Genetic Stability Analysis of Transgenic AM79-EPSPS Glyphosate-resistant Soybean (Glycine max)
WENG Jia-Hui1, 2, LOU Yi-Yuan2, XU Jing2, ZHOU Jun2, JIANG Hong-Ye2, ZHAO Zhen-Ning2, HE Jun-Guang2, *, LIU Yong-Li1, *
1 College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310063, China; 2 Zhejiang Xin'an Chemical Industry Group Co., Ltd., Hangzhou 311600, China
Abstract：Breeding new herbicide-tolerant transgenic soybean (Glycine max) varieties is effective means to balance supply and demand of soybean in China. However, the safety evaluation of new varieties of transgenic crops should be carried out before they are commercialized, and genetic stability is one of the important evaluation factors. A new glyphosate-resistant gene AM79-EPSPS with national intellectual property rights has been cloned by Chinese researchers, but there is no report on its application in transgenic soybean. In this study, the progenies of A6 and A8, two transgenic AM79-EPSPS soybean lines, were taken as the research objects in order to analyze the genetic stability of T1 and T2 generation transformant materials based on DNA level, transcription level, translation level and tolerance level of the transformant to glyphosate, respectively. PCR was used to detect the major functional elements (including promoter, target gene and terminator) of the inserted fragments in two generation plants (T1 and T2) of A6 and A8. The main functional elements, the 35S promoter, AM79-EPSPS gene and nos terminator could amplify the target band, indicating that the main functional elements were inserted completely in different generations. Southern blot analysis was performed to detect the insertion copy number of the target gene AM79-EPSPS in T1 and T2 generation plants of A6 and A8 transformants. The result showed that the target genes at different generations were inserted through a single-copy insertion into the genomes of the 2 transformants, and the integration of every generation was consistent with each other. Transcription levels of AM79-EPSPS were detected by RT-PCR, and the result showed that AM79-EPSPS gene was expressed in different tissue of T1 and T2 generation plants in A6 and A8 transformants, indicating that the gene expression of AM79-EPSPS in A6 and A8 transformants remained stable between generations. The protein expression of AM79-EPSPS in T1 and T2 generation of A6 and A8 transformants was detected by Western blot and ELISA. The results of Western blot showed that in the T1 and T2 generation of A6 and A8, exogenous protein AM79-EPSPS could be labeled by EPSPS antibody, and the molecular weight of AM79 antibody-labeled protein was within the range of 47~50 kD, which was consistent with the predicted size of the protein. The results of ELISA analysis showed that the expression levels of AM79-EPSPS protein were different in the same tissues of A6 and A8 in different generations, but there was no significant difference. Therefore, the expression level of the target protein AM79-EPSPS was relatively stable between different generations. Verification of stable inheritance of transformant tolerance to glyphosate by spraying glyphosate. The results showed that the plants of different generations of transformants A6 and A8 could tolerate glyphosate at seedling stage, and there was no significant difference in tolerance between the 2 generations, but the negative control died. Therefore it could be concluded that the transformants demonstrate intergenerational stability in terms of the tolerance to glyphosate in herbicides. This study indicated that these 2 transformants were expected to become materials for future spreading and application,and that AM79-EPSPS gene could be used as a candidate gene for breeding commercial glyphosate-resistant soybean and other crops. Furthermore, this study also provides a methodological basis for the national genetically modified crops safety assessment, and promotes the development process of domestic genetically modified crops industry.
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