转cry1Ab-vip3Af2和cp4-epsps基因的抗虫耐除草剂水稻的研究

doi:10.3969/j.issn.1004-1524.20230491

浙江农业学报 ›› 2023, Vol. 35 ›› Issue (8): 1823-1833.DOI: 10.3969/j.issn.1004-1524.20230491

转cry1Ab-vip3Af2和cp4-epsps基因的抗虫耐除草剂水稻的研究

张思雨¹(), 林朝阳¹^,², 叶雨轩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:2023-04-18 出版日期:2023-08-25 发布日期:2023-08-29
作者简介:张思雨(1998—),女,山西晋城人,硕士研究生,主要从事转基因水稻研究。E-mail:13327562932@163.com
通讯作者: *沈志成,E-mail:zcshen@zju.edu.cn
基金资助:
农业农村部转基因重大专项(2016ZX08010-003)

Characterization of transgenic insect resistance and glyphosate tolerance rice expressing cry1Ab-vip3Af2 and cp4-epsps

ZHANG Siyu¹(), LIN Chaoyang¹^,², YE Yuxuan¹^,², SHEN Zhicheng¹^,^*()

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

Received:2023-04-18 Online:2023-08-25 Published:2023-08-29

摘要/Abstract

摘要：

为培育抗虫耐除草剂转基因水稻(Oryza sativa L.),本研究将抗虫融合基因cry1Ab-vip3Af2与耐草甘膦基因cp4-epsps构建在同一T-DNA中,并利用农杆菌侵染法导入秀水134水稻。对获得的转化体的分子特征及抗虫耐除草剂性能进行分析研究,从众多转化体中筛选得到优良转化体VB3。High efficiency TAIL-PCR (Hi-Tail PCR)的分析结果表明,VB3转化体中外源T-DNA的插入位点未编码已知或预测的功能基因。DNA杂交印迹结果显示,VB3转化体的外源T-DNA以单拷贝的形式整合在水稻基因组中。酶联免疫吸附反应和蛋白质免疫印迹分析结果表明,T₁、T₂代VB3转化体的不同组织中均检测到较高的外源蛋白表达量,且不同世代之间表达量无明显差异。室内生测结果显示,VB3转化体对大螟(Sesamia inferens)、二化螟(Chilo suppressalis)和稻纵卷叶螟(Cnaphalocrocis medinalis)均表现出较高的抗性。三种靶标昆虫的幼虫取食VB3植物组织后,48 h的死亡率达到90%左右,72 h内全部死亡。草甘膦耐受性能测定结果表明,VB3转化体能够耐受20、40和80 mL·hm^-2的草甘膦。综上所述,本研究筛选出的VB3转化体具有较好的生产应用前景。

关键词: 转基因, 害虫防治, 抗虫水稻, 耐除草剂水稻

Abstract:

In order to develop a transgenic rice (Oryza sativa L.) with insect resistance and herbicide tolerance, a T-DNA with both expression cassettes of the insecticidal fusion gene cry1Ab-vip3Af2 and glyphosate tolerance gene cp4-epsps was constructed and transferred into japonica rice Xiushui 134. A leading event named VB3 was selected based on molecular characterization, insect-resistance and herbicide-tolerance assays. Hi-Tail PCR suggested that the T-DNA insertion site of VB3 does not encode any known or predicted functional genes. Southern blotting suggested that the VB3 was a single copy insertion event. The enzyme-linked immunosorbent assay and Western blotting showed that the exogenous proteins were detected at high levels in different tissues of VB3, and had no significant difference among different generations. The laboratory bioassay showed that the VB3 was highly resistant to Sesamia inferens, Chilo suppressalis and Cnaphalocrocis medinalis. The mortality of three target insect larvae on VB3 was about 90% in 48 hours and 100% in 72 hours. The glyphosate tolerance test showed that the VB3 showed no visible damage with 20、40 and 80 mL·hm^-2 of recommended glyphosate dosage respectively. In collusion, the VB3 event has potential value for possible commercial development.

Key words: transgenic, pest control, insect resistance rice, herbicide tolerance rice

中图分类号:

S435.11

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.

ZHANG Siyu, LIN Chaoyang, YE Yuxuan, SHEN Zhicheng. Characterization of transgenic insect resistance and glyphosate tolerance rice expressing cry1Ab-vip3Af2 and cp4-epsps[J]. Acta Agriculturae Zhejiangensis, 2023, 35(8): 1823-1833.

图/表 13

图1 T-DNA结构示意图

Fig.1 Diagram of the T-DNA structure

表1 特异性PCR用到的引物及序列

Table 1 Primers of PCR

引物 Primer	引物序列 Primer sequences(5'→3')
cry1Ab-vip3Af2-F	ACCTTCTTCTTGTTCAGGTCGATCTC
cry1Ab-vip3Af2-R	CATCAACACCATGCTGCACATCTACC
cp4-epsps-F	TTCATGAGGACGTTCAGGATGGTCAC
cp4-epsps-R	AGATGGGCGTACAGGTCAAATCCGA

表2 Hi-Tail PCR用到的引物及序列

Table 2 Primers of Hi-Tail PCR

引物 Primer	引物序列 Primer sequences(5'→3')
LAD1	ACGATGGACTCCAGAGCGGCCGCVNVNNNGGAA
LAD2	ACGATGGACTCCAGAGCGGCCGCBNBNNNGGTT
LAD3	ACGATGGACTCCAGAGCGGCCGCVVNVNNNCCAA
LAD4	ACGATGGACTCCAGAGCGGCCGCBDNBNNNCGGT
RB-1	GGCACTGGCCGTCGTTTTACAACGTCGT
RB-2	ACGATGGACTCCAGTCCGGCCGCGTTACCCAACTTAA- TCGCCTTGCAGC
RB-3	GCACCGATCGCCCTTCCCAACAGTTGC
LB-1	TTTCTCCATAATAATGTGTGAGTAGTTCCC
LB-2	ACGATGGACTCCAGTCCGGCCCTCATGTGTTGAGCAT- ATAAGAAACCCTTAG
LB-3	CTAAAACCAAAATCCAGTACTAAAATCC
AC1	ACGATGGACTCCAGAG

表3 靶标害虫室内生物活性测定

Table 3 The laboratory bioassay of target pests

靶标昆虫 Target pests	被试组织 Subject tissue	幼虫数量 Quantity of larvae	水稻生长期 Rice growth stage	观察时间 Observation time/h
大螟 Sesamia inferens	VB3叶片 VB3 leaves	20	分蘖期、抽穗期、灌浆期 Tillering stage, heading stage, filling stage	24
	秀水134叶片 Xiushui 134 leaves
二化螟 Chilo suppressalis	VB3叶片 VB3 leaves	20	分蘖期、抽穗期、灌浆期 Tillering stage, heading stage, filling stage	24
	秀水134叶片 Xiushui 134 leaves
稻纵卷叶螟 Cnaphalocrocis medinalis	VB3叶片 VB3 leaves	20	分蘖期、抽穗期、灌浆期 Tillering stage, heading stage, filling stage	24
	秀水134叶片 Xiushui 134 leaves

表4 草甘膦耐受性能测定

Table 4 Glyphosate tolerance determination

除草剂剂量 Herbicide concentration/ (mL·hm^-2)	被试组织 Subject tissue	观察时间 Observation time/d
0	VB3幼苗期3~5叶 3-5 leaves at seedling stage of VB3	7
	秀水134幼苗期3~5叶 3-5 leaves at seedling stage of Xiushui 134
20	VB3幼苗期3~5叶 3-5 leaves at seedling stage of VB3	7
	秀水134幼苗期3~5叶 3-5 leaves at seedling stage of Xiushui 134
40	VB3幼苗期3~5叶 3-5 leaves at seedling stage of VB3	7
	秀水134幼苗期3~5叶 3-5 leaves at seedling stage of Xiushui 134
80	VB3幼苗期3~5叶 3-5 leaves at seedling stage of VB3	7
	秀水134幼苗期3~5叶 3-5 leaves at seedling stage of Xiushui 134

图2 T0代水稻转化体中外源蛋白的表达量 A,Cry1Ab-Vip3Af2蛋白的表达量,图中虚线所示Cry1Ab-Vip3Af2的表达量为5 μg·g-1,以此为筛选标准;B,CP4-EPSPS蛋白的表达量,图中虚线所示CP4-EPSPS的表达量为150 μg·g-1,以此为筛选标准。

Fig.2 The expression level of exogenous proteins in T0 transformants of rice A, The expression level of Cry1Ab-Vip3Af2, the dotted line showed the Cry1Ab-Vip3Af2 expression level was 5 μg·g-1, which was used as the screening standard; B, The expression level of CP4-EPSPS, the dotted line showed the CP4-EPSPS expression level was 150 μg·g-1, which was used as the screening standard.

图3 T1、T2代VB3水稻转化体的PCR检测结果 A,cry1Ab-vip3Af2基因的PCR检测结果;B,cp4-epsps基因的PCR检测结果;M,DNA分子量标准;+,阳性质粒;-,非转基因水稻对照;1~3,VB3的T1代和T2代样品。

Fig.3 PCR analysis of T1, T2 generations of VB3 A, PCR analysis of cry1Ab-vip3Af2; B, PCR analysis of cp4-epsps; M, DNA molecular marker;-, Non-transgenic rice control;+, Positive plasmids; 1-3, Samples of T1 and T2 generations of VB3.

图4 VB3边界序列鉴定及分析 A,对VB3边界的三轮Hi-Tail PCR电泳结果。R1,第一轮PCR反应产物;R2,第二轮PCR反应产物;R3,第三轮PCR反应产物;M,DNA分子量标准。B,VB3边界的PCR鉴定结果。M,DNA分子量标准。

Fig.4 Identification and analyzation of VB3 border sequences A, The electrophoresis result of Hi-Tail PCR about VB3 border sequences. R1, Round 1 products; R2, Round 2 products; R3, Round 3 products; M, DNA molecular marker. B, The identification of VB3 border sequences. M, DNA molecular marker.

图5 VB3的Southern blot杂交结果 A,以cry1Ab全长DNA序列为探针的杂交图;B,以cp4-epsps全长DNA序列为探针的杂交图;+,阳性质粒;NT,以相同酶切的阴性水稻对照组;M,DNA分子量标准。

Fig.5 Southern bloting of transgenic rice VB3 A, Hybridization with cry1Ab full-length gene as probe; B, Hybridization with cp4-epsps full-length gene as probe;+, Positive plasmids; NT, Non-transgenic rice control digested with the same enzyme; M, DNA molecular maker.

图6 VB3各组织中外源蛋白的表达量 A,VB3各组织中Cry1Ab-Vip3Af2蛋白的表达量;B,VB3各组织中CP4-EPSPS蛋白的表达量。

Fig.6 The expression level of exogenous proteins in different tissues of VB3 A, The expression level of Cry1Ab-Vip3Af2 in different tissues of VB3; B, The expression level of CP4-EPSPS in different tissues of VB3.

图7 T1、T2代VB3水稻中Cry1Ab-Vip3Af2蛋白和CP4-EPSPS蛋白的Western blotting检测 A,Cry1Ab-Vip3Af2蛋白的Western blotting检测结果;B,CP4-EPSPS蛋白的Western blotting检测结果;M,标准蛋白分子量;+,原核表达的蛋白;-,非转基因水稻对照组;L,叶片组织;St,茎秆组织;Sd,籽粒。

Fig.7 Western blotting about Cry1Ab-Vip3Af2 and CP4-EPSPS in T1 and T2 generations of VB3 A, Western blotting of Cry1Ab-Vip3Af2; B, Western blotting of CP4-EPSPS; M, Protein pageruler;+, Protein expressed by E.coli;-, Non-transgenic rice control; L, Leaves; St, Stem; Sd, Seed.

表5 大螟、二化螟、稻纵卷叶螟幼虫取食VB3和非转基因水稻叶片的死亡率

Table 5 Mortality of Sesamia inferens, Chilo suppressalis, Cnaphalocrocis medinalis fed on VB3 and non-transgenic rice leaves

时期 Stage	水稻株系 Rice line	大螟Sesamia inferens			二化螟Chilo suppressalis			稻纵卷叶螟Cnaphalocrocis medinalis
		死亡率Mortality/%			死亡率Mortality/%			死亡率Mortality/%
		24 h	48 h	72 h	24 h	48 h	72 h	24 h	48 h	72 h
分蘖期 Tillering stage	VB3 VB3	29.37± 3.00^**	91.30± 2.50^**	100^**	31.40± 2.44^**	85.07± 2.04^**	100^**	32.57± 2.68^**	92.10± 1.97^**	100^**
	秀水134 Xiushui 134	3.06± 0.57	5.80± 1.45	7.33± 0.81	2.80± 0.46	3.77± 0.45	7.60± 0.56	3.30± 0.44	5.57± 0.76	8.50± 0.53
抽穗期 Heading stage	VB3 VB3	23.63± 2.65^**	88.73± 1.23^**	100^**	22.53± 1.78^**	86.73± 2.87^**	100^**	23.43± 1.25^**	87.03± 1.69^**	100^**
	秀水134 Xiushui 134	3.47± 0.40	4.67± 0.40	8.30± 0.9	2.57± 0.23	3.97± 0.42	6.87± 0.25	3.03± 0.23	5.67± 0.57	7.83± 0.81
灌浆期 Filling stage	VB3 VB3	30.37± 2.57^**	82.97± 2.32^**	100^**	32.60± 2.04^**	93.77± 1.17^**	100^**	26.53± 0.96^**	89.00± 2.26^**	100^**
	秀水134 Xiushui 134	2.77± 0.42	4.57± 0.61	7.37± 0.55	4.00± 0.46	5.17± 0.45	7.50± 0.17	2.80± 0.36	4.67± 0.32	8.17± 0.25

图8 温室条件下VB3对草甘膦的耐受能力测定

Fig.8 The glyphosate tolerance of VB3 in green house

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转cry1Ab-vip3Af2和cp4-epsps基因的抗虫耐除草剂水稻的研究

Characterization of transgenic insect resistance and glyphosate tolerance rice expressing cry1Ab-vip3Af2 and cp4-epsps

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