轉雙Bt基因巨霸楊外源基因表達及抗蟲性檢測1)

趙潔　杜沙沙　邱彤　楊敏生　王進茂

(河北農業大學，保定，071000)

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轉雙Bt基因巨霸楊外源基因表達及抗蟲性檢測1)

趙潔杜沙沙邱彤楊敏生王進茂

(河北農業大學，保定，071000)

摘要為提高轉基因楊樹中Bt基因的表達效率并擴大抗蟲譜，以同時轉入Cry1Ac和Cry3A基因的轉雙Bt基因巨霸楊(Populus deltocdes ‘55/56’×P. deltocdes ‘2KEN8’)株系1年生苗為材料，對外源基因的表達和抗蟲性進行檢測。經PCR檢測，證明目的基因已被整合到巨霸楊基因組中。利用熒光定量PCR和ELISA技術檢測了4個轉基因株系葉片中Bt基因的轉錄豐度和毒蛋白表達量。結果表明：不同株系間Bt基因的轉錄豐度存在顯著差異，Cry3A基因的轉錄豐度顯著高于Cry1Ac基因的轉錄豐度；2種Bt毒蛋白表達量在各株系間存在顯著差異，Cry3A毒蛋白質量分數均顯著高于Cry1Ac毒蛋白質量分數。各株系對鱗翅目害蟲美國白蛾(Hyphantria cunea)幼蟲抗蟲效果不明顯，且無顯著差異；對鞘翅目害蟲柳藍葉甲(Plagiodera versicolora)表現出極高抗蟲效果，且均顯著高于轉單基因Cry3A 741楊高抗株系CC84，不同株系間存在顯著差異。

關鍵詞巨霸楊；雙Bt基因；轉錄豐度；毒蛋白；抗蟲性

分類號S718.43；Q965.9

Exogenous Gene Expression and Insect Resistance Detection of TransgenicPopulusdeltocdes‘55/56’×P.deltocdescv‘2KEN8’ with DoubleBtGene

Zhao Jie, Du Shasha, Qiu Tong, Yang Minsheng, Wang Jinmao

(Agricultural University of Hebei, Baoding 071000, P. R. China)//Journal of Northeast Forestry University，2016,44(3)：47-51.

To improve the efficiency of genetically modified (gm) polar Bt gene expression and expand the insect-resistant spectrum, we chose thePopulusdeltocdes‘55/56’×P.deltocdescv‘2KEN8’ with doubleBtgene ofCry1AcandCry3Aof one-year seedlings as materials, and tested the expression of exogenous genes and insect resistance. The purpose gene was integrated into the genome in theP.deltocdes‘55/56’×P.deltocdescv‘2KEN8’ by PCR identification. We used the fluorescent quantitative PCR technique and ELISA to detect the four gm strains transcription abundanceBttoxic protein gene expression content in the leaves. The difference existed between the twoBtgene transcription abundance in different strains:Cry3Agene transcription abundance was significantly higher thanCry1Ac. Two kinds of Bt toxic protein expression quantity were significantly different between each strain.Cry3Atoxic protein levels in each strain were significantly higher thanCry1Ac. Each strains’ insect-resistant effect to lepidoptera pests, larvae (Hyphantriacunea) was not obvious with no significant difference between different strains. The strains showed very high insect-resistant effect to coleoptera pests (Plagioderaversicolora), and were significantly higher than that of single geneCry3Apoplars high strain CC84, and there was significant difference between different strains.

KeywordsPopulus deltocdes ‘55/56’×P. deltocdescv ‘2KEN8’; Double Bt gene; Transcript abundance; Toxic protein; Insect resistance

楊樹栽培歷史悠久，用途廣泛，但是近年來蟲害嚴重，給農林業造成巨大損失，使用傳統的化學防治消滅害蟲，雖可一時緩解蟲害，但是會造成環境污染。利用植物基因工程可以將殺蟲蛋白基因轉入植物，使植物獲得抗蟲性。甄志先等將BtCry3A基因轉入741楊，獲得的轉基因株系對鞘翅目害蟲的致死率很低，但是對幼蟲的發育起到了明顯的抑制作用[1]。王穎等將部分改造的Cry1Ac基因與慈菇蛋白酶抑制劑(API)基因構建的載體導入三倍體毛白楊，獲得的轉基因植株明顯抑制了鱗翅目幼蟲的生長發育，且不同轉基因株系飼養的幼蟲，其生長發育存在顯著差異[2]。……