抗虫与耐草甘膦转基因棉花株系的抗性评价与遗传稳定分析

doi:10.3864/j.issn.0578-1752.2025.12.002

摘要/Abstract

摘要：

【目的】棉铃虫和田间杂草是制约大田棉花高产的重要因素，现有单一抗虫或耐除草剂品种难以满足高效生产需求，培育兼具抗虫与耐除草剂复合性状的转基因棉花品种，为棉花抗逆育种提供高效种质资源。【方法】通过农杆菌介导法将抗虫融合基因cry1Ac-vip3Da与耐草甘膦基因g10-epsps导入棉花品种R15中，利用PCR鉴定再生株系，对阳性株系持续多代自交纯合，筛选抗性优良的稳定株系；利用qRT-PCR、ELISA分析转基因株系不同组织中目标基因的表达情况；并通过生物活性测定和草甘膦耐受试验分别鉴定不同转基因株系T₄—T₆代际间抗虫与耐草甘膦抗性的遗传稳定性；同时，综合分析不同转基因株系的农艺性状。【结果】PCR鉴定获得8个抗虫耐草甘膦转基因株系，其中，3个转基因株系CA-6、CA-7、CA-17的抗性表现较优；qRT-PCR分析发现cry1Ac-vip3Da和g10-epsps在3个转基因株系各组织中均高表达，且在同株系不同组织中的表达量有所差异；经ELISA检测，在3个转基因株系各组织之间，Cry1Ac-Vip3Da和G10-EPSPS蛋白的含量差异显著，其中，叶片中的蛋白含量最高，且随生育期（四叶期至吐絮期）逐渐下降，但T₄—T₆代际间蛋白表达稳定；生物活性测定和草甘膦耐受试验表明，3个转基因棉花株系T₄—T₆代的校正死亡率为65.12%—82.75%，可耐受4倍以上推荐剂量的草甘膦，且抗性在世代间无衰减；农艺性状分析表明，与对照相比，转基因株系在株高、果枝数、单株铃数、铃重、衣分和籽棉、皮棉产量等农艺性状方面无显著差异。【结论】外源基因cry1Ac-vip3Da和g10-epsps在转基因株系CA-6、CA-7和CA-17中稳定遗传，协同赋予抗虫性与草甘膦耐受性，且农艺性状未受外源基因影响。

关键词: cry1Ac-vip3Da, g10-epsps, 转基因棉花, 抗虫性, 草甘膦耐受性, 稳定遗传

Abstract:

【Objective】Cotton bollworm (Helicoverpa armigera) and field weeds are major constraints to high-yield cotton production. Existing varieties with single traits (insect resistance or herbicide tolerance) fail to meet the demands of efficient cultivation. Developing transgenic cotton varieties with combined insect resistance and glyphosate tolerance will provide high-efficiency germplasm resources for stress-resistant cotton breeding.【Method】The insect-resistant fusion gene cry1Ac-vip3Da and glyphosate-tolerant gene g10-epsps were introduced into cotton R15 through Agrobacterium-mediated method, regenerated transgenic plants were screened via PCR, positive lines underwent multi-generation self-pollination to achieve homozygosity, and stable lines with superior resistance were selected. The expression of target genes in different tissues of transgenic lines was analyzed using qRT-PCR and ELISA. Bioactivity assays and glyphosate tolerance tests were conducted to evaluate the genetic stability of insect resistance and herbicide tolerance across generations (T₄-T₆). Agronomic traits of transgenic lines were comprehensively assessed. 【Result】Eight positive transgenic lines with dual resistance were identified through PCR screening, and CA-6, CA-7 and CA-17 lines exhibited higher resistance. qRT-PCR revealed high expression of cry1Ac-vip3Da and g10-epsps in all tissues of these lines, and expression levels varied significantly among tissues. ELISA analysis demonstrated significant differences in Cry1Ac-Vip3Da and G10-EPSPS protein content across tissues of the three transgenic lines, with the highest levels observed in leaves. Protein accumulation gradually decreased during the developmental stages (from the four-leaf stage to boll-opening stage), but remained stable across T₄-T₆ generations. Bioactivity assays and glyphosate tolerance tests demonstrated that three transgenic cotton lines (T₄-T₆ generations) exhibited corrected mortality rates of 65.12%-82.75%, tolerated glyphosate at over four times the recommended dosage, and showed no attenuation of resistance across generations. There were no significant differences in plant height, number of fruit branches, number of bells per plant, bell weight, lint percentage, seed cotton yield, and lint cotton yield between transgenic lines and R15.【Conclusion】The exogenous genes cry1Ac-vip3Da and g10-epsps were stably inherited across generations in transgenic lines CA-6, CA-7, and CA-17, conferring dual insect resistance and glyphosate tolerance without compromising agronomic performance.

Key words: cry1Ac-vip3Da, g10-epsps, transgenic cotton lines, insect resistance, glyphosate tolerance, stable genetics

文晋, 宁艳芳, 秦欣, 刘圆, 张晓玲, 朱永红, 田时敏, 马燕斌. 抗虫与耐草甘膦转基因棉花株系的抗性评价与遗传稳定分析[J]. 中国农业科学, 2025, 58(12): 2291-2302.

WEN Jin, NING YanFang, QIN Xin, LIU Yuan, ZHANG XiaoLing, ZHU YongHong, TIAN ShiMin, MA YanBin. Resistance Evaluation and Genetic Stability Analysis of Insect- Resistant and Glyphosate-Tolerant Transgenic Cotton Lines[J]. Scientia Agricultura Sinica, 2025, 58(12): 2291-2302.

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引物名称 Primer name	引物序列 Primer sequence (5′-3′)	用途 Usage
g10-epsps-F	CTCTTCTTCCTGACGGACTC	PCR
g10-epsps-R	GACTTTCTGATGATGTGGTGTGC
cry1Ac-vip3Da-F	CCATCTCACGCCATACTTCC
cry1Ac-vip3Da-R	CACCTTGATTGTCCCTGGTC
Histone3-QF	GGTGGTGTGAAGAAGCCTCAT	qRT-PCR
Histone3-QR	AATTTCACGAACAAGCCTCTGGAA
cry1Ac-vip3Da-QF	GAGGTACGAGGTGACTGCCAA
cry1Ac-vip3Da-QR	TCCTGTACTCAGCCTCACTGCT
g10-epsps-QF	CTCCCTTATCGGTTTCTCTGAAG
g10-epsps-QR	TGTAGTTCTTGGATGGCTGTGC

表型参数 Phenotypic parameters	R15	CA-6			CA-7			CA-17
表型参数 Phenotypic parameters	R15	T₄	T₅	T₆	T₄	T₅	T₆	T₄	T₅	T₆
叶片受害率 Leaf damage rate (%）	100.00	10.14±0.35b	11.68±0.85a	10.93±0.84ab	3.76±0.28e	3.97±0.33e	3.56±0.56e	5.38±0.55d	6.54±0.63c	6.38±0.35cd
成活率 Survival rate (%)	0.00	100.00	100.00	100.00	100.00	100.00	100.00	100.00	100.00	100.00

材料 Material	株高 Plant height (cm)	果枝数 Fruit branches	单株铃数 Bolls number	铃重 Boll weight (g)	籽棉产量 Seed cotton yield (kg·hm^-2)	皮棉产量 Lint yield (kg·hm^-2)	衣分 Lint percentage (%)
R15	98.33±9.01	13.56±2.13	25.8±1.8	5.60±0.34	4231.56	1760.33	41.60±2.12
CA-6	100.43±4.51	14.10±1.93	25.6±3.2	5.90±0.65	4236.26	1745.34	41.20±2.06
CA-7	99.27±4.95	14.00±2.21	28.4±2.1*	5.40±1.01	4396.89	1798.33	40.90±2.25
CA-17	96.17±5.92	13.30±1.49	24.1±1.9	5.70±0.74	4191.50	1680.79	40.10±2.40