转多基因107杨对目标害虫的抗性检测

doi:10.11707/j.1001-7488.20211109

Abstract

Abstract:

Objective: The purpose of this study was to explore the stability and expression of exogenous gene in transgenic poplar 107(Populus×euramericana 'Neva') and to screen transgenic lines with double resistance to Lepidoptera and Coleoptera pests, providing valuable reference for breeding new varieties of transgenic poplar with multiple genes resistant to pests. Method: PCR, toxin protein content detection and insect resistance detection were performed in 2-year-old field seedlings of three lines (ECAA1-3) transformed with Cry1Ac-Cry3A-BADH genes and three lines (ECAB1-3) transformed with Cry1Ac-Cry3A-NTHK1 genes. Result: PCR gel electrophoresis test proved that the target gene was still in the genome of transgenic poplar 107. The detection of Bt toxin of transgenic poplar 107 showed that the 2-year-old transgenic lines in August all contained Cry1Ac and Cry3A toxin, but the content of Cry3A toxin was relatively high. The toxic protein content of Cry1Ac was 3.60-20.76 ng·g^-1, and the toxic protein content of Cry3A was 3 479.66-7 230.29 ng·g^-1. Indoor insect feeding test showed that the transgenic poplar 107 had resistance to both Hyphantria cunea and Plagiodera versicolora larvae, among which ECAB1 had the highest lethal rate to H. cunea (100%), and ECAA1, ECAA2 and ECAA3 significantly inhibited the growth and development of H. cunea larvae of the first instar. With the exception of transgenic line ECAA2, all the other lines showed high insect resistance to P. versicolora, with the lethal rate of 100% for the first instar larvae, 85%-100% for the first and second instar mixed larvae, and over 66% for adults. Conclusion: The exogenous genes of transgenic poplar 107 were still present and stably expressed, and their insect resistance was significantly improved compared with that of non-transgenic controls. However, there were significant differences among different transgenic lines, the strains ECAA3 and ECAB1 showed high insect resistance to the larvae of H. cunea and P. versicolora.

Key words: Populus×euramericana 'Neva', Bt gene, polygenic transformation, gene expression, insect resistance

CLC Number:

Kunjin Han,Juanjuan Guo,Ziqing Lü,Yuyan Li,Shijie Wang,Minsheng Yang,Jinmao Wang. Detection of Resistance of Multi-Gene Transgenic Populus×euramericana 'Neva' to Target Pests[J]. Scientia Silvae Sinicae, 2021, 57(11): 85-93.

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目的基因 Gene	引物序列 Primer sequence (5′—3′)		扩增片段 Fragment size/bp
Cry1Ac	F: ATGGATAACAATCCGAACATCA	R: CCACCTTTGTCCAAACACTGAA	546
Cry3A	F: CACTGTTCCCACTGTACGATGT	R: ATGTTGAAGAAGTCCACGCTCT	667

日期 Date	杨树株系 Poplar line	2龄幼虫取食叶面积率 Leaf feeding ratio by larvae(%)	2龄幼虫死亡数 Deaths
08-06—08-07	ECAA1	9.29±2.39 a	0
	ECAA2	8.74±0.70 a	1
	ECAA3	9.85±0.58 a	1
	ECAB1	10.07±0.57 a	0
	ECAB2	9.57±0.39 a	0
	CK	10.36±0.70 a	0
08-10—08-11	ECAA1	19.80±5.65 a	0
	ECAA2	54.58±7.86 a	2
	ECAA3	40.43±11.64 a	2
	ECAB1	26.35±18.26 a	2
	ECAB2	29.05±10.61 a	4
	CK	40.11±8.96 a	1
08-14—08-15	ECAA1	64.89±10.69 ab	0
	ECAA2	81.46±11.54 a	4
	ECAA3	71.81±14.45ab	4
	ECAB1	63.63±20.56 ab	8
	ECAB2	2.17±1.22 b	8
	CK	100.00±0.00 a	4

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Detection of Resistance of Multi-Gene Transgenic Populus×euramericana 'Neva' to Target Pests

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测试天数 Days	幼虫死亡数Larval deaths
测试天数 Days	ECAA1	ECAA2	ECAA3	ECAB1	ECAB2	ECAB3	CK
3	13	12	28	11	7	13	9
6	1	9	11	50	2	11	4
9	3	3	1	8	2	12	0
12	0	3	3	0	4	7	1
14	4	6	8	0	6	7	0
总死亡数 Total deaths	21	33	51	69	21	50	14
测试总虫数 Total tests	71	40	59	69	67	115	74
校正死亡率 Adjusted mortality(%)	13.25±0.51 d	78.14±4.21 b	83.23±5.37 b	100.00±0.00 a	14.97±0.68 d	31.14±1.37 c	0.00±0.00 e