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.

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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

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