转多基因欧美杨Bt基因表达特征  

Expression Characteristics of Bt Gene in Transgenic Poplar Transformed by Different Multi-Gene Vectors

作　　者：张超 王进茂 赵洁 庞丁玮 张德健[4] 杨敏生 Zhang Chao;Wang Jinmao;Zhao Jie;Pang Dingwei;Zhang Dejian;Yang Minsheng(Key Laboratory of Germplasm Resources of Forest Trees and Forest Protection of Hebei College of Forestry,Hebei Agricultural University Baoding 071000;Hebei Qianmo City Planning and Design Consulting Co.,Ltd.Shijiazhuang 050061;Hebei Wuling Mountain National Nature Reserve Administration Chengde 067300;School of Life Sciences,Inner Mongolia University Hohhot 010020)

机构地区：[1]河北农业大学林学院河北省林木种质资源与森林保护重点实验室,保定071000 [2]河北阡陌城市规划设计咨询有限公司,石家庄050061 [3]河北雾灵山国家级自然保护区管理局,承德067300 [4]内蒙古大学生命科学学院,呼和浩特010020

出　　处：《林业科学》2019年第9期61-70,共10页Scientia Silvae Sinicae

基　　金：国家自然科学基金项目(31660211);转基因生物新品种培育重大专项(2018ZX08020002)

摘　　要：【目的】探究转多基因欧美杨107杨中外源Bt基因是否稳定存在及表达,探索转基因107杨不同时间、不同部位Bt毒蛋白表达规律,研究多基因转化的载体结构及基因互作对外源基因表达稳定性和高效性的影响。【方法】选取1年生转Cry1Ac-Cry3A-NTHK1基因107杨3个株系(A1、A2、A3)和转Cry1Ac-Cry3A-BADH基因107杨3个株系(B1、B2、B3),通过PCR技术对转基因107杨中外源基因进行检测和验证,通过实时荧光定量PCR对Bt基因的转录丰度进行检测,利用ELISA技术对转基因107杨不同时间、不同部位毒蛋白含量进行检测。【结果】PCR检测结果显示,转基因107杨均能扩增出与阳性对照大小一致的特异性条带,阴性对照未扩增出特异性条带,证明外源基因在转基因107杨中稳定存在;实时荧光定量PCR检测结果表明,2种Bt基因在转基因107杨中均能稳定表达,Cry1Ac基因的转录丰度在2.1×10^4~5.1×10^4之间,Cry3A基因的转录丰度在2.8×10^6~5.6×10^7之间,Cry1Ac基因和Cry3A基因转录丰度无相关性;ELISA技术检测结果显示,转基因107杨中均检测到Cry1Ac和Cry3A毒蛋白存在。2种Bt基因转录丰度和毒蛋白含量无相关性,但2种Bt毒蛋白含量之间呈现出正相关关系。转2种不同载体的107杨6、7月份2种Bt毒蛋白的含量较低,8月份急剧上升,Cry1Ac毒蛋白的含量均在9月份达到峰值,Cry3A毒蛋白含量均在8月份达到峰值,10月急剧下降。8月份不同部位(上、中、下)的叶片Bt毒蛋白含量未表现出一致性规律,不同部位(上、中、下)木质部的Bt毒蛋白含量也未呈现出一致性规律。【结论】转Cry1Ac-Cry3A-NTHK1基因107杨和转Cry1Ac-Cry3A-BADH基因107杨不同株系间2种Bt基因的转录丰度存在显著差异,Cry3A基因的转录丰度显著高于Cry1Ac基因;2种Bt毒蛋白在各株系间存在显著差异,Cry1Ac毒蛋白含量极低,Cry3A毒蛋白含量极显著高于Cry1Ac,与转录水平检测结果一致。2种不同载体�【Objective】 This paper was aimed to explore the stability of existence and expression of the exogenous Bt gene in transgenic poplar 107(Populus×euramericana ‘74/76’), and to explore the expression patterns of Bt toxin in different parts of transgenic poplar 107 at different times, and to investigate the effects of gene interaction and multi-gene vector structure on the stability and efficiency of foreign gene expression in polygenic transformation vectors.【Method】 Three strains of one-year-old transgenic poplar 107 with genes of Cry1 Ac-Cry3 A-NTHK1(A1, A2, A3) and three strains with genes of Cry1 Ac-Cry3 A-BADH(B1, B2, B3) were selected to detect and validate foreign genes in the transgenic poplar trees using PCR and to detect transcript abundance of Bt gene usng fluorescence quantitative PCR, and to detect toxic protein contents in different parts of the transgenic poplar trees at different times using ELISA.【Result】 The PCR detection showed that the transgenic poplar 107 could amplify a specific band at the same size as that of the positive control, and the negative control did not amplify the specific band, which proves that the foreign gene is stable in transgenic poplar 107;The fluorescence quantitative PCR showed that two Bt genes were stably expressed in each line, and the transcript abundance of Cry1 Ac gene was ranged from 2.1×10^4 to 5.1×10^4, the transcriptional abundance of Cry3 A gene ranged from 2.8×10^6 to 5.6×10^7. There was no correlation between transcript abundance of Cry1 Ac gene and transcriptional abundance of Cry3A. The ELISA detection showed existence of both Cry1 Ac and Cry3 A toxic proteins in each line of the transgenic poplar 107. There was no correlation between transcript abundance and toxic protein content of the two Bt genes. But there was a positive correlation between toxic protein content of the two Bt genes. The content of two types of Bt toxic proteins was lower in June and July of the two different vectors, and increased sharply in August. The content of

关 键 词：转基因杨树 多基因载体 基因互作 外源基因表达 BT毒蛋白 

分 类 号：S718.46[农业科学—林学]