马铃薯SGT3基因表达及其启动子功能分析

doi:10.11686/cyxb20140224

摘要/Abstract

摘要： 鼠李糖转移酶(rhamnosyltransferase SGT3)是植物糖苷生物碱合成的关键酶,它主要调控α-茄碱和α-查茄碱从其β形式的转化。本文研究马铃薯栽培种SGT3基因的表达特点及其启动子的功能。实时定量PCR结果发现在红光照射24 h后,SGT3的表达量是黑暗处理的26.8倍,说明红光显著诱导了SGT3的表达;为进一步分析该基因的光调控机理,本项研究克隆到SGT3上游长度为2449 bp的启动子序列,通过分析发现该启动子的转录起始位点位于翻译起始位点上游-152 bp,同时确定了该启动子核心序列及上游增强子、抑制子序列,受病原菌、损伤、干旱、ABA 激素及一系列光调控的顺式元件。构建不同长度(349,572,979,1312和1870 bp)的该启动子驱动报告基因GUS的植物表达载体并转化烟草。结果证明,不同长度的SGT3启动子都可以启动GUS表达,但没有CMV 35S启动的GUS表达量高;其中在P572和P979的表达强度较高,这可能与该片段含有启动子的正调控元件(GATA BOX,5′UTRPY-RICH STRETCH)有关,GUS表达强度在P1312和P1870中明显减弱,预测到该区段存在抑制基因表达的负调控元件(WRKY710S);SGT3启动子的组织特异性实验表明GUS染色主要集中在烟草叶片的叶脉部分,茎中的表皮、韧皮部及木质部,但髓部几乎不表达,根中主要分布在根冠、分生区以及维管束组织中。上述结果为将来研究SGT3在糖苷生物碱合成过程中的调节功能提供了依据。

Abstract: Rhamnosyltransferase (SGT3) is a key enzyme involved in synthesis of plant glycoalkaloids, and mainly catalyzes the transformation of α-solanine and α-chaconine from their β-form. We studied the SGT3 gene expression and its promoter function in potato cultivars (Solanum tuberosum), and found that after 24 hours red light treatment, the transcriptional expression of SGT3 was increased 26.8 fold compared with control (under dark). The data thus suggested that red light induced the expression of SGT3. To further understand the regulation mechanism of SGT3, the putative SGT3 promoter region (2449 bp upstream of the open reading frame) was cloned and the sequence was analyzed to predict the cis-elements including promoter core sequences, enhancer sequences, inhibiting sequences, pathogen- responsive element, drought- and ABA- responsive element, and a few light-regulated elements. The transcription start site of the promoter is located 152 bp upstream of the translation start site. To study the function of the SGT3 promoter, the GUS expression vector driven by SGT3 or CMV 35S promoter were constructed and transformed to tobacco leaves to analyze the transient expression and stable expression. The results demonstrated that the SGT3 promoter can drive the expression of GUS, but the expression level was less than that driven by the CMV 35S promoter. The highest GUS expression was detected in tobacco leaves transformed by P572 and P979 but the GUS expression was decreased with P1312 and P1870. More positive regulatory elements were observed within 1000 bp upstream of the start code. The tissue specificity analysis indicated that the SGT3 driven GUS expression was mainly concentrated in the veins of the tobacco leaves, but was not detected in mesophyll cells. In the root, GUS was detected in meristem and vascular tissues. In the stem, GUS was detected in epidermis, xylem and phloem of tobacco stems.

中图分类号:

崔同霞,白江平,魏桂民,赵旭,王蒂,张金文. 马铃薯SGT3基因表达及其启动子功能分析[J]. 草业学报, 2014, 23(2): 196-206.

CUI Tong-xia, BAI Jiang-ping, WEI Gui-ming, ZHAO Xu, WANG Di, ZHANG Jin-wen. Red light induced SGT3 gene expression and functional analysis of the SGT3 promoter in potato[J]. Acta Prataculturae Sinica, 2014, 23(2): 196-206.

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