转基因水稻E1C9K-18的获得及其事件特异性检测方法的建立

doi:10.3969/j.issn.1674-7968.2020.12.015

Abstract
Figure/Table
References
Related Citation (15)

Download: PDF (4526 KB) HTML (1 KB)
Export: BibTeX | EndNote (RIS)

Abstract The herbicide resistance and Lepidopteran resistance of rice (Oryza sativa) play important roles in weeds management and reduction of yield loss by insect pests, and the event-specific detection method is an essential technique for supervision of transgenic rice. In this study, the expression vector was constructed firstly, in which the Lepidopteran resistance gene Cry1Ca^# (gene coding insecticidal crystal protein Cry1Ca) was driven by wound-induced promoter and the herbicide resistance gene Epsps^# (gene coding 5-enolpyruvylshikimate 3-phosphate synthase) was driven by constitutive promoter, and a single copy transgenic event E1C9K-18 was developed by Agrobacterium-mediated genetic transformation from 9K19-5 (O. sativa subsp. indica), which possessed elite glyphosate resistance. Then, the hiTAIL-PCR (high-efficiency thermal asymmetric interlaced PCR) was adopted to reveal the flanking sequence of transgenic rice E1C9K-18, and the right flanking sequence with 420 bp length was discovered. Comparing the right flanking sequence with rice genome database, it was found the exogenous fragment was inserted behind the nucleotide residue No. 33 189 510 of chromosome 4. The left flanking sequence with 613 bp length was amplified using the primers that were designed according to the sequence of integration site on rice genome and left side sequence of T-DNA. Compared the left flanking sequence with rice genome database, it was found the exogenous fragment was inserted before the nucleotide residue No. 33 189 480 of chromosome 4. However, the insertion of T-DNA resulted in a deletion of 29 nucleotide residues in rice genome. Based on the left and right flanking sequences, the event-specific PCR detection method and tri-primers PCR method were developed for E1C9K-18, which will provide technical supports for identification of this transgenic event and rapidly selection of homozygote from segregation population.

Key words： Transgenic rice Flanking sequence Event-specific detection Genotyping

Received: 25 February 2020

ZTFLH:

S-3

Corresponding Authors: *xiaoguoying@isa.ac.cn

	Service

	E-mail this article
	Add to my bookshelf
	Add to citation manager
	E-mail Alert
	RSS
	Articles by authors
	XU Li-Kui
	DENG Li-Hua
	LI Hua
	XIAO Guo-Ying

Cite this article:

XU Li-Kui,DENG Li-Hua,LI Hua, et al. Development of Transgenic Rice (Oryza sativa) E1C9K-18 and Establishment of Its Event-specific Detection Method[J]. 农业生物技术学报, 2020, 28(12): 2250-2260.

URL:

http://journal05.magtech.org.cn/Jwk_ny/EN/10.3969/j.issn.1674-7968.2020.12.015 OR http://journal05.magtech.org.cn/Jwk_ny/EN/Y2020/V28/I12/2250

[1] 陈利钢, 2009. 拟南芥WRKY基因对SERRATE的调控和拟南芥WRKY8, 28, 48和71的基因功能分析[D]. 博士学位论文, 中国科学院研究生院, 导师: 余迪求, pp. 28.
(Chen L G, 2009. Functional analysis of AtWRKY8, 28, 48 and 71 and the regulation of SERRATE by WRKY super family in Arabidopsis[D]. Thesis for Ph.D., University of Chinese Academy of Sciences, Supervisor: Yu D Q, pp. 28.)
[2] 崔帅, 王作平, 于江辉, 等. 2018. 转基因水稻BPL9K-2事件特异性检测方法的建立[J]. 中国生物工程杂志, 38(11): 32-41.
(Cui S, Wang Z P, Yu J H, et al.2018. Event-specific detection methods of genetically modified rice BPL9K-2[J]. China Biotechnology, 38(11): 32-41.)
[3] 邓力华, 2013. Epsps、Gox和CrylCa基因的优化以及在水稻中的表达研究[D]. 博士学位论文,中国科学院大学, 导师: 肖国樱, pp. 50, 97-98.
(Deng L H, 2013. Optimizing and expressing of Epsps, Gox and Cry1Ca genes in rice[D]. Thesis for Ph.D., University of Chinese Academy of Sciences, Supervisor: Xiao G Y, pp. 50, 97-98.)
[4] 杜春芳, 李朋波, 李润植. 2004. 一种快速鉴定转基因植物纯合体的新方法[J]. 生物技术通讯, 15(6): 585-587.
(Du C F, Li P B, Li R Z.2004. A new method for the rapid identification of homozygous transgenic plants[J]. Letters In Biotechnology, 15(6): 585-587.)
[5] 郭超, 何行健, 邓力华, 等. 2017. 转基因水稻BarKasalath-01事件特异性检测[J]. 分子植物育种, 15(11): 4466-4475.
(Guo C, He X J, Deng L H, et al.2017. Event-specific detection of genetically modified rice BarKasalath-01[J]. Molecular Plant Breeding, 15(11): 4466-4475.)
[6] 胡文彬, 2017. 多抗型转基因水稻的培育及特性研究[D]. 博士学位论文, 中国科学院大学, 导师: 肖国樱, pp. 52-54.
(Hu W B, 2017. Breeding and characteristic analysis of multi-resistance genetically modified rice[D]. Thesis for Ph.D., University of Chinese Academy of Sciences, Supervisor: Xiao G Y, pp. 52-54.)
[7] 蒋利平, 翁绿水, 肖国樱. 2013. 转基因水稻B2A68事件特异性检测方法的建立[J]. 杂交水稻, 28(5): 60-67.
(Jiang L P, Weng L S, Xiao G Y.2013. Establishment of an event-specific method to detect transgenic rice B2A68[J]. Hybrid Rice, 28(5): 60-67.)
[8] 刘发央, 侯路珍, 谢小冬. 2005. 启动子Actin1的克隆及植物表达载体的构建[J]. 草原与草坪, 5(3): 66-71.
(Liu F Y, Hou L Z, Xie X D.2005. Cloning of monomial promoter of Actin1 gene and construction of plant expression vector[J]. Grassland and Turf, 5(3): 66-71.)
[9] 王昌涛, 梁粤, 王欢, 等. 2006. 玉米Ubiquitin启动子的克隆及功能鉴定[J]. 沈阳农业大学学报, 37(1): 9-12.
(Wang C T, Liang Y, Wang H, et al.2006. Cloning and function identification of maize ubiquitin promoter[J]. Journal of Shenyang Agricultural University, 37(1): 9-12.)
[10] 王恒波, 肖乃衍, 张华, 等. 2018. 转基因番木瓜55-1转化事件定性PCR检测方法的建立[J]. 热带作物学报, 39(9): 1751-1757.
(Wang H B, Xiao N Y, Zhang H, et al.2018. Establishment of a qualitative PCR detection method for transgenic Papaya 55-1[J]. Chinese Journal of Tropical Crops, 39(9): 1751-1757.)
[11] 王良超, 2014. OsLSR: 一个调控免疫反应和花发育的水稻基因[D]. 博士学位论文, 浙江大学,导师: 凃巨民, pp. 28-29.
(Wang L C, 2014. OsLSR: A rice gene that regulates immune response and floral differentiation[D]. Thesis for Ph.D., Zhejiang University, Supervisor: Tu J M, pp. 28-29.)
[12] 魏岁军, 邓力华, 肖国樱. 2014. 转基因水稻EB7001S事件特异性检测方法的建立[J]. 农业生物技术学报, 22(5): 621-631.
(Wei S J, Deng L H, Xiao G Y.2014. Establishment of an event-specific method to detect transgenic rice (Oryza sativa) EB7001S[J]. Journal of Agricultural Biotechnology, 22(5): 621-631.)
[13] 闫建俊. 2019. 转基因马铃薯外源基因插入位点分析及检测方法的建立[J/OL]. 分子植物育种: 1-7 (2019-12-01).
(Yan J J.2019. Analysis of insertion site of transgenic potato exogenous gene and the establishment of detection method[J/OL]. Molecular Plant Breeding: 1-7 (2019-12-01)).
[14] 曾崇华, 陈芬, 孟秋成, 等. 2016. 水稻HD9802S/Kasalath后代中高组织培养力家系的筛选[J]. 杂交水稻, 31(1): 51-56.
(Zeng C H, Chen F, Meng Q C, et al.2016. Selection of families with high tissue culture ability in filial generation of HD9802S/Kasalath in rice[J]. Hybrid Rice, 31(1): 51-66.)
[15] 曾强, 孟秋成, 邓力华, 等. 2019. 抗草甘膦、抗螟虫水稻E1C608的鉴定和重要表型特征分析[J]. 中国生物工程杂志, 39(11): 31-38.
(Zeng Q, Meng Q C, Deng L H, et al.2019. Identification and analysis of important phenotypes of E1C608 with glyphosate resistance and lepidopteran resistance in rice[J]. China Biotechnology, 39(11): 31-38.)
[16] 张斌, 何福林. 2017. 三引物法鉴定转基因水稻U5纯合体[J]. 分子植物育种, 15(11): 4476-4482.
(Zhang B, He F L.2017. Identification of transgenic rice U5 homozygote by three primers[J]. Molecular Plant Breeding, 15(11): 4476-4482.)
[17] 张斌, 阮颖. 2018. 转基因水稻U41三引物检测方法的建立[J]. 基因组学与应用生物学, 37(3): 1308-1314.
(Zhang B, Ruan Y.2018. Establishment of three primer detection method for transgenic rice U41[J]. Genomics and Applied Biology, 37(3): 1308-1314.)
[18] 张焕春, 汪小福, 李玥莹, 等. 2012. 转Cry1Ab水稻纯合体快速准确的PCR鉴定方法[J]. 浙江农业学报, 24(4): 549-554.
(Zhang H C, Wang X F, Li Y Y, et al.2012. A rapid and accurate PCR method for homozygous lines screening for genetically modified rice containing Cry1Ab[J]. Acta Agriculturae Zhejiangensis, 24(4): 549-554.)
[19] Anayol E, Bakhsh A, Karakoç Ö C, et al.2016. Towards better insect management strategy: Restriction of insecticidal gene expression to biting sites in transgenic cotton[J]. Plant Biotechnology Reports, 10(2): 83-94.
[20] Chen L, Zhang L, Yu D Q.2010. Wounding-induced WRKY8 is involved in basal defense in Arabidopsis[J]. Molecular Plant-Microbe Interactions, 23(5): 558-565.
[21] Christensen A H, Quail P H.1996. Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants[J]. Transgenic Research, 5(3): 213-218.
[22] He Y, Ning T T, Xie T T, et al.2011. Large-scale production of functional human serum albumin from transgenic rice seeds[J]. Proceedings of the National Academy of Sciences USA, 108(47): 19078-19083.
[23] Liu Y G, Chen Y.2007. High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences[J]. Biotechniques, 43(5): 649-656.
[24] Marmiroli N, Maestri E, Gulli M, et al.2008. Methods for detection of GMOs in food and feed[J]. Analytical and Bioanalytical Chemistry, 392(3): 369-384.
[25] McElroy D, Zhang W G, Cao J, et al.1990. Isolation of an efficient actin promoter for use in rice transformation[J]. The Plant Cell, 2(2): 163-171.
[26] Surzycki S.2000. DNA transfer and hybridization[M]. Berlin Heidelberg, Springer, pp. 233-262.
[27] Toki S, Hara N, Ono K, et al.2006. Early infection of scutellum tissue with Agrobacterium allows high-speed transformation of rice[J]. Plant Journal, 47(6): 969-976.
[28] Wang Z P, Deng L H, Weng L S, et al.2017. Transgenic rice expressing a novel phytase-lactoferricin fusion gene to improve phosphorus availability and antibacterial activity[J]. Journal of Integrative Agriculture, 16(4): 774-788.