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CN-122011148-A - Use of insect inhibitory proteins and methods for increasing plant resistance

CN122011148ACN 122011148 ACN122011148 ACN 122011148ACN-122011148-A

Abstract

The invention relates to application of Col05, col06 or Col29 protein for inhibiting or killing insects, wherein the Col05, col06 or Col29 protein respectively has an amino acid sequence shown as SEQ ID NO. 1, SEQ ID NO. 3 or SEQ ID NO. 5, dicotyledonous plant tobacco is selected as a first host, monocotyledonous plant corn is selected as a second host, and plant codon optimization is carried out on the Col05, col06 or Col29 protein to respectively obtain a nucleotide sequence shown as SEQ ID NO. 2, SEQ ID NO. 4 or SEQ ID NO. 6. Compared with the prior art, the application of the insect inhibition proteins Col05, col06 or Col29 and the encoding genes provides a more effective insect inhibition method, in particular a method for preventing and controlling the double-spotted fluorescent leaf beetles, which can effectively reduce the use of pesticides and obviously reduce ecological risks. The invention provides a brand new inhibition method of double-spotted fluorescent leaf beetles, the existing gene of the double-spotted fluorescent leaf beetles is mainly RNAi, and the method of the invention is completely different from the method of using protein to resist insects in insect resistance mechanism.

Inventors

  • HUANG CHAOLONG
  • TAO MENGMENG
  • LIU CONGCONG
  • WANG SHENGYONG
  • ZHAO XINYU
  • Du Haiye

Assignees

  • 国投种业科技有限公司

Dates

Publication Date
20260512
Application Date
20260415

Claims (10)

  1. 1. The application of the insect inhibitory protein is characterized in that the protein is Col05, col06 or Col29 protein, and is used for inhibiting or killing insects invading plants; The Col05 protein has an amino acid sequence shown as SEQ ID NO. 1, the Col06 protein has an amino acid sequence shown as SEQ ID NO. 3, and the Col29 protein has an amino acid sequence shown as SEQ ID NO. 5; The insect is diabrotica; the plant is tobacco.
  2. 2. The use according to claim 1, wherein the coding gene of the Col05 protein has a nucleotide sequence shown as SEQ ID NO.2, the coding gene of the Col06 protein has a nucleotide sequence shown as SEQ ID NO. 4, and the coding gene of the Col29 protein has a nucleotide sequence shown as SEQ ID NO. 6.
  3. 3. The method according to claim 2, wherein the dicotyledonous plant tobacco is selected as the first host, the monocotyledonous plant maize is selected as the second host, and the Col05, col06 or Col29 protein is subjected to plant codon optimisation to obtain the nucleotide sequence as shown in SEQ ID NO.2, SEQ ID NO. 4 or SEQ ID NO. 6, respectively.
  4. 4. A method for improving insect resistance of a plant by using an insect inhibitory protein, wherein the protein is Col05, col06 or Col29 protein, the method comprises the step of expressing the Col05, col06 or Col29 protein coding gene in the plant, wherein the Col05 protein has an amino acid sequence as shown in SEQ ID NO. 1, the Col06 protein has an amino acid sequence as shown in SEQ ID NO. 3, and the Col29 protein has an amino acid sequence as shown in SEQ ID NO. 5; the plant is tobacco.
  5. 5. The method according to claim 4, wherein the encoding gene of the Col05 protein has a nucleotide sequence shown in SEQ ID NO.2, the encoding gene of the Col06 protein has a nucleotide sequence shown in SEQ ID NO. 4, and the encoding gene of the Col29 protein has a nucleotide sequence shown in SEQ ID NO. 6; Selecting dicotyledonous plant tobacco as a first host, and monocotyledonous plant corn as a second host, and carrying out plant codon optimization on Col05, col06 or Col29 proteins to obtain nucleotide sequences shown as SEQ ID NO. 2, SEQ ID NO. 4 or SEQ ID NO. 6 respectively.
  6. 6. The method according to claim 5, wherein the method comprises the steps of constructing a transient expression vector using a Col05, col06 or Col29 protein-encoding gene, introducing Agrobacterium to obtain Agrobacterium expressing the gene, and applying Agrobacterium to the plant to express the Col05, col06 or Col29 protein-encoding gene.
  7. 7. The method of claim 6, wherein the applying the Agrobacterium to the plant is applying the Agrobacterium to a plant leaf.
  8. 8. The method of claim 6 or 7, wherein the transient expression vector comprises two expression cassettes, the first cassette comprising a Col05, col06 or Col29 protein encoding gene, and the second cassette is used to screen the marker expression units to identify whether the Col05, col06 or Col29 protein is expressed in plant cells.
  9. 9. The method according to claim 4 to 8, wherein the insect is a coleopteran pest, preferably diabrotica.
  10. 10. A plant obtainable according to the method of any one of claims 4 to 9, said plant being tobacco.

Description

Use of insect inhibitory proteins and methods for increasing plant resistance Technical Field The present invention relates to the use of insect inhibitory proteins and methods for increasing plant resistance. In particular, the invention relates to the use of the Col05, col06 or Col29 proteins for inhibiting coleoptera pests, in particular for inhibiting diabrotica, and to methods of increasing the resistance of plants, in particular tobacco, to coleoptera pests, in particular diabrotica. Background Corn is used as a pillar crop of Chinese agriculture, the planting area and the yield stably occupy the front of the grain crop for a long time, and the corn is not only an important basic stone for guaranteeing the national grain safety, but also a core resource for supporting the development of animal husbandry and processing industry. In recent years, along with the continuous expansion of the test spot area of transgenic corn, farmers gradually recognize the remarkable advantages of transgenic insect-resistant corn in the aspects of planting management and yield improvement. This transition has led scientists to note that, in addition to the effective control of lepidopteran pests in first-generation transgenic insect-resistant corn, coleopteran double-spotted diabrotica beetles are becoming increasingly more prevalent year by year, and have become a new challenge for industry development. Currently, agricultural technology popularization centers in rural areas focus on the fact that double-spot fluorite leaf beetles seriously damage corns and soybeans in northern corn main cultivation areas from Heilongjiang in the east to Xinjiang in the west and from inner Mongolia in the north to Henan in the south and the like, and the production of the corns and the soybeans is seriously affected. The double-spotted fluorescent leaf beetles are used as representatives of coleopteran pests, the life cycle of the double-spotted fluorescent leaf beetles is obviously different from that of lepidopteran pests, the pests only generate one generation in one year, wintering is carried out in soil in the form of diapause eggs, and the diapause eggs can resist extreme environments to maintain the population scale of the second year. In spring, the larvae break the soil and eat the corn roots, so that the root systems of the seedlings are damaged, the development is bad, the early growth of the corn is seriously affected, in summer, the adults eat corn leaves and filaments instead, so that serious problems of kernel loss, tip bursting and the like are caused, and the corn yield is directly threatened. In order to effectively damage the double-spot firefly beetles of the corn, the agricultural and technical departments in each place guide farmers to carry out deep ploughing in winter. However, the diapause eggs of the double-spotted fluorescent leaf beetles have extremely strong environmental tolerance, the method has limited prevention and control effects, and the centralized flying prevention and pesticide application of the tissue villages and towns in summer has hysteresis quality and serious environmental and ecological problems. Currently, the genes of the double-spotted fluorescent leaf beetles are mainly RNAi, and the method is completely different from the method using protein insect resistance in insect resistance mechanism. No effective coleopteran pest proteins, particularly anti-diabrotica proteins, have been found. Disclosure of Invention To solve the problems of the prior art, in a first aspect of the present invention there is provided the use of an insect inhibitory protein, the protein being the Col05, col06 or Col29 protein, for inhibiting or killing insects which attack plants. The Col05 protein has an amino acid sequence shown as SEQ ID NO. 1, namely ：MKELQEITNAWGAWKAKQLGTSLHFTASTKYGGNSHLSPYTKYEVQSDLEAGGFHFVNKRELPDGVGFDLSHTVHNSTGEKQTMDVTFTEKKTSELTISVTEAVKIGNKVSGGVNVPFFADGKVEITAELSVSSTQSWKNSKDNGLSIKVPTVVPAHSSVLVKSTWVINRLQADWVADVVMRGYVAVRFNDYVYYDNYKENGWHALWFIPIKSVFDEIIQNNIIDTTGYIRQYNSVIAQAKGTLETATTAFGKTSFSPIDSASVIGNVRYNKEEPVDYVLIGADNEEGV. The Col06 protein has an amino acid sequence shown as SEQ ID NO. 3, namely ：MNSLQQITDAWGKWYSQQHGTTCRFTASTDYSSQSFLDDYHQYQVSTTAQNIVYDDNSLPTNGSEIAFKTIYNNNTQAANQQSLIETATSTQSFEWSITEAVSIGVEISATEGVPAVASSTQKVTVNLSLSSTQKSTVTNTQSWSVNTILTIPPQSTIKADIVIGTQSYNINFTLSVMLNGYVAIWNNDKVNGHWLWFIPITQVFSDCIANNIIDTSGYDFVGGGISTTASGVFTGSQGISVGVNTTQYPLNSNTDAAKEPGFIDTPAVSKIVAMAGKE. The Col29 protein has an amino acid sequence shown as SEQ ID NO. 5, namely ：MESNNQQNKMVEVNGTTSNSFILSPITSNIITDVDQQMNKISDYYYNNNLKLKDIGDYYHIIRLENKNTTMSFDLNADDIKNLHYNDLQPQYIGENEFKNTTDQEQTFTTASYSQAVTNSVSSTVIQGFKATSTTSLLKIPILLPGGINLNAEFNSASNTTTTNTTTETLTAPPQNIKVPAGRTYKVEVNLLKKKFTGDIDFHGKGTNVKSNLKVRATYYGPGFPRPTKYPTYTYSTADMWRGLTTEQKKQITGVNFNNNKDLTIDGTTKVEGIYGSNLEVVVYDITNKNTPKIVETRTFK. The insect is double-spotted fluorescent leaf beetles, and the plant is tobacco. In a preferred embodiment, the gene encoding the Col05 protein has the n