Application of myb transcription factor gene in regulating resistance of spodoptera frugiperda to vip3a protein

Abstract

The application provides application of a Myb transcription factor gene in regulation of Spodoptera frugiperda resistance to Vip3A protein, and relates to the technical field of biotechnology.The inventors have found that the Myb transcription factor is significantly related to the resistance of Spodoptera frugiperda to Vip3A, and the sensitivity of Spodoptera frugiperda to Vip3A can be regulated by changing the expression of the Myb transcription factor; meanwhile, the Myb transcription factor can also be used as a marker for the resistance of Spodoptera frugiperda to Vip3A, and is used for monitoring the resistance level of Spodoptera frugiperda to Vip3A in the field.The present study provides a train of thought for clarifying the action mechanism of toxins and identifying pest resistance genes, and the research results can be used for research and development of new Bt crops, field detection and comprehensive management of pest resistance to Bt crops / Bt preparations, and have important theoretical and practical significance for sustainable application of Bt insecticidal proteins.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the application of the Myb transcription factor gene in regulating the resistance of the fall armyworm to the Vip3A protein. Background Technology

[0002] The fall armyworm (Spodoptera frugiperda), also known as the common armyworm, belongs to the family Noctuidae in the order Lepidoptera. It feeds on and damages more than 300 crops, including corn, rice, sorghum, and cotton. Native to tropical and subtropical regions of the Americas, this pest has now invaded Africa, Asian countries, and Australia. Due to its high reproductive rate, voracious feeding behavior, and difficulty in control, it poses a serious threat to global agricultural production. Bt insecticidal proteins have shown good control effects against the fall armyworm and have become the main method of control in many regions. However, with the continued large-scale planting of Bt crops, the fall armyworm has gradually developed resistance to various Cry proteins in regions such as Puerto Rico, Florida, North Carolina, Brazil, and Argentina. Vip3 protein is another type of Bt insecticidal protein produced by Bacillus thuringiensis during its vegetative growth stage; it does not share the same sequence homology or binding site as Cry proteins. Compared to Cry1Ab and Cry1F, Vip3A exhibits higher insecticidal activity against the fall armyworm and also shows high toxicity in fall armyworms resistant to Cry toxins, indicating no cross-resistance. In the control of lepidopteran pests, the introduction of this novel insecticidal protein can effectively supplement the toxicity of Cry-based insecticidal proteins, thus delaying the evolution of Bt resistance in pests and controlling lepidopteran pests resistant to Cry insecticidal proteins. Crops expressing Vip3A, such as corn and cotton, are already commercially cultivated in Brazil, the United States, and Argentina. Currently, the fall armyworm has not developed widespread resistance to Vip3A in the field, but individuals carrying the Vip3A resistance allele have been found in the United States and Brazil. Proactively implementing resistance management strategies before widespread resistance to Vip3A develops in pests is of great significance for the sustainable use of the Vip3A protein. Furthermore, elucidating its still unclear insecticidal and resistance mechanisms will provide crucial information for resistance management strategies.

[0003] In view of this, the present invention is hereby proposed. Summary of the Invention

[0004] The first objective of this invention is to provide the application of the Myb transcription factor gene in regulating the resistance of fall armyworm to Vip3A protein, in order to solve at least one of the above-mentioned problems.

[0005] A second objective of this invention is to provide a biomarker for resistance to the Vip3A protein in the fall armyworm.

[0006] A third objective of this invention is to provide the application of the above-mentioned markers in detecting resistance of the fall armyworm to the Vip3A protein.

[0007] A fourth objective of this invention is to provide a reagent for detecting resistance of the fall armyworm to the Vip3A protein.

[0008] The fifth objective of this invention is to provide the application of the above-mentioned reagent in the preparation of products for detecting the resistance of fall armyworm to Vip3A protein.

[0009] The sixth objective of this invention is to provide a reagent kit.

[0010] In a first aspect, the present invention provides the application of the Myb transcription factor gene in regulating the resistance of fall armyworm to Vip3A protein, the nucleic acid sequence of which is shown in SEQ ID NO.1.

[0011] As a further technical solution, the expression of the Myb transcription factor gene was inhibited to improve the resistance of the fall armyworm to the Vip3A protein.

[0012] As a further technical solution, methods to inhibit the expression of Myb transcription factor genes include RNA interference.

[0013] Secondly, the present invention provides a biomarker for resistance of the fall armyworm to the Vip3A protein, said biomarker being selected from any one of the following:

[0014] a. Myb transcription factor gene;

[0015] b. RNA transcribed from the Myb transcription factor gene;

[0016] c. Myb transcription factor;

[0017] The CDS sequence of the Myb transcription factor gene is shown in SEQ ID NO.1;

[0018] The amino acid sequence of the Myb transcription factor is shown in SEQ ID NO.2.

[0019] Thirdly, the present invention provides the application of the above-mentioned markers in detecting the resistance of fall armyworm to Vip3A protein.

[0020] Fourthly, the present invention provides a reagent for detecting resistance of fall armyworm to Vip3A protein, the reagent comprising primers for detecting Myb transcription factor genes, primers for detecting Myb transcription factor RNA, or antibodies for detecting Myb transcription factors;

[0021] The CDS sequence of the Myb transcription factor gene is shown in SEQ ID NO.1;

[0022] The amino acid sequence of the Myb transcription factor is shown in SEQ ID NO.2.

[0023] As a further technical solution, the sequences of the primers used to detect Myb transcription factor RNA are shown in SEQ ID NO.5 and SEQ ID NO.6.

[0024] Fifthly, the present invention provides the application of the above-mentioned reagent in the preparation of products for detecting the resistance of fall armyworm to Vip3A protein.

[0025] As a further technical solution, the product includes a PCR kit or an immunochromatographic kit.

[0026] In a sixth aspect, the present invention provides a reagent kit comprising the above-described reagents.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The inventors' research revealed a significant correlation between the Myb transcription factor and Vip3A resistance in the fall armyworm. Altering the expression of the Myb transcription factor can regulate the fall armyworm's sensitivity to Vip3A. Furthermore, the Myb transcription factor can serve as a biomarker for Vip3A resistance in the fall armyworm, used to monitor resistance levels in the field. This research provides insights into clarifying the mechanism of action of toxins and identifying pest resistance genes. The findings can be applied to the development of novel Bt crops, field detection and integrated management of pest resistance to Bt crops / Bt formulations, and have significant theoretical and practical implications for the sustainable application of Bt insecticidal proteins. Attached Figure Description

[0029] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0030] Figure 1 A site significantly associated with Vip3A resistance was located in the genome-wide association study (GWAS);

[0031] Figure 2 To identify candidate genes based on the narrowed 190Kb region;

[0032] Figure 3 The effects of injecting dsRNA or dsGFP on the fall armyworm. Detailed Implementation

[0033] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0034] In a first aspect, the present invention provides the application of the Myb transcription factor gene in regulating the resistance of the fall armyworm to the Vip3A protein, the nucleic acid sequence of which is shown in SEQ ID NO.1:

[0035]

[0036] The amino acid sequence of the Myb transcription factor is shown in SEQ ID NO.2 as follows:

[0037] MEKNEESSMDEEKGTKSYLQLQIERLKVLQAKLSTEAEEDYVIIKEERSGYDSETSEYSEDSAAAYEDVQAATKSSGPRKNINRGRWTKEEDKKLKACVNIYNENWDLISAQFADRSDVQCQQRWTKVVNPELVKGPWTKEEDEKVMELVAKYGPKKWTLIARHLKGRIGKQCRERWHNHLNPSIKKTAWTEHEDRVIYQAHKQLGNQWAKIAKLLPGRTDNAIKNHWNSTMRRKYEPDL LDSFETLRRKQTRVKPRELTDSSVMQEKVQVAYNDQSWSDPFNESAQSNSSNTSQPPSKHVLHFRQLLKDQLNSLTNQHQVIPKSPDRGGLVAADEMEIVDTPFKFVNIETLPTDSPLRNYLSSASAGTENNQDTVTYAVQAEASKEIEVPSAMYTSPRKKGGETSSPPPILRRKTKKLPVATTNTNNTNAWTDTLSKALEYRESGVTPIKALPFSPSQFLNSPAGISFSTIDANSTPLRHAAKEWSASPLLQTPTPVNHVTPGGPNPVKSNTPKTPTPFKIAMAELGKKSGLKYEPSSPGLLVEDITMMMKRENSDSTVQLNDSLLSTVADQENGNVQINDSGIGSLKRGSESGKENVPGHKKARKALAPAWAALTSTPHAREHTPTLVPDVSYIETPSKTLAGDSSVMFSPPSIVKHSLLEESTSLHSLISAENTPDTSKYEDIDIEAVITEKTKVQHRSLLLTNPEPNTLGPTDIIYPEYEITPFKDITNLDNPSFSRLNANRLRTITTGNELACDTLIAPKQVLANALADHIYKVTNQKPSTSRIDEILTNKIMNKNDYAQKENQWWTVDKDTGQTDDVFSNDYYMFANM(SEQ ID NO.2).

[0038] Myb transcription factor (MYB transcription factor), also known as a trans-acting factor, contains a highly conserved DNA-binding sequence—the MYB motif—and is ubiquitous in plants and animals. It is widely involved in the growth, development, and metabolic regulation of plants and animals, such as cell morphology and pattern building, regulation of secondary metabolism, and responses to biotic and abiotic stresses. Furthermore, the regulation of Myb transcription factors is diverse, including protein-protein interactions, polymerase regulation, redox reactions, phosphorylation, and ubiquitination.

[0039] The inventors discovered that the Myb transcription factor is significantly associated with the resistance of the fall armyworm to Vip3A. By inhibiting the expression of the Myb transcription factor gene, the resistance of the fall armyworm to the Vip3A protein can be improved.

[0040] In some alternative implementations, methods for inhibiting Myb transcription factor gene expression include, but are not limited to, RNA interference, or other methods well known to those skilled in the art.

[0041] Secondly, the present invention provides a biomarker for resistance of the fall armyworm to the Vip3A protein, said biomarker being selected from any one of the following:

[0042] a. Myb transcription factor gene;

[0043] b. RNA transcribed from the Myb transcription factor gene;

[0044] c. Myb transcription factor;

[0045] The CDS sequence of the Myb transcription factor gene is shown in SEQ ID NO.1;

[0046] The amino acid sequence of the Myb transcription factor is shown in SEQ ID NO.2.

[0047] The inventors discovered that the Myb transcription factor is significantly correlated with the resistance of the fall armyworm to Vip3A. Therefore, the Myb transcription factor can be used as a marker of the fall armyworm's resistance to Vip3A to monitor the resistance level of the fall armyworm to Vip3A in the field.

[0048] Thirdly, the present invention provides the application of the above-mentioned markers in detecting the resistance of fall armyworm to Vip3A protein.

[0049] Since the above-mentioned biomarkers are significantly correlated with the resistance of fall armyworm to Vip3A, the resistance level of fall armyworm to Vip3A protein can be obtained by detecting the expression level of the above-mentioned biomarkers.

[0050] Fourthly, the present invention provides a reagent for detecting resistance of fall armyworm to Vip3A protein, the reagent comprising primers for detecting Myb transcription factor genes, primers for detecting Myb transcription factor RNA, or antibodies for detecting Myb transcription factors;

[0051] The CDS sequence of the Myb transcription factor gene is shown in SEQ ID NO.1;

[0052] The amino acid sequence of the Myb transcription factor is shown in SEQ ID NO.2.

[0053] The reagents provided by this invention can be used to detect the resistance of the fall armyworm to Vip3A protein.

[0054] In some alternative embodiments, the sequences of the primers used to detect Myb transcription factor RNA are shown in SEQ ID NO. 5 and SEQ ID NO. 6.

[0055] q-Myb-F:GAAGATGAGAAGGTGATG (SEQ ID NO.5);

[0056] q-Myb-R: TTATAGACGGGTTTAGGT (SEQ ID NO. 6).

[0057] Fifthly, the present invention provides the application of the above-mentioned reagent in the preparation of products for detecting the resistance of fall armyworm to Vip3A protein.

[0058] As a further technical solution, the product includes a PCR kit or an immunochromatographic kit.

[0059] For example, primers for detecting the Myb transcription factor gene or primers for detecting Myb transcription factor RNA are used to prepare a PCR kit; antibodies for detecting Myb transcription factors are used to prepare an immunochromatographic kit.

[0060] In a sixth aspect, the present invention provides a reagent kit comprising the above-described reagents.

[0061] The reagents described above were used to prepare a kit that can be used to detect the resistance level of fall armyworm to Vip3A protein.

[0062] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0063] Example 1

[0064] 1. GWAS analysis:

[0065] Resistant females (female fall armyworms resistant to Vip3A protein) were crossed with susceptible males (male fall armyworms susceptible to Vip3A protein). The F1 offspring (30 females and 30 males) were self-crossed to obtain the F2 offspring. The F2 generation larvae were treated with an artificial diet containing Vip3Aa, and weighed after 7 days to obtain phenotypic data associated with the resistance trait. The F2 generation larvae were sequenced, and the sequencing data were aligned to the fall armyworm reference genome using the default parameters of BWA software. SAMtools was used to filter the data. SNP loci were then identified using GATK software with the following parameters: QD 563 < 2.0 || MQ < 40.0 || FS > 60.0 || MQRankSum < -12.5 || ReadPosRankSum < -8.0. A linear model (LMM) association analysis was performed using GEMMA software to analyze the genotype and phenotypic data. The significance threshold was determined using Bonfeeoni correction (corrected P-value = 0.05 / N).

[0066] Association analysis between fall armyworm resequencing data and Vip3a resistance was performed using GWAS, identifying a locus significantly associated with resistance. Fine mapping narrowed this locus to a 190 kb region. This region contained 11 annotated genes, three of which were expressed in the midgut of fall armyworm. Of these three genes, only the Myb gene showed a significant difference in expression levels between resistant and susceptible populations. Figure 1 ( Figure 1 The red arrows in the middle indicate significantly associated sites; local LD ​​analysis initially narrowed down the linkage intervals of the sites. Figure 2 As shown.

[0067] 2. Interference of the myb gene in the fall armyworm:

[0068] 2.1 Preparation of Myb gene dsRNA

[0069] 2.1.1 Design of target gene primers:

[0070] RNA was extracted from *Fall Armyworm* (Bt protein sensitive) using Trizol reagent. First-strand cDNA synthesis was performed using the SuperScript™ III First-Stand Synthesis System reverse transcription kit. Primers containing the T7 promoter (Primer-F:) were designed using Primer Premier 5 software based on the CDS sequence of the *Fall Armyworm* Myb gene. TAATACGACTCACTATAGGG GCCTACGAAGATGTCCAG (SEQ ID NO.3), Primer-R: TAATACGACTCAC TATAGGGGGAGTTCCAATGGTTCTTTA (SEQ ID NO.4) was sent to Shanghai Sangon Biotech for primer synthesis.

[0071] 2.1.2 PCR amplification of the target fragment and preparation of dsRNA:

[0072] The fall armyworm cDNA was used as a template to amplify the gene using FastPfu enzyme. The reaction system is shown in Table 1 below:

[0073] Table 1

[0074]

[0075] After mixing the above reaction solutions, briefly centrifuge, and then perform the reaction in a PCR instrument according to the following procedure (see Table 2):

[0076] Table 2

[0077]

[0078]

[0079] After PCR amplification, the PCR products were recovered according to the Wizard SV Gel and PCR Clean-Up System kit instructions. Subsequently, dsRNA was synthesized according to the instructions of the Nanjing Novizan T7 High Yield RNA Transcription Kit. The reaction system is shown in Table 3 below:

[0080] Table 3

[0081]

[0082] Gently mix all components with a pipette, briefly centrifuge, and incubate at 37°C for 4 hours. Add 1 μL of DNase I to the reaction system and incubate at 37°C for 15 minutes to digest the transcribed DNA template. The synthesized dsRNA is analyzed by electrophoresis, purified, and stored for later use.

[0083] 2.2dsRNA injection

[0084] Third-instar larvae of a sensitive population with uniform body size were selected and starved for 4 hours before being placed on ice. 2.5 μg of dsRNA was injected into the space between the second and third penultimate leg segments on the abdomen of the larvae using a capillary tube pulled out by a needle puller. 24 larvae were injected per treatment, with three replicates. dsGFP was injected as a control.

[0085] 2.2.1 Measurement of body weight gain of fall armyworms fed Vip3A diet after dsRNA injection

[0086] After injection, the larvae were placed in 24-well plates containing normal feed for 24 hours to recover. Their weight was then recorded as M1, and they were transferred to feed containing 2 μg / ml Vip3A. The control (CK) feed served as the standard control. M2 was recorded after 24 hours, and M3 after 48 hours. The relative growth rate after 24 hours was calculated as (M2-M1) / M1, and the relative growth rate after 48 hours was (M3-M1) / M1. The results showed that the weight gain of larvae injected with dsMyb was significantly higher than that of the control group (dsGFP) (p<0.05). Figure 3 The value of A (relative weight growth rate of fall armyworms after injection of dsRNA or dsGFP feeding on Vip3A feed ([final weight - initial weight] / initial weight); t-test comparison: *P<0.05, **P<0.01) is shown in the figure.

[0087] 2.2.2 Silence Efficiency Detection

[0088] 24 and 72 hours after dsRNA injection, samples were prepared and the silencing efficiency of the target gene was detected using qPCR. The primers for Myb quantitative PCR were designed as follows: q-Myb-F: GAAGATGAGAAGGTGATG (SEQ ID NO.5); q-Myb-R: TTATAGACGGGTTTAGGT (SEQ ID NO.6). The internal reference gene was β-actin: q-β-actin-F: CCTGGTATTGCTGACCGTATGC (SEQ ID NO.7); q-β-actin-R: CTGTTGGAAGGTGGAGAGGGAA (SEQ ID NO.8). All assays included three biological replicates, each containing five samples. Significant difference analysis was performed using the independent samples t-test in SPSS software. Figure 3 The B in the figure represents the interference efficiency of the Myb gene dsRNA on its target gene; where SfMyb is an abbreviation of the Latin name of the fall armyworm, referring to Myb.

[0089] The results showed that the target gene silencing efficiency reached 58% and 49% 24 hours and 48 hours after dsRNA injection, respectively, which is good for Lepidoptera.

[0090] In summary, genome-wide association analysis and fine mapping identified a Myb transcription factor significantly associated with Vip3A resistance in the fall armyworm. RNAi-induced reduction of Myb gene expression significantly decreased the susceptibility of fall armyworm larvae to Vip3A. This study provides insights into clarifying the mechanism of action of toxins and identifying pest resistance genes. The findings can be applied to the development of novel Bt crops, field detection and integrated management of pest resistance to Bt crops / Bt formulations, and have significant theoretical and practical implications for the sustainable application of Bt insecticidal proteins.

[0091] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of the Myb transcription factor gene in regulating the resistance of fall armyworm to Vip3A protein, characterized in that, The CDS sequence of the Myb transcription factor gene is shown in SEQ ID NO.1; Inhibiting the expression of the Myb transcription factor gene enhances the resistance of fall armyworm to Vip3A protein.

2. The application according to claim 1, characterized in that, Methods to suppress Myb transcription factor gene expression include RNA interference.

3. The application of biomarkers in detecting resistance to Vip3A protein in fall armyworm, characterized in that, The marker is selected from any one of the following ac: a. Myb transcription factor gene; b. RNA transcribed from the Myb transcription factor gene; c. Myb transcription factor; The CDS sequence of the Myb transcription factor gene is shown in SEQ ID NO.1; The amino acid sequence of the Myb transcription factor is shown in SEQ ID NO.

2.

4. Application of the reagent in the preparation of products for detecting resistance of fall armyworm to Vip3A protein; The reagents include primers for detecting the Myb transcription factor gene or primers for detecting Myb transcription factor RNA. The CDS sequence of the Myb transcription factor gene is shown in SEQ ID NO.

1.

5. The application according to claim 4, characterized in that, The sequences of the primers used to detect Myb transcription factor RNA are shown in SEQ ID NO.5 and SEQ ID NO.

6.

6. The application according to claim 4, characterized in that, The products include PCR kits.

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