马铃薯多基因质体转化载体及其在光呼吸支路构建中的应用

Potato Polygenesic Plastid Transformation Carrier and Its Application in the Construction of Photorespiratory Bypass

为了将大肠杆菌的乙醇酸代谢途径引入马铃薯叶绿体构建光呼吸代谢支路,同时规避目标蛋白不能通过叶绿体信号肽定位的风险,研究采用了Cre-Lox P多基因组装、融合蛋白表达、多顺反子表达等技术构建多基因质体转化载体,一次性将目标基因引入叶绿体基因组。研究成功构建了多基因质体转化载体,将编码壮观霉素抗性筛选标记氨基葡糖苷腺苷转移酶aad A、绿色荧光蛋白GFP、大肠杆菌乙醇酸脱氢酶(EcGLC)的3个亚基,乙醛酸聚醛酶(EcGCL)和羟基丙二酸半醛还原酶(Ec TSR)的7个基因添加到受体载体,最终的载体大小为18.1 kb。在构建的过程中,研究发现供体受体质粒的大小比例、Prrn启动子在细菌中的启动基因的表达等问题严重影响重组的效率。以上研究结果为光呼吸的代谢改造研究提供了一个新的思路,并解除了质体转化载体构建中基因数量的限制,对于叶绿体中复杂的代谢途径构建具有借鉴意义。

In order to introduce the glycolic acid metabolism pathway of Escherichia coli into the chloroplast of potato to construct the photorespiration pathway, and meanwhile avoid the risk that the target protein can not be located through the chloroplast signal peptide, polygene plastid transformation vector was constructed by Cre-Lox P polygene assembly, fusion protein expression and polycis trans expression technology in this study, and the target gene was introduced into chloroplast genome at one time. The polygenic plastid transformation carrier was successfully built, which was used to encode the three subunits of aminoglycoside adenyltransferase（aad A）,green fluorescent protein（GFP）, and glycolate dehydrogenase of E. coli（Ec GLC）, and add the seven genes of glyoxylate carboligase（EcGCL） and tartronate semialdehyde reductase（Ec TSR） to acceptor vector, and the final plasmid size was 18.1 kb. In the construction process, we were also found the size ratio of acceptor and donor plastids, and the expression of promoter gene in Prrn promoter in bacteria seriously affect the efficiency of recombination. The above research provided a new idea for the future photorespiratory metabolism study, and relieve the limitation of gene number in plastid transformation vector construction, which was of great significance for the construction of complex metabolic pathways in chloroplasts.