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Acta Agronomica Sinica ›› 2024, Vol. 50 ›› Issue (1): 89-99.doi: 10.3724/SP.J.1006.2024.33016

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

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 Online:2024-01-12 Published: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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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.)

Table 1

Criteria for evaluation of insect resistance in laboratory bioassay"

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

Fig. 1

Domain prediction of M2cryAb-vip3Aa protein"

Fig. 2

Detection of target genes of LG11 by PCR A: m2cryAb-vip3Aa, the expected size of the target band is 958 bp; B: bar, the expected size of the target band is 506 bp. M: DL2000 DNA marker; N: pure water; 1: positive control plasmid; 2: negative control (containing Chang 7-2) genomic DNA; 3: BC4F2 transformation LG11 genomic DNA; 4: BC5F2 transformation LG11 genomic DNA; 5-6: genomic DNA of other transformants with the same vector."

Fig. 3

Southern hybridization of target genes m2cryAb-vip3Aa and bar A: Southern hybridization of target gene m2cryAb-vip3Aa by Hind III digestion; B: Southern hybridization of target gene m2cryAb-vip3Aa by BamH I digestion; C: Southern hybridization of target gene bar by Hind III digestion; D: Southern hybridization of target gene bar by BamH I digestion. M: DNA marker, the size is labeled at the side; CK: negative control (containing Chang 7-2); 1: positive control plasmid; 2: BC4F2 transformation LG11 genomic DNA; 3: BC5F1 transformation LG11 genomic DNA; 4: BC5F2 transformation LG11 genomic DNA."

Table 2

Relative expression pattern of the exogenous genes in LG11 transformants"

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 —

Fig. 4

Laboratory bioassay of maize leaves at heart leaf stage and maize silks on Ostrinia furnacalis (5 days after insect ingestion) A: Chang 7-2 leaves at heart leaf stage; B: LG11 leaves at heart leaf stage; C: Chang 7-2 silks; D: LG11 silks."

Table 3

Laboratory bioassay of Ostrinia furnacalis and Spodoptera frugiperda"

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 — —

Table 4

Field identification of resistance of LG11 to Ostrinia furnacalis"

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

Fig. 5

Insect resistance of silk grafted with Ostrinia furnacalis and Spodoptera frugiperda at ear stage A: Chang 7-2 grafted with Ostrinia furnacalis; B: LG11 grafted with Ostrinia furnacalis; C: Chang 7-2 grafted with Spodoptera frugiperda; D: LG11 grafted with Spodoptera frugiperda."

Table 5

Field identification of resistance of LG11 to Spodoptera frugiperda"

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

Fig. 6

Tolerance of LG11 and control Chang 7-2 after one week of spraying with glufosinate A: Chang 7-2 at 0 times dose; B: Chang 7-2 at 1 times dose; C: LG11 at 0 times dose; D: LG11 at 1 time dose; E: LG11 at 2 times dose; F: LG11 at 4 times dose."

Table 6

Tolerance of LG11 of BC5F2 generation to glufosinate herbicide"

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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