一种龙眼快速遗传转化方法的建立  

Establishment of a rapid transgenic method on longan seedling

作　　者：陈裕坤[1] 程春振[1] 张梓浩[1] 赖钟雄[1] CHEN Yukun;CHENG Chunzheng;ZHANG Zihao;LAI Zhongxiong(Institute of Horticultural Biotechnology,Fujian Agriculture and Forestry University,Fuzhou 350002,China)

机构地区：[1]福建农林大学园艺植物生物工程研究所,福州350002

出　　处：《应用与环境生物学报》2020年第6期1540-1545,共6页Chinese Journal of Applied and Environmental Biology

基　　金：国家自然科学基金项目(31572088,31672127)资助。

摘　　要：针对龙眼遗传转化传统方法存在转化效率低、周期长、转化的胚状体再生困难以及转化植株生长异常等问题,以龙眼土播实生幼苗为受体材料,采用去顶芽(保留真叶)和去顶法(切除带叶的上部)进行创伤,利用根癌农杆菌介导法侵染伤口,通过抗性筛选获得正常生长发育的再生植株,经PCR扩增、目标片段测序及qRT-PCR检测鉴定阳性转化植株.结果显示,去顶芽法获得的抗性芽GUS基因阳性检测率33.84%、特异片段(35S-mft-intron)的阳性率为20.83%,去顶法获得的抗性芽GUS基因阳性检测率32.5%;经测序验证,PCR扩增产物的序列与载体特异片段的相似度为100%,外源基因已经整合到龙眼基因组中;qRT-PCR表明外源基因在转基因龙眼植株中表达量较高,在未转化龙眼植株中不表达,说明外源基因在龙眼植株内进行转录,建立了一种龙眼快速转基因方法.该方法可快速获得大量转基因植株,具有操作简便、成本低、周期短、转化效率较高、成活率高、可周年生产等优点,对龙眼的基因功能研究和种质创新具有重要的意义.Traditional longan genetic transformation methods have a series of problems,such as low transformation efficiency,long cycles,transformed embryoids that are difficult to regenerate,and abnormal growth of transformed plants.In this study,we transformed the target genes into longan soil seedlings by Agrobacterium tumefaciens-mediated transformation through infection of a wound,generated by removing the apical bud(keep true leaf)or the top(remove the upper part of the true leaf)of the seedling.Many normal,resistant buds were obtained through hygromycin selection,and successfully transformed plants were identified by polymerase chain reaction(PCR),sequencing,and quantitative reverse-transcription PCR(qRT-PCR).PCR detection of target genes showed that the successful transformation rate of GUS in resistant shoots was 33.84%and 32.5%following removal of the apical bud and the top of the seedling,respectively.The successful transformation rate of a specific fragment(35S-mft-intron)in resistant shoots was 20.83%by removing the apical bud of the seedling.It was verified by sequencing that the similarity between the PCR amplification product and the specific fragment of the vector was 100%,indicating that the target genes were integrated into longan gDNA.qRT-PCR analysis suggested that exogenous genes were highly expressed in transgenic longan plants but not in the negative control,which confirmed that the target transformation genes in longan were being transcribed.This method can produce a large number of transgenic longan trees rapidly,and it may play a significant role in gene functional studies and the creation of longan germplasm,owing to its easy,rapid,and efficient production,as well as the high survival rate of transgenic plants.

关 键 词：龙眼 实生幼苗 根癌农杆菌介导 遗传转化 

分 类 号：S667.2[农业科学—果树学]