转基因本氏烟草中CtDXR基因拷贝数及耐盐耐高温功能的初步分析  

Preliminary analysis of CtDXR gene copy number,salt tolerance and heat tolerance in Transgenic Nicotiana benthamiana

作　　者：田春尧 冀慧玥 丁润月 郑乔木 周嘉裕[1] 廖海[1] TIAN Chunyao;JI Huiyue;DING Runyue;ZHENG Qiaomu;ZHOU Jiayu;LIAO Hai(School of Life and Science,Southwest Jiaotong University,Chengdu 610031,China)

机构地区：[1]西南交通大学生命科学与工程学院,成都610031

出　　处：《生物学杂志》2024年第4期17-23,共7页Journal of Biology

基　　金：四川省科技项目(2017JY0222,2018SZ0061,2021ZHFP0170);四川省中医药管理局面上项目(2021MS116);成都市科技局项目(2022-YF05-01357-SN);西南交通大学医工结合项目(2682021ZTPY017)。

摘　　要：评估转基因本氏烟草中CtDXR基因的拷贝数,并初步分析其耐盐与耐高温能力。首先利用Southern Blot技术确定野生型本氏烟草中Actin基因的拷贝数,并以Actin基因为内参基因,以转CtDXR基因本氏烟草为材料,采用实时荧光定量PCR方法,检测转基因植株中CtDXR基因的拷贝数。最后,初步分析转基因植株的耐盐与耐高温能力。基于PCR方法,随机检测10株T1代转基因本氏烟草植株均能扩增出目的条带,表明它们均已成功转入目的基因CtDXR;Actin基因在本氏烟草基因组中为单拷贝基因;以Actin基因为内参基因,最终确定80%的转CtDXR基因植株为单拷贝或低拷贝株系;盐胁迫下,转CtDXR基因植株的株高(P<0.01)、侧根数(P<0.01)与鲜重(P<0.001)优于野生型植株,高温胁迫下,转CtDXR基因植株的株高(P<0.01)、叶片数(P<0.01)与鲜重优于野生型植株,表明转基因植株具有更强的耐盐和耐高温能力。利用研究建立的转基因本氏烟草外源基因拷贝数的检测方法,对转CtDXR基因植株完成外源基因拷贝数的鉴定,且初步鉴定了CtDXR基因具有耐盐与耐高温功能。To evaluate the copy number of CtDXR gene in transgenic Nicotiana benthamiana and further analyze its tolerance to salt and heat stress,Southern Blot was applied to determine the copy number of the Actin gene in wild-type N.benthamiana and the copy number of the CtDXR gene in CtDXR transgenic N.benthamiana was detected by real-time PCR with Actin gene as reference gene.Then,the salt and heat tolerance of CtDXR transgenic N.benthamiana was analyzed.According to the PCR-dependent method,10 of T1 transgenic N.benthamiana were randomly detected and all of which obtained positive results,indicating that CtDXR has been successfully transformed.The Actin gene is a single-copy gene in the genome of wild-type N.benthamiana.80%of transgenic N.benthamiana were determined as single-copy or low-copy lines.CtDXR transgenic N.benthamiana showed better performance,such as plant height(P<0.01),number of lateral roots(P<0.01),and fresh weight(P<0.001),than wild-type N.benthamiana under salt stress,whereas CtDXR transgenic N.benthamiana exhibited better performance,such as plant height(P<0.01),number of leaves(P<0.01),and fresh weight,than wild-type N.benthamiana under heat stress.Taken together,CtDXR conferred salt and heat tolerance to N.benthamiana.In this study,a method to detect copy number of foreign gene in transgenic N.benthamiana was established,by which the copy number of CtDXR gene in transgenic N.benthamiana was accomplished.Moreover,CtDXR gene was preliminarily identified to have salt and heat tolerance.

关 键 词：本氏烟草 1-脱氧-D-木酮糖-5-磷酸还原异构酶 拷贝数 Southern Blot 实时荧光定量PCR 

分 类 号：Q78[生物学—分子生物学]