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CN-122024823-A - Unified evaluation method for multi-pest resistance of corn co-transformed by adaptive multi-source pest-resistant protein

CN122024823ACN 122024823 ACN122024823 ACN 122024823ACN-122024823-A

Abstract

The invention discloses an evaluation method of the insect-resistant effect of transgenic corn, which is characterized in that a standardized planting system is constructed, unified insect-receiving of multiple pests is realized by adopting special powdery flocculent feed, investigation indexes and calculation logic are standardized, data processing and insect-resistant grade judgment are completed by combining a Python automatic analysis system, and a complete evaluation system of standardized planting, unified insect-receiving, unified investigation and automatic analysis is formed.

Inventors

  • WANG YUNPENG
  • CHEN YINGNAN
  • WANG BAIFENG
  • LI SHIJIE
  • WU FENGCI
  • YIN JUNQI
  • WANG DAMING
  • JIANG ZHILEI
  • SONG XINYUAN

Assignees

  • 吉林省农业科学院(中国农业科技东北创新中心)
  • 海南吉科农业科技有限公司

Dates

Publication Date
20260512
Application Date
20251215

Claims (10)

  1. 1. The method for evaluating the insect-resistant effect of the transgenic corn is characterized by comprising the following steps of: (1) The standard planting, namely randomly and sectionally planting the transgenic corn material to be evaluated and the control corn variety without spraying insecticide in the whole growth period; (2) The method comprises the following steps of uniformly inoculating multiple pests, namely inoculating the pests with special flocculent feed after corn grows to a 4-leaf stage, wherein the special flocculent feed comprises the following components in percentage by mass of 50+/-10% of corncob powder, 20+/-5% of soybean meal, 15+/-5% of wheat bran, 5+/-2% of yeast powder, 5+/-2% of sucrose, 1+/-0.5% of compound vitamin and 4+/-2% of mineral mixture, adding antibiotics for preservation, and spraying 15+/-5% of sterile water for humidifying before use; (3) Uniformly investigating and calculating indexes, namely randomly investigating a plurality of corn plants after 14 days of insect inoculation, and recording data; (4) And (5) data processing and insect resistance grade judgment.
  2. 2. The method according to claim 1, wherein the step (2) of inoculating the pests with the special feed in the form of a powder flocculent is performed by inoculating each corn with a quantity (e.g. 10ml, 20 ml) of the powder flocculent so that each corn is inoculated with a quantity of pests.
  3. 3. The method of claim 2, wherein the inoculated pests include larvae of the species armyworm, larva of the asian corn borer, and/or larva of the spodoptera frugiperda, wherein each of the species armyworm or asian corn borer is inoculated with 10ml (including 35±5 larvae) of the floc feed and each of the species spodoptera frugiperda is inoculated with 20ml (including 70±10 larvae) of the floc feed.
  4. 4. The method according to claim 3, wherein the grading standard of the leaf grade in the heart leaf stage of the myxose is that corn leaves are not damaged or only have needle-shaped insect holes (less than or equal to 1 mm) on the leaves, then the leaf grade is 1, corn only has small number of insect holes (less than or equal to 5 mm) on the individual leaves, then the leaf grade is 2, corn minority leaves have insect holes (less than or equal to 5 mm) on the leaf grade, then the leaf grade is 3, corn individual leaves have nicks (less than or equal to 10 mm) on the leaf grade is 4, corn minority leaves have nicks (less than or equal to 10 mm) on the leaf grade, then the leaf grade is 5, corn minority leaves have nicks (less than or equal to 10 mm) on the leaf grade is 6, corn individual leaf parts are eaten, then the leaf grade is 7, corn minority leaves are eaten with large pieces (less than or equal to 10 mm) on the leaf part leaves, then the leaf grade is 8, and the leaf grade is eaten with the leaf grade is 9; The resistance evaluation criteria of the armyworm are that the average leaf level is 1.0-2.0 and 2.1-4.0 is high, 4.1-6.0 is medium, 6.1-8.0 is sense, and 8.1-9.0 is high.
  5. 5. The method of claim 3 wherein the leaf grade grading criteria for the heart stage of Asian corn borer is 1 for 1-2 apertures of 1mm or less on individual leaves of corn, 2 for 3-6 apertures of 1mm or less on individual leaves of corn, 3 for 7 or more apertures of 1mm or less on few leaves of corn, 4 for 1-2 apertures of 2mm or less on individual leaves of corn, 5 for 3-6 apertures of 2mm or less on few leaves of corn, 6 for 7 or more apertures of 6 for leaf grade, 1-2 apertures of 2mm or more on few leaves of corn, 7 for leaf grade, 3-6 apertures of >2mm or more on few leaves of corn, 8 for leaf grade, 7 or more apertures of 2mm or more on most leaves of corn, 9 for leaf grade; the resistance evaluation criteria of the Asiatic corn borer are that the average leaf feeding grade is 1.0-2.9 as high resistance, 3.0-4.9 as high resistance, 5.0-6.9 as medium resistance, 7.0-8.9 as sense and 9.0 as high sense.
  6. 6. The method according to claim 3, wherein the classification standard of the leaf level in the heart leaf period of spodoptera frugiperda is that the corn leaf is not damaged or only has needle-like (less than or equal to 1 mm) worm holes on the leaf, the leaf level is 1, the corn only has small number of worm holes with the size (less than or equal to 5 mm) worm holes on the individual leaf, the leaf level is 2, the leaf level is 3 when the small number of corn leaves have the worm holes with the size (less than or equal to 5 mm), the leaf level is 4 when the small number of corn leaves have the notch (less than or equal to 10 mm), the leaf level is 5 when the small number of corn leaves have the notch (less than or equal to 10 mm), the leaf level is 6 when the small number of corn leaves have the large notch (less than 10 mm), the leaf level is 7 when the small number of corn leaves have the large notch (less than 10 mm), the leaf level is 8 when the small number of corn leaves have the large notch (less than 10 mm), and the leaf level is 9 when the small number of corn leaves have the small number of corn leaves; The resistance evaluation criteria of spodoptera frugiperda are that the average leaf feeding grade is 1.0-2.0 and is high in resistance, 2.1-4.0 is medium in resistance, 4.1-6.0 is medium in resistance, 6.1-8.0 is sense, and 8.1-9.0 is high in sense.
  7. 7. The method of claim 1, wherein the step (4) of data processing and insect-resistance level determination is automated data processing and insect-resistance level determination.
  8. 8. The method of claim 7, wherein step (4) is performed by calculating average leaf level and standard deviation using a Python analysis system using pandas script, performing Tukey HSD significance test (P < 0.05) using MultiComparison tool, and determining the insect resistance level of transgenic corn against resistance evaluation criteria.
  9. 9. The method of claim 1, wherein the transgenic corn material is a single or multiple insect-resistant protein expressed transgenic corn material.
  10. 10. The method of claim 9, wherein the insect-resistant protein comprises ecry1gb.1ig, cry1a.105, cry2Ab2, vip3Aa19, vip3Aa-k1, cry1Ab, cry1a.105, or vip3Aa.

Description

Unified evaluation method for multi-pest resistance of corn co-transformed by adaptive multi-source pest-resistant protein Technical Field The invention relates to the technical field of biology, in particular to an evaluation method of insect-resistant effect of transgenic corn. Background The current transgenic corn research and development has been carried out from single insect-resistant protein expression, and the technology is comprehensively developed to a new phase of co-expression of multi-source insect-resistant proteins (such as serial Cry proteins, vip proteins and other proteins of different families), and the technology is upgraded, so that the insect-resistant spectrum can be remarkably widened, the development of insect resistance can be delayed, and the problem of resistance identification to be solved urgently is brought to the industry. The insect-resistant spectrum identification dimension is greatly expanded, the multiple insect-resistant protein corn is required to finish resistance verification aiming at various target pests (such as Asian corn borer, armyworm, spodoptera frugiperda and the like), the identification range is extended from single pest to multiple pest combination, the detection task amount is multiplied, the experimental material processing complexity is remarkably improved, the number of the multiple insect-resistant protein corn materials is continuously increased, the resistance evaluation of different materials to different pests is required to be synchronously carried out, and the complexity of sample management and operation flow is far higher than that of the traditional single protein material identification. More importantly, in the traditional identification system, the insect-receiving methods (such as larva inoculation amount and feed type) and investigation indexes (such as pest grading standards) of different pests are lack of unified standards, so that the experimental period is long, the data comparability is low, even the result deviation occurs, and the large-scale and efficient identification requirement of the multi-insect-resistant protein corn is difficult to adapt, so that the method becomes a core technical bottleneck for restricting the rapid promotion of the transgenic corn industry. Therefore, a set of standardized and efficient transgenic corn insect resistance evaluation systems is needed to be constructed. Disclosure of Invention Accordingly, the main object of the present invention is to provide a method for evaluating the pest-resistant effect of transgenic corn. In order to achieve the above purpose, the technical scheme of the invention is realized as follows: in one aspect, the invention provides a method for evaluating the insect-resistant effect of transgenic corn, which comprises the following steps: (1) Standard planting, namely randomly partitioning and planting the transgenic corn material to be evaluated and the control corn variety; (2) The method comprises the following steps of uniformly inoculating multiple pests, namely inoculating the pests with special flocculent feed after corn grows to a 4-leaf stage, wherein the special flocculent feed comprises the following components in percentage by mass of 50+/-10% of corncob powder, 20+/-5% of soybean meal, 15+/-5% of wheat bran, 5+/-2% of yeast powder, 5+/-2% of sucrose, 1+/-0.5% of compound vitamin and 4+/-2% of mineral mixture, adding antibiotics for preservation, and spraying 15+/-5% of sterile water for humidifying before use; (3) Uniformly investigating and calculating indexes, namely randomly investigating a plurality of corn plants after 14 days of insect inoculation, and recording data; (4) And (5) data processing and insect resistance grade judgment. In one embodiment, the special flocculent feed comprises 50% of corncob powder, 20% of soybean powder, 15% of wheat bran, 5% of yeast powder, 5% of sucrose, 1% of compound vitamin and 4% of mineral mixture by mass, and antibiotics are added for preservation. In one embodiment, the step (1) is standardized planting, namely randomly partitioning planting the transgenic corn material to be evaluated and the control corn variety, setting 3 times of repetition, setting a cell area of 30m <2 >, a row spacing of 60cm and a plant spacing of 25cm, setting a 2m interval between cells, and not spraying insecticide in the whole growth period. In one embodiment, the step (2) is characterized in that multiple pests are inoculated uniformly, wherein after corn grows to a 4-leaf stage, the pests are inoculated with special flocculent feed, the special flocculent feed comprises, by mass, 50% of corncob powder, 20% of soybean meal, 15% of wheat bran, 5% of yeast powder, 5% of sucrose, 1% of compound vitamin and 4% of mineral mixture, antibiotic is added for preservation, 15% of sterile water is sprayed before use for conditioning, the inoculated pests comprise first hatched larvae of armyworms, first hatched larvae of Asian corn borers and/