Papaya CpMYB114L gene and application thereof

Abstract

The invention belongs to the technical field of gene engineering, particularly relates to a papaya CpMYB114L gene, and further discloses application of the papaya CpMYB114L gene in regulating and controlling the sweetness of papaya fruits. The CpMYB114L gene is screened and confirmed to play an important role in secondary metabolism of papaya in the maturing period by observing metabolic behaviors of organic acids and saccharides in the maturing process of papaya, and the CpMYB114L gene participates in metabolism of malic acid and synthesis of glucose, fructose and cane sugar in the maturing process of papaya fruits. The positive activation effect is achieved on the regulation and control of the sweetness of the papaya fruits.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to a papaya CpMYB114L gene, and further discloses its use for regulating the sweetness of papaya fruits. Background Art

[0002] Papaya (Carica papaya L.), also known as pawpaw, milk fruit, and longevity fruit, belongs to the genus Carica of the family Caricaceae, and is one of the most important fruit crops in tropical and subtropical regions. It is known as one of the "three major herbaceous fruit trees in the tropics" together with bananas and pineapples. Papaya is used as medicine with its fruit. It is an evergreen soft-wooded large perennial herbaceous plant or an evergreen soft-wooded small tree in tropical and subtropical regions. Papaya fruits are rich in vitamins, dietary fiber, and trace elements, etc., and have high nutritional and medicinal values. It is listed as the top ten most valuable fruits by the World Health Organization and has the reputations of "king of beneficial fruits", "king of fruits", and "longevity melon".

[0003] Papaya is native to South America and was introduced into China in the 17th century, with a planting history of 300 years. It is planted in Guangdong, Guangxi, Hainan, Fujian, Taiwan and other places. Research shows that papaya is rich in more than 17 kinds of amino acids, calcium, iron, etc., and also contains papain, carpaine, etc. Half of a medium-sized papaya is sufficient to supply an adult's daily requirement of vitamin C. The fruits of papaya can not only be used as fruits and vegetables, but also have various medicinal values. The latex of immature papaya can be used to extract papain, which is an excellent raw material for making cosmetics and has the effect of beautifying and whitening. The carpaine contained in papaya has the effect of anti-tumor, and also has antibacterial, anti-parasitic, blood pressure lowering and other effects; the papain contained can help with protein digestion, and can be used for chronic indigestion and gastritis, etc. Papain also has anticoagulant effects, etc.

[0004] Papaya belongs to fruits with a relatively low fructose content. Many scientific research scholars have been committed to breeding papayas with a high fructose content. At present, the fructose content in papaya pulp is an important index for evaluating the quality of papaya, and thus has become an important factor considered by breeders when breeding excellent papaya plants. At present, for the selection and breeding of this index, it is necessary to wait for the plants to flower and bear fruit and then measure the fructose content of the fruits to screen excellent plants, with a long selection and breeding time and a relatively high selection and breeding cost. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to provide a papaya CpMYB114L gene and the protein encoded thereby;

[0006] The second technical problem to be solved by the present invention is to provide the use of the above-mentioned papaya CpMYB114L gene as a gene for regulating the sweetness of papaya fruits.

[0007] To solve the above technical problems, a papaya CpMYB114L gene of the present invention has a full-length cDNA sequence of 684 bp;

[0008] The cDNA sequence of the CpMYB114L gene is shown as SEQ ID No.1.

[0009] The present invention also discloses a protein encoded by the papaya CpMYB114L gene, and the protein contains 227 amino acids;

[0010] The protein includes the amino acid sequence shown as SEQ ID No.2.

[0011] The present invention also discloses a recombinant expression vector, and the expression vector contains the papaya CpMYB114L gene;

[0012] Optionally, the recombinant expression vector is the CpMYB114-L vector OE-CpMYB114L containing the 35S promoter-driven.

[0013] The present invention also discloses a recombinant bacterium containing the papaya CpMYB114L gene or the recombinant expression vector.

[0014] The present invention also discloses a stable crop strain stably genetically expressed from the recombinant bacterium.

[0015] The present invention also discloses a method for cultivating a high-sweetness papaya crop, including the step of transforming the papaya CpMYB114L gene into a target crop.

[0016] The present invention also discloses the use of the papaya CpMYB114L gene as a papaya fruit sweetness regulation gene.

[0017] Specifically, the sweetness regulation includes increasing the sugar synthesis amount and / or enhancing the acid metabolism rate;

[0018] Optionally, the sugars include glucose, fructose, and sucrose;

[0019] Optionally, the acids include malic acid.

[0020] Specifically, the target genes for the CpMYB114L gene to regulate the papaya fruit sweetness include CpIDH5 and / or CpME2.

[0021] The present invention also discloses the application of the papaya CpMYB114L gene in the field of papaya breeding improvement.

[0022] The present invention also discloses the application of the papaya CpMYB114L gene in at least one of the following fields (1)-(3):

[0023] (1) The CpMYB114L gene is involved in the metabolism of malic acid during the ripening process of papaya fruits;

[0024] (2) The CpMYB114L gene is involved in the synthesis of glucose, fructose and / or sucrose during the ripening process of papaya fruits;

[0025] (3) The CpMYB114L gene positively activates the expression of CpIDH5 and / or CpME2.

[0026] By observing the metabolic behaviors of organic acids and sugars during the ripening process of papaya, the present invention screened and confirmed that the CpMYB114L gene plays an important role in secondary metabolism during papaya ripening and has a positive activation effect on the regulation of papaya fruit sweetness.

[0027] In order to identify the role of CpMYB114L in the ripening process of papaya, the present invention constructed a CpMYB114-L vector (OE-CpMYB114L) driven by the 35S promoter, transformed Agrobacterium tumefaciens, and injected the transformed Agrobacterium suspension into papaya fruits for transient gene overexpression experiments. The results of RT-qPCR analysis showed that the CpMYB114L gene was involved in the metabolism of malic acid and the synthesis of glucose, fructose and sucrose during the ripening process of papaya fruits.

[0028] The present invention further verified that the target genes directly regulated by CpMYB114L include CpIDH5 and CpME2, and the CpMYB114L can positively activate the expression of CpIDH5 and CpME2, which has positive significance for the breeding improvement of papaya. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to make the content of the present invention easier to be clearly understood, the following further describes the present invention in detail according to the specific embodiments of the present invention and in combination with the drawings, wherein,

[0030] Figure 1 are the results of ethylene release amount, respiration rate and sugar and acid contents of papaya fruits at different ripening stages; wherein, (A) papaya fruits at different ripening stages; (B) ethylene release amount; (C) respiration rate; (D) soluble solid content; (E) acid content; (F) ratio of soluble solid to acid; (G) glucose content; (H) fructose content; (I) sucrose content; (J) malic acid content; (K) citric acid content; (L) tartaric acid content;

[0031] Figure 2Transcriptome analysis of papaya fruits at different ripening stages; among them, (A) Venn diagram; (B) Number of up- and down-regulated genes; (C) GO enrichment analysis; (D) KEGG enrichment analysis;

[0032] Figure 3 Based on the expression of 35 MYB genes in papaya fruits at different ripening stages; among them, (A) Heatmap of the expression of 35 MYB genes; (B) Fluorescent quantitative analysis of 8 selected MYB genes;

[0033] Figure 4 Based on the homologous alignment analysis and subcellular localization of the CpMYB114L gene; among them, (A) Phylogenetic tree analysis of the homologous alignment of the CpMYB114L gene-encoded protein; (B) Alignment analysis of the CpMYB114L gene-encoded protein with the 8 protein sequences with the highest homology; (C) Subcellular localization of the CpMYB114L gene-encoded protein;

[0034] Figure 5 Based on the functional verification results of CpMYB114L; among them, (A) Papaya fruits injected with CpMYB114L overexpression and empty vector; (B) qRT-PCR verification of the expression level of CpMYB114L in papaya fruits; (C) Glucose, fructose and sucrose contents in papaya fruits; (D) Malic acid and citric acid contents in papaya fruits; (E) CpMYB114L-overexpressing papaya callus; (F) qRT-PCR verification of the expression level of CpMYB114L in callus; (G) Glucose, fructose and sucrose contents in callus; (H) Malic acid and citric acid contents in callus;

[0035] Figure 6 Based on the DAP-seq data analysis of CpMYB114L; among them, (A) Categories and proportions of CpMYB114L-binding sequences; (B) Conserved sequences bound by CpMYB114L; (C) KEGG enrichment analysis of CpMYB114L target genes;

[0036] Figure 7 To verify the interaction results of CpMYB114L with the promoter sequences of CpIDH5 and CpME2; among them, (A) Construction of CpIDH5 and CpME2 promoter reporter vectors and CpMYB114L effector vectors; (B) Y1H and (C) EMSA experiments to prove the interaction of CpMYB114L with the promoters of CpIDH5 and CpME2;

[0037] Figure 8 Results of yeast one-hybrid verification of sugar and acid metabolism-related genes where CpMYB114L does not interact with the promoter;

[0038] Figure 9To verify the positive regulation of CpMYB114L on the expression of CpIDH5 and CpME2; among them, (A) Schematic diagram of the construction of the CpIDH5 and CpME2 promoter reporter vectors and the CpMYB114L effector vector; (B) LUC enzyme activity detection; (C) LUC fluorescence imaging; (D) Relative expression levels of the CpIDH5 and CpME2 genes in papaya fruits with transient overexpression of CpMYB114L; (D) Relative expression levels of the CpIDH5 and CpME2 genes in calli of papaya overexpressing CpMYB114L;

[0039] Figure 10 It is the functional verification result of CpIDH5 and CpME2 in Example 7. Specific implementation manners

[0040] 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.

[0041] Example 1

[0042] This example studies the ethylene release amount, respiration rate and sugar and acid contents of papaya fruits at different maturity stages. Among them, the ethylene and respiration rate are measured with a portable ethylene detector and a carbon dioxide release detector, and the soluble solids and acid contents are measured with a portable sugar-acid meter. The test method is the standard method.

[0043] During the ripening process of papaya fruits, their peel color changes from green to yellow. According to the maturity, in this example, similar-sized and healthy fruits are divided into the green ripening stage (GS), the color-breaking stage (CB), the ripening stage (HY) and the over-ripening stage (FY) (as Figure 1 shown in A).

[0044] The test results of this example show that during the ripening process of papaya fruits, the ethylene release amount, respiration rate and soluble solids content (SSC) gradually increase, while the acid content (AC) gradually decreases, and the ratio of SSC / AC also increases (as Figure 1 shown in B-F).

[0045] In this example, in order to observe the metabolic behaviors of organic acids and sugars during the ripening process of papaya, the contents of glucose, fructose, sucrose, malic acid, citric acid and tartaric acid were measured respectively using an HPLC system. The results prove that compared with GS fruits, the contents of glucose, fructose, sucrose and citric acid in CB, HY and FY are significantly increased, while the contents of malic acid and tartaric acid are significantly decreased (as Figure 1Among the CB, HY, and FY fruits, the HY fruit had the highest ethylene release rate, glucose content, and SSC / AC ratio, and the lowest sucrose content; while the CB fruit had the lowest malic acid content and the highest citric acid content. However, the tartaric acid content in papaya fruits was much lower than that of malic acid and citric acid.

[0046] Example 2

[0047] This example focuses on the transcriptome analysis and candidate gene screening of papaya fruits at different maturity stages.

[0048] In this example, 12 fruit cDNA libraries were constructed and sequenced to analyze the transcriptional expression of papaya at the GS, CB, HY, and FY stages.

[0049] In this example, a total of 34.93 Gb of high-throughput sequencing data was obtained, with an average of 2.91 Gb of data and 4.09×10 7 read lengths per sample. Approximately 85.77% of the reads were successfully mapped to the papaya reference genome (as shown in Table 1 below).

[0050] Table 1 Transcriptome data of papaya fruit samples

[0051]

[0052]

[0053] In the above comparison groups of CB vs. GS, GS vs. HY, and GS vs. FY, a total of 10,099 differentially expressed genes (DEGs) were identified in this example. Among them, there were 2,205, 2,591, and 2,892 up-regulated genes, and 3,691, 5,513, and 5,009 down-regulated genes, respectively (as shown in Figure 2 Appendix A and B). It can be seen that during the ripening process of papaya, the number of DEGs gradually increases.

[0054] In this example, GO enrichment analysis showed that these differential genes were mainly enriched in terms such as 'Binding', 'Catalytic activity', 'Cellular process', 'Metabolic process', and 'Cellular anatomical entity' (as shown in Figure 2 Appendix C). In addition, KEGG pathway enrichment analysis showed that DEGs were highly enriched in the 'Metabolic pathways' and 'Biosynthesis of secondary metabolites' pathways (as shown in Figure 2 Appendix D).

[0055] In this example, to further analyze the expression pattern of the MYB gene, the transcriptome data was further compared with the Phytozome v13 database, and a total of 35 MYB genes were identified (see Table 2 below).

[0056] Table 2 Annotation of 35 MYB genes obtained from transcriptome data

[0057]

[0058]

[0059] Furthermore, a clustering heatmap of the expression levels was constructed using the TPM values of these 35 MYB genes, and there were significant differences in the expression levels of 11 MYB genes (as shown in A in Figure 3 ). The relative expression levels of these 11 MYB genes were verified by qRT-PCR. Since the TPM values of CpMYB305-like, CpMYB26, and CpMYB52 were less than 1, they were considered not to be expressed and were not verified here. The qRT-PCR results were consistent with the transcriptome data. In particular, the transcriptional expression level and relative expression level of CpMYB114-like (CpMYB114L) were the highest (as shown in B in Figure 3 ), which means that CpMYB114L may play an important role in secondary metabolism during papaya ripening, so it was selected as a candidate gene.

[0060] After verification, the full-length cDNA sequence of CpMYB114L was 684 bp, encoding a protein containing 227 amino acids. The cDNA sequence of the CpMYB114L gene is shown in SEQ ID No.1, and its encoded protein includes the amino acid sequence shown in SEQ ID No.2.

[0061] SEQ ID No.1:

[0062] ATGGGAGGAGCTGCATGGACTAAAGAAGAAGATTACTTGCTCAGAAAATC

[0063] TGTAATGCAATATGGAGAAGGGAAATGGCATCTCATCCCTCTATTGGCTGGT

[0064] CTAAACAGATGCCGGAAAAGCTGTAGACTGAGATGGTTAAACTATTTACGT

[0065] CCAAACATCAAGAGAGGGAGTTTTGCAGAAGATGAAGTGGATCAAATCAT

[0066] TGAGCTCCACAGACTTTTTGGTAACAGGTGGTCACTAATTGCTGCTAGACT

[0067] ACCAGGAAGAACAGCTAATGATGTGAAGAACTATTGGAATTACCATTTGAG

[0068] TAGAAGATTCAATTCACAAGATGAAAACAAACACAAACAAATCACTAGAA

[0069] AAAATACTAATATTCATGCAAAACCAAATTCTTCTACCAATTCCAAATCAATT

[0070] AGAGAAATCTCACAAGTTAAACAACAAGATATCAGCACATCAACAGCACC

[0071] AATGGAGGGCTCTGGTCAACTGGTAGTTGAAGCAGAATGCAGTGATCAGC

[0072] AGTTGCAGGCCGATGAAGATGGTACAAATGGAGGCAATCAAAACTTTGAA

[0073] GCCAAAAATTTGTGCGATGAGGATTTCAAGATTCAAGATGTTAGAGATGAT

[0074] AGAATTGAGGGTTTTAGCCAGTGGGATTGGGATGATTGGATACTGGGTATGGATTTGTGGAATGGAACATTGTGA。

[0075] SEQ ID No.2:

[0076] MGGAAWTKEEDYLLRKSVMQYGEGKWHLIPLLAGLNRCRKSCRLRWLNYL

[0077] RPNIKRGSFAEDEVDQIIELHRLFGNRWSLIAARLPGRTANDVKNYWNYHLSR

[0078] RFNSQDENKHKQITRKNTNIHAKPNSSTNSKSIREISQVKQQDISTSTAPMEGS

[0079] GQLVVEAECSDQQLQADEDGTNGGNQNFEAKNLCDEDFKIQDVRDDRIEGFSQWDWDDWILGMDLWNGTL*。

[0080] Forty-nine homologous proteins of 43 species were downloaded from the NCBI website, and a phylogenetic tree was constructed using MEGA X for analysis. The results showed that CpMYB114L had the highest homology with the proteins PpMYB90 (XP_007205727.1), PdMYB113L (XP_034220695.1), PmMYB90L (XP:008218386.1), Paverl (XP_021800783.1), RrMYB10.1 (QBP33436.1), RrMYB8L (XP-062006805.1), RcMYB113 (XP_024190887.1) and MsMYB1L (XPO_0501428.1) (as shown in Figure 4 Figure A). According to the neighbor-joining algorithm results, CpMYB114L and eight of these MYB transcription factors all contain two conserved SANT domains, indicating that these MYB transcription factors may belong to the R2R3 class of MYB proteins (as shown in Figure 4 Figure B).

[0081] Furthermore, a CpMYB114L-GFP fusion vector driven by the CaMV35S promoter was constructed and transformed into Arabidopsis protoplasts for expression. The cDNA of the constructed CpMYB114L was ligated to the PAN582-GFP vector. Select the leaves of Arabidopsis thaliana that have not flowered, cut them into leaf strips 0.5-1 mm wide with a blade, place them in a cellulase digestion solution, rinse and purify them with W5 solution to obtain protoplasts, add the constructed vector, induce it to transfer into the protoplasts with PEG, then dilute and wash it once with W5 solution, centrifuge, add WI solution, and culture it at room temperature in the dark for 20 hours, and observe the GFP signal with a laser confocal microscope. The results showed that the GFP-labeled CpMYB114L protein was localized in the nucleus (as shown in Figure 4 Figure C).

[0082] Example 3

[0083] Based on the previously screened CpMYB114L gene, this example further verified the biological function of CpMYB114L and screened for target genes.

[0084] In this example, to identify the role of CpMYB114L during papaya fruit ripening, a vector (OE-CpMYB114L) containing CpMYB114-L driven by the 35S promoter was constructed, transformed into Agrobacterium tumefaciens, and the transformed Agrobacterium suspension was injected into papaya fruits for transient gene overexpression experiments. The cDNA of CpMYB114L constructed was ligated to the PRI101 vector. It was then transferred into competent GV3101 Agrobacterium. The Agrobacterium was diluted with the suspension (containing 200 μM acetosyringone) to an OD600 of 0.5 - 0.8. Three holes with a depth of 5 mm were made in the papaya fruits using a syringe needle, and 1 ml of the suspension was injected into them. They were cultured for 5 days at room temperature in the dark, and the tissues around the injection sites were taken for detection. The results are as shown in Figure 5 Figure A.

[0085] The results of RT-qPCR analysis showed that in OE-CpMYB114L papaya fruits, the transcriptional level of CpMYB114L was significantly higher than that of the empty vector (as shown in Figure 5 Figure B). Further, the HPLC system was used to determine the contents of glucose, fructose, sucrose, malic acid, and citric acid in OE-CpMYB114L and empty vector samples. The results are as shown in Figure 5 Figures C and D. The contents of glucose, fructose, and sucrose in the OE-CpMYB114L samples were significantly higher than those of the empty vector, while the malic acid content was significantly lower than that of the empty vector. There was no significant difference in the citric acid content between the OE-CpMYB114L and empty vector samples.

[0086] In addition, the overexpression vector was transformed into the callus of 'Zihui' papaya for further verification (as shown in Figure 5 Figure E). The results of RT-qPCR showed that in the OE-CpMYB114L callus, the expression level of CpMYB114L increased significantly (as shown in Figure 5 Figure F). Compared with the empty vector callus, the papaya callus overexpressing CpMYB114L had higher contents of glucose, fructose, and sucrose, while the malic acid content was lower than that of the empty vector (as shown in Figure 5 Figures G and H).

[0087] The above results indicate that CpMYB114L is involved in the metabolism of malic acid and the synthesis of glucose, fructose, and sucrose during papaya fruit ripening.

[0088] Example 4

[0089] In this example, to identify the target genes directly regulated by CpMYB114L, DNA affinity purification sequencing (DAP-seq) of the CpMYB114L transcription factor was carried out. The results are shown in Appendix Figure 6 .

[0090] The above results showed that a total of 20,989 enrichment peaks were obtained. Among them, 44.08% of the peaks were located on the gene body, 21.85% were located on the promoters of open reading frames (ORFs) (promoters <2 kbp), 32.36% were distributed in the intergenic regions, and the other 1.71% belonged to the downstream of ORFs (<3 kbp) (as shown in Figure 6 A). And through MEME and DREME analyses, short consensus motifs of these enrichment peaks were calculated and obtained, as shown in Figure 6 B.

[0091] Furthermore, KEGG enrichment analysis was performed on these enrichment peaks. As shown in the results in Figure 6 C, the top 10 KEGG enrichment pathways were listed, mainly pathways related to sugar or organic acid metabolism, including 'Carbon fixation in photosynthetic organisms' and 'TCA cycle', which contained genes such as CpMDH2, CpOGDH, CpDLAT, CpIDH5, CpMDH1, CpIDCH1, CpGOT1, CpGGAT2, CpME2, and CpPPC2L.

[0092] Example 5

[0093] In this example, the performance of the CpMYB114L gene was verified through a yeast one-hybrid (Y1H) experiment. The results showed that CpMYB114L interacted with the promoters of CpIDH5 and CpME2 (as shown in Figure 7 A and B).

[0094] In this example, through electrophoretic mobility shift (EMSA) analysis, the results showed that CpMYB114L bound to the sequence region from -308 to -322 bp in the CpIDH5 promoter (MBS1: TCTTCACCTCTCCC) and the sequence region from -1288 to -1302 bp in the CpME2 promoter (MBS2: CCTGGTTTGCGAGT) (as shown in Figure 7 C and D).

[0095] It can be seen that the results of the Y1H experiment showed that there was no interaction between the promoter regions of the genes CpMDH2, CpOGDH, CpDLAT, CpMDH1, CpIDCH1, CpGOT1, CpGGAT2, and CpPPC2L predicted based on DAP-seq and CpMYB114L (as shown in Figure 8 ).

[0096] Example 6

[0097] In this example, transient dual-luciferase (LUC) assays were further performed on tobacco leaves to verify the effect of CpMYB114L on the promoter activities of CpIDH5 and CpME2.

[0098] Their promoter sequences were respectively fused to the upstream of the LUC reporter gene in the pGreenII 0800-LUC vector, and the full-length CDS of CpMYB114L was inserted into the pGreenI 62-SK vector (as shown in Figure 9 A). The results of enzyme activity and fluorescence detection showed that the enzyme activities and fluorescence amounts of the co-expression of 35S::CpMYB114L with proCpIDH5::LUC and 35S::CpMYB114L with proCpME2::LUC were respectively higher than those of the single-vector expression of proCpIDH5::LUC and proCpME2::LUC (as shown in Figure 9 B and C).

[0099] In addition, in papaya fruits and calli overexpressing CpMYB114L, the expression levels of CpIDH5 and CpME2 were higher than those of the empty vector (as shown in Figure 9 D and E). These results all indicated that CpMYB114L positively activates the expression of CpIDH5 and CpME2.

[0100] Example 7

[0101] In this example, to further verify the functions of CpIDH5 and CpME2, the cDNAs of CpIDH5 and CpME2 were ligated to the PRI101 overexpression vector, transformed into competent Agrobacterium tumefaciens GV3101, and cultured with shaking at 160 rpm until OD600 = 0.5 - 0.8. After centrifugation, the pellet was resuspended. Three 5-mm deep holes were pricked into the fruits with a syringe needle. Using a 1-ml syringe, 200 μL of the resuspended solution was slowly injected into the holes. The samples were cultured at room temperature in the dark for 5 days, and the tissues around the injection holes were taken for detection. The results are as shown in the appendix Figure 10 . Among them, Figure 10 A and D in the appendix are papaya fruits of OE-CpIDH5 and OE-CpME2 respectively.

[0102] The results of RT-qPCR analysis showed that in the papaya fruits of OE-CpIDH5 and OE-CpME2, the transcriptional levels of CpIDH5 and CpME2 were significantly higher than those of the empty vector (as shown in Figure 10 B and E). Further, the contents of glucose, fructose, sucrose, malic acid, and citric acid in the samples of OE-CpIDH5, OE-CpME2, and their empty vectors were determined using the HPLC system. The results are as shown in Figure 10 C. There were no significant differences in the contents of glucose, fructose, sucrose, malic acid, and citric acid between the samples of OE-CpIDH5 and the empty vector. The results are as shown inFigure 10 As shown in Figure F, the contents of glucose, fructose and sucrose in the OE-CpME2 sample were significantly higher than those in the empty vector, while the content of malic acid was significantly lower than that in the empty vector. There was no significant difference in the content of citric acid between the OE-CpME2 sample and the empty vector sample. It is indicated that CpMYB114L regulates the sugar and acid contents of papaya fruits by positively activating CpME2.

[0103] In summary, the present invention screened and confirmed that the CpMYB114L gene plays an important role in secondary metabolism during papaya ripening. The CpMYB114L gene is involved in the metabolism of malic acid and the synthesis of glucose, fructose and sucrose during the papaya fruit ripening process, and has a positive activation effect on the regulation of papaya fruit sweetness.

[0104] The embodiments of the present invention have been described in detail above. Specific examples are used herein to illustrate the principle and implementation manner of the present invention. The description of the above embodiments is only used to help understand the method and its core idea of the present invention; at the same time, for those of ordinary skill in the art, according to the idea of the present invention, there will be changes in the specific implementation manner and application scope. In summary, the content of this specification should not be construed as a limitation to the present invention.

Claims

1. A papaya CpMYB114L gene, characterized in that: The cDNA sequence of the CpMYB114L gene is 684 bp in length; The cDNA sequence of the CpMYB114L gene is shown as SEQ ID No.

1.

2. A protein encoded by the papaya CpMYB114L gene according to claim 1, characterized in that: The protein contains 227 amino acids; The protein comprises the amino acid sequence shown in SEQ ID No.

2.

3. A recombinant expression vector, characterized in that: The expression vector comprises the papaya CpMYB114L gene according to claim 1; Optionally, the recombinant expression vector is a CpMYB114-L vector OE-CpMYB114L driven by a 35S promoter.

4. A recombinant bacterium or crop strain containing the papaya CpMYB114L gene according to claim 1 or the recombinant expression vector according to claim 3.

5. A method for cultivating high-sweetness papaya crops, characterized in that: The method comprises the step of transforming the papaya CpMYB114L gene according to claim 1 into target crops.

6. Use of the papaya CpMYB114L gene according to claim 1 as a papaya fruit sweetness regulating gene.

7. The use according to claim 7, characterized in that The sweetness regulation includes increasing the amount of carbohydrate synthesis and / or improving the acid metabolism rate; Optionally, the sugars include glucose, fructose, and sucrose; Optionally, the acid comprises malic acid.

8. The use according to claim 6 or 7, characterized in that The target genes of the CpMYB114L gene regulating the sweetness of papaya fruit include CpIDH5 and / or CpME2.

9. Application of the papaya CpMYB114L gene according to claim 1 in the field of papaya breeding and improvement.

10. Use of the papaya CpMYB114L gene of claim 1 in at least one of the following fields (1) to (3): (1) The CpMYB114L gene is involved in the metabolism of malic acid during papaya fruit ripening; (2) The CpMYB114L gene is involved in the synthesis of glucose, fructose, and / or sucrose during papaya fruit ripening; (3) The CpMYB114L gene positively activates the expression of CpIDH5 and / or CpME2.

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