耐草甘膦和啶嘧磺隆的转基因水稻研究

doi:10.3969/j.issn.1004-1524.20240340

浙江农业学报 ›› 2024, Vol. 36 ›› Issue (9): 1957-1968.DOI: 10.3969/j.issn.1004-1524.20240340

耐草甘膦和啶嘧磺隆的转基因水稻研究

吴浩峰¹(), 林朝阳¹^,², 沈志成¹^,^*()

1.浙江大学昆虫科学研究所,浙江杭州 310058
2.浙江大学新农村发展研究院,浙江杭州 310058

收稿日期:2024-04-12 出版日期:2024-09-25 发布日期:2024-09-30
作者简介:*沈志成,E-mail: zcshen@zju.edu.cn
吴浩峰(1998—),男,浙江湖州人,硕士,主要从事转基因水稻研究。E-mail: 13666508089@163.com
通讯作者: *沈志成,E-mail: zcshen@zju.edu.cn
基金资助:
浙江省“领雁”研发攻关计划(2023C02033)

A transgenic rice resistant to glyphosate and flazasulfuron

WU Haofeng¹(), LIN Zhaoyang¹^,², SHEN Zhicheng¹^,^*()

1. Instittute of Insect Sciences, Zhejiang University, Hangzhou 310058, China
2. The Rural Development Academy, Zhejiang University, Hangzhou 310058, China

Received:2024-04-12 Online:2024-09-25 Published:2024-09-30

摘要/Abstract

摘要：

杂草防治是水稻生产中的重要环节。该研究将耐草甘膦基因CP4-EPSPS和耐除草剂基因P450-N-Z1转入粳稻品种秀水134中,筛选获得一个对草甘膦以及啶嘧磺隆均具有良好耐受性的转化体GF-9。田间试验结果显示,该转化体能够耐受1 800 g·hm^-2(以酸当量计)草甘膦酸和30 g·hm^-2(以有效成分计)啶嘧磺隆活性成分的复合除草剂。分子特征鉴定结果表明,GF-9是单拷贝T-DNA转化体,其整合位点不在已知的注释基因中。田间初步试验表明,GF-9的田间表现型与非转基因对照相比无显著差异。综上所述,GF-9是具有潜在商业价值的转基因耐除草剂水稻转化体。

关键词: 水稻, 转基因, 耐除草剂

Abstract:

Weed control is an important aspect of rice production. In this study, a transgenic herbicide tolerant rice was created by expressing the glyphosate tolerant gene CP4-EPSPS and multiple-herbicide tolerant gene P450-N-Z1. The transformant event GF-9 was selected by screening with two herbicides, glyphosate and flazasulfuron. Field trial demonstrated that theGF-9 could tolerate combined herbicides at rate of 1 800 g·hm^-2 (based on acid equivalent) glyphosate and 30 g·hm^-2 (based on active ingredients) flazasulfuron. Southern blot and high-efficiency thermal asymmetric interlaced PCR results showed that the GF-9 was a single copy T-DNA insertion event and its insertion site was not inside a known or predicted functional gene. The field agronomic performance of GF-9 showed no significant difference compared to non-transgenic controls. In summary, the transgenic herbicide tolerant event GF-9 is a potential commercial event for development of herbicide tolerant rice.

Key words: rice, transgenic, herbicide tolerant

中图分类号:

S511

吴浩峰, 林朝阳, 沈志成. 耐草甘膦和啶嘧磺隆的转基因水稻研究[J]. 浙江农业学报, 2024, 36(9): 1957-1968.

WU Haofeng, LIN Zhaoyang, SHEN Zhicheng. A transgenic rice resistant to glyphosate and flazasulfuron[J]. Acta Agriculturae Zhejiangensis, 2024, 36(9): 1957-1968.

图/表 18

图1 T-DNA结构示意图

Fig.1 Diagram of the T-DNA structure

表1 复合除草剂成分表

Table 1 List of compound herbicide ingredients

除草剂浓度 Herbicide concentration	41%草甘膦异丙胺盐水剂用量 41% Glyphosate isopropylamine aqueous solution dosage	25%啶嘧磺隆水分散粒剂用量 25% Flazasulfuron water dispersible granules dosage
0(清水Water control)	0	0
1倍浓度Single concentration	5 mL·L^-1(900 g·hm^-2)	0.1 g·L^-1(15 g·hm^-2)
2倍浓度Double concentration	10 mL·L^-1(1 800 g·hm^-2)	0.2 g·L^-1(30 g·hm^-2)
4倍浓度Quadruple concentration	20 mL·L^-1(3 600 g·hm^-2)	0.4 g·L^-1(60 g·hm^-2)

表2 外源基因的PCR引物序列

Table 2 PCR primer sequences for the foreign genes

引物 Primer	引物序列 Primer sequences(5'→3')
CP4-F	ATGGCGGCGACCATGGCGTCCAACG
CP4-R	TCAAGCGGCCTTCGTGTCAGACAGTTC
N-Z1-F	ATGGATAAGGCCTACGTGGCCCTCC
N-Z1-R	TCAGAGCTCCTGCAAAACCTCACGC

表3 高效热不对称交错PCR引物序列

Table 3 Primers sequences for high-efficiency thermal asymmetric interlaced PCR

引物Primer	引物序列Primer sequences(5'→3')
LB-SPI	TTTCTCCATAATAATGTGTGAGTAGTTCCC
LB-SP2a	ACGATGGACTCCAGTCCGGCCCTCATGTGTTGAGCATATAAGAAACCCTTAG
LB-SPIII	CTAAAACCAAAATCCAGTACTAAAATCC
RB-0b	CGTGACTGGGAAAACCCTGGCGTT
RB-1b	ACGATGGACTCCAGTCCGGCCCAACTTAATCGCCTTGCAGCACATC
RB-2b	GAAGAGGCCCGCACCGATCGCCCTT
LAD1	ACGATGGACTCCAGAGCGGCCGCVNVNNNGGAA
LAD2	ACGATGGACTCCAGAGCGGCCGCBNBNNNGGTT
LAD3	ACGATGGACTCCAGAGCGGCCGCVVNVNNNCCAA
LAD4	ACGATGGACTCCAGAGCGGCCGCBDNBNNNCGGT
AC1	ACGATGGACTCCAGAG

表4 边界验证引物序列

Table 4 Boundary validation primer sequence

引物 Primer	引物序列 Primer sequences(5'→3')
L1	CTCCCCCATTCTGGCTTGGAA
L2	CATTGTGAAGCCCTCGAGTAATTGG
L3	ACCGGAGACATATAGTGCTGGTTT
R1	TGGGCGCTCTTCGACGGATT
R2	GGCCATCGCAGCCATTAAATCG
R3	TCCTCTGTGGGGCTTTGGAC

图2 转化体的温室草甘膦耐受试验结果 Water,清水处理;Glyphosate,900 g·hm-2(以酸当量计)的草甘膦处理;TG,GF系列转基因水稻转化体;NT,非转基因对照组。

Fig.2 Glyphosate tolerance test results of transgenic rice events in greenhouse Water, Water control; Glyphosate, Spary glyphosate at a rate of 900 g·hm-2 (based on acid equivalent); TG, GF series transgenic rice events; NT, Non-transgenic control.

图3 转化体的温室啶嘧磺隆耐受试验结果 Water,清水处理;Flazasulfuron,15 g·hm-2(以有效成分计)的啶嘧磺隆处理;TG,GF系列转基因水稻转化体;NT,非转基因对照组。

Fig.3 Flazasulfuron tolerance test results of transgenic rice events in greenhouse Water, Water control; Flazasulfuron, Spary flazasulfuron at a rate of 15 g·hm-2 (based on active ingredient); TG, GF series transgenic rice events; NT, Non-transgenic control.

图4 复合除草剂稻田除草效果图 A,复合除草剂喷施7 d后稻田杂草发生情况;B,复合除草剂喷施28 d后稻田杂草发生情况;TG,GF系列转基因水稻转化体;NT,非转基因对照组;1倍,1倍浓度复合除草剂处理;2倍,2倍浓度复合除草剂处理;4倍,4倍浓度复合除草剂处理。

Fig.4 Images of weed control effects in paddy fields using compound herbicides A, Occurrence of weeds in rice fields 7 days after application of a mixed herbicide;B, Occurrence of weeds in rice fields 28 days after application of a mixed herbicide; TG, GF series transgenic rice events; NT, Non-transgenic control; Single, Single concentration compound herbicide treatment; Double, Double concentration compound herbicide treatment; Quadruple, Quadruple concentration compound herbicide treatment.

图5 GF-9在不同浓度除草剂处理28 d后的受害情况 TG,GF系列转基因水稻转化体;NT,非转基因对照组;1倍,1倍浓度复合除草剂处理;2倍,2倍浓度复合除草剂处理;4倍,4倍浓度复合除草剂处理。

Fig.5 The damage situation of GF-9 after 28 days of treatment with different concentrations of herbicide TG, GF series transgenic rice events; NT, Non-transgenic control; Single, Single concentration compound herbicide treatment; Double, Double concentration compound herbicide treatment; Quadruple, Quadruple concentration compound herbicide treatment.

表5 GF-9在不同浓度除草剂处理下的株高

Table 5 Plant height for GF-9 under different herbicide concentrations

除草剂浓度 Herbicide concentration	GF-9	秀水134 XS-134
0(清水Water control)	83.57±2.44 Aa	86.16±3.67 A
1倍浓度Single concentration	82.03±2.38 a	—
2倍浓度Double concentration	78.50±2.36 b	00.00±0.00
4倍浓度	78.20±2.57 b	—
Quadruple concentration

表6 GF-9在不同浓度除草剂处理下田间表现型

Table 6 Agronomic performance of GF-9 under different concentrations of herbicide

试验材料 Material	除草剂浓度 Herbicide concentration	穗长 Panicle length/cm	分蘖数 Tiller number	百粒重 Hundred-grain weight/g	结实率 Setting rate%
GF-9	0(清水Water control)	14.82±0.72 Aa	9.00±1.18 Aa	2.536±0.027 Aa	68.29±4.60 Aa
	1倍浓度Single concentration	15.84±0.81 a	10.20±2.35 a	2.575±0.025 a	71.01±7.52 a
	2倍浓度Double concentration	15.72±1.05 a	10.20±2.30 a	2.519±0.048 a	69.12±5.49 a
	4倍浓度Quadruple concentration	15.47±1.21 a	8.58±2.50 a	2.254±0.042 b	55.20±2.60 b
秀水134 XS-134	0(清水Water control)	14.32±1.57 A	8.50±1.35 A	2.543±0.051 A	65.00±5.70 A

图6 不同浓度除草剂处理下GF-9的穗长与结实率情况 NT,清水处理下的非转基因对照;1倍,1倍浓度复合除草剂处理;2倍,2倍浓度复合除草剂处理;4倍,4倍浓度复合除草剂处理。

Fig.6 Panicle length and fruiting rate of GF-9 under different concentrations of herbicide NT, Non-transgenic control under water treatment; Single, Single concentration compound herbicide treatment; Double, Double concentration compound herbicide treatment; Quadruple, Quadruple concentration compound herbicide treatment.

图7 GF-9的外源基因PCR检测结果 A,CP4-EPSPS基因的PCR检测结果;B,P450-N-Z1基因的PCR检测结果;M,DNA标准分子量;+,阳性质粒;-,非转基因对照;1,GF-9 T0代样品基因组;2~4,GF-9 T1代样品基因组;3~7,GF-9 T2代样品基因组。

Fig.7 PCR detection results of the transgenes in GF-9 A, PCR results of CP4-EPSPS gene; B, PCR results of P450-N-Z1 gene; M, DNA Marker; +, Positive plasmid; -, Non-transgenic control; 1, GF-9 T0 generation sample genome; 2-4, GF-9 T1 generation sample genome; 3-7, GF-9 T2 generation sample genome.

图8 水稻转化体GF-9的边界验证结果 A,GF-9的左边界PCR验证结果;B,GF-9的右边界PCR验证结果。M,DNA长度标记;L1~L3,T-DNA左边界外三个不同距离的验证引物分别与LB-SPI的PCR结果;R1~R3,T-DNA右边界外3个距离的验证引物分别与RB-0b的PCR结果;-,阴性水稻对照组。

Fig.8 Boundary verification results of the event GF-9 A, PCR validation results of the left border of GF-9; B, PCR validation results of the right border of GF-9. M, DNA Marker; L1-L3, PCR results of the verification primers for three different distances outside the left border of T-DNA, respectively with LB-SPI; R1-R3, PCR results of the verification primers for three different distances outside the right border of T-DNA, respectively with RB-0b; -, Negative rice control.

图9 GF-9的外源T-DNA基因组整合模型及边界测序验证结果 L1~L3,T-DNA左边界验证引物;R1~R3,T-DNA右边界验证引物。

Fig.9 Integration model of the foreign T-DNA in GF-9 and verification results of the boundary sequencing L1-L3, Primers for validating the left border of T-DNA.; R1-R3, Primers for validating the right border of T-DNA.

图10 GF-9的限制性内切酶和CP4探针的位置和预期杂交信号带大小示意图 Probe,制作探针所用的基因序列。

Fig.10 Schematic diagram of GF-9 restriction enzyme and CP4 probe location and expected hybridization signal band size Probe, Gene sequence used to make probe.

图11 GF-9的Southern blot杂交结果图 +,阳性质粒;NT,以相同限制性内切酶酶切的阴性水稻对照组;M,DNA长度标记。

Fig.11 Southern blot hybridization of GF-9

图12 CP4-EPSPS蛋白的Western blot检测结果 M,蛋白标准分子量;+,原核表达的CP4-EPSPS蛋白;-,非转基因水稻对照组;1~7,T1代转化体GF-9的7棵植株。

Fig.12 Western blot results of CP4-EPSPS protein M, Protein pageruler; +, CP4-EPSPS protein expressed in prokaryotes; -, Non-transgenic rice control group; 1-7, Seven independent plants of T1 generation transgenic line GF-9.

参考文献 16

[1]	AKBAR N, EHSANULLAH, JABRAN K, et al. Weed management improves yield and quality of direct seeded rice[J]. Australian Journal of Crop Science, 2011, 5(6): 688-694.
[2]	DUKE S O, POWLES S B. Glyphosate: a once-in-a-century herbicide[J]. Pest Management Science, 2008, 64(4): 319-325. DOI PMID
[3]	BENTLEY R. The shikimate pathway: a metabolic tree with many branches[J]. Critical Reviews in Biochemistry and Molecular Biology, 1990, 25(5): 307-384.
[4]	DILL G M. Glyphosate-resistant crops: history, status and future[J]. Pest Management Science, 2005, 61(3): 219-224. DOI PMID
[5]	陈世国, 强胜, 毛婵娟. 草甘膦作用机制和抗性研究进展[J]. 植物保护, 2017, 43(2): 17-24.
	CHEN S G, QIANG S, MAO C J. Mechanism of action of glyphosate and research advances in glyphosate resistance[J]. Plant Protection, 2017, 43(2): 17-24. (in Chinese with English abstract)
[6]	沈国辉, 杨烈, 钱振官, 等. 秀百宫: 暖季型草坪广谱性除草剂[J]. 草原与草坪, 2001, 21(3): 49-51.
	SHEN G H, YANG L, QIAN Z G, et al. Shibagen: a broad spectrum herbicide for weed control in warm season turfgrass[J]. Grassland and Turf, 2001, 21(3): 49-51. (in Chinese with English abstract)
[7]	范文政. 啶嘧磺隆: 葡萄、柑橘、橄榄、甘蔗和非耕地的新型除草剂[J]. 世界农药, 2004, 26(1): 48-49.
	FAN W Z. Flazasulfuron:the new herbicides for grapes, citrus, olives, sugarcane, and non-agricultural fields.[J]. World Pesticide, 2004, 26(1): 48-49. (in Chinese)
[8]	ZHENG T, YU X X, SUN Y Z, et al. Expression of a cytochrome P450 gene from Bermuda grass Cynodon dactylon in soybean confers tolerance to multiple herbicides[J]. Plants, 2022, 11(7): 949.
[9]	HIEI Y, KOMARI T, KUBO T. Transformation of rice mediated by Agrobacterium tumefaciens[J]. Plant Molecular Biology, 1997, 35(1): 205-218.
[10]	HEALEY A, FURTADO A, COOPER T, et al. Protocol: a simple method for extracting next-generation sequencing quality genomic DNA from recalcitrant plant species[J]. Plant Methods, 2014, 10(1): 21.
[11]	LIU Y G, CHEN Y L. High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences[J]. BioTechniques, 2007, 43(5): 649-650, 652, 654.
[12]	胡江博, 任正鹏, 丁翔, 等. 稻田除草剂应用现状与抗除草剂水稻育种研究进展[J]. 中国稻米, 2023, 29(4): 13-19. DOI
	HU J B, REN Z P, DING X, et al. Application of herbicides in rice fields and research progress on herbicide-resistant rice varieties breeding[J]. China Rice, 2023, 29(4): 13-19. (in Chinese with English abstract) DOI
[13]	刘庆虎, 陈国奇, 张玉华, 等. 不同叶龄千金子、稗和马唐对氰氟草酯和五氟磺草胺的敏感性[J]. 南京农业大学学报, 2016, 39(5): 771-776.
	LIU Q H, CHEN G Q, ZHANG Y H, et al. Sensitivities of Leptochloa chinensis, Echinochloa crusgalli and Digitaria sanguinalis at different leaf stages to cyhalofop-butyl and penoxsulam[J]. Journal of Nanjing Agricultural University, 2016, 39(5): 771-776. (in Chinese with English abstract)
[14]	DAMALAS C A, KOUTROUBAS S D. Herbicide-resistant barnyardgrass (Echinochloa crusgalli) in global rice production[J]. Weed Biology and Management, 2023, 23(1): 23-33.
[15]	KAWAHIGASHI H, HIROSE S, OHKAWA H, et al. Herbicide resistance of transgenic rice plants expressing human CYP1A1[J]. Biotechnology Advances, 2007, 25(1): 75-84. PMID
[16]	HAN H P, YU Q, BEFFA R, et al. Cytochrome P450 CYP81A10v7 in Lolium rigidum confers metabolic resistance to herbicides a cross at least five modes of action[J]. The Plant Journal, 2021, 105(1): 79-92.

耐草甘膦和啶嘧磺隆的转基因水稻研究

A transgenic rice resistant to glyphosate and flazasulfuron

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 18

参考文献 16

相关文章 15

编辑推荐

Metrics

本文评价

[1]	颜晶莹, 倪亮, 沈星宇, 李玉. 热处理对转基因秸秆中重组蛋白和重组DNA的降解作用[J]. 浙江农业学报, 2024, 36(9): 2079-2088.
[2]	展梦琪, 苏傲雪, 侯倩, 张皓宇, 姜欣蕊, 徐艳. 水稻对林丹的吸收累积与代谢组学研究[J]. 浙江农业学报, 2024, 36(9): 2110-2121.
[3]	邵亚旭, 刘涛, 王事成, 晏磊. 秸秆-有机肥育秧基质的配比筛选与成型工艺[J]. 浙江农业学报, 2024, 36(8): 1856-1866.
[4]	董爱琴, 陈院华, 杨涛, 徐昌旭, 程丽群, 谢杰. 紫云英和石灰配施对水稻镉吸收的影响[J]. 浙江农业学报, 2024, 36(3): 600-612.
[5]	郑涵, 丁文金, 何招亮, 侯凡, 戴彬凤, 钟列权, 张海鹏, 杨勇. 穗分化期高温对水稻生长发育的影响及缓解措施研究进展[J]. 浙江农业学报, 2024, 36(2): 470-480.
[6]	杨西帆, 郭彬, 裘高扬, 刘俊丽, 童文彬, 杨海峻, 祝伟东, 毛聪妍. 不同钝化产品对水稻生产中镉、铅、砷的钝化效果[J]. 浙江农业学报, 2024, 36(1): 1-8.
[7]	罗英杰, 崔维军, 王忠华, 吴月燕, 林宏友, 周洁, 严成其, 王栩鸣. 水稻泛素连接酶D3与抗病相关蛋白VOZ2的互作分析[J]. 浙江农业学报, 2024, 36(1): 9-17.
[8]	张思雨, 林朝阳, 叶雨轩, 沈志成. 转cry1Ab-vip3Af2和cp4-epsps基因的抗虫耐除草剂水稻的研究[J]. 浙江农业学报, 2023, 35(8): 1823-1833.
[9]	杨坤, 侯冠军, 赵秀侠, 方婷, 王利军. 水生动植物协同净化系统对鳜鱼养殖池塘水质与经济效益的影响[J]. 浙江农业学报, 2023, 35(7): 1709-1719.
[10]	王鑫彤, 万祖粱, 杨振中, 王国骄. 秸秆秋季湿耙还田对水稻不同生育时期叶片-土壤生态化学计量特征的影响[J]. 浙江农业学报, 2023, 35(6): 1243-1252.
[11]	刘光瑞, 宗渊, 李云, 曹东, 刘宝龙, 包雪梅, 李建民. 当归转录因子AsMYB44的克隆与功能研究[J]. 浙江农业学报, 2023, 35(6): 1253-1264.
[12]	张雪楠, 王乐乐, 钮铭轩, 詹妮, 任浩杰, 徐浩聪, 杨昆, 武立权, 柯健, 尤翠翠, 何海兵. 基于叶片反射光谱和叶绿素荧光估测水稻叶片含水量[J]. 浙江农业学报, 2023, 35(6): 1265-1277.
[13]	张超正, 张旭鹏, 陈丹玲. 劳动力老龄化、耕地细碎化必然导致水稻生产成本增加吗?——基于鄂东南地区的微观调查[J]. 浙江农业学报, 2023, 35(5): 1211-1222.
[14]	夏小东, 张晓波, 施勇烽, 许如根. 水稻致死突变体基因克隆与分子机制研究进展[J]. 浙江农业学报, 2023, 35(5): 1223-1234.
[15]	蒋莹莹, 张华, 雷志伟, 徐恒, 张恒, 朱英. 茉莉酸信号关键转录因子OsMYC2影响水稻愈伤诱导和分化的功能初探[J]. 浙江农业学报, 2023, 35(5): 973-982.