| 摘要: |
| 【目的】构建中国水仙ZDS反义基因表达载体，为进一步研究ZDS基因的功能、改良中国水仙花色、培育花色新品种提供参考。【方法】以中国水仙花为材料，采用RT-PCR方法克隆其ZDS基因，通过PCR、双酶切、连接等方法将ZDS基因反向互补插入到pCAMBIA1301双GUS植物表达载体中，构建ZDS反义基因表达载体P1301-ZDS，再通过冻融法将重组质粒P1301-ZDS导入根癌农杆菌。采用根癌农杆菌介导法将ZDS反义基因转化中国水仙带盘鳞茎，通过直接、间接、延迟筛选法3种方法对侵染的带盘鳞茎进行抗性筛选，采用直接筛选法和逐步降低选择压(质量浓度)方法对抗性鳞茎进行增殖和分化培养，采用GUS组织化学和hyg基因的PCR检测方法对中国水仙转基因植株进行鉴定。【结果】成功构建了中国水仙ZDS反义基因表达载体P1301-ZDS；延迟筛选法为根癌农杆菌侵染带盘鳞茎的有效筛选方法，转化效率达到28.54%；逐步降低选择压法为抗性小鳞茎增殖和分化培养的有效培养方法；将65株抗性小苗在1/2 MS+0.2 mg/L NAA+10 mg/L Hyg +100 mg/L Cef 生根培养基中培养，获得了12株再生植株，转化率达到18.46％；检测结果表明，10株抗性植株hyg基因和gus基因已经整合到中国水仙的基因组中并稳定表达；1株抗性植株稳定表达hyg基因但gus基因未稳定表达，1株抗性植株2个基因均未稳定表达。【结论】构建中国水仙ZDS反义基因表达载体并侵染中国水仙带盘鳞茎，经抗性筛选后获得12株中国水仙转基因植株。 |
| 关键词: 中国水仙 ZDS基因 载体构建 遗传转化 |
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| 基金项目:国家“十一五”科技支撑计划项目(2007BAD07B00)；福建农林大学科技创新团队基金项目(cxtd12013) |
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| Construction of expression vector with ZDS antisense gene and regeneration of transgenic plants of Narcissus tazetta var.Chinensis |
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FENG Ying,ZHOU Xiang,PAN Teng-fei,et al
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| Abstract: |
| 【Objective】This study constructed expression vector of ZDS antisense gene to provide reference for studying function of ZDS gene,improving and cultivating new color varieties of Narcissus tazetta var.Chinensis.【Method】ZDS gene was cloned from flower with RT-RCR method,and ZDS gene was reversely inserted into plant expression vector pCAMBIA1301 with double GUS by PCR,double digestion and connection method to obtaining expression vector P1301 ZDS with ZDS antisense gene.Then recombinant plasmid P1301-ZDS was transformed into Agrobacterium through freeze-thawing method.ZDS antisense gene was transformed into bulbs by Agrobacterium mediated transformation method and resistant bulbs were selected by direct selection method,indirect selection method and intermittent selection method.The multiplication and differentiation of resistant bulbs were cultured with direct selection method and gradual reduction of hyg concentration method,and transgenic plants were identified by GUS histochemical assay and PCR assays.【Result】Expression vector with ZDS antisense gene was obtained.Resistant bulbs were obtained by retarded selection and transgenic rate was up to 28.54%.The multiplication and differentiation of resistant bulbs were cultured on medium with gradual reduction of hyg concentration.A total of 65 plants rooted on 1/2 MS+0.2 mg/L NAA+10 mg/L Hyg+100 mg/L Cef,and 12 resistant plantlets were obtained with the transgenic rate of 18.46%.The assays of transgenic plants proved that gus gene and hyg gene were integrated into the genome of 10 transgenic plants and stably expressed,hyg gene was expressed but gus gene was not expressed in one transgenic plant,and both genes were not expressed in the other plant.【Conclusion】Expression vector with ZDS antisense gene was constructed and infected into bulb,and 12 transgenic plantlets were obtained. |
| Key words: Narcissus tazetta var.Chinensis ZDS gene expression vector construction transformation |
