农杆菌介导虎杖芪合酶基因遗传转化壶瓶枣的研究

罗在柒; 郭辉力; 杨亚东; 杨明峰; 马兰青; 王有年

摘要

【目的】白藜芦醇作为植物次生代谢产物不但能提高果树抗真菌能力，还能改善果品质量。遗传转化芪合酶基因能够增强植物的抗真菌能力，虎杖芪合酶基因具有较高的催化合成白藜芦醇的效率，研究虎杖芪合酶基因遗传转化壶瓶枣，以期获得具有抗真菌能力且改善枣果实品质的遗传转化新材料。【方法】通过组织培养再生体系与目的基因转化技术，优化获得茎诱导丛生芽，构建植物表达载体，优化遗传转化体系，采用农杆菌介导法将虎杖白藜芦醇生物合成关键酶基因 PcPKS5遗传转化壶瓶枣。【结果】优化壶瓶枣茎段诱导得到高分化率的丛生芽遗传转化体系，再生增植率为11.0，为壶瓶枣成功实现遗传转化奠定了基础。将壶瓶枣0.8～1.0 cm 含茎尖和茎段的外植体材料置于农杆菌浓度 OD600=0.6时侵染菌液中浸泡15.0 min，然后置于培养基上避光共培养3天；随后转入含 Cb 300 mg·L －1，AS 60 mg·L －1的丛生芽诱导分化培养基中培养5～6周。将分化丛生芽转接至含4.0 mg·L －1 Basta 的培养基中，获遗传转化植株173株；经 Basta 筛选， GUS显色、gDNA PCR、RT-PCR 等检测证实，成功获得3个阳性转基因株系，荧光实时定量检测表明株系2表达效率较高。经植物化学成分分析，转化植株中目的基因得以表达，生成了目标产物白藜芦醇，其含量为0.45μg·g －1(鲜质量)。【结论】本研究成功实现虎杖芪合酶基因遗传转化壶瓶枣，获得壶瓶枣遗传转化新材料。且 PcPKS5在枣树中异源表达，转基因材料中能够代谢合成白藜芦醇，有望提高转 PcPKS5基因壶瓶枣抗枣树病原真菌病能力。白藜芦醇是否在壶瓶枣遗传转化材料果实中积累，及转基因植株果实品质的影响仍需深入研究。%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.

农杆菌介导虎杖芪合酶基因遗传转化壶瓶枣的研究

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