Construction, Expression And Catalytic Activity Identification Of 4CL-STS Fusion Gene And Its Overexpression In Ziziphus Jujube Mill

Zyziphus jujube Mill.is an ancient fruiter which originates in China. It has become the highest yield of fruiter in China. The fungal diseases have seriously affected the yield and quality of fruit. Resveratrol can inhibit fungal diseases because it is phytoalexin. It exerts anti-carcinogenic, cardiovascular protection and anti-oxidative efferts on the human body. The key gene of resveratrol biosynthesis pathway was integrated into the Hupin jujube using the basic principle and technology of plant genetic engineering, which not only could improve antagonistic fungal diseases, but also may improve the quality of jujube fruit.More rate-limiting steps exist in the metabolic pathway of resveratrol. Overexpression of a key enzyme may increase the accumulation of resveratrol.But the high level accumulation of resveratrol is very difficult because of the limitation of other steps. In order to increase the content of resveratrol in transgenic plants, multiple genes co-transformation is required, and constructing fusion gene is preferred. Fusion protein can greatly improve the catalytic efficiency due to the "proximity effect". The construction of fusion gene and its genetic transformation are expected to greatly improve the content of resveratrol in transgenic jujube.Resveratrol is the product of stilbene synthase (STS), the different STS have difference catalytic efficiency.4-coumaroyl-CoA ligase (4CL) and STS are rate-limiting enzymes in resveratrol biosynthesis pathway. Firstly, two different STS from Vitis vinifera and Polygonum cuspidatum Sieb. et Zucc were investigated. Then, five different length flexible peptides were used as linkers to construct 4CL-linker-STS fusion enzymes. The enzyme kinetics was analysed to determine the best molecular design. Meanwhile, the ability of synthesis of resveratrol was determined through prokaryotic expression system. The plant expression vector was constructed using the highest activity of the fusion enzyme gene. The recombinant plasmid was integrated into jujube, and overexpressed in transgenic plants. The level of accumulation of resveratrol and the ability of the fungal antagonist of transgenic plants were determined. The main results and conclusions are as follows.1. Vitis vinifera stilbene synthase gene (VvSTS) was amplified according to overlap PCR protocol with genomic DNA as template.VvSTS and PcSTS were analyzed through heterologous expression in Escherichia coli. The expression products were purified with Ni-NTA sepharose affinity chromatography and desalted through PD-10 column. The molecular weight of the two fusion proteins were about 43 kDa. Enzyme reaction and product analysis showed that both products were all resveratrol. The enzyme kinetic analysis showed that the catalyze efficiency (Kcat/Km) of PcPKS5 was 2.4 times of the VvSTS. Our findings confirm that STS from certain plants has much higher catalytic capability. The possible causes were analyzed from the active sites and conserved sites of the type III polyketide synthases.2. The flexible peptides (GSG)1, (GSG)2, (GSG)3, (GSG)4, (GSG)5 were used as linkers to construct fusion genes:4CL-xa-STS (x=3,6,9,12,15. a represents amino acid residues). The fusion genes were subcloned into prokaryotic expression vector pET30a(+), then recombinant vectors pET30a-4CL-xa-STS were introduced into Escherichia coli expression strain Rosetta. The expression products were purified with Ni-NTA sepharose affinity chromatography and desalted through PD-10 column. The molecular weight of the five fusion proteins were about 104 kDa. Enzyme reaction and product analysis showed that the five fusion enzymes constructed with different linkers displayed the activities of both 4-coumaroyl-CoA ligase and stilbene synthase. The five products were all resveratrol. The enzyme kinetic analysis showed that the catalyze efficiency (Kcat/Km) of 4CL-3a-STS was the highest. The catalytic efficiency of the fusion proteins decreased gradually with increasing linker length. Homology model analysis of the fusion protein showed that no strong polar interactions such as hydrogen bonds or salt bridges existed between the 4CL and STS domains. Physical distance between active sites may be the only factor affecting fusion protein activity. A longer linker would increase the physical distance between both enzyme active sites which may in turn decrease the activity of the fusion protein.3. The plant expression vector was constructed using 4CL-3a-STS. The overexpression of 4CL-3a-STS in Hupin jujube was studied by gene transformation via Agrobacterium.Five transgenic plants were obtained after antibiotic selection and molecular identification. PCR, PCR-Southern and Northern analyses of transgenic plants showed that the fusion gene 4CL-3a-STS was integrated into the Hupin jujube genome and expressed at the transcriptional level. The content of resveratrol in transgenic plants increased significantly. The average content was 2.07 μg/g fresh weight. Antibacterial activity determination showed that the extracts of transgenic plants could obviously inhibit the comom fungal diseases Isariopsis imdica and Alternaria alternate.