油菜叶绿体乙酰辅酶A羧化酶单交换表达载体的构建

Costruction of single-cross expression vector for chloroplast acetyl coenzyme A carboxylase in Brassica napus

关键词:  油菜  叶绿体  乙酰辅酶A羧化酶  单交换  表达载体  构建

Key words:  Brassica napus  chloroplast  acetyl coenzyme A carboxylase  single-cross  expression vector  construction

摘要:

根据已知序列设计引物,通过PCR扩增获得质体定位的乙酰辅酶A羧化酶的4个亚基的基因序列。先将该酶4个亚基的基因进行拼接,然后将这4个拼接好的片段,克隆到pMD18-T载体上,得到质粒pHBM714。再以质粒pHBM714 DNA为模板,用分别带有Cpo I和Asc I酶切位点的引物进行PCR扩增,PCR产物在dTTP的保护下经T4 DNA聚合酶处理,与将质粒pHBM720 DNA纯化后经Cpo I和Asc I双酶切后得到的大片段连接,连接产物转化大肠杆菌Xl10-gold,得到正确的重组子命名为pHBM726。此质粒pHBM726,即为带有壮观霉素抗性基因(aadA)筛选标记的质体定位的乙酰辅酶A羧化酶基因油菜叶绿体单交换表达载体; 在此载体中壮观霉素抗性基因(aadA)、乙酰辅酶A羧化酶的4个亚基的基因(ACC)和绿色荧光蛋白基因(gfp)共6个基因串联在一起,共用一个启动子序列,一起来进行表达; 通过酶切检测、PCR验证和测序验证,均表明该表达载体构建成功。最后此载体在大肠杆菌中表达时,发现重组菌能够在含壮观霉素的培养基上生长,且在可见光下,能看到绿色荧光,表明壮观霉素抗性基因和绿色荧光蛋白基因均在大肠杆菌中成功表达; 表达产物通过Western印迹验证表明组成乙酰辅酶A羧化酶的4个亚基的基因在大肠杆菌中成功表达。以上结果表明,该表达载体中串联排列的这6个基因均在大肠杆菌中成功表达。该研究结果可为质体定位的乙酰辅酶A羧化酶转叶绿体的研究奠定基础,为油菜油脂代谢研究提供参考。

Abstract:

This study aimed to construct Brassica napus chloroplast acetyl coenzyme A carboxylase single cross-over expression vector,to lay the foundation for chloroplast transformation research of B. napus acetyl coenzyme A carboxylase,at the same time,to provide a good reference for the research of oilrape lipid metabolism. According to the known sequences form GenBank,the corresponding primers were designed,the gene sequences of four subunits for plastid acetyl coenzyme A carboxylase were amplified by Polymerase chain reaction. The form of acetyl coenzyme A carboxylase of Escherichia coli and the form of acetyl coenzyme A carboxylase of plastid were similar,therefore the gene sequences of four subunits of plastid acetyl coenzyme A carboxylase were spliced according to the form of acetyl coenzyme A carboxylase of Escherichia coli. And the plasmid pHBM714 was obtained by cloned the gene fragments of chloroplast acetyl coenzyme A carboxylase into pMD18-T vector. Then the DNA sequence of plasmid pHBM714 was taken as the templaste,and the front of primers were added to the gene sequence of restriction enzyme sites of Cpo I and the gene sequence of restriction enzyme sites of Asc I respectively. At last,the sequence of chloroplast acetyl coenzyme A carboxylase was amplified by Polymerase chain reaction. These products of Polymerase chain reaction were processed by T4 DNA polymerase in the protection of dTTP,and which was jointed with the larget fragment that was got by the plasmid of pHBM720 DNA digested by restriction enzyme of Cpo I and restriction enzyme of Asc I. The ligation products was transformed into Escherichia coli Xl10-gold. The recombinant that was verified by amplification of Polymerase chain reaction and restriction enzymes digested correctly was named the plasmid of pHBM726. The recombinant plasmid of pHBM726 was the single cross-over expression vector for chloroplast acetyl coenzyme A carboxylase in Brassica napus which was obtained with selection marker of spectinomycin resistance gene(aadA)(-Prrn-SD-aadA-ACC-gfp-psbA3’-RbcL-Ampr+Ori -ACCD-). And it consisted of spectinomycin resistance gene(aadA),biotin carboxylase gene(BC),biotin carboxyl carrier protein gene(BP4),carboxyltransferase beta subunit gene(β-CT),carboxyl transferase alpha subunit gene(α-CT)and green fluorescent protein gene(gfp),furthermore,the six genes that were connected in series,and these genes which were expressed together with a common promoter sequence in Escherichia coli. The plasmid of pHBM726 was constructed successfully that were verified by restriction enzyme digestion,Polymerase chain reaction and sequencing. Finally,the recombinant plasmid of pHBM726 was expressed in Escherichia coli,with the recombinant plasmid pHBM726 of Escherichia coli could grow well on the medium plate containing spectinomycin,and the single colony was able to emit green fluorescence under visible light excitation,which indicated that spectinomycin resistance gene and green fluorescent protein gene were expressed successfully in Escherichia coli; the expression products of four subunits of plastid targeted acetyl coenzyme A carboxylase gene were all detected by Western blotting,it showed that four subunits of plastid targeted acetyl coenzyme A carboxylase gene were expressed successfully in Escherichia coli. All the results showed that four subunits of plastid targeted acetyl coenzyme A carboxylase gene,spectinomycin resistance gene and green fluorescent protein gene which were successfully expressed in Escherichia coli. This study constructed single cross-over expression vector for Brassica napus chloroplast acetyl coenzyme A carboxylase,and will lay the solid foundation for the chloroplast transformation research of Brassica napus.