油茶CoSAD基因载体的构建、鉴定及功能分析

摘要

为揭示油茶( Camellia oleifera Abel)硬脂酰-ACP脱饱和酶( SAD)基因(即CoSAD基因)的功能,构建了该基因的原核表达载体pET28b-CoSAD、植物表达载体pBI121-CoSAD和RNA干扰载体pBI121-CoSAD RNAi,并采用PCR扩增及双酶切方法对3类载体进行鉴定；在此基础上,对原核表达载体中的CoSAD基因进行诱导表达分析,并对pBI121-CoSAD转化的拟南芥〔Arabidopsis thaliana ( Linn.) Heynh.〕sad突变体植株和pBI121-CoSAD RNAi转化的拟南芥野生型植株进行转基因鉴定和主要脂肪酸成分含量分析。 PCR扩增和双酶切结果显示：从 pET28b-CoSAD、pBI121-CoSAD和pBI121-CoSAD RNAi 载体的阳性克隆中均可获得目的条带,表明这3类载体均构建成功；用1 mmol·L-1 IPTG分别诱导0.5、1.0、2.0、3.0、4.0和5.0 h,CoSAD基因均能够在pET28b-CoSAD转化的大肠杆菌BL21感受态细胞中正常表达,能够获得与预测结果相符的相对分子质量约47000的特异目的蛋白条带,且蛋白活性随诱导时间的延长而升高。从pBI121-CoSAD转化的拟南芥突变体植株和pBI121-CoSAD RNAi转化的拟南芥野生型植株中也均可扩增出目的条带。 GC-MS分析结果显示：与拟南芥野生型植株相比,其突变体植株的硬脂酸和棕榈酸含量较高、油酸和棕榈油酸含量较低；但突变体植株经pBI121-CoSAD转化后,硬脂酸和棕榈酸含量降低而油酸和棕榈油酸含量提高；野生型植株经过pBI121-CoSAD RNAi转化后,硬脂酸和棕榈酸含量提高、油酸和棕榈油酸含量降低,表明pBI121-CoSAD转化能够促进拟南芥sad突变体植株体内饱和脂肪酸向不饱和脂肪酸转化,而pBI121-CoSAD RNAi转化对拟南芥SAD基因的表达有明显的抑制作用,这2种重组质粒均可影响拟南芥植株的脂肪酸含量。研究结果表明：油茶CoSAD基因具有调控饱和脂肪酸(硬脂酸和棕榈酸)向不饱和脂肪酸(油酸和棕榈油酸)转化的功能,对茶油的脂肪酸组成具有关键的调控作用。%In order to reveal the function of stearoyl-ACP desaturase ( SAD) gene from Camellia oleifera Abel, that is CoSAD gene, its prokaryotic expression vector pET28b-CoSAD, plant expression vector pBI121-CoSAD and RNA interference vector pBI121-CoSAD RNAi were constructed, and three types of vectors were identified by PCR amplification and double enzyme digestion methods. On this basis, induced expression analysis on CoSAD gene in prokaryotic expression vector was carried out, transgenic identification and analysis on main fatty acid composition contents in sad mutant plant of Arabidopsis thaliana ( Linn.) Heynh. transformed by pBI121-CoSAD and its wild type plant transformed by pBI121-CoSAD RNAi were also carried out. Results of PCR amplification and double enzyme digestion show that target bands all can be obtained from positive clones of pET28b-CoSAD, pBI121-CoSAD and pBI121-CoSAD RNAi vectors, meaning that these three types of vectors all are constructed successfully. Using1 mmol·L-1 IPTG induced for 0. 5, 1. 0, 2. 0, 3. 0, 4. 0 and 5. 0 h, respectively, CoSAD gene can express normally in E. coli BL21 competent cell transformed by pET28b-CoSAD, and can obtain a specific target protein band with relative molecular mass about 47 000 , which is consistent with predicted value, and the protein activity increases with prolonging of induction time. Also, target bands all can be amplified from mutant plant of A. thaliana transformed by pBI121-CoSAD and its wild type plant transformed by pBI121-CoSAD RNAi. GC-MS analysis result shows that compared with wild type plant of A. thaliana, contents of stearic acid and palmitic acid in mutant plant are higher, those of oleic acid and palmitoteic acid are lower. But, after transformed by pBI121-CoSAD, contents of stearic acid and palmitic acid in mutant plant decrease while those of oleic acid and palmitoteic acid increase. After transformed by pBI121-CoSAD RNAi, contents of stearic acid and palmitic acid in wild type plant of A. thaliana increase while those of oleic acid and palmitoteic acid decrease, meaning that transformation of pBI121-CoSAD can promote the conversion of saturated fatty acids to unsaturated fatty acids in sad mutant plant of A. thaliana, while transformation of pBI121-CoSAD RNAi has an obviously inhibition to expression of SAD gene from A. thaliana, these two recombinant plasmids both can influent fatty acid content in A. thaliana plant. It is suggested that CoSAD gene has the function in regulating saturated fatty acids of stearic acid and palmitic acid to transform into unsaturated fatty acids of oleic acid and palmitoteic acid, and plays a key regulatory role in fatty acid composition of tea oil.