抗草甘膦、抗螟虫水稻E1C608的鉴定和重要表型特征分析 <sup>*</sup>

doi:10.13523/j.cb.20191104

中国生物工程杂志

2019, Vol. 39

Issue (11): 31-38 DOI: 10.13523/j.cb.20191104

研究报告

抗草甘膦、抗螟虫水稻E1C608的鉴定和重要表型特征分析 ^*

曾强^1,²,孟秋成¹,邓力华¹,李锦江¹,于江辉¹,翁绿水¹,肖国樱^1,³(

)

1 中国科学院亚热带农业生态研究所亚热带农业生态过程重点实验室长沙 410125
2 中国科学院大学北京 100049 3 中国科学院种子创新研究院北京 100101

Identification and Analysis of Important Phenotypes of E1C608 with Glyphosate Resistance and Lepidopteran Resistance in Rice

ZENG Qiang^1,²,MENG Qiu-cheng¹,DENG Li-hua¹,LI Jin-jiang¹,YU Jiang-hui¹,WENG Lu-shui¹,XIAO Guo-ying^1,³(

)

1 Key Laboratory of Agro-ecological Processes in Subtropical Region, Institute of Subtropical Agriculture,Chinese Academy of Sciences, Changsha 410125, China
2 University of Chinese Academy of Sciences, Beijing 100049, China

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摘要：

草害和虫害是造成水稻减产的两个重要因素,培育转基因抗除草剂、抗虫水稻是解决这两个问题的有效途径之一。分子鉴定结果表明,以恢复系R608为受体的遗传转化获得了3个独立转基因事件,其中1个为单拷贝转化体,命名为E1C608-3。用ELISA方法检测外源抗草甘膦和抗螟虫蛋白含量的结果表明,E1C608-3的T₂代分蘖期的根、茎、叶中EPSPS蛋白含量为120.16~1 223.28μg/g,CRY1C蛋白含量为1.23~8.72μg/g,且两个外源蛋白在不同器官中的表达量均表现为叶>茎>根(P<0.01)。T₃代测定结果显示,E1C608-3转化体在秧苗期草甘膦的耐受浓度至少为16g/L,其耐受浓度至少是转化受体R608的16倍。T₃代抗虫实验结果显示,E1C608-3对稻纵卷叶螟幼虫平均致死率为95.56%,表现出良好的螟虫抗性。T₄代农艺性状测定结果显示,E1C608-3与转化受体存在较大差异,但都处在水稻天然种质资源的变异范围之内。以上结果表明,E1C608-3是一个抗除草剂、抗螟虫的水稻新种质。

关键词： 水稻; 转基因; 抗草甘膦; 抗螟虫; 农艺性状

Abstract:

Weeds and insect pests are two important biotic stresses to result in yield loss in rice, and breeding bioengineered rice with herbicide resistance and insect resistance is one of the effective ways to solve these two problems. And for that, the glyphosate resistance and Lepidopteran resistance as well as molecular and some agronomic characteristics of transgenic line E1C608 in rice were identified. Results of molecular identification showed that three independent transformation events were obtained, in which one single copy insertion event was named as E1C608-3. The transformant E1C608-3 of T₂ generation was used to detect exogenous protein contents in root, stem and leaf at tillering stage by ELISA. The protein contents of EPSPS and CRY1C in different organs of E1C608-3 were significantly different both in descending order of leaf>stem>oot (P<0.01), ranged from120.16μg/g to 1 223.28μg/g and from 1.23μg/g to 8.72μg/g, respectively. The glyphosate tolerable concentration at seedling stage of E1C608-3 in T₃ generation reached at least 16g/L, which was sixteen times higher than that of the transformation receptor R608. The larva mortality of rice leaf roller fed on leaves of E1C608-3 in T₃ generation for five days was 95.56%, that showed the E1C608-3 with an excellent resistance to Lepidopteran insects. The data suggested that some agronomic traits were significantly different between E1C608-3 in T₄ generation and R608 (P<0.01), but all of the variations were within the variation range of natural germplasm resource in rice. Overall, a novel germplasm with glyphosate resistance and Lepidopteran resistance was verified to be developed in rice.

Key words: Rice Bioengineering Glyphosate resistance Lepidopteran resistance Agronomic traits

收稿日期: 2019-03-28 出版日期: 2019-12-17

ZTFLH:

Q819

基金资助: * 国家转基因生物新品种培育科技重大专项(2016ZX08001003-001)

通讯作者: 肖国樱 E-mail: xiaoguoying@isa.ac.cn

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引用本文:

曾强,孟秋成,邓力华,李锦江,于江辉,翁绿水,肖国樱. 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, 2019, 39(11): 31-38.

ZENG Qiang,MENG Qiu-cheng,DENG Li-hua,LI Jin-jiang,YU Jiang-hui,WENG Lu-shui,XIAO Guo-ying. Identification and Analysis of Important Phenotypes of E1C608 with Glyphosate Resistance and Lepidopteran Resistance in Rice. China Biotechnology, 2019, 39(11): 31-38.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20191104 或 https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I11/31

图1 转化载体pC3300-Epsps-35SCry1Ca的T-DNA结构

表1 转基因植株分子检测引物

图2 T1代转基因株植株的分子鉴定

图3 EPSPS和CRY1C蛋白的含量测定

图4 转化体E1C608-3的T3代植株喷施不同浓度的草甘膦实验

图5 转化体对稻纵卷叶螟抗性的鉴定

表2 转基因水稻E1C608农艺性状表现及评价级别

[1]	Muthayya S, Sugimoto J D, Montgomery S , et al. An overview of global rice production, supply, trade, and consumption. Annals of the New York Academy of Sciences, 2014,1324(1):7-14. doi: 10.1111/nyas.12539 pmid: 25224318
[2]	Oerke E C . Crop losses to pests. Journal of Agricultural Science, 2006,144(144):31-43. doi: 10.1007/s11274-019-2781-z pmid: 31832786
[3]	Edwards W J . Weed control in roundup ready TM rice . Proceedings of the California Weed Science, 2001,53(3):133-135.
[4]	翁绿水, 蒋利平, 肖国樱 . 抗虫抗除草剂转基因水稻恢复系B2A68的培育. 杂交水稻, 2013,28(1):63-67.
	Weng L S, Jiang L P, Xiao G Y . Development of an insect-resistant and herbicide-resistant transgenic restorer line B2A68 in rice. Hybrid Rice, 2013,28(1):63-67.
[5]	胡利华, 吴慧敏, 周泽民 , 等. 利用农杆菌介导法将柠檬酸合成酶基因(CS)导入籼稻品种明恢86. 分子植物育种, 2006,4(2):160-166.
	Hu L H, Wu H M, Zhou Z M , et al. Introduction of citrate synthase gene (CS) into an elite indica rice restorer line minghui 86 by Agrobacterium-mediated method. Molecular Plant Breeding, 2006,4(2):160-166.
[6]	Deng L H, Weng L S, Xiao G Y . Optimization of Epsps gene and development of double herbicide tolerant transgenic PGMS rice. Journal of Agricultural Science & Technology, 2014,16(1):217-228.
[7]	Padgette S R, Kolacz K H, Delannay X , et al. Development, identification, and characterization of a glyphosate-tolerant soybean line. Crop Science, 1995,35(5):1451-1461. doi: 10.2135/cropsci1995.0011183X003500050032x
[8]	吴慧敏 . 抗除草剂水稻培育及应用研究. 武汉:华中农业大学, 2005.
	Wu H M . Study on cultivation and application of herbicide resistant rice. Wuhan: Huazhong Agricultural University, 2005.
[9]	肖国樱 . 作物对除草剂的抗性及其在杂种优势利用中的应用策略的探讨. 杂交水稻, 1997,12(5):1-3.
	Xiao G Y . The view of crop herbicide resistance for heterosis utilization. Hybrid Rice, 1997,12(5):1-3.
[10]	Chen H, Lin Y J, Zhang Q F . Review and prospect of transgenic rice research. Science Bulletin, 2009,54(22):4049-4068. doi: 10.16288/j.yczz.18-213 pmid: 30369468
[11]	Fujimoto H, Itoh K, Yamamoto M , et al. Insect-resistant rice generated by introduction of a modified δ-endotoxin gene of Bacillus thuringiensis. Biocontrol Science & Technology, 1993,11(10):1151-1155. doi: 10.3390/md17120695 pmid: 31835449
[12]	Datta K, Vasquez A, Jumin T , et al. Constitutive and tissue-specific differential expression of the cry1A(b) gene in transgenic rice plants conferring resistance to rice insect pest. Theoretical and Applied Genetics, 1998,97(1-2):20-30. doi: 10.1007/s001220050862
[13]	Zheng X S, Yang Y J, Xu H X , et al. Resistance performances of transgenic Bt rice lines T2A-1 and T1c-19 against Cnaphalocrocis medinalis (Lepidoptera: Pyralidae). Journal of Economic Entomology, 2011,104(5):1730-1735. doi: 10.1603/EC10389
[14]	Zaidi M A, Ye G Y, Yao H W , et al. Transgenic rice plants expressing a modified cry1Ca1 gene are resistant to Spodoptera litura and Chilo suppressalis. Molecular Biotechnology, 2009,43(3):232-242. doi: 10.1007/s12033-009-9201-9
[15]	邓力华, 邓晓湘, 魏岁军 , 等. 抗虫抗除草剂转基因水稻B1C893的获得与鉴定. 杂交水稻, 2014,29(1):67-75.
	Deng L H, Deng X X, Wei S J , et al. Development and identification of herbicide and insect resistant transgenic plant B1C893 in rice. Hybrid Rice, 2014,29(1):67-66.
[16]	Hu W B, Deng X Y, Deng X X , et al. Characteristic analysis of tetra-resistant genetically modified rice. Journal of Integrative Agriculture, 2018,17(3):493-506. doi: 10.1016/S2095-3119(17)61722-2
[17]	钟其全, 廖翠猛, 盘毅 , 等. 广适性高产两系杂交中稻新组合C两优608. 杂交水稻, 2011,26(4):79-81.
	Zhong Q Q, Liao C M, Pan Y , et al. C liangyou 608, a new two-line medium hybrid rice combination with high yield and wide adaptability. Hybrid Rice, 2011,26(4):79-81.
[18]	Toki S, Hara N, Ono K , et al. Early infection of scutellum tissue with Agrobacterium allows high-speed transformation of rice. The Plant Journal, 2006,47(6):969-976. doi: 10.1111/j.1365-313X.2006.02836.x pmid: 16961734
[19]	Lin Y J, Zhang Q F . Optimizing the tissue culture conditions for high efficiency transformation of indica rice. Plant Cell Reports, 2005,23(8):540-547. doi: 10.1007/s00299-004-0843-6
[20]	Murray M G, Thompson W F . Rapid isolation of high molecular weight plant DNA. Nucleic Acids Research, 1980,8(19):4321-4325. doi: 10.1093/nar/8.19.4321 pmid: 7433111
[21]	Yang Z, Chen H, Tang W , et al. Development and characterization of transgenic rice expressing two Bacillus thuringiensis genes. Pest Management Science, 2011,67(4):414-422. doi: 10.1002/ps.2079
[22]	韩龙植, 魏兴华, 曹桂兰 , 等. 水稻种质资源描述规范和数据标准. 北京: 中国农业出版社, 2006: 66-69.
	Han L Z, Wei X H, Cao G L , et al. Descriptors and data standard for rice. Beijing: China Agricultural Press, 2006: 66-69.
[23]	黄大年, 李敬阳, 章善庆 . 用抗除草剂基因快速检测和提高杂交稻纯度的新技术. 科学通报, 1998,43(1):67-70.
	Huang D N, Li J Y, Zhang S Q . A new technology for rapid detection of herbicide resistance genes and improving purity of hybrid rice. Chinese Science Bulletin, 1998,43(1):67-70.
[24]	熊绪让, 唐俐, 邓晓湘 , 等. 抗除草剂两系杂交水稻香125S/Bar68-1栽培试验初报. 杂交水稻, 2004,19(5) : 41-43.
	Xiong X R, Tang L, Deng X X , et al. A Preliminary report on the experiments of herbicide-resistant two-line hybrid rice xiang 125S/Bar68-1. Hybrid Rice, 2004,19(5) : 41-43.
[25]	Zhao T, Lin C Y, Shen Z C . Development of transgenic glyphosate-resistant rice with G6 gene encoding 5 enolpyruvylshikimate-3-phosphate synthase. Agricultural Sciences in China, 2011,10(9):1307-1312. doi: 10.1016/S1671-2927(11)60123-5
[26]	赵艳, 邓春泉, 邓丽蝶 . 洁净DNA转化获得2mG2-epsps基因单拷贝整合的抗草甘膦水稻. 中国水稻科学, 2014,28(1):15-22. doi: 10.3969/j.issn.10017216.2014.01.003
	Zhao Y, Deng C Q, Deng L D . Generation of glyphosate-resistant transgenic rice harboring single copy of 2mG2-epsps gene by clean DNA transformation. Chinese Journal of Rice Science, 2014,28(1):15-22. doi: 10.3969/j.issn.10017216.2014.01.003
[27]	Chhapekar S, Raghavendrarao S, Pavan G , et al. Transgenic rice expressing a codon-modified synthetic CP4-EPSPS, confers tolerance to broad-spectrum herbicide, glyphosate. Plant Cell Reports, 2015,34(5):721-731. doi: 10.1007/s00299-014-1732-2 pmid: 25537885
[28]	Oono K . Putative homologous mutations in regenerated plants of rice. Molecular and General Genetics, 1985,198(3):377-384. doi: 10.1007/BF00332926
[29]	Bregitzer P, Dahleen L S, Neate S , et al. A single backcross effectively eliminates agronomic and quality alterations caused by somaclonal variation in transgenic barley. Crop Science, 2008,48(2):471-479. doi: 10.2135/cropsci2007.06.0370

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