基于微滴式数字PCR定量检测转基因玉米'CC-2'外源基因和内参基因在土壤中的存留

doi:10.3969/j.issn.1674-7968.2022.05.018

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Abstract Monitoring the persistence and dynamic changes of transgenes in the soil from genetically modified (GM) crop is an important issue in assessing the potential risks of GM crops. The present study applied a novel droplet digital polymerase chain reaction (ddPCR) method to quantitatively detect the copy number concentrations of exogenous gene CP4-EPSPS (Agrobacterium tumefaciens strain CP4 5-enolpyruvyl shikimate-3-phosphate synthase) and reference gene zSSIIb (Zea mays starch synthase isoform zSTSII-2) fragments in the rhizosphere soil of herbicide-resistant transgenic maize (Zea mays) 'CC-2' and its non-transgenic control line 'Zheng-58', at different growth stages of maize (seedling stage, jointing stage, silking stage, milk-ripe stage and full-ripe stage). The limit of quantitation (LOQ) of ddPCR for the CP4-EPSPS and zSSIIb system was (0.48±0.07) and (0.22±0.04) copies/µL, respectively. The copy number concentrations of CP4-EPSPS were lower than the limit of quantitation in the rhizosphere soil of GM maize 'CC-2' at all growth stages, and the positive detection rate of CP4-EPSPS obviously declined with increasing growth stage. There was no significant difference between CP4-EPSPS and zSSIIb concentrations in rhizosphere soil of 'CC-2'. At different growth stage, there was a similar change tendency in the positive detection rate and concentration of zSSIIb gene fragments in rhizosphere soil of 'CC-2' and 'Zheng-58', but the copy number concentration of zSSIIb in rhizosphere soil of 'Zheng-58' was significantly higher than that of 'CC-2' at the jointing stage (P<0.05). Test results showed that the transgene fragments of GM maize could enter into the soil environment through root exudates, but the concentration was very low and showed a decreasing trend with the growth stage, which indicates that the ddPCR method is suitable for sensitive and precise quantitative analysis of the persistent plant DNA fragments in soil environment. This study provided a novel method and reference for quantitative detection of transgene from GM crops in soil.

Key words： Genetically modified maize 'CC-2' Droplet digital PCR (ddPCR) Agrobacterium tumefaciens strain CP4 5-enolpyruvyl shikimate-3-phosphate synthase (CP4-EPSPS) Horizontal gene transfer Soil ecosystem

Received: 01 August 2021

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Corresponding Authors: **liuyan@nies.org

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	DONG Shan-Shan
	XIAO Ze-Hua
	ZHANG Di-Ni
	LIU Yan

Cite this article:

DONG Shan-Shan,XIAO Ze-Hua,ZHANG Di-Ni, et al. Quantitative Detection of Soil Persistence of Exogenous and Reference Gene from Genetically Modified Maize (Zea mays) 'CC-2' by Droplet Digital PCR[J]. 农业生物技术学报, 2022, 30(5): 1014-1022.

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http://journal05.magtech.org.cn/Jwk_ny/EN/10.3969/j.issn.1674-7968.2022.05.018 OR http://journal05.magtech.org.cn/Jwk_ny/EN/Y2022/V30/I5/1014

[1] 陈利, 肖靖秀, 郑毅 . 2016. 间作玉米大豆根系分泌物中有机酸的变化特征[J]. 西南林业大学学报 , 36(5): 78-83.
(Chen L, Xiao J X, Zheng Y.2016. Characteristics of organic acids from root exudation in maize and soybean intercropping[J]. Journal of Southwest Forestry University, 36(5): 78-83.)
[2] 邓婷婷, 黄文胜, 葛毅强, 等 . 2019. 基于微滴式和芯片式数字 PCR 技术的转基因克螟稻成分的定量检测[J]. 食品科学, 40(8): 311-318.
(Deng T T, Huang W S, Ge Y Q, et al.2019. Quantification of genetically modified rice (Oryza sativa L.) event KMD by digital PCR based on droplet and microfluidic chip[J]. Food Science, 40(8): 311-318.)
[3] 冯兆民, 舒跃龙 . 2017. 数字 PCR 技术及其应用进展[J]. 病毒学报 , 33(01): 103-107.
(Feng Z M, Shu Y L.2017. An overview of digital PCR[J]. Chinese Journal of Virology, 33(1): 103-107.)
[4] 国际农业生物技术应用服务组织 .2021. 2019 年全球生物技术/转基因作物商业化发展态势[J]. 中国生物工程杂志, 41(1): 114-119.
(International service for the acquisi‐tion of agri-biotech applications.2021. The global status of commercialized biotech/GM crops in 2019[J]. China Biotechnology, 41(1): 114-119.)
[5] 李刚, 修伟明, 赵建宁, 等 . 2012. 转基因抗虫棉花重组 DNA在土壤中分布的实时定量 PCR 分析[J]. 农业环境科学学报, 31(10): 1933-1940.
(Li G, Xiu W M, Zhao J N, etal.2012. Real time PCR assays for the distribution of recombinant DNA of a transgenic insect-resistant cotton in soil[J]. Journal of Agro-Environment Science, 31(10): 1933-1940.)
[6] 李荣田, 高士童, 高祎, 等 . 2020. Bt 早粳稻对土壤微生物数量影响及其外源基因的转移[J]. 中国农学通报, 36(9): 56-64.
(Li R T, Gao S T, Gao W, et al.2020. Effect of Bt early japonica rice on soil microorganism quantity and transfer of exogenous genes of rice[J]. Chinese Agricultural Science Bulletin, 36(9): 56-64.)
[7] 刘晓, 朱鹏宇, 王垚, 等 . 2018. 数字 PCR 在功能核酸精准检测中的研究进展[J]. 生物技术通报 , 34(9): 149-162.
(Liu X, Zhu P Y, Wang Y, et al.2018. Development progress of digital PCR in the precise detection of functional nucleic acid[J]. Biotechnology Bulletin, 34(9): 149-162.)
[8] 马晓星, 孙伟博, 魏辉, 等 . 2018. 转 PeTLP 基因'南林 895'杨对土壤微生物的影响及外源基因分子检测[J]. 浙江林业科技, 38(4): 28-37.
(Ma X X, Sun W B, Wei H, et al.2018. Effect of transgenic Populus deltoides × P. euramericanna cv. 'Nanlin 895' with PeTLP on soil microbes and molecular analysis on exogenous genes[J]. Journal of Zhejiang Forestry Science Technology, 38(4): 28-37.)
[9] 吴元凤, 李刚, 冀国桢, 等 . 2015. 土壤环境中转基因植物重组 DNA 持留与水平转移研究进展[J]. 生态学杂志, 34(03): 878-884.
(Wu Y F, Li G, Ji G Z, et al.2015. Re‐search progress of persistence and horizontal gene transfer of recombinant DNA from genetically modified plants in soil environment[J]. Chinese Journal of Ecology, 34(3): 878-884.)
[10] 原霖, 董浩, 倪建强, 等 . 2019. 非洲猪瘟病毒微滴数字 PCR检测方法的建立[J]. 畜牧与兽医, 51(7): 81-84.
(Yuan L, Dong H, Ni J Q, et al.2019. Development of droplet digital PCR for detection of African swine fever virus[J]. Animal Husbandry and Veterinary Medicine, 51(7): 81-84.)
[11] 张福锁 .1992. 根分泌物及其在植物营养中的作用(综述)[J]. 北京农业大学学报, 18(04): 353-356.
(Zhang F S.1992. Root exudates and their role in plant nutrition (a review)[J]. Acta Agriculturae Universitatis Pekinensis, 18(4): 353-356.)
[12] Bosman K J, Nijhuis M, van Ham P M, et al.2015. Compari‐son of digital PCR platforms and semi-nested qPCR as a tool to determine the size of the HIV reservoir[J]. Scientific Reports, 5(1): 1-9.
[13] Cao Y, Raith M R, Griffith J F.2015. Droplet digital PCR for simultaneous quantification of general and human-associated fecal indicators for water quality assessment[J]. Water Research, 70: 337-349.
[14] Cleaves H J, Crapster-Pregont E, Jonsson C M, et al.2011. The adsorption of short single-stranded DNA oligomers to mineral surfaces[J]. Chemosphere, 83(11): 1560-1567.
[15] de Vries J, Heine M, Harms K, et al.2003. Spread of recombinant DNA by roots and pollen of transgenic potato plants, identified by highly specific biomonitoring using natural transformation of an Acinetobacter sp[J]. Applied and Environmental Microbiology, 69(8): 4455-4462.
[16] del Pozo O, Lam E.1998. Caspases and programmed cell death in the hypersensitive response of plants to pathogens[J]. Current Biology, 8(20): 1129-1132.
[17] Dong S S, Zhang D N, Yu C G, et al.2021. Using droplet digital PCR to detect plant DNA in tissues of zebrafish (Danio rerio) fed genetically modified maize[J]. Aquaculture Research, 52(9): 4467-4474.
[18] Douville M, Gagné F, Blaise C, et al.2007. Occurrence and persistence of Bacillus thuringiensis (Bt) and transgenic Bt corn cry1Ab gene from an aquatic environment[J]. Ecotoxicology and Environmental Safety, 66(2): 195-203.
[19] Dubelman S, Fischer J, Zapata F, et al.2014. Environmental fate of double-stranded RNA in agricultural soils[J]. PLOS ONE, 9(3): e93155.
[20] Hay I, Morency M J, Séguin A.2002. Assessing the persistence of DNA in decomposing leaves of genetically modified poplar trees[J]. Canadian Journal of Forest Research, 32(6): 977-982.
[21] Hunter M E, Dorazio R M, Butterfield J S S, et al.2017. Detection limits of quantitative and digital PCR assays and their influence in presence -absence surveys of environmental DNA[J]. Molecular Ecology Resources, 17(2): 221-229.
[22] Iijima M, Griffiths B, Bengou G.2000. Sloughing of cap cells and carbon exudation from maize seedling roots in compacted sand[J]. New Phytologist, 145(3): 477-482.
[23] Lerat S, Gulden R H, Hart M M, et al.2007. Quantification and persistence of recombinant DNA of Roundup Ready corn and soybean in rotation[J]. Journal of Agricultural and Food Chemistry, 55(25): 10226-10231.
Levy-Booth D, Campbell R, Gulden R, et al.2008. Real-time polymerase chain reaction monitoring of recombinant DNA entry into soil from decomposing roundup ready leaf biomass[J]. Journal of Agricultural and Food Chemistry, 56(15): 6339-6347.
[24] Lo C C, Chen S C, Yang J Z.2007. Use of real-time polymerase chain reaction (PCR) and transformation assay to monitor the persistence and bioavailability of transgenic genes released form genetically modified papaya expressing nptⅡ and PRSV genes in the soil[J]. Agricul‐tural and Food Chemistry, 55(18): 7534-7540.
[25] Morisset D, Štebih D, Milavec M, et al.2013. Quantitative analysis of food and feed samples with droplet digital PCR[J]. PLOS ONE, 8(5): e62583.
[26] Mulero S, Boissier J, Allienne J F, et al.2020. Environmental DNA for detecting Bulinus truncatus: A new environ‐mental surveillance tool for schistosomiasis emergence risk assessment[J]. Environmental DNA, 2(2): 161-174.
[27] Pontiroli A, Ceccherini M T, Pote J, et al.2010. Long-term persistence and bacterial transformation potential of transplastomic plant DNA in soil[J]. Research in Microbiology, 161(5): 326-334.
[28] Pontiroli A, Simonet P, Frostegard A, et al.2007. Fate of transgenic plant DNA in the environment[J]. Environmental Biosafety Research, 6(1-2): 15-35.
[29] Rizzi A, Raddadi N, Sorlini C, et al.2012. The stability and degradation of dietary DNA in the gastrointestinal tract of mammals: Implications for horizontal gene transfer and the biosafety of GMOs[J]. Critical Reviews in Food Science and Nutrition, 52(2): 142-161.
[30] Tepfer D, Garcia-Gonzales R, Mansouri H, et al.2003. Homology-dependent DNA transfer from plants to a soil bacterium under laboratory conditions: Implications in evolution and horizontal gene transfer[J]. Transgenic Research, 12(4): 425-437.
[31] Zhang F L, Niu B, Chang L J, et al.2018. A new construct specific real-time PCR method for screening GMO in‐gredients with gat-tpin Ⅱ cassette in foods, feeds and seeds[J]. Food Control, 86(4): 266-274.
[32] Zhu B, Ma B L, Blackshaw R E.2010. Development of real time PCR assays for detection and quantification of transgene DNA of a Bacillus thuringiensis (Bt) corn hybrid in soil samples[J]. Transgenic Research, 19(5): 765-774.
[33] Zuo L H, Yang R L, Zhen Z X, et al.2018. A 5-year field study showed no apparent effect of the Bt transgenic 741 poplar on the arthropod community and soil bacterial diversity[J]. Scientific Reports, 8(1): 1-13.