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作物学报 ›› 2024, Vol. 50 ›› Issue (1): 89-99.doi: 10.3724/SP.J.1006.2024.33016

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

转基因抗虫耐除草剂玉米自交系LG11的获得及抗性分析

岳润清*(), 李文兰, 孟昭东   

  1. 山东省农业科学院玉米研究所 / 小麦玉米国家工程实验室 / 农业农村部黄淮海北部玉米生物学与遗传育种重点实验室, 山东济南250100
  • 收稿日期:2023-03-16 接受日期:2023-05-24 出版日期:2024-01-12 网络出版日期:2023-05-29
  • 通讯作者: *岳润清, E-mail: yuerunqing@126.com
  • 基金资助:
    泰山学者工程项目和财政部和农业农村部国家现代农业产业技术体系建设专项(CARS-02-09)

Acquisition and resistance analysis of transgenic Maize Inbred Line LG11 with insect and herbicide resistance

YUE Run-Qing*(), LI Wen-Lan, MENG Zhao-Dong   

  1. Maize Research Institute, Shandong Academy of Agricultural Sciences / National Engineering Laboratory of Wheat and Maize / Key Laboratory of Biology and Genetic Improvement of Maize in Northern Yellow-Huai Rivers Plain, Ministry of Agriculture and Rural Affairs, Jinan 250100, Shandong, China
  • Received:2023-03-16 Accepted:2023-05-24 Published:2024-01-12 Published online:2023-05-29
  • Contact: *E-mail: yuerunqing@126.com
  • Supported by:
    Taishan Scholars Program and China Agriculture Research System of MOF and MARA(CARS-02-09)

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摘要：

虫害是影响我国玉米产量和品质的重要限制因子, 转Bt (Bacillus thuringiensis)基因玉米具有很好的抗虫性, 能有效减少化学杀虫剂的使用, 深受种植者欢迎。但是大面积持续种植转基因玉米, 会引发靶标害虫产生抗性, “多基因”策略是阻止或延缓靶标害虫抗性种群发生的主要治理策略之一。本课题组利用人工合成方法将Cry1Ab和Vip3Aa蛋白的主要结构域组合形成融合蛋白M2cryAb-vip3Aa, 这2种杀虫蛋白在进化上没有同源性且杀虫机理存在差别, 能有效降低靶标害虫产生抗性的几率。本研究拟通过转基因方法将m2cryAb-vip3Aa和bar基因串联导入受体材料, 并以骨干自交系昌7-2为回交亲本进行回交转育, 获得抗虫耐除草剂且拥有优良农艺性状的转基因玉米新材料, 并对抗虫性和耐除草剂抗性进行分析。研究结果将丰富现有的抗虫耐除草剂玉米种质资源, 并为玉米田间害虫防治和杂草治理提供新的解决方案。

关键词: m2cryAb-vip3Aa, bar, 转基因, 玉米

Abstract:

Insect pests are an important limiting factor affecting the yield and quality of maize. Bacillus thuringiensis transgenic maize has good insect resistance and can reduce the use of chemical insecticides effectively, which is very popular among growers. However, a large area of continuous planting of transgenic corn will lead to the resistance of target pests. “Multi-gene” strategy is one of the management strategies to prevent or delay the occurrence of resistant populations of target pests. The anti-insect vector constructed in this study is a fusion protein M2cryAb-vip3Aa formed by combining the main structural domains of Cry1Ab and Vip3Aa proteins using synthetic methods. These two insecticidal proteins had no evolutionary homology and different insecticidal mechanisms, which could effectively reduce the resistance probability of target pests. In this study, to obtain a new transgenic maize material with insect resistance and herbicide resistance and excellent agronomic traits, and analyze its insect resistance and herbicide resistance, m2cryAb-vip3Aa and bar genes were introduced into the recipient materials in series by transgenic method, and Chang 7-2, a backbone inbred line, was used as the backcross parent for backcross transfer. The results of this study enriches the existing germplasm resources of insect-resistant and herbicide-tolerant maize, and provides new solutions for pest control and weed control in maize fields.

Key words: m2cryAb-vip3Aa, bar, transgenic, maize (Zea mays L.)

表1

室内生物测定抗虫性评价标准"

抗虫性水平
Insect resistance level		 幼虫校正死亡率y
Adjusted larval mortality y (%)
高抗High resistance y ≥ 90
抗Resistance 90 > y ≥ 60
中抗Medium resistance 60 > y ≥ 40
低抗Low resistance 40 > y ≥ 20
感Sensitivity 20 > y

图1

M2cryAb-vip3Aa蛋白的结构域预测"

图2

转化体LG11目的基因的PCR检测 A: m2cryAb-vip3Aa基因, 目标条带预期大小958 bp; B: bar基因, 目标条带预期大小506 bp。M: DL2000 DNA marker; N: 纯水; 1: 阳性对照质粒; 2: 阴性对照(含昌7-2)基因组DNA; 3: BC4F2代转化体LG11基因组DNA; 4: BC5F2代转化体LG11基因组DNA; 5~6: 同载体其他转化体基因组DNA。"

图3

目的基因m2cryAb-vip3Aa和bar的Southern杂交结果 A: 目的基因m2cryAb-vip3Aa的Hind III酶切杂交图谱; B: 目的基因m2cryAb-vip3Aa的BamH I酶切杂交图谱; C: 目的基因bar的Hind III酶切杂交图谱; D: 目的基因bar的BamH I酶切杂交图谱。M: DNA marker, 大小标注在旁; CK: 阴性对照(含昌7-2); 1: 阳性对照质粒; 2: BC4F2代转化体LG11基因组DNA; 3: BC5F1代转化体LG11基因组DNA; 4: BC5F2代转化体LG11基因组DNA。"

表2

转化体LG11外源基因的表达模式分析"

检测基因
Gene name		 生育期
Growth stage		 组织
Tissue		 BC5F2代LG11
BC5F2 transformation LG11		 昌7-2
Chang 7-2
m2cryAb-vip3Aa 苗期Seedling stage 根Root 36.43±7.42 c —
茎Stem 38.73±3.30 b —
叶Leaf 70.01±9.19 a —
吐丝期Silking stage 根Root 2.38±0.05 f 未检出Not detected
茎Stem 1.11±0.07 g 未检出Not detected
成熟期Maturity stage 根Root 2.77±10.56 e —
叶Leaf 1.08±0.07 g —
籽粒Kernel 10.06±1.77 d —
bar 苗期Seedling stage 根Root 2.86±1.15 a —
茎Stem 2.74±0.35 a —
叶Leaf 1.51±0.62 b —
吐丝期Silking stage 根Root 0.24±0.01 b 未检出Not detected
成熟期Maturity stage 根Root 1.41±0.49 b —

图4

玉米心叶期叶片和玉米花丝对玉米螟室内生测结果(接虫后5 d) A: 昌7-2心叶期叶片; B: LG11心叶期叶片; C: 昌7-2花丝; D: LG11花丝。"

表3

玉米螟和草地贪夜蛾室内生测"

靶标害虫
Target pest		 材料
Material name		 叶片Leaf 花丝Silk
死亡率
Mortality rate (%)		 校正死亡率
Adjusted mortality rate (%)		 抗性
Resistance		 死亡率
Mortality
rate
(%)		 校正死亡率
Adjusted mortality
rate (%)		 抗性
Resistance
玉米螟
Ostrinia
ubilalis		 BC5F2代LG11
BC5F2 transformation LG11		 100.00±0.00 a 100.00 高抗
High resistance		 100.00±0.00 a 100.00 高抗
High resistance
昌7-2
Chang 7-2		 10.00±1.50 b — — 7.50±4.80 b — —
草地贪夜蛾
Spodoptera frugiperda		 BC5F2代LG11
BC5F2 transformation LG11		 97.50±5.00 a 97.44 高抗
High resistance		 100.0±0.0 a 100.00 高抗
High resistance
昌7-2
Chang 7-2		 2.50±0.50 b — — 7.50±5.00 b — —

表4

LG11对玉米螟的田间抗性鉴定"

材料
Material name		 心叶期Heart leaf stage 吐丝期Silking stage
食叶级别
Level of leaf feeding		 虫害级别
Level of pest infestation		 抗性级别
Resistance		 雌穗被害级别
Level of female panicle damage		 抗性级别
Resistance
BC5F2代LG11
BC5F2 transformation LG11		 1.33±0.24 b 1 高抗
High resistance		 1.40±0.06 b 高抗
High resistance
昌7-2
Chang 7-2		 7.73±0.31 a 7 感
Sensitivity		 6.50±0.71 a 感
Sensitivity

图5

花丝接玉米螟和草地贪夜蛾后的穗期抗虫效果 A: 昌7-2接玉米螟; B: LG11接玉米螟; C: 昌7-2接草地贪夜蛾; D: LG11接草地贪夜蛾。"

表5

LG11对草地贪夜蛾的田间抗性鉴定"

材料
Material		 心叶期Heart leaf stage 吐丝期Silking stage
食叶级别
Level of leaf feeding		 虫害级别
Level of pest infestation		 抗性级别
Resistance		 雌穗被害级别
Level of female
panicle damage		 抗性级别
Resistance
BC5F2代LG11
BC5F2 transformation LG11		 1.09±0.09 b 1 高抗
High resistance		 1.41±0.41 b 高抗
High resistance
昌7-2
Chang 7-2		 7.83±0.24 a 7 感
Sense		 6.20±0.57 a 感
Sense

图6

喷施草铵膦1周后LG11和对照昌7-2的耐受性 A: 0倍剂量下昌7-2; B: 1倍剂量下昌7-2; C: 0倍剂量下LG11; D: 1倍剂量下LG11; E: 2倍剂量下LG11; F: 4倍剂量下LG11。"

表6

BC5F2代LG11对草铵膦除草剂耐受性"

药后时间
Time after herbicide spraying		 材料
Material		 剂量(倍)
Dose (times)		 成苗率
Planting rate (%)		 受害率
Damage rate (%)		 株高
Plant height (cm)
1 week 昌7-2
Chang 7-2		 1× 0.00 b 100.00±0.00 a —
0× 100.00±0.00 a 0.00 c 66.80±1.73 b
LG11 0× 100.00±0.00 a 0.00 c 74.80±2.25 a
1× 100.00±0.00 a 0.00 c 76.07±2.04 a
2× 100.00±0.00 a 4.57±3.64 b 69.40±2.80 ab
4× 100.00±0.00 a 4.69±2.15 b 74.27±3.45 a
2 weeks 昌7-2
Chang 7-2		 1× 0.00 b 100.00±0.00 a —
0× 100.00±0.00 a 0.00 b 105.60±3.50 b
LG11 0× 100.00±0.00 a 0.00 b 113.87±0.23 a
1× 100.00±0.00 a 0.00 b 116.07±3.52 a
2× 100.00±0.00 a 0.00 b 113.47±1.22 ab
4× 100.00±0.00 a 0.00 b 114.33±5.03 a
4 weeks 昌7-2
Chang 7-2		 1× 0.00 b 100.00±0.00 a —
0× 100.00±0.00 a 0.00 b 165.60±1.40 a
LG11 0× 100.00±0.00 a 0.00 b 163.93±2.01 a
1× 100.00±0.00 a 0.00 b 161.13±1.68 a
2× 100.00±0.00 a 0.00 b 164.13±0.76 a
4× 100.00±0.00 a 0.00 b 163.20±1.22 a
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