ZHAO Huan-huan,WU Xing,ZHANG Feng,et al.Cloning and Plant Expression Vector Construction of AtLACS9 Gene from Arabidopsis Thaliana[J].Soybean Science,2011,30(02):190-193.[doi:10.11861/j.issn.1000-9841.2011.02.0190]
拟南芥AtLACS9基因的克隆及其植物表达载体构建
- Title:
- Cloning and Plant Expression Vector Construction of AtLACS9 Gene from Arabidopsis Thaliana
- 文章编号:
- 1000-9841（2011）02-0190-04
- 关键词:
- 长链脂酰辅酶A合成酶; 基因克隆; 载体构建
- 分类号:
- S565.1
- 文献标志码:
- A
- 摘要:
- 将大豆种子凝集素基因的启动子（lec）从pBI-lec载体上酶切下来，连接到植物表达载体pCAMBIA3301（p3301）的多克隆位点上，然后运用RT-PCR方法扩增了拟南芥AtLACS9基因的编码区cDNA，并将其克隆到改造后的p3301载体的lec启动子之后，为进一步转化大豆、获得油份含量提高的转基因大豆做准备。结果表明：p3301载体改造成功，命名为p3301-lec；AtLACS9基因的编码区cDNA由2076 bp组成，编码含691个氨基酸残基的蛋白，其种子特异性表达载体p3301-lec-AtLACS9构建成功，可进行后续研究。
- Abstract:
- Triacylglycerol (TAG) is the major form of storage lipid in oilseeds, and acyl-coenzyme A (CoA) and glycerol-3-phosphate are precursors for the synthesis of TAG. The long-chain acyl-CoA synthetases (LACSs) catalyze the synthesis of acyl-CoA molecules and therefore influence TAG content in oilseeds. In this paper, the soybean seed-specific lectin promoter(lec), which was digested with restriction enzymes from vector pBI-lec, was inserted into the multiple cloning site of the plant expression vector pCAMBIA 3301(p3301), and then the complete coding region cDNA of Arabidopsis thaliana LACS9(AtLACS9) gene was amplified by reverse transcription PCR（RT-PCR）and subcloned into the reconstructed vector p3301 downstream of the lec promoter. This work aimed at laying a foundation for future transformation of soybean with the gene and then obtaining transgenic soybean with enhanced oil content. The results showed that the vector p3301 was successully reconstructed by inserting the lec promoter and was named p3301-lec; AtLACS9 had an open reading frame of 2076 bp and the corresponding protein consists of 691 amino acids; the seed-specific expression vector of AtLACS9, namely p3301-lec-AtLACS9, was constructed successfully and could be used in the next experiments.
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备注/Memo
基金项目：转基因生物新品种培育科技重大专项资助项目（2009ZX08004-009B）。