A method for creating a transgenic papaya strain highly resistant to papaya ringspot virus and papaya mosaic virus.

Abstract

This invention provides a method for creating a transgenic papaya strain highly resistant to papaya ringspot virus and papaya mosaic virus. The method involves using PRSV and PLDMV of papaya... CP The fusion gene fragment expression cassette and the selection marker gene expression cassette were placed in two separate T-DNA domains and inserted into a plant expression vector. This vector was then introduced into papaya to obtain a papaya line without the selection marker; alternatively, PRSV and PLDMV were used. CP A fusion gene expression cassette and its reverse sequence were tandemly inserted into a plant expression vector, which was then introduced into papaya to obtain... CP A papaya strain with inversely linked gene expression cassettes. The marker-free papaya of this invention does not contain selection markers, effectively improving safety; CP Papaya with inversely linked gene expression cassettes does not require further homozygosity to enhance disease resistance.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to plant transgenic biotechnology breeding, specifically to a method for creating a transgenic papaya strain that is highly resistant to papaya ringspot virus and malformation mosaic virus. Background Technology

[0002] papaya( Carica papaya L. ) belongs to the class Dicotyledons ( Dicotyledons Caricaceae ( Caricaceae ) Papaya genus ( Carica Papaya, originally from South America, is now widely distributed in the tropical and subtropical regions of the Americas, Asia, Africa, and Oceania. It is one of the important consumer goods and export tropical fruits in China and the global tropics. Along with bananas and pineapples, papaya is known as one of the "three major tropical herbaceous fruit trees." Its fruit is rich in nutrients, including amino acids, vitamins, and trace elements; the flesh is sweet and juicy. Papaya can also be processed into products such as canned fruit, dried fruit, and preserved fruit; the seeds can be pressed for oil, and the sap from the green fruit is processed into papain, which is widely used in the food, cosmetics, pharmaceutical, and leather industries.

[0003] Papaya ringspot virus (PRSV) is the most serious viral disease of papaya, primarily affecting its leaves, stems, and fruits. This disease spreads rapidly and affects a wide area, becoming widespread in papaya-growing regions of my country and posing a serious threat to papaya production, thus becoming one of the major bottlenecks restricting the development of the global papaya industry. Currently, the most effective method for controlling papaya viral diseases is transgenic disease-resistant breeding. Therefore, developing virus-resistant transgenic papaya varieties has become an urgent need for the healthy development of the papaya industry.

[0004] Scientists have developed several transgenic papaya varieties (strains) resistant to PRSV. The first transgenic papaya variety was the Sunset variety, in which the coat protein (CP) gene of Hawaiian PRSV 55-1 was transferred into it. It exhibited good resistance in the field and was commercially available in Hawaii in 1997. Subsequently, the transgenic papaya strain X17-2 from the University of Florida was also commercially available in 2009. Transgenic papaya breeding in my country began in 1996, and the country developed the PRSV-resistant strain Huanong No. 1 and the resistant strain YK 16-0-1. These transgenic papayas utilize transgenic technology to introduce some target genes of the virus into the papaya, thereby inducing post-transcriptional gene silencing (PTGS) to acquire viral resistance. The PTGS induced by transgenes specifically initiates a cytoplasmic mRNA degradation mechanism, causing the mRNA of viral target genes to be rapidly and specifically degraded into small RNA fragments. PTGS typically occurs effectively when the transgene is transcribed from a strong promoter or when multiple copies of the transgene are present.

[0005] Papaya leaf distortion mosaic virus (PLDMV) is another major virus that has seriously threatened papaya production in recent years. It was first discovered in the Ryukyu Islands of Japan in 1945. PLDMV belongs to the same genus as PRSV (Potato Virus Y) and is transmitted by aphids. PLDMV infection in papaya can cause symptoms such as yellowing, mosaic, and curling of leaves, often resulting in significant yield reduction or even complete crop failure. The symptoms of this virus on papaya fruit are very similar to those of PRSV. Because PLDMV and PRSV are not related and have low sequence homology, transgenic papaya lines resistant to PRSV do not possess good resistance to PLDMV. Therefore, there is an urgent need to cultivate papaya lines that are highly resistant to both PRSV and PLDMV. Existing transgenic papaya varieties have been bred by fusing the CP genes of PRSV and PLDMV to create dual-resistant papaya lines that are resistant to both PRSV and PLDMV. However, the disease resistance of these lines still needs further improvement. The purpose of this invention is to further enhance the disease resistance of dual-resistant papaya lines. Simultaneously, these lines contain selection markers, and their safety for consumption and environmental protection also needs further improvement. Another objective of this invention is to create dual-resistant papaya lines without selection markers. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a method for creating a transgenic papaya strain that is highly resistant to papaya ringspot virus and papaya mosaic virus.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A method for creating a transgenic papaya variety highly resistant to papaya ringspot virus and papaya mosaic virus, wherein the method involves using PRSV and PLDMV of papaya... CP The fusion gene fragment expression cassette and the selection marker gene expression cassette were placed in two separate T-DNA domains and inserted into a plant expression vector. The vector was then introduced into papaya to obtain a transgenic papaya line that was highly resistant to papaya ringspot virus and papaya mosaic virus without the selection marker.

[0009] Furthermore, the creation method includes the following steps:

[0010] Synthetic PRSV and PLDMV CP gene fragments PR-CP and PL-CP , connection as PL-CP-PR-CP Fusion gene fragments; among which, PR-CP The sequence of the gene fragment is shown in SEQ ID NO: 1. PL-CP The sequence of the gene fragment is shown in SEQ ID NO: 2;

[0011] Will PL-CP-PR-CP Fusion gene fragment expression cassettes and selection marker genes NPTII The expression cassettes were placed in two independent T-DNA domains and inserted into the plant expression vector pBI121 to obtain the recombinant vector pBI121-CP1 containing dual T-DNA.

[0012] The recombinant vector pBI121-CP1 containing double T-DNA was introduced into papaya, and transgenic papaya lines with high resistance to papaya ringspot virus and malformation mosaic virus without selection markers were screened from the transgenic progeny.

[0013] Furthermore, the specific method for constructing the recombinant vector pBI121-CP1 containing dual T-DNA is to first... PL-CP- PR-CP The GUS gene in the pBI121 vector was replaced with a fusion gene fragment to obtain the recombinant vector pBI121-CP, which contains... PL-CP-PR- CP Fusion gene fragments, their upstream CaMV 35S promoter and downstream NOS The sequence of terminators is PL-CP-PR-CP The fusion gene fragment expression cassette; in the resulting recombinant vector pBI121-CP NPTII downstream of the expression box and PL-CP-PR-CP LB T-DNA and RB T-DNA sequences were inserted upstream of the expression cassette of the fusion gene fragment, respectively, to obtain the recombinant vector pBI121-CP1 containing dual T-DNA.

[0014] Furthermore, the specific process of vector introduction into papaya involves introducing the vector into Agrobacterium, transforming the resulting recombinant strain into papaya, and obtaining a transgenic papaya strain that is highly resistant to papaya ringspot virus and malformation mosaic virus without selection markers.

[0015] Agrobacterium strain AGL1 was preferred, and papaya was transformed into papaya embryogenic callus.

[0016] A transgenic papaya strain highly resistant to papaya ringspot virus and papaya mosaic virus, wherein the transgenic papaya strain highly resistant to papaya ringspot virus and papaya mosaic virus is obtained using the above-mentioned creation method;

[0017] The transgenic papaya lines highly resistant to papaya ringspot virus and papaya mosaic virus contain PRSV and PLDMV. CP Gene expression cassettes of gene fragments caused PTGS in the transgenic papaya lines highly resistant to papaya ringspot virus and malformation mosaic virus, as well as invading PRSV and PLDMV viruses. CP Posttranscriptional gene silencing prevents the invading virus from accumulating in the transgenic papaya lines that are highly resistant to papaya ringspot virus and malformation mosaic virus, thus conferring them resistance to PRSV and PLDMV.

[0018] A method for creating a transgenic papaya variety highly resistant to papaya ringspot virus and papaya mosaic virus, wherein the method involves using PRSV and PLDMV... CP A fusion gene expression cassette and its reverse sequence were tandemly inserted into a plant expression vector, which was then introduced into papaya to obtain... CP The transgenic papaya strain with high resistance to papaya ringspot virus and malformation mosaic virus, which incorporates a fusion gene fragment expression cascade in reverse tandem.

[0019] Furthermore, the creation method includes the following steps:

[0020] Synthetic PRSV and PLDMV CP gene fragments PR-CP and PL-CP , connection as PL-CP-PR-CP Fusion gene fragments; among which, PR-CP The sequence of the gene fragment is shown in SEQ ID NO: 1. PL-CP The sequence of the gene fragment is shown in SEQ ID NO: 2; PL-CP-PR-CP The fusion gene fragment replaces the GUS gene in the pBI121 vector, resulting in a product... CaMV 35S Startup Driver and NOS Termination of sub-termination PL-CP-PR-CP The gene fragment expression cassette was fused, and the resulting recombinant vector was pBI121-CP;

[0021] The recombinant vector pBI121-CP NPTII Expression box — PL-CP-PR-CP Sequence of expression boxes (containing) NPTII Expression box and PL-CP-PR-CP The reverse sequence corresponding to the expression frame sequence is inserted into pBI121-CP. PL-CP-PR-CP Downstream of the expression cassette, the recombinant vector pBI121-CP2 was obtained;

[0022] The recombinant vector pBI121-CP2 was introduced into papaya to obtain a transgenic papaya line with the target gene reversed tandem, which is highly resistant to papaya ringspot virus and papaya mosaic virus.

[0023] Furthermore, the specific method for constructing the recombinant vector pBI121-CP2 is to first... PL-CP-PR-CP The GUS gene in the pBI121 vector was replaced by a fusion gene fragment to obtain the recombinant vector pBI121-CP. Then, the recombinant vector pBI121-CP... NPTII—PL-CP-PR-CP The reverse sequence corresponding to the sequence is inserted into pBI121-CP. PL-CP-PR-CP Downstream of the expression frame, the recombinant vector pBI121-CP2 is obtained.

[0024] Furthermore, the specific process of vector introduction into papaya involves introducing the vector into Agrobacterium, and then transforming the resulting recombinant strain into papaya to obtain the transgenic papaya strain that is highly resistant to papaya ringspot virus and malformation mosaic virus.

[0025] Agrobacterium strain AGL1 was preferred, and papaya was transformed into papaya embryogenic callus.

[0026] A transgenic papaya strain highly resistant to papaya ringspot virus and papaya mosaic virus, wherein the transgenic papaya strain highly resistant to papaya ringspot virus and papaya mosaic virus is obtained using the above-mentioned creation method;

[0027] The transgenic papaya lines highly resistant to papaya ringspot virus and papaya mosaic virus contain PRSV and PLDMV. CP The fusion of gene fragment expression cassettes caused PTGS in the transgenic papaya lines highly resistant to papaya ringspot virus and malformation mosaic virus, as well as invading PRSV and PLDMV viruses. CP Posttranscriptional gene silencing prevents the invading virus from accumulating in the transgenic papaya lines that are highly resistant to papaya ringspot virus and malformation mosaic virus, thus conferring them resistance to PRSV and PLDMV.

[0028] The beneficial effects of the method for creating a transgenic papaya variety with high resistance to papaya ringspot virus and papaya mosaic virus of the present invention are as follows:

[0029] The disease resistance of the marker-free transgenic papaya heterozygous lines created in this invention is comparable to that of existing double-resistant papaya lines (the papaya lines disclosed in patent number TW201125981A), while the disease resistance of the homozygous lines is superior to that of existing double-resistant papaya lines (the papaya lines disclosed in patent number TW201125981A). Furthermore, the marker-free transgenic papaya of this invention does not contain any selection markers, which can effectively improve the food safety and environmental safety of transgenic plants, making them more acceptable to consumers.

[0030] The reverse-tandem transgenic papaya strains created in this invention exhibit significantly better resistance to PLDMV than existing dual-resistant papaya strains, whether heterozygous or homozygous. Furthermore, these dual-resistant strains achieve good disease resistance even when heterozygous, eliminating the need for further homozygous treatment to enhance resistance. Therefore, this method can significantly shorten the disease resistance breeding time for papaya and improve breeding efficiency, making it of significant application value in improving papaya resistance to Hainan PRSV and PLDMV.

[0031] The present invention discloses CP Fusion gene fragments PL-CP-PR-CP The double T-DNA method and reverse tandem method provide new approaches for creating new papaya varieties resistant to PRSV and PLDMV. Attached Figure Description

[0032] Figure 1 This is a structural diagram of the plant expression vector pBI121 in Example 1 of the present invention;

[0033] Figure 2 This is a structural diagram of the recombinant vector pBI121-CP1 in Embodiment 1 of the present invention;

[0034] Figure 3 This is a structural diagram of the recombinant vector pBI121-CP2 in Embodiment 1 of the present invention;

[0035] Figure 4 This is the PCR identification of the T0 generation of some regenerated plants CP1 in Example 2 of the present invention; where M is DL2000 Marker, the unit is bp, and the molecular weights from top to bottom are 2000, 1000, 750, 500, 250 and 100, K+ is the positive control (i.e., recombinant vector pBI121-CP1), K- is the negative control of non-transgenic papaya, K is the water control, and 1~20 represent 20 regenerated plants CP1 respectively;

[0036] Figure 5This is the PCR identification of the T0 generation of some regenerated plants CP2 in Example 2 of the present invention; where M is DL2000 Marker, the unit is bp, the molecular weight from top to bottom is 2000, 1000, 750, 500, 250 and 100, K+ is positive control (i.e. recombinant vector pBI121-CP2), K- is non-transgenic papaya negative control, K is water control, and 1~8 represent 8 regenerated plants CP2 respectively;

[0037] Figure 6 These are field disease resistance phenotypes of the transgenic papaya lines CP1-9 (hybrid), CP1-9 (homozygous), CP2-4 (hybrid), CP2-4 (homozygous), TW (dual-resistant), and Tainong 2, as described in Example 4 of this invention. Figure A shows the field disease resistance phenotype of the transgenic papaya line CP1-9 (hybrid); Figure B shows the field disease resistance phenotype of the transgenic papaya line CP1-9 (homozygous); Figure C shows the field disease resistance phenotype of the transgenic papaya line CP2-4 (hybrid); Figure D shows the field disease resistance phenotype of the transgenic papaya line CP2-4 (homozygous); Figure E shows the field disease resistance phenotype of the dual-resistant line TW; and Figure F shows the field disease resistance phenotype of Tainong 2.

[0038] Figure 7 This is a PCR image of PRSV detection for the transgenic papaya lines CP1-9 (heterozygous), CP1-9 (homozygous), CP2-4 (heterozygous), CP2-4 (homozygous), and the double-resistant TW line in Example 4 of this invention; where M represents DL2000. Markers, in bp, have molecular weights of 2000, 1000, 750, 500, 250, and 100 from top to bottom. K+ represents the Tainong 2 infected plant control, K- represents the Tainong 2 uninfected papaya control, and K represents the water control. TW represents the PRSV detection PCR image of the double-resistant line TW, CP1-9 heterozygous represents the PRSV detection PCR image of the CP1-9 heterozygous transgenic papaya line, CP1-9 homozygous represents the PRSV detection PCR image of the CP1-9 homozygous transgenic papaya line, CP2-4 heterozygous represents the PRSV detection PCR image of the CP2-4 heterozygous transgenic papaya line, and CP2-4 homozygous represents the PRSV detection PCR image of the CP2-4 homozygous transgenic papaya line.

[0039] Figure 8This is a PLDMV detection PCR image of the transgenic papaya lines CP1-9 (hybrid), CP1-9 (homozygous), CP2-4 (hybrid), CP2-4 (homozygous), and TW (double-resistant) in Example 4 of this invention; where M is the DL2000 Marker, in bp, and the molecular weights from top to bottom are 2000, 1000, 750, 500, 250, and 100. K+ represents the infected control plant of Tainong 2, K- represents the uninfected papaya control plant of Tainong 2, and K represents the water control; TW represents the PLDMV detection PCR image of the double-resistant line TW, CP1-9 heterozygous represents the PLDMV detection PCR image of the heterozygous transgenic papaya line CP1-9, CP1-9 homozygous represents the PLDMV detection PCR image of the homozygous transgenic papaya line CP1-9, CP2-4 heterozygous represents the PLDMV detection PCR image of the heterozygous transgenic papaya line CP2-4, and CP2-4 homozygous represents the PLDMV detection PCR image of the homozygous transgenic papaya line CP2-4.

[0040] Figure 9 This is a heatmap of the disease resistance observation of the transgenic papaya lines in the field in Example 4 of the present invention; wherein, CP1-9Het represents the heterozygous plant of the transgenic papaya line CP1-9, CP1-9Hom represents the homozygous plant of the transgenic papaya line CP1-9, CP2-4Het represents the heterozygous plant of the transgenic papaya line CP2-4, CP2-4Hom represents the homozygous plant of the transgenic papaya line CP2-4, TW represents the double-resistant line TW, and WT represents Tainong No. 2;

[0041] Figure 10 This refers to the siRNA expression levels of PRSV CP in the following transgenic papaya lines in Example 5 of this invention: CP1-9 heterozygous, CP1-9 homozygous, CP2-4 heterozygous, CP2-4 homozygous, and the double-resistant line TW. Specifically, TW represents the siRNA expression level of PRSV CP in the double-resistant line TW; CP1-9 heterozygous represents the siRNA expression level of PRSV CP in the transgenic papaya line CP1-9 heterozygous; CP1-9 homozygous represents the siRNA expression level of PRSV CP in the transgenic papaya line CP1-9 homozygous; CP2-4 heterozygous represents the siRNA expression level of PRSV CP in the transgenic papaya line CP2-4 heterozygous; and CP2-4 homozygous represents the siRNA expression level of PRSV CP in the transgenic papaya line CP2-4 homozygous.

[0042] Figure 11This refers to the siRNA expression levels of PLDMV CP in the following transgenic papaya lines in Example 5 of this invention: CP1-9 heterozygous, CP1-9 homozygous, CP2-4 heterozygous, CP2-4 homozygous, and the double-resistant TW line. Specifically, TW represents the siRNA expression level of PLDMV CP in the double-resistant TW line; CP1-9 heterozygous represents the siRNA expression level of PLDMV CP in the CP1-9 heterozygous line; CP1-9 homozygous represents the siRNA expression level of PLDMV CP in the CP1-9 homozygous line; CP2-4 heterozygous represents the siRNA expression level of PLDMV CP in the CP2-4 heterozygous line; and CP2-4 homozygous represents the siRNA expression level of PLDMV CP in the CP2-4 homozygous line. Detailed Implementation

[0043] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can also be implemented in other ways different from those described herein. Those skilled in the art can make similar extensions without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below. The present invention will be further described in detail below with reference to specific embodiments to enable those skilled in the art to understand it.

[0044] Furthermore, for specific techniques or conditions not specified in the detailed embodiments disclosed below, please refer to the techniques or conditions described in the literature in this field (e.g., refer to *Molecular Cloning: A Laboratory Manual*, 3rd edition, translated by Sambrook et al., Science Press) or the product instructions. Reagents whose manufacturers are not specified are all commonly available products.

[0045] Example 1: Construction of Papaya pBI121-CP1 and pBI121-CP2 Recombinant Vectors

[0046] Based on PRSV and PLDMV strains collected in Sanya City, Hainan Province CP Gene fragments, and referenced global PRSV and PLDMV viral strains. CP Gene sequences, synthesis of PRSV and PLDMV CP gene fragments PR-CP and PL-CP , PR-CP The gene fragment is 801 bp in size, and its sequence is shown in SEQ ID NO: 1. PL-CP The gene fragment is 703 bp in size, and its sequence is shown in SEQ ID NO: 2. PL-CP in front,PR-CP In the subsequent order, the synthesis link is... PL-CP-PR-CP fusion gene fragments (i.e. PL-CP-PR-CP (fusion gene fragment).

[0047] Using conventional technology, PL-CP-PR-CP The fusion gene fragment replaces the GUS gene in the plant expression vector pBI121 (plant expression vector pBI121 is as follows). Figure 1 As shown in the figure, the recombinant vector pBI121-CP was obtained. PL-CP-PR-CP Fusion gene fragments are CaMV 35S Startup Sub-Driver, by NOS Termination of sub-termination.

[0048] In the recombinant vector pBI121-CP NPTII Downstream of the expression box and PL-CP-PR-CP An LB T-DNA sequence (as shown in SEQ ID NO: 3) and an RB T-DNA sequence (as shown in SEQ ID NO: 4) were inserted upstream of the fusion gene fragment expression cassette, respectively, to obtain the recombinant vector pBI121-CP1 containing dual T-DNA (recombinant vector pBI121-CP1 is shown in SEQ ID NO: 4). Figure 2 (As shown).

[0049] The sequence of the NPTII expression cassette—PL-CP-PR-CP expression cassette in the recombinant vector pBI121-CP (containing...) NPTII Expression box and PL-CP-PR-CP The reverse sequence corresponding to the sequence of the expression box (the reverse sequence is shown in SEQ ID NO: 5) is inserted into pBI121-CP. PL-CP-PR-CP Downstream of the expression cassette, the recombinant vector pBI121-CP2 was obtained (recombinant vector pBI121-CP2 is shown below). Figure 3 (As shown).

[0050] Positive clones of recombinant vectors pBI121-CP1 and pBI121-CP2 were screened and sent to a sequencing company for sequencing. Positive clones with correct sequencing were preserved, and plasmids were extracted. They were then transformed into Agrobacterium AGL1 cells using the liquid nitrogen freeze-thaw method to obtain AGL1 cells containing pBI121-CP1 and pBI121-CP2 plasmids, respectively. The cells were stored in glycerol at -80°C for later use.

[0051] Example 2: Transformation of Papaya with Recombinant Vector

[0052] Using stem segments of Papaya Tainong 2 seedlings as explants, embryogenic callus was induced, and recombinant vector strains were transformed. The specific steps included:

[0053] 1) Induction of embryogenic callus from papaya

[0054] The papaya callus induction medium contained 2.2 g / L MS dry powder, 70.0 g / L sucrose, 0.1 g / L inositol, 1 mL / L MS vitamin (1000x), 0.01 g / L 2,4-dichlorophenoxyacetic acid, and 4.0 g / L plant gel, with a pH of 5.8.

[0055] Sterilize papaya seeds in a 1.1% sodium hypochlorite solution for 1 hour, rinse with sterile water, and shake in 50 mL of 1M KNO3 aqueous solution for 24 hours. Discard floating seeds, suspend the remaining seeds in 100 mL of sterile water, and stir at 32°C for about 5 days until the seed coat ruptures. Sow the germinated seeds on 1% (w / v) water agar and grow under a cool white fluorescent lamp at 24–26°C. Seedlings after about 2 weeks can be used as explants. Cut the hypocotyls of the seedlings into 2–3 mm long segments and spread them flat on callus induction medium. Incubate in the dark at 27°C for 6–8 weeks until embryogenic callus can be observed on the hypocotyl segments. Most hypocotyl segments will produce embryogenic callus within 2 weeks. Subsequently, replace the induction medium every 3 weeks to continue inducing embryogenic callus, obtaining papaya embryogenic callus.

[0056] 2) Preparation of Agrobacterium-containing bacterial solution

[0057] LB medium contains 10.0 g / L peptone, 5.0 g / L yeast extract and 10.0 g / L NaCl.

[0058] Take LB medium and add 50.0 μg carbenicillin and 50.0 μg kanamycin per milliliter to obtain LB plates containing carbenicillin and kanamycin.

[0059] The papaya infection solution contained 2.2 g / L MS dry powder, 30.0 g / L sucrose, 0.002 g / L 2,4-dichlorophenoxyacetic acid and 0.04 g / L acetylsyringone, with a pH of 5.8.

[0060] Take the bacterial suspensions of AGL1 bacteria containing pBI121-CP1 plasmid and pBI121-CP2 plasmid, stored at -80℃ respectively, and streak them onto LB agar plates containing carbenicillin and kanamycin, respectively. Incubate in the dark at 28℃ for 3 days to isolate the corresponding single colonies. Pick 3-5 of the corresponding single colonies and place them into LB broth containing 50 μg / mL carbenicillin and 50 μg / mL kanamycin, respectively. Incubate at 28℃ with shaking until the OD of each bacterial suspension is reached. 600 The concentration was set to 0.6, centrifuged at 5000 rpm for 10 min, the culture medium was discarded, the corresponding bacterial cells were collected, and then papaya infection solution was added, shaken well, and the OD was adjusted. 600The value was 0.6, and the corresponding bacterial infection solutions were obtained (labeled as pBI121-CP1 bacterial infection solution and pBI121-CP2 bacterial infection solution, respectively).

[0061] 3) Infection and co-cultivation

[0062] The papaya co-culture medium contained 2.2 g / L MS dry powder, 30.0 g / L sucrose, 0.002 g / L 2,4-dichlorophenoxyacetic acid, 0.04 g / L acetylsyringone, and 4.0 g / L plant gel, with a pH of 5.8.

[0063] Each bacterial inoculation solution was activated and cultured at 24℃ and 150 rpm for 2-3 hours to obtain the corresponding activated bacterial solutions. Papaya embryogenic callus tissue was collected into 50 mL sterile centrifuge tubes, and each activated bacterial solution was poured into a separate tube. The tubes were then gently shaken at 24℃ and 150 rpm for 30 minutes for infection. After infection, the bacterial solutions were discarded, and the infected papaya callus tissue was poured into sterile petri dishes containing multiple layers of filter paper and spread out to air dry. The dried papaya callus was then transferred to a papaya co-culture medium lined with a layer of sterile filter paper, sealed with sealing film, and incubated in the dark at 24℃ for 2 days to obtain the corresponding co-cultured papaya callus tissue.

[0064] 4) Antibacterial and screening

[0065] The papaya selection medium contained 2.2 g / L MS dry powder, 70.0 g / L sucrose, 0.1 g / L inositol, 1 mL / L MS vitamin (1000x), 0.01 g / L 2,4-dichlorophenoxyacetic acid, 0.5 g / L termethin, 0.1 g / L kanamycin, and 4.0 g / L plant gel, with a pH of 5.8.

[0066] After co-culturing, the papaya callus tissues were transferred to papaya selection medium. The callus tissues were gently pushed into the papaya selection medium to ensure good contact between the callus and the selection agent in the medium. The culture was carried out under light at 28°C, and the callus tissues were subcultured every 3-4 weeks for a total of 3 months to obtain the corresponding resistant callus tissues.

[0067] 5) Differentiation and rooting

[0068] The papaya differentiation medium contained 4.4 g / L MS dry powder, 30.0 g / L sucrose, 0.002 g / L 6-benzylaminopurine, 0.002 g / L naphthaleneacetic acid, 0.5 g / L termethin, 0.1 g / L kanamycin and 4.0 g / L plant gel, with a pH of 5.8.

[0069] The papaya rooting medium contained 2.2 g / L MS dry powder, 20.0 g / L sucrose, 0.0005 g / L indolebutyric acid, 0.5 g / L termethin, 0.1 g / L kanamycin and 4.0 g / L plant gel, with a pH of 5.8.

[0070] The resistant callus tissues were transferred to papaya differentiation medium and cultured at 28°C for 12h / 12h light / dark cycles, with subculture every 3-4 weeks until shoots approximately 1cm long formed, a process that takes about 2 months. The shoots were then isolated and transferred to papaya rooting medium for rooting, and cultured in the dark at 28°C for one week. The rooted shoots were then transferred to vermiculite (50mL Erlenmeyer flasks) and 1 / 2 MS medium containing 0.0005g / L indolebutyric acid were added. Rooting should be completed within approximately one month. The corresponding rooted papaya plants were then transferred to pots for room temperature cultivation.

[0071] 6) PCR identification of regenerated plants

[0072] The regenerated papaya plants transformed with the recombinant vector pBI121-CP1 were named CP1, and the regenerated papaya plants transformed with the recombinant vector pBI121-CP2 were named CP2. Leaves from CP1 and CP2 were collected, and genomic DNA was extracted using the MEGA Plant Genomic DNA Rapid Extraction Kit (D3187-02). Using this DNA as a template, partial sequences of the recombinant vectors in the regenerated plants CP1 and CP2 were detected by PCR. The detection primers are shown in Table 1. One regenerated plant, CP1, was... CP Genes and NPTII Genetic testing was performed to select plants that tested positive for both genes; CP2 was then tested on the regenerated plants. CP Gene detection was performed, and positive plants were selected. The enzyme used for PCR detection was Vazyme's 2 x Rapid Taq Master Mix. The reaction system consisted of 8.5 μL sterile water, 1 μL each of primers (10 μM), 12.5 μL Taq Mix, and 2 μL DNA template. The PCR amplification program was as follows: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 90 s, for 35 cycles; and a final extension at 72℃ for 7 min. PCR products were separated and detected by 2% agarose gel electrophoresis. Electrophoresis images of PCR products from partially regenerated plants CP1 and CP2 are shown below. Figure 4 and Figure 5 .

[0073] from Figure 4 It can be seen that samples 1, 2, 4–7, 9–12, 14, 15, and 17–20 of some regenerated CP1 plants can amplify approximately 300 bp. NPTIIBands 1, 4, 5, 11, and 15 all amplified to approximately 1300 bp. CP The bands were the same size as the positive control, while the negative control and water control showed no target bands, indicating that these samples were all transgenic positive plants. Among them, samples 1, 4, 5, 11, and 15 could amplify both genes. Further planting of the next generation and selection of plants containing only these genes... CP The plant.

[0074] from Figure 5 It can be seen that some regenerated plants can amplify bands of about 1300bp at CP2 1-3 and 5-7, which are the same size as the positive control, while the negative control and water control do not have this band, indicating that these samples are all transgenic positive plants.

[0075] Table 1 Primers for positive detection of transgenic organisms

[0076]

[0077] Example 3: Analysis of pBI121-CP1 and pBI121-CP2 insertion sites in papaya

[0078] Select CP1 only CP The specific insertion sites of the recombinant vectors pBI121-CP1 and pBI121-CP2 into the papaya genome were analyzed using next-generation sequencing technology. The specific steps are as follows:

[0079] (1) DNA extraction and library construction. Genomic DNA was extracted using the CTAB method. The genomic DNA was randomly fragmented into fragments of about 350 bp in length using a fragment disruptor. After end repair and A-tailing, adapters were ligated to both ends of the fragments to prepare a DNA library.

[0080] (2) Library testing. The library was initially quantified using Qubit 2.0, and then the insert size of the library was detected using Agilent 2100. After the insert size met the expectations, the effective concentration (3nM) of the DNA library was accurately quantified using qPCR to ensure the quality of the library.

[0081] (3) Sequencing, sequencing result analysis, and data filtering. Novaseq X Plus platform was used for sequencing based on the effective library concentration and data output requirements, with a PE150 strategy. Raw image data was converted into raw sequencing reads through base calling analysis and then filtered.

[0082] (4) Sequencing data quality assessment. Valid sequencing data are aligned to the reference genome using BWA to obtain the initial alignment results in BAM format. Then, the alignment results are sorted using SAMtools; and repeat reads are marked using Picard. Finally, the alignment results after repeat marking are used to perform statistics on coverage, depth, etc.

[0083] (5) The effective clean reads obtained after quality control filtering were compared with the non-transgenic papaya sunset reference genome GWHBFSD00000000 using BWA software to obtain the comparison results (bam format). The comparison results were sorted using SAMtools software, and SNP and InDel detection and filtering were performed based on the sorted comparison results.

[0084] (6) Align the InDel-related reads with the T-DNA sequence and the genome sequence respectively. Based on the alignment results, select the position of the inserted fragment relative to the reference genome.

[0085] Sequencing analysis revealed that 75 InDel-related reads in the CP1 plant containing only CP were aligned to bases 850145-851062 on chromosome 9 of the papaya genome. This site is a non-functional region, and the line was named the transgenic papaya line CP1-9.

[0086] The transgenic CP2 plant had 111 InDel-related reads that aligned to the 2038793-2039374 base pairs on chromosome 4 of the papaya genome. This site is also a non-functional region. The line was named the transgenic papaya line CP2-4.

[0087] Example 4: Field disease resistance test of transgenic papaya lines

[0088] Homozygous and heterozygous progeny of transgenic papaya lines CP1-9 and CP2-4, as well as the double-resistant line TW (the papaya line disclosed in patent number TW201125981A), were planted in an experimental papaya field chronically diseased (PRSV and PLDMV) areas. Tainong 2 was used as the wild-type control (WT). The field resistance of the homozygous and heterozygous progeny of transgenic papaya lines CP1-9 and CP2-4 to the double-resistant line TW and the wild-type control WT was compared. Disease severity of PRSV and PLDMV was investigated at different growth stages of the papaya, once every month, for a total of 5 investigations. Disease severity was classified according to Table 2.

[0089] Table 2 Classification of Papaya Virus Disease Caused by Virus

[0090]

[0091] Simultaneously, fresh leaf samples were collected from each transgenic papaya line, total RNA was extracted, and PRSV or PLDMV-specific sequences were amplified to determine whether the plants contained PRSV or PLDMV viruses. Primers used for PCR detection are shown in Table 3. The enzyme used was Vazyme's 2 x Rapid Taq Master Mix. The reaction system consisted of 8.5 μL sterile water, 1 μL each of primers (10 μM), 12.5 μL TaqMix, and 2 μL DNA template. The PCR amplification program was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, annealing at 60℃ (PRSV) or 56℃ (PLDMV) for 30 s, extension at 72℃ for 45 s, for 35 cycles; and a final extension at 72℃ for 5 min. PCR products were separated and detected by 2% agarose gel electrophoresis.

[0092] Table 3 Primers for PRSV and PLDMV virus detection

[0093]

[0094] Disease investigation revealed that the wild control group, Tainong 2, exhibited obvious mosaic patterns, deformities, and "green islands" in the early stages. Some heterozygous plants of the dual-resistant TW strain and the transgenic papaya strain CP1-9 also showed leaf distortion and water-soaked streaks on the petioles. Homozygous plants of the transgenic papaya strain CP1-9, heterozygous plants of the transgenic papaya strain CP2-4, and homozygous plants of the transgenic papaya strain CP2-4 showed no obvious disease symptoms (see Table 4). Figure 6 PCR detection results of PRSV and PLDMV sequences in various transgenic papaya plants and wild-type control WT ( Figure 7 and Figure 8 The results showed that PRSV and PLDMV viruses were detected in the wild-type control WT, and PLDMV virus was also detected in symptomatic plants of the double-resistant strain TW and the transgenic papaya strain CP1-9. PLDMV virus was not detected in any of the remaining plants. PRSV virus was not detected in any of the papaya plants. Based on the viral PCR test results, this invention selected 10 plants from each transgenic strain for disease severity heatmap analysis. The results are shown below. Figure 9 It can be seen that the dual-resistant strain TW and the heterozygous transgenic papaya strain CP1-9 have good resistance to PRSV, but weak resistance to PLDMV; while the homozygous transgenic papaya strain CP1-9, the heterozygous transgenic papaya strain CP2-4, and the homozygous transgenic papaya strain CP2-4 all show good resistance to both PRSV and PLDMV.

[0095] This indicates that the disease resistance of heterozygous plants of the marker-free transgenic papaya line CP1-9 is comparable to that of the dual-resistant line TW, while the disease resistance of homozygous plants of the transgenic papaya line CP1-9 is stronger than that of the dual-resistant line TW. Furthermore, the marker-free transgenic papaya line CP1-9, whether homozygous or heterozygous, does not contain any selection markers, which can effectively improve the food safety and environmental safety of transgenic plants.

[0096] Therefore, the method of reverse tandem of viral gene expression frames can shorten breeding time and improve breeding efficiency.

[0097] Table 4. Survey on PRSV and PLDMV infection status in transgenic papaya.

[0098]

[0099] Example 5: siRNA detection in transgenic papaya lines

[0100] The target sequences of PRSV CP and PLDMV CP introduced into each transgenic papaya are cleaved into small interfering RNA (siRNA) after transcription to perform their functions. Therefore, small RNA sequencing was used to analyze the expression levels of siRNA produced by PRSV CP and PLDMV CP. The specific method is as follows: Tissue culture seedlings from the double-antibiotic line TW, heterozygous plants of transgenic papaya lines CP1-9, homozygous plants of transgenic papaya lines CP1-9, heterozygous plants of transgenic papaya lines CP2-4, and homozygous plants of transgenic papaya lines CP2-4 were collected, and total RNA was extracted from leaves for small RNA sequencing. Libraries were constructed using the NEB Next® MultiplexSmall RNA Library Prep Set (NEB E7300L). Utilizing the special structures at the 3' and 5' ends of small RNA, adapters were directly added to both ends of the small RNA starting sample, followed by reverse transcription to synthesize cDNA. Subsequently, PCR amplification and PAGE gel electrophoresis were performed to separate the target DNA fragments. The recovered cDNA library was obtained by gel extraction. Library integrity and insert size were detected using AATI, and effective library concentration was detected by qPCR. After passing the library tests, different libraries were pooled according to effective concentration and target data volume requirements before sequencing. The sequencing platform was Novaseq 6000, and the strategy was SE50. FastP software was used to remove sequencing adapters from raw reads, filtering out reads containing poly-N and low-quality reads. sRNAminer software was used to remove adapter sequences from raw reads, filtering out organelle and ribosomal RNA sequences to obtain clean reads. These clean reads were then back-linked to PRSV CP sequences, PLDMV CP sequences, papaya genome sequences, and T-DNA sequences to obtain PRSV_CP_rc, PLDMV_CP_rc, genome_rc, and T_DNA_rc, which were used to calculate siRNA expression levels (RP10M). Sequencing results showed that siRNA was produced by the double-antibiotic line TW, heterozygous plants of transgenic papaya line CP1-9, homozygous plants of transgenic papaya line CP1-9, heterozygous plants of transgenic papaya line CP2-4, and homozygous plants of transgenic papaya line CP2-4, and was concentrated in PRSV. CP and PLDMV CP Several regions of a gene sequence.

[0101] For PRSV CP The expression levels of siRNA were calculated, and the results are shown in [the table]. Figure 10The study found that the siRNA expression levels of heterozygous CP1-9 transgenic papaya lines were comparable to those of the double-resistant TW line, with no significant difference. However, the siRNA expression levels of homozygous CP1-9 transgenic papaya lines were approximately twice that of heterozygous CP1-9 lines. Similarly, the siRNA expression levels of heterozygous CP2-4 transgenic papaya lines were also comparable to those of homozygous CP1-9 lines, with no significant difference. However, the siRNA expression levels of homozygous CP2-4 transgenic papaya lines were again approximately twice that of heterozygous CP2-4 lines, showing a significant difference. All five transgenic lines exhibited extremely strong resistance to PRSV in field cultivation, indicating that even relatively low siRNA expression levels constituted strong PRSV resistance.

[0102] For PLDMV CP The expression levels of siRNA were calculated, and the results are shown in [the table]. Figure 11 The study found that the siRNA expression levels of heterozygous CP1-9 transgenic papaya lines were comparable to those of the double-resistant TW line, with no significant difference. However, the siRNA expression levels of homozygous CP1-9 transgenic papaya lines were approximately twice that of heterozygous CP1-9 transgenic papaya lines, showing a significant difference. Similarly, the siRNA expression levels of heterozygous CP2-4 transgenic papaya lines were also comparable to those of homozygous CP1-9 transgenic papaya lines, with no significant difference. However, the siRNA expression levels of homozygous CP2-4 transgenic papaya lines were again approximately twice that of heterozygous CP2-4 transgenic papaya lines, showing a significant difference. Of the five transgenic papaya lines, approximately half of the heterozygous CP1-9 and the double-resistant TW line were infected with PLDMV in field planting, indicating weak resistance to PLDMV. In contrast, the homozygous CP1-9, heterozygous CP2-4, and homozygous CP2-4 lines exhibited extremely strong resistance to PLDMV, suggesting that a high level of siRNA expression is required to produce strong resistance to PLDMV.

[0103] In addition, PLDMV CP The siRNA expression level in these five transgenic lines was approximately the same as that of PRSV. CP The siRNA expression level was 2-3 times higher, possibly due to PLDMV. CP The gene is located directly downstream of the promoter, while PRSV CP The gene is located in PLDMV CP Downstream of the gene, the level of transcriptional expression is relatively low.

[0104] Meanwhile, considering the disease resistance phenotypes observed in field cultivation, these transgenic papaya lines showed PRSV...CP and PLDMV CP The expression level of gene siRNA is positively correlated with its disease resistance phenotype in field planting.

[0105] The above results indicate that the siRNA expression level of the marker-free transgenic papaya line CP1-9 homozygous plants created in this invention is higher than that of the double-resistant line TW, and the disease resistance is stronger than that of the double-resistant line TW. Moreover, it does not contain selection markers, which can effectively improve the food safety and environmental safety of transgenic plants.

[0106] The siRNA expression levels of the inversely tandem transgenic papaya line CP2-4 heterozygous and homozygous plants were significantly higher than those of the double-resistant line TW. This indicates that the inversely tandem transgenic papaya line CP2-4 heterozygous plants can achieve the same disease resistance as the CP1-9 homozygous line, exhibiting higher disease resistance than the double-resistant line TW, without the need for further homozygosity to enhance disease resistance.

[0107] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. Those skilled in the art can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for creating a transgenic Carica papaya line highly resistant to Papaya ring spot virus and Cucumber mosaic virus, characterized by, The creation method is to insert the fusion gene fragment of PRSV and PLDMV of Carica papaya into a plant expression vector, and then introduce the vector into Carica papaya to obtain a transgenic Carica papaya line with high resistance to Carica papaya ring spot virus and mosaic virus without a screening marker. CP The fusion gene fragment of PRSV and PLDMV of Carica papaya is inserted into a plant expression vector, and then the vector is introduced into Carica papaya to obtain a transgenic Carica papaya line with high resistance to Carica papaya ring spot virus and mosaic virus without a screening marker. Synthetic PRSV and PLDMV CP Fusion gene fragments PR-CP and PL-CP , are connected as PL-CP-PR-CP Fusion gene fragments; wherein, PR-CP the sequence of the gene fragment is shown as SEQ ID NO: 1, PL-CP the sequence of the gene fragment is shown as SEQ ID NO: 2; The transgenic Carica papaya line with high resistance to Papaya ring spot virus and Cucumber mosaic virus without screening marker is a homozygous transgenic Carica papaya line with high resistance to Papaya ring spot virus and Cucumber mosaic virus without screening marker.

2. The method for creating a transgenic papaya strain highly resistant to papaya ringspot virus and malformation mosaic virus according to claim 1, characterized in that, The creation method comprises the following steps: Will PL-CP-PR-CP Fusion gene fragment expression cassettes and selection marker genes NPTII The expression cassettes were placed in two independent T-DNA domains and inserted into the plant expression vector pBI121 to obtain the recombinant vector pBI121-CP1 containing dual T-DNA. The recombinant vector pBI121-CP1 containing double T-DNA is introduced into Carica papaya, and the transgenic Carica papaya line with high resistance to Papaya ring spot virus and Cucumber mosaic virus without screening marker is screened from the transgenic offspring.

3. The method of creating a transgenic papaya line highly resistant to carposeris papaya ring spot virus and potyvirus according to claim 2, characterized in that, The specific method for constructing the recombinant vector pBI121-CP1 containing dual T-DNA is to first... PL- CP-PR-CP The GUS gene in the pBI121 vector was replaced with a fusion gene fragment to obtain the recombinant vector pBI121-CP, which contains... PL-CP- PR-CP Fusion gene fragments, their upstream CaMV 35S promoter and downstream NOS The sequence of terminators is PL-CP-PR-CP The fusion gene fragment expression cassette; in the resulting recombinant vector pBI121-CP NPTII downstream of the expression box and PL-CP-PR-CP LB T-DNA and RB T-DNA sequences were inserted upstream of the expression cassette of the fusion gene fragment, respectively, to obtain the recombinant vector pBI121-CP1 containing dual T-DNA; The LB T-DNA sequence is shown in SEQ ID NO: 3, and the RB T-DNA sequence is shown in SEQ ID NO:

4.

4. The method for creating a transgenic Carica papaya line highly resistant to Carica papaya ringspot virus and Cassava vein mosaic virus according to any one of claims 1 to 3, characterized in that, The specific process of introducing the vector into Carica papaya is to introduce the vector into Agrobacterium, and the obtained recombinant strain is used to transform Carica papaya, so as to obtain the transgenic Carica papaya line with high resistance to Papaya ring spot virus and Cucumber mosaic virus without screening marker.

5. A method of creating a transgenic Carica papaya line with high resistance to Papaya ring spot virus and Cucumber mosaic virus, characterized in that, The creation method involves combining PRSV and PLDMV. CP The gene fragment expression cassette and its reverse sequence were tandemly inserted into a plant expression vector, and then the vector was introduced into papaya to obtain the transgenic papaya line with the target gene reverse tandemly tandemly resistant to papaya ringspot virus and malformation mosaic virus. Synthetic PRSV and PLDMV CP gene fragments PR-CP and PL-CP , are connected as PL-CP-PR-CP a fusion gene fragment; wherein, PR-CP the sequence of the gene fragment is shown as SEQ ID NO: 1, PL-CP the sequence of the gene fragment is shown as SEQ ID NO:

2.

6. The method of creating a transgenic papaya line highly resistant to carposeris papaya ring spot virus and potyvirus according to claim 5, characterized in that, The creation method comprises the following steps: Will PL-CP-PR-CP The GUS gene in the plant expression vector pBI121 was replaced by a fusion gene fragment to obtain the recombinant vector pBI121-CP. The sequence of the expression frame in the recombinant vector pBI121-CP2 is as follows: NPTII The sequence of the expression frame in the recombinant vector pBI121-CP2 is as follows: PL-CP-PR-CP The sequence of the expression frame in the recombinant vector pBI121-CP2 is as follows: PL-CP-PR-CP The sequence of the expression frame in the recombinant vector pBI121-CP2 is as follows: The recombinant vector pBI121-CP2 is introduced into Carica papaya, and the transgenic Carica papaya line with high resistance to Papaya ring spot virus and Cucumber mosaic virus is obtained.

7. The method for creating a transgenic papaya strain highly resistant to papaya ringspot virus and malformation mosaic virus according to claim 6, characterized in that, The specific method for constructing the recombinant vector pBI121-CP2 is to first... PL-CP-PR-CP The GUS gene in the pBI121 vector was replaced by a fusion gene fragment to obtain the recombinant vector pBI121-CP. Then, the recombinant vector pBI121-CP... NPTII-PL-CP-PR-CP The reverse sequence corresponding to the sequence is inserted into pBI121-CP. PL-CP-PR-CP Downstream of the expression cassette, the recombinant vector pBI121-CP2 is obtained.

8. The method for creating a transgenic Carica papaya line highly resistant to Carica papaya ringspot virus and Cassava vein mosaic virus according to any one of claims 5 to 7, characterized in that, The specific process of introducing the vector into Carica papaya is to introduce the vector into Agrobacterium, and the obtained recombinant strain is used to transform Carica papaya, so as to obtain the transgenic Carica papaya line with high resistance to Papaya ring spot virus and Cucumber mosaic virus.

Images