甘蓝型油菜BnLEC1.A07基因启动子在不同含油量品系中的克隆与活性分析

Cloning and activity of BnLEC1.A07 gene promoter in rapeseed(Brassica napus L.)lines with different oil content

为了进一步研究BnLEC1基因对油脂生物合成的影响,以高油品系61616和低油品系51070为材料,进行了转录组和定量PCR分析。结果表明,BnLEC1.A07基因在61616品系中的表达水平明显高于51070品系。对启动子P_(LEC1)61616(1829 bp)和P_(LEC1)51070(1824 bp)进行了克隆和序列比较。结果表明,两个启动子之间存在大量的SNP和InDel,其中以单碱基SNP最为丰富。顺式作用元件分析表明,P_(LEC1)61616比P_(LEC1)51070多1个CAAT-box和2个茉莉酸反应元件,缺少1个细胞周期调控元件和2个非生物胁迫反应元件。随后我们构建了包含该启动子的GUS融合载体和LUC融合载体。烟草叶片瞬时转化后的GUS染色、LUC荧光显像和荧光素酶活性分析显示,P_(LEC1)61616的活性高于P_(LEC1)51070。综合结果表明,BnLEC1.A07基因在不同品系中的表达水平与其含油量呈正相关,P_(LEC1)61616比P_(LEC1)51070具有更强的启动子活性。本研究为进一步探讨脂肪合成基因BnLEC1.A07在不同油菜品系中的调控机制提供了理论依据。

In order to further study the gene BnLEC1 on oil biosynthesis,high-oil variety 61616 and low-oil variety 51070 were used as materials to carry out transcriptome and quantitative PCR analysis.The results showed that the expression level of BnLEC1.A07 in 61616 was significantly higher than that in 51070.Cloning and sequence comparison of the promoter P_(LEC1)61616(1829 bp)and P_(LEC1)51070(1824 bp)were performed respectively.The results showed that a large number of SNPs and InDels between the two promoters,and single-base SNPs were abundant.Cis-acting elements analysis showed that P_(LEC1)61616 had one CAAT-box and two jasmonic acid response elements more than those of P_(LEC1)51070,and one cell cycle control element and two abiotic stress response elements were missing.The promoter fusion vectors with GUS and LUC were constructed respective.GUS staining,LUC fluorescence imaging in vivo after transient transformation of tobacco leaves and luciferase activity assay showed that the activity of P_(LEC1)61616 was higher than that of P_(LEC1)51070.The comprehensive results showed that the expression level of BnLEC1.A07 gene in different lines was positively correlated with oil content.P_(LEC1)61616 had stronger priming activity than P_(LEC1)51070.This study provides a theoretical basis for further exploring the regulation mechanism of the lipid synthesis gene BnLEC1.A07 in different rapeseed lines.