Cotton anti-thrip transcription factor GhMYC2 gene and application thereof

Abstract

The invention discloses a cotton anti-thrip transcription factor GhMYC2 gene and application thereof, and relates to the technical field of genetic engineering. The nucleotide sequence of the cotton anti-thrip transcription factor GhMYC2 gene is as shown in SEQ ID NO. 1. The GhMYC2 gene encodes a GhMYC2 protein, and the amino acid sequence of the GhMYC2 protein is as shown in SEQ ID NO. 2. According to the cotton anti-thrip transcription factor GhMYC2 gene and the application thereof, the GhMYC2 gene is knocked out through CRISPR / Cas9, an anti-thrip cotton plant can be obtained, and a foundation is laid for cultivation of a new cotton anti-thrip variety.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, in particular to a cotton thrips resistance transcription factor GhMYC2 gene and application thereof. Background Art

[0002] Cotton is an important fiber and oil crop in the world. It is often attacked by various insects during the entire growth process, which directly affects the yield and quality of cotton. In recent years, with the large-scale promotion and planting of transgenic insect-resistant cotton, lepidopteran insects such as cotton bollworm have been reduced to secondary pests, while the harm of secondary pests such as cotton thrips has increased significantly, especially cotton thrips not only in the seedling stage, but also in the flowering stage. Thrips mostly concentrate on the tender heads and backs of cotton plants to absorb sap, causing the growth points to burn, multiple cotton heads, broken leaves and rust spots to appear, delaying the development period of cotton plants, reducing bolls and reducing quality. Thrips have a wide host range, and there are no very good new varieties of thrips-resistant cotton on the market. At present, chemical pesticides are more commonly used for the prevention and control of thrips, and excessive use of pesticides will cause greater pollution to the soil, air and water, seriously affecting human health. Therefore, cultivating insect-resistant varieties and exploring insect-resistant genes are the most economical, effective and environmentally friendly ways to improve cotton yield and quality.

[0003] Plants have developed a variety of defense mechanisms against herbivorous insects during the evolution process, which are generally divided into constitutive defense and induced defense. Constitutive defense mechanisms refer to physical and chemical factors that originally exist in plants and hinder insect feeding, mainly including morphological insect resistance and biochemical insect resistance. Morphological insect resistance mainly refers to the morphological characteristics of the plant itself, which can inhibit or hinder insect feeding, movement, egg laying or survival. The length and density of cotton hairs are important factors affecting insect activity and are closely related to the insect resistance of plants. Relevant reports point out that cotton leaf hairs are related to green stink bugs, and hairless or less hairs have a certain resistance to them, and the incidence of aphids in hairless or less hairy varieties is lower. The length of cotton midrib hairs is significantly negatively correlated with thrips resistance, and there are more cotton bollworms in hairy varieties than in less hairy varieties. Plant biochemical insect resistance refers to certain metabolites produced during plant growth, which have a certain effect on plant insect resistance. Related reports show that some plant volatile secondary metabolites have a certain attraction effect on thrips, including compounds such as benzene rings and pyridines. Rosemary and geranium have a repellent effect on thrips. In addition to the repellent effect mediated by volatile secondary metabolites, plants can also inhibit insect egg laying and feeding through non-volatile or semi-volatile secondary metabolites, or even poison them. For example, the essential oils or extracts of marjoram, lavender, and mint have a significant repellent effect on thrips.

[0004] Unlike constitutive defense mechanisms, induced resistance is associated with relatively minor changes in plant metabolism. All plants, whether resistant or susceptible, respond to insect attack by coordinating defense strategies through endogenous phytohormone signaling, followed by activation of biochemical cascades of local or systemic defense mechanisms. Among the early responses of plants, jasmonic acid (JA) functions as a major regulator of induced insect defense.

[0005] GhMYC2 is a bHLH-MYC2 transcription factor that plays a decisive role in the formation of pigment glands in cotton. Inhibiting the expression of GhMYC2 in glandular cotton by gene silencing or knockout results in a glandless phenotype in the plant. In recent years, the function and mechanism of GhMYC2 in regulating the formation of gossypol and pigment glands have been extensively studied. Transposons were inserted into the GhMYC2 coding sequence in the A subgenome of glandless cotton, and mutations occurred in the homologous promoter in the D subgenome, ultimately leading to a glandless phenotype. GhMYC2 regulates the expression of terpenoid biosynthesis genes by directly regulating the transcription factors GoHSFA4a, GoNAC42, and GhJUB1, thereby regulating the biosynthesis of gossypol. In addition, it has been demonstrated that the formation of glands and the biosynthesis of terpenoids are controlled by two different pathways in which GhMYC2 participates. Summary of the invention

[0006] The purpose of the present invention is to provide a cotton thrips-resistant transcription factor GhMYC2 gene and an application thereof. By knocking out the GhMYC2 gene through CRISPR / Cas9, thrips-resistant cotton plants can be obtained, laying a foundation for the cultivation of new thrips-resistant cotton varieties.

[0007] To achieve the above object, the present invention provides a cotton thrips resistance transcription factor GhMYC2 gene, and the nucleotide sequence of the GhMYC2 gene is shown in SEQ ID NO.1.

[0008] The present invention also provides a cotton thrips-resistant GhMYC2 protein. The coding gene of the GhMYC2 protein is the GhMYC2 gene shown in SEQ ID NO.1, and the amino acid sequence of the GhMYC2 protein is shown in SEQ ID NO.2.

[0009] The present invention also provides application of the cotton anti-thrips transcription factor GhMYC2 gene in regulating cotton anti-thrips.

[0010] Furthermore, the GhMYC2 gene was knocked out by CRISPR / Cas9 to improve cotton's resistance to thrips.

[0011] Furthermore, after knocking out the GhMYC2 gene through CRISPR / Cas9, the anthocyanin content of the plant was reduced.

[0012] The advantages and positive effects of the cotton anti-thrips transcription factor GhMYC2 gene and its application described in the present invention are:

[0013] 1. The present invention provides a cotton anti-thrips transcription factor GhMYC2 gene, which can be used to regulate the formation of anthocyanins in cotton, and provide insights into the mechanism of anthocyanin synthesis in cotton and other food crops, vegetables, fruits and other plants.

[0014] 2. The present invention provides a cotton thrips-resistant transcription factor GhMYC2 gene. By knocking out the GhMYC2 gene through CRISPR / Cas9, thrips-resistant cotton plants can be obtained, laying the foundation for the cultivation of new thrips-resistant cotton varieties.

[0015] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The metabolite analysis results of the thrips-resistant and susceptible cotton varieties in Example 1 of the present invention, wherein (a) is the enrichment analysis of non-target metabolic pathways, (b) is the differential metabolites of the flavonoid and anthocyanin pathways, (c) is the flavonoid content in the thrips-resistant and susceptible individual plants of the F2 segregation population, (d) is the flavonoid content in the thrips-resistant and susceptible varieties, (e) is the anthocyanin content in the thrips-resistant and susceptible individual plants of the F2 segregation population, and (f) is the anthocyanin content in the thrips-resistant and susceptible varieties;

[0017] Figure 2 The results of the thrips selective feeding experiment in Example 1 of the present invention, wherein (a) is the cottonpol content in the resistant and susceptible thrips individual plants of the F2 segregation population, (b) is the cottonpol content in the resistant and susceptible thrips varieties, (c) is the selective feeding of thrips after the cotyledons are injected with cottonpol, (d) is the selective feeding of thrips after the cotyledons are injected with flavonoids, (e) is the selective feeding of thrips after the cotyledons are injected with anthocyanins, (f) is the statistics of the number of thrips at different times of selective feeding injected with different metabolites, (g) is the anthocyanin extract of green / purple cabbage, (h) is the anthocyanin content of green / purple cabbage, (i) is the selective feeding of thrips on green / purple cabbage, and (j) is the statistics of the number of thrips at different times of selective feeding on green / purple cabbage;

[0018] Figure 3 The reduced anthocyanin content of the GhMYC2 mutant ghmyc2 in Example 3 of the present invention can enhance thrips resistance, wherein (a) is the sequencing comparison of the mutation site sequence of the ghmyc2 mutant, (b) is the anthocyanin extract of the wild type and the ghmyc2 mutant, (c) is the anthocyanin content of the wild type and the ghmyc2 mutant, (d) is the thrips resistance of the wild type and the ghmyc2 mutant, and (e) is the thrips pest index of the wild type and the ghmyc2 mutant;

[0019] Figure 4 The figure shows the change of anthocyanin content in the cotton callus tissue overexpressing GhMYC2 in Example 3 of the present invention, wherein (a) is the appearance of the positive callus tissue, and (b) is the comparison of anthocyanin content in the callus tissue;

[0020] Figure 5 This is the effect of overexpressing GhMYC2 in Arabidopsis on thrips resistance according to Example 3 of the present invention, wherein (a) is the WB detection of overexpressed Arabidopsis, (b) is the anthocyanin content of wild type and overexpressed Arabidopsis, (c) is the thrips resistance of wild type and overexpressed Arabidopsis, (d) is the fresh weight of wild type and overexpressed Arabidopsis 20 days after inoculation with thrips, and (e) is the dry weight of wild type and overexpressed Arabidopsis 20 days after inoculation with thrips. DETAILED DESCRIPTION

[0021] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0022] Unless otherwise defined, technical or scientific terms used in the present invention shall have the common meanings understood by one having ordinary skills in the field to which the present invention belongs.

[0023] Unless otherwise defined, the instruments, equipment and reagents used in the present invention are conventionally commercially available.

[0024] Escherichia coli Top10 and Agrobacterium tumefaciens GV3101 were purchased from Sangon Biotechnology Co., Ltd. with catalog numbers B528412 and B528430, respectively.

[0025] The pGreenII0800-LUC vector is used for transient expression of LUC in tobacco, and the pCAMBIA1304 vector is used for transient expression of GUS in tobacco. Both vectors are commercial vectors and were previously preserved by the laboratory; pCambia2300-Cas9 is used to construct CRISPR / Cas9 gene editing vector, from the article "Ge XY, et al. Efficient genotype-independent cotton genetic transformation and genome editing. Journal of Integrative Plant Biology, 2023, 65(4): 901-917"; pGreen0229 is used to construct Arabidopsis overexpression vector.

[0026] All cotton varieties used are from germplasm resources collected and preserved in the laboratory, from the article "Ma ZY, et al. High-quality genome assembly and resequencing of modern cotton cultivarsprovide resources for crop improvement. Nature Genetics, 2021, 53(9): 1385-1391"; Arabidopsis thaliana is the Columbia wild type Arabidopsis thaliana, and tobacco is Nicotiana benthamiana, which was previously preserved in the laboratory.

[0027] Example 1 The anthocyanin content in thrips-resistant cotton varieties is significantly lower than that in thrips-susceptible varieties

[0028] 1. Non-target metabolic analysis of thrips-resistant cotton varieties:

[0029] The applicant conducted non-target metabolic analysis of 5 biological replicates on the leaves of the thrips-resistant variety Wu 374 and the thrips-susceptible variety Zaoyang Bu Luolei. Through enrichment analysis of the measured metabolites, flavonoids and their downstream anthocyanin pathways were significantly enriched, and the 24 metabolites of this pathway in the thrips-resistant varieties were significantly lower than those in the thrips-susceptible varieties. In order to further verify the non-target metabolic results, the flavonoid and anthocyanin contents of multiple resistant and susceptible varieties and resistant and susceptible individual plants of the F2 segregation population constructed from resistant and susceptible parents were determined. The results were consistent with the non-target metabolic results, that is, the flavonoid and anthocyanin contents of the thrips-resistant materials were significantly lower than those of the thrips-susceptible materials ( Figure 1 shown).

[0030] 2. Thrips prefer to feed on leaves with high anthocyanin content:

[0031] Due to the inevitable relationship between pigment glands and gossypol, the content of gossypol in multiple resistant and susceptible varieties and F2 populations was further detected. Almost no gossypol was detected in thrips-resistant varieties or strains. It is reported that flavonoids, anthocyanins and gossypol are involved in the interaction between plants and insects. In order to further confirm the relationship between these three metabolites and thrips resistance, the cotyledons of Zhongwu 374 were injected with three metabolites and sterile water (as a control) through the lower epidermis using a needle-free syringe, and the cotyledons injected with different metabolites were selectively fed by thrips. The results showed that thrips were more inclined to feed on cotyledons injected with anthocyanins, while the cotyledons injected with gossypol and flavonoids were not much different from the control. In order to exclude the influence of cotton genetic background, a selective feeding experiment was further carried out with freeze-dried green cabbage soaked with different metabolites. The results showed that anthocyanins, as important secondary metabolites of plants, can attract thrips to feed, but have little to do with gossypol and flavonoids. As we all know, purple cabbage has a much higher content of anthocyanins than green cabbage. In order to further verify that anthocyanins are the main factor in plant resistance to thrips, fresh leaves of purple cabbage and green cabbage were prepared. The thrips feeding experiment showed that thrips prefer to feed on purple cabbage ( Figure 2 Taken together, these results suggest that anthocyanins are major negative regulators of plant thrips resistance, whereas no linear relationship exists between thrips resistance and gossypol and flavonoids.

[0032] Example 2 GhMYC2 negatively regulates thrips resistance

[0033] 1. Knockout of GhMYC2 enhances cotton thrips resistance:

[0034] The CRISPR / Cas9 gene-edited mutant GhMYC2 strain with a nucleotide sequence inserted in both the At and Dt subgenomes was obtained by Agrobacterium-mediated genetic transformation screening. The anthocyanin content of WT and GhMYC2 was first determined. Compared with WT, the anthocyanin content of GhMYC2 was significantly reduced. The results of thrips resistance identification showed that the thrips resistance of GhMYC2 was significantly improved ( Figure 3 shown).

[0035] 2. Overexpression of GhMYC2 reduces resistance to thrips in cotton:

[0036] Similarly, an overexpression vector driven by the Ubi promoter to express GhMYC2 was constructed for genetic transformation of cotton. After obtaining positive calli, it was found that the anthocyanin content of the positive calli was significantly increased. Unfortunately, due to the excessive accumulation of secondary metabolites such as anthocyanins in the positive calli, the callus tissue could not further differentiate into seedlings ( Figure 4As shown). In view of this, GhMYC2 was further transformed into Arabidopsis by the floral dipping method, and the obtained T3 generation pure Arabidopsis was identified by WB using HA tag antibody. Three strains with effective protein expression were selected for anthocyanin content determination. Compared with WT Arabidopsis, the anthocyanin content of the three overexpression strains was significantly increased, and the thrips resistance was significantly reduced ( Figure 5 shown).

[0037] Therefore, the present invention adopts the above-mentioned cotton anti-thrips transcription factor GhMYC2 gene and its application, and knocks out the GhMYC2 gene through CRISPR / Cas9 to obtain thrips-resistant cotton plants, laying the foundation for the cultivation of new cotton thrips-resistant varieties.

[0038] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A cotton anti-thrips transcription factor GhMYC2 gene, characterized in that: The nucleotide sequence of the GhMYC2 gene is shown in SEQ ID NO.

1.

2. Cotton anti-thrips GhMYC2 protein, characterized in that: The gene encoding the GhMYC2 protein is the GhMYC2 gene shown in SEQ ID NO.1, and the amino acid sequence of the GhMYC2 protein is shown in SEQ ID NO.

2.

3. Use of the cotton anti-thrips transcription factor GhMYC2 gene as claimed in claim 1 in regulating cotton resistance to thrips.

4. The use according to claim 3, characterized in that: Knocking out the GhMYC2 gene through CRISPR / Cas9 improves cotton's resistance to thrips.

5. The use according to claim 4, characterized in that: After knocking out the GhMYC2 gene through CRISPR / Cas9, the anthocyanin content of the plant decreased.

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