转GbVe1基因在棉花基因组中的整合与定位分析

doi:10.11963/1002-7807.ctzzbl.20190105

摘要/Abstract

摘要：

【目的】明确转GbVe1基因棉花的插入位点序列特征。【方法】Southern杂交筛选低拷贝基因插入的转基因棉花株系,以hiTAIL-PCR（Polymerase chain reaction）获取其T-DNA侧翼序列,然后根据获得的T-DNA侧翼序列设计特异PCR引物,验证插入位点的准确性。【结果】Southern杂交候选了T-DNA低拷贝插入的3个转基因棉花株系,hiTAIL-PCR分离到RB端侧翼序列（119~1 018 bp）、LB端侧翼序列（243~516 bp）; 侧翼序列的AT碱基含量在63%以上。转基因株系7/100826-152和12/100826-393插入位点都位于Gohir.D01G157600.1内含子上。转基因株系1/w-ch14插入位点分别位于Gohir.D01G157600.1内含子和A12染色体的基因间隔区中。T-DNA在Gohir.D01G157600.1内含子的插入事件造成了21 bp碱基的基因组序列缺失。T-DNA到侧翼序列的PCR产物证明Gohir.D01G157600.1上的插入位点真实可靠。【结论】hiTAIL-PCR获取了转GbVe1基因棉花的T-DNA侧翼序列,提供了T-DNA插入位点位于Gohir.D01G157600.1基因内含子的特异性检测引物。

关键词: 侧翼序列; GbVe1; 棉花; 插入位点

Abstract:

[Objective] The aim of this study was to obtain the flanking sequences of T-DNA in the transgenic cotton containing a GbVe1 over-expression cassette. [Method] The T-DNA insertion copy number in the transgenic GbVe1 cotton was analyzed by southern blot. Flanking sequences of the transgenic lines with putative single T-DNA insertion copy were obtained using high-efficiency Thermal asymmetric interlaced polymerase chain reaction (hiTAIL-PCR). The T-DNA insertion sites were further confirmed by PCR with specific primers. [Result] RB-flanking sequences (119-1 018 bp) and LB-flanking sequences (243-516 bp) were obtained from three transgenic lines with low copy number of T-DNA insertion. The AT content was more than 63% in these flanking sequences. A same single insertion site in the intron of Gohir.D01G157600.1 was found in the two transgenic lines 7/100826-152 and 12/100826-393, while two separated insertion sites, one also in the intron of Gohir.-D01G157600.1 and the other in the intergenic region of A12 chromosome, were found in the transgenic line 1/w-ch14. A deletion of 21 bp was found in the insertion site in the intron of Gohir.D01G157600.1. The T-DNA insertion in the intron of Gohir.D01G157600.1 was further confirmed by the specific PCR. [Conclusion] The flanking sequences of T-DNA in the transgenic GbVe1 cotton were obtained and the specific transformation event in the intron of Gohir.D01G157600.1 was further confirmed by PCR.

Key words: flanking sequence; GbVe1; Gossypium hirsutum; insertion site

陈天子, 凌溪铁, 杨郁文, 张保龙. 转GbVe1基因在棉花基因组中的整合与定位分析[J]. 棉花学报, 2019, 31(1): 1-11.

Chen Tianzi, Ling Xitie, Yang Yuwen, Zhang Baolong. The integration and insertion site of GbVe1 gene in the genome of transgenic cotton (Gossypium hirsutum)[J]. Cotton Science, 2019, 31(1): 1-11.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: http://journal.cricaas.com.cn/Jweb_mhxb/CN/10.11963/1002-7807.ctzzbl.20190105

http://journal.cricaas.com.cn/Jweb_mhxb/CN/Y2019/V31/I1/1

图/表 8

图1

表1

本研究中所用的引物"

引物名称 Primer name	引物序列 Sequences of primers	引物位置 Location of primers
LAD1-1	5'-ACGATGGACTCCAGAGCGGCCGC(G/C/A)N(G/C/A)NNNGGAA-3'	无
LAD1-2	5'-ACGATGGACTCCAGAGCGGCCGC (G/C/T)N(G/C/T)NNNGGTT-3'	无
LAD1-3	5'-ACGATGGACTCCAGAGCGGCCGC (G/C/A)(G/C/A)N(G/C/A)NNNCCAA-3'	无
LAD1-4	5'-ACGATGGACTCCAGAGCGGCCGC (G/C/T)(G/A/T)N(G/C/T)NNNCGGT-3'	无
AC1	5'-ACGATGGACTCCAGAG- 3'	无
LB35STR-1	5'- AAAACCAAAATCCAGTACTAAAATCCAG -3'	LB端35S polyA(5'->3') 41-14 bp,距LB 105 bp
LB35STR-2	5' -CGCTCATGTGTTGAGCATATAAGAAACCCT- 3'	LB端35S polyA(5'->3') 124-95 bp,距LB 186 bp
LB35STR-3C1	5'-ACGATGGACTCCAGTCCGGCCAATAATGTGTGAGTAGTTCCCAGATAAGGG-3'	LB端35S polyA(5'->3') 176-147 bp,距LB 238 bp
LB35STR-4	5' -CTCCATAATAATGTGTGAGTAGTTCCCAGATAAGG -3'	LB端35S polyA(5'->3') 182-148 bp,距LB 239 bp
RB-2a	5'-GTAATGCATGACGTTATTTATGAGATGGGTT- 3'	RB端nos 终止子,距RB 175 bp
RB-0a	5'-GGCAATAAAGTTTCTTAAGATTGAATCCTGT- 3'	RB端nos 终止子,距RB 272 bp
RBGUS-4C	5'-ACGATGGACTCCAGTCCGGCCCAAGCTAGCCACCACCACCACCACCAC- 3'	GUS CDS(5'->3') 1816-1842 bp,距RB 380 bp
RBGUS-2	5' -GCGTTGGCGGTAACAAGAAAGGGAT- 3'	GUS CDS(5'->3') 1694-1718 bp,距RB 501 bp
GUS-1F	5'-CAGCGCGAAGTCTTTATACCGAAAGGTTGGGCAGG- 3'	GUS CDS(5'->3') 432-457 bp
GUS-1R	5'-ACTGCTTTTTCTTGCCGTTTTCGTCGGTAATCACC- 3'	GUS CDS(5'->3') 690-656 bp
GUS-2F	5'-GCCGGGAATGGTGATTACCGACGAAAACGGCAAGA- 3'	GUS CDS(5'->3') 649-681 bp
GUS-2R	5'-GACATGCGTCACCACGGTGATATCGTCCACCCAG- 3'	GUS CDS(5'->3') 794-761 bp
Gbvdr-1F	5'-GGTTGTTGCGTCAACTTGAAAGCAGCTTCAGCTAC- 3'	Gbve1 CDS(5'->3') 126-160 bp
Gbvdr-1R	5'-CCCGTAGCTGATATATTTACTCCATCAAGATAGAG- 3'	Gbve1 CDS(5'->3') 618-584 bp
Gbvdr-2F	5'-CTTTGAAGGGCGAATACCAGAAGTCATTGGAAC- 3'	Gbve1 CDS(5'->3') 2 645-2 879 bp
Gbvdr-2R	5'-CAAATCCCACCCCCAGGAATATGAACAGCCAATCT- 3'	Gbve1 CDS(5'->3') 3 059-3 025 bp
nptii-1F	5'-TCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATG- 3'	nptii CDS(5'->3') 36-70 bp
nptii-1R	5'-GCTGCCTCGTCCTGGAGTTCATTCAGGGCACCGG- 3'	nptii CDS(5'->3') 197-164 bp
R4	5'-GTATTTGGGGCTTTTTTGGACGAATCATTTCCGGC - 3'	见图4示意
R2	5'-GGCAATATATAAACTGGTACATCTCTACCATCATTTT- 3'	见图4示意
R1	5'-AATATTTTGTGCTTTGAGCCGCAAAGCTGCATAAT - 3'	见图4示意
S11	5'-AAGCTATCCAAATTGGCCAACCTTGCAATGACCTC- 3'	见图4示意
S21	5'-TATTAGTTCACCTACTTTGCAACCAACACTCACT- 3'	见图4示意

表1

图2

图3

表2

表3

图4

图5

参考文献 17

[1]	郭三堆, 王远, 孙国清,等. 中国转基因棉花研发应用二十年[J]. 中国农业科学, 2015, 48(17): 3372-3387.
	Guo Sandui, Wang Yuan, Sun Guoqing, et al. Twenty years of research and application of transgenic cotton in China[J]. Scientia Agricultura Sinica, 2015, 48(17): 3372-3387.
[2]	James C.Global status of commercialized biotech/GM crops: 2016. ISAAA Brief, No. 52. Ithaca, NY: ISAAA, 2016.
[3]	杨立桃, 蒋玲曦, 沈凯琳,等. 转基因棉花MON88913转化体特异性定性、定量PCR检测方法(英文)[J]. 食品安全质量检测学报, 2009, 1(1): 18-27.
	Yang Litao, Jiang Lingxi, Shen kailin, et al. Event specific qualitative and quantitative PCR detection methods for genetically modified cotton MON88913[J]. Food Safety and Quality Detection Technology, 2009, 1(1): 18-27.
[4]	汪秀秀, 杨捷琳, 宋青,等. 转基因棉花GHB119品系特异性定量PCR检测方法的建立[J]. 农业生物技术学报, 2014, 22(3): 380-388.
	Wang Xiuxiu, Yang Jielin, Song Qing, et al. Establishment of a novel event- specific quantitative PCR method for genetically modified cotton (Gossypium sp.) GHB119 detection[J]. Journal of Agricultural Biotechnology, 2014, 22(3): 380-388.
[5]	杨阳, 王叶, 范金杰,等. 转基因棉花MON757转化体特异性PCR检测方法及应用[J]. 农业生物技术学报, 2016, 24(6): 908-918.
	Wang Ye, Fan Jinjie, ea al. Event-specific PCR detection methods of genetically modified cotton (Gossypium hirsutum) MON757 and their application[J]. Journal of Agricultural Biotechnology, 2016, 24(6): 908-918.
[6]	李忆, 尹全, 刘勇. 利用多重PCR技术同时检测6种棉花转化体的方法研究[J]. 棉花学报, 2017, 29(5): 487-494.
	Li Yi, Yin Quan, Liu Yong. Development of a multiplex polymerase chain reaction method for simultaneous detection of six events in genetically modified cotton[J]. Cotton Science, 2017, 29(5): 487-494.
[7]	侯娜, 贺辉群, 董美,等. 转基因抗虫棉外源DNA插入整合结构分析和转化事件特异性检测方法的建立[J]. 分子植物育种, 2012, 10(3): 317-323.
	Hou Na, He Huiqun, Dong Mei, et al. The exogenous gene integrated structure and event-specific detection of insect resistant transgenic cotton[J]. Molecular Plant Breeding, 2012, 10(3): 317-323.
[8]	汪巧, 谢家建, 苏长青,等. 转cry1Ac基因抗虫棉鄂杂棉1号的旁侧序列和品系特异性检测[J]. 农业生物技术学报, 2011, 19(3): 427-433.
	Wang Qiao, Xie Jiajian, Su Changqing, et al. The genome flanking sequence and event-specific qualitative detection of the transgenic crylAc cotton Ezamian 1[J]. Journal of Agricultural Biotechnology, 2011, 19(3): 427-433.
[9]	孙爻, 谢家建, 范荫荫,等. 转基因抗虫棉中cry1Aa 基因表达盒的序列测定和检测[J]. 棉花学报, 2011, 23(3): 224-227.
	Sun Yao, Xie Jiajian, Fan Yinyin, et al. The sequence and detection of the cry1Aa gene expression cassette in transgenic Bt cotton[J]. Cotton Science, 2011, 23(3): 224-227.
[10]	王叶, 谢家建, 黄春蒙,等. 转cry1Aa基因抗虫棉整合结构解析及转化体特异性检测方法的建立[J]. 棉花学报, 2017, 29(4): 307-315.
	Wang Ye, Xie Jiajian, Huang Chunmeng, et al. Integrated structure of the modified cry1Aa gene in cotton and its event-specific detection[J]. Cotton Science, 2017, 29(4): 307-315.
[11]	Zhang Baolong, Yang Yuwen, Chen Tianzi, et al. Island cotton Gbve1 gene encoding a receptor-like protein confers resistance to both defoliating and non-defoliating isolates of Verticillium dahliae[J]. PLoS ONE, 2012, 7(12): e51091. doi: 10.1371/journal.pone.0051091
[12]	Liu Yaoguang, Chen Yuanling. High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences[J]. BioTechniques, 2007, 43(5): 649-656. pmid: 18072594
[13]	Porebski S, Bailey L G, Baum B R. Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components[J]. Plant Molecular Biology Reporter, 1997, 15(1): 8-15. doi: 10.1007/BF02772108
[14]	左开井, 张献龙, 聂以春,等. 转基因抗虫棉Bt基因插入区碱基组成分析[J]. 遗传学报, 2002, 29(8): 735-740. pmid: 12200866
	Zuo Kaijing, Zhang Xianlong, Nie Yichun, et al. Sequence analysis of Bt insertion flanking fragments in transgenic Bt cotton[J]. Acta Genetica Sinica, 2002, 29(8): 735-740. pmid: 12200866
[15]	王晓波, 蒋凌雪, 魏利,等. 外源抗草甘膦EPSPs基因在大豆基因组中的整合与定位[J]. 作物学报, 2010, 36(3): 365-375. doi: 10.3724/SP.J.1006.2010.00365
	Wang Xiaobo, Jiang Lingxue, Wei Li, et al. Integration and insertion site of EPSPs gene on the soybean genome in genetically modified glyphosate-resistant soybean[J]. Acta Agronomica Sinica, 2010, 36(3): 365-375. doi: 10.3724/SP.J.1006.2010.00365
[16]	李彦龙. 棉花T-DNA插入突变体侧翼序列分析与双T-DNA载体系统构建[D]. 武汉: 华中农业大学, 2016.
	Li Yanglong. Analysis of T-DNA flanking sequences and structure twin T-DNA vector system in cotton (Gossypium hirsutum L.)[D]. Wuhang: Huangzhong Agricultral University, 2016
[17]	Forsbach A, Schubert D, Lechtenberg B, et al. A comprehensive characterization of single-copy T-DNA insertions in the Arabidopsis thaliana genome[J]. Plant Molecular Biology, 2003,

片段^a Fragments^a	长度^b Length^b /bp	T-DNA^c T-DNA^c	未知序列^d Unknown sequence^d /bp	侧翼序列 ^e Flanking sequence^e /bp
1	1 300	N	605,1 015	0
2	450	Y	119,216	119,216
3	1 200	Y	1 018	1 018
4	470	Y	180	180
5	1 500	Y	1 207	1 207
6	1 000	Y	780	780
7	1 200	Y	1 018	1 018
8	1 200	Y	1 008,1 010	1 008,1 010
9	1 200	Y	1 007,1 009	1 007,1 009
10	750	Y	513,516	513,516
11	250	N	0	0
12	250	N	0	0
13	700	Y	516	516
14	250	N	0	0
15	700	Y	243,516	243,516
16	500	N	0	0

检索项 Query id	目标项 Subject id	相同率 Identity /%	比对长度 Alignment length /bp	错配Mismatches /bp	空缺 Gap opens /bp	检索项起始位置 Query start position /bp	检索项终止位置 Query end position /bp	目标项起始位置 Subject start position /bp	目标项终止位置Subject end position /bp	E	分值 Bit score
15-1	D01 G. hirsutum_er	100	243	0	0	57	299	53 897 337	53 897 095	1.39E-120	439
15-1	A01 G. hirsutum_er	84.364	275	11	2	57	299	90 666 064	90 665 790	2.69E-85	322
15-2	D01 G. hirsutum_er	99.612	516	2	0	238	753	53 897 337	53 896 822	0	922
15-2	A01 G. hirsutum_er	90.328	548	21	2	238	753	90 666 064	90 665 517	0	769
8-1	D01 G. hirsutum_er	99.008	1 008	8	2	59	1 065	53 898 365	53 897 359	0	1 766
8-1	A01 G. hirsutum_er	89.877	1 057	49	6	59	1 065	90 667 134	90 666 086	0	1 449
8-2	D01 G. hirsutum_er	99.307	1 010	6	1	56	1 064	53 898 368	53 897 359	0	1 786
8-2	A01 G. hirsutum_er	89.906	1 060	48	6	56	1 064	90 667 137	90 666 086	0	1 456
9-1	D01 G. hirsutum_er	99.603	1 007	4	0	59	1 065	53 898 365	53 897 359	0	1 799
9-1	A01 G. hirsutum_er	90.255	1 057	45	6	59	1 065	90 667 134	90 666 086	0	1 467
9-2	D01 G. hirsutum_er	99.504	1 009	3	1	59	1 067	53 898 365	53 897 359	0	1 795
9-2	A01 G. hirsutum_er	90.35	1 057	46	5	59	1 067	90 667 134	90 666 086	0	1 474