‘蜂蜜罐’枣遗传转化条件的优化及Bt基因的导入

‘蜂蜜罐’枣遗传转化条件的优化及Bt基因的导入

张燕征;张梦洋;李继东;谭彬;王腾飞;郑先波;叶霞;冯建灿

【摘要】The establishment of a high efficient and stable genetic transformation system is the basis for genetic improvement of plant germplasm by genetic engineering. In this study, the binary vector Pbi121 containing gus (β-glucuronidase) gene and the neomycin phosphotransferase Ⅱ (NPTⅡ) gene was introduced into jujube

'Fengmiguan' (Ziziphus jujuba Mill) by Agrobacterium-mediated transformation using epicotyls and cotyledons as explants. The effects of different bacterial concentra-tions,time of infection and co-cultivation,the concentration of acetosyringone (AS) and the infection pattern on the transient expression rate of guswere investigated. Bt (Bacillus thuringiensis) gene was also introduced into jujube 'Fengmiguan' by using the optimized genetic transformation system. The results showed that the most appropriate genetic transformation system by gus staining for the epicotyls and cotyledons was when bacterial culture reached OD600= 0.5 and infected for 10 min under vacu-um and then co-cultivated in a medium supplemented with 20 mg·L-1,10 mg·L-1of AS for 3 d, resepctively, the rate ofgus transient expression on epicotyls was significantly higher than cotyledons. Bt gene introduced into 'Fengmiguan' was carried out by using the optimized genetic transformation system and epicotyls and cotyledons were used as explants. After co-cultivation in a selective medium, the resistant shoots with the height of 1 ~1.5 cm were analyzed

by PCR. Total 24 transgenic shoots were obtained and elongated from epicotyls and cotyledons. The transgenic plantlets with Bt gene were eventually obtained. Thus would provide a possibility for creating insect-resistant germplasm.%为建立‘蜂蜜罐’枣高效遗传转化体系,以‘蜂蜜罐’枣胚培苗上胚轴和子叶为外植体进行转化,研究了不同菌液浓度、侵染时间、共培养时间、AS(乙酰丁香酮)浓度和侵染方式对gus(β-葡糖醛酸糖苷酶)基因瞬时表达率的影响.结果表明,当菌液OD600值为0.5,真空辅助侵染10 min,共培养3 d,上胚轴和子叶外植体的共培养培养基中分别添加20和10 mg·L-1乙酰丁香酮时,其gus瞬时表达率均最高,为最佳遗传转化体系.利用已优化的遗传转化体系进行Bt基因转化,共获得24个PCR(聚合酶链式反应)检测呈阳性的再生芽;对PCR检测呈阳性的不定芽进行伸长培养和不定根诱导,最终获得完整转基因植株.本研究初步获得了转Bt(苏云金芽孢杆菌)基因的枣植株,为通过转基因手段进行枣抗虫新种质选育提供可能.

【期刊名称】《河南农业大学学报》

【年(卷),期】2018(052)001

【总页数】7页(P43-49)

【关键词】'蜂蜜罐'枣;上胚轴;子叶;gus瞬时表达率;Bt基因

【作者】张燕征;张梦洋;李继东;谭彬;王腾飞;郑先波;叶霞;冯建灿

【作者单位】河南农业大学林学院,河南郑州450002;河南省果树瓜类生物学重点实验室,河南郑州450002;河南农业大学园艺学院,河南郑州450002;河南省果树瓜类生物学重点实验室,河南郑州450002;河南农业大学林学院,河南郑州450002;河南省果树瓜类生物学重点实验室,河南郑州450002;河南农业大学园艺学院,河南

郑州450002;河南省果树瓜类生物学重点实验室,河南郑州450002;河南农业大学园艺学院,河南郑州450002;河南省果树瓜类生物学重点实验室,河南郑州450002;河南农业大学园艺学院,河南郑州450002;河南省果树瓜类生物学重点实验室,河南

郑州450002;河南农业大学园艺学院,河南郑州450002;河南省果树瓜类生物学重

点实验室,河南郑州450002;河南农业大学林学院,河南郑州450002;河南农业大

学园艺学院,河南郑州450002;河南省果树瓜类生物学重点实验室,河南郑州450002

【正文语种】中文

【中图分类】S665.1

枣(Ziziphus jujuba Mill.)原产于中国，种质资源丰富，栽培历史悠久，其产量是

居中国干果类第1位的果树[1]。遗传转化是枣育种和分子生物学研究的重要手段，利用根癌农杆菌介导的转化体系，将外源基因导入枣基因组，可以改良其性状，或进行基因功能研究。目前，何叶华等[2-3]在‘茶陵沙枣’、‘鸡蛋枣’、‘宝玉’，黄建[4]在‘木枣’和‘沾化冬枣’[5-6]，尚霄丽[7]在‘灰枣’，徐明[8]

在‘哈密大枣’及罗在柒等[9]在‘壶瓶枣’等枣品种上均开展了遗传转化的研究。何业华等[2-3]选取再生能力较强的‘茶陵沙枣’的茎段和下胚轴为试验材料，进

行根癌农杆菌介导的ACC合成酶反义基因转化，转化率为2.4%～4%，经Southern blot检测获得6株转化植株。GU[6]以‘沾化冬枣’茎尖为试材，建立了遗传转化体系，gus瞬时表达率仅为0.7%～3.2%。因此，建立一个高效稳定的遗传转化体系是利用基因工程手段进行枣种质改良的基础。此外，在现有的枣遗传转化研究中，多以叶片、茎段、茎尖等作为转化的外植体，由于各品种的基因型不同，转化条件也有一定差异。而以胚培养获得的各种外植体如上胚轴、子叶、下胚