Application and method of CmGH9B3 gene in promoting grafting healing of muskmelon

Abstract

The invention relates to the technical field of grafting cultivation, and particularly discloses application and a method of a CmGH9B3 gene in promoting grafting healing of muskmelons, the nucleotide sequence of the CmGH9B3 gene is shown as SEQ ID NO.6, and the amino acid sequence coded by the CmGH9B3 gene is shown as SEQ ID NO.7; by regulating and controlling the overexpression of the CmGH9B3 gene in the muskmelon scion, the healing of the graft union after the muskmelon is grafted with the pumpkin is promoted. Through overexpression of the CmGH9B3 gene in the muskmelon scion, the physical adhesion capacity of the muskmelon scion and the grafted seedling of the pumpkin rootstock is improved, the conduit absorption capacity of the grafted seedling is improved, and healing of a graft union after the muskmelon is grafted with the pumpkin is promoted.

Description

Technical Field

[0001] The present invention relates to the technical field of grafting cultivation, and specifically to the application and method of the CmGH9B3 gene in promoting the graft healing of melons. Background Art

[0002] Grafting is a propagation method in which a bud or branch of one plant is grafted onto another plant so that the two joined parts grow into a complete plant. The bud or branch grafted on is called the scion, and the plant to which it is grafted is called the rootstock. The key to successful grafting is to closely combine the cambiums of the scion and the rootstock. Cambium cells have the ability to divide. They can divide outward to produce phloem with sieve tubes inside, and divide inward to produce xylem with vessels inside. In this way, the vascular tissues of the two plants are united into a whole, which can not only ensure the upward transport of water and inorganic salts from the roots, but also ensure the downward transport of organic nutrients produced by photosynthesis to the roots. The grafted plant can then survive and carry out normal physiological functions. Affinity refers to the ability of the rootstock and scion to heal after grafting. Generally, the closer the genetic relationship, the stronger the affinity and the higher the graft survival rate. It can be seen that the graft healing ability directly affects the graft survival rate. In actual production, the healing process of grafted seedlings is mainly regulated by controlling environmental conditions such as light, temperature, and humidity in the facility to ensure the healing efficiency of grafted seedlings. However, due to large environmental differences between different regions and facilities, management methods are also different and there is no unified standard. Therefore, it is difficult to ensure efficient graft healing.

[0003] At present, grafting cultivation has become one of the main cultivation modes in melon production. Efficient cultivation of melon grafted seedlings is an important basis for ensuring the effect of melon grafting cultivation. However, in actual production, due to differences in rootstock-scion combinations, grafting methods, and environmental regulation, the cultivation cost of melon grafted seedlings is relatively high, and the quality is uneven, seriously affecting the effect of melon grafting cultivation and the economic benefits of the industry. Therefore, creating key technologies for cultivating melon grafted seedlings with high healing efficiency is of great value to the sustainable and efficient development of the melon industry. It is necessary to explore a new way to improve the graft healing ability of melons. Summary of the Invention

[0004] To develop a new way to improve the graft healing ability of melons, the present invention provides the application and method of the CmGH9B3 gene in promoting the graft healing of melons. By overexpressing the CmGH9B3 gene in the melon scion, the present invention improves the physical adhesion ability of the grafted seedlings of melon scions and pumpkin rootstocks and the catheter absorption ability of the grafted seedlings, thereby promoting the rapid healing of the graft interface of melon grafted to pumpkin.

[0005] The present invention provides the application of the CmGH9B3 gene in promoting the graft healing of melons. The nucleotide sequence of the CmGH9B3 gene is as shown in SEQ ID NO.6, and the amino acid sequence encoded by it is as shown in SEQ ID NO.7; by regulating the overexpression of the CmGH9B3 gene in the melon scion, the healing of the graft union after grafting melons onto pumpkins is promoted.

[0006] By means of genetic engineering, the present invention enables the overexpression of the CmGH9B3 gene in the melon scion, improves the physical adhesion ability of the grafted seedlings of the melon scion and the pumpkin rootstock, and the catheter absorption ability of the grafted seedlings, and promotes the healing of the graft union of melons grafted onto pumpkins.

[0007] Furthermore, the overexpression of the CmGH9B3 gene in the melon scion is used to improve the physical adhesion ability of the grafted seedlings of the melon scion and the pumpkin rootstock and to improve the catheter absorption ability of the grafted seedlings.

[0008] The present invention also provides an overexpression recombinant vector of the CmGH9B3 gene, which is obtained by ligating the CmGH9B3 gene to the BAMHI and SALI sites of the pCAMBIA1300 vector.

[0009] Furthermore, the primers for amplifying the CmGH9B3 gene are as shown in SEQ ID NO.4 to SEQ ID NO.5.

[0010] The present invention also provides a recombinant Agrobacterium, which contains the overexpression recombinant vector of the CmGH9B3 gene.

[0011] The present invention also provides a method for rapidly promoting the healing of the graft union between melons and pumpkins, including the following steps:

[0012] Ligate the CmGH9B3 target gene to the BAMHI and SALI sites of the pCAMBIA1300 vector to construct an overexpression recombinant vector of the CmGH9B3 gene, transform the competent cells to obtain positive clones and transfer them into the competent cells of Agrobacterium rhizogenes K599 to obtain recombinant Agrobacterium;

[0013] Use the recombinant Agrobacterium to infect the hypocotyls of melons and culture to obtain an overexpression melon line of the CmGH9B3 gene;

[0014] When a true leaf of the overexpression melon line of the CmGH9B3 gene is fully expanded, use it as a melon scion to graft onto a pumpkin.

[0015] Furthermore, the melon is the thin-skinned melon variety "Huabao".

[0016] Furthermore, the pumpkin is the Chinese pumpkin variety "Shengzhen No.1".

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] Through genetic transformation, the present invention enables overexpression of the CmGH9B3 gene in the melon scion. Compared with injecting the empty vector, it significantly improves the physical adhesion ability of the grafted seedlings of the melon scion and the pumpkin rootstock, as well as the catheter absorption ability of the grafted seedlings, and enhances the healing rate of the grafting interface of the melon grafted onto the pumpkin. It provides technical support for the melon grafting technology. Brief Description of the Drawings

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0020] Figure 1 It is the expression level of CmGH9B3 in the CmGH9B3 transient silencing line and the CmGH9B3 overexpression line of the present invention;

[0021] In the figure, A is the expression level of CmGH9B3 in the CmGH9B3 transient silencing line constructed in Example 1;

[0022] B is the expression level of CmGH9B3 in the CmGH9B3 overexpression line constructed in Example 2.

[0023] Figure 2 It is the quantification diagram of the expression level of CmGH9B3 during the grafting healing process of the melon / pumpkin grafted seedlings in the silencing line of the present invention.

[0024] Figure 3 It is the quantification diagram of the expression level of CmGH9B3 during the grafting healing process of the melon / pumpkin grafted seedlings in the overexpression line of the present invention.

[0025] Figure 4 It is the influence of the CmGH9B3 gene on the healing efficiency of the melon / pumpkin grafting interface;

[0026] In the figure, A is the test of the physical adhesion rate and the catheter connection rate after melon / pumpkin grafting

[0027] Among them, a represents cutting the melon scion and the pumpkin rootstock; b represents fixing the melon / pumpkin grafted seedlings with a grafting clip; c represents removing the grafting clip and counting the number of melon scions separated from the pumpkin rootstock; d represents the operation method of the acid fuchsin absorption test; e represents the phenotype diagram of the unconnected melon / pumpkin grafting interface; f represents the phenotype diagram of the connected melon / pumpkin grafting interface;

[0028] B is the influence of the CmGH9B3 gene on the physical adhesion rate after melon / pumpkin grafting;

[0029] C is the effect of the CmGH9B3 gene on the conduit connectivity rate after grafting melon / pumpkin. Detailed implementation manners

[0030] The following details the specific implementation manners of the present invention, but it should be understood that the protection scope of the present invention is not limited by the specific implementation manners. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0031] Example 1: Effect of CmGH9B3 gene silencing on the healing efficiency of the grafting interface between melon scions and pumpkin rootstocks.

[0032] 1. Construction of pTRV2 recombinant vector and recombinant Agrobacterium

[0033] Extract the RNA of the melon variety "Huabao" (provided by Shaanxi Yangling Qianjin Agricultural Development Co., Ltd.), and reverse transcribe it into cDNA. Using the cDNA as a template, use CmGH9B3-pTRV2-F and CmGH9B3-pTRV2-R as primers to amplify the target fragment: the CmGH9B3 gene. The target fragment is shown in SEQ ID NO.3 and has a size of 300 bp.

[0034] CmGH9B3-pTRV2-F: 5'-CAATGTAAAATTCAATCTTCCGATGG-3' (SEQ ID NO.1);

[0035] CmGH9B3-pTRV2-R: 5'-AACATCCGATCCTGGGTTC-3' (SEQ ID NO.2).

[0036] SEQ ID NO.3:

[0037] ATGGCTTTCACAACCACAATGCTTTCATGGGGAGCACTCGAGTACGGGGCGCGTATGGGTAGCGAATTAGGCAACACACGAGCCGCCATCCGTTGGGCCACCGATTACCTTCTCAAGTGCGCGACCGCCACTCCAGGCAAGCTCTACGTCGGCGTGGGAGACCCTCACGCCGACCACAAGTGCTGGGAACGGCCTGAGGACATGGATACTGTTCGAACCGTATACTCTGTTTCTGCCGGGAACCCAGGATCGGATGTTG。

[0038] Amplification reaction system: 25 μL of high-fidelity DNA polymerase, 2 μL of CmGH9B3-pTRV2-F, 2 μL of CmGH9B3-pTRV2-R, 1 μL of cDNA, 20 μL of RNase-Free ddH2O.

[0039] Amplification reaction procedure: 5 min at 95 °C for 1 cycle; 30 s at 95 °C, 30 s at 55 °C, 40 s at 72 °C for 35 cycles; 10 min at 72 °C.

[0040] Take 2 μL of the amplification product, 3 μL of the pTRV2 vector, 5 μL of 2xInflusion (Shenyang Wanze Biotechnology Co., Ltd., product number Code#CU201-03), and connect in a metal bath at 50 °C for 30 min. According to the above ligation system, ligate the target fragment of the CmGH9B3 gene with the EOCRI and BAMHI sites of the pTRV2 vector to obtain the pTRV2 recombinant vector. Transform the pTRV2 recombinant vector into Escherichia coli DH5α competent cells, pick single colonies for colony PCR identification, and sequence alignment to obtain correct positive clones. Transfer the obtained positive clones into GV3101 Agrobacterium competent cells to obtain recombinant Agrobacterium for subsequent melon cotyledon infection experiments.

[0041] 2. Construction of transient silencing melon lines of the CmGH9B3 gene

[0042] Using the melon variety "Huabao" as the test material, transform the pTRV2 recombinant vector into melon self-rooted seedlings by the method of slowly injecting the prepared infection solution through the back of a 5-ml syringe into the melon cotyledons to obtain transient silencing materials. The specific steps are as follows:

[0043] (1) When the melon seedlings transplanted in the substrate as explants grow to the stage where the cotyledons are fully unfolded, use the recombinant Agrobacterium carrying the pTRV2 recombinant vector with an OD 600 of approximately 0.7 to infect the melon cotyledons.

[0044] (2) After inoculation, culture in an artificial light incubator. First, keep it in the dark for 18 h, and then have normal day-night alternation. The light culture temperature is 26 °C and the dark culture temperature is 18 °C. Obtain the transient silencing melon line of the CmGH9B3 gene, denoted as TRV-CmGH9B3.

[0045] Example 2: Effect of overexpression of the CmGH9B3 gene on the graft union healing efficiency of melon scions and pumpkin rootstocks.

[0046] 1. Construction of the pCAMBIA1300 recombinant vector and recombinant Agrobacterium

[0047] Extract the RNA of the melon cultivar "Huabao" (provided by Shaanxi Yangling Qianjin Agricultural Development Co., Ltd.), and reverse transcribe it into cDNA. Using the cDNA as a template, use CmGH9B3-1300-F and CmGH9B3-1300-R as primers to amplify the target fragment: the CmGH9B3-OE gene. The target fragment is shown in SEQ ID NO.6 and its size is 1488 bp.

[0048] CmGH9B3-1300-F: 5'-ATGGCTTCTCCCATCTCAAAT-3' (SEQ ID NO.4); CmGH9B3-1300-R: 5'-ATGTTTGCCCGAGAAGAATG-3' (SEQ ID NO.5).

[0049] CmGH9B3-OE gene:

[0050]

[0051] The amino acid sequence encoded by the CmGH9B3-OE gene is shown in SEQ ID NO.7.

[0052] SEQ ID NO.7:

[0053] MASPISNSSSTLYSLFFYFGLLLSFSFAGRARANPNYRDALAKSILFFEGQRSGRIPANQRITWRSNSGLYDGELAHVDLTGGYYDAGDNVKFNLPMAFTTTMLSWGALEYGARMGSELGNTRAAIRWATDYLLKCATATPGKLYVGVGDPHADHKCWERPEDMDTVRTVYSVSAGNPGSDVAGETAAALAAASLVFRRVDRKYSRVLLATAKKVMEFALEHRGSYSDSLSSAVCPFYCSYSGYKDELVWGAAWLLRATNDVKYFNLLKSLGGDDVTDIFSWDNKFAGAHVLLSRRSLLNNDKNFDLYKQEAEAFMCRILPNSPSSSTKYTQGRLMFKLPESNLQYVTSITFLLTTYSKYMSAGKHTFNCGNLVVTPASLKNLAKIQVDYILGVNPLKMSYMVGYGKNFPKRIHHRGSSLPSKATHPQAIACDGGFQPFFYSYNPNPNILIGAVVGGPNQSDGFPDDRTDYSHSEPATYINAALVGPLAFFSGKH.

[0054] Amplification reaction system: 25 μL of high-fidelity DNA polymerase, 2 μL of CmGH9B3-1300-F, 2 μL of CmGH9B3-1300-R, 1 μL of cDNA, 20 μL of RNase-Free ddH2O.

[0055] Amplification reaction procedure: 5 min at 95°C, 1 cycle; 30 s at 95°C, 30 s at 55°C, 100 s at 72°C, 35 cycles; 10 min at 72°C.

[0056] Take 2 μL of the amplified product of the target fragment, 3 μL of the pCAMBIA 1300 vector plasmid, 5 μL of 2xInflusion, and connect them in a metal bath at 50 °C for 30 min. Connect the target fragment to the BAMHI and SALI sites of the pCAMBIA 1300 vector to obtain the 1300 recombinant vector. Transform the 1300 recombinant vector into competent Escherichia coli DH5α cells, pick single colonies for colony PCR identification, and sequence alignment to obtain correct positive clones. Transfer the obtained positive clones into competent Agrobacterium rhizogenes K599 to obtain recombinant Agrobacterium, which is used for subsequent melon hypocotyl infection experiments.

[0057] 2. Construction of CmGH9B3 gene overexpression melon lines

[0058] Using the melon cultivar "Huabao" as the test material, the 1300 recombinant vector was transformed into melon self-rooted seedlings by the root induction method of slanting the hypocotyls of melon seedlings, soaking the cut hypocotyls in the prepared infection solution for 30 min, transplanting them into the substrate, and injecting 1 ml of the infection solution into the substrate to obtain overexpression materials. The specific steps are as follows:

[0059] (1) When the melon seedlings transplanted as explants into the substrate grow until the cotyledons are fully unfolded, use recombinant Agrobacterium carrying the 1300 recombinant vector with an OD 600 of approximately 0.7 to infect the melon hypocotyls.

[0060] (2) After infection, culture in an artificial light incubator, first in the dark for 18 h, and then with normal day-night alternation. The light culture temperature is 26 °C and the dark culture temperature is 18 °C. Obtain CmGH9B3 gene overexpression melon lines, denoted as CmGH9B3-OE.

[0061] Identify the transient silencing melon lines of the CmGH9B3 gene constructed in Example 1 and the overexpression melon lines of the CmGH9B3 gene constructed in Example 2, and analyze the effect of the CmGH9B3 gene on the graft union healing efficiency. The test methods and results are as follows.

[0062] I. Identification of the transient silencing melon lines of the CmGH9B3 gene constructed in Example 1 and the overexpression melon lines of the CmGH9B3 gene constructed in Example 2 by qRT-PCR analysis

[0063] (1) Extract the RNA from the leaves of the transiently silenced melon lines of the CmGH9B3 gene and the overexpressed melon lines of the CmGH9B3 gene, and perform qRT-PCR analysis on the CmGH9B3-silenced plants. The transiently silenced melon lines were compared with the melon leaves injected with the mixed infection solution of the empty vectors pTRV1 and pTRV2, and the overexpressed melon lines were compared with the melon leaves injected with the infection solution of the empty vector pCAMBIA 1300. Design real-time quantitative primers for the CmGH9B3-silenced and overexpressed plants for detection.

[0064] The sequences of the real-time quantitative primers are as follows:

[0065] CmGH9B3-F: 5'-GAACCCAGGATCGGATGTTG-3' (SEQ ID NO.8);

[0066] CmGH9B3-R: 5'-GCAGAGGAAAGCGAATCACTAT-3' (SEQ ID NO.9).

[0067] The results are as Figure 1 shown. The expression level in the CmGH9B3 transiently silenced lines was significantly reduced compared with the control, approximately by 6-fold, while the expression level in the CmGH9B3 overexpressed lines was significantly increased compared with the control, approximately by 2-fold. This indicates that both the CmGH9B3 transiently silenced and overexpressed vector plasmids have been successfully transformed into melons and can significantly regulate the expression of CmGH9B3 in the melon stem tissue.

[0068] (2) Grafting can be carried out when a true leaf of the transiently silenced melon lines and the overexpressed melon lines is fully expanded, and samples are taken 6 days after grafting.

[0069] II. Effects of transient silencing and overexpression of CmGH9B3 on the expression level of CmGH9B3 during the graft union healing process

[0070] To clarify the effects of transient silencing and overexpression of CmGH9B3 on the expression level of CmGH9B3 during the graft union healing process of melons and pumpkins, we took the melon stem tissue of the identified positive plant grafted seedlings, compared with the grafted seedlings injected with the empty vector normally, extracted RNA and reverse transcribed it into cDNA. Using the real-time quantitative primers of CmGH9B3 and taking the 18S gene as the internal reference, qRT-PCR analysis was performed.

[0071] The sequences of the real-time fluorescence quantitative primers are the same as above. The results are as Figure 2 shown. During the graft union healing process, the expression level of CmGH9B3 in the silenced plants was significantly reduced compared with the control. From Figure 3It can be seen that the expression level of CmGH9B3 in the overexpressing plants was significantly increased compared with the control. This indicates that the CmGH9B3-silenced plants can significantly inhibit the expression of CmGH9B3 in the stem tissue of the melon scion; on the contrary, the CmGH9B3-overexpressing plants can significantly promote the expression of CmGH9B3 in the stem tissue of the melon scion.

[0072] III. Effects of Transient Silencing and Overexpression of CmGH9B3 on the Grafting Healing Efficiency of Melon / Pumpkin

[0073] (1) Two days after grafting, the grafting clips were removed, and the physical adhesion rate of the grafted seedlings was counted. As can be seen from Figure 4 it, transient silencing of CmGH9B3 can significantly reduce the physical adhesion ability of the grafted seedlings of the melon scion and the pumpkin rootstock. While overexpression of CmGH9B3 can significantly improve the physical adhesion ability of the grafted seedlings of the melon scion and the pumpkin rootstock.

[0074] (2) Six days after grafting, the stem segments 1 cm above and below the grafting interface were transversely cut, and the pumpkin rootstock was placed vertically downward in 1% acid fuchsin solution for 40 min. The stem segment 2.5 mm above the grafting joint was transversely cut by hand, and the absorption of acid fuchsin was observed under a microscope to count the vessel connection ability of the grafted seedlings. As can be seen from Figure 4 it, transient silencing of CmGH9B3 can significantly reduce the vessel absorption ability of the grafted seedlings, while overexpression of CmGH9B3 can significantly improve the vessel absorption ability of the grafted seedlings. It can be seen that CmGH9B3 plays a key role in improving the grafting healing ability of the melon scion and its grafting healing efficiency with the pumpkin rootstock.

[0075] The melon used in the present invention is the thin-skinned melon variety "Huabao", and the pumpkin used is the Chinese pumpkin variety "Shengzhen No. 1".

[0076] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic creative concepts.

[0077] Obviously, those skilled in the art can make various changes and deformations to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and deformations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and deformations.

Claims

1. Application of the CmGH9B3 gene in promoting graft healing of melons, characterized in that, The nucleotide sequence of the CmGH9B3 gene is shown in SEQ ID NO.6, and the amino acid sequence encoded by it is shown in SEQ ID NO.7; by regulating the overexpression of the CmGH9B3 gene in melon scions, the healing of the graft union after grafting melons onto pumpkins is promoted.

2. Use of the CmGH9B3 gene according to claim 1 in promoting healing after grafting of melons, characterized in that, The overexpression of the CmGH9B3 gene in melon scions is used to improve the physical adhesion ability of grafted seedlings of melon scions and pumpkin rootstocks and to improve the duct absorption ability of grafted seedlings.

3. An overexpression recombinant vector of the CmGH9B3 gene, characterized in that, It is obtained by ligating the CmGH9B3 gene to the BAMHI and SALI sites of the pCAMBIA1300 vector.

4. The CmGH9B3 gene overexpression recombinant vector according to claim 3, characterized in that, The primers for amplifying the CmGH9B3 gene are shown in SEQ ID NO.4 to SEQ ID NO.

5.

5. A recombinant Agrobacterium, characterized in that, The recombinant Agrobacterium contains the CmGH9B3 gene overexpression recombinant vector described in claim 3.

6. A method for improving the rapid healing after grafting melons and pumpkins, characterized in that, It includes the following steps: The CmGH9B3 target gene is ligated to the BAMHI and SALI sites of the pCAMBIA1300 vector to construct an overexpression recombinant vector of the CmGH9B3 gene, positive clones are obtained by transformation of competent cells and transferred into the competent cells of Agrobacterium rhizogenes K599 to obtain recombinant Agrobacterium; The melon hypocotyls are infected with the recombinant Agrobacterium, and overexpression melon lines of the CmGH9B3 gene are obtained by cultivation; When a true leaf of the overexpression melon line of the CmGH9B3 gene is fully expanded, it is used as a melon scion and grafted onto a pumpkin.

7. The method for improving the rapid healing after grafting of melon and pumpkin according to claim 6, characterized in that, The melon is the thin-skinned melon variety "Huabao".

8. The method for improving the rapid healing after grafting melons and pumpkins according to claim 6, characterized in that, The pumpkin is the Chinese pumpkin variety "Shengzhen No.1".

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