作物学报 ›› 2019, Vol. 45 ›› Issue (9): 1440-1445.doi: 10.3724/SP.J.1006.2019.84167
ZHOU Xiang-Yang1,ZHAO Liang2,DI Jia-Chun2,CHEN Xu-Sheng2,*()
摘要:
以中美2个抗虫棉品种GK19与33B为试验材料, 利用检测中美Bt基因的特异性引物, 分别对抗虫棉亲本GK19和33B进行PCR扩增, 并通过SSR分子标记技术对其Bt基因进行分子鉴定与染色体定位, 旨在从外源基因转化事件的视角探究中美转基因抗虫棉差异的分子基础。结果表明, GK19为中国转Bt基因抗虫棉, 33B为美国转Bt基因抗虫棉; GK19的Bt基因被定位在棉花Chr.20上, 共16对SSR多态性标记与其Bt基因连锁, 两侧的分子标记为NAU3907和NAU2579, 其遗传距离分别为2.4 cM和1.5 cM; 33B的Bt基因被定位在棉花Chr.26上, 共20对SSR多态性标记与Bt基因连锁, 目标Bt基因位于标记NAU460和dc40260之间, 其遗传距离分别为3.6 cM和2.0 cM。以上结果表明GK19和33B属于不同的遗传转化事件。
[1] | Schnepf H E, Whiteley H R . Cloning and expression of the Bacillius thuringiensis crystal protein gene in Escherichia coli. Proc Natl Acad Sci USA, 1981,78:2893-2897. |
[2] | Perlak F J, Deaton R W, Armstrong T A, Fuchs R L, Sims S R, Greenplate J T, Fischhoff D A . Insect resistant cotton plants. Biotechnology, 1990,8:939-943. |
[3] | Perlak F J, Fuchs R L, Dean D A, McPherson S L, Fischhoff D A . Modification of the coding sequence enhances plant expression of insect control protein genes. Proc Natl Acad Sci USA, 1991,88:3324-3328. |
[4] | 于凤玲, 秦新敏, 蒋晓茹 . 美国保铃抗虫棉33B的生育特点及栽培技术要点. 中国棉花, 1999, (8):42-43. |
Yu F L, Qin X M, Jiang X L . Growth characteristics and key points of cultivation techniques of boll-guard cotton 33B from American. China Cotton, 1999, (8):42-43 (in Chinese). | |
[5] | 陈旭升, 狄佳春, 刘剑光, 许乃银, 肖松华 . 转基因棉花育种进展及其产业化前景分析. 中国农学通报, 2002,18(2):72-74. |
Chen X S, Di J C, Liu J G, Xu N Y, Xiao S H . Progress and industrialization prospects of genetically modified cotton breeding. Chin Agric Sci Bull, 2002,18(2):72-74 (in Chinese). | |
[6] | 谢道昕, 范云六, 倪万潮, 黄骏麒 . 苏云金芽孢杆菌(Bt)杀虫晶体蛋白基因导入棉花获得转基因植株. 中国科学( B), 1991, ( 4):367-373. |
Xie D X, Fan Y L, Ni W C, Huang J Q . Bacillus thuringiensis(Bt) insecticidal crystal protein gene was introduced into cotton to obtain transgenic plants. Chin Sci(B), 1991, (4):367-373 (in Chinese). | |
[7] | 郭三堆 . CrylA杀虫基因的人工合成. 中国农业科学, 1993,26(2):85-86. |
Guo S D . Synthesis of CrylA insecticidal gene. Sci Agric Sin, 1993,26(2):85-86 (in Chinese). | |
[8] | 郭三堆, 王远, 孙国清, 金石桥, 周焘, 孟志刚, 张锐 . 中国转基因棉花研发应用二十年. 中国农业科学, 2015,48:3372-3387. |
Guo S D, Wang Y, Sun G Q, Jin S Q, Zhou T, Meng Z G, Zhang R . Twenty years of research and application of transgenic cotton in China. Sci Agric Sin, 2015,48:3372-3387 (in Chinese with English abstract). | |
[9] | 匡猛, 杨伟华, 许红霞, 王延琴, 周大云, 冯新爱 . 单粒棉花种子DNA快速提取方法. 分子植物育种, 2010,8:827-831. |
Kuang M, Yang W H, Xu H X, Wang Y Q, Zhou D Y, Feng X A . A rapid method of DNA extraction from single cotton seed. Mol Plant Breed, 2010,8:827-831 (in Chinese with English abstract). | |
[10] | 王奕海, 谢家建, 张永军, 王锡锋, 彭于发 . 一种检测抗虫棉中不同Bt基因表达盒结构的PCR方法. 农业生物技术学报, 2009,17:914-919. |
Wang Y H, Xie J J, Zhang Y J, Wang X F, Peng Y F . A PCR method to detect different Bt gene expression cassettes in transgenic Bt cotton. J Agric Biotechnol, 2009,17:914-919 (in Chinese with English abstract). | |
[11] | 景超, 马晓杰, 狄佳春, 陈旭升 . 陆地棉超矮杆突变体基因的初步定位. 遗传, 2011,33:1393-1397. |
Jing C, Ma X J, Di J C, Chen X S . Gene mapping of an ultra- dwarf mutant in upland cotton. Hereditas, 2011,33:1393-1397 (in Chinese with English abstract). | |
[12] | 张军, 武耀廷, 郭旺珍, 张天真 . 棉花微卫星标记的PAGE/银染快速检测. 棉花学报, 2000,12:267-269. |
Zhang J, Wu Y T, Guo W Z, Zhang T Z . Fast screening of microsatellite markers in cotton with PAGE /silver staining. Cotton Sci, 2000,12:267-269 (in Chinese with English abstract). | |
[13] |
燕树锋, 祝水金, 刘海芳, 卢彩霞, 铁双贵 . 转EPSPS基因抗草甘膦棉花的遗传分析. 华北农学报, 2015,30(3):54-57.
doi: 10.7668/hbnxb.2015.03.010 |
Yan S F, Zhu S J, Liu H F, Lu C X, Tie S G . Genetic analysis of transgenic glyphosate-resistance cotton with EPSPS gene. Acta Agric Boreali-Sin, 2015,30(3):54-57 (in Chinese with English abstract).
doi: 10.7668/hbnxb.2015.03.010 |
|
[14] | 杨晓杰, 刘传亮, 张朝军, 武芝霞, 张雪妍, 刘坤, 房卫平, 李付广 . 不同转化方法获得的转基因棉花外源基因拷贝数分析. 农业生物技术学报, 2011,19:221-229. |
Yang X J, Liu C L, Zhang C J, Wu Z X, Zhang X Y, Liu K, Fang W P, Li F G . Comparative analysis of exogenous gene copy numbers in transgenic cotton transformed by different methods. J Agric Biotechnol, 2011,19:221-229 (in Chinese with English abstract). | |
[15] | 宋敏, 刘丽军, 苏颖异 . 主要农作物转化事件的专利保护及对我国的启示. 华北农学报, 2011, 26(增刊):285-290. |
Song M, Liu L J, Su Y Y . Patent protection for transgenic events of major crops and policy implications for China. Acta Agric Boreali-Sin, 2011, 26(suppl):285-290 (in Chinese with English abstract). | |
[16] | Parker J D, Rebinovitch P S, Burmer G C . Targeted gene walking polymerase chain reaction. Nucl Acids Res, 1991,19:3055-3060. |
[17] | Liu Y G, Whitter R F . Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics, 1995,25:674-681. |
[18] | Liu Y G, Chen Y L . High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences. Biotechniques, 2007,43:649-656. |
[19] | 侯娜, 贺辉群, 董美, 徐荣旗, 宛煜嵩, 金芜军, 刘好宝 . 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. 分予植物育种, 2012,10:317-323. |
Hou N, He H Q, Dong M, Xu R Q, Wan Y S, Jin W J, Liu H B . The exogenous gene integrated structure and event specific detection of insect resistant transgenic cotton. Mol Plant Breed, 2012,10:317-323 (in Chinese with English abstract). | |
[20] | 郭金英, 张天真 . TAIL-PCR方法研究花粉管通道法转化机理初探. 河北农业大学学报, 2010,33(4):5-9 |
Guo J Y, Zhang T Z . Study on the mechanism of pollen-tube pathway-mediated transformation method with TAIL-PCR. J Agric Univ Hebei, 2010,33(4):5-9 (in Chinese with English abstract). | |
[21] | Li F, Fan G, Wang K . Genome sequence of the cultivated cotton Gossypium arboreum. Nat Genet, 2014,46:567-572. |
[22] | Zhang T, Hu Y, Jiang W . Sequencing of allotetraploid cotton (Gossypium hirsutum L. acc. TM-1) provides a resource for fiber improvement. Nat Biotechnol, 2015,33:531-537. |
[23] | 董志强, 赵明, 舒文华, 张保明, 郝红晶 . 转Bt基因棉Bt蛋白的亚细胞结构定位. 作物学报, 2006,32:1924-1926. |
Dong Z Q, Zhao M, Shu W H, Zhang B M, Hao H J . The sub-cellular localization of the Bt crystal protein in transgenic Bt cotton cell. Acta Agron Sin, 2006,32:1924-1926 (in Chinese with English abstract). | |
[24] | 肖松华, 狄佳春, 刘剑光, 许乃银, 陈旭升, 黄骏麒 . 转基因抗虫棉Bt基因的遗传连锁分析. 棉花学报, 2002,14:134-137. |
Xiao S H, Di J C, Liu J G, Xu N Y, Chen X S, Huang J Q . The analysis of linkage for Bt gene in transgenic bollworm-resistant cotton. Cotton Sci, 2002,14:134-137 (in Chinese with English abstract). | |
[25] | Guo W, Cai C, Wang C . A microsatellite-based, gene-rich linkage map reveals genome structure, function and evolution in Gossypium. Genetics, 2007,176:527-541. |
[26] | Zhao L, Lyu Y D, Cai C P . Toward allotetraploid cotton genome assembly: integration of a high-density molecular genetic linkage map with DNA sequence information. BMC Genomics, 2012,13:539-570. |
[27] | 刘吉焘, 马晓杰, 狄佳春, 陈旭升 . 棉花草甘膦抗性基因CP4-EPSPS的初步定位. 江苏农业学报, 2013,29:480-484. |
Liu J T, Ma X J, Di J C, Chen X S . Mapping of glyphosate-resistant gene CP4-EPSPS in cotton. Jiangsu J Agric Sci, 2013,29:480-484 (in Chinese with English abstract). | |
[28] | 安百伟, 赵亮, 狄佳春, 陈旭升 . 陆地棉Bt抗虫基因类型鉴定与染色体定位. 江苏农业学报, 2016,32:262-266. |
An B W, Zhao L, Di J C, Chen X S . Identification and chromosomal mapping of insect-resistant gene Bt in upland cotton. Jiangsu J Agric Sci, 2016,32:262-266 (in Chinese with English abstract). | |
[29] | 周向阳, 赵亮, 狄佳春, 陈旭升 . 抗虫杂交棉苏杂6号抗虫亲本的Bt基因鉴定与染色体定位. 江苏农业学报, 2016,32:987-999. |
Zhou X Y, Zhao L, Di J C, Chen X S . Identification and chromosomal mapping of Bt gene in the insect-resistant parents of hybrid cotton Suza 6. Jiangsu J Agric Sci, 2016,32:987-999 (in Chinese with English abstract). |
[1] | 陈旭升,狄佳春,周向阳,赵亮. 陆地棉高秆突变体的激素变化与Tp基因的染色体定位[J]. 作物学报, 2017, 43(06): 935-939. |
[2] | 丁检,吴双,蔡彩平,郭旺珍*. 棉花溶血磷酸酯酰转移酶(LPAT)家族基因的发掘和表达分析[J]. 作物学报, 2015, 41(03): 378-385. |
[3] | 乔麟轶,李欣,畅志坚,张晓军,詹海仙,郭慧娟,李建波,常建忠,郑军. 粗山羊草全基因组Aux/IAA基因家族的分离、染色体定位及序列分析[J]. 作物学报, 2014, 40(12): 2059-2069. |
[4] | 李丽,汪顺峰,刘芳,唐世义,谭兆云,张建,滕中华,刘大军,张正圣. 陆地棉转录因子的染色体定位[J]. 作物学报, 2012, 38(08): 1361-1368. |
[5] | 薛飞,王长有,张丽华,张宏,李浩,王亚娟,刘新伦,吉万全. 来自野生二粒小麦的抗白粉病基因PmAS846及其染色体定位和分子标记分析[J]. 作物学报, 2012, 38(04): 589-595. |
[6] | 苏长青, 谢家建, 孙爻, 彭于发. 一种适合转基因棉CpTI和cry1A基因剂量测定的标准质粒的构建和应用[J]. 作物学报, 2011, 37(09): 1533-1539. |
[7] | 佘茂云, 陈朵朵, 冯晨, 杜丽璞, 叶兴国. 小麦亚硝酸还原酶基因及调控序列克隆、定位和表达分析[J]. 作物学报, 2011, 37(01): 28-39. |
[8] | 秦伟,赵光耀,曲志才,张立超,段佳磊,李爱丽,贾继增,孔秀英. 小麦白粉病菌诱导的TaWRKY34基因的鉴定与分析[J]. 作物学报, 2010, 36(2): 249-255. |
[9] | 辛承松, 董合忠, 罗振, 唐薇, 张冬梅, 李维江, 孔祥强. 黄河三角洲盐渍棉花施用氮、磷、钾肥的效应研究[J]. 作物学报, 2010, 36(10): 1698-1706. |
[10] | 辛承松;董合忠;唐薇;张冬梅;罗振;李维江. 滨海盐渍土抗虫棉养分吸收和干物质积累特点[J]. 作物学报, 2008, 34(11): 2033-2040. |
[11] | 庄丽芳;宋立晓;冯祎高;钱保俐;徐海滨;裴自友;亓增军. 小麦EST-SSR标记的开发和染色体定位及其在追踪黑麦染色体中的应用[J]. 作物学报, 2008, 34(06): 926-933. |
[12] | 张磊;张宝石;周荣华;高丽峰;赵光耀;宋彦霞;贾继增. 小麦细胞分裂素氧化/脱氢酶基因(TaCKX2)的克隆及其遗传作图[J]. 作物学报, 2007, 33(09): 1419-1425. |
[13] | 王润奇;高俊华;关中波;毛丽萍. 谷子几种农艺性状基因染色体定位及连锁关系的初步研究[J]. 作物学报, 2007, 33(01): 9-14. |
[14] | 王长有;吉万全;张改生;王秋英;蔡东明;薛秀庄. 小麦种质N9134抗白粉病基因的SSR标记和染色体初步定位[J]. 作物学报, 2007, 33(01): 163-166. |
[15] | 李韬;张增艳;林志珊;陈孝;高珊;辛志勇. 小麦抗白粉病新基因的AFLP和SSR标记及其染色体定位[J]. 作物学报, 2005, 31(09): 1105-1109. |
|