关键词: 转基因玉米, 复合性状, m2cryAb-vip3A, bar, 分子特征, 抗性评价

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