CN116179591A

CN116179591A - Application of strawberry endogenous growth regulating gene combination in genetic transformation

Info

Publication number: CN116179591A
Application number: CN202310205951.1A
Authority: CN
Inventors: 唐雅君; 周军会; 苑海迪
Original assignee: Institute Of Modern Agriculture Peking University
Current assignee: Institute Of Modern Agriculture Peking University
Priority date: 2023-03-06
Filing date: 2023-03-06
Publication date: 2023-05-30

Abstract

The invention provides an application of strawberry endogenous growth regulating gene combination in genetic transformation, wherein the strawberry endogenous growth regulating 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 as follows: (a) a polypeptide as set forth in SEQ ID NO: 1-3. The strawberry endogenous growth regulating gene combination is transferred into the strawberry by utilizing genetic transformation methods such as an agrobacterium infection method, so that the bud regeneration rate can be improved, the genetic transformation seedling rate and rooting rate of the strawberry can be improved, the genetic transformation time is greatly shortened, and a foundation is laid for large-scale genetic transformation screening of functional genes and molecular breeding.

Description

Application of strawberry endogenous growth regulating gene combination in genetic transformation

Technical Field

The invention relates to the technical field of genetic transformation, in particular to application of a strawberry endogenous growth regulating gene combination in genetic transformation.

Background

Strawberry is an important economic crop, and has wide cultivation area and large cultivation area, and is planted in many countries. The strawberry has relatively short growth cycle, has the potential of being used as a non-respiratory jump type fruit research mode, is used for theoretically establishing a strawberry fruit development mode, identifying and excavating genes related to the quality characters of the strawberries, reveals a genetic control mechanism of the strawberries, and is beneficial to screening of good germplasm resources in production. At present, the whole genome and the genome of the strawberry are sequenced, a plurality of potential regulatory genes which can regulate the fruit yield, the fruit quality, the fruit shape, the disease resistance and the stress tolerance of the strawberry are discovered, and the molecular breeding process is expected to be assisted and accelerated by applying a gene editing technology.

The traditional breeding method is used for variety improvement, the period is long, some characters are difficult to obtain, and the genetic function verification and variety improvement are required to be completed through genetic engineering technology. With the continuous development of biotechnology, genetic material is widely applied to various plants by using a gene editing technology to perform artificial operation, and a new direction is provided for creating new germplasm. At present, a genetic transformation system is established in strawberries, but the total transformable variety is few, the genetic transformation efficiency is low, and especially the regeneration rate of the genetic transformation buds of the strawberries cultivated by octaploids is only 4-11%, the period is long, and the experiment material can be obtained in 15 months for analysis, so that a large amount of manpower and material resources are consumed.

The overall low genetic transformation efficiency of strawberries is mainly reflected by low regeneration rate of genetic transformation buds and low rate of genetic transformation seedlings. In the prior art, the transformation efficiency of the method is mostly reflected by the bud regeneration rate, but the vitrification phenomenon is easy to occur after the bud regeneration, so that the explant can bud without meaning that the explant can smoothly grow into a positive plant and finish stable transformation. Therefore, the positive seedling rate is used as the standard of the overall genetic transformation efficiency of the strawberries more accurately. In addition, due to long genetic transformation time period, the overlong culture process also can cause pollution in the culture process, poor growth of explants and the like, and the experimental operation has adverse effects on the genetic transformation efficiency. Therefore, how to provide a genetic transformation method which can complete genetic transformation into seedlings quickly and has high seedling rate is very important to accelerate molecular breeding in the field of strawberries.

Disclosure of Invention

The invention mainly aims to provide an application of strawberry endogenous growth regulating gene combination in genetic transformation, so as to solve the problem of low seedling rate of strawberry genetic transformation in the prior art.

In order to achieve the above object, according to a first aspect of the present invention, there is provided the use of a strawberry endogenous growth regulatory gene combination in genetic transformation, the strawberry endogenous growth regulatory gene combination being 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 as follows: (a) the sequence set forth in SEQ ID NOs: 1-3; or (b) encodes a polypeptide having the sequence of SEQ ID NOs:4-6, nucleotide sequences of strawberry endogenous growth factors FveGRF3, fveGRF8 and FveGIF1; or (c) a nucleotide sequence which has homology of 85% or more with the nucleotide sequence of any one of (a) and (b) and has a function of improving the conversion efficiency of strawberry.

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

In order to achieve the above object, according to a third aspect of the present invention, there is provided a recombinant vector comprising a strawberry endogenous growth regulatory gene combination and a driving element for driving 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 use of claim 1.

Further, the driving element includes a promoter and a terminator; preferably, the promoter comprises a constitutive promoter or an inducible promoter; preferably, the constitutive promoter comprises an arabidopsis UBQ10 promoter or a tobacco mosaic virus 35S promoter; preferably, the inducible promoter comprises an estradiol inducible promoter; preferably, the terminator comprises a tobacco mosaic virus Nos terminator; preferably, the recombinant vector comprises the SunTag system and the XVE system.

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

Further, the above host cell includes E.coli or Agrobacterium.

In order to achieve the above object, according to a fifth aspect of the present invention, there is provided a genetic transformation method of strawberry, the genetic transformation method comprising: transforming the strawberry endogenous growth regulating gene combination into a target plant material using the host cell; preferably, the strawberry endogenous growth regulating gene combination is selected from any one or more of the following: fveGRF8 and FveGIF1; or FveGRF3 and FveGIF1; preferably, the number of strawberry endogenous growth regulating gene combinations is each independently 1 or more.

Further, the genetic transformation method includes any one of the following transformation methods: agrobacterium infection, gene gun, PEG-mediated protoplast transformation, plant virus-mediated transformation, pollen tube passage, and ovary injection; preferably, the agrobacterium infection method comprises: introducing the recombinant vector into an agrobacterium strain to obtain recombinant bacteria; preparing recombinant bacteria into a dye dip; placing target plant materials into a dip dyeing liquid for dip dyeing to obtain an infected explant; co-culturing the infected explant and recombinant bacteria to obtain a co-cultured explant; positive screening is carried out on the co-cultured explants to obtain positive tissue culture seedlings; preferably, the agrobacterium strain is GV3101; preferably, the target plant material comprises strawberry material; preferably, the strawberry material is selected from any one of the following: petals, pollen, cotyledons, cotyledonary nodes, leaves, stems or protoplasts.

Further, the positive selection includes: screening the co-cultured explants for antibiotic resistance to obtain positive transformants; performing bud growth and rooting culture on the positive transformant to obtain a seedling plant; carrying out gene detection on the seedling plants to obtain positive tissue culture seedlings; preferably, the gene detection comprises PCR, generation sequencing and/or high throughput sequencing.

In order to achieve the above object, according to a sixth aspect of the present invention, there is provided the use of the above recombinant vector, or the above host cell, or the above genetic transformation method for genetic transformation of strawberry.

By applying the technical scheme of the invention, the strawberry endogenous growth regulating gene provided by the application is utilized to combine FveGRF8 and FveGIF1 or FveGRF3 and FveGIF1, so that the genetic transformation seedling rate of the strawberry can be improved. The strawberry endogenous growth regulating gene combination is transferred into the strawberry by utilizing genetic transformation methods such as an agrobacterium infection method, so that the bud regeneration rate can be improved, the genetic transformation seedling rate of the strawberry can be improved, the strawberry can be rapidly rooted to 3-4cm, the genetic transformation time (transformation sprouting time and seedling time) is greatly shortened, and meanwhile, a foundation is laid for large-scale genetic transformation screening of functional genes and molecular breeding.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention. In the drawings:

FIG. 1 shows a schematic representation of a recombinant vector of an endogenous growth regulating gene of strawberry of the present application.

FIG. 2 shows a schematic diagram of the sequence structure of an endogenous growth regulating gene of strawberry;

FIG. 3 shows a schematic representation of regenerated shoots obtained during genetic transformation of strawberry;

fig. 4 shows a schematic representation of the rooted plants obtained during the genetic transformation of strawberry (bar=1 cm);

FIG. 5 shows a schematic representation of strawberry genetic transformation tissue culture;

FIG. 6 shows the statistical results of the genetic transformation of strawberry into seedling time;

FIG. 7 shows the statistical results of strawberry genetic transformation into seedlings;

FIG. 8 shows the total number statistics of FveGRF and FveGIF in the FveGRF-FveGIF combination in 900 positive transgenic strawberries;

FIG. 9 shows FveGRF-FveGIF combination statistics in 80 single combination positive transgenic strawberries.

Detailed Description

It should be noted that, in the case of no conflict, the embodiments and features in the embodiments may be combined with each other. The invention will be described in detail below with reference to the drawings in connection with embodiments.

As described in the background art, the existing technology related to strawberry genetic transformation has the defects of lower overall genetic transformation efficiency, only 4-11% and longer period. In addition, most of the calculation of genetic transformation efficiency focuses on the germination efficiency of explants, i.e., the bud regeneration rate, rather than the seedling rate. Thus, many transformation methods exist to statistically obtain only the data of the sprouting stage of the explants, and for practical strawberry breeding, the transformant needs to be capable of being grown into seedlings, so that stable inheritance of the second generation is possible. Because vitrification phenomenon easily occurs after bud regeneration, not all transformants which complete bud emergence can be grown, and the seedling rate after bud emergence is not clearly counted in the prior art. Therefore, it is important to pay attention to the seedling rate in genetic transformation in actual strawberry breeding, and based on the inventor of the application, a gene combination capable of truly improving the seedling rate of the genetic transformation of strawberries and shortening the transformation period is proposed.

In a first exemplary embodiment of the invention, there is provided the use of a strawberry endogenous growth regulating gene combination for genetic transformation, 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 as follows: (a) the sequence set forth in SEQ ID NOs: 1-3; or (b) encodes a polypeptide having the sequence of SEQ ID NOs:4-6, nucleotide sequences of strawberry endogenous growth factors FveGRF3, fveGRF8 and FveGIF1; or (c) a nucleotide sequence which has homology of 85% or more with the nucleotide sequence of any one of (a) and (b) and has a function of improving the conversion efficiency of strawberry.

SEQ ID NO:1: (DNA sequence of FveGRF 3)

GAAAGTATAAGTTTCTTAGAAACACAGATCTGAGTTAAATACGATTCTTACTGTACTTGCAATCTAAATCCAATGTCTTGGGAAAACCTAACACAGCATACATACTCGTACTTACCGCACAAGAAAAGAATATTAACCTTGAAAATGGGGGTGGTAAAGCCTGGGCTCGGCTGCAATGTAACTGCAAAGTCGGCTTGAAACCCTCTCCGCCAGTTATATAATCATGCATATCATCTGAGTCTAGGGTTTAGGTTACATTACTTAATGGGGTATAGTTATGATTAAACTAAACCAATTTAAGCCTAATAGAAACAGTTTGAGTGCTAGTTCCAAGGCAAAACAGAGAGACAGGAAAGACTGCAGGTTCTTTGCTCTCTCTACTAGTCTAGCTCTTCCATCTATTCATCATTTTTCGAAAATTTTTAATCTCACTTTTTTCGCTCTCAACTGTCTTGTCCGAGTGTCTTTTTCTCAAGTGCCCTCAGGCTTCTTCACTGGCTTAAACCAACTTTGCAGGCTTTAGTTTTTCATTTTTCCATTAAGTTTTTCTTTTAATGTCAGAATGTGAAGGTATCTGCCAAAGCTATAAAAGAACCACAGAGGCCTCTGCAGAAAGCTTGAGCAAACTTTCTCTTCACATAAACCCAACCAAACACCCACCCAGACAAGGATAAGAGTCTGAGATATAAATAAACCAAAGCCAACCAAGTTAAAACTCAAACCCCACTTTTCTCCTTTCTCTATGGACTTTCATCTCAAGCAATGGAGAAACCAGCAGCAGCATGAGTCAGAGGAACAACAGCCCTATCCTTCTGCTGCAAAGATACCAAAACTTGAGACCCAAACACACCCTGAACCAGTTTCTGGGTATGCTCTCCCTTTGTTTGTACCTGAACCAAACACCAAAGTCATCAGCACCCTGTCAGCATTTTCTGAATCTACTACCACCACCACCGCCCCAGCTTCTTCTGCCACCAGATTTCCCAGAATGGGGAGCTTCTTCAGCATGAGTCAGTGGCAAGAGCTTGAGCTACAGGCTTTGATATTCAGGTACATGCTGGCTGGTGCTGCTGTTCCTCCTGAACTTCTTCAGCCAATCAAGAGAAGCCTTCTGCATTCTCAATTCTTCTTCCAACACCATCTTCAACATTACCCTGCTTTGTTGCAATCCGGGTATTGGGGAAGAGGGTCCATGGATCCGGAGCCGACCAGGTGCCGGAGGACAGATGGCAAGAAATGGCGGTGCTCTAAAGACGTGGTGGCCGGTCAGAAGTACTGCGAGCGCCACGTGCACCGTGGACGAAACCGTTCAAGAAAGCCTGTGGAAGTCACCACCGCCACCACAAACGCCGCCGGAGGCAGCGGAGCTGCTACCGACAACTCGGTCGTGACTGTTGCCTCGTCGTTGGGAAAAGGGTCGAATGGTACCCACTTTGCTCTTTCTGGGTCATCACCATCCATTGATCTGCTCCAGCTCAGTCAGAATCAAAGGGAAGTTTCGAGAGGTGTTCAATCTGACAGTCATGTCTTGCGGCCGTTTTTTGATGACTGGCCGGGACAGCTCCAAGAACCGGACAATGGGAGAAGCAATGCTACGTCAAATAACAATGCCACATCCCTGTCCATTTCGCTGAAGTTGTCTACCGGCAATGGAGTAGAGCCAGGCCAAGAGAGCGAGCAGCCACAGTTGAATTGGCCCATGGGATGGGGATCAAACCATATGGTTTCGATGGGAGGGCCTCTTGCAGAGGCGCTGCGGTCATCCAACTCCAACTCCTCACCCACCAGTGTTCTTCATCAGTTGCCTCGCGGCTCTGTCTCTGAAACCAGCTTCATCAGCACCTGA。

SEQ ID NO:2: (DNA sequence of FveGRF 8)

ATCTACTTTATTAACTTGAAATTAGTAAAGTAAAGCAACGTTTATGTTGTCGATACCGTTCTCACAACATCTGAAGTTTGAACTCAACGACGACCGTGCGTACGCGGAGCAAGGAAATCACGGTAAACGGACATCTAGAGCGACTGTTACATAGATGAAGAGGTTTCTTGTTCACAATGGAAGAAGAGAAATGCGAAAGGATGAGAACGGTGGTTGTTTTAGGGTGACATGCAGAAAACGATAGCTACCGCAAATAATAACAATGATGAGTCATGAGAAAGAGAGAGCAGCAGTGGTGGTGGGTTGTGGTGGCTCCCTTCTCTCTTTTGCTTTGTCAACTAGCTACGCTACTAGTACATGAGATGGTGTGCGAATGTTGGAAGCATGGAAGCAAATCACAGGCTAAGAATGGCGGCAACCCCTTACAATCAAATACTATCATAAAAGAAAAAGAAAAAGAAAAAGAGTTTTCATTTTATTTTTTTATTCCAAAAGGGCTGAGATAATAGGATATGGGGGTCTATATCTTTGCTCTGCAACCCATGAAAGCCAGAGCACAAAGCTTTTCTCGGAAGCAGCAGCAGAAGCAGAACCAAAAAGTTGAGGTTCCCCCATTTGTGAATTTTATGCTTGTTCGATTGGAAAGGTAGTTGTTTCCCAAAATAGGTCCATTCATTATTAGGCTTCCAAGACAAGGAAACGAAATCGCAGAGCTAGCTTGGTTTTTGAGTAGTAGTAACTAAGGAAGAAGAAGAAGATGATGATGATGGTCCATCATGACAACCGTGGTCGTCCAGATCAAAGACGTGACGGTGGCGATGGTCCCACGTGTAACAGACCCCCTGGTGTTGCAGAACCAGTAGCTGGTAGTTCTTCTAGTAATAGTACTAGTGGAGGTTCTGTCTTGAGAGGCTTGCAGCCTTTTCAGTGCTTATCTAATACAAATACTCATCATCACAACTCAACCTCTCTCAGATCCCCAGGAGGGATGACGACAGCAGCAGCAGCTTTGGGTTTTCCTTTCACTAATCAACAGTGGAAGGAGCTTGAGAGACAAGCCATGATTTACAAGTACATGATGGCTTCTCTCCCTGTGCCTCGTGACCTCCTCTTTCCCACCACCACCACCTCTTCATCCTCTATTTCTCAATCTCCATTGAGCGGCGGAGGGTTCAATCTGAGGCTGTCGAATAGCACTGATCCGGAGCCGGGGCGGTGCAAAAGGACGGACGGGAAGAAATGGAGGTGTTCGAGAGATGTTGCGCCGGACCAGAAGTACTGTGAGCGCCACATGCACCGAGGCCGTCCCCGTTCAAGAAAGCATGTGGAAATTCATGCCAACAATACCACCACCACCGCCGCTACTATGACCACCGCCAAGAAGCTTCGCCGTGACAACAATAGCAACAATATCCCACCAATATGTGCTCATAATCTGCCGAATCCCGAGATGAGCAAGAACGGCTACCCGACTCAGTTTTTCGGATCTACACTGCAAACATTTCATCAAACTCCGGTGTCTTTGGACAAGTCCGGTGTGAAGTCTGCTACTTTTGGTGGTGGTGTAAACTCTGTTTCATCACACAGAGGAGTCAGAGGCATGGAGTGGATGATGAAAAGTGAAGCGCCGGCGATAAGTAGCTCCGATGCGCAATGGCAAAATTTGATGCATAGCAATAAAACAGAGCTGAGTAGCTGCTCTAGAATCCCCTACTTTGATGCAAATACCTCTATTTTCGGCCAGCGTTCAGAGCAAGAGTGTTACCTGAATCTAAATTCTTATACAAGTTTCAACGCCGGAGATGCTCGTCATCAGCAGGATGATAATGACTGTACCATGTTCTTGAGTCCCGATGTGGTAGCTTTGGAAAACCCTCTTCTTGAAGAAACCCCAAGAAGCTTCATCGATGCTTGGTCCAAGAACGACAATGGAGACAACATCACCTCAGTTCCATCAAGCGGGCATCTCTCGCCGTCTTCGTTGACACTATCAATGGGAGGGTACAACTCCATAAGTGACGAAATGGGGCAGACACAGATGAGCTTGGGATGTAACGATGGGAGTGGAAATGAGACTAGGCCTCCACAAATCACCACCTGGCTCACTCCATCTTCTTGGGTTTCCTCTCCGCCTGGTGGGCCTTTAGCTGAGGTGTTGAAGCCCGCCACCAGCGCCGCCTCAACACCGTCATCTCCGATCACCACTAACGGTAACCACGGCGAGTTTAGTAGCCCATTGGGGACTACTACGGCGTCGTCACCGTCTGGGGTGCTTCAGAAAACGCTAGCTTCATTCTCTGATAGCAGTGGGAATAGTAGCCCAAATCTTACAAGTTCCAAGCCCAAAACAGAGGCTGTTTCTCTGTGGCTGAATCAGAGCAAATCATAG。

SEQ ID NO:3: (DNA sequence of FveGIF 1)

CACTTGGTACTTTTATTACAGATGAATGAGCTTATTGGGGAAACCTCCACTCCCATATGGCAAAACGCCAAAACCAATTAAACGCTTAATTCACTGTCAAAAAATTTATTGCCTCTTTCCGTAATTTTTCTATTTCCCATTTTTGGTGAGGCACCTCACTGGATTGACCCTCTCAAATTGTTATGCAGCTCCTAATATCCAAAGCATTGCACTTTGGCTTTGGTTGTAGAGGAGCTTTCATGGAATCTCTTGACAAAGCCTAGACGTCATCACCATCAATAAGAGAGACTCGAAGAGTTTGGACCCAAGAGAGGCCCTATAGTCAGTCCAAACTTACCCAACAGACATTGTCACGAACGATGTGAGTGACCTATCACCACCTATGGATTCAATTCAATGTCATTTGTTTTGTGCTTAATAGAATAATATCGTAAGAGTGTTGATTATAGAATTTGAATTGAATTGATCTTCTGGGTAGATTTTTATTTGTGGATTTGTGGGTCTTTGAATAATTTACAGCAGCAGGTCACGTTCTGAGAGAGAGAGAGAGAGATAAGAAAACCAAAAACCCACAAAGCCAAAAAACCAAGCAAAACCCCAAAAAGGGCTCTGTTTTTCATATATACCCAACTCAACTCTCTCTCTATCTCTGTGTCCCTATATCTCTGTCTGAGCGGTGGGAGTGTCATTGTGTGTTGGAAGGGAAAGAGAGAGAGTGAAAACAGAGAGCAAAAAAAGGCAAAGAAGGGCAGAGCAGTAGTGAGTGTGGTCTCTCAACTCTCTGTGAGATAAACCTGCTGGGGACTTTTAGGAGTGAGAAAAGTGTGTGTTTTTTGAGTGAGAAAAGATAGAAGTGTCTTTGTGTGTGTAACTAGTGATATAAAAGAGAGATGCAGCAGCACCTGATGCAGATGCAGCCCATGATGGCAGGCTACTATCCCAACAGTGTCACTACTGATCACATTCAACAGTATTTGGACGAGAACAAGTCATTGATTCTGAAGATTGTTGAGAGCCAGAATTCAGGGAAATTGAGTGAATGTGCAGAGAACCAAGCAAGGCTACAGCGAAATCTGATGTACCTTGCTGCCATTGCTGATTCCCAACCCCAACCTCCCACTATGCATCCTCAGTACCCTTCCGGTGGCATGATGCAACCAGGAGCAAGTTACATGCAGCAACAAGCAGCTCAACAGATGTCACCTCAATCTCTCATGGCTGCACGCAATTCCATGATGTACAACCAGCAGCCATTTTCAGCTATGCAACAGCAAGCCCTGCATAGCCAACTTGCCATGAGCTCTGGAGGAAGTGGGGGACTTCACATGCTCCAAAATGAGGCAAACAATGCAGGAGGCAGTGGACAACTTGGGGCTGGAGGATTTTCTGATTTTGGACGCGGAGAAGGCATGCATAGGAGGATGGGCAGTGGAAGTAAGCACGATCTTGGTTCTTCTGATGGTCGGGGTGGGAGCTCTGGAGGCCATGGTGGAGATGGGGGTGAGACTCTTTACTTGAAATCCGCTGATGATTAA。

SEQ ID NO:4: (amino acid sequence of FveGRF 3)

MDFHLKQWRNQQQHESEEQQPYPSAAKIPKLETQTHPEPVSGYALPLFVPEPNTKVISTLSAFSESTTTTTAPASSATRFPRMGSFFSMSQWQELELQALIFRYMLAGAAVPPELLQPIKRSLLHSQFFFQHHLQHYPALLQSGYWGRGSMDPEPTRCRRTDGKKWRCSKDVVAGQKYCERHVHRGRNRSRKPVEVTTATTNAAGGSGAATDNSVVTVASSLGKGSNGTHFALSGSSPSIDLLQLSQNQREVSRGVQSDSHVLRPFFDDWPGQLQEPDNGRSNATSNNNATSLSISLKLSTGNGVEPGQESEQPQLNWPMGWGSNHMVSMGGPLAEALRSSNSNSSPTSVLHQLPRGSVSETSFIST。

SEQ ID NO:5: (amino acid sequence of FveGRF 8)

MMMMVHHDNRGRPDQRRDGGDGPTCNRPPGVAEPVAGSSSSNSTSGGSVLRGLQPFQCLSNTNTHHHNSTSLRSPGGMTTAAAALGFPFTNQQWKELERQAMIYKYMMASLPVPRDLLFPTTTTSSSSISQSPLSGGGFNLRLSNSTDPEPGRCKRTDGKKWRCSRDVAPDQKYCERHMHRGRPRSRKHVEIHANNTTTTAATMTTAKKLRRDNNSNNIPPICAHNLPNPEMSKNGYPTQFFGSTLQTFHQTPVSLDKSGVKSATFGGGVNSVSSHRGVRGMEWMMKSEAPAISSSDAQWQNLMHSNKTELSSCSRIPYFDANTSIFGQRSEQECYLNLNSYTSFNAGDARHQQDDNDCTMFLSPDVVALENPLLEETPRSFIDAWSKNDNGDNITSVPSSGHLSPSSLTLSMGGYNSISDEMGQTQMSLGCNDGSGNETRPPQITTWLTPSSWVSSPPGGPLAEVLKPATSAASTPSSPITTNGNHGEFSSPLGTTTASSPSGVLQKTLASFSDSSGNSSPNLTSSKPKTEAVSLWLNQSKS。

SEQ ID NO:6: (amino acid sequence of FveGIF 1)

MQQHLMQMQPMMAGYYPNSVTTDHIQQYLDENKSLILKIVESQNSGKLSECAENQARLQRNLMYLAAIADSQPQPPTMHPQYPSGGMMQPGASYMQQQAAQQMSPQSLMAARNSMMYNQQPFSAMQQQALHSQLAMSSGGSGGLHMLQNEANNAGGSGQLGAGGFSDFGRGEGMHRRMGSGSKHDLGSSDGRGGSSGGHGGDGGETLYLKSADD。

Although studies in wheat and citrus show that the GRF-GIF chimeric genes of different combinations can improve the bud regeneration efficiency of the callus to different degrees, whether the growth regulating genes can improve the strawberry transformation efficiency is unknown, and especially the seedling efficiency of explants is unknown. Therefore, the inventor of the application performs a series of screening tests on GRF-GIF gene combinations in strawberries, not only proves that not all GRF-GIF chimeric genes of different combinations in strawberries can improve the bud regeneration efficiency of callus, but also the GRF-GIF combinations capable of improving the seedling rate are screened. Furthermore, the GRF-GIF combination can be applied to the genetic transformation of strawberries, so that the seedling rate of the genetic transformation of the strawberries can be improved, and the GRF-GIF combination has more value in the practical breeding application of the strawberries. Furthermore, the genetic transformation of strawberry using these GRF-GIF combinations also enabled shortening of the growth regulatory gene combinations (40 days) of the strawberry transformation cycle.

In a second exemplary embodiment of the invention, a recombinant protein is provided, which is encoded by a strawberry endogenous growth regulatory gene combination in the above-mentioned applications. The expression of the recombinant protein in the strawberries is beneficial to improving the seedling efficiency of strawberry inheritance.

In a third exemplary embodiment 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 above application. The strawberry endogenous growth regulating gene combination can improve the genetic transformation efficiency of a target gene to be transformed, so that the seedling efficiency of strawberry inheritance can be improved when the recombinant vector carrying the gene combination is introduced into strawberries.

In the recombinant vector, the driving element includes a promoter sequence and a terminator sequence, and specifically, may be appropriately selected from among promoters and terminators in known commercial recombinant vectors, or may be obtained by modifying known promoters and terminators.

Any driving element capable of completing expression of a combination of strawberry endogenous growth regulatory genes in a recombinant vector is suitable for use in the present application, including but not limited to promoters, translation leader sequences, introns and polyadenylation recognition sequences. In a preferred embodiment, the driving element comprises a promoter and a terminator. The different driving elements in the recombinant vector are capable of manipulating the expression of the gene fragment introduced thereto, such as a promoter capable of controlling when the target gene is expressed or a promoter capable of overexpressing the target gene, etc.

Any promoter capable of accomplishing expression of the gene of interest in a recombinant vector is suitable for use in the present application, and in a preferred embodiment, the promoter comprises a constitutive promoter or an inducible promoter. Constitutive promoters refer to promoters that will generally cause a gene to be expressed in most cases in most cell types. And refers to promoters that are expressed primarily, but not necessarily exclusively, in a tissue or organ, but may also be expressed in a particular cell or cell type. In a preferred embodiment, the constitutive promoter comprises an arabidopsis UBQ10 promoter or a tobacco mosaic virus 35S promoter.

Inducible promoters refer to promoters of the type that are capable of substantially increasing the level of transcription of a gene upon stimulation by certain specific physical or chemical signals. In a preferred embodiment, the inducible promoter comprises an estradiol inducible promoter. In a preferred embodiment, the terminator comprises a tobacco mosaic virus Nos terminator.

The recombinant vector described above comprises the SunTag system and the XVE system, and in a preferred embodiment, the recombinant vector comprises the fusion gene LexA-VP16-ER, the expression induced by β -estradiol, designated pDV. The SunTag system carries dCAS9 protein, but does not edit the target, and can cooperate with other proteins to co-target the target according to gRNA; the XVE system is an estrogen-induced system that can regulate gene expression.

In a fourth exemplary embodiment of the present invention, a host cell transformed with the above recombinant vector is provided. The host cell includes E.coli or Agrobacterium.

In a fifth exemplary embodiment of the present invention, there is provided a genetic transformation method of strawberry, the genetic transformation method comprising: utilizing the host cell, transforming the strawberry endogenous growth regulating gene combination into target plant material.

The genetic transformation method comprises any one of the following transformation methods: agrobacterium infection, gene gun, PEG-mediated protoplast transformation, plant virus-mediated transformation, pollen tube passage, and ovary injection. Each of the above-described genetic transformation methods has advantages, for example, that Agrobacterium-mediated genetic transformation methods can obtain more low-copy insertion transformants, that protoplast transformation is used for transient detection, and the like.

In a preferred embodiment, the combination of strawberry endogenous growth regulating genes described above 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 improved genetic transformation of strawberry are used with the present application, and in a preferred embodiment, the number of strawberry endogenous growth regulating gene combinations is each independently 1 or more. In performing genetic transformation, more than one gene combination may be transformed into plant material, and the number of each gene combination may be one or more, depending on the actual application requirements.

In a preferred embodiment, the agrobacterium infection method comprises: introducing the recombinant vector into an agrobacterium strain to obtain recombinant bacteria; preparing recombinant bacteria into a dye dip; placing target plant materials into a dip dyeing liquid for dip dyeing to obtain an infected explant; co-culturing the infected explant and recombinant bacteria to obtain a co-cultured explant; and (5) carrying out positive screening on the co-cultured explants to obtain positive tissue culture seedlings.

The specific agrobacterium strain is also not particularly limited as long as it can infect strawberry and introduce a recombinant vector into strawberry. In a preferred embodiment, the agrobacterium strain is GV3101.

The target plant material includes strawberry material. The strawberry material can be reasonably selected according to actual needs, and the strawberry material comprises, but is not limited to, any one of the following: petals, pollen, cotyledons, cotyledonary nodes, leaves, stems or protoplasts.

The positive selection includes: screening the co-cultured explants for antibiotic resistance to obtain positive transformants; performing bud growth and rooting culture on the positive transformant to obtain a seedling plant; and (5) carrying out gene detection on the seedling plants to obtain positive tissue culture seedlings. Any method capable of performing a positive tissue culture seedling test is suitable for use in the present application, and in a preferred embodiment, the gene test comprises PCR, first generation sequencing and/or high throughput sequencing.

In a sixth exemplary embodiment of the present invention, there is provided the use of a recombinant vector as described above, or a host cell as described above, or a genetic transformation method as described above, for the genetic transformation of strawberry.

The present application is described in further detail below in conjunction with specific embodiments, which should not be construed as limiting the scope of the claims.

Example 1 construction of strawberry Gene editing-inducible activation System

First, the pDV plasmid (obtained from the university of Beijing, professor Liu Qikun) was used. The plasmid was composed of a combination of the SunTag system and the XVE system (LexA-VP 16-ER, expressed by beta-esctratiol induction), and was designated pDV. The vector has I-SceI and I-CeuI cleavage sites for addition of growth regulatory genes. Gibson assembly experiments were performed using the Vazyme company ClonExpress II One Step Cloning Kit (C112-01) kit and the reaction system is shown in Table 1. After the reaction system was added according to Table 1, the mixture was allowed to stand at 37℃for 30 minutes, and the ligation product was transformed into E.coli to obtain pDV-FveGRF/FveGIF positive clones.

TABLE 1

Component (A)	Add volume (μL)
		pDV (I-SceI, I-CeuI cleavage)	3
AtU6-FveGRF sgRNA-AtU-FveGIF sgRNA fragment	3
		5CE II Buffer	2
Exnase II	1
		ddH ₂ O	1
Make up to the total volume	10

Specifically, the pDV vector was a control vector, and did not contain a strawberry growth regulatory gene.

For the pDV-FveGRF/FveGIF vector, the inventor recombines FveGRF/FveGIF onto the pDV vector according to the identified FveGRF and FveGIF gene sequences (SEQ ID NO:1-SEQ ID NO: 3) as shown in figure 2 to obtain the corresponding pDV-FveGRF/FveGIF vector, and the related primer sequences used in the cloning process are detailed in Table 2, and the schematic diagram of the vector is detailed in figure 1.

The inventors designed 2 sgrnas per gene, and obtained a total of 20 pDV-FveGRF/FveGIF combinations, completing the construction of GRF1-10 and GIF1-2 12 cloning vectors, only the relevant primers for constructing GRF3, 8 and GIF1 being shown here.

TABLE 2

/>

Example 2 transformation of strawberry explants

The agrobacterium-mediated genetic transformation of the forest strawberry variety Yellow word (YW 5AF 7) is exemplified. YW5AF7 is a diploid strawberry material planted in the laboratory in advance. The cultivated strawberry variety is a Chaptera crassis variety, and the test material is planted in a laboratory greenhouse.

(1) Preparation of YW explants

Selecting seeds with plump forest strawberries YW5AF7 and normal appearance, sterilizing with 75% alcohol for 30s and 2% sodium hypochlorite solution for 10min, and culturing in MS solid culture medium at 23deg.C under illumination. After growing for 50 days, plants with good growth vigor are selected, tender green leaves are cut on an ultra clean bench, and the leaves are cut into small blocks with the length of 2.0mm multiplied by 5.0mm to be used as explants.

(2) Preparation of recombinant Agrobacterium

The vector of example 1 was transformed into competent cells of Agrobacterium GV3101 to obtain recombinant bacteria.

(3) Preparation of the dyeing liquor

Activating and culturing the recombinant strain in the step 2, wherein the concentration of the strain is OD ₆₀₀ When the ratio is between 0.6 and 0.8, centrifuging, collecting thalli, re-suspending the thalli by using an MS liquid sterile culture medium, and infiltrating the OD of the dye solution ₆₀₀ All 0.2-0.3.

(4) Infestation of the human body

Placing the explant obtained in the step (1) into the infection solution containing the recombinant bacteria obtained in the step (2), immersing the explant in the solution, placing the infection process on a small shaking table for shaking at a low speed, infecting for 30 minutes at room temperature and avoiding light, taking out the explant, and sucking the solution by using sterile filter paper to obtain the infected explant.

(5) Co-cultivation

After the completion of the step (4), the infected explant was dark-cultured in a co-culture medium (MS+3.4 mg/L6-BA+0.3 mg/LIBA+250mg/L Tim (timentin) +200mg/L Car (carbenicillin), 3.5g/L Phytagel, 20g/L sucrose, pH 5.8) for 3 days to obtain a co-cultured explant.

(6) Screening

After the completion of step (5), the co-cultured explants were cultured on a screening medium (MS+3.4 mg/L6-BA+0.3mg/L IBA+2mg/L hygromycin+250mg/L Tim+200mg/L Car+40. Mu.M/. Beta. -estradiol,3.5g/L Phytagel, 20g/L sucrose, pH 5.8) and about 23 explants were placed on each plate. The medium was changed every three weeks.

(7) Bud culture (as shown in FIG. 5)

After the completion of step (6), the plant tissue or bud containing the bud is cultured in a bud elongation medium (MS+2.0 mg/L6-BA+0.3mg/L IBA+2mg/L hygromycin+250mg/L Tim+200mg/L Car+40. Mu.M/L beta. -estradiol,3.5g/L Phytagel, 20g/L sucrose, pH 5.8) until the bud grows.

(8) Rooting

After the step (7) is completed, the obtained plant with the complete aerial parts (comprising stems, leaves and dead-end growth points) is placed in a rooting culture medium for culturing (MS+0.1 mg/L IBA+2mg/L hygromycin+250mg/L Tim+200mg/LCar+40 mu M/L beta-esctranol, 3.5g/L Phytagel, 20g/L sucrose, and pH is 5.8) until the young buds root to grow into complete seedling plants, and the strawberry plant is obtained.

Steps 6-8 were all performed at 16h day/8 h night photoperiod with a light intensity of about 7000lx and a temperature of 23 ℃.

In the transformation experiment process, statistics of the regeneration time and regeneration efficiency of the strawberry genetic transformation are carried out, so that the transformation efficiency of the explant expressing the strawberry endogenous growth regulating gene is obviously higher than that of the non-expressed control group.

Identification of experimental detection strawberry positive tissue culture seedlings

Regeneration rate is total number of explants to bud, total number of explants to positive seedling. The seedling standard is that the seedlings grow. In addition, the rooting efficiency is observed and counted, and the rooting standard is that the root grows to 3-4cm.

The identification test is to mix the constructed 20 pDV-FveGRF/FveGIF combinations into a library, and infect strawberry explants at the same time so as to screen the FveGRF-FveGIF combination with the strongest regeneration capacity at the same time. Wherein 951 strawberry explants are initially infected, 951 strawberry explants capable of sprouting normally are planted, 900 strawberry explants capable of sprouting normally grow after sprouting, and the sprouting rate is 95%.

1. Comparison of regeneration time and regeneration Rate

During the differentiation culture, the growth of the explant transformed with pDV-FveGRF/FveGIF vector was monitored, and the bud differentiation of the explant was observed, and the state shown in FIG. 3 was the normal growth state of the differentiated bud. The explants in the state are counted, and the transformation of the explants combined by the 20 pDV-FveGRF/FveGIF vectors can be 100% sprouted, the sprouting time is generally about 30-40 days (shown in figure 3), and the sprouting time is shortened by about 40 days compared with 56-84 days of the sprouting time of the conventional transformation technology. And the germination rate of the conventional transformation technology is only about 3-10%, and the germination rate is improved to 100%.

2. Comparison of seedling time and seedling rate

As shown in FIG. 4, taking an explant transformed with pDV-FveGRF/FveGIF vector as an example, leaves differentiated into seedling plants were taken, DNA was extracted by CTAB method, detected by primers (pDV-F1/R1, pDV-F2/R2) of Table 2, and the combination type of different FveGRF-FveGIF was analyzed by combining Sanger method sequencing and second generation sequencing. From the results of FIG. 7 (observed at 140 days of transformation), it can be seen that the seedling rate of the explants transformed with the pDV-FveGRF/FveGIF vector is generally significantly improved over that of the conventional transformation technique.

Since the genetic transformation process for performing the test effect of the present application is a co-cultivation process in which 20 combinations of FveGRF-FveGIF are mixed, some of the seedlings are not only the result of a single action of a certain combination. Sequencing all the seedling plant combinations to obtain the statistical result of the number of each combination of single pDV-FveGRF/FveGIF combination or combined action of multiple pDV-FveGRF/FveGIF combinations. From the data of FIGS. 8 and 3, it can be initially seen that the number of positive transgenic plants for the combinations FveGRF8-FveGIF1 and FveGRF3-FveGIF1 is higher, when the total number of combinations obtained above is combined.

And then, screening 80 seedling plants affected by a single combination, counting the sequencing results, and obtaining the number of positive transgenic plants with single effect of the combination of FveGRF-FveGIF through the data of FIG. 9 and Table 4, wherein the number of positive transgenic plants with the combination of FveGRF8-FveGIF1 and FveGRF3-FveGIF1 is higher, so that the two combinations can be used for remarkably improving the genetic transformation efficiency of strawberries, especially the seedling rate under the condition of single combination effect.

And the observation of the seedling time of each infected explant can be seen from fig. 6 that the seedling time of the explant co-cultured with FveGRF-FveGIF is about 40 days, generally faster than 140 days of the time of the conventional transformation technology, the transformation time is shortened by two times, and the rooting time of the combination of FveGRF8-FveGIF1 and FveGRF3-FveGIF is further improved with other FveGRF-FveGIF combinations. The regeneration time and rooting time of FveGRF8-FveGIF1 and FveGRF3-FveGIF were 35 days and 50 days, respectively.

TABLE 3 number of combinations of 900 Positive seedlings

TABLE 4 number of combinations in 80 Positive seedlings with single combination

From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: the strawberry endogenous growth regulating gene combination FveGRF8 and FveGRF 1 or FveGRF3 and FveGRF 1 provided by the application are utilized for genetic transformation operation, so that the genetic transformation efficiency of strawberries can be greatly improved in three aspects of bud regeneration, seedling formation and rooting, the genetic transformation period of the strawberries is greatly shortened, the practical breeding application of the strawberries is significant, and a foundation is laid for large-scale genetic transformation screening functional genes and molecular breeding of the strawberries.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of a strawberry endogenous growth regulating gene combination in genetic transformation, characterized in that the strawberry endogenous growth regulating gene combination is selected from any one of the following groups:

FveGRF8 and FveGIF1; or (b)

FveGRF3 and FveGIF1;

wherein, the nucleotide sequences of FveGRF3, fveGRF8 and FveGIF1 are as follows:

(a) As set forth in SEQ ID NOs: 1-3; or (b)

(b) Encoding a polypeptide having the sequence of SEQ ID NOs:4-6, nucleotide sequences of strawberry endogenous growth factors FveGRF3, fveGRF8 and FveGIF1; or (b)

(c) A nucleotide sequence having 85% or more homology with the nucleotide sequence of any one of (a) and (b) and having a function of improving strawberry transformation efficiency.

2. A recombinant protein encoded by the strawberry endogenous growth regulating gene combination of claim 1 for use.

3. A recombinant vector comprising a strawberry endogenous growth regulatory gene combination of the combination of FveGRF8 and FveGIF1 or FveGRF3 and FveGIF1 for use according to claim 1 and a driving element for driving the expression of the strawberry endogenous growth regulatory gene combination.

4. The recombinant vector according to claim 3, wherein the driving element comprises a promoter and a terminator;

preferably, the promoter comprises a constitutive promoter or an inducible promoter;

preferably, the constitutive promoter comprises an arabidopsis UBQ10 promoter or a tobacco mosaic virus 35S promoter;

preferably, the inducible promoter comprises an estradiol inducible promoter;

preferably, the terminator comprises a tobacco mosaic virus Nos terminator;

preferably, the recombinant vector comprises the SunTag system and the XVE system.

5. A host cell transformed with the recombinant vector of claim 3 or 4.

6. The host cell of claim 5, wherein the host cell comprises escherichia coli or agrobacterium.

7. A method of genetic transformation of strawberries, comprising: transforming the strawberry endogenous growth regulating gene combination of the use of claim 1 into a plant material of interest using the host cell of claim 5;

preferably, the strawberry endogenous growth regulating gene combination is selected from any one or more of the following:

FveGRF8 and FveGIF1; or FveGRF3 and FveGIF1;

preferably, the number of strawberry endogenous growth regulating gene combinations is each independently 1 or more.

8. The genetic transformation method according to claim 7, wherein the genetic transformation method comprises any one of the following transformation methods: agrobacterium infection, gene gun, PEG-mediated protoplast transformation, plant virus-mediated transformation, pollen tube passage, and ovary injection;

preferably, the agrobacterium infection method comprises:

introducing the recombinant vector into an agrobacterium strain to obtain recombinant bacteria;

preparing the recombinant bacteria into a leaching solution;

placing target plant materials into the dip dyeing liquid for dip dyeing to obtain an infected explant;

co-culturing the infected explant and the recombinant bacteria to obtain a co-cultured explant;

positive screening is carried out on the co-culture explant to obtain positive tissue culture seedlings;

preferably, the agrobacterium strain is GV3101;

preferably, the target plant material comprises strawberry material;

preferably, the strawberry material is selected from any one of the following: petals, pollen, cotyledons, cotyledonary nodes, leaves, stems or protoplasts.

9. The genetic transformation method according to claim 8, wherein the positive selection comprises:

performing antibiotic resistance screening on the co-culture explant to obtain a positive transformant;

performing bud growth and rooting culture on the positive transformant to obtain a seedling plant;

carrying out gene detection on the seedling plants to obtain positive tissue culture seedlings;

preferably, the gene detection comprises PCR, first generation sequencing and/or high throughput sequencing.

10. Use of the recombinant vector of any one of claims 3 to 4, or the host cell of claim 5, or the genetic transformation method of any one of claims 7 to 9, in the genetic transformation of strawberry.