A functional gene PaYUC10 for regulating fruit storage stability and its application

Abstract

The present invention discloses a functional gene PaYUC10 for increasing the storage stability of fruits and its application, wherein the nucleotide sequence of the functional gene PaYUC10 is shown in SEQ ID No.1; or the functional gene PaYUC10 is a coding gene for a protein composed of an amino acid sequence shown in SEQ ID No.2. The functional gene PaYUC10 for increasing the storage stability of fruits is used to increase the storage stability of fruits. The functional PaYUC10 gene in the present invention can be used for variety improvement of fruits that are not storage-resistant.

Description

Technical Field

[0001] The invention relates to the technical field of fruit storage stability, and specifically to a functional gene PaYUC10 for increasing the storage stability of fruits and its application. Background Art

[0002] Apricot (Prunus armeniaca) is a perennial woody plant in the Rosaceae family and an important economic crop in the world. Apricot fruit is rich in sugars, organic acids, carotenoids and polyphenols, which are very beneficial to the human body and have great market development potential. Apricot fruit is generally not resistant to storage, which seriously affects the storage, transportation and sales of apricot fruit. Apricot is a respiratory climacteric fruit. After commercial harvest, the respiration rate suddenly increases, a respiratory peak appears, and it reaches maturity and then ages rapidly, losing its commercial value. It is of great significance to develop functional genes that can inhibit the respiration intensity of apricot fruit after harvest and prolong the storage time, thereby laying the foundation for the breeding of storage-resistant apricot varieties. The expression pattern and functional role of the YUC10 gene in apricot fruit are still unclear, and few people have reported changes in storage time caused by upregulating the respiration intensity of the fruit. Summary of the invention

[0003] Therefore, the technical problem to be solved by the present invention is to provide a functional gene PaYUC10 for increasing the storage tolerance of fruits and its application, so as to increase the storage tolerance of storage-intolerant fruits.

[0004] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0005] A functional gene PaYUC10 for increasing fruit storage stability, wherein the nucleotide sequence of the functional gene PaYUC10 is shown as SEQ ID No.1; or the functional gene PaYUC10 is a gene encoding a protein composed of the amino acid sequence shown as SEQ ID No.2.

[0006] An application of a functional gene PaYUC10 for increasing the storability of fruits, using the functional gene PaYUC10 for increasing the storability of fruits to increase the storability of fruits.

[0007] The application of the functional gene PaYUC10 for upregulating the storage tolerance of fruits is to first insert the functional gene PaYUC10 into an overexpression vector to form a recombinant plasmid, then transform the recombinant plasmid into Agrobacterium, and finally use the Agrobacterium transformed with the recombinant plasmid to infect plants with fruits that are not resistant to storage, so that it is overexpressed in the plants.

[0008] The application of the functional gene PaYUC10 for increasing the storage stability of fruits comprises the following steps:

[0009] Step (1), using a high-fidelity enzyme to perform a PCR reaction to construct a recombinant plasmid pCambia2301-PaYUC10;

[0010] Step (2), using the recombinant plasmid pCambia2301-PaYUC10 to transform Agrobacterium to obtain transformed Agrobacterium:

[0011] Step (3), first pre-culture the explants of the storage-intolerant plants; then place the pre-cultured explants in a culture dish with transformation Agrobacterium for transformation culture, and after the transformation culture is completed, transfer the explants back to the pre-culture medium for co-culture; after the co-culture is completed, transfer the explants to a screening culture medium for selection culture to form callus tissue and / or adventitious buds;

[0012] Step (4) transferring the young shoots or adventitious shoots obtained by subculture of the callus tissue to a rooting medium to obtain complete plants.

[0013] In the application of the functional gene PaYUC10 for upregulating fruit storage stability, in step (1), the high-fidelity enzyme is KOD-401 high-fidelity enzyme.

[0014] In the above application of the functional gene PaYUC10 for upregulating fruit storage stability, in step (3), the explant is a true leaf, cotyledon or hypocotyl.

[0015] In the application of the functional gene PaYUC10 for upregulating fruit storage stability, in step (3), the tips and petioles of the true leaves and cotyledons are cut off, and the remaining parts are cut into leaf pieces of 0.5 cm × 0.5 cm; the hypocotyls are cut into segments of 0.5 to 0.6 mm in length and placed horizontally on the preculture medium, with 15 to 20 pieces placed in each preculture medium; the preculture time is 1 day;

[0016] The concentration of transformed Agrobacterium in the culture dish is 0.4 to 0.6 at OD600, the transformation culture time is 30 minutes, and the co-culture time is 1 to 2 days.

[0017] In the application of the functional gene PaYUC10 for upregulating fruit storage stability, in step (4), when the young shoots grow to 0.8-1.2 cm, they are transferred to a rooting medium for cultivation.

[0018] In the application of the functional gene PaYUC10 for upregulating the storage stability of fruits, the overexpression vector is pCambia2301 and the Agrobacterium strain is GV3101.

[0019] In the application of the functional gene PaYUC10 for upregulating the storage tolerance of fruits, the plants whose fruits are not tolerant to storage are tomato plants.

[0020] The technical solution of the present invention achieves the following beneficial technical effects:

[0021] 1. The present invention cloned the functional gene PaYUC10 from 'Pearl Oil Apricot'. By measuring the expression of PaYUC10 during fruit storage, it was found that the expression level of the functional gene was higher than that of storage-intolerant varieties. The functional gene PaYUC10 may be involved in the upregulation of fruit respiration intensity. Since the respiration intensity can affect fruit ripening and senescence, it is of great significance for its variety improvement.

[0022] 2. The present invention constructs an overexpression vector pCambia2301-PaYUC10 of the functional gene PaYUC10, and successfully obtains a transgenic positive strain of PaYUC10 gene overexpression by Agrobacterium tumefaciens-mediated heterologous transformation of tomatoes. The storage time and respiration intensity tests of transgenic positive tomatoes and negative plant fruits are conducted, and it is found that the respiration intensity of the transgenic strain is reduced and the storage time is extended. Therefore, the PaYUC10 gene can be used for variety improvement of storage-intolerant fruits. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a schematic diagram showing the change in the expression level of the PaYUC10 gene in Pearl Oil Apricot and Golden Sun with storage time in the embodiment of the present invention;

[0024] Figure 2 is a map of the overexpression vector pCambia2301-PaYUC10 of the PaYUC10 gene constructed in the embodiment of the present invention;

[0025] Figure 3 The MCS region sequence of the overexpression vector pCambia2301-PaYUC10 of the PaYUC10 gene constructed in the embodiment of the present invention;

[0026] Figure 4 This is a schematic diagram of the positive identification results of the DNA level of the tomato plants transformed by PaYUC10 in the embodiment of the present invention;

[0027] Figure 5 A comparison diagram of the storage conditions of the tomato fruits transformed with PaYUC10 and the tomato fruits of the control group in the embodiment of the present invention;

[0028] Figure 6 This is a curve diagram of the change in respiration intensity during storage of tomato fruits transformed with PaYUC10 and tomato fruits in the control group in the example of the present invention. DETAILED DESCRIPTION

[0029] In this example, storable apricot ('Zhenzhuyouxing'-ZZYX) and non-storable apricot ('Jintaiyang'-JTY) were used as research objects, and postharvest physiological measurements and transcriptomic analysis were performed, resulting in the acquisition of a candidate gene PaYUC10. The PaYUC10 gene was cloned from the apricot variety 'Zhenzhuyouxing', and its nucleotide sequence is shown in SEQ ID No.1; the protein encoded by the PaYUC10 gene is named PaYUC10 protein, and its amino acid sequence is shown in SEQ ID No.2.

[0030] The nucleotide sequence of SEQ ID No.1 is as follows:

[0031] atgatgtcccatcccttccgaacctccattttcatgtcgaaagacgacttcattcgttacctggacgagtacgtggccaggttcaacgtgaaccctcgctactgcagtgacgtggaggaggctgtttacgatgaagaggaagccaagtggagggtgacagtgaaagacaccgcttccgggacggtgtatgagatcgcatcggatttcttggtggttgcaactggagaaaatagccagccttttctccccagcgacttgcctgggatggagaccttcccaggaaaggtggttcattccagtgactataaaaacggggccgtttttaaagatgaaaaggttttggtaattggctgtggcaattctgggatggaaatcagcaacgatcttgcagacaatggagcacatacgtccattgttgttagaagccaaattcatgtgctgagcaaagaaatggttggcattggcatgcttctgtcgaattacctcccactgaaaatagtggacaagtttgtgtcactgcttgccaagttgagctacagtgatttgtccagccatggcattcatcgcccagttgaagggccatttctctttaaagcacttactggaaagactccagtaattgatcgtggaaccatcgagaaaattcgctccaaaaggattcaggtttttccagatgtagtgagtataaaccagaagactgtggaattcaaagatggtaaacgacggtgttttgatgctattgtgttagcaactggttacaaaagcgtagcacataaatggttgaaggactacaagttccttcttacagatgatggaaagcctaaagggaagtacccaaaccactggaagggcgagaaaggtgtttattgtgttggattaacagggaagggactgccagggattttcaaagattcaaaggctgtaactgaagacatctatcaacttacactaatggcacagaagtag

[0032] The amino acid sequence of SEQ ID No.2 is as follows:

[0033] MMSHPFRTSIFMSKDDFIRYLDEYVARFNVNPRYCSDVEEAVYDEEEAKWRVTVKDTASGTVYEIASDFLVVATGENSQPFLPSDLPGMETFPGKVVHSSDYKNGAVFKDEKVLVIGCGNSGMEISNDLADNGAHTSIVVRSQIHVLSKEMVGIGMLLSN YLPLKIVDKFVSLLAKLSYSDLSSHGIHRPVEGPFLFKALTGKTPVIDRGTIEKIRSKRIQVFPDVVSINQKTVEFKDGKRRCFDAIVLATGYKSVAHKWLKDYKFLLTDDGKPKGKYPNHWKGEKGVYCVGLTGKGLPGIFKDSKAVTEDIYQLTLMAQK

[0034] 1. Expression analysis of gene PaYUC10

[0035] During the postharvest storage period of apricot fruits at room temperature (D1-D6), transcriptomics and real-time fluorescence quantitative PCR experiments were used to detect and analyze the changes in the expression of PaYUC10 in the flesh of the storage-resistant variety 'Zhenzhuyouxing' and the storage-intolerant variety 'Jin Taiyang' to verify its function.

[0036] This example uses real-time fluorescence quantitative PCR (qPCR). The primers are designed according to the primer design principles of qPCR and Primer Premier 5 is used to design the primers online. The primers designed in this example are:

[0037] qYUC10F (GTGGAGGAGGCTGTTTACGA); qYUC10R (TGCAACCACCAAGAAATCCG). Actin was used as the internal reference and ddH2O was used as the negative control.

[0038] In this embodiment, the PCR system configuration (15 μL system) is shown in Table 1:

[0039] Table 1

[0040]

[0041] In this embodiment, the PCR reaction program settings are shown in Table 2:

[0042] Table 2

[0043]

[0044] The comparative Ct value method was used to analyze the data. In this example, Prism 5 statistical analysis software was used for data statistical analysis. Figure 1 As shown in the figure, during the storage of apricot fruit, PaYUC10 was highly expressed in the flesh of the storage-resistant variety 'Zhenzhuyouxing', which was always higher than the expression level in the flesh of the storage-intolerant variety 'Jin Taiyang', and its peak value was 4.95 times that of 'Jin Taiyang'. The functional gene PaYUC10 may have changed its amino acid sequence due to gene sequence variation, which in turn affects the storage time of apricot fruit, and is of great significance for its variety improvement.

[0045] 2. Construction of PaYUC10 overexpression vector for tomato transformation

[0046] Construction of the PaYUC10 gene overexpression vector pCambia2301-PaYUC10. The vector map is shown in Figure 2 , MCS region sequence see Figure 3 The function of PaYUC10 gene was further verified by Agrobacterium tumefaciens-mediated heterologous transformation of tomato. The specific construction steps are as follows:

[0047] (1) Construction of plasmid

[0048] The plasmid was constructed by PCR reaction using a high-fidelity enzyme (KOD-401, TOYOBO). The restriction fragment was recovered by electrophoresis (GK2042, Jierui Biotechnology), mixed with the empty vector recovered by restriction digestion, and added to the EasyGenoDNA recombination system for connection (#VI201-02, Tiangen Biotechnology) to construct the plasmid, obtaining pCambia2301-PaYUC10.

[0049] (2) Cultivation of Agrobacterium tumefaciens

[0050] Take the competent Agrobacterium stored at -80℃ and place it at room temperature or in the palm of your hand for a while until it partially melts. When it is in an ice-water mixing state, insert it into ice. Add 0.1 μg (volume ≤10 uL) plasmid DNA pCambia2301-PaYUC10 to every 100 μL competent state, stir the bottom of the tube by hand to mix, and then stand on ice for 5 min, liquid nitrogen for 5 min, 37℃ water bath for 5 min, and ice bath for 5 min. Add 700 μL of LB liquid medium without antibiotics and culture at 28℃ and 200 rpm for 2-3 h. Centrifuge at 6000 rpm for 1 min to collect the bacteria, keep about 100 μL of supernatant, gently blow and resuspend the bacteria block, apply it on the LB plate containing kanamycin kan, and invert it in a 28℃ incubator for 2-3 days. Randomly select 2-3 single colonies for colony PCR. Use a sterile pipette to pick up the correctly identified Agrobacterium monoclone and inoculate it into 5 mL LB liquid medium containing kanamycin kan (50 mL centrifuge tube) and culture it at 28℃, 200 rpm for 24 h. Centrifuge at 20℃, 4000 rpm for 15 min to collect the bacteria; pipette the bacteria evenly with transformation buffer and resuspend them to about OD600=0.5.

[0051] (3) Tomato genetic transformation

[0052] Select true leaves, cotyledons, and hypocotyls of tomatoes as explants for transformation. Cut off the tips and petioles of true leaves and cotyledons, and cut the rest into leaf pieces of 0.5 cm × 0.5 cm; cut the hypocotyls into segments of about 0.5-0.6 mm in length and place them horizontally on the pre-culture medium (the pre-culture medium uses MS as the basic culture medium, adds zeatin ZT to its concentration of 2.0 mg / L, and adds auxin IAA to its concentration of 0.2 mg / L), 15-20 pieces per dish. Put it in the pre-culture for 1 day under the conditions of 16 hours of light per day, light intensity of 1600-1800lx, and temperature of (24±2)℃ to obtain explants.

[0053] The explants were removed from the pre-culture medium and placed in a culture dish containing Agrobacterium diluted to about OD600=0.5 with transformation buffer. The transformation was carried out for 30 min. The explants were removed and blotted dry on sterile paper and placed back in the pre-culture medium for 1-2 days.

[0054] The co-cultivated explants were transferred to the selection medium (MS as the basic medium, with zeatin ZT2.0 mg / L, auxin IAA 0.2 mg / L, kanamycin Kan 100 mg / L, carbenicillin Cb 500 mg / L added) for selection culture. After a few days of selection culture, the cotyledons began to thicken and the hypocotyls began to thicken. The transformed explants will form callus and adventitious buds on the selection medium; subculture once every two weeks.

[0055] The callus with shoot primordium was cut into small pieces and transferred to stem elongation medium (MS as basic medium, supplemented with zeatin ZT 1.0 mg / L, auxin IAA 0.05 mg / L, kanamycin Kan 100 mg / L, and carbenicillin Cb 500 mg / L) for subculture, once every two weeks, or shorter if necessary.

[0056] When the adventitious buds grow to about 1 cm, select healthy regenerated buds, completely remove the base callus tissue and culture medium, and transfer them to rooting medium (MS as the basic culture medium, supplemented with 0.1 mg / L of auxin IAA, 50 mg / L of kanamycin Kan, and 300 mg / L of Cb carbenicillin) to culture them to form complete plants.

[0057] After the seedlings grow lateral roots, open the bottle cap, pour a small amount of sterile water into the culture bottle (overflowing the culture medium by 3-5 mm), and place the culture bottle in a cool and ventilated place for seedling hardening. After 3 days, wash the root culture medium, transfer the seedlings to the soil, cover the seedlings with transparent plastic film for the first 7 days, and cultivate them in weak light for a period of time to allow the regenerated seedlings to adapt to the transition from culture medium to nutrient matrix. After the plants are alive, move them to the natural environment for seedling hardening.

[0058] (4) DNA extraction from transformed tomato seedlings and PCR identification

[0059] Table 3

[0060]

[0061] Table 4

[0062]

[0063] The amplified products were identified by agarose gel electrophoresis. Figure 4 As shown, plants 3, 4, 6, 7, 9, and 10 were PCR positive, and the remaining 6 plants (plants 1, 2, 5, 8, 11, and 12) were negative.

[0064] (5) Observation on the storage time of transgenic tomatoes overexpressing PaYUC10

[0065] The harvested tomato fruits were stored in a constant temperature incubator at 25°C and 75% humidity to observe the differences in storage time. Figure 5 As shown. The transgenic tomato fruits overexpressing PaYUC10 (PaYUC10) showed obvious wrinkles and reached the shelf life in 13 days, which was much longer than the control tomato fruits (WT) (7 days). During storage, the peak respiration intensity of the transgenic tomato fruits overexpressing PaYUC10 was only 44.01% of that of the control tomatoes (see Figure 6 ); This indicates that the PaYUC10 gene can be used to regulate the storage time of fruits, which is of great significance for variety improvement.

[0066] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the claims of this patent application.

Claims

1. A functional gene PaYUC10 for upregulating fruit storability, characterized in that: The nucleotide sequence of the functional gene PaYUC10 is shown as SEQ ID No.1; or the functional gene PaYUC10 is a gene encoding a protein consisting of the amino acid sequence shown as SEQ ID No.

2.

2. An application of the functional gene PaYUC10 for increasing the storage stability of fruits, characterized in that: The functional gene PaYUC10 for increasing the storability of fruits as claimed in claim 1 is used to increase the storability of fruits.

3. The use of the functional gene PaYUC10 for increasing the storability of fruits according to claim 2, characterized in that: First, the functional gene PaYUC10 is inserted into an overexpression vector to form a recombinant plasmid, and then the recombinant plasmid is transformed into Agrobacterium. Finally, the Agrobacterium transformed with the recombinant plasmid is used to infect plants that are not tolerant to storage fruits, so that it is overexpressed in the plants.

4. The use of the functional gene PaYUC10 for increasing the storability of fruits according to claim 2, characterized in that: The steps include: Step (1), using a high-fidelity enzyme to perform a PCR reaction to construct a recombinant plasmid pCambia2301-PaYUC10; Step (2), using the recombinant plasmid pCambia2301-PaYUC10 to transform Agrobacterium to obtain transformed Agrobacterium: Step (3), first pre-culture the explants of the storage-intolerant plants; then place the pre-cultured explants in a culture dish with transformation Agrobacterium for transformation culture, and after the transformation culture is completed, transfer the explants back to the pre-culture medium for co-culture; after the co-culture is completed, transfer the explants to a screening culture medium for selection culture to form callus tissue and / or adventitious buds; Step (4) transferring the young shoots or adventitious shoots obtained by subculture of the callus tissue to a rooting medium to obtain complete plants.

5. The use of the functional gene PaYUC10 for increasing the storability of fruits according to claim 4, characterized in that: In step (1), the high-fidelity enzyme is KOD-401 high-fidelity enzyme.

6. The use of the functional gene PaYUC10 for increasing the storability of fruits according to claim 4, characterized in that: In step (3), the explant is a true leaf, a cotyledon or a hypocotyl.

7. The use of the functional gene PaYUC10 for increasing the storability of fruits according to claim 6, characterized in that: In step (3), the tips and petioles of the true leaves and cotyledons are cut off, and the remaining parts are cut into leaf pieces of 0.5 cm × 0.5 cm; the hypocotyls are cut into segments of 0.5 to 0.6 mm in length and placed horizontally on the preculture medium, with 15 to 20 pieces placed in each preculture medium; the preculture time is 1 day; the concentration of the transformed Agrobacterium in the culture dish is 0.4 to 0.6 at OD600, and the transformation culture time is 30 minutes; the co-culture time is 1 to 2 days.

8. The use of the functional gene PaYUC10 for increasing the storability of fruits according to claim 6, characterized in that: In step (4), when the young shoots grow to 0.8-1.2 cm, they are transferred to rooting medium for cultivation.

9. The use of the functional gene PaYUC10 for increasing the storability of fruits according to claim 3, characterized in that: The overexpression vector was pCambia2301 and the Agrobacterium strain was GV3101.

10. The use of the functional gene PaYUC10 for increasing the storability of fruits according to claim 3, characterized in that: The plant that is not tolerant of storage fruit is the tomato plant.

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