|
|
|
| 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.
|
|
Received: 02 January 2019
|
|
|
|
Corresponding Authors:
hejunguang121@126.com; liuyongli@zju.edu.cn
|
|
|
|
1 陈晟, 郭丽琼, 宋景深, 等. 2012. T5代γ-亚麻酸转基因大豆的遗传稳定性分析[J]. 大豆科学, 31(1): 24-28.
(Chen S, Guo L Q, Song J S, et al.2012. Genetic stability analysis of the fifth generation of transgenic soybeans expressing γ-linolenic acid[J]. Soybean Science, 31(1): 24-28.)
2 韩庆梅, 孙石, 侯文胜, 等. 2015. 高蛋氨酸转基因大豆的鉴定和遗传稳定性分析[J]. 中国油料作物学报, 37(6): 789-796.
(Han Q M, Sun S, Hou W S, et al.2015. Identification and genetic stability analysis of transgenic soybean with enhanced level of methionine[J]. Chinese Journal of Oil Crop Sciences, 37(6): 789-796.)
3 梁海生, 李梦桃, 李圣彦, 等. 2018. 转Bt基因抗虫玉米HGK60的农艺性状分析[J]. 生物技术通报, 34(7): 92-100.
(Liang H S, Li M T, Li S Y, et al.2018. Agronomic traits analysis of transgenic Bt cry1Ah maize HGK60 line[J]. Biotechnology Bulletin, 34(7): 92-100.)
4 林敏, 梁爱敏, 陆伟, 等. 2007. 高耐受草甘膦的EPSP合酶及其编码序列. 中国, 200710177090.1 [P].
(Lin M, Liang A M, Lu W, et al.2007. EPSP synthase with high glyphosate tolerance and its coding sequence. China, 200710177090.1 [P].)
5 刘建凤, 崔栗, 安利佳. 2012. 转AlNHX1基因大豆后代遗传稳定性及耐盐性分析[J]. 河北大学学报(自然科学版), 32(2): 173-179.
(Liu J F, Chui L, An L J, 2012. Genetic stability analysis and salt tolerance of the progenies of soybean transformed AlNHX1 gene[J]. Journal of Hebei University (Natural Science Edition), 32(2): 173-179.)
6 王海慧, 初可嘉, 管晓燕, 等. 2017. 嵌合基因gtfB-ctxB在防龋用转基因生菜的遗传稳定性研究[J]. 环境与健康杂志, 34(5): 401-403.
(Wang H H, Chu K J, Guan X Y, et al.2017. Genetic stability of anti-caries gene (gtfB-ctxB) in progenies of transgenic lettuce[J]. Journal of Environment and Health, 34(5): 401-403.)
7 王玉, 王建华, 刘允军. 2017. AM79-EPSPS蛋白的体外模拟胃肠液消化稳定性研究[J]. 生物技术通报, 33(6): 176-181.
(Wang Y, Wang J H, Liu Y J, Digestive stability of AM79-EPSPS protein in simulative gastric and intestinal fluid[J]. Biotechnology Bulletin, 33(6): 176-181.)
8 魏毅东, 罗曦, 吴方喜, 等. 2017. 利用qPCR法检测OsPIMT1转基因水稻中的外源片段拷贝数[J]. 福建稻麦科技, 35(4): 39-42.
(Wei Y D, Luo X, Wu F X, et al.2017. Determination of the copy number of exogenous fragment in OsPIMT1 transgenic rice by quantitative real-time PCR[J]. Fujian Science and Technology of Rice and Wheat, 35(4): 39-42.)
9 翁绿水, 王作平, 肖国樱. 2018. 转基因水稻B2A68中抗虫蛋白Cry2Aa的表达特征和抗性分析[J]. 农业生物技术学报, 26(5): 756-763.
(Weng L S, Wang Z P, Xiao G Y.2018.Expression profile of insecticidal protein Cry2Aa and lepidopteran resistance in transgenic rice (Oryza sativa) B2A68[J]. Journal of Agricultural Biotechnology, 26(5): 756-763.)
10 吴江敏, 孙亚东, 梁燕, 等. 2011. 番茄红素-β-环化酶(LYC-b)反义基因在番茄转基因后代的表达及其遗传稳定性[J]. 农业生物技术学报, 19(5): 801-807.
(Wu J M, Sun Y D, Liang Y, et al.2011. Expression and genetic stability of Lycopene-β-cyclase (LYC-b) antisense gene in the progeny of transgenic Tomato[J]. Journal of Agricultural Biotechnology, 19(5): 801-807.)
11 许兰珍, 何永睿, 彭爱红, 等. 2013. 抗菌肽ShivaA基因在转基因柑橘无性繁殖后代中的遗传与表达分析[J]. 农业生物技术学报, 21(7): 820-827.
(Xu L Z, He Y R, Peng A H, et al.2013. Analysis of the inheritance and expression of antibacterial peptide gene ShivaA in transgenic citrus (Citrus sinensis) during its asexual propagation[J]. Journal of Agricultural Biotechnology, 21(7): 820-827.)
12 杨华, 彭城, 肖英平, 等. 2018. 转基因大豆SHZD32-1转化体普通PCR和qRT-PCR检测方法的研究[J]. 农业生物技术学报, 26(3): 492-501.
(Yang H, Peng C, Xiao Y P, et al.2018. Study of conventional PCR and qRT-PCR detection methods for genetically modified soybean (Glycine max) SHZD32-1[J]. Journal of Agricultural Biotechnology, 26(3): 492-501.)
13 Bonny, Sylvie.2016. Genetically modified herbicide-tolerant crops, weeds, and herbicides: Overview and impact[J]. Environmental Management, 57(1): 31-48.
14 Brune P D, Culler A H, Ridley W P, et al.2013. Safety of GM crops: Compositional analysis[J]. Journal of Agricultural & Food Chemistry, 61(35): 8243-8247.
15 Dasmahapatra K K, Silvavásquez A, Chung J W, et al.2007. Genetic analysis of a wild-caught hybrid between non-sister Heliconius butterfly species[J]. Biology Letters, 3(6): 660-663.
16 de Vos C J, Swanenburg M.2017. Health effects of feeding genetically modified (GM) crops to livestock animals: A review[J]. Food and Chemical Toxicology, 117(8): 3-12.
17 Henao Muñoz L M.2015. Acute toxicity and sublethal effects of the mixture glyphosate (Roundup® Active) and Cosmo-Flux®411F to anuran embryos and tadpoles of four Colombian species[J]. Revista de Biologia Tropical, 63(1): 223-233. |
|
|
|
