花生二酰基甘油酰基转移酶基因克隆与功能研究
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山东省农业科学院青年基金(2007YQN008);山东省自然科学基金(ZR2009DM013)


Cloning and Functional Analysis of Diacylglycerol Acyltransferase Gene in Arachis hypogaea
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    摘要:

    利用RT-PCR方法,从花生品种‘鲁花14’未成熟种子中克隆了二酰基甘油酰基转移酶(Diacylglycerol acyltransferase,DGAT)基因AhDGAT3A和AhDGAT3B的cDNA,序列分析显示,两者在编码区核苷酸序列有96%相似性,氨基酸序列有94%相似,并对其中的AhDGAT3A进一步研究。(1)半定量RT-PCR分析显示,AhDGAT3A在花生根、茎、叶、花和种子中均有表达,且花中表达量最高,茎中表达量非常低;在花生果针入土60 d时种子中的表达量较其它发育时期有所提高。(2)利用染色体步移技术克隆了AhDGAT3A 5′上游1 588 bp调控区,在线软件分析发现该调控区除包括启动子核心元件外,还包含多个调控花粉中表达的顺式元件、光信号调控元件和胁迫相关元件等。(3)在烟草中过量表达AhDGAT3A基因,转基因烟草种子粗脂肪和主要脂肪酸含量较对照均有所下降,推测这一结果可能由转基因共抑制作用导致。

    Abstract:

    Two types of cDNA (named AhDGAT3A and AhDGAT3B) encoding diacylglycerol acyltransferase were isolated from the developing seeds of Arachis hypogaea Luhua 14 by RT-PCR.The similarities of the nucleotide and putative amino acid sequences of the two genes were 96% and 94%,and further analyses of AhDGAT3A were performed from different aspects:(1)By semi-quantitative RT-PCR analysis,it was showed that the transcription of AhDGAT3A mRNA was detectable in all organs analyzed,and the expression level of this gene was the highest in flower,and the lowest in stem;in seed,the expression level of this gene at the developmental stage of 60 d after pegging was higher than that at the other developing stages.(2)Using the method of chromosome walking,a 1 588 bp length of DNA fragment from 5′ upstream regulation region of AhDGAT3A was cloned,and the cis-acting elements were analyzed with the online software.It was found that in the promoter region,additionally,the core elements,there were several other pupative regulation elements which involved in the expression in pollens,the responsiveness of light and stress.(3)The investigation of the crude lipid content and the main fatty acids content was found that,due to the influence of homologous co-suppression,the seeds in all transgenic tobacco lines overexpressing AhDGAT3A contain less crude lipid and main fatty acids than those in controls.

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唐桂英,柳展基 ,徐平丽 ,等.花生二酰基甘油酰基转移酶基因克隆与功能研究[J].西北植物学报,2013,33(5):857-863

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  • 在线发布日期: 2013-06-05
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