基因枪法介导的辣椒花药遗传转化技术研究  

The Genetic Transformation of Pepper Anthers by Particle Bombardment

作　　者：张晓芬[1] 王国云 杜和山 陈斌[1] 温长龙 耿三省[1] ZHANG Xiaofen;WANG Guoyun;DU Heshan;CHEN Bin;WEN Changlong;GENG Sansheng(Beijing Vegetable Research Center,Beijing Accademy of Agriculture and Forestry Sciences,Beijing 100097)

机构地区：[1]北京市农林科学院蔬菜研究中心,北京100097

出　　处：《核农学报》2018年第11期2081-2087,共7页Journal of Nuclear Agricultural Sciences

基　　金：国家重点研发计划(2017YFD0101904);北京市农林科学院科技创新能力建设专项(KJCX20170102-14);北京市农林科学院蔬菜种质创新与新品种选育院级科技创新团队(JNKYT201601)

摘　　要：为建立基因枪法介导的辣椒遗传转化体系,以小孢子胚胎发生率较高的辣椒品种L5的花药为受体,利用基因枪法将绿色荧光蛋白(GFP)基因导入L5花药小孢子中,并进行花药培养诱导小孢子胚胎发生,探讨基因枪法转化辣椒花药小孢子的适宜参数。结果表明,辣椒小孢子处于单核靠边期的花药在9℃下暗培养4 d时为最佳受体;CaMV35S启动子能启动GFP基因在辣椒花药培养的小孢子胚胎中表达;基因枪各轰击参数对小孢子胚胎发生有显著影响(P<0.05),随着基因枪轰击次数和轰击压力的增加以及轰击距离的减小,小孢子胚胎发生数量逐渐减少;最适的轰击参数为基因枪轰击2次、轰击压力1 350 psi和轰击距离9 cm,此时获得的转化胚胎数量最多;在最适的轰击参数下辣椒花药小孢子的平均转化效率为4.2%;体式荧光显微镜下共检测到14个转化胚胎。本研究结果为进一步建立基因枪介导的辣椒花药稳定遗传转化体系提供了一定的理论基础和技术支撑。In order to establish the genetic transformation system of pepper（ Capsicum annuumL.） using particle bombardment,the anthers of high embryogenesis pepper variety ‘L5 ＇were used as receptors,and the gene of green fluorescent protein（ GFP） were transformed into the microspores of anthers. Then the microspore embryogenesis was induced by anther culture,the best parameters of particle bombardment for the genetic transformation of pepper have been preliminarily established. In the study,the best receptors for the particle bombardment were the anthers which were pre-cultured in the dark at 9℃ temperature for four days. The bombarding intensity had significant effect on the embryogenesis from anthers（ P〈0. 05）. The numbers of embryos were decreased with the increase of bombardment pressure and bombardment times,and decreased with the decrease of target distance. The best parameters of particle bombardment were shooting two times with 1350 psi rupture pressure and target distance of 9 cm. With which average transformation efciency was 4. 2%,and fourteen transformation embryos were identified using the Leica-microscope,but no transformation plants were obtained in this study. These results could be used in the stable genetic transformation of pepper using particle bombardment in the future.

关 键 词：辣椒 花药培养 小孢子 基因枪法 GFP 

分 类 号：S641.3[农业科学—蔬菜学]