大豆二酰甘油酰基转移酶基因GmDGAT1A启动子的克隆与功能分析

Cloning and Functional Analysis of Promoter of Diacylglycerol Acyltransferase Gene GmDGAT1A in Soybean

二酰甘油酰基转移酶(DGAT)是催化三酰甘油生物合成的关键酶,在三酰甘油的合成和积累过程中具有重要调控作用。为了研究大豆DGAT基因表达调控的分子机制,以大豆品种科丰1号为材料,通过PCR方法对GmDGAT1A的启动子(promoter-GmDGAT1A,pGmDGATIA)进行克隆,并通过转化拟南芥和GUS组织定位研究其功能。结果表明:以大豆叶片DNA为模板,成功克隆到GmDGAT1A基因ATG上游2192 bp启动子序列。序列分析表明,pGmDGAT1A除具有启动子所必需的TATA-box和CAAT-box等基本顺式作用元件外,还含有多个响应于光、赤霉素和脱落酸等顺式作用元件。以GUS为报告基因,成功构建了植物表达载体pCAMBIA1381Z-pGmDGAT1A,并转化野生型拟南芥获得转基因植株。对转基因拟南芥植株进行PCR检测,能扩增到2192 bp目标条带,表明已获得含有pGmDGAT1A的转基因拟南芥阳性植株。GUS组织化学染色结果显示,转基因拟南芥幼苗的叶脉和根染色较深,但是主根和侧根的根尖部分未染色;成熟期转基因拟南芥植株的根、叶脉以及角果内的隔膜和珠柄染色较深,茎和发育的种子未染色,表明pGmDGAT1A驱动的GUS主要在转基因拟南芥的根、叶脉以及角果内的隔膜和珠柄中表达。综上,克隆的大豆GmDGAT1A启动子具有活性,能够驱动下游目标基因的表达,有望应用于转基因育种。

Diacylglycerol acyltransferase(DGAT)is a key enzyme in the biosynthesis of triacylglycerol,which plays an important role in the synthesis and accumulation of triacylglycerol.In order to study the molecular mechanism of DGAT gene expression regulation in soybean,the promoter of GmDGAT1A(promoter-GmDGAT1A,pGmDGATIA)was cloned from the variety Kefeng 1 by using PCR amplification,and its function was analyzed by transforming Arabidopsis thaliana and GUS tissue localization.The results showed that:With leaf DNA as template,the 2192 bp promoter fragment of GmDGAT1A was successfully cloned from the variety Kefeng 1.Sequence analysis showed that pGmDGAT1A not only had the basic cis-acting elements such as TATA-box and CAAT-box,but also contained a number of cis-acting elements responsive to light,gibberellin and abscisic acid.The plant expression vector pCAMBIA1381Z-pGmDGAT1A was successfully constructed using GUS as a reporter gene and transformed into wild type Arabidopsis thaliana to obtain transgenic plants.The 2192 bp band could be amplified from the transgenic Arabidopsis thaliana plants by PCR,indicating that a transgenic line containing pGmDGAT1A was obtained.GUS histochemical staining of transgenic Arabidopsis thaliana seedlings showed that the veins and roots were deeply stained,but the root tips of main roots and lateral roots were not stained.For mature Arabidopsis thaliana plants,enhanced GUS staining was observed in roots,veins and diaphragms and funiculi of siliques,while the stems and seeds were not stained.These suggested that pGmDGAT1A-driven GUS was mainly expressed in roots,veins and diaphragms and funiculi of siliques.In conclusion,the cloned promoter of GmDGAT1A has the activity,can drive the expression of downstream target gene,and may be used in transgenic breeding.