关键词: 遗传转化, 虎杖芪合酶基因, 枣树, 农杆菌介导法, 白藜芦醇

Abstract: [Objective] Huping jujube is widely cultivated in northern China. A resveratrol synthase (STS) gene, PcPKS5, contains all functionally divergent plant specific type III PKSs and is involved in resveratrol synthesis. The resveratrol synthase genes are expressed in many transgenic crops such as rapeseed and wheat, successfully generating transgenic plants that have enhanced anti-fungal functions.To allow resveratrol accumulation in fruit organs and improve resistance of jujube to fungal pathogens, the PcPKS5 was transformed into Huping jujube under the control of the CaMV 35S promoter, and the obtained transgenic plants were tested if they increased resveratrol accumulation. This study aimed to assess the effects of heterologous overexpression of the resveratrol synthase gene (PcPKS5 ) in Huping jujube plant resistance and nutritional quality. [Method] Stems with leaves and shoot tips of Huping jujube were infected with agrobacterium carrying PcPKS5 and GUS, and three positive plants were identified. [Result] The PcPKS5 gene previously cloned from Polygonum cuspidatum in our laboratory was amplified in the TOP10 bacterial strain and ligated to the pMD 18-T vector. Two primers were designed based on the gene bank sequence EU647245 and synthesized by Sangon Biotech Shanghai Co. Ltd. to clone the STS gene for plant expression plasmid construction.Agrobacterium strain EHA105 harboring the pCAMBIA3301-121 plasmid with the PcPKS5 genes controlled by the cauliflower mosaic virus (CaMV) 35S promoter and termination sequences was used as the vector system for transformation. The infection lasted 15 min, a high percentage of GUS positive leaves was observed. The optimized conditions for transformation were 15 min infection and 2 days co-culture in the dark. The control bacterial concentration was OD₆₀₀ 0.6 as well, and AS was added at 60 mg·L^-1. Experimental result showed that a total of 197 plants regenerated from nearly 20 000 buds were obtained during the glufosinate-ammonium resistance screening. However, only three actual resistant transgenic plants were finally acquired by rescreening after rejuvenation. Thus the genetic transformation rate was 1.52 %. GUS staining was positive for these three plants, indicating that the GUS gene was integrated into Huping jujube's genome. Also, a band corresponding to the PcPKS5 gene was detected from both genomic DNA and cDNA from the transgenic plants, further indicating that the PcPKS5 gene had been integrated into the genome. Transgenic plants of line 2 were selected for further studies and successfully produced resveratrol. Resveratrol showed an elution time of 16.92 min in HPLC analysis, which was used to identify the presence of resveratrol in transgenic plants. The resveratrol content was calculated to be 0.45 μg·g^-1 fresh plant material, using standard curve analysis of the peak at 16.92 min, according to Y=67 354X+62 755 (R²=0.999 8). The m/z value of the compound collected by HPLC for this peak was 228.9 as determined by LCMS. This is in complete agreement with the m/z of the resveratrol reference standard. These findings demonstrated that the product isolated from the transgenic plants was resveratrol.[Conclusion] Here, we successfully transformed the STS gene PcPKS5, which was cloned from Polygonum cuspidatum, into Huping jujube. With a constitutive promoter, transgenic Huping jujube plants produced resveratrol. It is noteworthy that resveratrol production was relatively low in transgenic Huping jujube compared with other plants. One possible reason for this is that resveratrol may exist in other forms in the transgenic plants, e.g. resveratrol glucoside. New metabolic pathways have the potential to affect disease resistance. Therefore, the metabolic pathway of resveratrol derserves for further study. Although it takes long to obtain fruits from the transgenic Huping jujube plants, we finally obtained a new germplasm with insect and fungal resistance, establishing a new jujube variety. This study provides a basis for improving the quality of the jujube and adjusting resveratrol levels in the fruit.

Key words: genetic transformation system, resveratrol synthase gene (PcPKS5) from Polygonum cuspidatum, Zizyphus jujuba, agrobacterium mediated transformation, resveratrol