Maize CYP92C5 gene and its application in breeding varieties resistant to Asian corn borer

Abstract

The present invention discloses a corn CYP92C5 gene and its application in breeding varieties resistant to Asian corn borer, and relates to the technical field of plant genetic engineering. The nucleotide sequence of the CYP92C5 gene is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2. Studies have shown that mutants after the CYP92C5 gene is knocked out have significantly reduced attraction to Asian corn borer. The present invention develops a new insect-resistant material based on the new function of the corn CYP92C5 gene, providing a genetic basis for the breeding of corn resistant corn borer varieties. The CYP92C5 mutation confers resistance to corn borer, which makes it possible to produce various types of corn borer-resistant germplasms using CRISPR-mediated corn breeding genome editing technology.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, and in particular to a corn CYP92C5 gene and an application thereof in breeding varieties resistant to Asian corn borer. Background Art

[0002] Corn is one of the most important food and feed crops in the world, and is also a valuable raw material for industry and biofuels. Therefore, improving the quality and yield of corn is crucial to ensuring global food security. Insect pests have long been considered one of the main harmful factors limiting corn growth and production. During the life cycle of a plant, different parts of the plant are inevitably attacked by various insect populations. Among them, the Asian corn borer is a major corn pest belonging to the order Lepidoptera. It is distributed in Asia, the Western Pacific, and Australia. It mainly infects the leaves and stems of corn, causing significant damage to the corn and ultimately causing huge yield losses.

[0003] The current field management of Asian corn borer mainly relies on the application of chemical pesticides. However, since the larvae of Asian corn borer will burrow inside the plant when feeding, this leads to an increase in the use of pesticides, thereby threatening environmental safety and human health. Therefore, breeding resistant crop strains is a green, cost-saving and environmentally friendly means of controlling Asian corn borer. In this regard, the present invention proposes a corn CYP92C5 gene and its application in breeding varieties resistant to Asian corn borer. Summary of the invention

[0004] The purpose of the present invention is to provide a corn CYP92C5 gene and its application in breeding varieties resistant to Asian corn borer, to provide new materials for biological control of corn borer, and to provide a theoretical basis and gene resources for developing crop lines resistant to corn borer.

[0005] The present invention achieves the above-mentioned purpose through the following technical solutions:

[0006] The first object of the present invention is to provide a corn CYP92C5 gene, the full length of the CYP92C5 gene CDS is 1596bp, and its nucleotide sequence is shown in SEQ ID NO.1. The gene can be directly cloned from crops or obtained by artificial synthesis.

[0007] A further improvement is that the amino acid sequence encoded by the CYP92C5 gene is shown in SEQ ID NO.2, and the CYP92C5 gene encodes 531 amino acids.

[0008] The second object of the present invention is to provide an application of a corn CYP92C5 gene in breeding plant varieties resistant to Asian corn borer.

[0009] A further improvement is that the application is to knock out the CYP92C5 gene in the plant through gene knockout technology, so that the plant loses its attractiveness to the larvae of Asian corn borer and enhances the plant's ability to resist corn borer.

[0010] A further improvement is that the application is to use CRISPR knockout technology or screen homozygous corn varieties with missing CYP92C5 gene function from natural ecotypes during plant breeding to obtain plant varieties resistant to Asian corn borer.

[0011] A further improvement is that the plant includes at least one of corn, rice, wheat, rapeseed or sorghum.

[0012] The present invention has the following beneficial effects:

[0013] (1) The present invention knocks out the CYP92C5 gene by CRISPR to obtain a CYP92C5 gene mutant. The preference and dual-host selectivity of corn borer larvae were detected by biological tests on corn CYP92C5 gene mutants. The results confirmed that after the CYP92C5 gene was knocked out, the synthesis of terpenes DMNT and TMTT was reduced, and the attraction to Asian corn borer was significantly reduced. In addition, in the dual-host selection experiment, the degree to which the mutant after gene knockout was found and eaten by corn borer was significantly lower than that of the wild type.

[0014] (2) The present invention has developed a new insect-resistant material based on the new function of the corn CYP92C5 gene, providing a genetic basis for the breeding of corn varieties resistant to corn borers. The CYP92C5 mutation confers resistance to corn borers, which makes it possible to produce various types of corn borer-resistant germplasms using CRISPR-mediated corn breeding genome editing technology.

[0015] (3) The CYP92C5 gene provided by the present invention has good colinearity with homologous genes of other crops and has high research and application value, providing important gene resources for the development of natural insect-resistant strains of crops such as rice, wheat, rapeseed, and sorghum. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The electrophoresis diagram of the full-length amplification of CYP92C5 CDS (in the figure: lane 1 is a DNA marker (300bp-10000bp), lane 2 is a negative control, and lane 3 is a CYP92C5 gene fragment);

[0017] Figure 2 CRISPR site editing status;

[0018] Figure 3The results are as follows: The results of DMNT content detection in B104 and CYP92C5 gene mutants (c5-s1 and c5-s2 mutants) (Figure A is a comparison between B104 and c5-s1 mutants; Figure B is a comparison between B104 and c5-s2 mutants);

[0019] Figure 4 The results are as follows: The results are as follows: The results are as follows: The results are as follows: Detection of TMTT content in B104 and CYP92C5 gene mutants (c5-s1, c5-s2 mutants) (Figure A is a comparison between B104 and c5-s1 mutants; Figure B is a comparison between B104 and c5-s2 mutants);

[0020] Figure 5 Schematic diagram of the preference test structure for B104 and CYP92C5 gene mutant Asian corn borer larvae;

[0021] Figure 6 The results of preference of B104 and CYP92C5 gene mutants of Asian corn borer larvae (Figure A is a comparison between B104 and c5-s1 mutants; Figure B is a comparison between B104 and c5-s2 mutants);

[0022] Figure 7 The results of the preference of the Asian corn borer for pure DMNT and TMTT to plant seedlings (Figure A is the comparison between corn B73 and DMNT; Figure B is the comparison between corn B73 and TMTT).

[0023] Figure 8 The results of dual host selection of B104 and CYP92C5 gene mutant Asian corn borer larvae (the arrows in Figure A indicate the broken parts eaten by insects, and the circles indicate the traces of insects eating; Figure B shows the number of corn borer larvae on B104 and c5-s2 mutant strains within a certain period of time); DETAILED DESCRIPTION

[0024] The present application is further described in detail below in conjunction with the accompanying drawings. It is necessary to point out here that the following specific implementation methods are only used to further illustrate the present application and cannot be understood as limiting the scope of protection of the present application. Technical personnel in this field can make some non-essential improvements and adjustments to the present application based on the above application content.

[0025] 1. Materials and reagents

[0026] Unless otherwise specified, the materials and reagents used in this example can be obtained through commercial channels.

[0027] 2. Methods

[0028] No specific experimental method is specified in this embodiment, and all experimental methods can be carried out according to conventional methods.

[0029] 2.1 Cloning of the Maize CYP92C5 Gene

[0030] 2.1.1. Using wild-type corn variety B73 as material, RNA was extracted, and the extracted total RNA was reverse transcribed into cDNA as a template for PCR amplification.

[0031] 2.2.2. According to the nucleotide sequence of the corn CYP92C5 gene shown in SEQ ID NO.1, specific amplification primers were designed, and the designed specific primers were used for amplification to obtain the corn CYP92C5 gene fragment (the electrophoresis result is shown in Figure 1 The cloned protein was ligated to the T cloning vector PEASY-T3 to obtain T3-CYP92C5, which was transformed into Escherichia coli. The positive clones were picked and sequenced, and the sequencing results were consistent with the predicted results.

[0032] The primer sequences are shown in SEQ ID NO.3 and SEQ ID NO.4:

[0033] SEQ ID NO.3: CYP92C5-F: (5'>ATGGAGCTGGCATCAACA<3');

[0034] SEQ ID NO.4: CYP92C5-R: (5'>TCATTCGGTCGCGCTGTA<3').

[0035] 2.2 Identification of maize CRISPR / Cas9 knockout mutants

[0036] 2.2.1. Construction of CYP92C5 mutant strain

[0037] The CYP92C5 gene can synthesize two terpene homologues, DMNT and TMTT. The CYP92C5 gene was knocked out by gene editing using CRISPR / Cas9 technology. The oligonucleotide sequence of the sgRNA was 5'>GCTCTTCTCCGCGCG CCGCG<3'. Finally, two CYP92C5 mutant strains were obtained in the B104 background and named c5-s1 (c5, the abbreviation of CYP 92C5, s, the abbreviation of single mutation, and the Arabic number 1 represents the row number), c5-s2 ( Figure 2 ).

[0038] 2.2.2 Enrichment of corn volatiles by SPME

[0039] In the headspace vial, 0.1 g of leaf sample and 1 μL of 10 ng / mL bromodecane (as an internal standard) were added and thoroughly mixed with 5 mL of saturated sodium chloride aqueous solution to enhance the release of volatile compounds in the sample. The headspace vial was stabilized in a 30°C water bath for 10 minutes, followed by a 40-minute SPME extraction. An Agilent 8890A gas chromatograph and 5977B mass spectrometer were used to separate and identify the volatile compounds extracted by SPME. The volatile compounds were separated on a HP-5M (S30 m×0.25 mm×0.25 μm) capillary column with helium as the carrier gas (purity >99.999%). The results are shown in Figure 2. Figure 3 , 4 As shown, the presence of DMNT and TMTT was almost undetectable in the gene-edited mutants (c5-s1, c5-s2), and the content was extremely low compared with the control (B104), proving that the obtained c5-s1 and c5-s2 were CYP92C5 gene knockout strains.

[0040] 2.3 Effects of CRISPR / Cas9 knockout mutants on the behavior of Ostrinia furnacalis

[0041] 2.3.1. Indoor insect preference test device to measure the preference of Asian corn borer larvae

[0042] According to the experimental requirements, the control (B104) and CYP92C5 mutant strains (c5-s1, c5-s2) were placed in two glass jars, and the two glass jars were connected to the two ends of a glass test tube with an odorless plastic tube. The number of larvae moving to either end of the glass tube was recorded within 10-60 minutes and compared to illustrate the preference of the larvae. The device structure is as follows Figure 5 As shown, the results are Figure 6 shown.

[0043] In addition, based on Figure 4 The device structure shown in the figure, the compounds (DMNT and TMTT) and plant seedlings (corn B73 variety) are placed in two glass jars, and the two glass jars are connected to the two ends of a glass test tube with odorless plastic tubes. The number of larvae moving to either end of the glass tube is recorded within 10-60 minutes and compared to illustrate the preference of the larvae. The results are shown in Figure 7 shown.

[0044] 2.3.2. Experiment on dual host selection of corn borer larvae

[0045] The experiment was carried out in a mesh box with a length of 91 cm, a width of 59 cm and a height of 61 cm. The mesh is breathable. Different corn materials were placed on both sides of the tray (B104 control on one side and CYP92C5 mutant strain c5-s2 on the other side). The tray consisted of 8 holes per row and 4 holes per column, with a depth of about 11 cm and a hole diameter of about 5.8 cm. The tops of the remaining empty holes were covered with paper to make it easier for corn borer larvae to move freely. After a certain period of time, the number of larvae on different corn materials was counted. Different biological replicates were separated by a distance of more than 5 meters to avoid potential influences between different replicates or plant materials. The results are shown in the figure. Figure 8 As shown, B104 was found and eaten by insects to a significantly higher extent than the CYP92C5 mutant strain c5-s2.

[0046] 3. Conclusion

[0047] After the CYP92C5 gene was edited and knocked out, the DMNT and TMTT contents were extremely low. Compared with the control (B104), the gene-edited mutant significantly reduced its attractiveness to the larvae of the Asian corn borer. In the dual-host selection experiment, the knockout of CYP92C5 significantly affected the ability of the Asian corn borer larvae to find corn. Compared with the control group, the mutant significantly reduced the degree of gnawing by the Asian corn borer at the same time, indicating that the loss of the CYP92C5 gene can improve the resistance of corn to the Asian corn borer, providing a new direction for the cultivation of varieties resistant to the Asian corn borer.

[0048] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. An application of a corn CYP92C5 gene in breeding plant varieties resistant to Asian corn borer, characterized in that: The nucleotide sequence of the CYP92C5 gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2; The application is at least one of the following: (1) Knock out the CYP92C5 gene in corn through gene knockout technology, making corn lose its attractiveness to Asian corn borer larvae and enhancing corn's ability to resist corn borer; (2) In the corn breeding process, CRISPR knockout technology is used or homozygous corn varieties with missing CYP92C5 gene function are screened from natural ecotypes to obtain corn varieties resistant to Asian corn borer.

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