Application of endogenous growth regulatory gene combinations in strawberry genetic transformation

Abstract

The present invention provides an application of a strawberry endogenous growth regulatory gene combination in genetic transformation, wherein the strawberry endogenous growth regulatory gene combination is selected from any one of the following groups: FveGRF8 and FveGIF1; or FveGRF3 and FveGIF1; wherein the nucleotide sequences of FveGRF3, FveGRF8 and FveGIF1 are: (a) the nucleotide sequence shown in SEQ ID NO: 1-3. The strawberry endogenous growth regulatory gene combination is transferred into strawberries by genetic transformation methods such as Agrobacterium infection, which can not only improve the bud regeneration rate, but also improve the genetic transformation seedling rate and rooting rate of strawberries, greatly shorten the genetic transformation time, and also lay a foundation for large-scale genetic transformation screening of functional genes and molecular breeding.

Description

Technical Field

[0001] The invention relates to the technical field of genetic transformation, and in particular to an application of a strawberry endogenous growth regulatory gene combination in genetic transformation. Background Art

[0002] Strawberry is an important economic crop with a wide cultivation area and a large cultivated area. It is grown in many countries. The strawberry growth cycle is relatively short, and it has the potential to be used as a research model for non-respiratory climacteric fruits, in order to theoretically establish a strawberry fruit development model, identify and explore genes related to strawberry quality traits, and reveal their genetic regulatory mechanisms, which will be beneficial to the screening of excellent strawberry germplasm resources in production. At present, the whole genome and pan-genome sequencing of strawberries have been completed, and many potential regulatory genes that may regulate strawberry fruit yield, fruit quality, fruit shape, disease resistance and stress tolerance have been discovered. In the future, it is expected that gene editing technology will be used to assist and accelerate the molecular breeding process.

[0003] Variety improvement through traditional breeding methods has a long cycle, and some traits are difficult to obtain. Genetic engineering technology is needed to complete gene function verification and variety improvement. With the continuous development of biotechnology, the use of gene editing technology to manipulate genetic material has begun to be widely used in various plants, providing a new direction for the creation of new germplasm. At present, a genetic transformation system has been established in strawberries, but the overall number of transformable varieties is small and the genetic transformation efficiency is relatively low, especially for octoploid cultivated strawberries, whose genetic transformation bud regeneration rate is only 4-11%, and the cycle is long. It takes about 15 months to obtain experimental materials for analysis, which requires a lot of manpower and material resources.

[0004] The overall genetic transformation efficiency of strawberry is relatively low, mainly reflected in the low genetic transformation bud regeneration rate and genetic transformation seedling rate. In the prior art, the transformation efficiency of the method is often reflected by the bud regeneration rate, but vitrification phenomenon is prone to occur after bud regeneration, so the fact that the explant can sprout does not mean that it can smoothly grow into a positive plant and complete stable transformation. Therefore, it is more accurate to use the positive seedling rate as the standard of the overall genetic transformation efficiency of strawberry. In addition, due to the long genetic transformation time cycle, its excessively long cultivation process will also cause the adverse effects of experimental operations on the genetic transformation efficiency such as pollution in the cultivation process and poor growth of the explant. Therefore, how to provide a genetic transformation method that can quickly complete genetic transformation seedlings and has a high seedling rate is very important to accelerate molecular breeding in the field of strawberry. Summary of the invention

[0005] The main purpose of the present invention is to provide an application of a strawberry endogenous growth regulatory gene combination in genetic transformation to solve the problem of low seedling rate of strawberry genetic transformation in the prior art.

[0006] In order to achieve the above-mentioned object, according to the first aspect of the present invention, there is provided an application of a strawberry endogenous growth regulatory gene combination in genetic transformation, wherein the strawberry endogenous growth regulatory gene combination is selected from any one of the following groups: FveGRF8 and FveGIF1; or FveGRF3 and FveGIF1; wherein the nucleotide sequence of FveGRF3, FveGRF8, and FveGIF1 is: (a) a nucleotide sequence as shown in SEQ ID NOs: 1-3; or (b) a nucleotide sequence encoding strawberry endogenous growth factors FveGRF3, FveGRF8, and FveGIF1 having an amino acid sequence as shown in SEQ ID NOs: 4-6; or (c) a nucleotide sequence having more than 85% homology with the nucleotide sequence of any one of (a) and (b) and having the function of improving the transformation efficiency of strawberry.

[0007] In order to achieve the above object, according to a second aspect of the present invention, a recombinant protein is provided, wherein the recombinant protein is encoded by the strawberry endogenous growth regulatory gene combination in the above application.

[0008] In order to achieve the above-mentioned object, according to the third aspect of the present invention, a recombinant vector is provided, which comprises a strawberry endogenous growth regulatory gene combination and a driving element for driving the expression of the strawberry endogenous growth regulatory gene combination, wherein the strawberry endogenous growth regulatory gene combination is a combination of FveGRF8 and FveGIF1 or a combination of FveGRF3 and FveGIF1 in the application of claim 1.

[0009] Furthermore, the above-mentioned driving element includes a promoter and a terminator; preferably, the promoter includes a constitutive promoter or an inducible promoter; preferably, the constitutive promoter includes Arabidopsis UBQ10 promoter or tobacco mosaic virus 35S promoter; preferably, the inducible promoter includes estradiol-inducible promoter; preferably, the terminator includes tobacco mosaic virus Nos terminator; preferably, the recombinant vector includes SunTag system and XVE system.

[0010] In order to achieve the above object, according to the fourth aspect of the present invention, a host cell transformed with the above recombinant vector is provided.

[0011] Furthermore, the host cell includes Escherichia coli or Agrobacterium.

[0012] In order to achieve the above-mentioned purpose, according to the fifth aspect of the present invention, a genetic transformation method of strawberry is provided. The genetic transformation method comprises: using the above-mentioned host cell to transform the above-mentioned strawberry endogenous growth regulatory gene combination into the target plant material; preferably, the strawberry endogenous growth regulatory gene combination is selected from any one or more of the following: FveGRF8 and FveGIF1; or FveGRF3 and FveGIF1; preferably, the number of the strawberry endogenous growth regulatory gene combination is independently 1 or more.

[0013] Furthermore, the above-mentioned genetic transformation method includes any one of the following transformation methods: Agrobacterium infection method, gene gun method, PEG-mediated protoplast transformation, plant virus-mediated transformation, pollen tube channel method and ovary injection method; preferably, the Agrobacterium infection method includes: introducing a recombinant vector into an Agrobacterium strain to obtain a recombinant bacterium; preparing the recombinant bacterium into an impregnation solution; placing the target plant material in the impregnation solution for impregnation to obtain an infected explant; co-culturing the infected explant and the recombinant bacteria to obtain a co-cultured explant; positively screening the co-cultured explant to obtain a positive tissue culture seedling; preferably, the Agrobacterium strain is GV3101; preferably, the target plant material includes a strawberry material; preferably, the strawberry material is selected from any one of the following: petals, pollen, cotyledons, cotyledon nodes, leaves, stems or protoplasts.

[0014] Furthermore, the above-mentioned positive screening includes: screening the co-cultured explants for antibiotic resistance to obtain positive transformants; performing bud growth and rooting culture on the positive transformants to obtain seedlings; performing genetic testing on the seedlings to obtain positive tissue culture seedlings; preferably, the genetic testing includes PCR, first-generation sequencing and / or high-throughput sequencing.

[0015] In order to achieve the above object, according to the sixth aspect of the present invention, there is provided the application of the above recombinant vector, or the above host cell, or the above genetic transformation method in strawberry genetic transformation.

[0016] By applying the technical scheme of the present invention, the strawberry endogenous growth regulatory gene combination FveGRF8 and FveGIF1 or FveGRF3 and FveGIF1 provided by the present application can be used to improve the genetic transformation seedling rate of strawberries. The strawberry endogenous growth regulatory gene combination is transferred into strawberries by genetic transformation methods such as Agrobacterium infection, which can not only improve the bud regeneration rate, but also improve the genetic transformation seedling rate of strawberries, and can quickly take root to 3-4cm, greatly shortening the genetic transformation time (transformation budding time and seedling time), and also laying a foundation for large-scale genetic transformation screening of functional genes and molecular breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0018] Figure 1 A schematic diagram of the recombinant vector of the strawberry endogenous growth regulatory gene of the present application is shown.

[0019] Figure 2 A schematic diagram showing the sequence structure of strawberry endogenous growth regulatory genes;

[0020] Figure 3 A schematic diagram showing the regenerated shoots obtained during the genetic transformation of strawberry;

[0021] Figure 4 Schematic diagram showing rooted plants obtained during genetic transformation of strawberry (bar=1 cm);

[0022] Figure 5 A schematic diagram of strawberry genetic transformation tissue culture is shown;

[0023] Figure 6 The statistical results of strawberry genetic transformation seedling time are shown;

[0024] Figure 7 The statistical results of strawberry genetic transformation seedling rate are shown;

[0025] Figure 8 The total number of FveGRF and FveGIF in the FveGRF-FveGIF combination in 900 positive transgenic strawberries is shown;

[0026] Fig. 9 The statistical results of the FveGRF-FveGIF combination in 80 positive transgenic strawberries with single combination effects are shown. DETAILED DESCRIPTION

[0027] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0028] As described in the background technology, the existing technology of strawberry genetic transformation has the defects of relatively low overall genetic transformation efficiency, only 4-11%, and long cycle. In addition, when calculating the genetic transformation efficiency, most of the attention is paid to the budding efficiency of the explant, i.e. the bud regeneration rate, rather than the seedling rate. Therefore, the data obtained by statistics of many existing transformation methods are only the data of the explant budding stage, and for actual strawberry breeding, it is necessary that the transformant can be seedled, and the stable inheritance of the second generation can be completed. Due to the vitrification phenomenon that is easy to occur after the bud regeneration, not all transformants that have completed budding can be seedled, and the seedling rate after budding is not clearly counted in the prior art. Therefore, it is very important to pay attention to the seedling rate in genetic transformation in actual strawberry breeding. Based on this, the inventor of the present application has proposed a gene combination that can really improve the seedling rate of strawberry genetic transformation and shorten the transformation cycle.

[0029] In a first typical embodiment of the present invention, an application of a strawberry endogenous growth regulatory gene combination in genetic transformation is provided, wherein the strawberry endogenous growth regulatory gene combination is selected from any one of the following groups: FveGRF8 and FveGIF1; or FveGRF3 and FveGIF1; wherein the nucleotide sequences of FveGRF3, FveGRF8, and FveGIF1 are: (a) nucleotide sequences as shown in SEQ ID NOs: 1-3; or (b) nucleotide sequences encoding strawberry endogenous growth factors FveGRF3, FveGRF8, and FveGIF1 having amino acid sequences as shown in SEQ ID NOs: 4-6; or (c) nucleotide sequences having more than 85% homology with the nucleotide sequences of any one of (a) and (b) and having the function of improving strawberry transformation efficiency.

[0030] SEQ ID NO: 1: (DNA sequence of FveGRF3)

[0031]

[0032] SEQ ID NO: 2: (DNA sequence of FveGRF8)

[0033]

[0034] SEQ ID NO: 3: (DNA sequence of FveGIF1)

[0035]

[0036] SEQ ID NO: 4: (Amino acid sequence of FveGRF3)

[0037] MDFHLKQWRNQQQHESEEQQPYPSAAKIPKLETQTHPEPVSGYALPLFVPEPNTKVISTLSAFSESTTTTTAPASSATRFPRMGSFFSMSQWQELELQALIFRYMLAGAAVPPELLQPIKRSLLHSQFFFQHHLQHYPALLQSGYWGRGSMDPEPTRCRRTDGKKWRCSKDVVAGQKYCERHVHRGRNRSRKPVEVTTATTNAAGGSGAATDNSVVTVASSLGKGSNGTHFALSGSSPSIDLLQLSQNQREVSRGVQSDSHVLRPFFDDWPGQLQEPDNGRSNATSNNNATSLSISLKLSTGNGVEPGQESEQPQLNWPMGWGSNHMVSMGGPLAEALRSSNSNSSPTSVLHQLPRGSVSETSFIST。

[0038] SEQ ID NO: 5: (Amino acid sequence of FveGRF8)

[0039] MMMMVHHDNRGRPDQRRDGGDGPTCNRPPGVAEPVAGSSSSNSTSGGSVLRGLQPFQCLSNTNTHHHNSTSLRSPGGMTTAAAALGFPFTNQQWKELERQAMIYKYMMASLPVPRDLLFPTTTTSSSSISQSPLSGGGFNLRLSNSTDPEPGRCKRTDGKKWRCSRDVAPDQKYCERHMHRGRPRSRKHVEIHANNTTTTAATMTTAKKLRRDNNSNNIPPICAHNLPNPEMSKNGYPTQFFGSTLQTFHQTPVSLDKSGVKSATFGGGVNSVSSHRGVRGMEWMMKSEAPAISSSDAQWQNLMHSNKTELSSCSRIPYFDANTSIFGQRSEQECYLNLNSYTSFNAGDARHQQDDNDCTMFLSPDVVALENPLLEETPRSFIDAWSKNDNGDNITSVPSSGHLSPSSLTLSMGGYNSISDEMGQTQMSLGCNDGSGNETRPPQITTWLTPSSWVSSPPGGPLAEVLKPATSAASTPSSPITTNGNHGEFSSPLGTTTASSPSGVLQKTLASFSDSSGNSSPNLTSSKPKTEAVSLWLNQSKS。

[0040] SEQ ID NO: 6: (Amino acid sequence of FveGIF1)

[0041] MQQHLMQMQPMMAGYYPNSVTTDHIQQYLDENKSLILKIVESQNSGKLSECAENQARLQRNLMYLAAIADSQPQPPTMHPQYPSGGMMQPGASYMQQQAAQQMSPQSLMAARNSMMYNQQPFSAMQQQALHSQLAMSSGGSGGLHMLQNEANNAGGSGQLGAGGFSDFGRGEGMHRRMGSGSKHDLGSSDGRGGSSGGHGGDGGETLYLKSADD。

[0042] Although studies in wheat and citrus have shown that different combinations of GRF-GIF mosaic genes can improve the bud regeneration efficiency of callus tissue to varying degrees, it is still unknown whether growth regulatory genes can improve strawberry transformation efficiency, especially for explant seedling efficiency. Therefore, the inventors of the present application have carried out a series of screening tests on the GRF-GIF gene combination in strawberry, not only proving that not all different combinations of GRF-GIF mosaic genes can improve the bud regeneration efficiency of callus tissue in strawberry, but also screened out the GRF-GIF combination that can improve the seedling rate. And then these GRF-GIF combinations are applied to the genetic transformation of strawberry to improve the seedling rate of strawberry genetic transformation, which is more valuable in the actual breeding application of strawberry. In addition, these GRF-GIF combinations are utilized to carry out the genetic transformation of strawberry, and the growth regulatory gene combination (40 days) of the transformation cycle of strawberry can also be shortened.

[0043] In a second typical embodiment of the present invention, a recombinant protein is provided, which is encoded by the strawberry endogenous growth regulatory gene combination in the above application. The expression of the recombinant protein in strawberry helps to improve the genetic seedling efficiency of strawberry.

[0044] In a third typical embodiment of the present invention, a recombinant vector is provided, the recombinant vector comprising a strawberry endogenous growth regulatory gene combination and a driving element for driving the expression of the strawberry endogenous growth regulatory gene combination, the strawberry endogenous growth regulatory gene combination being a combination of FveGRF8 and FveGIF1 or a combination of FveGRF3 and FveGIF1 in the above application. The strawberry endogenous growth regulatory gene combination of the present application can improve the genetic transformation efficiency of the target gene to be transformed, so when the recombinant vector carrying the gene combination is introduced into strawberries, the genetic seedling efficiency of strawberries can also be improved.

[0045] In the recombinant vector, the driving element includes a promoter sequence and a terminator sequence. Specifically, they can be reasonably selected from promoters and terminators in known commercial recombinant vectors, or obtained by modifying known promoters and terminators.

[0046] Any driving element capable of expressing the strawberry endogenous growth regulatory gene combination in a recombinant vector is suitable for the present application, and the driving element includes but is not limited to a promoter, a translation leader sequence, an intron, and a polyadenylation recognition sequence. In a preferred embodiment, the driving element includes a promoter and a terminator. Different driving elements in the recombinant vector can manipulate the expression of the gene fragment introduced therein, such as a promoter capable of controlling when to express the target gene or a promoter capable of overexpressing the target gene, etc.

[0047] Any promoter capable of achieving target gene expression in a recombinant vector is suitable for the present application. In a preferred embodiment, the promoter includes a constitutive promoter or an inducible promoter. A constitutive promoter refers to a promoter that generally causes a gene to be expressed in most cell types in most cases. It also refers to a promoter that is primarily, but not necessarily exclusively, expressed in a tissue or organ, and can also be expressed in a specific cell or cell type. In a preferred embodiment, the constitutive promoter includes the Arabidopsis UBQ10 promoter or the tobacco mosaic virus 35S promoter.

[0048] Inducible promoter refers to a promoter that can significantly increase the transcription level of a gene under the stimulation of certain specific physical or chemical signals. In a preferred embodiment, the inducible promoter includes an estradiol inducible promoter. In a preferred embodiment, the terminator includes the tobacco mosaic virus Nos terminator.

[0049] The above-mentioned recombinant vector includes the SunTag system and the XVE system. In a preferred embodiment, the recombinant vector includes the fusion gene LexA-VP16-ER, which is induced to express by β-estradiol and named pDV. The SunTag system carries the dCas9 protein, but does not edit the target site. It will target the target site in coordination with other proteins according to gRNA; the XVE system is a system for regulating gene expression induced by estrogen.

[0050] In a fourth typical embodiment of the present invention, a host cell is provided, wherein the host cell is transformed with the above-mentioned recombinant vector, and the host cell comprises Escherichia coli or Agrobacterium.

[0051] In a fifth typical embodiment of the present invention, a strawberry genetic transformation method is provided, the genetic transformation method comprising: using the above-mentioned host cell to transform the strawberry endogenous growth regulatory gene combination into the target plant material.

[0052] The above genetic transformation method includes any of the following transformation methods: Agrobacterium infection method, gene gun method, PEG-mediated protoplast transformation, plant virus-mediated transformation, pollen tube channel method and ovary injection method. Each of the above genetic transformation methods has its own advantages. For example, the Agrobacterium-mediated genetic transformation method can obtain more low-copy insertion transformants, and protoplast transformation is used for transient detection.

[0053] In a preferred embodiment, the above-mentioned strawberry endogenous growth regulatory gene combination is selected from any one or more of the following: FveGRF8 and FveGIF1; or FveGRF3 and FveGIF1. Any number of gene combinations that can achieve the improvement of strawberry genetic transformation is used in this application. In a preferred embodiment, the number of strawberry endogenous growth regulatory gene combinations is independently 1 or more. In the process of genetic transformation, more than one gene combination can be transformed into the plant material, and the number of each gene combination can be one or more, depending on the actual application requirements.

[0054] In a preferred embodiment, the Agrobacterium infection method includes: introducing a recombinant vector into an Agrobacterium strain to obtain a recombinant bacterium; preparing the recombinant bacterium into an impregnation solution; placing the target plant material in the impregnation solution for immersion to obtain an infected explant; co-culturing the infected explant with the recombinant bacterium to obtain a co-cultured explant; and performing positive screening on the co-cultured explant to obtain a positive tissue culture seedling.

[0055] There is no particular limitation on the specific Agrobacterium strain, as long as it can infect strawberries and introduce the recombinant vector into strawberries. In a preferred embodiment, the Agrobacterium strain is GV3101.

[0056] The target plant material includes strawberry material. The strawberry material can be reasonably selected according to actual needs. The strawberry material of the present application includes but is not limited to any one of the following: petals, pollen, cotyledons, cotyledon nodes, leaves, stems or protoplasts.

[0057] The above-mentioned positive screening includes: screening the co-cultured explants for antibiotic resistance to obtain positive transformants; performing bud growth and rooting culture on the positive transformants to obtain seedlings; and performing gene detection on the seedlings to obtain positive tissue culture seedlings. Any method capable of completing the detection of positive tissue culture seedlings is applicable to the present application. In a preferred embodiment, the gene detection includes PCR, first-generation sequencing and / or high-throughput sequencing.

[0058] In a sixth typical embodiment of the present invention, there is provided an application of the above-mentioned recombinant vector, or the above-mentioned host cell, or the above-mentioned genetic transformation method in genetic transformation of strawberry.

[0059] The present application is further described in detail below in conjunction with specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed in the present application.

[0060] Example 1: Construction of an inducible activation system for gene editing in strawberry

[0061] First, the pDV plasmid (obtained from Professor Liu Qikun of Peking University) was used. This plasmid is composed of the SunTag system and the XVE system (LexA-VP16-ER, induced by β-estradiol) and is named pDV. The vector has I-SceI and I-CeuI restriction sites for adding growth regulatory genes. The Gibson assembly experiment used the ClonExpress IIOne Step Cloning Kit (C112-01) from Vazyme, and the reaction system is shown in Table 1. After adding the reaction system according to Table 1, place it at 37°C for 30 minutes, and then transform the ligation product into Escherichia coli to obtain pDV-FveGRF / FveGIF positive clones.

[0062] Table 1

[0063] Components Addition volume (μL) pDV (I-SceI, I-CeuI digested) 3 AtU6-FveGRF sgRNA-AtU6-FveGIF sgRNA fragment 3 5CE II Buffer 2 Exnase II 1 <![CDATA[ddH2O]]> 1 Fill to total volume 10

[0064] Specifically, the pDV vector is a control vector and does not contain a strawberry growth regulatory gene.

[0065] For the pDV-FveGRF / FveGIF vector, the inventors identified the FveGRF and FveGIF gene sequences (SEQ ID NO: 1-SEQ ID NO: 3), such as Figure 2 As shown, FveGRF / FveGIF was recombined into the pDV vector to obtain the corresponding pDV-FveGRF / FveGIF vector. The relevant primer sequences used in the above cloning process are shown in Table 2, and the vector schematic diagram is shown in Figure 1 .

[0066] The inventors designed two sgRNAs for each gene, obtained a total of 20 pDV-FveGRF / FveGIF combinations, and completed the construction of 12 cloning vectors GRF1-10 and GIF1-2. Only the relevant primers for constructing GRF3, 8 and GIF1 are shown here.

[0067] Table 2

[0068]

[0069]

[0070] Example 2: Transformation of strawberry explants

[0071] Take the genetic transformation of forest strawberry variety Yellow Wonder (YW5AF7) mediated by Agrobacterium as an example. YW5AF7 is a diploid strawberry material planted in the laboratory in the early stage. The cultivated strawberry variety is the Akihime variety, and the experimental material is the material planted in the laboratory greenhouse.

[0072] (1) Preparation of YW explants

[0073] Select plump and normal-looking seeds of forest strawberry YW5AF7, disinfect them with 75% alcohol for 30 seconds, 2% sodium hypochlorite solution for 10 minutes, and culture them in MS solid medium under 23°C light conditions. After growing for 50 days, select plants with good growth, cut their tender green leaves on a clean bench, and cut the leaves into small pieces of 2.0 mm × 5.0 mm as explants.

[0074] (2) Preparation of recombinant Agrobacterium

[0075] The vectors in Example 1 were transformed into Agrobacterium GV3101 competent cells to obtain recombinant bacteria.

[0076] (3) Preparation of infection solution

[0077] The recombinant bacteria in step 2 were activated and cultured at a strain concentration of OD 600 =0.6-0.8, centrifuge, collect the bacteria, and resuspend the bacteria in MS liquid sterile culture medium. 600 Both are 0.2-0.3.

[0078] (4) Infection

[0079] The explant obtained in step (1) is placed in the infection solution containing the recombinant bacteria obtained in step 2, and the explant is immersed in the liquid. During the infection process, the explant is placed on a small shaker and shaken at a low speed. The infection is carried out at room temperature and away from light for 30 minutes. Then the explant is taken out and the liquid is absorbed with sterile filter paper to obtain the infected explant.

[0080] (5) Co-culture

[0081] After step (4) is completed, the infected explants are placed in a co-culture medium (MS + 3.4 mg / L 6-BA + 0.3 mg / L IBA + 250 mg / L Tim (Timentin) + 200 mg / L Car (carbenicillin), 3.5 g / L Phytagel, 20 g / L sucrose, pH 5.8) and cultured in the dark for 3 days to obtain co-cultured explants.

[0082] (6) Screening

[0083] After step (5) is completed, the co-cultivated explants are cultured on a screening medium (MS+3.4mg / L 6-BA+0.3mg / L 1BA+2mg / L hygromycin+250mg / L Tim+200mg / L Car+40μM / Lβ-estradiol, 3.5g / L hytagel, 20g / L sucrose, pH 5.8), with about 23 explants placed on each plate. The medium is replaced every three weeks.

[0084] (7) Bud culture (such as Figure 5 (shown)

[0085] After step (6) is completed, the plant tissue or buds containing buds are cultured in a bud elongation medium (MS+2.0mg / L6-BA+0.3mg / L IBA+2mg / L hygromycin+250mg / L Tim+200mg / L Car+40μM / Lβ-estradiol, 3.5g / L Phytagel, 20g / L sucrose, pH 5.8) until the young buds grow up.

[0086] (8) Rooting

[0087] After step (7) is completed, the obtained plant with complete above-ground parts (including stems, leaves and fixed end growth points) is placed in a rooting medium for culture (MS+0.1mg / L IBA+2mg / L hygromycin+250mg / L Tim+200mg / LCar+40μM / Lβ-estradiol, 3.5g / L Phytagel, 20g / L sucrose, pH 5.8) until the young shoots take root and grow into complete seedlings, thereby obtaining strawberry plants.

[0088] Steps 6-8 were all carried out under a 16 h day / 8 h night photoperiod, a light intensity of about 7000 lx, and a temperature of 23°C.

[0089] During the transformation experiment, the strawberry genetic transformation regeneration time and regeneration efficiency were statistically analyzed, and it can be seen that the transformation efficiency of the explants expressing the strawberry endogenous growth regulatory gene of the present application was significantly higher than that of the non-expressing control group.

[0090] Identification of positive tissue culture seedlings of strawberry in experimental detection

[0091] The regeneration rate is the total number of explants that sprouted / the total number of explants, and the seedling rate is the total number of explants with positive seedlings / the total number of explants. The seedling standard is that the seedlings grow into seedlings. In addition, the rooting efficiency is observed and counted, and the rooting standard is that the root length grows to 3-4 cm.

[0092] The identification test is to mix the constructed 20 pDV-FveGRF / FveGIF combinations into a library, and infect strawberry explants at the same time to simultaneously screen the FveGRF-FveGIF combination with the strongest regeneration ability. Among them, 951 strawberry explants were initially infected, 951 strawberry explants were able to sprout normally, and 900 strawberry explants grew normally into seedlings after sprouting, with a seedling rate of 95%.

[0093] 1. Comparison of regeneration time and regeneration rate

[0094] During the differentiation culture, the growth of the explants transformed with the pDV-FveGRF / FveGIF vector was monitored to observe the budding and differentiation of the explants. Figure 3 The state shown is a normal growth state of differentiation and budding. Statistics of explants in this state showed that 100% of the explants transformed with the above 20 pDV-FveGRF / FveGIF vector combinations of this application can bud, and the budding time is generally around 30-40 days (such as Figure 3 As shown in FIG. 1 , the germination time of conventional transformation technology is shortened by about 40 days, which is 56-84 days. The germination rate of conventional transformation technology is only about 3-10%, while the present application increases the germination rate to 100%.

[0095] 2. Comparison of seedling time and seedling rate

[0096] like Figure 4 As shown, taking the explant transformed with pDV-FveGRF / FveGIF vector as an example, the leaves differentiated into seedlings were taken, DNA was extracted by CTAB method, and primers (pDV-F1 / R1, pDV-F2 / R2) in Table 2 were used for detection, and different FveGRF-FveGIF combination types were analyzed by Sanger sequencing and second-generation sequencing. Figure 7 The results (observed on the 140th day after transformation) show that the seedling rate of explants transformed with pDV-FveGRF / FveGIF vector is generally significantly improved compared with the conventional transformation technology.

[0097] Since the genetic transformation process for testing the effect of this application is a co-cultivation process of mixing 20 FveGRF-FveGIF combinations, some seedlings are not only the result of a single combination. All combinations of seedlings were sequenced to obtain the statistical results of the number of combinations of a single pDV-FveGRF / FveGIF combination or multiple pDV-FveGRF / FveGIF combinations. Figure 8It can be preliminarily seen from the data in Table 3 that the number of positive transgenic plants in the combinations of FveGRF8-FveGIF1 and FveGRF3-FveGIF1 is higher.

[0098] Then, 80 seedlings affected by a single combination were selected and their sequencing results were statistically analyzed. Fig. 9 From the data in Table 4, we can see the number of positive transgenic plants produced by the single action of the combination of FveGRF-FveGIF, among which the number of positive transgenic plants produced by the combinations of FveGRF8-FveGIF1 and FveGRF3-FveGIF1 is higher. It can be seen that the two combinations can significantly improve the genetic transformation efficiency of strawberry, especially the seedling rate, when acting as a single combination.

[0099] The seedling time of each infected explant was observed. Figure 6 It can be seen that the seedling time of explants co-cultured with FveGRF-FveGIF is about 40 days, which is generally faster than the 140 days of conventional transformation technology, shortening the transformation time by two times, and the rooting time of the combination FveGRF8-FveGIF1 and FveGRF3-FveGIF is further improved compared with other FveGRF-FveGIF combinations. The regeneration time and rooting time of FveGRF8-FveGIF1 and FveGRF3-FveGIF are 35 days and 50 days, respectively.

[0100] Table 3 Number of combinations among 900 positive seedlings

[0101]

[0102]

[0103] Table 4 Number of positive seedlings of each combination among 80 plants with single combination effect

[0104]

[0105]

[0106] From the above description, it can be seen that the above-mentioned embodiments of the present invention achieve the following technical effects: using the strawberry endogenous growth regulatory gene combination FveGRF8 and FveGIF1 or FveGRF3 and FveGIF1 provided in the present application for genetic transformation operations can greatly improve the genetic transformation efficiency of strawberries in terms of bud regeneration, seedling formation and rooting, and greatly shorten the genetic transformation cycle of strawberries, which is of great significance for the actual breeding application of strawberries, and also lays the foundation for large-scale genetic transformation screening of functional genes and molecular breeding of strawberries.

[0107] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An application of a strawberry endogenous growth regulatory gene combination in strawberry genetic transformation, characterized in that: The strawberry endogenous growth regulatory gene combination is selected from any one of the following groups: FveGRF8 and FveGIF1 ;or FveGRF3 and FveGIF1 ; Among them, the FveGRF3 , FveGRF8, Said FveGIF1 The nucleotide sequence is: (a) the nucleotide sequence shown in SEQ ID NOs: 1-3; or (b) Nucleotide sequences encoding the strawberry endogenous growth factors FveGRF3, FveGRF8 and FveGIF1 having the amino acid sequences shown in SEQ ID NOs: 4-6.

2. A recombinant vector, characterized in that: The recombinant vector comprises a strawberry endogenous growth regulatory gene combination and a driving element for driving the expression of the strawberry endogenous growth regulatory gene combination, wherein the strawberry endogenous growth regulatory gene combination is the one in the application of claim 1. FveGRF8 and FveGIF1 A combination of FveGRF3 and FveGIF1 combination.

3. The recombinant vector according to claim 2, characterized in that The driving element includes a promoter and a terminator.

4. The recombinant vector according to claim 3, characterized in that The promoter is a constitutive promoter or an inducible promoter.

5. The recombinant vector according to claim 4, characterized in that The constitutive promoter is Arabidopsis UBQ10 promoter or tobacco mosaic virus 35S promoter.

6. The recombinant vector according to claim 4, characterized in that The inducible promoter is an estradiol inducible promoter.

7. The recombinant vector according to claim 3, characterized in that The terminator is the tobacco mosaic virus Nos terminator.

8. The recombinant vector according to claim 3, characterized in that The recombinant vectors include the SunTag system and the XVE system.

9. A non-animal or plant host cell, characterized in that: The non-animal or plant host cell is transformed with the recombinant vector according to any one of claims 2-8.

10. The non-animal or plant host cell according to claim 9, characterized in that The non-animal or plant host cell includes Escherichia coli or Agrobacterium.

11. A method for genetic transformation of strawberry, characterized in that: The genetic transformation method comprises: using the host cell of claim 9 to transform the strawberry endogenous growth regulatory gene combination in the application of claim 1 into strawberry materials.

12. The genetic transformation method according to claim 11, characterized in that The strawberry endogenous growth regulating gene combination is selected from any one or more of the following: FveGRF8 and FveGIF1 ;or FveGRF3 and FveGIF1 .

13. The genetic transformation method according to claim 12, characterized in that: The number of the strawberry endogenous growth regulatory gene combinations is independently one or more.

14. The genetic transformation method according to claim 11, characterized in that: The genetic transformation method includes any one of the following transformation methods: Agrobacterium infection method, gene gun method, PEG-mediated protoplast transformation, plant virus-mediated transformation, pollen tube channel method and ovary injection method.

15. The genetic transformation method according to claim 14, characterized in that The Agrobacterium infection method comprises: Introducing the recombinant vector into the Agrobacterium strain to obtain a recombinant bacterium; preparing the recombinant bacteria into an impregnation solution; placing the strawberry material in the impregnation solution for impregnation to obtain an infected explant; co-culturing the infected explants with the recombinant bacteria to obtain co-cultured explants; The co-cultured explants are subjected to positive screening to obtain positive tissue culture seedlings.

16. The genetic transformation method according to claim 15, characterized in that The Agrobacterium strain is GV3101.

17. The genetic transformation method according to claim 11, characterized in that: The strawberry material is selected from any one of the following: petals, pollen, cotyledons, cotyledon nodes, leaves, stems or protoplasts.

18. The genetic transformation method according to claim 15, characterized in that: The positive screening includes: performing antibiotic resistance screening on the co-cultured explants to obtain positive transformants; Performing bud growth and rooting culture on the positive transformant to obtain seedling plants; The seedlings are subjected to gene testing to obtain positive tissue culture seedlings.

19. The genetic transformation method according to claim 18, characterized in that: The gene detection includes PCR, first-generation sequencing and / or high-throughput sequencing.

20. Use of the recombinant vector according to any one of claims 2 to 8, or the host cell according to claim 9, or the genetic transformation method according to any one of claims 11 to 19 in genetic transformation of strawberry.

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