Application and method of a gene affecting the length of papaya flower spike and fruit stalk and fruit setting rate

Abstract

The present invention provides an application and method of a gene that affects the length of papaya flower spike and fruit stalk and fruit setting rate, belonging to the field of biotechnology. CpPUP11 Gene expression level to increase the length of papaya flower spikes and fruit stalks and the fruit setting rate; the papaya breeding method is to increase the CpPUP11 Gene expression level to obtain papaya strains that affect the length of papaya flower spikes and fruit stalks and fruit setting rate; the method is to plant CpPUP11 A papaya strain with high gene expression is used to increase the length of papaya flower spikes and fruit stalks and the fruit setting rate. CpPUP11 The gene affects the development of papaya flower spikes and fruit stalks, as well as the fruit setting efficiency, and has an important impact on papaya yield and manual picking. It has important application value and provides a research basis for the breeding of new excellent papaya varieties.

Description

Technical Field

[0001] The invention belongs to the field of plant biotechnology and relates to an application and method of a gene affecting the length of papaya flower spikes and fruit stalks and the fruit setting rate. Background Art

[0002] Papaya is native to South America and is now widely planted in tropical and warmer subtropical regions around the world. Papaya is planted in Guangdong, Hainan, Guangxi, Yunnan, Fujian and other places in my country. It is an important economic fruit in my country and tropical regions around the world. Its fruit has beautiful appearance, thick and delicate flesh, abundant juice, sweet and delicious taste, rich in amino acids, vitamins and trace elements, etc. It is known as the "fruit of a hundred benefits", "the king of fruits" and "the melon of longevity", and is one of the four famous fruits in Lingnan. Papaya not only has important edible value, but can also be used for canned food, preserved fruit and dried fruit processing. The green fruit milk is rich in papain and is widely used in cosmetics, pharmaceuticals and other industries, with more important economic value.

[0003] The length of the flower spike and fruit stalk affects the density of papaya fruiting, which in turn affects the fruit yield and quality. At the same time, the length of the fruit stalk is also related to the difficulty of manual picking. Purine permease PUPs is an important transporter of cytokinins in plants. It plays an important role in plant seed germination, pollen development, fruit size, etc. At present, there are no reports on the effects of the purine permease PUPs gene in papaya on the length of the flower spike and fruit stalk and the fruiting rate. Summary of the invention

[0004] In view of the above problems, the present invention provides an application and method of a gene that affects the length of papaya flower spike and fruit stalk and the fruit setting rate.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is:

[0006] An application of genes that affect the length of papaya flower spikes and fruit stalks and fruit setting rate, the application is to increase CpPUP11 Gene expression to increase the length of papaya flower spikes and fruit stalks and fruit setting rate;

[0007] CpPUP11 Gene for papaya purine permease 11 The gene, with the gene accession number being LOC110812563 and the gene sequence being shown in SEQ ID NO: 1, is a purine permease. The gene is 1502 bp in length and contains 2 exons. The CDS coding region is 1137 bp in length. The CDS coding sequence is shown in SEQ ID NO: 2, encoding 378 amino acids. The amino acid sequence is shown in SEQ ID NO: 3.

[0008] Furthermore, the application is to use gene overexpression technology or gene editing technology to increase CpPUP11Gene expression level.

[0009] Furthermore, the gene overexpression technique is to CpPUP11 Gene overexpression vector or containing CpPUP11 The recombinant strain of the gene overexpression vector was transformed into papaya, so that CpPUP11 Gene overexpression;

[0010] The recombinant strain is preferably Agrobacterium;

[0011] Specifically, the gene overexpression technology is to construct CpPUP11 Gene overexpression vector CpPUP11 Gene overexpression vector was transformed into papaya to make CpPUP11 Gene overexpression.

[0012] Furthermore, CpPUP11 Gene overexpression vector is CpPUP11 The PCR amplification product of the gene was connected to the PBI121-GUS backbone vector to obtain the overexpression vector PBI121-GUS-CpPUP11.

[0013] Furthermore, CpPUP11 The PCR amplification product of the gene was obtained by PCR amplification using the cDNA of papaya as a template and primers PUP11--F / PUP11--R;

[0014] Among them, PUP11--F:5'-CG GGATCC ATGGAGGCGAGTAAAT-3′;

[0015] PUP11--R:5'-TCC CCCGGG TTAAACATGAGCC-3'.

[0016] Furthermore, the gene overexpression technology comprises the following steps:

[0017] Build CpPUP11 Gene overexpression vector;

[0018] use CpPUP11 Gene overexpression vector, construct containing CpPUP11 Recombinant strains of gene overexpression vectors;

[0019] Utilize the included CpPUP11 The recombinant strain of the gene overexpression vector was transformed into papaya to make CpPUP11 Gene overexpression.

[0020] Furthermore, the papaya transformation is carried out by using papaya seedling stem segments as explants to induce embryonic callus, and then using CpPUP11The recombinant strain containing the gene overexpression vector was transformed to obtain CpPUP11 Gene overexpression papaya lines.

[0021] Furthermore, the gene editing technology includes but is not limited to CRISPR-Cas mediated enhancer knock-in technology, promoter region editing technology or 5'UTR editing technology.

[0022] A sort of CpPUP11 Gene editing vector, the gene editing vector is based on papaya CpPUP11 The gene knockout target was designed based on the gene CDS sequence and connected to the vector to obtain the gene editing vector pTRANS_220d-CpPUP11;

[0023] The two gene knockout target sequences are shown in SEQ ID NO: 4 and SEQ ID NO: 5.

[0024] A kind of CpPUP11 The recombinant strain of the gene editing vector is Agrobacterium tumefaciens AGL1.

[0025] Utilize the included CpPUP11 The recombinant strain of the gene editing vector was transformed into papaya to obtain a transformant with extremely shortened flower spikes. The flowers of the transformant naturally fell off in the late flowering period and could not set fruit.

[0026] Although the present invention is for better verification CpPUP11 The effect of gene expression on the length of papaya spike and fruit stalk and fruit setting rate was designed to reduce CpPUP11 Gene editing vectors for gene expression. CpPUP11 Based on the premise that the gene expression amount affects the regulation of papaya flower spike and fruit stalk length and fruit setting rate, technicians in this field use conventional gene editing technology to adjust and design a method to improve CpPUP11 Gene editing vectors that increase gene expression CpPUP11 Gene expression level of gene editing vector, transformed papaya, to obtain CpPUP11 A papaya strain with high gene expression level can improve the length of the papaya flower spike and fruit stalk and the fruit setting rate.

[0027] At the same time, the present invention proves CpPUP11 Under the premise of the regulatory effect of gene expression on the length of papaya flower spike and fruit stalk and fruit setting rate, those skilled in the art can also process papaya by other gene editing technologies, such as promoter region editing, 5'UTR editing and other technologies, to obtain CpPUP11 A papaya strain with high gene expression level can improve the length of the papaya flower spike and fruit stalk and the fruit setting rate.

[0028] A papaya breeding method, the papaya breeding method is to increase the CpPUP11 Gene expression levels to obtain papaya strains that affect the length of papaya flower spikes and fruit stalks and fruit setting rate;

[0029] CpPUP11 The gene sequence of the gene is shown in SEQ ID NO: 1;

[0030] Specifically, increase the CpPUP11 The gene expression method can be to use gene overexpression technology to increase the gene expression in papaya. CpPUP11 Gene expression can also be increased by gene editing technology in papaya CpPUP11 Gene expression level.

[0031] A method for regulating the length of papaya flower spikes and fruit stalks and the fruit setting rate, the method comprising planting CpPUP11 Papaya strains with high gene expression levels to increase the length of papaya flower spikes and fruit stalks and fruit setting rate;

[0032] CpPUP11 The gene sequence of the gene is shown in SEQ ID NO: 1;

[0033] Specifically, CpPUP11 Papaya strains with high gene expression levels can be obtained using gene overexpression technology CpPUP11 Papaya strains with high gene expression can also be obtained using gene editing technology CpPUP11 Papaya strains with high gene expression.

[0034] The beneficial effects of the application and method of a gene affecting the length of papaya flower spike and fruit stalk and fruit setting rate of the present invention are:

[0035] The present invention also constructs papaya CpPUP11 Gene overexpression vectors and CpPUP11 Gene editing vector, obtained after genetic transformation of papaya CpPUP11 Gene overexpression strains and gene editing strains, the papaya flower spikes and fruit stalks of the overexpression strains were significantly longer, and the fruit setting rate was improved; while the papaya of the gene editing strains had almost no flower spikes, and the flowers fell off naturally, resulting in no fruit setting; indicating CpPUP11 The gene can positively regulate the development of papaya flower spikes and fruit stalks, as well as the fruit setting rate, and has an important impact on papaya yield and manual picking, and has important application value;

[0036] The present invention CpPUP11 Genes affect the development of papaya flower spikes and fruit stalks, as well as fruit setting efficiency, providing a research basis for breeding new high-quality papaya varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1It is constructed in Example 1 of the present invention CpPUP11 Gene overexpression vector PBI121-GUS-CpPUP11;

[0038] Figure 2 In Example 2 of the present invention CpPUP11 Gene editing vector pTRANS_220d-CpPUP11;

[0039] Figure 3 Papaya in Example 3 of the present invention CpPUP11 PCR identification results of gene overexpression strains; M represents the molecular weight marker DL2000; numbers 1 to 8 represent the corresponding CpPUP11 Gene overexpression strains 1 to 8, among which strains 1 to 4 and strains 6 to 7 are CpPUP11 Gene overexpression positive strain; K+ is the vector plasmid, used as a positive control; K- is the non-transgenic papaya Tainong No. 2, used as a negative control; K is sterile water, used as a blank control;

[0040] Figure 4 Papaya in Example 3 of the present invention CpPUP11 PCR identification results of gene-edited strains; M represents the molecular weight marker DL2000; numbers 1 to 6 represent the corresponding CpPUP11 Gene-edited strains 1 to 6, of which strains 3, 4, and 6 are CpPUP11 Gene editing positive strain; K+ is the vector plasmid, used as a positive control; K- is the non-transgenic papaya Tainong No. 2, used as a negative control; K is sterile water, used as a blank control;

[0041] Figure 5 In Example 4 of the present invention CpPUP11 The flower spike length phenotype of gene overexpression positive strain 3 and non-transgenic control papaya; among them, ck in the upper row represents the flower spike of non-transgenic control papaya, and strain 3 in the lower row is CpPUP11 Flower spike of gene overexpression positive line 3;

[0042] Figure 6 In Example 4 of the present invention CpPUP11 The length of the spike of gene overexpression positive strains 1 to 3 and the spike of non-transgenic control papaya. ck represents the spike length of non-transgenic control papaya; strains 1 to 3 represent CpPUP11 Flower spikes of gene overexpression positive strains 1 to 3;

[0043] Figure 7 In Example 4 of the present invention CpPUP11The fruit stalk length phenotype of the gene overexpression positive strain and the non-transgenic control papaya; ck represents the non-transgenic control papaya; A~F represent CpPUP11 Gene overexpression positive strains 1 to 4, and strains 6 to 7;

[0044] Figure 8 In Example 4 of the present invention CpPUP11 Fruit stalk length bar graph of gene overexpression positive strains; ck represents non-transgenic control papaya; strains 1 to 4, 6 to 7 represent CpPUP11 Gene overexpression positive strains 1 to 4, and strains 6 to 7;

[0045] Fig. 9 It is a phenotype diagram of the flower spike of gene-edited strain No. 6 and the flower spike of non-transgenic control papaya in Example 4 of the present invention; wherein the upper row is ck, representing the flower spike of non-transgenic control papaya, and the lower row is the flower spike of gene-edited strain No. 6;

[0046] Fig.10 In Example 4 of the present invention CpPUP11 The length bar graph of the flower spike of gene-edited positive strain 6 and the flower spike of the non-transgenic control; ck represents the flower spike of the non-transgenic control papaya; strain 6 represents CpPUP11 Flower spike of gene-edited positive line 6;

[0047] Fig.11 In Example 5 of the present invention CpPUP11 Gene overexpression positive strains CpPUP11 Gene expression bar graph; ck represents the non-transgenic papaya control; strains 1 to 4, 6 to 7 represent CpPUP11 Gene overexpression positive strains 1 to 4, and strains 6 to 7;

[0048] Fig.12 In Example 6 of the present invention CpPUP11 PCR identification results of editing sites of gene-edited positive strains 3, 4, and 6; M represents the molecular weight marker DL2000; band numbers 1 to 3 correspond to CpPUP11 PCR identification results of gene editing positive strains 3, 4, and 6;

[0049] Fig.13 In Example 7 of the present invention CpPUP11 Single fruit stalk comparison chart of gene overexpression positive strain 3 and non-transgenic papaya control; among them, ck on the left represents the single fruit stalk chart of non-transgenic papaya control, and the single fruit stalk charts in the middle and right are CpPUP11 A single fruit stalk of gene overexpression positive strain 3; in the figure, the red arrow points to CpPUP11 The gene overexpression positive strain 3 had two fruits on a single fruit stalk. DETAILED DESCRIPTION

[0050] The technical solutions in the embodiments of the present invention are described clearly and completely below. In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention can also be implemented in other ways different from those described herein, and those skilled in the art can make similar generalizations without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below. The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand.

[0051] In addition, if no specific techniques or conditions are specified in the specific examples disclosed below, the techniques or conditions described in the literature in this field (for example, refer to "Molecular Cloning Experiment Guide" written by Sambrook et al., translated by Huang Peitang et al., third edition, Science Press) or the product instructions are used. If the manufacturer of the reagents used is not specified, they are all conventional products that can be purchased.

[0052] Example 1 Papaya CpPUP11 Construction of gene overexpression vector

[0053] papaya purine permease 11 The gene, gene accession number is LOC110812563, the gene sequence is shown in SEQ ID NO: 1, it is a purine permease, the full length of the gene is 1502 bp, contains 2 exons, the gene sequence is shown in SEQ ID NO: 1, the CDS coding region is 1137 bp, the CDS coding sequence is shown in SEQ ID NO: 2, encoding 378 amino acids, the amino acid sequence is shown in SEQ ID NO: 3. The restriction sites BamHI and SmaI were added to both ends of the gene to design primers (Table 1). Total RNA was extracted from the leaves of Tainong No. 2 papaya, and the reverse transcribed cDNA was used as a template to perform PCR amplification with the above primers. The reaction system was: 2× PrimeSTAR Max Premix 12.5 μL, primers (10 μM) 1 μL each, cDNA template 2 μL, sterile water 8.5 μL. The PCR amplification program was as follows: 94°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 60°C annealing for 30 s, 72°C extension for 60 s, 35 cycles; 72°C extension for 7 min. The resulting PCR product was ligated with the PBI121-GUS backbone vector to obtain the overexpression vector PBI121-GUS-CpPUP11 (see Figure 1 ), which is CpPUP11 Gene overexpression vector. CpPUP11The gene overexpression vector was transformed into Agrobacterium AGL1 to obtain AGL1 bacteria containing PBI121-GUS-CpPUP11 plasmid, which was stored at -80°C with glycerol for later use.

[0054] Table 1 Primers for cloning papaya PUP11 gene

[0055]

[0056] Example 2 Papaya CpPUP11 Construction of gene editing vector

[0057] S1. The CDS sequence of the gene was analyzed on the gene editing target online design website (http: / / crispor.tefor.net / ). According to the specificity, editing efficiency, off-target rate and position of the target, a double target site knockout was designed, and two suitable gene knockout targets (sgRNA1 and sgRNA2) were selected. The two target sequences are shown in SEQ ID NO: 4 (sgRNA1 target sequence) and SEQ ID NO: 5 (sgRNA2 target sequence). Then the two selected targets were input into the online primer design software Multi-gRNA Array Assembly-Primer Design and MapConstruction (http: / / cfans-pmorrell.oit.umn.edu / CRISPR _Multiplex / assembly.php), and the target vector (pMOD_B2301), promoter system (35S), restriction endonuclease (Esp3Ⅰ), and splicing system (tRNA-Gly) were selected. The software will automatically generate three pairs of primers (see Table 2).

[0058] Table 2 Gene editing target primers

[0059]

[0060] S2. The above three pairs of primers were used as templates for PCR amplification using the intermediate vector pMOD_B2301 plasmid. The reaction system was: 2× PrimeSTAR Max Premix 12.5 μL, primers (10 μM) 1 μL each, pMOD_B2301 plasmid (100 ng / μL) 1 μL, sterile water 9.5 μL. The PCR amplification program was: 98°C / 1 min; 98°C / 10 s, 60°C / 15 s, 72°C / 15 s, 30 cycles; 72°C / 2 min.

[0061] The three amplified fragments obtained were placed in the same PCR tube with the pMOD_B2301 plasmid, and digested and ligated with endonucleases SapⅠ and Esp3Ⅰ and T4 ligase to obtain the ligation product. The obtained ligation product was transformed into E. coli DH5α competent cells, and a single clone was selected for bacterial liquid PCR identification (primers are shown in Table 3) and sequenced to verify the correct single clone. The plasmid pMOD_B2301-PUP11 was extracted, and it was digested with AarI and ligated with T4 ligase with the plasmids of the other two intermediate vectors pMOD_A0101 and pMOD_C3001 and the backbone vector pTRANS_220d in the same PCR tube. The obtained ligation product was transformed into E. coli DH5α competent cells, and a single clone was selected for bacterial liquid PCR identification (primers are shown in Table 3). The positive clones were screened and sent to the sequencing company for sequence determination. The positive clones with correct sequencing were saved, and the plasmid was extracted and named pTRANS_220d-CpPUP11, which is CpPUP11 Gene editing vector; CpPUP11 The gene editing vector was transformed into Agrobacterium AGL1 to obtain AGL1 bacteria containing the pTRANS_220d-CpPUP11 plasmid, which was stored at -80°C with glycerol for future use.

[0062] Table 3 Primers for bacterial liquid PCR identification

[0063]

[0064] Example 3 Transformation of papaya with overexpression vector and gene editing vector

[0065] 1) Using papaya Tainong No. 2 as the test material, the seeds were sterilized and then subjected to germination test. After about 2 weeks, the grown seedling stems were cut into uniform segments to induce embryonic callus, which were used as transformation explants. Then, Agrobacterium containing the overexpression vector PBI121- GUS-CpPUP11 and Agrobacterium containing the gene editing vector TRANS-220d-CpPUP11 were used to mediate embryonic callus for genetic transformation. Kanamycin was used to screen transformants in papaya transformation, and the results were as follows: CpPUP11 Gene overexpression lines and CpPUP11 Gene-edited strains.

[0066] 2) The above genetic transformation resulted in 8 CpPUP11 Gene overexpression lines and 6 CpPUP11Gene-edited strains. Leaf tissues of each strain were taken, and DNA was extracted using the MAGEN Plant Genomic DNA Extraction Kit, which was then used as a template for PCR detection. The primers for positive plant identification are shown in Table 4. The vector plasmid was used as the positive control, the genomic DNA of papaya Tainong No. 2 was used as the negative control, and sterile water was used as the blank control. The enzyme used for PCR detection was Vazyme's 2 x Rapid Taq MasterMix, and the reaction system was: 9.5 μL of sterile water, 1 μL of each primer (10 μM), 12.5 μL of Taq Mix, and 1 μL of DNA template. The PCR amplification program was: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 30 s, and 35 cycles; extension at 72°C for 2 min. The PCR amplification product was detected by 2% agarose gel electrophoresis, and the results are shown in the table. Figure 3 and Figure 4 .from Figure 3 It can be seen that M is DL2000 Marker, and numbers 1 to 8 represent the corresponding CpPUP11 Gene overexpression strains 1 to 8, among which strains 1 to 4 and strains 6 to 7 are CpPUP11 Gene overexpression positive strain; K+ is the vector plasmid, as a positive control; K- is the non-transgenic papaya Tainong No. 2, as a negative control; K is sterile water, as a blank control. Figure 4 It can be seen that M is DL2000 Marker, and numbers 1 to 6 represent the corresponding CpPUP11 Gene-edited strains 1 to 6, of which strains 3, 4, and 6 are CpPUP11 Gene-edited positive strain; K+ is the vector plasmid, used as a positive control; K- is the non-transgenic papaya Tainong No. 2, used as a negative control; K is sterile water, used as a blank control.

[0067] Table 4 Primers for detecting transgenic positive plants

[0068]

[0069] Example 4 CpPUP11 Gene overexpression positive strains and CpPUP11 Measurement of the length of flower spikes and fruit stalks of gene-edited positive lines

[0070] The above CpPUP11 Gene overexpression positive strains and CpPUP11The gene-edited positive strains and the control non-transgenic papaya Tainong No. 2 (labeled ck) were planted in isolation nets and cultivated conventionally. The length of the flower spike was measured during the flowering period, and the length of the fruit stalk of each strain was measured during the fruiting period. The measured data were analyzed by one-way analysis of variance using PASW Statistics18 software, and the Duncan's new multiple range method was used to detect significant differences and make annotations. CpPUP11 The results of the measurements of the flower spikes and fruit stalks of the gene overexpression lines are shown in Tables 5, 6 and Figure 5~Figure 8 The results show that CpPUP11 The length of the panicle of gene overexpression positive line 3 was significantly longer than that of the non-transgenic papaya control. CpPUP11 The fruit stalk length of gene overexpression positive strains was significantly longer than that of non-transgenic papaya controls.

[0071] Table 5 Part CpPUP11 Flower spike length of gene overexpression positive lines

[0072]

[0073] Table 6 CpPUP11 Fruit stalk length of gene overexpression positive strains

[0074]

[0075] and, CpPUP11 The flower spike of gene-edited positive strain 6 became shorter, significantly shorter than the flower spike length of the non-transgenic control papaya. The results are shown in Tables 7 and Fig. 9 , Fig.10 As shown, the flowers fall off naturally in the late flowering period, resulting in no fruiting.

[0076] Table 7 CpPUP11 Flower spike length of gene-edited positive strain 6

[0077]

[0078] Example 5 Papaya overexpression strain CpPUP11 Determination of gene expression

[0079] design CpPUP11 qPCR primers for genes in papaya Cpa03g018830 The gene is an internal reference gene ( Cpa03g018830 The gene CDS coding sequence is shown in SEQ ID NO: 6). CpPUP11 Gene overexpression positive strains CpPUP11 The gene expression level was detected, and the non-transgenic papaya was used as the control (expression level was 1) to calculate CpPUP11 Gene overexpression positive strains CpPUP11 Gene expression level. CpPUP11qPCR primer sequences of genes and Cpa03g018830 The qPCR primer sequences of the genes are shown in Table 8. The details are as follows:

[0080] Pick CpPUP11 Total RNA was extracted from leaf samples of gene overexpression positive lines and non-transgenic control papayas using the Polysaccharide Polyphenol Plant Total RNA Extraction Kit (Tiangen Biochemical Technology, Beijing), and reverse transcribed into cDNA using the PrimeScriptTM RT Reverse Transcription Kit (Takara, Dalian). The cDNA was used as a template for qPCR. CpPUP11 qPCR primers for genes and Cpa03g018830 The qPCR primers of the gene were used for qPCR reaction in QuantStudioTM 1 Plus real-time fluorescence quantitative PCR instrument (Thermo Fisher Scientific, Shanghai). The enzyme used for qPCR was NOVA's Taq SYBR® Green qPCR Premix, and the reaction system was: Taq SYBR® Green qPCR Premix (universal) 10.0 μL, primers (10.0 μM) 0.5 μL each, cDNA template 1.0 μL, Nuclease-Free Water 8.0 μL. CpPUP11 and Cpa03g018830 The qPCR reaction program was: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 10 s, 58℃ annealing for 30 s, and 40 cycles. Each sample was biologically replicated three times, and the measured data were analyzed by one-way ANOVA using PASW Statistics18 software. Duncan's new multiple range method was used to detect significant differences and annotated. The results are shown in Figure 2. Fig.11 As shown, 6 CpPUP11 Gene overexpression positive strains CpPUP11 The gene expression levels were significantly increased compared with the control.

[0081] Table 8 Fluorescence quantitative PCR primers

[0082]

[0083] Example 6 CpPUP11 Identification and analysis of mutations at editing sites in gene-edited strains

[0084] PCR identification primers PUP11-F and PUP11-R (sequences shown in Table 9) were designed near the upstream and downstream of the editing site. CpPUP11The editing sites of gene-edited positive strains (strain 3, strain 4, and strain 6) were amplified by PCR and sequenced for identification. The enzyme used for PCR was Vazyme's 2 x Rapid Taq Master Mix. The PCR reaction system was: 9.5 μL of sterile water, 1 μL of each primer (10 μM), 12.5 μL of Taq Mix, and 1 μL of DNA template. The PCR amplification program was: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 30 s, and 35 cycles; finally extension at 72°C for 7 min. The PCR amplification products were detected by 2% agarose gel electrophoresis, and the 3 gene-edited positive plants were CpPUP11 The electrophoresis of PCR products at the gene editing site is shown in Fig.12 .from Fig.12 It can be seen that the three gene editing positive strains can amplify a 461bp band. The PCR products were sent to the company for sequencing. The editing effect was judged according to the sequencing peak graph and sequencing sequence, and the mutation of the editing site was obtained. The sequencing peak graph shows that the two target sites of strains 3 and 4 were not edited, and strain 6 was a multi-peak graph, which was a non-homozygous mutation. Hi-TOM sequencing was performed on papaya samples with non-homozygous mutations to clarify the mutation form of non-homozygous mutations. The specific method is: primers are designed near the upstream and downstream of the editing site, and Hi-TOM adapter primers are added to the 5' ends of the upstream and downstream primers respectively to obtain Hi-TOM sequencing primers Hi-PUP11-F and Hi-PUP11-R (sequences are shown in Table 9), and PCR reactions are performed on sample DNA with non-homozygous mutations with this primer. The enzyme used for PCR is Vazyme's 2 x Rapid Tag Master Mix. The PCR reaction system was as follows: 9.5 μL sterile water, 1 μL primers (10 μM), 12.5 μL Taq Mix, and 1 μL DNA template. The PCR amplification program was as follows: 94°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 56°C annealing for 30 s, 72°C extension for 30 s, 35 cycles; and finally 72°C extension for 7 min. The PCR amplification product was confirmed to have the target band by 2% agarose gel electrophoresis and then sent to Beijing Qingke Biotechnology Co., Ltd. for Hi-TOM sequencing.

[0085] According to Hi-TOM sequencing of non-homozygous mutations, the mutation forms of the editing sites of gene-edited strain 6 were analyzed, and the results are shown in Table 10. As can be seen from Table 10, strain 6 is a mosaic mutation, and there are five editing methods, namely: inserting only one base (A) at the first target site; inserting one base (A) at the first target site and inserting one base (T) at the second target site; inserting one base (A) at the first target site and deleting 4 bases at the second target site (AAAC); deleting 4 bases at the second target site (AAAC); inserting only one base (T) at the second target site.

[0086] Table 9 Primers for editing site identification

[0087]

[0088] Note: The bold part indicates the adapter primer.

[0089] Table 10 Editing site mutation results of gene editing positive strain 6

[0090]

[0091] Example 7 Papaya CpPUP11 Measurement of fruit setting rate in gene overexpression lines

[0092] Planting papaya CpPUP11 Gene overexpression positive strain 3 and papaya Tainong 2, papaya at harvest CpPUP11 Gene overexpression positive strain 3 can produce 1 to 2 fruits on a single fruit stalk (e.g. Fig.13 As shown in the figure, the number of fruit stalks per plant was 18, and the number of fruits set was 22; the number of fruits set per fruit stalk of the non-transgenic control plant (i.e., papaya Tainong No. 2) was fixed at 1 during the same harvest period, the number of fruit stalks per plant was 16, and the number of fruits set was 16. CpPUP11 The fruit setting rate of gene overexpression positive line 3 can be increased by more than 30% compared with the non-transgenic control plants.

[0093] In summary, the present invention fully proves CpPUP11 The gene can positively regulate the development of papaya flower spikes and fruit stalks, as well as the fruit setting rate. Those skilled in the art can use gene overexpression technology or gene editing technology to improve the CpPUP11 Gene expression level, thereby increasing the length of papaya flower spike and fruit stalk and the fruit setting rate.

[0094] Although the present invention is for better verification CpPUP11 The effect of gene expression on the length of papaya spike and fruit stalk and fruit setting rate was designed to reduce CpPUP11 Gene editing vectors for gene expression. CpPUP11Based on the premise that the gene expression amount affects the regulation of papaya flower spike and fruit stalk length and fruit setting rate, technicians in this field use conventional gene editing technology to adjust and design a method to improve CpPUP11 Gene editing vectors that increase gene expression CpPUP11 Gene expression level of gene editing vector, transformed papaya, to obtain CpPUP11 A papaya strain with high gene expression level can improve the length of the papaya flower spike and fruit stalk and the fruit setting rate.

[0095] At the same time, the present invention proves CpPUP11 Under the premise of the regulatory effect of gene expression on the length of papaya flower spike and fruit stalk and fruit setting rate, those skilled in the art can also process papaya by other gene editing technologies, such as promoter region editing, 5'UTR editing and other technologies, to obtain CpPUP11 A papaya strain with high gene expression level can improve the length of the papaya flower spike and fruit stalk and the fruit setting rate.

[0096] Therefore, the present invention can use gene editing technology including but not limited to CRISPR-Cas mediated enhancer knock-in technology, promoter region editing technology or 5'UTR editing technology to treat papaya to increase the expression of CpPUP11 Gene expression level.

[0097] Although the above embodiments have been described in detail, they are only a part of the embodiments of the present invention, rather than all the embodiments. A person skilled in the art can also obtain other embodiments based on the embodiments without inventiveness, and these embodiments all belong to the protection scope of the present invention.

Claims

1. An application of a gene that affects the length of papaya flower spikes and fruit stalks and fruit setting rate, characterized in that: The application is achieved by increasing CpPUP11 Gene expression to increase the length of papaya flower spikes and fruit stalks and fruit setting rate; CpPUP11 The gene sequence of the gene is shown in SEQ ID NO:

1.

2. The use of the gene affecting the length of papaya flower spike and fruit stalk and fruit setting rate according to claim 1, characterized in that: The application is to use gene overexpression technology or gene editing technology to increase CpPUP11 Gene expression level.

3. The use of the gene affecting the length of papaya flower spike and fruit stalk and fruit setting rate according to claim 2, characterized in that: The gene overexpression technique is to CpPUP11 Gene overexpression vector or containing CpPUP11 The recombinant strain of the gene overexpression vector was transformed into papaya, so that CpPUP11 Gene overexpression.

4. The use of the gene affecting the length of papaya flower spike and fruit stalk and fruit setting rate according to claim 3, characterized in that: CpPUP11 Gene overexpression vector is CpPUP11 The amplified product of the gene is connected to the backbone vector to obtain.

5. The use of the gene affecting the length of papaya flower spike and fruit stalk and fruit setting rate according to claim 4, characterized in that: CpPUP11 The amplified product of the gene was obtained by PCR amplification using the cDNA of papaya as a template and primers PUP11--F / PUP11--R; Among them, PUP11--F:5'-CG GGATCC ATGGAGGCGAGTAAAT-3′; <h2 style=";text-align:left;direction:ltr">PUP11--R:5'-TCC<h2 style=";text-align:left;direction:ltr"> CCCGGG <h2 style=";text-align:left;direction:ltr"> TTAAACATGAGCC-3'.

6. Use of a gene affecting the length of papaya flower spike and fruit stalk and fruit setting rate according to any one of claims 2 to 5, characterized in that: The gene overexpression technique comprises the following steps: Build CpPUP11 Gene overexpression vector; use CpPUP11 Gene overexpression vector, construct containing CpPUP11 Recombinant strains of gene overexpression vectors; Utilize the included CpPUP11 The recombinant strain of the gene overexpression vector was transformed into papaya to make CpPUP11 Gene overexpression.

7. The use of the gene affecting the length of papaya flower spike and fruit stalk and fruit setting rate according to claim 6, characterized in that: The papaya transformation method uses papaya seedling stem segments as explants to induce embryonic callus, and then uses CpPUP11 The recombinant strain containing the gene overexpression vector was transformed to obtain CpPUP11 Gene overexpression papaya lines.

8. The use of the gene affecting the length of papaya flower spike and fruit stalk and fruit setting rate according to claim 2, characterized in that: The gene editing technology includes CRISPR-Cas mediated enhancer knock-in technology, promoter region editing technology or 5'UTR editing technology.

9. A papaya breeding method, characterized in that: The papaya breeding method is to increase the CpPUP11 Gene expression levels to obtain papaya strains that affect the length of papaya flower spikes and fruit stalks and fruit setting rate; CpPUP11 The gene sequence of the gene is shown in SEQ ID NO:

1.

10. A method for regulating the length of papaya flower spikes and fruit stalks and the fruit setting rate, characterized in that: The method is to plant CpPUP11 Papaya strains with high gene expression levels to increase the length of papaya flower spikes and fruit stalks and fruit setting rate; CpPUP11 The gene sequence of the gene is shown in SEQ ID NO:1.

Images