8年生嫁接转基因杨树Bt毒蛋白的表达与运输

8年生嫁接转基因杨树Bt毒蛋白的表达与运输
陈盼飞;任亚超;张军;王进茂;杨敏生
【期刊名称】《林业科学》
【年(卷),期】2016(052)007
【摘要】Objective]This paper tries to study whether the exogenous Bt gene stably existed and expressed in 8-year-old 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 BtCry1Ac 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 BtCry1Ac 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 BtCry1Ac 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 be transported 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 non-transgenic ground stems,we also found that the Bt toxin protein could be expressed in roots and stem base and be transported to and accumulated in the outer crown mainly through phloem. It showed that the content of Bt toxin protein gradually decreased upward along the non-transgenic trunk,and it increased from the inside tree crown to the
outer.[Conclusion]The transportation of Bt toxin protein in the perennial grafted poplar showed a similar trend though the grafting methods were different. Additionally,the Bt toxin protein could be transported from transgenic part to non-transgenic part,and the content of toxin protein performed a similar transportation and accumulation trend from source to library. Transgenic poplars could be widely applied in production by a traditional way of grafting,which would improve the ecological safety of transgenic trees.%【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BtCry1Ac 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BtCry1Ac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】BtCry1Ac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
现出类似由源向库运输和积累的趋势。

转基因杨树可以通过传统嫁接方式在生产上应用，以提高转基因林木的生态安全性。

【总页数】7页(P46-52)
【作者】陈盼飞;任亚超;张军;王进茂;杨敏生
【作者单位】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;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;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;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;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
【正文语种】中文
【中图分类】S718.46;S722.3+6
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