外源基因插入位点对抗虫棉Bt蛋白含量的影响

doi:10.13304/j.nykjdb.2024.0885

中国农业科技导报 ›› 2025, Vol. 27 ›› Issue (7): 44-53.DOI: 10.13304/j.nykjdb.2024.0885

外源基因插入位点对抗虫棉Bt蛋白含量的影响

赵贵元¹^,²(), 王永强², 刘建光², 耿昭², 张寒霜², 吴立强¹, 王省芬¹(), 张桂寅¹()

^1.河北农业大学农学院，华北作物改良与调控国家重点实验室，河北省作物种质资源重点实验室，河北保定 071000
^2.河北省农林科学院棉花研究所，农业农村部黄淮海半干旱区棉花生物学与遗传育种重点实验室，石家庄 050051

收稿日期:2024-10-28 接受日期:2024-11-27 出版日期:2025-07-15 发布日期:2025-07-11
通讯作者: 王省芬,张桂寅
作者简介:赵贵元 E-mail：zhaogy0302@163.com
基金资助:
农业生物育种重大项目(2023ZD04039-05);国家棉花产业技术体系建设项目(CARS-15-03);河北省棉花产业协同创新中心建设项目;河北省省级科技计划项目(21326314D);河北省现代农业产业技术体系棉花产业创新团队项目(HBCT2024100203);河北省农林科学院创新工程项目(2022KJCXZX-MHS-5)

Effect of Exogenous Gene Insertion Site on Bt Protein Content in Insect-resistant Cotton

Guiyuan ZHAO¹^,²(), Yongqiang WANG², Jianguang LIU², Zhao GENG², Hanshuang ZHANG², Liqiang WU¹, Xingfen WANG¹(), Guiyin ZHANG¹()

^1.State Key Laboratory of North China Crop Improvement and Regulation，Key Laboratory for Crop Germplasm Resources of Hebei，College of Agronomy，Hebei Agricultural University，Hebei Baoding 071000，China
^2.Key Laboratory of Cotton Biology and Genetic Breeding in Huanghuaihai Semiarid Area of Ministry of Agriculture and Rural Affairs，Institute of Cotton，Hebei Academy of Agriculture and forestry Sciences，Shijiazhuang 050051，China

Received:2024-10-28 Accepted:2024-11-27 Online:2025-07-15 Published:2025-07-11
Contact: Xingfen WANG,Guiyin ZHANG

摘要/Abstract

摘要：

外源基因的插入位点能影响受体材料的基因表达及性状表现。为明确外源基因插入位点对抗虫棉Bt蛋白含量的影响，选用29个抗虫棉品系进行Bt蛋白含量测定以及全基因组重测序分析。结果表明，不同品系间的Bt蛋白含量存在显著差异，其中K3品系蛋白含量最高。通过全基因组重测序分析和PCR鉴定发现，Bt基因在29个品系均为单拷贝但插入位点存在差异，其中K3品系的Bt基因位于A02染色体2 791 303~2 791 335 bp（A02型），而其他品系的Bt基因则位于D12染色体（D12型）。为进一步验证插入位点对Bt蛋白含量的影响，利用K3品系与对照新棉33B进行杂交并构建F₂群体，进一步分析F₂群体的Bt蛋白含量发现，Bt基因插入位点位于A02染色体的F₂单株（A02型）Bt蛋白含量极显著高于D12型单株。综上所述，Bt抗虫蛋白含量受插入位点影响，其位于A02染色体更利于Bt基因的高效表达。以上结果可为培育抗虫棉新品种提供种质资源和理论支撑。

关键词: 抗虫棉, 外源基因, 插入位点, Bt蛋白

Abstract:

The insertion sites of exogenous genes can affect the gene expression and character expression of recipient materials. In order to determine the effect of exogenous gene insertion sites on Bt protein content of insect-resistant cotton， 29 insect-resistant cotton lines were selected for Bt protein content determination and whole genome resequencing analysis. The results showed that there were significant differences in Bt protein content among different lines， and the K3 line was the highest. Whole-genome resequencing analysis and PCR identification revealed that the Bt gene was a single copy in 29 lines， but the insertion sites were different. The Bt gene of K3 line was located in the 2 791 303~2 791 335 bp of A02 chromosome， while the Bt gene of other lines were located in D12 chromosome. To further verify the effect of insertion site on Bt protein content， the K3 line was hybridization with control Xinmian 33B， and F₂ population was constructed. The results showed that the Bt protein content of F₂ individual plants with the insertion site of Bt gene in A02 chromosome was significantly higher than that D12 type individual plants. In summary， the Bt protein content was affected by the insertion site of Bt gene， and its location on chromosome A02 was more conducive to the efficient expression of Bt gene. Above results could provide germplasm resources and theoretical support for breeding new varieties of insect-resistant cotton.

Key words: insect-resistant cotton, exogenous gene, insertion site, Bt protein

中图分类号:

S562

赵贵元, 王永强, 刘建光, 耿昭, 张寒霜, 吴立强, 王省芬, 张桂寅. 外源基因插入位点对抗虫棉Bt蛋白含量的影响[J]. 中国农业科技导报, 2025, 27(7): 44-53.

Guiyuan ZHAO, Yongqiang WANG, Jianguang LIU, Zhao GENG, Hanshuang ZHANG, Liqiang WU, Xingfen WANG, Guiyin ZHANG. Effect of Exogenous Gene Insertion Site on Bt Protein Content in Insect-resistant Cotton[J]. Journal of Agricultural Science and Technology, 2025, 27(7): 44-53.

图/表 9

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品系 Line	亲本 Parent	Bt基因 Bt gene	品系 Line	亲本 Parent	Bt基因 Bt gene
K1	ND0901×33B	+	K21	ND0921×33B	+
K3	ND0903×33B	+	K22	ND0922×33B	+
K4	ND0904×33B	+	K23	ND0923×33B	+
K5	ND0905×33B	+	K24	ND0924×33B	+
K6	ND0906×33B	+	K27	ND0927×33B	+
K8	ND0908×33B	+	K28	ND0928×33B	+
K9	ND0909×33B	+	K29	ND0929×33B	+
K10	ND0910×33B	+	K30	ND0930×33B	+
K11	ND0911×33B	+	K31	ND0931×33B	+
K12	ND0912×33B	+	K32	ND0932×33B	+
K13	ND0913×33B	+	K33	ND0933×33B	+
K17	ND0917×33B	+	K34	ND0934×33B	+
K18	ND0918×33B	+	K35	ND0935×33B	+
K19	ND0919×33B	+	K26（33B）	爱字90×岱字50 Aizi 90×Daizi 50	+
K20	ND0920×33B	+	CCRI12	乌干达4号×邢台6871 Wuganda 4×Xingtai 6871	-

品系 Line	亲本 Parent	Bt基因 Bt gene	品系 Line	亲本 Parent	Bt基因 Bt gene
K1	ND0901×33B	+	K21	ND0921×33B	+
K3	ND0903×33B	+	K22	ND0922×33B	+
K4	ND0904×33B	+	K23	ND0923×33B	+
K5	ND0905×33B	+	K24	ND0924×33B	+
K6	ND0906×33B	+	K27	ND0927×33B	+
K8	ND0908×33B	+	K28	ND0928×33B	+
K9	ND0909×33B	+	K29	ND0929×33B	+
K10	ND0910×33B	+	K30	ND0930×33B	+
K11	ND0911×33B	+	K31	ND0931×33B	+
K12	ND0912×33B	+	K32	ND0932×33B	+
K13	ND0913×33B	+	K33	ND0933×33B	+
K17	ND0917×33B	+	K34	ND0934×33B	+
K18	ND0918×33B	+	K35	ND0935×33B	+
K19	ND0919×33B	+	K26（33B）	爱字90×岱字50 Aizi 90×Daizi 50	+
K20	ND0920×33B	+	CCRI12	乌干达4号×邢台6871 Wuganda 4×Xingtai 6871	-

引物名称 Primer name	引物序列 Sequence （5’-3’）	扩增片段长度 Amplified fragment length/bp	退火温度 Annealing temperature/℃
D12-LB-F	CACCAAAGAGAAACCCCAATC	456	59.7
D12-LB-R	CGCTGATTGTTCTGTTCCTCC	456	59.7
D12-RB-F	GTCAATACCGCAGGGCACTTA	639	59.0
D12-RB-R	AGTCAAAGGAGCTTCATGGGT	639	59.0
A02-LB-F	CACCAAAGAGAAACCCCAATC	289	58.6
A02-LB-R	CACGGACAACTCAGGCAAGTA	289	58.6
A02-RB-F	CCCAGGCTTGTCCACATCATC	692	57.1
A02-RB-R	TCCCTCCTGAGCTACCATGTC	692	57.1

引物名称 Primer name	引物序列 Sequence （5’-3’）	扩增片段长度 Amplified fragment length/bp	退火温度 Annealing temperature/℃
D12-LB-F	CACCAAAGAGAAACCCCAATC	456	59.7
D12-LB-R	CGCTGATTGTTCTGTTCCTCC	456	59.7
D12-RB-F	GTCAATACCGCAGGGCACTTA	639	59.0
D12-RB-R	AGTCAAAGGAGCTTCATGGGT	639	59.0
A02-LB-F	CACCAAAGAGAAACCCCAATC	289	58.6
A02-LB-R	CACGGACAACTCAGGCAAGTA	289	58.6
A02-RB-F	CCCAGGCTTGTCCACATCATC	692	57.1
A02-RB-R	TCCCTCCTGAGCTACCATGTC	692	57.1

品系 Line	染色体 Chromosome	插入起点 Insert starting point/bp	插入终点 Insertion end point/bp	碱基缺失 Base deletion/bp	拷贝数 Copies number
K1	ChrD12	49 127 447	49 127 527	80	1
K3	ChrA02	2 791 303	2 791 335	32	1
K4	ChrD12	49 127 447	49 127 527	80	1
K5	ChrD12	49 127 447	49 127 527	80	1
K6	ChrD12	49 127 447	49 127 527	80	1
K8	ChrD12	49 127 447	49 127 527	80	1
K9	ChrD12	49 127 447	49 127 527	80	1
K10	ChrD12	49 127 447	49 127 527	80	1
K11	ChrD12	49 127 447	49 127 527	80	1
K12	ChrD12	49 127 447	49 127 527	80	1
K13	ChrD12	49 127 433	49 127 527	94	1
K17	ChrD12	49 127 447	49 127 527	80	1
K18	ChrD12	49 127 447	49 127 527	80	1
K19	ChrD12	49 127 447	49 127 527	80	1
K20	ChrD12	49 127 447	49 127 527	80	1
K21	ChrD12	49 127 447	49 127 527	80	1
K22	ChrD12	49 127 447	49 127 527	80	1
K23	ChrD12	49 127 447	49 127 550	103	1
K24	ChrD12	49 127 447	49 127 527	80	1
K27	ChrD12	49 127 447	49 127 527	80	1
K28	ChrD12	49 127 447	49 127 527	80	1
K29	ChrD12	49 127 447	49 127 527	80	1
K30	ChrD12	49 127 447	49 127 527	80	1
K31	ChrD12	49 127 447	49 127 527	80	1
K32	ChrD12	49 127 447	49 127 527	80	1
K33	ChrD12	49 127 447	49 127 527	80	1
K34	ChrD12	49 127 447	49 127 527	80	1
K35	ChrD12	49 127 447	49 127 527	80	1
K26（33B）	ChrD12	49 127 447	49 127 527	80	1

外源基因插入位点对抗虫棉Bt蛋白含量的影响

Effect of Exogenous Gene Insertion Site on Bt Protein Content in Insect-resistant Cotton

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Metrics

F₁单株 F₁individual line	Bt蛋白含量 Bt protein content/（ng·g^-1）	F₁单株代号 F₁ individual line	Bt蛋白含量 Bt protein content/（ng·g^-1）	F₁单株 F₁ individual line	Bt蛋白含量 Bt protein content/（ng·g^-1）
1	2 620.96	35	1 900.34	69	1 959.75
2	2 343.26	36	2 620.28	70	2 282.87
3	2 080.38	37	2 003.94	71	2 375.58
4	1 719.96	38	2 708.17	72	2 582.07
5	1 901.95	39	2 509.09	73	2 681.05
6	1 567.73	40	1 739.91	74	1 778.00
7	2 710.37	41	1 986.55	75	1 979.19
8	2 160.84	42	2 003.78	76	2 425.47
9	2 458.44	43	2 452.15	77	2 592.63
10	2 206.88	44	2 689.70	78	2 749.04
11	2 030.17	45	2 006.63	79	2 552.07
12	2 616.94	46	1 740.00	80	2 032.94
13	1 804.79	47	2 092.82	81	1 752.55
14	2 380.22	48	2 293.19	82	2 506.93
15	2 519.91	49	2 493.81	83	1 766.27
16	2 521.00	50	1 569.09	84	2 384.92
17	1 839.19	51	1 937.93	85	2 458.43
18	2 658.52	52	1 600.92	86	2 156.09
19	1 980.21	53	2 034.36	87	2 629.90
20	2 159.45	54	2 320.95	88	1 717.48
21	1 589.29	55	2 641.93	89	1 983.71
22	2 728.60	56	1 710.80	90	2 697.81
23	1 904.49	57	1 846.32	91	2 124.19
24	1 786.33	58	2 500.14	92	2 477.61
25	2 058.37	59	2 240.53	93	2 530.88
26	2 744.91	60	2 588.66	94	2 214.65
27	2 762.57	61	2 657.60	95	2 265.45
28	2 133.94	62	2 463.61	96	2 119.77
29	1 591.75	63	1 885.70	97	1 962.63
30	1 710.46	64	2 138.93	98	1 747.97
31	2 150.90	65	2 274.05	99	1 857.94
32	2 238.87	66	1 957.03	100	2 001.26
33	2 691.36	67	2 034.69	101	2 410.54
34	1 790.58	68	2 470.77	102	2 401.90

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