Molecular Characteristics and Functional Evaluation of Transgenic Maize BFL4-1 With Stacked Insect and Herbicide Resistance Traits

doi:10.13304/j.nykjdb.2020.0111

Abstract

Abstract:

Ostrinia furnacalis （Asian corn borer） and weeds seriously affect maize growth， development and production in China. Transgenic stacking maizes with insect and herbicide resistance traits have become the main trend of transgenic maize breeding. cry1Ab， cry1F and cp4epsps were introduced into maize Hi-II mediated by Agrobacteria. After backcrossing with maize inbred line Zheng 58 and then self-pollination， one transgenic line named BFL4-1 was obtained. Through whole genome sequencing and flanking sequence analysis， there was one insertion in BFL4-1 and the insertion fragment was located in the intergenic region of chromosome 3. In BFL4-1 plants， cry1Ab， cry1F and cp4epsps were detected in different tissues on transcription and protein levels. Field experiments revealed that the BFL4-1 transgenic plants were highly resistant to Asian corn borer and more tolerant to glyphosate. There were no significant differences between BFL4-1 and the control （Zheng 58） on agronomic traits. Above results showed that BFL4-1 line could be served as a new maize transgenic line with stacked traits showing high resistance to insect and glyphosate.

Key words: transgenic maize, insect resistance, herbicide resistance, stacking trait, molecular characterization, stability of inheritance

摘要：

玉米螟和田间杂草严重影响我国玉米的生长发育，导致玉米产量和品质下降。培育具有抗虫、耐除草剂等复合性状的转基因玉米成为转基因玉米育种的一个趋势。将cry1Ab、cry1F和cp4epsps构建在同一载体上并利用农杆菌转化到玉米Hi-II中，通过与玉米自交系郑58进行回交转育，获得了具有郑58遗传背景并兼具抗虫和耐除草剂性状的转基因玉米株系BFL4-1。全基因组高通量测序和侧翼序列测序分析表明，BFL4-1中目的基因插入片段位于第3染色体基因间区，为单一插入位点。转录水平和蛋白水平检测结果表明，cry1Ab、cry1F和cp4epsps在不同组织中均能稳定表达。田间玉米螟接虫和喷施草甘膦试验表明，BFL4-1株系高抗玉米螟并且对草甘膦具有较好的耐受性，与对照相比在农艺性状方面无显著差异。由此表明，获得的BFL4-1为具有抗玉米螟和耐草甘膦除草剂复合性状的转基因玉米新材料。

关键词: 转基因玉米, 抗虫, 耐除草剂, 复合性状, 分子特征, 遗传稳定性

CLC Number:

S513

Junjie ZOU, Miaoyun XU, Lan ZHANG, Hongyan ZHENG, Lei WANG. Molecular Characteristics and Functional Evaluation of Transgenic Maize BFL4-1 With Stacked Insect and Herbicide Resistance Traits[J]. Journal of Agricultural Science and Technology, 2022, 24(1): 31-37.

邹俊杰, 徐妙云, 张兰, 郑红艳, 王磊. 转基因复合抗虫耐除草剂玉米BFL4-1的分子特征及功能评价[J]. 中国农业科技导报, 2022, 24(1): 31-37.

Figures/Tables 6

References 18

1	孙卓婧，张安红，叶纪明.转基因作物研发现状及展望［J］. 中国农业科技导报， 2018， 20（7）：11-18.
	SUN Z J， ZHANG A H， YE J M. Research and development status and prospect of genetically modified crops ［J］. J. Agric. Sci. Technol.， 2018， 20（7）： 11-18.
2	沈平，章秋艳，林友华，等. 推进我国转基因玉米产业化的思考［J］. 中国生物工程杂志， 2016， 36（4）：24-29.
	SHENG P， ZHANG Q Y， LIN Y H， et al.. Thinking to promote the industrialization of genetically modified corn of our country ［J］. China Biotechnol.， 2016， 36（4）： 24-29.
3	国际农业生物技术应用服务组织. 2018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， 2019， 39（8）：1-6.
	ISAAA. Global status of commercialized biotech/gm crops in 2018 ［J］. China Biotech.， 2019， 39（8）： 1-6.
4	QUE Q， CHILTON M-D M， DE FONTES C M， et al.. Trait stacking in transgenic crops： challenges and opportunities ［J］. GM Crops， 2010， 1（4）： 220-229.
5	王旭静，张欣，刘培磊，等. 复合性状转基因植物的应用现状与安全评价［J］. 中国生物工程杂志，2016，36（4）：18-23.
	WANG X J， ZHANG X， LIU P L， et al.. The application and safe assessment of stacked transgenic plants ［J］. China Biotech.， 2016， 36（4）： 18-23.
6	沈平，武玉花，梁晋刚，等. 转基因作物发展及应用概述［J］. 中国生物工程杂志， 2017， 37（1）：119-128.
	SHENG P， WU Y H， LIANG J G， et al.. The overview in development and application of genetically modified crops ［J］. China Biotech.， 2017， 37（1）：119-128.
7	束长龙，张风娇，黄颖，等. Bt杀虫基因研究现状与趋势［J］. 中国科学：生命科学， 2016， 46（5）：548-555.
	SHU C L， ZHANG F J， HUANG Y， et al.. Current status and research trends of Bt insecticidal gene ［J］. Sci. Sin. Vitae， 2016， 46（5）： 548-555.
8	FRAME B R， MCMURRAY J M， FONGER T M， et al.. Improved Agrobacterium-mediated transformation of three maize inbred lines using MS salts ［J］. Plant Cell Rep.， 2006， 25（10）：1024-1034.
9	GUTTIKONDA S K， MARRI P， MAMMADOV J， et al.. Molecular characterization of transgenic events using next generation sequencing approach ［J/OL］. PLoS ONE， 2016， 11（2）： e0149515 ［2021-03-19］. .
10	梁海生，李梦桃，李圣彦，等. 转Bt基因抗虫玉米HGK60的农艺性状分析［J］. 生物技术通报， 2018， 34（7）：92-100.
	LIANG H S， LI M T， LI S Y， et al.. Agronomic traits analysis of transgenic Bt cry1Ah maize HGK60 line ［J］. Biotechnol. Bull.， 2018， 34（7）： 92-100.
11	赫福霞，郎志宏，张杰，等. 转Bt cry1Ah/2mG2-epsps双价基因抗虫/耐除草剂玉米研究［J］. 作物杂志，2012，6：29-33.
	HE F X， LANG Z H， ZHANG J， et al.. Insect-resistant and glyphosate-tolerant transgenic maize transformed by Bt cry1Ah and 2mG2-epsps genes ［J］. Crop J.， 2012， 6： 29-33.
12	余桂容，曹颖，杜文平，等. 抗虫耐草甘膦双价基因表达载体的构建及玉米转化［J］. 西南农业学报， 2015， 25（2）：475-479.
	YU G R， CAO Y， DU W P， et al.. Construction of binary vector obtaining insect and herbicide resistance genes and transformation of maize ［J］. Southwest China J. Agric. Sci.， 2015， 25（2）： 475-479.
13	杨小艳，翁建峰，谢树章，等. 转cry1Ab_Ma基因抗虫玉米植株的获得及其后代分析［J］.分子植物育种， 2015， 13（9）：1943-1948.
	YANG X Y， WENG J F， XIE S Z， et al.. Construction and genetic transformation of plant expression vector harboring insect resistant gene cry1Ab-Ma in maize ［J］. Mol. Plant Breed.， 2015， 13（9）： 1943-1948.
14	刘小丹，李淑华，徐国良，等. 转基因玉米育种研究进展［J］. 玉米科学， 2012， 20（6）：1-8.
	LIU X D， LI S H， XU G L， et al.. Review of genetically modified maize ［J］. J. Maize Sci.， 2012， 20（6）： 1-8.
15	孙红炜，徐晓辉，李凡，等. 抗虫耐除草剂玉米杂交种瑞丰1号-双抗12-5’对3种主要鳞翅目害虫的抗性［J］. 山东农业科学， 2018， 50 （5）：109-114.
	SUN H， XU X， LI F， et al.. Resistances of transgenic maize hybrid ‘Ruifeng 1-double resistance 12-5’ with insect and herbicide resistance against three major lepidopteran pests ［J］. Shandong Agric. Sci.， 2018， 50（5）：109-114.
16	孙越，刘秀霞，李丽莉，等. 兼抗虫、除草剂、干旱转基因玉米的获得和鉴定［J］. 中国农业科学， 2015， 48（2）：215-228.
	SUN Y， LIU X X， LI L L， et al.. Production of transgenic maize germplasm with multi-traits of insect-resistance， glyphosate-resistance and drought-tolerance ［J］. Sci. Agric. Sin.， 2015， 48（2）： 215-228.
17	李国平，刘冰，黄建荣，等. 转聚合cry1A.105、cry2Ab2和cp4epsps基因抗虫耐除草剂玉米的田间抗性评价［J］. 植物保护， 2019， 45 （1）：142-147.
	LI G P， LIU B， HUANG J R， et al.. Field evaluation of Bt corn expression cry1A.105 + cry2Ab2 + cp4epsps against Lepidoptera pests and tolerance to glyphosate ［J］. Plant Protect.， 2019， 45（1）： 142-147.
18	柳青，刘洋，郭嘉，等. 新型高抗玉米螟虫、耐除草剂转基因玉米和非转基因玉米营养成分的比较分析［J］. 东北农业科学， 2018， 43 （4）：27-31.
	LIU Q， LIU Y， GUO J， et al.. Comparison and analysis of nutrients in new high resistant to corn borer， herbicide-tolerant transgenic corn and no-GM corn ［J］. J. Northeast Agric. Sci.， 2018， 43（4）： 27-31.

引物名称 Primer name	引物序列 Primer sequence（5’-3’）	扩增片段大小Fragment size/bp	注释 Reference
cry1Ab-1F cry1Ab-1R	CTACTTGTACCAGAAGATCGAC GACCGGCAACAGGATTCAATC	356	cry1Ab 鉴定cry1Ab identification
cry1F-1F cry1F-1R	CTTACGTTCCAGTCCTTCTCC CTCAACACATGAGCGAAACCC	403	cry1F鉴定cry1F identification
cp4epsps-1F cp4epsps-1R	TCGATCACCGTATCGCTATGA CAGACTCGCAAGAACTCGCAC	254	cp4epsps 鉴定cp4epsps identification
BFL4-1-TF BFL4-1-TR	TAACCGTAGCAGGGGTAGTTAGTT TGTGTGAGTAGTTCCCAGATAAG	548	BFL4-1特异性检测 Specific detection of BFL4-1
cry1Ab-qF cry1Ab-qR cry1AF-qF cry1AF-qR cp4epsps-qF cp4epsps-qR zSSIIb-qF zSSIIb-qR	CTACTTGTACCAGAAGATCGAC TCAGTCCTCGTTCAGGTCGGTG CTTACGTTCCAGTCCTTCTC TCACTCATCTAGGCAGAACTCG TCGATCACCGTATCGCTATGA TCAAGCAGCCTTAGTGTCGGAG CGGTGGATGCTAAGGCTGATG AAAGGGCCAGGTTCATTATCCTC	256	qRT-PCR
		312
		164
		88

引物名称 Primer name	引物序列 Primer sequence（5’-3’）	扩增片段大小Fragment size/bp	注释 Reference
cry1Ab-1F cry1Ab-1R	CTACTTGTACCAGAAGATCGAC GACCGGCAACAGGATTCAATC	356	cry1Ab 鉴定cry1Ab identification
cry1F-1F cry1F-1R	CTTACGTTCCAGTCCTTCTCC CTCAACACATGAGCGAAACCC	403	cry1F鉴定cry1F identification
cp4epsps-1F cp4epsps-1R	TCGATCACCGTATCGCTATGA CAGACTCGCAAGAACTCGCAC	254	cp4epsps 鉴定cp4epsps identification
BFL4-1-TF BFL4-1-TR	TAACCGTAGCAGGGGTAGTTAGTT TGTGTGAGTAGTTCCCAGATAAG	548	BFL4-1特异性检测 Specific detection of BFL4-1
cry1Ab-qF cry1Ab-qR cry1AF-qF cry1AF-qR cp4epsps-qF cp4epsps-qR zSSIIb-qF zSSIIb-qR	CTACTTGTACCAGAAGATCGAC TCAGTCCTCGTTCAGGTCGGTG CTTACGTTCCAGTCCTTCTC TCACTCATCTAGGCAGAACTCG TCGATCACCGTATCGCTATGA TCAAGCAGCCTTAGTGTCGGAG CGGTGGATGCTAAGGCTGATG AAAGGGCCAGGTTCATTATCCTC	256	qRT-PCR
		312
		164
		88

农艺性状Agronomic trait	郑58 Zheng 58	BFL4-1
生育周期Growth period/d	110±2.0	108±2.0
抽雄期Tasseling stage/d	59±2.0	58±2.0
株高Plant height/m	1.83±0.25	1.78±0.2
穗位高Ear height/m	0.85±0.15	0.84±0.1
穗行数Rows per era	14±0	14±0
行粒数Kernels per row	36.0±2.0	35.0±2.0
百粒重100-kernel weight/g	29.8±2.1	28.8±2.2
萌发率Germination rate/%	95.0±1.6	96.0±2.0

农艺性状Agronomic trait	郑58 Zheng 58	BFL4-1
生育周期Growth period/d	110±2.0	108±2.0
抽雄期Tasseling stage/d	59±2.0	58±2.0
株高Plant height/m	1.83±0.25	1.78±0.2
穗位高Ear height/m	0.85±0.15	0.84±0.1
穗行数Rows per era	14±0	14±0
行粒数Kernels per row	36.0±2.0	35.0±2.0
百粒重100-kernel weight/g	29.8±2.1	28.8±2.2
萌发率Germination rate/%	95.0±1.6	96.0±2.0

[1]	LIU Jianfeng1, YAO Ying1, ZHAO Jie1, SU Zhi2, WEI Jianrong1*. Mechanism of Insect Resistance of Seabuckthorn (Hippophae rhamnoides L.)and Progress on Molecular Breeding [J]. Journal of Agricultural Science and Technology, 2020, 22(8): 108-115.
[2]	MA Shuo1, JIAO Yue2, WANG Xujing1, ZHAI Yong2, WANG Zhixing1*. Genome Sequencing Application on Molecular Characterization Assessment of Transgenic Plant [J]. Journal of Agricultural Science and Technology, 2020, 22(5): 6-14.
[3]	XU Xiaoli1, JIANG Yuanyuan1, WANG Pengfei2, LAI Yongmin1, CHEN Xiaoyun1, XU Junfeng1*. Resistance Evaluation of Genetically Modified Maize GAB-3 Expressing Cry1Ab and Cry2Ab Against Four Major Lepidopteran Pests [J]. Journal of Agricultural Science and Technology, 2020, 22(12): 97-104.
[4]	YANG Yang1,2, LIANG Chengzhen2, MENG Zhigang2, CHEN Quanjia1, LUO Shuping1, ZHANG Rui2, GUO Sandui2*. Construction of Insect and Herbicide Resistant Expression Vector Based on GR79 EPSPS Screening Marker and Identification of Resistance [J]. Journal of Agricultural Science and Technology, 2018, 20(11): 22-28.
[5]	MEI Chuang1, YAN Peng1, Mamat A1, MA Kai1, HAN Li-qun1, XU Zheng2, . The Relationship between Bark Thickness and Diameter Class on Agrilus mali Damage in Xinjiang Wild Apple [J]. Journal of Agricultural Science and Technology, 2016, 18(4): 24-30.
[6]	MAN Kun1,2§, YANG Bing1§, XUE Jing1§, QIN Xiang\\|jie1,2, ZHOU Hong\\|zhuan1, XU F. Expression of Core Neutralizing Epitope Gene of Porcine Epidemic Diarrhea Virus in Maize [J]. , 2014, 16(6): 28-35.
[7]	YANG Zhao-jun1, LANG Zhi-hong1, ZHANG Jie2, SONG Fu-ping2, HE Kang-lai2, HUANG D. Studies on Insect-resistant Transgenic Maize (Zea mays L.) Harboring Bt cry1Ah and cry1Ie Genes [J]. , 2012, 14(4): 39-45.
[8]	LI Sheng-yan1,2, LANG Zhi-hong2, ZHU Li2, LI Xiu-ying1,2, ZHANG Jie3, HE Kang-la. Improvement of Bt cry1Ah Gene Expression in Transgenic Maize (Zea mays L.) through Codon Optimization [J]. , 2011, 13(6): 20-26.
[9]	WANG Xin1, KUANG Wei1,2, WAN Fang-Hao1 . Research Advances in the Effect of Resistance Gene Mi-1 in Tomato [J]. , 2009, 11(5): 23-29.
[10]	WANG Xiu-jun, LANG Zhi-hong\| LU Wei, LIN Min\| SHAN An-shan, HUANG Da-fang . Transferring Glyphosate-tolerant mG2-epsps Gene into Maize \|Inbred Line via Pollen-tube Pathway [J]. , 2008, 10(4): 56-62.