棉花学报 ›› 2019, Vol. 31 ›› Issue (1): 1-11.doi: 10.11963/1002-7807.ctzzbl.20190105
• 研究与进展 • 下一篇
收稿日期:
2018-03-30
发布日期:
2019-01-15
通讯作者:
张保龙
E-mail:actzi0503@gmail.com;zhbl2248@hotmail.com
作者简介:
陈天子(1981―),男,博士, 基金资助:
Chen Tianzi(),Ling Xitie,Yang Yuwen,Zhang Baolong*(
)
Received:
2018-03-30
Published:
2019-01-15
Contact:
Zhang Baolong
E-mail:actzi0503@gmail.com;zhbl2248@hotmail.com
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。
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.
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 |
R2 | 5'-GGCAATATATAAACTGGTACATCTCTACCATCATTTT- 3' | 见 |
R1 | 5'-AATATTTTGTGCTTTGAGCCGCAAAGCTGCATAAT - 3' | 见 |
S11 | 5'-AAGCTATCCAAATTGGCCAACCTTGCAATGACCTC- 3' | 见 |
S21 | 5'-TATTAGTTCACCTACTTTGCAACCAACACTCACT- 3' | 见 |
表2
hiTAIL-PCR 获取的转GbVe1基因棉花T-DNA侧翼序列"
片段a Fragmentsa | 长度b Lengthb /bp | T-DNAc T-DNAc | 未知序列d Unknown sequenced /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 |
表3
转基因株系12/100826-393的T-DNA侧翼序列与棉花TM-1基因组的blast结果"
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 |
[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, |
[1] | 李鸣凤,彭文勇,何华,刘新伟,赵竹青. 外施不同形态硼对棉花吸收利用硼及其他矿质元素的影响[J]. 棉花学报, 2021, 33(5): 385-392. |
[2] | 王燕,张谦,王树林,韩硕,冯国艺,董明,钱玉源,祁虹. 耕层重构对棉田土壤养分、微生物数量与酶活性的影响[J]. 棉花学报, 2021, 33(5): 422-434. |
[3] | 张岚,程琦,梁士辰,邓雨潇,潘玉欣. 棉花UGPase基因鉴定与生物信息学分析[J]. 棉花学报, 2021, 33(4): 337-346. |
[4] | 马怡茹,吕新,祁亚琴,张泽,易翔,陈翔宇,鄢天荥,侯彤瑜. 基于无人机数码图像的机采棉脱叶率监测模型构建[J]. 棉花学报, 2021, 33(4): 347-359. |
[5] | 苟浩琦,马常凯,张迁,范术丽,马启峰,张朝军. 棉花光敏雄性不育系psm5的培育及其育性转变规律[J]. 棉花学报, 2021, 33(4): 360-367. |
[6] | 徐鹏,郭琪,徐珍珍,孟珊,陈天子,沈新莲. 基于重测序鉴定SbHKT基因在陆地棉基因组中的插入位点[J]. 棉花学报, 2021, 33(4): 377-383. |
[7] | 王林, 张强, 马江锋, 朱玉永, 田英, 李红, 毕显杰, 宋敏, 王海标, 雷天翔, 李召虎, 田晓莉, 杜明伟, 张立祯, 赵冰梅. 新疆棉区植保无人机喷施棉花脱叶催熟剂效果研究[J]. 棉花学报, 2021, 33(3): 200-208. |
[8] | 王金刚, 姜艳, 田甜, 朱永琪, 杨振康, 周天航, 张文旭, 佟炫梦, 孙嘉祺, 王海江. 减氮配施生物刺激素对棉花产量及氮肥吸收利用的影响[J]. 棉花学报, 2021, 33(3): 209-223. |
[9] | 易翔, 张立福, 吕新, 张泽, 田敏, 印彩霞, 马怡茹, 范向龙. 基于无人机高光谱融合连续投影算法估算棉花地上部生物量[J]. 棉花学报, 2021, 33(3): 224-234. |
[10] | 孙璘, 海艳, 唐晓雪, 祖丽皮亚·艾买, 焦瑞莲, 任毓忠, 李国英. 新疆棉花茎腐病的病原鉴定及其生物学特性研究[J]. 棉花学报, 2021, 33(3): 235-246. |
[11] | 党文芳, 刘萍, 管力慧, 杨红梅, 牛新湘, 李萍, 楚敏, 娄恺, 史应武. 土壤环境因子对棉花根际与内生拮抗细菌存活数量的影响[J]. 棉花学报, 2021, 33(3): 247-257. |
[12] | 杨可心, 陈秀叶, 刘畅, 鹿秀云, 郭庆港, 马平. 棉花枯萎病菌新生理型菌株毒素鉴定及其活性测定[J]. 棉花学报, 2021, 33(3): 258-268. |
[13] | 安杰,韩迎春,张正贵,冯璐,雷亚平,杨北方,王国平,李小飞,王占彪,邢芳芳,熊世武,辛明华,李亚兵. 不同熟性棉花品种冠层温度分布特点[J]. 棉花学报, 2021, 33(2): 134-143. |
[14] | 孟浩峰,雷长英,张旺锋,张亚黎. 系统调控下棉花比叶重的变化机制[J]. 棉花学报, 2021, 33(2): 144-154. |
[15] | 张友昌,黄晓莉,胡爱兵,李洪菊,冯常辉,李蔚,张贤红,罗艳萍,杨国正. 长江流域麦/油后直播棉花播种时间下限研究[J]. 棉花学报, 2021, 33(2): 155-168. |
|