8年生嫁接转基因杨树Bt毒蛋白的表达与运输  

Expression and Transportation of Bt Toxic Protein in 8-Year-Old Grafted Transgenic Poplar

作　　者：陈盼飞 任亚超[1] 张军[1] 王进茂[1] 杨敏生[1] 

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

出　　处：《林业科学》2016年第7期46-52,共7页Scientia Silvae Sinicae

基　　金：国家自然科学基金项目(31370663);国家林业局科技发展中心项目(JC-2014-04)

摘　　要：【目的】研究经历多年生长和对复杂自然环境适应后,嫁接的8年生转基因741杨中外源Bt基因是否稳定存在及表达,探索成年树木Bt毒蛋白的运输部位、运输量、运输的方向性和积累等规律。【方法】利用转Bt Cry1Ac基因741杨与未转基因741杨互为砧木和接穗进行嫁接,在自然条件下生长8年后,对2种不同嫁接方式杨树砧木和接穗的Bt Cry1Ac基因进行PCR检测和验证,利用ELISA技术对成年嫁接杨树的不同部位、不同组织进行毒蛋白含量检测。【结果】Bt Cry1Ac基因的PCR检测结果表明,Pb29/741嫁接杨中接穗部分和741/Pb29嫁接杨中砧木部分均扩增出与阳性对照大小一致的特异性条带,其余非转基因部分和阴性对照未扩增出特异性条带,证明Bt基因在嫁接杨树的转基因部分稳定存在,未发现外源基因丢失现象。ELISA检测表明,2种不同嫁接方式处理的741杨砧木和接穗的叶片、韧皮部、木质部和髓中均检测到Bt毒蛋白存在,证明Bt基因在8年生转基因嫁接741杨中稳定表达,且Bt毒蛋白可以在成年嫁接741杨的砧木和接穗间运输。Pb29/741成年株中,根部为非转基因部分,地上枝干部分为转基因组织,地上转基因组织能够表达Bt毒蛋白,其含量呈现出树冠外侧向内侧逐渐升高,树干部分向下又逐渐降低的趋势,且可以向根和树干基部非转基因组织运输和积累,以韧皮部运输为主。741/Pb29成年株中,根部为转基因部分,地上枝干为非转基因组织,根和树干基部组织也可以表达Bt毒蛋白,且可以由根部和干基部由枝干向树冠外侧运输并积累,以韧皮部运输为主;非转基因枝干部分呈现出树干向上逐渐降低,树冠内侧向外侧逐渐升高的趋势。【结论】尽管嫁接方式不同,嫁接的转基因杨树经历8年生长和复杂自然环境的影响和适应后,Bt毒蛋白的运输都呈现出由转基因组织向非转基因组织运输现象,毒蛋白含量呈现出【Objective】This paper tries to study whether the exogenous Bt gene stably existed and expressed in 8-yearold grafted transgenic 741 poplar [Populus alba ×（ P. davidiana ＋ P. simonii） × P. tomentosa],which have adapted to the complex natural environment many years after planting,and to explore patterns of sites,volume and direction of transportation and accumulation of Bt toxin protein in adult poplar.【Method】Poplar 741（ 741） and transgenic poplar 741（ Pb29） with Bt Cry1 Ac gene were grafted reciprocally as scion and stock. 8 years after planting in natural conditions,the two types of grafted poplars（ 741 / Pb29,Pb29 /741） were used for detection and validation of Bt Cry1 Ac gene by PCR. In addition,the content of toxin protein in different tissues and different parts of adult grafted poplar was detected by means of ELISA technical system.【Result】The PCR test of Bt Cry1 Ac gene showed that specific bands at the same size as that of the positive control were detected for both the scion of Pb29 /741 and the rootstock of 741 / Pb29,but not for the rest of non-transgenic parts and the negative control,proving that the Bt gene existed stably in the transgenic tissue of grafted poplar and no gene loss happened. Bt toxin protein was detected in all of the leaves,phloem,xylem and pith by the ELISA test,proving that Bt gene were expressed stably in 8-year-old transgenic grafted poplar 741,and Bt toxin can betransported between scion and stock in adult grafted transgenic poplar 741. As for the Pb29 /741 adult plants,which had non-transgenic parts underground and transgenic parts above ground,we found that the Bt toxin protein could be expressed in above ground tissue and be transported to and accumulated in the non-transgenic tissue of roots and stem base mainly through phloem. And the content of the toxic protein gradually increased from outer to the inner of the crown,and decreased downward along the trunk progressively. While for the 741 / Pb29 adult plants,which with transgenic roots and

关 键 词：转基因杨树 嫁接 BT毒蛋白 基因表达 蛋白运输 

分 类 号：S718.46[农业科学—林学] S722.36