Papaya CpYYL gene, protein, primer pair, expression recombinant vector, transformant and application

Abstract

The invention provides a papaya CpYYL gene, a protein, a primer pair, an expression recombinant vector, a transformant and application, and belongs to the technical field of papaya gene breeding. The nucleotide sequence of the papaya CpYYL gene is shown as SEQ ID No.1. YYh and YhYh plants are obtained by expressing the papaya CpYYL gene, and excellent male and female same-flower plants (XYh) without sex separation can be obtained after the YYh and YhYh plants are hybridized with XX plants, so that the problem of sex separation of papaya in production is thoroughly solved, and the economic value of papaya is remarkably improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of papaya gene breeding, and particularly relates to a papaya CpYYL gene, protein, primer pair, expression recombinant vector, transformant and application. BACKGROUND

[0002] Papaya (Carica papaya L.) belongs to the Caricaceae family of the Brassicales order, and is mainly distributed in tropical and subtropical regions. Papaya has high nutritional value, and its nutritional value ranks first among 34 kinds of fruits according to the assessment of 8 kinds of vitamins and minerals necessary for the human body. Papain is widely used in the food industry and medical field.

[0003] The three sexes of papaya are female (XX), male (XY) and hermaphrodite (XY h ), the compact pear-shaped fruit shape of the hermaphrodite papaya plant is lower in transportation cost than the round female fruit, the yield is higher because each tree of the hermaphrodite plant bears fruit, and the hermaphrodite papaya plant is self-pollinated, so the hermaphrodite (XY h ) plant is generally planted in production.

[0004] The hermaphrodite (XY h ) papaya plant is self-pollinated to produce seeds containing 2 / 3 hermaphrodite (XY h ) plants and 1 / 3 female (XX) plants, Y h Y h genotype seeds are naturally sterile. Commercial varieties are hybrid varieties, and the female plant is the female parent and the hermaphrodite plant is the male parent for seed production, 1 / 2 of the seeds are female plants. In the current papaya planting mode, 5 seedlings are usually planted in each hole to ensure that at least one seedling is a hermaphrodite plant. These seedlings need to grow to flowering to identify the hermaphrodite plant and then cut off the female plant and the extra hermaphrodite plant. This planting mode greatly increases the planting cost, and the water and fertilizer utilization efficiency is reduced due to the dense planting in the seedling stage, so the sex separation of papaya is a major problem that needs to be solved. SUMMARY

[0005] The present application aims to provide a papaya CpYYL gene, protein, expression recombinant vector, transformant and application, by expressing the CpYYL gene in papaya, YY h and Y h Y h plants are obtained, and after hybridization with XX type plants, excellent hermaphrodite plants (XY h), thereby completely solving the low efficiency and high cost of cultivation mode (dense planting in seedling stage, thinning after flowering, and processing to cut off 80% of trees in the ground) of papaya due to gender separation, resulting in the consequences of increase of irrigation, fertilizer, and labor costs, affected root system and crown development of papaya trees, and delayed harvest period. In this study, the Y h Y h genotype, and then the XX female plant is crossed with the Y h Y h plant to obtain 100% non-separation XY h plant, which eliminates the labor cost of dense planting and thinning, reduces the cost of irrigation and fertilization, has developed root system and crown, enhances the ability of plants to resist adversity and diseases and pests, and has early results and harvest period, improves yield and quality. In order to achieve the above purposes, the present application provides the following technical solutions.

[0006] The present application provides a papaya CpYYL gene, and the nucleotide sequence of the papaya CpYYL gene is shown in SEQ ID No. 1.

[0007] The present application also provides a CpYYL protein encoded by the papaya CpYYL gene, and the amino acid sequence of the CpYYL protein is shown in SEQ ID No. 2.

[0008] The present application also provides a primer pair for amplifying the papaya CpYYL gene, and the nucleotide sequence of the upstream primer is shown in SEQ ID No. 3, and the nucleotide sequence of the downstream primer is shown in SEQ ID No. 4.

[0009] The present application also provides an expression recombinant vector, which comprises an initial expression vector and the papaya CpYYL gene.

[0010] The present application also provides a transformant, which comprises the expression recombinant vector and a competent Agrobacterium cell.

[0011] The present application also provides an application of the papaya CpYYL gene in cultivating fertile papaya YY h or Y h Y h plant.

[0012] The present application also provides an application of overexpression of the papaya CpYYL gene in cultivating papaya XY

[0013] The present application also provides a method for cultivating fertile papaya Y h Y h plant, which comprises the following steps:

[0014] (1) the expression recombinant vector is transformed into the papaya callus, culture is carried out, and the transgenic seedling ProYYL::YYL / ZH XX T1 is obtained;

[0015] (2) the transgenic seedling ProYYL::YYL / ZH XX T1 obtained in step (1) is crossed with papaya SunUp, Zhongbai or Hongling, and the hermaphroditic F1 generation plant ProYYL::YYL / SunUpXY h ;

[0016] (3) the hermaphroditic F1 generation plant ProYYL::YYL / SunUpXY h obtained in step (2) is selfed, and the fertile papaya YY h Y h plant is obtained.

[0017] The application further provides a method for cultivating the fertile papaya YY h plant, comprising the following steps:

[0018] the hermaphroditic F1 generation plant ProYYL::YYL / SunUpXY h obtained is emasculated, and is crossed with the male papaya XY plant, so that the fertile papaya YY h plant is obtained.

[0019] The application further provides a method for cultivating the papaya gender non-separation hermaphroditic XY h plant, comprising the following steps:

[0020] the fertile papaya YY h Y h plant obtained is crossed with the female papaya XX plant, so that the papaya gender non-separation hermaphroditic XY h plant is obtained.

[0021] Compared with the prior art, the application has the beneficial effects that:

[0022] The application is based on the sex chromosome structure of papaya, and structural variation analysis is carried out on the genes of the Y chromosome (MSY) specific to the male of papaya and the Y h chromosome gender determination region (HSY) specific to the hermaphroditic plant, a homologous gene CpYYL of the Arabidopsis thaliana embryo abortion gene EMB506 is finally screened, the gene is located in the gender determination region, is a complete functional gene on X, and part of sequences of the first exon and the first intron on HSY become pseudogenes due to deletion, and it is speculated that the gene may be the cause of the papaya Y h Y hCandidate genes for genotype abortion. Therefore, this invention, by combining bioinformatics analysis of the sex-determining region of papaya with gene function research, verifies the key role of the gene CpYYL in the embryonic development of papaya, providing important gene resources for functional genomics research of papaya, and offering new molecular tools and methods for papaya breeding.

[0023] This invention obtains Y chromosomes that can develop normally through genetic modification. h Y h When this plant is crossed with an XX-type plant, superior hermaphroditic plants (XY) can be obtained, where male and female flowers do not separate. h This invention completely solves the problem of sex segregation in papaya production, significantly increasing the economic value of papaya. Furthermore, it provides reference and technical support for the regulation of embryonic development in papaya and other hermaphroditic plants, and for plant genetic improvement to address sex segregation in the offspring of self-pollination in hermaphroditic plants. Simultaneously, this invention provides important technical support for solving the major problem of sex segregation in papaya by developing reliable molecular markers for identifying female and hermaphroditic papaya plants.

[0024] This invention improves the Y- of papaya h Y h The expression level of the CpYYL gene in papaya affects the genotype Y. h Y h The embryos do not fail, allowing for the acquisition of normally developing papaya Y chromosomes. h Y h The plant was hybridized with XX-type plants to obtain superior hermaphroditic plants (XY) that do not separate male and female flowers. h This invention completely solves the problem of sex separation in papaya production, thereby increasing the economic value of papaya. At the same time, based on the method of this invention, new papaya varieties with excellent traits can be bred, which can meet the diversified market demand and promote the development of the papaya industry. In addition, it also helps to accelerate the development of new varieties and the improvement of traditional breeding methods. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1The gene structure and conserved protein domain of the CpYYL alleles in the sex-determining regions of papaya X and HSY are shown, where a is the gene structure of the CpYYL alleles CpYYL-HSY and CpYYL-X; and b is the conserved domain of the CpYYL alleles CpYYL-HSY and CpYYL-X.

[0027] Figure 2 The images show actual specimens from the cultivation of ProCpYYL::CpYYL transgenic plants. a) shows the embryo extracted from a sterilized mature papaya seed; b) shows callus growth from the embryo after 25 days of culture in M13 medium; c) shows callus after 40 days of culture in M13 medium; d) shows well-growing embryogenic callus on Ci medium; e) shows co-culturing the embryogenic callus with the target vector on filter paper; f) shows the co-cultured embryogenic callus on differentiation medium; g) shows resistant green shoots selected on selection medium; h) shows the growth of ProCpYYL::CpYYL resistant shoots; i) shows the validated transgenic plant ProCpYYL::CpYYL / ZH XX T1, scale bar = 1 cm.

[0028] Figure 3 To verify the PCR results of the transgenic papaya lines, two pairs of primers were used to perform PCR analysis on the DNA of 30 transgenic papaya lines. The YYL-F / R primers were designed to amplify the CDS sequence of PapYYL, and the YYL-vector-F / R primers were used to amplify the partial CDS sequence and vector sequence of YYL.

[0029] Figure 4 To verify whether the samples were ProCpYYL::CpYYL transgenic plants using the transgenic verification primers YYL-F / R and YYL-vector-F / R, agarose gel electrophoresis image was obtained, identifying 16 transgenic lines. Lanes 1-20 contain plant DNA samples, with the control group being Y... h Y h DNA samples from plants, as well as female, male, and hermaphroditic plants;

[0030] Figure 5 To utilize X-copy specific primers Intron1X-specific-F / R, Y h Specific primer TE-1-Y h -specific-F / R and Y-specific primers TE-2-Y-specific-F / R primers were used to verify whether the sample was Y. h Y h And YY h Agarose gel electrophoresis images of genotyped plants; lanes 1-20 are plant DNA samples, and control is Y. h Yh DNA samples from plants, as well as female, male, and hermaphroditic plants;

[0031] Figure 6 For Papaya Y h Y h And YY h Images showing the phenotypic characteristics of genotyped plants, where a represents a yellow-colored male plant (ZHXY); and b represents a white hermaphroditic plant (ZBXY). h c represents the YY plants obtained by screening the ProCpYYL::CpYYL vector. h ;d represents the Y plants obtained after screening following transformation with the ProCpYYL::CpYYL vector. h Y h . Detailed Implementation

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0034] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0035] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0036] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0037] This invention provides a papaya CpYYL gene, the nucleotide sequence of which is shown in SEQ ID No. 1.

[0038]

[0039] The present invention also provides a CpYYL protein encoded by the papaya CpYYL gene, the amino acid sequence of which is shown in SEQ ID No. 2.

[0040] In this invention, the amino acid sequence of the CpYYL protein is: MASWATTVFTANYFLPRLSTVVTASASPSSFENGAFKVLGRLRSGVFGLLATQRESVVRTCVFGSKDRKPRLLIHQEFREEPDDGSGSDYEEEGEEMGKNDLDFESDWEDERGNAMAMVISVEEPTTNPYEEELAKEVEQLLGPEERAIFQKNVIPNLGNLSTEKWKPLHTLALSGQIPFIDQLLENGLDINAVDKDGLTALHKAIIGKNETVISHLLRKGASPNVQDRDGATPLHYAVEVGVIQTVKLLIKYNVDVNVADNEGWTPLHVAVQSRNRDITKVLLITGSDKTRRNKDGRTPLDLSFCYGKDFKSYDLAKLLKIIATDQYP (SEQ ID No. 2).

[0041] The present invention also provides primer pairs for amplifying the aforementioned papaya CpYYL gene, wherein the nucleotide sequence of the upstream primer is shown in SEQ ID No. 3 and the nucleotide sequence of the downstream primer is shown in SEQ ID No. 4.

[0042] In this invention, the nucleotide sequence of the upstream primer is: AAAACTGCAGATGGCCTCGTGGGCTACGACAGTT (SEQ ID No. 3); the nucleotide sequence of the downstream primer is: CCCAACGCGTTTATGGATATTGGTCAGTTGCTATGATC (SEQ ID No. 4).

[0043] The present invention also provides an expression recombinant vector, comprising an initial expression vector and the aforementioned papaya CpYYL gene.

[0044] In this invention, the initial expression vector is preferably pOX.

[0045] The present invention also provides a transformant comprising the aforementioned expression recombinant vector and competent Agrobacterium cells.

[0046] This invention also provides the application of the aforementioned papaya CpYYL gene in the cultivation of fertile papaya YY. h Or Y h Yh Applications in plants.

[0047] This invention also provides the application of overexpressing the aforementioned papaya CpYYL gene in the cultivation of papaya plants with hermaphroditic and non-separating male and female flowers.

[0048] This invention also provides a method for cultivating fertile papaya Y. h Y h The method for treating plants includes the following steps:

[0049] (1) The expression recombinant vector was transferred into papaya callus and cultured to obtain transgenic seedlings ProYYL::YYL / ZH XX T1;

[0050] (2) The transgenic seedlings ProYYL::YYL / ZH XX T1 obtained in step (1) were crossed with papaya SunUp, Zhongbai or Hongling to obtain the hermaphroditic F1 generation plants ProYYL::YYL / SunUpXY h ;

[0051] (3) The hermaphroditic F1 generation plants ProYYL::YYL / SunUpXY obtained in step (2) h Self-pollination yields fertile papaya Y-type seeds. h Y h .

[0052] This invention also provides a method for cultivating fertile papaya YY. h The method for treating plants includes the following steps:

[0053] The obtained hermaphroditic F1 generation plants ProYYL::YYL / SunUpXY h Male papaya plants were emasculated and crossed with male papaya plants XY to obtain fertile papaya plants YY. h Plant.

[0054] This invention also provides a method for cultivating papaya plants with non-separating sexes and hermaphroditic flowers (XY). h The method for treating plants includes the following steps:

[0055] The obtained fertile papaya Y h Y h The plant was crossed with a female papaya XX plant to obtain the aforementioned papaya species that are hermaphroditic and do not separate sexes (XY). h Plant.

[0056] Example 1: Construction of CpYYL expression vector

[0057] Seeds of papaya varieties SunUp, Zhonghuang (ZH), and Zhongbai (ZB) are preserved resources at the Genomics Center of Fujian Agriculture and Forestry University. Papaya seedlings were cultivated in greenhouses at Fujian Agriculture and Forestry University at a temperature maintained at 28℃ with a photoperiod of 16h / 8h. After two months of cultivation, the seedlings were planted in papaya greenhouses in Yongchun, Fujian, and in experimental plots on the Fujian Agriculture and Forestry University campus.

[0058] Preparation of cDNA as a cloning template: Take 100mg of SunUp leaves as a sample, grind them thoroughly in liquid nitrogen, extract total RNA according to the instructions of the RNA extraction kit (Plant RNA Kit, Omega R6827), and reverse transcribe it into cDNA for later use.

[0059] Cloning of the CpYYL gene: The primer sequence designed for amplifying the CpYYL gene with a vector adapter is as follows: YYL-CDS-PstF:

[0060] 5'-AAAACTGCAGATGGCCTCGTGGGCTACGACAGTT-3' (SEQ ID No. 3), YYL-CDS-MluR:

[0061] 5'-CCCAACGCGTTTATGGATATTGGTCAGTTGCTATGATC-3' (SEQ ID No. 4),

[0062] Using the cDNA prepared above as a template, PCR amplification was performed using the primers described above. The reaction system is shown in Table 1.

[0063] Table 1 PCR reaction system

[0064] Component Amount PrimeSTAR Max DNA Polymerase 25 μL Primer-F / R 1 μL cDNA 1 μL ddH2O to 50 μL

[0065] The PCR amplification program was as follows: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 30 s, for 34 cycles. After the cycle, the final extension was performed at 72℃ for 5 min. The PCR product was subjected to agarose gel electrophoresis and the target fragment was recovered using a DNA gel recovery kit.

[0066] The pOX vector was linearized using the restriction enzymes PstI and MluI, and digested at 37°C for 1 h. The reaction system is shown in Table 2.

[0067] Table 2 Linearized reaction system

[0068]

[0069]

[0070] The target fragment was ligated to the pOX vector and the ligation was carried out at room temperature for 2 hours. The reaction system is shown in Table 3.

[0071] Table 3. Reaction system for target fragment ligation.

[0072] Component Amount T4 DNA Ligase Buffer (10X) 2 μL T4 DNA Ligase 1 μL Linearized Vector 200 ng Target Fragment 40 ng ddH2O to 20 μL

[0073] Add 5 μL of the ligation product to 50 μL of E. coli DH5α competent cells, gently mix, and incubate on ice for 30 min; heat shock at 42℃ for 60 s; stand on ice for 2 min; add 700 μL of antibiotic-free LB liquid medium (Coolaber); incubate at 200 rpm and 37℃ on a shaker for 1 h; centrifuge at 5000 rpm for 1 min; discard the supernatant, resuspend the bacterial cells, and spread an appropriate amount of the bacterial suspension onto LB solid medium (Coolaber) containing 50 mg / L kanamycin resistance, incubate at 37℃ upside down overnight until single colonies grow, and perform positive clone detection and sequencing to obtain the nucleotide sequence of the CpYYL gene as shown in SEQ ID NO.1 and the amino acid sequence as shown in SEQ ID NO.2.

[0074] Example 2: Construction of the ProCpYYL::CpYYL expression vector

[0075] DNA was extracted from the Sunup papaya variety using the CTAB method: Fresh papaya leaves (about the size of a fingernail) were placed in 2.0 ml centrifuge tubes. A 5 mm steel ball was added to each tube, and the tubes were clearly labeled. The tubes were then flash-frozen in liquid nitrogen and disrupted using a tissue homogenizer at 20 rpm for 1 min. 700 μL of CTAB extraction buffer was added to each tube, vortexed, and heated at 65°C for 1 h. An equal volume of chloroform was added, and the tubes were centrifuged at 13000 rpm for 10 min. The supernatant (450 μL) was transferred to a new 1.5 mL centrifuge tube, and an equal volume of pre-chilled isopropanol was added. The tubes were inverted several times until the solution was clear, and the solution was precipitated at -20°C for 1 h. The supernatant was discarded after centrifugation at 13000 rpm for 10 min, and the tubes were washed twice with 70% ethanol. The tubes were then centrifuged at 13000 rpm for 5 min, and the supernatant was discarded. The tubes were then centrifuged empty for 2 min, and any excess liquid except for the precipitate was removed. Open the lid, place it in a 65℃ oven and dry for 5 minutes to allow the alcohol to evaporate completely. Add 50μL ddH2O to each precipitate tube and store at -20℃ for later use.

[0076] Designed for amplifying the CpYYL promoter with a vector adapter (ProCpYYL)

[0077]

[0078] 5'-ACCGGAATTCATCGCGACTTGTAGGTGCCATAGT ACTCTAGAACTAGTTAATTAAATGGAGTTTGAGGATCAAGATGAGC-3' (SEQ ID No. 5), ProCpYYL-R: 5'-AACCCTGCAGTGATTGCCAACTTGTTTATTCAACCCTTGTGT-3' (SEQ ID No. 6).

[0079] The expression vector was constructed using the same method as in Example 1, except that: the DNA prepared in this example was used as a template, and the promoter sequence was obtained by PCR amplification using the primer set shown in SEQ ID No. 5 and SEQ ID No. 6. The pOX vector was then linearized using the restriction enzymes EcoRI and PstI, and the promoter ProCpYYL was ligated into the pOX vector to obtain the ProCpYYL::CpYYL expression vector.

[0080] Example 3: Cultivation of transgenic plants

[0081] Transformation of competent Agrobacterium cells: Remove GV3101 transformed competent Agrobacterium cells stored at -80℃ and freeze-thaw them on ice. Add 100 ng of the ProCpYYL::CpYYL vector plasmid constructed in Example 2, mix well, and incubate on ice for 5 min, in liquid nitrogen for 5 min, in a 37℃ water bath for 5 min, and in an ice bath for 5 min. Add 700 μL of LB liquid medium and incubate on a shaker at 28℃ and 200 rpm for 2 h. Centrifuge at 6000 rpm, discard the supernatant, resuspend the cells, and plate them on LB solid medium containing 50 mg / L rifampicin and 50 mg / L kanamycin. Incubate at 28℃ in the dark for 2 days. Pick single colonies and add them to a test tube containing 5 ml of the corresponding antibiotic-containing LB liquid medium (both antibiotics and LB medium were purchased from Coolaber). Incubate at 28℃ in the dark until the bacterial culture reaches OD. 600 Once the value reaches 1.0, the bacterial culture is verified by PCR and then stored at -80℃ with glycerol for later use.

[0082] Seed disinfection: In a clean bench, remove mature papaya ZH seeds, wash twice with sterile water, disinfect with 75% alcohol for 1 minute, wash four times with sterile water, disinfect with 3.5% NaClO3 for 20 minutes, wash four times with sterile water, disinfect with 0.1% mercury for 10 minutes, and wash four times with sterile water. After seed disinfection, blow until there are no obvious water marks, then remove the embryos. Place the removed intact embryos in K4 medium and culture for one month to allow them to expand. Figure 2 c is the display.

[0083] Callus induction and propagation: After one month of culture in K4 medium, callus was transferred to M13 medium to induce callus formation. Subculture was performed monthly, and callus tissue began to grow after approximately five months. The callus tissue was then selected and inoculated into Ci medium for propagation. After four generations of callus propagation, it was ready for infection and transformation. Figure 2 d shows the healing process.

[0084] Callus infection and transformation: Infection: Agrobacterium should ideally turn a bright orange color when shaken. Centrifuge at 3500 rpm for 10 min, discard the supernatant, and resuspend the bacteria in 1 / 2 MS liquid medium until the Agrobacterium OD value is 0.8. Add approximately 10 mL of embryogenic callus to 40 mL of bacterial culture and infect for 20 min (including 4 min of vigorous shaking). Stand the callus upright to settle, discard the supernatant, blot the callus dry with filter paper, and blow dry for 20 min, trying to dry it as much as possible, but do not blow until the callus color turns dark brown. Co-culture: Place the callus on Ci medium lined with sterile filter paper and incubate in the dark for 24 h. Antimicrobial inhibition: After co-culture, slightly dry the callus and transfer it to Ci double antibody (Cb 200 mg / L and Cef 200 mg / L) medium for dark incubation. After 7 days, transfer it again to Ci double antibody medium for dark incubation. This is the critical period for antimicrobial inhibition. During this period, observe frequently for bacterial growth. If bacterial growth occurs, transfer and treat it promptly. Screening: Callus that had been placed twice on Ci antibiotics was transferred to MBNTH medium (6BA and NAA at 0.2 mg / L, termethin at 100 mg / L, and hygromycin at 50 mg / L) and screened under light. During this period, the growth of bacteria was frequently observed; if growth was observed, the callus was transferred promptly; otherwise, it was transferred to MBNTH medium once a month. After three months of screening, the selected yellow-green shoots were picked out and placed on fresh MBNTH medium. Figure 2 g shows callus in differentiation.

[0085] Rooting of transgenic seedlings: Take healthy, positive tissue culture seedlings over 2cm in length, cut off the white tissue block at the bottom and the messy leaves, leaving a small section of stem that can be inserted into the soil. Soak the seedlings in a 0.01% strong rooting powder solution for about 3 hours. Mix the rooting solution with clay to form a mud, wrap it around the stem end left at the bottom of the tissue culture seedling, and then bury the seedling with the mud in the mixed nutrient soil. Water with a small amount of 0.2% methyl thiophanate solution, then water thoroughly, cover, and place in a greenhouse. Rooting will begin in about 2 weeks. Water regularly during this period, but do not remove the cover until roots have formed. Transplant the seedlings into larger pots as soon as they grow.

[0086] The formulation of the culture medium involved in this embodiment is shown in Table 4.

[0087] Table 4. Formulation composition of different culture media

[0088]

[0089]

[0090] Example 4Y h Y h And YY h Acquisition and identification of plants

[0091] 4.1 Identification of the transgenic positive seedlings obtained in Example 3 by PCR amplification using specific primers. Primers YYL-F (CAGTAGTGAGGACTTGTGTGTTTGG(SEQ ID No. 8)) and YYL-R (TATCTCTATTCCTACTCTGGACTGC(SEQ ID No. 9)) can amplify the CpYYL gene, and primers YYL-vector-F (GAATGTGATTCCTAACCTTGGCA(SEQ ID No. 10)) and YYL-vector-R (ACCGGCAACAGGATTCAATC(SEQ ID No. 11)) can amplify the CpYYL gene and a sequence segment on the vector. The presence of bands on both primer pairs indicates that the plant is a transgenic plant. A total of 30 lines were screened, resulting in 30 transgenic lines of ProCpYYL::CpYYL / ZH XX T1 generation. Figure 3 As shown in Table 5.

[0092] Table 5 PCR reaction system

[0093] Component Volume 2x Rapidly Master Mix 10 μL Primer-F / R 0.5 μL ddH2O 8 μL DNA 1 μL

[0094] The PCR amplification program was as follows: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 30 s, 34 cycles, followed by 72℃ extension for 5 min after each cycle.

[0095] 4.2 In order to obtain fertile Y chromosomes h Y h Plants, with ProCpYYL::CpYYL / ZH XX T1 generation and papaya SunUpXY h The F1 generation was obtained through hybridization, and transgenic hermaphroditic F1 plants ProCpYYL::CpYYL / SunUpXY were selected. h The plant was self-pollinated to obtain F2 generation segregating plants. The presence of Y chromosome in the F2 segregating population was detected using molecular markers. h Y h Genotype of plant.

[0096] At the same time, ProCpYYL::CpYYL / SunUpXY h After emasculating the plants, they were crossed with Papaya XY to obtain the F2 population. YY was then selected using molecular markers. h Genotype of plant.

[0097] Y h Y h And YY h The method for identifying transgenic plants was as follows: DNA was extracted from the leaves of the obtained F2 generation segregators using the CTAB method. Two pairs of transgenic verification primers, YYL-F / R and YYL-vector-F / R, were used to identify whether the plants were transgenic. Twenty F2 generation plants were selected for identification, and the results are as follows: Figure 4 As shown.

[0098] The results showed that the PCR results of 16 F2 progeny plants (3 / 4 / 5 / 6 / 8 / 9 / 10 / 11 / 12 / 14 / 15 / 16 / 17 / 18 / 19 / 20) showed bands for both primer pairs YYL-F / R and YYL-vector-F / R, indicating that these lines were all transgenic lines.

[0099] 4.3 Sequence alignment of the X copy and HSY copy of the CpYYL gene revealed that the HSY copy of the CpYYL gene lacks partial sequences of the first exon and the first intron. Therefore, primers Intron1X-specific-F (TTTGTAGGTGTATCCTACTACTTCG (SEQ ID No. 12)) and Intron1X-specific-R (GCTCAACCACGCAATGACTGAAGAT (SEQ ID No. 13)) were designed on the first intron of the CpYYL_X copy to serve as molecular markers for identifying plants containing the X chromosome. Combined with the Papaya Y chromosome... h Specific verification primer TE-1-Y h -specific-F(CGAGAGCAAGTGGCACGCAAGG(SEQ ID No.14)) and TE-1-Y h -specific-R (GTCACCTGTCTCGCCGACGTG (SEQ ID No. 15)) and Y-specific primer TE-2-Y-specific-F

[0100] (GATGATTAAATCAATTATTAGTCGCT(SEQ ID No. 16)) and TE-2-Y-specific-R(AATAAGAGCAACATCGGCATCG(SEQ ID No. 17)) can distinguish whether offspring plants contain a Y chromosome or a Y chromosome. h Chromosome. Y chromosome identified. h Y h And YY h Genetically modified plants.

[0101] The removal of samples containing the X chromosome was verified using 20 F2 progeny plants with Intron1X-specific-F / RX copy-specific primers. TE-1-Y was also used. h Validation was performed using TE-2-Y-specific-F / R and TE-2-Y-specific-F / R primers, which can distinguish Y h Y h And YY h Plant. Identification results as follows: Figure 5 The image shown is a gelatin print.

[0102] The results showed that when these 20 strains were verified using Intron1X-specific-F / R primers, those that did not produce a band were suspected Y-cell lines. h Y h And YY h Based on the transgenic verification results, seven F2 progeny plants (6 / 11 / 12 / 14 / 16 / 17 / 18) were identified as suspected Y-type plants. h Y h And YY h Plants; using TE-1-Y h The TE-2-Y-specific-F / R and TE-2-Y-specific-F / R primers were used for verification, and it was finally confirmed that 6 / 17 was Y. h Y h Plants, 11 / 12 / 14 / 16 / 18 are YY h Plant.

[0103] 4.4 The obtained Y h Y h YY h Phenotypic observation was performed on genotyped plants. Results are as follows: Figure 6 As shown in c and d.

[0104] The results show that YY h The flower's phenotypic characteristics include a long inflorescence stalk, and the flower having only stamens, making it a male flower, while Y... h Y h The phenotype is hermaphroditic, with both male and female flowers.

[0105] As can be seen from the above embodiments, the present invention provides a papaya CpYYL gene, protein, expression recombinant vector, transformant, and application. The present invention increases the expression level of the CpYYL gene in papaya, thereby increasing the expression level of the papaya genotype Y. h Y h The embryos do not fail, and fertile papaya Y chromosomes are obtained. h Y h The plant was hybridized with XX-type plants to obtain superior hermaphroditic plants (XY) that do not separate male and female flowers. hThis will completely solve the problem of sex separation in papaya production and increase the economic value of papaya.

[0106] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A papaya CpYYL gene, characterized in that, The nucleotide sequence of the papaya CpYYL gene is shown in SEQ ID No.

1.

2. A CpYYL protein encoded by the papaya CpYYL gene, characterized in that, The amino acid sequence of the CpYYL protein is shown in SEQ ID No.

2.

3. A primer pair for amplifying the papaya CpYYL gene as described in claim 1, characterized in that, The nucleotide sequence of the upstream primer is shown in SEQ ID No. 3, and the nucleotide sequence of the downstream primer is shown in SEQ ID No.

4.

4. A recombinant expression vector, characterized in that, It includes the initial expression vector and the papaya CpYYL gene as described in claim 1.

5. A transformant, characterized in that, It includes the recombinant expression vector and competent Agrobacterium cells as described in claim 4.

6. The papaya CpYYL gene as described in claim 1 in the cultivation of fertile papaya YY h Or Y h Y h Applications in plants.

7. The application of overexpression of the papaya CpYYL gene as described in claim 1 in the cultivation of papaya plants with hermaphroditic flowers that do not separate.

8. A method for cultivating fertile papaya Y h Y h The method of using plants is characterized by, Includes the following steps: (1) The expression recombinant vector described in claim 4 was transferred into papaya callus and cultured to obtain transgenic seedling ProYYL::YYL / ZH XX T1; (2) The transgenic seedlings ProYYL::YYL / ZH XX T1 obtained in step (1) were crossed with papaya SunUp, Zhongbai or Hongling to obtain the hermaphroditic F1 generation plants ProYYL::YYL / SunUpXY h ; (3) The hermaphroditic F1 generation plants ProYYL::YYL / SunUpXY obtained in step (2) h Self-pollination yields fertile papaya Y-type seeds. h Y h Plant.

9. A method for cultivating fertile papaya YY h The method of using plants is characterized by, Includes the following steps: The hermaphroditic F1 generation plant ProYYL::YYL / SunUpXY obtained according to claim 8 h Male papaya plants were emasculated and crossed with male papaya plants XY to obtain fertile papaya plants YY. h Plant.

10. A method for cultivating papaya plants with non-separation of sexes, hermaphroditic XY h The method of using plants is characterized by, Includes the following steps: The fertile papaya Y obtained according to claim 8 h Y h The plant was crossed with a female papaya XX plant to obtain the aforementioned papaya species that are hermaphroditic and do not separate sexes (XY). h Plant.

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