Scientia Agricultura Sinica ›› 2025, Vol. 58 ›› Issue (7): 1269-1283.doi: 10.3864/j.issn.0578-1752.2025.07.002

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

Molecular Characteristics and Resistance Evaluation of Transgenic Maize LD05 with Stacked Insect and Herbicide Resistance Traits

YUE RunQing(), LI WenLan, DING ZhaoHua, MENG ZhaoDong   

  1. Maize Research Institute, Shandong Academy of Agricultural Sciences/Shandong Key Laboratory of Maize Biological Breeding/ National Engineering Center of Wheat and Maize, Jinan 250100
  • Received:2024-08-19 Accepted:2024-09-26 Online:2025-04-08 Published:2025-04-08

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Abstract:

【Objective】To clarify the molecular characteristics and the effectiveness of target traits of transgenic maize LD05 with composite insect and herbicide resistance, and to provide data basis, technical support and product reserve for industrial application.【Method】Using biological information analysis, we designed and modified the proprietary insect-resistant fusion gene m2cryAb-vip3A, and selected BC4F3, BC4F4 and BC4F5 generations of the newly created transgenic hybrid insect-resistant and herbicide-tolerant maize LD05 to carry out experimental research. Specific PCR and Southern blot were used to analyze the stability of genomic integration. qRT-PCR and ELISA were used to analyze the expression stability. The resistance to target pests was evaluated by bioassay and field trials, and the herbicide tolerance was tested by field spraying of glufosinate. 【Result】A new insect-resistant fusion gene m2cryAb-vip3A with independent property right was discovered and designed, and a multivalent insect-resistant and herbicide resistant maize transformant LD05 was created. The exogenous T-DNA was integrated into the maize genome in the form of a single copy. The qRT-PCR results indicated that m2cryAb-vip3A and bar were both expressed in various tissues and organs across three generations, and the variation trend of expression quantities was largely consistent. Specifically, the expression level of m2cryAb-vip3A was the highest in the leaves at the seedling stage of the three consecutive generations, with an average expression quantity of 36.73, while the expression level was the lowest in the cob at the mature stage, with an average of merely 0.91. The expression pattern of bar was similar to that of m2cryAb-vip3A, with the highest expression level in the leaves at the seedling stage, averaging 7.35, and the expression level decreased after the jointing stage. The ELISA results demonstrated that M2CryAb-VIP3A could stably accumulate in different organs and at different periods in the three generations, and the protein accumulation amounts in different generations were similar. Among them, the accumulation amount was the highest in the leaves at the seedling stage of different generations, all exceeding 19.67 μg·g-1 fresh weight. The expression of the targeted protein at a relatively high level could be detected in different tissues of the PAT transgenic plants of three consecutive generations, and there was no significant difference in the expression quantity between different generations. Among them, the expression level was the highest in the leaves at the seedling stage of different generations, with an average content of 16.61 μg·g-1 fresh weight, while the accumulation amount was the lowest in the roots at the mature stage, with an average content of 0.30 μg·g-1 fresh weight. The bioassay result showed that the corrected mortality of Ostrinia furnacalis, Spodoptera fragiperda and Mythimna separata reached 100% after feeding on V5 maize leaf tissue of LD05 for 96 h, which was a high resistance level. The results of field trials showed that LD05 transformants had high resistance to Ostrinia furnacalis at V5 stage and silking stage, to Mythimna separata at V5 stage, and to Helicoverpa armigera at silking stage. The results of glufosinate tolerance test showed that transgenic maize LD05 could tolerate 4-fold glufosinate. Agronomic character investigation showed that there was no difference between transgenic maize LD05 and control maize Zheng 58.【Conclusion】A novel insect-resistant fusion gene m2cryAb-vip3A with independent property rights was developed, and a transgenic hybrid insect-resistant and herbicide-tolerant maize LD05 was created with clear molecular characteristics, genetic stability and outstanding functional traits.

Key words: transgenic maize, stacked trait, m2cryAb-vip3A, bar, molecular characteristic, resistance evaluation

Table 1

Primers were used in this study"

引物 Primers 序列 Primer sequence (5′-3′) 用途 Purpose
23A0496 CGCATAGAAACAACAGAAGTG 左边界Left boundary
23A0497 TCCTAAAACCAAAATCCAGTA
23A0498 GGCAGAGGCATCTTCAACG 右边界Right boundary
23A0499 AAACCCCAAGTCCAAGTAAC
LC0797 ATCTCGTCCAGCGTCAGGT 检测m2cryAb-vip3A
Detection of m2cryAb-vip3A
LC0798 TCAACATCGGCATCAACAA
LCH001 TTGCCCAGCTATCTGTCACTTT 检测m2cryAb-vip3A启动子CaMV 35S promoter
Detection of m2cryAb-vip3A promoter CaMV 35S promoter
LCH002 TGTTCGGGTTGTTGTCCATTCT
LCH003 AAAACCCATCTCATAAATAACG 检测m2cryAb-vip3A终止子NOS terminator
Detection of m2cryAb-vip3A terminator NOS terminator
LCH004 GAACAACAACTTGGAGGACTAC
LC0234 CTGAAGTCCAGCTGCCAGAA 检测bar
Detection of bar
LC0235 ATGAGCCCAGAACGACGC
LCH005 GAGGAAGGTCTTGCGAAGGATAGT 检测bar启动子CaMV35S promoter (enhanced)
Detection of bar promoter CaMV 35S promoter (enhanced)
LCH006 AGCATAAAGTGTAAAGCCTGGGGT
LC0787 TTATCTGGGAACTACTCAC 检测bar终止子CaMV poly(A)
Detection of bar terminator CaMV poly(A)
LC0788 GACTGGGCTCCACGCTCTA
23A0349 GTTCTTGGTGACGCTCTTC m2cryAb-vip3A的Southern blot探针合成
Synthesis of m2cryAb-vip3A by Southern blot probe
23A0350 GCTCAGCAGCACGCTCTAC
23A0345 GGCTGGCTGGTGGCAGGATATATT bar的Southern blot探针合成
Synthesis of bar by Southern blot probe
23A0346 ACGAAGTTCGAGAAGGACA
23C073 GTTTCCTTTACCGGGGACGA m2cryAb-vip3A定量PCR
qRT-PCR primers of m2cryAb-vip3A
23C074 ACCACCCCCTTCAACTTCAG
23C007 CAGGAACCGCAGGAGTGGAC bar定量PCR
qRT-PCR primers of bar
23C008 CTTCAGCAGGTGGGTGTAGAGC
LC0378 CGGTGGATGCTAAGGCTG zSSⅡb定量PCR
qRT-PCR primers of zSSⅡb
LC0379 AAAGGGCCAGGTTCATTATCCTC

Fig. 1

Domain prediction of M2cryAb-vip3A protein A: Predicted conserved domains of M2CryAb-VIP3A; B: Prediction of ubiquitination site of M2CryAb-VIP3A protein; C: Prediction of phosphorylation site of M2CryAb-VIP3A protein; D: Prediction of N-glycosylation site of M2CryAb-VIP3A protein"

Fig. 2

Structure diagram of T-DNA insertion of D05 transformant and results of transformant specific PCR A: Structure diagram of T-DNA insertion of D05 transformant; B: Amplification results of LB; C: Amplification results of RB. M: DNA Marker; N: Water; 1: BC4F3 generation LD05 transformant; 2: BC4F4 generation LD05 transformant; 3: BC4F5 generation LD05 transformant; 4: Positive control plasmid pCAMBIA3300+m2cryAb-vip3A; 5: Zheng 58"

Fig. 3

Results of PCR detection of LD05 transformant target genes and elements A: m2cryAb-vip3A gene; B: m2cryAb-vip3A promoter CaMV 35S promoter; C: m2cryAb-vip3A terminator NOS terminator; D: bar gene; E: bar promoter CaMV35S promoter(enhanced); F: bar terminator CaMV poly(A). M: DNA Marker; N: Water; 1: Positive control plasmid pCAMBIA3300+m2cryAb-vip3A; 2: Zheng 58; 3: BC4F3 generation LD05 transformant; 4: BC4F4 generation LD05 transformant; 5: BC4F5 generation LD05 transformant"

Fig. 4

Results of Southern blot of target genes m2cryAb-vip3A and bar A: Hybridization results of target gene m2cryAb-vip3A by HindⅢ digestion; B: Hybridization results of target gene m2cryAb-vip3A by KpnI digestion; C: Hybridization results of target gene bar by HindⅢ digestion; D: Hybridization results of target gene bar by BamHI digestion; the arrow on the right of the figure marks the target band. M: DNA marker; N: Negative control; 1: Positive control plasmid; 2: BC4F4 transformation LD05; 3: BC4F5 transformation LD05"

Fig. 5

Relative expression pattern of the exogenous genes in LD05 transformants A: Heterologous gene m2cryAb-vip3A; B: Heterologous gene bar. The letters represent the significant difference (P<0.05). The same as below"

Fig. 6

Expression analysis of heterologous protein of LD05 A: Heterologous protein M2cryAb-VIP3A; B: Heterologous protein PAT"

Fig. 7

Results of laboratory bioassay analysis of target pests by LD05"

Fig. 8

Statistical results of laboratory bioassay on target pests A: Ostrinia furnacalis; B: Spodoptera fragiperda; C: Mythimna separata; D: Helicoverpa armigera"

Table 2

Analysis field trials of Ostrinia furnacalis, Mythimna separata, and Helicoverpa armigera"

材料
Material		 玉米螟 Ostrinia furnacalis 黏虫 Mythimna separata 棉铃虫 Helicoverpa armigera
心叶期 V5 stage 花丝期 Silking stage 心叶期 V5 stage 花丝期 Silking stage
危害级别
Damage level		 抗性
Resistance		 危害级别
Damage level		 抗性
Resistance		 危害级别
Damage level		 抗性
Resistance		 危害级别
Damage level		 抗性
Resistance
LD05（BC4F3） 1.00 高抗
High resistance		 1.00 高抗
High resistance		 1.02 高抗
High resistance		 1.00 高抗
High resistance
LD05（BC4F4） 1.00 高抗
High resistance		 1.00 高抗
High resistance		 1.01 高抗
High resistance		 1.00 高抗
High resistance
LD05（BC4F5） 1.00 高抗
High resistance		 1.00 高抗
High resistance		 1.03 高抗
High resistance		 1.00 高抗
High resistance
郑58
Zheng 58		 8.85 高感
High sense		 8.99 高感
High sense		 8.61 高感
High sense		 6.72 感
Sense
先达205
Xianda 205		 8.69 高感
High sense		 8.43 高感
High sense		 8.05 高感
High sense		 6.95 感
Sense

Table 3

Tolerance of LD05 of BC4F5 generation to glufosinate herbicide"

药后时间（周）
Time after herbicide spraying (weeks)		 材料
Material		 剂量（倍）
Dose(times)		 成苗率
Planting percent (%)		 受害率
Damage rate (%)
1 郑58
Zheng 58		 0× 100.00±0.00a -
1× 0.00±0.00b 100.00±0.00a
LD05 0× 100.00±0.00a -
1× 100.00±0.00a 0.00±0.00b
2× 100.00±0.00a 0.00±0.00b
4× 100.00±0.00a 10.67±6.93b
2 郑58
Zheng 58		 0× 100.00±0.00a -
1× 0.00±0.00b 100.00±0.00a
LD05 0× 100.00±0.00a -
1× 100.00±0.00a 0.00±0.00b
2× 100.00±0.00a 0.00±0.00b
4× 100.00±0.00a 0.00±0.00b
4 郑58
Zheng 58		 0× 100.00±0.00a -
1× 0.00±0.00b 100.00±0.00a
LD05 0× 100.00±0.00a -
1× 100.00±0.00a 0.00±0.00b
2× 100.00±0.00a 0.00±0.00b
4× 100.00±0.00a 0.00±0.00b

Table 4

Comparative analysis of agronomic characters between LD05 of BC4F5 generation and Zheng58"

农艺性状
Agronomic trait		 LD05
(BC4F5)		 郑58
Zheng58
抽雄期Tasseling stage (d) 59±1.0a 59±1.0a
生育期Growth stage (d) 108.7±1.5a 109.3±1.5a
株高Plant height (cm) 163.0±2.6a 164.0±2.0a
穗位高Ear height (cm) 56.0±2.0a 55.3±1.5a
穗行数Rows per ear 12±0a 12±0a
穗粗Ear diameter (cm) 3.8±0.1a 3.8±0.1a
百粒重100-kernel weight (g) 33.4±0.5a 33.5±0.7a
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