sl-PL13与Sl-miR482表达量在果实成熟不同阶段呈现此消彼长的关系。利用PCR扩增及酶切连接方法,成功构建STTM482沉默载体并转化农杆菌。Sl-miR482在果实成熟软化过程中发挥重要作用。

关键词: Sl-miR482, STTM, 果实成熟与软化, 调控机制, 载体构建

Abstract: The aim of this study is to investigate the specific expression pattern of tomato gene Sl-miR482 in fruit ripening stage,and to conduct bioinformatics analysis of its function and to construct STTM silencing vector. Quantitative PCR was used for gene expression detection,promoter analysis software and target gene prediction website was to perform bioinformatics analysis,PCR amplification,restriction enzyme digestion and ligation reaction were applied to construct STTM vector,and Agrobacterium was transformed. The expression level of Sl-miR482 increased gradually during flowering,and reached the peak in the immature green stage,and then gradually decreased. Further bioinformatics analysis showed that ethylene response elements were present in the Sl-miR482 promoter. The results of target gene prediction website showed that tomato pectin lyase Sl-PL13 was the target gene of Sl-miR482. Gene expression analysis showed that the expression levels of Sl-PL13 and Sl-miR482 demonstrated a trade-off relationship at different stages of fruit ripening. In sum,the STTM482 silencing vector is successfully constructed and transformed into Agrobacterium by PCR amplification and restriction enzyme ligation. The results of this study indicate that Sl-miR482 plays an important role in fruit ripening and softening.

Key words: Sl-miR482, STTM, fruit maturation and softening, regulatory mechanism, vector construction

莫显兰, 史列琴, 陆秋利, 王小敏, 任振新. Sl-miR482在番茄果实中的表达分析及STTM沉默载体的构建[J]. 生物技术通报, 2019, 35(12): 50-56.

MO Xian-lan, SHI Lie-qin, LU Qiu-li, WANG Xiao-min, REN Zhen-xin. Expression Analysis of Sl-miR482 in Tomato Fruit and the Construction of STTM Silencing Vector[J]. Biotechnology Bulletin, 2019, 35(12): 50-56.

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