sikFBA4基因启动子的克隆及低温表达模式分析

doi:10.11686/cyxb2017527

摘要/Abstract

摘要： 天山雪莲生长于常年积雪覆盖高海拔处的高山区域,具有很强的极端低温生境适应能力,属于研究植物低温适应的良好模式植物。前期的研究表明,sikFBA4基因可以显著提高番茄的抗寒能力。为进一步研究sikFBA4基因的耐寒响应模式,以天山雪莲为材料,采用实时荧光定量PCR分析sikFBA4在低温胁迫下的表达模式;利用高效热不对称PCR法(Hi-Tail PCR)克隆sikFBA4启动子序列并进行生物信息学分析。为分析PsikFBA4序列克隆的完整性及转录表达特性,将PsikFBA4与GUS基因融合在烟草中进行瞬时表达。结果表明,sikFBA4在低温胁迫条件下的表达发生瞬时显著上调,并在1 h达到峰值,而后表达下调。通过启动子克隆分析,在PsikFBA4序列-1648 bp位置处具有冷响应元件LTRE;进一步的GUS染色和酶活力测定结果表明,低温能够显著提高GUS基因的表达活性,说明sikFBA4属于低温诱导型基因。SikFBA4基因启动子的克隆、序列分析以及表达分析,为进一步探究雪莲果糖-1,6-二磷酸醛缩酶(sikFBAs)基因的表达与调控机制奠定了基础。

关键词: 天山雪莲, sikFBA4, 高效热不对称PCR, 启动子, GUS

Abstract: Saussurea involucrata tolerates severe abiotic stresses, especially cold conditions. Therefore, it is a useful plant for studies on cold adaptability. Previous studies have shown that the cold tolerance of Lycopersicon esculentum was significantly improved by over-expressing sikFBA4. To further analyze sikFBA4, changes in its transcript levels under a 4 ℃ treatment were determined by qRT-PCR. Hi-Tail PCR was used to clone the promoter sequence of sikFBA4 from genomic DNA, and PlantCARE software was used to predict and analyze promoter elements in PsikFBA4. To evaluate the activity and study the expression characteristics of PsikFBA4, we constructed GUS gene reporter constructs for transient expression in Nicotiana tabacum leaves. The results showed that sikFBA4 was strongly induced by low temperature. Its transcript levels peaked at 1 hour of cold treatment before declining slowly. The bioinformatics analysis indicated that a low temperature response element was located at -1648 bp in the gene promoter. The result that GUS activity was regulated by PsikFBA4 in response to a cold treatment confirmed that PsikFBA4 is a cold-inducible promoter. These results provide a foundation for further research on the expression and regulation mechanism of sikFBA genes.

Key words: Saussurea involucrata, sikFBA4, Hi-Tail PCR, promoter, GUS

张尧, 李忠晴, 王爱英, 祝建波. sikFBA4基因启动子的克隆及低温表达模式分析[J]. 草业学报, 2018, 27(12): 199-207.

ZHANG Yao, LI Zhong-qing, WANG Ai-ying, ZHU Jian-bo. Promoter cloning and analysis of low temperature-induced expression of sikFBA4 from Saussurea involucrata[J]. Acta Prataculturae Sinica, 2018, 27(12): 199-207.

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