Bt早粳稻对土壤微生物数量影响及其外源基因的转移

doi:10.11924/j.issn.1000-6850.casb20191000720

摘要/Abstract

摘要：

为了研究种植Bt早粳稻对土壤微生物数量影响以及Bt早粳稻中的外源DNA向土壤微生物水平转移情况,以Bt早粳稻各生长发育期田间土壤为试材,用平板菌落计数法、PCR法测得土壤微生物数量和Bt基因等DNA片段等数据。结果显示：在水稻生长发育的返青期、分蘖期、孕穗期、抽穗期和成熟期,种植Bt早粳稻‘HD1’、‘HD2’、‘HD3’和‘HD4’等品系的田间根际及行间土壤细菌、放线菌和真菌数量与种植其受体品种对应田间土壤微生物数量相比,均未见显著不同;未发现Bt早粳稻各品系外源DNA片段向土壤细菌、放线菌和真菌等微生物的水平转移现象。Bt早粳稻田间土壤微生物数量与普通水稻相比较没有显著差异,土壤微生物中也未见来自于Bt水稻的外源DNA。

关键词: Bt早粳稻, 基因水平转移, PCR, 土壤微生物, 环境安全性

Abstract:

To study the effect of transplanting Bt early japonica rice on soil microorganism number and the horizontal transfer of exogenous DNA of rice to soil microorganism, using field soil at different growth stages of rice as material, the number of soil microorganisms and exogenous DNA fragments such as Bt gene were measured with plate colony counting and PCR detection. The results showed that there were no significant differences among Bt early rice lines (‘HD1’, ‘HD2’) and corresponding recipient variety or among lines (‘HD3’, ‘HD4’) and their receptor in the number of bacteria, actinomycetes and fungus of rhizosphere and inter-row soil at rice growth and development stages of returning green, tillering, booting, heading and maturity. It was not found that exogenous DNA fragments of Bt early japonica rice lines were horizontally transferred to bacteria, actinomycetes and fungi of soil microorganism. There was no significant difference in the number of soil microorganisms between Bt early japonica rice and normal rice, and no exogenous DNA of Bt rice was found in soil microorganisms.

Key words: Bt early japonica rice, horizontal gene transfer, PCR, soil microorganism, environmental safety

中图分类号:

S511.22
Q786

李荣田, 高士童, 高祎, 刘长华. 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, 2020, 36(9): 56-64.

Rongtian Li, Shitong Gao, Yi Gao, Changhua Liu. Effect of Bt Early Japonica Rice on Soil Microorganism Quantity and Transfer of Exogenous Genes of Rice[J]. Chinese Agricultural Science Bulletin, 2020, 36(9): 56-64.

图/表 9

参考文献 40

[1]	王敏,关潇.不同转基因作物对土壤微生物的影响[J].山西农业科学,2015,43(02):229-232,243.
[2]	关潇,吴刚,王敏.转Bt基因水稻对土壤微生物群落结构的影响[J].湖北农业科学,2015,54(05):1046-1052,1058.
[3]	陈承利,廖敏,曾路生.污染土壤微生物群落结构多样性及功能多样性测定方法[J].生态学报,2006,26(10):3404-3412.
[4]	谷荣辉,成功,李建钦,等.转基因水稻对土壤性质及土壤生物的影响[J].云南农业大学学报:自然科学,2014,29(03):448-457.
[5]	Angle J S.Release of transgenic plants: biodiversity and population-level considerations[J]. Molecular ecology,1994,3(1):45-50.
[6]	王洪兴,陈欣,唐建军,等.释放后的转抗病虫基因作物对土壤生物群落的影响[J].生物多样性,2002,10(02):232-237.
[7]	王洪兴,陈欣,唐建军,等.转Bt基因水稻秸秆降解对土壤微生物可培养类群的影响[J].生态学报,2004,24(01):89-94.
[8]	赵佳,聂园军,黄静,等.转基因作物影响土壤微生物群落多样性的研究[J].山西农业科学,2017,45(10):1702-1705.
[9]	白耀宇,蒋明星,程家安.Bt水稻Cry1Ab杀虫蛋白表达的时间动态及其在水稻土中的降解[J].生态学报,2005,25(07):1583-1590.
[10]	Saxena D, Flores S, Stotzky G.Bt toxin is released in root exudates from 12 transgenic corn hybrids representing three transformation events[J]. Soil Biology and Biochemistry,2002,34(1):0-137.
[11]	Icoz I, Stotzky G.Fate and effects of insect-resistant Bt crops in soil ecosystems[J]. Soil Biology & Biochemistry,2008,40(3):559-586.
[12]	Baumgarte S, Tebbe C C.Field studies on the environmental fate of the Cry1Ab Bt-toxin produced by transgenic maize (MON810) and its effect on bacterial communities in the maize rhizosphere[J]. Molecular ecology,2005,14(8):2539-2551.
[13]	Rui Y K, Yi G X, Zhao J, et al.Changes of Bt Toxin in the Rhizosphere of Transgenic Bt Cotton and its Influence on Soil Functional Bacteria[J]. World Journal of microbiology & biotechnology,2005,266(1-2):77-89.
[14]	Stotzky G.Persistence and biological activity in soil of the insecticidal proteins from Bacillus thuringiensis, especially from transgenic plants[J]. Plant and Soil,2005,266(1-2):77-89.
[15]	陈晓雯,林胜,尤民生,等.转基因水稻对土壤微生物群落结构及功能的影响[J].生物安全学报,2011,20(02):151-159.
[16]	陈敏,应文荷.转Bt水稻与常规水稻根际土壤细菌类群的比较研究[J].杭州师范学院学报:自然科学版,2005,4(04):290-292.
[17]	Li Z L, Bu N S, Cui J, et al.Effects of long-term cultivation of transgenic Bt rice (Kefeng-6) on soil microbial functioning and C cycling[J]. Scientific Reports,2017,7(1):4647.
[18]	李本金,李玥仁,胡奇勇,等.抗真菌转基因水稻对根际土壤微生物群落的影响[J].福建农业大学学报,2006,35(03):319-323.
[19]	Sahoo R K, Ansari M W, Tuteja R, et al.Salt tolerant SUV3 overexpressing transgenic rice plants conserve physicochemical properties and microbial communities of rhizosphere[J]. Chemosphere,2015,119:1040-1047.
[20]	Wu W X, Ye Q F, Min H, et al.Bt-transgenic rice straw affects the culturable microbiota and dehydrogenase and phosphatase activities in a flooded paddy soil[J]. Soil Biology & Biochemistry, 2004,36(2):289-295.
[21]	杨永华. 转基因作物对土壤微生物群落的影响及主要研究策略[J].农业生物技术学报,2011,19(01):1-8.
[22]	唐影,李世东,缪作清.转基因作物对土壤微生物的影响[J].中国生物防治,2007,23(04):383-390.
[23]	吴迪,王秋玉.转基因植物对根际土壤生态系统的影响[J].中国生物工程杂志,2007,27(02):113-118.
[24]	刘华,常晓蕾,蒋玮,等.转基因棉花中选择性标记基因nptⅡ对土壤微生物的影响[J].上海农业学报,2017,33(02):7-12.
[25]	王倩倩,杜国强,师校欣.转CpTI基因苹果种植地外源基因残留及对土壤微生物的影响[J].分子植物育种,2014,12(06):1175-1180.
[26]	马晓星,孙伟博,魏辉,等.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,2018,38(04):28-37.
[27]	马晓星,孙伟博,魏辉,等.转AtSnRK2C基因杨树田间试验及生物安全性分析[J].分子植物育种,2019,17(11):3570-3578.
[28]	朱文旭,丁昌俊,张伟溪,等.8年生转基因库安托杨外源基因转移及对土壤微生物数量影响的检测[J].林业科学研究,2017,30(02): 349 353.
[29]	吕威,卢楠,侯荣轩,等.12年生转基因毛白杨外源基因转移及其对土壤微生物的影响[J].东北林业大学学报,2018,46(09):1-6.
[30]	吕威,孙宇涵,张华新,等.转基因三倍体毛白杨花粉活力及枯落物外源基因检测[J].北京林业大学学报,2019,41(07):91-100.
[31]	梁晋刚,张秀杰.转基因作物对土壤微生物多样性影响的研究方法[J].生物技术通报,2017,33(10):111-116.
[32]	李相全,张忠亮,陶波,等.抗草甘膦转基因大豆对土壤酶活性及微生物数量的影响[J].大豆科学,2015,34(04):648-652.
[33]	陶波,曹宝祥,韩玉军,等.转G2-EPSPS和GAT基因抗草甘膦大豆对土壤微环境的影响[J].中国生物防治学报,2019,35(02):203-208.
[34]	Xie M, Zhang Y J, Peng D L, et al.Field studies show no significant effect of a Cry1Ab/Ac producing transgenic cotton on the fungal community structure in rhizosphere soil[J]. European Journal of Soil Biology,2016,73:69-76.
[35]	吕秀华. 转基因银中杨对根际土壤微生物的影响[J].基因组学与应用生物学,2018,37(05):1965-1970.
[36]	Davison J.Genetic exchange between bacteria in the environment[J]. Plasmid,1999,42(2):73-91.
[37]	Gebhard F, Smalla K.Monitoring field releases of genetically modified sugar beets for persistence of transgenic plant DNA and horizontal gene transfer[J]. FEMS Microbiology Ecology,1999,28(3):261-272.
[38]	Paget E, Lebrun M, Freyssinet G, et al.The fate of recombinant plant DNA in soil[J]. European Journal of Soil Biology,1998,34(2):81-88.
[39]	周琴,孙明,李林,等.苏云金芽胞杆菌标记重组菌株的构建与杀虫基因水平转移[J].应用生态学报,2005,16(01):142-146.
[40]	Zuo L, Yang R, Zhen Z, et al.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,2018,8(1):1956.

DNA片段	序列		PCR产物/bp
DNA片段	上游引物	下游引物	PCR产物/bp
cry1C^*	5′TTCTACTGGGGAGGACATCG3′<break/>5′CGGTATCTTTGGGTGATTGG3′	5′CGGTATCTTTGGGTGATTGG3′	602
cry2A^*	5′CGTGTCAATGCGGACCTGAT3′<break/>5′GATGCCGGACAGGATGTAGT3′	5′GATGCCGGACAGGATGTAGT3′	600
bar	5′TCTCGGTGACGGGCAGGAC3′<break/>5′CCTGGAAGGCACGCAACG3′	5′CCTGGAAGGCACGCAACG3′	323
P_35S	5′GCTCCTACAAATGCCATCATTGA3′<break/>5′GATAGTGGGATTGTGCGTCATCCC3′	5′GATAGTGGGATTGTGCGTCATCCC3′	195
T_35S	5′GTGGTGTAAACAAATTGACGCTTAG3′<break/>5′GTTTCGCTCATGTGTTGAGCAT3′	5′GTTTCGCTCATGTGTTGAGCAT3′	206
NOS-cry2A^*	5′GAGCTCGAATTTCCCCGATCGTTC3′<break/>5′CCGATCTAGTAACATAGATGACACCG3′	5′CCGATCTAGTAACATAGATGACACCG3′	311
NOS-cry1C^*	5′GAATTTCCCCGATCGTTCAAACA3′<break/>5′CCGATCTAGTAACATAGATGACACCG3′	5′CCGATCTAGTAACATAGATGACACCG3′	305
ubi	5′GGACACCAACCAGCGAACCAG3′<break/>5′GTGCCGTGAGAGGAGGAGGAG3′	5′GTGCCGTGAGAGGAGGAGGAG3′	201
16S rDNA	5′GAGAGTTTGATCCTGGCTCAG3′<break/>5′CTACGGCTACCTTGTTACGA3′	5′CTACGGCTACCTTGTTACGA3′	约1400
18S rDNA	5′GTAGTCATATGCTTGTCTC3′<break/>5′TCCGCAGGTTCACCTACGGA3′	5′TCCGCAGGTTCACCTACGGA3′	约1800

DNA片段	序列		PCR产物/bp
DNA片段	上游引物	下游引物	PCR产物/bp
cry1C^*	5′TTCTACTGGGGAGGACATCG3′<break/>5′CGGTATCTTTGGGTGATTGG3′	5′CGGTATCTTTGGGTGATTGG3′	602
cry2A^*	5′CGTGTCAATGCGGACCTGAT3′<break/>5′GATGCCGGACAGGATGTAGT3′	5′GATGCCGGACAGGATGTAGT3′	600
bar	5′TCTCGGTGACGGGCAGGAC3′<break/>5′CCTGGAAGGCACGCAACG3′	5′CCTGGAAGGCACGCAACG3′	323
P_35S	5′GCTCCTACAAATGCCATCATTGA3′<break/>5′GATAGTGGGATTGTGCGTCATCCC3′	5′GATAGTGGGATTGTGCGTCATCCC3′	195
T_35S	5′GTGGTGTAAACAAATTGACGCTTAG3′<break/>5′GTTTCGCTCATGTGTTGAGCAT3′	5′GTTTCGCTCATGTGTTGAGCAT3′	206
NOS-cry2A^*	5′GAGCTCGAATTTCCCCGATCGTTC3′<break/>5′CCGATCTAGTAACATAGATGACACCG3′	5′CCGATCTAGTAACATAGATGACACCG3′	311
NOS-cry1C^*	5′GAATTTCCCCGATCGTTCAAACA3′<break/>5′CCGATCTAGTAACATAGATGACACCG3′	5′CCGATCTAGTAACATAGATGACACCG3′	305
ubi	5′GGACACCAACCAGCGAACCAG3′<break/>5′GTGCCGTGAGAGGAGGAGGAG3′	5′GTGCCGTGAGAGGAGGAGGAG3′	201
16S rDNA	5′GAGAGTTTGATCCTGGCTCAG3′<break/>5′CTACGGCTACCTTGTTACGA3′	5′CTACGGCTACCTTGTTACGA3′	约1400
18S rDNA	5′GTAGTCATATGCTTGTCTC3′<break/>5′TCCGCAGGTTCACCTACGGA3′	5′TCCGCAGGTTCACCTACGGA3′	约1800

性状	HD1	HD2	HD3	HD4	CK1	CK2
Basta抗感特性	+	+	+	+	–	–
Cry1C-蛋白	+	–	+	–	–	–
Cry2A-蛋白	–	+	–	+	–	–
bar-PCR	+	+	+	+	–	–
cry1C^*-PCR	+	–	+	–	–	–
cry2A^*-PCR	–	+	–	+	–	–

性状	HD1	HD2	HD3	HD4	CK1	CK2
Basta抗感特性	+	+	+	+	–	–
Cry1C-蛋白	+	–	+	–	–	–
Cry2A-蛋白	–	+	–	+	–	–
bar-PCR	+	+	+	+	–	–
cry1C^*-PCR	+	–	+	–	–	–
cry2A^*-PCR	–	+	–	+	–	–