Papaya CpWRKY57 gene and application thereof

Abstract

The invention belongs to the technical field of bioengineering, particularly relates to a papaya CpWRKY57 gene, and further discloses application of the CpWRKY57 gene in the fields of improving papaya anthracnose resistance and promoting plant dwarfing. According to the scheme, the CpWRKY57 gene is separated from papaya leaves for the first time, a CpWRKY57 instantaneous overexpression vector and a CpWRKY57 stable overexpression vector are constructed, postharvest papaya fruits and tomato explants are instantaneously converted respectively, instantaneous overexpression of the CpWRKY57 gene in the papaya fruits and heterologous overexpression of the CpWRKY57 gene in the tomatoes are achieved, and the CpWRKY57 gene can be applied to the field of genetic engineering. According to the invention, the CpWRKY57 gene is used as a marker, disease-resistant phenotype analysis is carried out through needling inoculation of colletotrichum gloeosporioides, the effect of the CpWRKY57 gene in the process of resisting anthracnose of papaya and the function of promoting plant dwarfing are defined, and a new gene resource and a theoretical basis are provided for disease-resistant and dwarfing breeding of papaya.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a papaya CpWRKY57 gene, and further disclosing the application of the CpWRKY57 gene in improving papaya anthracnose resistance and promoting plant dwarfing. Background Art

[0002] Papaya (Carica papaya L.), also known as papaya, milk melon, or longevity fruit, belongs to the genus Carica in the family Caricaceae. Papaya is one of the most widely cultivated and consumed tropical fruits in the world, and a specialty fruit of Lingnan in my country. It has high nutritional and medicinal value, and is listed by the World Health Organization as the most valuable of the ten fruits, earning it the reputation of "King of Fruits with a Hundred Benefits." Papaya not only has a short growing cycle and high yield, but it can also be used as a fruit and vegetable, resulting in less sales pressure and good economic benefits. Therefore, papaya plays an important role in supporting the structural reform of agricultural supply, especially in recent years with the continuous increase in demand for papaya, indicating a broad prospect for industrial development.

[0003] However, papaya fruit is extremely prone to rotting after harvesting, primarily due to anthracnose. Statistics show that anthracnose can cause 25-50% loss of marketable fruit, significantly impacting the industry. Currently, papaya production relies mainly on fungicides, essential oils, and heat treatment to treat harvested fruit. However, these methods often lead to increased pathogen resistance, poor fruit quality, and environmental pollution. More importantly, anthracnose bacteria typically infect the fruit before ripening in the field, making post-harvest control merely a remedial measure, increasing production costs without addressing the root cause. Furthermore, papaya grows rapidly; most cultivated varieties reach 2-3 meters in height within a year of planting, with perennial varieties reaching tens of meters. In many major growing areas, frequent windstorms and easy lodging, coupled with high temperature and humidity, lead to frequent outbreaks of anthracnose and other pests and diseases, making daily management extremely difficult. Therefore, in papaya cultivation and production, it is necessary to control the plant height to ensure the convenience of daily management.

[0004] Currently, the main method in production is to reduce apical dominance controlled by auxin by using oblique pulling to minimize plant height. However, this method is not only time-consuming and labor-intensive, but also has limited effect on height control and cannot solve the fundamental problem. Clearly, the ideal approach is to screen for disease-resistant and dwarfing genes in papaya, analyze their mechanisms of action, and then use molecular breeding to create new disease-resistant and dwarfing germplasm to fundamentally solve the problem. However, compared to other horticultural crops, there are few reports on gene mining, mechanism research, and molecular breeding studies related to anthracnose and plant height in papaya.

[0005] The rapid activation of the plant immune system is accompanied by the inhibition of plant growth and development; this process is known as the "growth-defense balance." Numerous studies have shown that WRKY plays a crucial role in regulating this balance in model plants. WRKY is one of the largest transcription factor families in plants, characterized by one or two WRKY domains (conserved motif WRKYGQK) at the N-terminus and a zinc finger-like domain at the C-terminus. These domains regulate the expression of stress-responsive and growth-related genes by binding to W-box(C / T)TGAC(C / T) within the promoter regions of these genes, thereby achieving dual regulation of plant defense and growth.

[0006] However, research on the function of WRKY transcription factors in horticultural crops has largely focused on their roles in individual physiological processes such as growth, development, or defense responses. For example, apple MdWRKY9 mediates dwarfing by directly inhibiting the transcription of the brassinolide (BR) rate-limiting synthase gene MdDWF4, thereby reducing BR production; tomato WRKY-B regulates lateral branching by targeting and binding to the branching gene BLIND (BL), the auxin export vector gene PIN4, and the AUX / IAA gene IAA15, thereby regulating the expression of these genes. Similarly, WRKY transcription factors also participate in the defense responses of horticultural crops, and, like in model plants, mostly function by mediating the JA, SA, and ET signaling pathways. For example, VviWRKY10 and VviWRKY30 play a role in regulating the defense response of grapes against powdery mildew by transcriptionally regulating different genes involved in the SA, ET, and ROS pathways; VvWRKY5 enhances grape resistance to white rot by regulating JA biosynthesis and signal transduction pathways; papaya CpWRKY50 enhances resistance to anthracnose fungus C. brevisporum by promoting the JA signaling pathway; and sugarcane ScWRKY4 negatively regulates the defense response against pathogens through JA signaling. However, studies on the dual role of WRKY genes in the growth, development, and defense response of horticultural crops are rarely reported. Summary of the Invention

[0007] Therefore, the technical problem to be solved by this invention is to provide a papaya CpWRKY57 gene, and further verify through a transient overexpression system that transient overexpression of CpWRKY57 in post-harvest papaya fruit can significantly enhance the anthracnose resistance of papaya; at the same time, using transgenic technology, CpWRKY57 is heterologously overexpressed in tomato to further verify the function of the CpWRKY57 gene in resisting anthracnose, as well as its role in regulating plant height;

[0008] The second technical problem to be solved by this invention is to provide the application of the above-mentioned papaya CpWRKY57 gene in the field of papaya breeding and improvement, especially in improving the anthracnose resistance of papaya and promoting plant dwarfing.

[0009] To address the aforementioned technical problems, the present invention provides a papaya CpWRKY57 gene, wherein the CDS region of the CpWRKY57 gene includes the nucleotide sequence shown in SEQ ID No. 1.

[0010] The present invention also provides a papaya CpWRKY57 protein encoded by the papaya CpWRKY57 gene, wherein the CpWRKY57 protein comprises the amino acid sequence shown in SEQ ID No. 2.

[0011] The present invention also provides an expression vector containing the papaya CpWRKY57 gene, the expression vector comprising the transient overexpression vector pRI101-AN-CpWRKY57, and / or the stable overexpression vector Super1300-CpWRKY57.

[0012] The present invention also provides a method for constructing the expression vector, comprising the following steps:

[0013] (1) Cloning of CpWRKY57 gene: Total RNA was extracted from papaya leaves and reversed into cDNA. Using this as a template, specific primers were designed for PCR amplification, and the product was recovered to obtain the CDS sequence of CpWRKY57 gene.

[0014] (2) Vector construction: The recovered product and the pRI101-AN vector were digested with XbaⅠ and KpnⅠ enzymes respectively, and then ligated to construct the pRI101-AN-CpWRKY57 transient overexpression vector; or, the recovered product and the Super1300-GFP vector were digested with XbaⅠ and KpnⅠ enzymes respectively, and then ligated to construct the Super1300-CpWRKY57 stable overexpression vector.

[0015] Specifically, this invention provides a transient overexpression vector pRI101-AN-CpWRKY57 for the CpWRKY57 gene. By transiently transforming post-harvest papaya fruit, anthracnose spores were inoculated at the CpWRKY57 overexpression sites to study the effect of CpWRKY57 overexpression on anthracnose resistance in papaya. First, total RNA was extracted from papaya leaves and reverse transcribed into cDNA. Using this cDNA as a template, specific primers were designed for PCR amplification. The amplified product was recovered via gel electrophoresis. The recovered product and the pRI101-AN vector were digested with XbaI and KpnI enzymes, respectively, and then ligated to construct the pRI101-AN-CpWRKY57 transient overexpression vector.

[0016] Specifically, this invention provides a stable overexpression vector for the CpWRKY57 gene, Super1300-CpWRKY57. Using Agrobacterium-mediated transformation with tomato explants as recipients, genetic transformation was performed. Stable CpWRKY57 overexpressing tomato lines were obtained through molecular identification. Anthracnose was then inoculated onto the fruits of the transgenic tomato lines to study their biological functions. First, total RNA was extracted from papaya leaves and reverse transcribed into cDNA. Using this cDNA as a template, specific primers were designed for PCR amplification. The amplified product was recovered via gel electrophoresis. The recovered product and the Super1300 vector were digested with XbaI and KpnI enzymes, respectively, and then ligated to construct the Super1300-CpWRKY57 overexpression vector.

[0017] The present invention also provides a cell line containing the papaya CpWRKY57 gene or the expression vector.

[0018] The present invention also provides a recombinant bacterium containing the papaya CpWRKY57 gene or the expression vector.

[0019] The present invention also discloses a method for constructing the recombinant bacteria, including the step of using the expression vector host strain to enable the vector to be effectively expressed in the host.

[0020] The present invention also provides a stable crop line obtained by stable genetic expression of the recombinant bacteria.

[0021] Specifically, the crops include papaya plants or tomato plants.

[0022] This invention also provides the application of the aforementioned papaya CpWRKY57 gene in the field of papaya breeding and improvement.

[0023] The present invention also provides the use of the papaya CpWRKY57 gene as a papaya anthracnose resistance regulatory gene and / or dwarfing regulatory gene.

[0024] The present invention also provides a method for cultivating papaya crops that combine high resistance to anthracnose and dwarfing, including the step of transforming the papaya CpWRKY57 gene into the target plant.

[0025] This invention also investigated the transient overexpression and heterologous expression functions of the papaya CpWRKY57 gene, with the specific steps as follows:

[0026] (1) Cloning of the CpWRKY57 gene: Total RNA was extracted from papaya leaves, reversed into cDNA, and specific primers were designed to clone the CDS sequence of the CpWRKY57 gene.

[0027] (2) Vector construction: The transient overexpression vector pRI101-AN-CpWRKY57 of CpWRKY57 was constructed, and the stable overexpression vector Super1300-CpWRKY57 was constructed at the same time.

[0028] (3) Preparation of engineered bacteria: The constructed pRI101-AN-CpWRKY57 and Super1300-CpWRKY57 were transformed into Agrobacterium EHA105 to prepare engineered bacteria;

[0029] (4) Transient overexpression and phenotypic analysis of CpWRKY57: Agrobacterium containing the CpWRKY57 overexpression vector pRI101-AN-CpWRKY57 was injected into post-harvest papaya fruit. After 3 days, the expression of CpWRKY57 was detected. It was found that the expression level of CpWRKY57 was significantly increased. Then, anthracnose suspension spores were inoculated into the overexpression site by needle puncture. After 3 days, there were no obvious symptoms in the overexpression site, while the control site with empty vector had obvious disease and rot. The statistical results of the number and size of lesions were consistent with the phenotype, verifying the function of CpWRKY57.

[0030] (5) Genetic transformation and phenotypic analysis of tomato (CpWRKY57 stable overexpression vector): The prepared Super1300-CpWRKY57 engineered bacteria were genetically transformed using tomato explants, and stable CpWRKY57 overexpression lines were obtained by molecular identification. Anthracnose suspension spores were inoculated onto the fruits of wild type and the three overexpression lines by needle prick. Two days later, the overexpression fruits showed no obvious symptoms, while the wild type showed obvious anthracnose lesions, further verifying the function of CpWRKY57 in enhancing the anthracnose resistance of papaya. At the same time, other agronomic traits were observed, and it was found that the CpWRKY57 overexpression tomato plants showed a significant dwarfing phenotype, with a plant height of about half that of wild type tomatoes, that is, the expression of CpWRKY57 promoted the dwarfing of plants.

[0031] This invention is based on a preliminary study of the response of papaya WRKY family genes to anthracnose infection. Surprisingly, the CpWRKY57 gene was discovered, exhibiting high induced expression levels in resistant varieties and essentially unchanged expression levels in susceptible varieties. Further in-depth research on this gene led to the first cloning of the papaya CpWRKY57 gene. Using a transient overexpression system, CpWRKY57 was transiently overexpressed in postharvest papaya fruit, further validating its ability to significantly enhance papaya's anthracnose resistance. Simultaneously, using transgenic technology, CpWRKY57 was heterologously overexpressed in tomato. This further validated the anthracnose resistance function of the CpWRKY57 gene and revealed that overexpression of the CpWRKY57 gene can also reduce plant height and promote dwarfing. The present invention effectively overcomes the current situation where there is limited research on the WRKY gene family in papaya, which is suffering from anthracnose. The papaya CpWRKY57 gene can be used as a target gene for in-depth research on anthracnose resistance improvement and plant dwarfing in papaya, and has high application and research value.

[0032] This invention is the first to isolate the CpWRKY57 gene from papaya leaves. By constructing a transient overexpression vector and a stable overexpression vector of CpWRKY57, and transiently transforming postharvest papaya fruits and tomato explants respectively, the invention achieved transient overexpression of the CpWRKY57 gene in papaya fruits and heterologous overexpression in tomatoes. Furthermore, by needle-sting inoculation with anthracnose, the disease resistance phenotype was analyzed, clarifying the role of the CpWRKY57 gene in papaya anthracnose resistance and its function in promoting plant dwarfing. This provides new gene resources and theoretical basis for disease resistance and dwarfing breeding of papaya.

[0033] This invention isolates and obtains the CDS sequence of the papaya CpWRKY57 gene. Using Agrobacterium-mediated transformation, the CpWRKY57 gene is successfully transiently overexpressed in postharvest papaya fruit. Simultaneously, the gene is overexpressed in tomato, resulting in a stable CpWRKY57-overexpressing tomato transgenic line. Anthracnose inoculation was then performed using both materials. Phenotypic analysis revealed that CpWRKY57 gene overexpression significantly enhances the resistance of papaya and tomato fruits to anthracnose and reduces plant height. This invention provides a new gene resource for disease resistance and dwarfing breeding of papaya.

[0034] The construction method described in this invention utilizes a simple and effective transient gene expression technique, which can effectively shorten the time required to study gene function. Furthermore, heterologous gene expression can play an important role in the study of gene function in species where transgenic transformation is not easily achieved, and is of great significance for revealing the function of papaya genes and molecular breeding. Attached Figure Description

[0035] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0036] Figure 1 The image shows the cloning of the CpWRKY57 gene and the structure of its encoded protein. A: Schematic diagram of the CpWRKY57 protein structure; B: Expression patterns of CpWRKY57 in different tissues; C: Subcellular localization of the CpWRKY57 protein; D: Homology comparison and domain analysis of CpWRKY57 with WRKY57 proteins from six other species; the green box represents the heptapeptide (WRKYGQK) domain, and the orange box represents the C2H2 zinc finger domain.

[0037] Figure 2 The results of colony PCR identification of the CpWRKY57 transient overexpression vector pRI101-CpWRKY57;

[0038] Figure 3 This study presents the phenotypic analysis of CpWRKY57 transient overexpression and the changes in CpWRKY57 expression levels. A and C represent the phenotypes of fruits inoculated with CpWRKY57 transient overexpression 0 days after inoculation (3 days after pRI101-CpWRKY57 transient transformation) and 3 days after inoculation (6 days after pRI101-CpWRKY57 transient transformation). Each fruit underwent two treatments: an empty vector control and pRI101-CpWRKY57 (CpWRKY57 overexpression) infection. The three middle wells in each treatment were injection points for either the empty vector control or the CpWRKY57 overexpression transformation vector, while the six outer wells were inoculation points for anthracnose spores. B and D represent the changes in CpWRKY57 expression levels after 3 days of transient transformation with the CpWRKY57 overexpression vector and empty vector. EF represents the statistical results of the number of bacterial colonies (E) and the maximum colony diameter (F) after inoculation with fruits inoculated with the CpWRKY57 overexpression vector and empty vector.

[0039] Figure 4 The colony PCR identification results of CpWRKY57 overexpression vector Super1300-CpWRKY57;

[0040] Figure 5This study aimed to identify tomato plants stably overexpressing CpWRKY57 and analyze their phenotypic results after inoculation with anthracnose. A: RT-PCR detection of CpWRKY57 expression in eight overexpression lines; M: D2000. B: Phenotypic analysis of non-transgenic tomatoes and CpWRKY57 overexpressing OE1, OE2, and OE3 transgenic tomatoes after needle prick inoculation. For each genotype, the left side of the tomato fruit was inoculated with ddH2O as a control, and the right side with anthracnose spores. White arrows indicate anthracnose lesions; Bar = 5 mm. C: Statistical results of the maximum lesion diameter and area after inoculation in overexpressing and wild-type tomatoes.

[0041] Figure 6 Phenotypic analysis of plant height in tomatoes stably overexpressing CpWRKY57; AC: comparison of plant height in wild-type and CpWRKY57 overexpressing OE1, OE2 and OE3 transgenic tomatoes during the vegetative growth stage (A), flowering stage (B) and fruiting stage (C), Bar = 1 cm; D: statistical results of plant height in wild-type and CpWRKY57 overexpressing OE1, OE2 and OE3 transgenic tomatoes during the vegetative growth stage. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0043] The materials involved in the following embodiments of the present invention include:

[0044] The papaya experimental material used conventional planting and field management, while the tomato material used was Micro-TOM.

[0045] The primers and sequencing used in the experiment were completed by Sangon Biotech (Shanghai) Co., Ltd.

[0046] XbaⅠ and KpnⅠ were acquired from NEB Company;

[0047] High-fidelity enzymes and T4 DNA ligase were purchased from TaKaRa Engineering Co., Ltd.

[0048] Escherichia coli competent cells DH5α and the EasyGenoDNA recombination system were purchased from Tiangen Biotech (Beijing) Co., Ltd.

[0049] The remaining molecular reagents were mainly purchased from Tiangen Biotech (Beijing) Co., Ltd. and Sangon Biotech (Shanghai) Co., Ltd.

[0050] Agrobacterium competent cells EHA105 were purchased from Beijing Huayueyang Biotechnology Co., Ltd.

[0051] In the following embodiments of the present invention, the cloning, identification, and real-time quantitative PCR (RT-qPCR) primer information used in the experiments is shown in Table 1 below.

[0052] Table 1 Primers used in the experiment for cloning, identification, and real-time quantitative PCR (RT-qPCR)

[0053] Primer names Primer sequence CpWRKY57-F 5'-ATGGACGACAGGGAGAGACCC-3' CpWRKY57-R 5'-TCAAATGTTTCGCATCCCAGG-3' CpWRKY57-F1 5'-GCTCTAGAATGGACGACAGGGAGAGA-3' CpWRKY57-R1 5'-GGGGTACCTCAAATGTTTCGCATCCC-3' qCpEIF4A-F 5'-AGGCAGGCAAGAGAAGAT-3' qCpEIF4A-R 5'-TTCATACCGAGTAGCGATTC-3' qCpWRKY57-F 5'-AGAGTGAGGTGGATCACTTAGA-3' qCpWRKY57-R 5'-GTAGCTCCTAGGGAATGGACTA-3'

[0054] Example 1

[0055] This embodiment focuses on the cloning and bioinformatics analysis of the CDS region of the CpWRKY57 gene.

[0056] Based on the papaya CpWRKY57 sequence from the NCBI website, primers CpWRKY57-F / CpWRKY57-R were designed using Primer Premier 5.0 (see Table 1). Total RNA was extracted from papaya leaves using the Trizol method, and genomic DNA contamination was removed from the total RNA using DNase I (Takara, Japan). PrimeScripffMII 1 was then used to further analyze the RNA. st The Strand cDNA Synthesis Kit (Takara, Japan) reverse transcribes extracted total RNA into single-stranded cDNA.

[0057] Using cDNA as a template and CpWRKY57-F / CpWRKY57-R as primers, the coding sequence CDS was cloned. The PCR product was subjected to 1.5% agarose gel electrophoresis. The product was recovered and linked to pMD19-T cells, which were then transformed into DH5α competent cells. Single colonies were selected for colony PCR. Positive bacteria were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing to obtain the CDS sequence of CpWRKY57, which was 942 bp in size. The CDS sequence of the CpWRKY57 gene was determined, as detailed in SEQ ID No. 1.

[0058] SEQ ID No.1 (CpWRKY57 CDS sequence):

[0059] ATGGACGACAGGGAGAGACCCGATCCAGGATCCAAGTACACCGCCGATTCCAGCTGGGGACTCGGGCTAACGGATTCCGACTCAGTCGACTACTTCATCGACTGCAGAGAGAGCACCATACTCGGCGAATTCGGTTGGAATCTGCACTTCGATGAGGAGAGCCGGTTCGCTGACCTTGACCGGATTGCAGTCGGCAGCAGATCCGATTCGGCGGCAAATCTCCATAGCAGCTTGTCGGAGAGTACTTGTGGCGATGGACCCTTGCTGCGGAGTTCTAGTAATGTCCCCTCTGATCCGAGCGGGTCTTGTTCTACCCCAGTTGCTGCCCTTGCTGACGTGTCTACCTCAAATCCTTCGGTATCTTCCAGCTCCAGCGAGGATCCGCCCGAGAAATCGACGGGCTCCGGCGGGAAACCGCCCGAGATACCGAATAAAGTCAGGAAGAAGGGACAAAAGCGAGTCCGGCAACCTCGTTTTGCATTTATGACTAAGAGTGAGGTGGATCACTTAGAGGATGGCTACCGATGGCGGAAATACGGACAAAAGGCTGTTAAAAATAGTCCATTCCCTAGGAGCTACTATCGCTGCACAAATAGCAAATGCAGTGTGAAGAAAAGGGTGGAACGCTCCTCTGAAGATCCAACAATCGTAATAACTACATACGAAGGCCAACACTGTCATCATACCGCCGGATTCCCTCGCAGTGGTATCATTACCCATGAGGCTAGCTATGCCGGAAGGCTAATGCCCGCAATTTCCCAATTTTGTTATCCCGGAATTCAATTGTCTCGGGACGCTCCACCTACTCAAAACTTGTCACGGTTGCACAGATTAGCTGGTGAAGCAGGAGAACCCCATATGCACTCGGAAACTTTCCCGCAGTTTCCAATTGATGAGGGGTTGCTAGGAGATATTGTGCCACCTGGGATGCGAAACATTTGA。

[0060] Example 2

[0061] This embodiment performs bioinformatics analysis on the CDS region of the CpWRKY57 gene.

[0062] The amino acid sequence of the CpWRKY57 protein encoded by the CpWRKY57 gene is shown in SEQ ID No. 2.

[0063] SEQ ID No.2 (CpWRKY57 protein):

[0064] MDDRERPDPGSKYTADSSWGLGLTDSDSVDYFIDCRESTILGEFGWNLHFDEESRFADLDRIAVGSRSDSAANLHSSLSESTCGDGPLLRSSSNVPSDPSGSCSTPVAALADVSTSNPSVSSSSSEDPPEKSTGSGGKPPEIPNKVRKKGQKRVRQP RFAFMTKSEVDHLEDGYRWRKYGQKAVKNSPFPRSYYRCTNSKCSVKKRVERSSEDPTIVITTYEGQHCHHTAGFPRSGIITHEASYAGRLMPAISQFCYPGIQLSRDAPPTQNLSRLHRLAGEAGEPHMHSETFPQFPIDEGLLGDIVPPGMRNI.

[0065] The conserved domains of CpWRKY57 and the physicochemical properties of the protein were analyzed using ExPaSy (http: / / web.expasy.org / protparam / ). For example... Figure 1 Figure A shows a schematic diagram of the CpWRKY57 protein structure. The CpWRKY57 gene encodes 313 amino acids. The theoretical molecular weight of CpWRKY57 is 34341.99, the theoretical isoelectric point is 6.21, and the instability index is 58.55. This protein contains 41 acidic amino acid residues (Asp+Glu) and 37 basic amino acid residues (Lys+Arg), with an aliphatic index of 57.03 and an average hydrophilicity (GRAVY) of -0.739, indicating that the CpWRKY57 protein is a hydrophilic and unstable protein.

[0066] According to protein structure prediction from the SMART website (http: / / smart.embl.de / smart / set_mode.cgi?NORMAL=1), CpWRKY57 contains a typical WRKY domain (containing a conserved heptapeptide WRKYGQK domain and a C2H2 zinc finger structure), located at positions 170-229 of the amino acid sequence, belonging to the class IIC WRKY family transcription factor (see...). Figure 1 (B)

[0067] Using the BLASTP tool in NCBI to search for homologous amino acid sequences of the CpWRKY57 protein, it was found that CpWRKY57 has high homology with WRKY57 of Arabidopsis thaliana (NP 001321561.1), Alnus glutinosa (XP062162194.1), Gossypium hirsutum (XP 016703893.1), Heveabrasiliensis (XP 021659547.2), Hibiscus syriacus (XP 039047189.1), and Populus alba (XP 034907193.1). Multiple sequence alignment revealed (see...) Figure 1 In the C), the WRKY domains of these proteins are highly conserved, with the (WRKYGQK) conserved domain having completely identical amino acid sequences. The C2H2 type zinc finger structure also shows only a few amino acid differences, indicating that the CpWRKY57 protein has high conservation in different plants.

[0068] Example 3

[0069] In this embodiment, a transient expression vector of CpWRKY57 was constructed and phenotypic analysis was performed.

[0070] Construction of CpWRKY57 transient overexpression vector

[0071] Based on the aforementioned CpWRKY57 coding sequence, a forward primer CpWRKY57-F1 containing an Xba I restriction site and a reverse primer CpWRKY57-R1 containing a KpnI restriction site were designed (as shown in Table 1). cDNA was used as a template for amplification. The PCR reaction program was: 94℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 56℃ annealing for 5 s, 72℃ extension for 10 s, 72℃ extension for 5 min, 30 cycles. After gel extraction, the recovered product and the transient overexpression vector pRI101 were digested with restriction endonucleases Xba I and KpnI (TaKaRa), respectively, and then ligated. The mixture was then transformed into DH5α competent cells, and single colonies were picked for colony PCR using CpWRKY57-F1 / CpWRKY57-R1 (as shown in Table 1). Figure 2 As shown in the figure, the positive colonies were shaken and sent to the company for sequencing. After the sequencing was correct, the plasmid was extracted, enzyme digestion and identification were performed to obtain the transient overexpression vector pRI101-CpWRKY57.

[0072] Molecular identification and phenotypic analysis of CpWRKY57 transient overexpression

[0073] Plasmids containing the CpWRKY57 overexpression vector (pRI101-CpWRKY57) and the empty vector (pRI101) were transformed into Agrobacterium EHA105, and colony PCR was performed for identification. Positive single colonies were selected to prepare engineered bacteria. The engineered bacteria containing the CpWRKY57 overexpression vector (pRI101-CpWRKY57) and the empty vector (pRI101) were injected into postharvest papaya fruits, respectively. Fluorescent real-time quantitative primers qCpWRKY57-F / qCpWRKY57-R were designed (as shown in Table 1). After 3 days, the expression of CpWRKY57 was detected by RT-qPCR.

[0074] The specific steps for RT-qPCR were as follows: Using the SYBR Green Pro TaqHS Premix (TaKaRa) kit, cDNA samples from fruits injected with overexpressed and empty vectors were used as templates, respectively. Eukaryotic translation initiation factor 4A (EIF4A) was used as an internal reference gene. RT-qPCR detection was performed using an Applied Biosystems 7500 real-time quantitative PCR instrument (primers are shown in Table 1: qCpEIF4A-F and qCpEIF4A-R). The reaction system consisted of: 10 μL of 2×SYBR Green Pro TaqHS Premix, 0.4 μL each of forward and reverse primers, 2 μL of cDNA template, 0.4 μL of 50×ROX Reference Dye, and 6.8 μL of ddH2O, for a total volume of 20 μL. The two-step amplification program was: 95℃ for 30 s; 95℃ for 5 s, 56℃ for 30 s, for 40 cycles. The standard melting curve program was: 95℃ for 15 s; 60℃ for 1 min; 95℃ for 15 s. The experiment was repeated 3 times, using 2 -ΔΔCt The method calculates the relative expression levels of genes.

[0075] The results showed that the expression level of CpWRKY57 was significantly increased in the fruit parts injected with the overexpression vector pRI101-CpWRKY57 (see...). Figure 3 (A) Then, anthrax suspension spores were inoculated into the empty vector and overexpression sites by needle puncture, and the phenotypes were observed. It was found that after 3 days, the overexpression treatment sites showed no obvious anthrax symptoms compared with before inoculation, while the empty vector control sites had obvious disease and rotting (see A). Figure 3 (B)

[0076] Example 4

[0077] This embodiment constructs a CpWRKY57 heterologous overexpression vector and performs phenotypic analysis.

[0078] Construction of CpWRKY57 heterologous overexpression vector

[0079] Based on the aforementioned CpWRKY57 coding sequence, PCR amplification was performed using cDNA as a template, employing the forward primer CpWRKY57-F1 containing an XbaI restriction site and the reverse primer CpWRKY57-R1 containing a KpnI restriction site (as shown in Table 1). The PCR reaction program was as follows: 94℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 56℃ annealing for 5 s, 72℃ extension for 10 s, 72℃ extension for 5 min, for 30 cycles. After gel extraction, the PCR product and the overexpression empty vector Super1300 were double-digested with XbaI and KpnI (TaKaRa), then recovered, ligated, transformed into DH5α competent cells, and single colonies were picked. Colony PCR identification was performed using the specific primers CpWRKY57-F1 / CpWRKY57-R1 (see Table 1). Figure 4 Positive bacterial colonies were shaken and sent to the company for sequencing. After the sequencing was confirmed to be correct, plasmids were extracted, enzyme digestion and identification were performed to obtain the recombinant overexpression vector Super1300-CpWRKY57.

[0080] Molecular identification of CpWRKY57 heterologous overexpression lines

[0081] Remove the leaf tips and petioles from the true leaves of Micro-Tom tomatoes, and cut the remaining parts into 0.5cm × 0.5cm leaf pieces. Place 15-20 pieces horizontally on pre-culture medium and pre-culture for 1 day. Simultaneously, transform the correctly sequenced Super1300-CpWRKY57 plasmid into Agrobacterium EHA105. Perform colony PCR identification using specific primers CpWRKY57-F / CpWRKY57-R. Select positive colonies and culture overnight, adjusting the OD of the bacterial culture. 600 =0.5.

[0082] Explants were removed and transformed in Agrobacterium for 30 minutes. The explants were then blotted dry on sterile paper and reintroduced into the pre-culture medium for 1-2 days. Afterward, the co-cultured explants were transferred to a selection medium for screening to obtain resistant callus and adventitious shoots. Callus with shoot primordia was cut into small pieces and transferred to stem elongation medium for subculture. When the adventitious shoots reached approximately 1 cm in length, healthy regenerated shoots were selected and transferred to rooting medium for further culture to obtain resistant regenerated plants.

[0083] RNA was extracted from five resistant regenerated plants and untransformed wild-type tomato plants, and reverse transcribed into cDNA. Amplification was performed using primers CpWRKY57-F / CpWRKY57-R, with the wild-type plant serving as a control. RT-PCR was used to detect CpWRKY57 expression in the five resistant regenerated plants. Plants that amplified the target fragment band were considered stable heterologous overexpression plants of CpWRKY57. Results are as follows: Figure 5As shown in Figure A, the target band could be detected in all five strains, with stronger expression levels in OE1 and OE3.

[0084] Phenotypic analysis of CpWRKY57 heterologous overexpression lines

[0085] Preparation concentration of 10 6 Anthracnose suspension spores at a concentration of / L were inoculated onto fruits of wild-type and three overexpression tomato lines (OE1, OE2, and OE3) using the needle-pricking method. Simultaneously, ddH2O was inoculated as a control. Phenotypic results were observed. Figure 5 As shown in Figures B and C, two days after inoculation, the overexpressing fruit showed no obvious anthracnose symptoms compared to before inoculation, while the control showed obvious anthracnose lesions. This demonstrates that the method of improving anthracnose resistance in papaya based on the CpWRKY57 gene of this invention can significantly improve the anthracnose resistance of papaya fruit.

[0086] In addition to the disease resistance phenotype, OE1, OE2, and OE3 tomato plants also exhibited a significant dwarfing phenotype. Figure 6 (A, B, and C) The plant height is about half that of the wild type. Figure 6 (D). The results in summary demonstrate that the method of improving anthracnose resistance and plant height of papaya based on the CpWRKY57 gene of papaya can significantly improve the anthracnose resistance of papaya fruit and may also achieve the goal of dwarfing the plant, thus having high application and research value.

[0087] The embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A papaya CpWRKY57 gene, characterized in that, The CDS region of the CpWRKY57 gene includes the nucleotide sequence shown in SEQ ID No.

1.

2. A papaya CpWRKY57 protein encoded by the papaya CpWRKY57 gene as described in claim 1, characterized in that, The CpWRKY57 protein comprises the amino acid sequence shown in SEQ ID No.

2.

3. An expression vector containing the papaya CpWRKY57 gene as described in claim 1, characterized in that, The expression vectors include the transient overexpression vector pRI101-AN-CpWRKY57 and / or the stable overexpression vector Super1300-CpWRKY57.

4. A method for constructing the expression vector of claim 3, characterized in that, The steps include: (1) Cloning of CpWRKY57 gene: Total RNA was extracted from papaya leaves and reversed into cDNA. Using this as a template, specific primers were designed for PCR amplification, and the product was recovered to obtain the CDS sequence of CpWRKY57 gene. (2) Vector construction: The recovered product and the pRI101-AN vector were digested with XbaⅠ and KpnⅠ enzymes respectively, and then ligated to construct the pRI101-AN-CpWRKY57 transient overexpression vector; or, the recovered product and the Super1300-GFP vector were digested with XbaⅠ and KpnⅠ enzymes respectively, and then ligated to construct the Super1300-CpWRKY57 stable overexpression vector.

5. A cell line containing the papaya CpWRKY57 gene of claim 1 or the expression vector of claim 3.

6. A recombinant bacterium containing the papaya CpWRKY57 gene of claim 1 or the expression vector of claim 3.

7. A stable crop line obtained by stable genetic expression of the recombinant bacteria of claim 6.

8. The application of the papaya CpWRKY57 gene as described in claim 1 in the field of papaya breeding and improvement.

9. The use of the papaya CpWRKY57 gene as a papaya anthracnose resistance regulatory gene and / or dwarfing regulatory gene according to claim 1.

10. A method for cultivating papaya crops that combines high resistance to anthracnose and dwarfing, characterized in that, The method includes the step of transforming the papaya CpWRKY57 gene of claim 1 into the target plant.

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