CN120310836A - Method for promoting plant regeneration efficiency of poplar haploid by increasing the expression of PtrWOX5.2 gene in poplar - Google Patents

Abstract

The present invention relates to the field of biotechnology, and specifically discloses a method for promoting the plant regeneration efficiency of poplar haploids by increasing the expression of the PtrWOX5.2 gene in poplars. The present invention transfers the PtrWOX5.2 gene into a poplar haploid cell line or callus induced by anther culture, and the transgenic poplar haploid cell line or callus overexpressing PtrWOX5.2 is compared with a control, and the cell proliferation is increased, the callus growth is accelerated, the callus differentiation rate is improved, and more clustered buds are differentiated. These results show that the overexpression of the PtrWOX5.2 gene can improve the regeneration efficiency of poplar haploid plants.

Description

Method for promoting plant regeneration efficiency of poplar haploid by improving PtrWOX5.2 gene expression in poplar

Technical Field

The invention relates to a method for promoting plant regeneration efficiency of poplar haploids by improving expression of PtrWOX5.2 genes in poplar in the technical field of biology.

Background

On one hand, the plant haploid breeding can quickly obtain homozygous diploid plants in one generation through chromosome doubling, can effectively control offspring character separation, greatly shortens breeding period, improves selection efficiency and quickens breeding process, and on the other hand, provides plant materials with homozygous backgrounds for genetic research and genomics research, and has unique effects in the fields of genetic group construction, gene function identification, transgenic plant rapid stability, genome sequencing and the like.

Forest is a highly heterozygous outcross species of genome, and has the characteristics of high genetic heterozygosity, large volume, strict cross pollination and the like, and it is difficult to obtain a homozygous line through the traditional multi-generation selfing means. For forest, haploid (DH line) is obtained by haploid induction and doubling technology, and is an effective way for culturing improved varieties of forest by using pure line.

Improving haploid induction efficiency and simplifying induction programs are key to haploid breeding. Anther culture is the most widely used haploid induction technique. However, many poplar varieties with excellent agronomic traits are difficult to obtain regenerated plants in anther culture, and there is a great need to resolve the molecular mechanisms of haploid production and overcome the regeneration problem caused by limitations caused by genotypes by optimizing the tissue culture system of anther culture. In the process of inducing the anther culture to form haploids, the pollen mother cells generate mononuclear pollen grains after meiosis, each pollen grain carries different genetic combinations, and the requirements of different pollen grains on culture conditions can be different, so that the conditions of the optimized culture medium are more complicated.

Key developmental regulators affecting plant regeneration, such as LEAFY COTYLEDON (LEC 1), LEC2, WUSCHEL (WUS) and BABY BOOM (BBM), GROWTH-REGULATING FACTOR 4 (GRF 4) -GRF-INTERACTING FACTOR 1 (GIF 1), etc., have been identified by extensive analysis of the cell lineages and molecular mechanisms of plant regeneration. Overexpression of these factors promotes somatic embryo production or bud regeneration. The regulatory factors can effectively improve the regeneration capacity of plants, solve the bottleneck problem of genotype dependence and provide a new strategy for improving the regeneration efficiency of haploid plants cultured by anthers.

Disclosure of Invention

The invention aims to solve the technical problem of how to improve the plant regeneration efficiency of poplar haploids.

In order to solve the above technical problems, the present invention provides a method for promoting plant regeneration efficiency of poplar haploids by increasing expression of PtrWOX5.2 gene in poplar, wherein PtrWOX5.2 gene is a gene encoding PtrWOX5.2 protein, and PtrWOX5.2 protein is a protein of A1, A2 or A3:

a1, the amino acid sequence is the protein of SEQ ID No.2 in the sequence table;

A2, substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID No.2 in the sequence table to obtain a protein which has more than 80% of identity with the protein shown in A1 and has similar functions;

A3, a fusion protein obtained by linking protein tags at the N terminal or/and the C terminal of A1 or A2.

In the above method, SEQ ID No.2 of the sequence Listing consists of 182 amino acid residues.

In the above method, identity refers to the identity of amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, by using blastp as a program, expect values are set to 10, all filters are set to OFF, BLOSUM62 is used as Matrix, gap existence cost, per residue gap cost and Lambda ratio are set to 11,1 and 0.85 (default values), respectively, and identity of a pair of amino acid sequences is searched for and calculated, and then the value (%) of identity can be obtained.

In the above method, the 80% identity or more may be at least 81%, 85%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

In the above method, the PtrWOX5.2 protein may be derived from poplar.

In the above method, the PtrWOX5.2 gene may specifically be a nucleic acid molecule whose coding sequence of the coding strand is SEQ ID No.1 of the sequence Listing.

In the above method, the poplar haploid is derived from a poplar haploid or non-haploid plant, such as a poplar haploid cell line or derived from a callus induced by anther culture of poplar.

In the above method, the plant regeneration efficiency of promoting poplar haploid is represented by any one or a combination of a plurality of Y1-Y3:

y1 promotes proliferation of cells;

Y2 accelerates the growth of callus;

Y3 promotes differentiation of clumped buds.

The method comprises the step of introducing the PtrWOX5.2 gene into a receptor poplar haploid cell line or anther culture induced callus to obtain a poplar with high haploid plant regeneration efficiency, thereby improving haploid induction efficiency. (the original non-differentiated or non-regenerated haploid cell line or anther culture induced callus can regenerate plants after PtrWOX5.2 gene is introduced, so that more haploid plants are obtained, and haploid induction efficiency is improved.)

In the above method, the PtrWOX5.2 gene can be modified as follows and then introduced into the receptor poplar to achieve better expression effect:

1) Modifying the gene sequence adjacent to the initiation methionine to effect initiation of translation, e.g., by utilizing sequences known to be effective in plants;

2) The choice of promoter will vary with the time and space requirements of expression and will also depend on the target species, e.g., tissue or organ specific expression promoters, depending on what period the desired receptor is to develop, promoters linked to facilitate expression thereof in plants, which may include constitutive, inducible, chronologically regulated, developmentally regulated, chemically regulated, tissue-preferred and tissue-specific promoters, although many promoters derived from dicots have been demonstrated to be functional in monocots and vice versa, dicot promoters are desirably selected for expression in dicots, monocot promoters for expression in monocots, promoter 35S is employed in one embodiment of the invention to drive the ptrwox5.2 gene;

3) The expression efficiency of the gene of the invention can also be increased by ligating a suitable transcription terminator, for example tml derived from CaMV, E9 derived from rbcS, any available terminator known to function in plants can be ligated to the gene of the invention;

4) Enhancer sequences such as intron sequences (e.g., derived from Adhl and bronzel) and viral leader sequences (e.g., derived from TMV, MCMV and AMV) are introduced.

The PtrWOX5.2 gene can be introduced into plant cells by conventional biotechnological methods using Ti plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, etc （ Weissbach, 1998, Method for Plant Molecular Biology VIII, Academy Press, New York, pp.411-463; Geiserson and Corey , 1998 , Plant Molecular Biology(2nd Edition).

The invention also provides a protein, which is PtrWOX5.2 protein.

The invention also provides biological materials related to the PtrWOX5.2 protein, which also belong to the protection scope of the invention.

The biological material related to PtrWOX5.2 protein provided by the invention is any one of the following B1 to B5:

b1, a nucleic acid molecule encoding PtrWOX5.2 protein;

b2, an expression cassette comprising a nucleic acid molecule as described in B1;

B3, a recombinant vector comprising the nucleic acid molecule of B1, or a recombinant vector comprising the expression cassette of B2;

b4, a recombinant microorganism comprising a nucleic acid molecule according to B1, or a recombinant microorganism comprising an expression cassette according to B2, or a recombinant microorganism comprising a recombinant vector according to B3;

b5, a transgenic plant cell line comprising a nucleic acid molecule according to B1, or a transgenic plant cell line comprising an expression cassette according to B2, or a transgenic plant cell line comprising a recombinant vector according to B3.

Wherein the nucleic acid molecule may be DNA such as cDNA, genomic DNA or recombinant DNA, or RNA such as mRNA or hnRNA.

In the above biological material, the nucleic acid molecule B1 is the PtrWOX5.2 gene, and specifically can be a nucleic acid molecule with a coding sequence of a coding chain being SEQ ID No.1 in a sequence table.

In the above biological material, the expression cassette of B2 (ptrwox 5.2 gene expression cassette) refers to DNA capable of expressing the ptrwox5.2 gene in a host cell, and the DNA may include not only a promoter for promoting transcription of the ptrwox5.2 gene but also a terminator for terminating transcription of the ptrwox5.2 gene. Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to, constitutive promoters, tissue, organ and development specific promoters, and inducible promoters. Examples of promoters include, but are not limited to, the constitutive promoter 35S of cauliflower mosaic virus, the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", chao et al (1999) Plant physiolog 120:979-992), the chemically inducible promoter from tobacco, pathogenesis-related 1 (PR 1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiohydroxy acid S-methyl ester)), the tomato protease inhibitor II promoter (PIN 2) or LAP promoter (both inducible with jasmonic acid a ester), the heat shock promoter (U.S. Pat. No.5,187,267) tetracycline inducible promoter (U.S. Pat. No.5,057,422), seed-specific promoters such as the millet seed-specific promoter pF128 (CN 101063139B (Chinese patent 2007 1 0099169.7)), seed storage protein specific promoters (e.g., legumin, C.L.5), The promoter of napin, oleosin and soybean beta conglycin (Beachy et al (1985) EMBO J. 4:3047-3053)). They may be used alone or in combination with other plant promoters. All references cited herein are incorporated by reference in their entirety. Suitable transcription terminators include, but are not limited to, the Agrobacterium nopaline synthase terminator (NOS terminator), the CaMV 35S terminator, the tml terminator, the CaMV, Pea rbcS E9 terminator and nopaline and octopine synthase terminators (see, e.g., odell et al (I ⁹⁸⁵) Nature 313:810; rosenberg et al (1987) Gene, 56:125; guerineau et al (1991) mol. Gen. Genet, 262:141; proudfoot (1991) Cell, 64:671; sanfacon et al Genes Dev., 5:141; mogen et al (1990) PLANT CELL, 2:1261; munroe et al (1990) Gene, 91:151; ballad et al (1989) Nucleic Acids Res. 17:7891; joshi et al (1987) Nucleic Acid Res., 15:9627).

The existing plant expression vector can be used for constructing a recombinant expression vector containing the PtrWOX5.2 gene expression cassette. The plant expression vector comprises a binary agrobacterium vector, a vector which can be used for plant microprojectile bombardment and the like. Such as pCAMBIA2301、pCAMBIA4301、pAHC25、pWMB123、pBin438、pCAMBIA1302、pCAMBIA1301、pCAMBIA1300、pBI121、pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Co.), etc. The plant expression vector may also comprise the 3' -untranslated region of a foreign gene, i.e., comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The polyadenylation signal may direct the addition of polyadenylation to the 3 'end of the mRNA precursor and may function similarly to the 3' transcribed untranslated regions of Agrobacterium tumefaciens induction (Ti) plasmid genes (e.g., nopaline synthase gene Nos) and plant genes (e.g., soybean storage protein genes). When the gene of the present invention is used to construct a plant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancers may be ATG initiation codon or adjacent region initiation codon, etc., but must be identical to the reading frame of the coding sequence to ensure proper translation of the entire sequence. The sources of the translational control signals and initiation codons are broad, and can be either natural or synthetic. The translation initiation region may be derived from a transcription initiation region or a structural gene. To facilitate identification and selection of transgenic plant cells or plants, the plant expression vectors used may be processed, for example by adding genes encoding enzymes or luminescent compounds which produce a color change (GUS gene, luciferase gene, etc.), antibiotic marker genes (such as nptII gene conferring resistance to kanamycin and related antibiotics, bar gene conferring resistance to the herbicide phosphinothricin, HPT gene conferring resistance to the antibiotic hygromycin, hph gene, and dhfr gene conferring resistance to methatrexate, EPSPS gene conferring resistance to glyphosate) or chemical reagent marker genes, etc. (such as herbicide resistance genes), mannose-6-phosphate isomerase gene providing mannose metabolizing ability, etc. From the safety of transgenic plants, transformed plants can be screened directly in stress without adding any selectable marker gene.

In the above biological material, the recombinant microorganism may specifically be yeast, bacteria, algae and fungi.

In order to solve the technical problems, the invention also provides a plant reagent for improving the regeneration efficiency of poplar haploids, wherein the active ingredients of the plant reagent are substances for promoting or improving the PtrWOX5.2 gene and improving the abundance of the PtrWOX5.2 protein. The substance may comprise the ptrwox5.2 protein and/or the biological material.

The active ingredients of the above plant agents may also contain other biological or/and non-biological ingredients, and the other active ingredients of the above plant agents may be determined by one skilled in the art based on the effect of improving the efficiency of reproduction of poplar haploids.

The invention also protects the method, or the PtrWOX5.2 protein, or the application of the biological material in poplar breeding.

Aiming at the problem that poplar haploid cells are difficult to regenerate buds, the callus induced by Lu Mao (LM 50) anther culture is taken as a receptor material, and the growth rate of the callus can be promoted and the bud regeneration efficiency can be improved through the overexpression of PtrWOX5.2 genes. In order to further verify the effect, the haploid cell line 1588 of the aspen which is identified as an anther culture source is taken as a receptor material, and through over-expression of PtrWOX5.2 genes, the growth of callus formed by induction of the haploid cell line can be accelerated, and the regeneration of buds can be effectively promoted. These results indicate that overexpression of the PtrWOX5.2 gene can improve the efficiency of regeneration of poplar haploids.

Drawings

FIG. 1 is a photograph of calli derived from transformation of LM50 anther culture with PtrWOX5.2 gene in example 1 of the present invention. Specifically, photographs of control EHA105/pCAMBIA2301 post-callus infection differentiation cultures 30 d (FIG. 1 a), 45 d (FIG. 1 c) and rooting cultures 15 d (FIG. 1 e), photographs of EHA105/pCAMBIA4301-PtrWOX5.2 post-callus infection differentiation cultures 15 d (FIG. 1 b), 30 d (FIG. 1 b) and rooting cultures 15 d (FIG. 1 f).

FIG. 2 shows the GUS expression results in example 1 of the present invention. Fig. 2 a, 3d, fig. 2 b, 7d, fig. 2 c, 14d, fig. 2d, 30d.

FIG. 3 shows the formation of callus and shoot regeneration in example 1 of the present invention. FIG. 3a shows the formation of callus blocks by transformation of the Populus parviflora 1588 cell line containing PtrWOX5.2 gene, FIG. 3 b shows the browning of the Populus parviflora 1588 cell line transformed with the control vector pCAMBIA2301, FIG. 3 c shows the bud regeneration of the callus blocks transformed with the PtrWOX5.2 gene, and FIG. 3d shows the failure of the callus transformed with the control vector pCAMBIA2301 to induce bud regeneration.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The quantitative tests in the following examples were all performed in triplicate, and the results were averaged.

The experimental methods in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Populus tomentosa Lu Mao (LM 50) was described in the following examples in the non-patent literature, "Miao Yu, gao Kai, huang Sai, an Xinmin. Populus tomentosa fine clone 'LM50' anther culture plant regeneration System was established. J.Biotechnology, 2022, 42 (5): 46-57", available to the public from the applicant for testing of duplicate inventions.

The following examples describe experiments for repeating the invention in non-patent document "Song J, Lu S, Chen Z, Lourenco R, Chiang V.Genetic Transformation of Populustrichocarpa Genotype Nisqually-1:A Functional Genomic Tool for Woody Plants. Plant Cell Physiol, 2006, 47 (11): 1582-1589.", publicly available from applicant.

The aspen 1588 haploid cell line in the examples described below was provided by the professor of the curculigo of the university of northeast forestry, and was obtained by the method for obtaining aspen haploid cell line in chinese patent application with application number "CN202210847072.4" entitled "method for obtaining aspen haploid cell line", and other aspen haploid cell lines obtained by the method can also be used for performing the test of example 2 of the present invention.

PCAMBIA2301 is described in the examples below in non-patent documents "Li Fei, cao Yongqiong, wang Gang, guo Yanbing, li Ziwei, wu Gongzhi. Agrobacterium tumefaciens mediates the establishment and optimization of genetic transformation systems for Calla helveticus. Southern agricultural journal, 2024, 55 (6): 1692-1699", available to the public from the applicant for testing of the repeated invention.

PCAMBIA4301 is described in the following examples in non-patent documents "Wang Bingfeng, wang Hongzhi, wei Jianhua. Functional analysis of the poplar transcription factor PtoMYB 057. Guangdong agricultural science 2015, 15:105-109.", publicly available from the applicant to replicate experiments of the present invention.

Example 1

PtrWOX5.2 Gene cloning

According to Phytozome database of CDS sequence information of the WOX5.2 gene of populus tomentosa, a PCR specific primer is designed for the gene sequence by using software Primer5.0, and an EcoRI enzyme digestion site is introduced, wherein the specific primer sequence is as follows:

WOX5-2-EcoRI-CDS-F: GAATTCATGGAAGAGAGAATGTCAGG (wherein the EcoRI enzyme recognition sequence is shown in italics);

WOX5-2-EcoRI-CDS-R: GAATTCCTACACAAAGCTTAATCGCAG (wherein the EcoRI enzyme recognition sequence is indicated in italics).

PCR amplification is carried out by taking extracted populus tomentosa cDNA as a template, and amplified products are recovered to obtain a gene PtrWOX5.2 gene fragment, T vector connection and escherichia coli competent transformation are carried out, and the gene fragment is sent to the Optimago company for DNA sequencing. The coding sequence of PtrWOX5.2 gene is shown as SEQ ID No.1, and the amino acid sequence of the coded protein PtrWOX5.2 is shown as SEQ ID No. 2.

SEQ ID No.1

ATGGAAGAGAGAATGTCAGGCTTTTGTATCACAAAAGCTGGACGTGGTGGTGGCAGTGGCAATAACTGTGGCACAGGAACTAAATGTGGGCGTTGGAATCCTACTACCGAACAAGTTAAACTTCTAACTGACTTGTTCAGGTCTGGGCTTCGAACCCCAAGTACTGATGAGATCCAGAACATCTCCACCCAGCTTAGTTTTTATGGCAAGATCGAGAGCAAGAACGTTTTTTACTGGTTTCAAAATCATAAAGCTAGAGAAAGACAGAAGCGGCGCAGGGTTTCTGTTGATGAGAAGGATGCCATGATTCGTAGAGATGACAGATTTTCTTCTGCGCGATATTTTACTGAGATCAATCATGTGAACGAACCAGAAAGAGTGATTGAGACTCTTCAACTTTTCCCTTTAAACTCCTTTGATGAAGCGGGACCAGAGAAATTCAGGTTCCAAGCAAATGAATGCAATGAAGCTGCAGCTGCATTTTCTTATAAATTTGGTACAGAAATGGATCATCCACATTTAGATCTGCGATTAAGCTTTGTGTAG

SEQ ID No.2

MEERMSGFCITKAGRGGGSGNNCGTGTKCGRWNPTTEQVKLLTDLFRSGLRTPSTDEIQNISTQLSFYGKIESKNVFYWFQNHKARERQKRRRVSVDEKDAMIRRDDRFSSARYFTEINHVNEPERVIETLQLFPLNSFDEAGPEKFRFQANECNEAAAAFSYKFGTEMDHPHLDLRLSFV

2. Anther culture induction medium and differentiation medium

Induction medium MS+2 mg/L2, 4-D+1 mg/L KT+30 g/L sucrose+3 g/L Gelrite

Differentiation medium MS+6-BA0.5 mg/L+ZT1.0 mg/L+IBA0.25 mg/L+30 g/L sucrose+7 g/L agar powder

Construction of PtrWOX5.2 Gene overexpression vector

Construction of an over-expression vector by GATEWAY method TOPO vector digested with EcoRI was ligated with the above-obtained gene PtrWOX5.2 fragment (containing the sequence shown in SEQ ID No. 1) using T ₄ DNA ligase, and an entry vector was constructed at 16℃overnight. And (3) replacing the target gene PtrWOX5.2 gene fragment on the entry vector with correct sequencing by using LR reaction on a plant target expression vector pCAMBIA4301 to obtain a recombinant expression vector pCAMBIA4301-PtrWOX5.2 for expressing PtrWOX5.2 protein. The recombinant expression vector pCAMBIA4301-PtrWOX5.2 contains GUS gene, and the GUS gene contains introns, so that the GUS gene can not be expressed in bacteria any more and can only be expressed in plants, and GUS staining can be used for detecting whether the PtrWOX5.2 expression vector is integrated into the genome of the plants.

Coli competent cells were transformed with the recombinant vector pCAMBIA4301-PtrWOX5.2 and colony PCR identified using attB1-F and attB1-R primers. The plasmid was extracted and sent to the department of Optimus of Optimimimus of Optimimimimimimimimimimimimimimimimimimimimimimimim. The vector pCAMBIA4301-PtrWOX5.2, which was sequenced correctly, was stored at-20 ℃.

The vector pCAMBIA4301-PtrWOX5.2 and the control vector pCAMBIA4301 are respectively introduced into the agrobacterium strain EHA105 by using an electric shock transformation method to obtain agrobacterium strain EHA105/pCAMBIA4301-PtrWOX5.2 and the control agrobacterium strain EHA105/pCAMBIA2301 for preservation at-80 ℃.

Agrobacterium tumefaciens-mediated transformation of 4

4.1 Preparation of a soaking solution and a culture medium

The frozen Agrobacterium strain EHA105/pCAMBIA4301-PtrWOX5.2 was dipped at-80℃in a YEP solid medium containing kanamycin (Kan) 50 mg/. Mu.L, tetracycline (Tet) 5 mg/L, rifampicin (Rif) 50mg/L, at 28℃and 250 rpm for 2 d. Positive monoclonal colonies are picked and inoculated into 10 mL YEP liquid culture medium containing 50 mg/mu L kanamycin (Kan), 5 mg/L tetracycline (Tet) and 50mg/L rifampicin (Rif), the culture is carried out at 28 ℃ and 250 rpm overnight, 2 mL bacterial liquid is sucked into 80 mL YEP liquid culture medium at 28 ℃,250 rpm is oscillated and cultured for 5-6 h, OD value is detected to reach about 0.3-0.6, the bacterial liquid is transferred into 50mL and 6300 rpm centrifugal 10min for bacterial collection, the YEP is poured out, 1/2 MS liquid culture medium containing 100 mu M Acetosyringone (AS) is utilized for re-suspending bacterial liquid of 50mL, and agrobacterium strain EHA105/pCAMBIA4301-PtrWOX5.2 is obtained for standby.

Replacing bacterial liquid of the agrobacterium strain EHA105/pCAMBIA2301 with bacterial liquid of the agrobacterium strain EHA105/pCAMBIA4301-PtrWOX5.2, and keeping other steps unchanged to obtain the agrobacteria strain EHA105/pCAMBIA2301 for later use.

The formula of the co-culture medium comprises MS+2 mg/L2, 4-D+1 mg/L KT+100 mu M AS+30 g/L sucrose+7 g/L agar powder.

The recovery culture medium formula comprises MS+2mg/L2, 4-D+1mg/L KT+300 mg/LTIMENTIN +200mg/L cephalosporin+30 g/L sucrose+3 g/L Gelrite

The formula of the screening induction culture medium is MS+2 mg/L2, 4-D+1 mg/L KT+30 mg/L kan+300 mg/LTIMENTIN +200mg/L cephalosporin+30 g/L sucrose+3 g/L Gelrite, and the pH value is 5.8.

The formula of the screening differentiation medium is MS+0.5 mg/L6-BA+1.0 mg/L ZT+0.25 mg/L IBA+30 mg/L kan+300 mg/LTIMENTIN +100mg/L cefuroxime axetil+30 g/L sucrose+7 g/L agar powder, and the pH value is 5.8.

The rooting culture medium is selected from MS+10mg/L VB1+0.25 mg/L IBA+30mg/L kan+300 mg/L TIMENTIN +30g/L sucrose+7g/L agar powder.

Wherein 2,4-D is 2, 4-dichlorophenoxyacetic acid, KT is kinetin, gelrite is plant gel, kan is kanamycin, timestin is Timentin, 6-BA is 6-benzylaminopurine, ZT is Zeatin (Zeatin), IBA is 3-indolebutyric acid.

4.2PtrWOX5.2 improving the ability of the LM50 anther culture of populus tomentosa to induce callus differentiation

The genotype Lu Mao (LM 50) of populus tomentosa is taken as a test material, and flowers are taken from an LM50 male plant of healthy populus tomentosa growing in the Shandong Guan County populus tomentosa gene library for 15-20 years. Taking anther with microspores in a mononuclear borderline period for inoculation culture, and inducing callus, wherein the method comprises the following steps:

Flower buds are sterilized, scales are stripped, anthers are inoculated on an induction medium, 30 anthers are inoculated on each culture dish, each treatment is repeated 3 times, and the flower bud is finished in a sterile environment. The inoculated culture dish is sealed, and the culture dish is placed in a tissue culture chamber for dark culture, and the callus induced by anther is transferred to a new induction culture medium every week during the culture period, wherein the culture time is about 1 month, and the culture condition is unchanged.

Transferring the callus to a screening differentiation medium for illumination culture, wherein the temperature of a tissue culture room is 23+/-2 ℃, the relative humidity is 60% -70%,16 h illumination/8 h darkness is realized, and the illumination intensity is about 200 mu mol/m ²/s².

As a result, after the differentiation culture of the calli obtained by the LM50 anther culture, regenerated shoots were not obtained, and green calli were obtained. The effect of the overexpression of PtrWOX5.2 gene on regeneration of poplar callus plants was studied using these green callus blocks, from which regenerated shoots were not obtained, as explants, as follows:

The green callus blocks of the explants are cut into small callus blocks with the length of about 0.8-1cm, and the small callus blocks are placed into an agrobacterium strain EHA105/pCAMBIA4301-PtrWOX5.2 infection liquid for infection, so that the small callus blocks are placed into the agrobacterium strain EHA105/pCAMBIA2301 infection liquid for infection to serve as an empty vector control.

Culturing 15min under dark conditions at 25 ℃ and 120 rpm. The surface of the explant is soaked with the sterilized absorbent paper and the explant is placed in a co-culture medium for 2-3 d in the dark at 23 + -2 ℃. Transferring the co-cultured explant into recovery culture based on 7 d of recovery culture under the dark condition of 23+/-2 ℃, continuing to transfer into screening induction culture medium for culture under the dark condition for 4-5 weeks, transferring the surviving callus into screening differentiation culture medium, and culturing under the illumination of 23+/-2 ℃ for about 8 weeks.

The callus after PtrWOX5.2 gene transformation has a high growth rate, clustered seedlings appear about 15 d after infection (b of FIG. 1), independent seedlings appear in 15 d after continuous culture (d of FIG. 1), and after the independent seedlings are transferred to a screening rooting medium, the seedlings can root after 15-20 d culture, and complete plants can be regenerated (f of FIG. 1). Control vector pCAMBIA2301 was able to form clumped seedlings after infection of the callus (a of fig. 1), but the clumped seedlings were weak in yellowing (c of fig. 1) and eventually failed to continue to grow and die (e of fig. 1). The result shows that PtrWOX5.2 gene can promote the growth rate of the callus cultured by the anther of poplar and improve the bud regeneration efficiency (see table 1).

TABLE 1 statistics of regeneration efficiency after transformation of callus with PtrWOX5.2 Gene

4.3PtrWOX5.2 promoting the formation of calli from the haploid cell line of Populus delbrueckii 1588 and regeneration of shoots

And (3) placing the haploid cell line of the aspen 1588 subjected to the subculture for 15-21 days into an agrobacterium strain EHA105/pCAMBIA4301-PtrWOX5.2 infection solution to infect, and placing the haploid cell line into the agrobacterium strain EHA105/pCAMBIA2301 infection solution to infect as an empty vector control.

Culturing 15 min under dark conditions at 25 ℃ and 120 rpm. The surface of the aspen 1588 haploid cell line is soaked in the dye solution and the explant is cultured in a co-culture medium at the temperature of 23+/-2 ℃ in the dark for 2-3 d. Transferring the co-cultured 1588 haploid cell line of the populus euphratica into recovery culture for 7 d under the dark condition of 23+/-2 ℃ and continuously transferring into the screening induction culture medium for 4-5 weeks.

In this process, after agrobacteria infection and co-cultivation of the calli of populus xidans, calli samples were selected and cultivated for 3 days, 7 days, 14 days, and 30 days, and GUS staining was performed thereon to examine whether PtrWOX5.2 expression vector was integrated into plant genome.

The results showed (Table 2) that the GUS gene was expressed at all time points (FIG. 2a, FIG. 2b, FIG. 2 c and FIG. 2 d). As the incubation time was prolonged, the GUS expression level showed a decreasing trend, with the highest expression level at 3 days (FIG. 2 a). Even at 30 days, the GUS gene could still be detected (FIG. 2 d), indicating that the vector had been stably transformed into callus.

TABLE 2 GUS expression detection

After infection with the agrobacterium strain EHA 105-mediated pCAMBIA4301-ptrwox5.2, the aspen 1588 haploid cell line carrying the ptrwox5.2 gene showed a faster growth rate, calli appeared in the screening induction medium after 7-15 days of dark culture, and large yellow-green calli blocks were partially formed after continued culture for 40 days (fig. 3 a), whereas the cell line transformed with the control vector grew slowly, mostly with browning (fig. 3 b). This result suggests that overexpression of PtrWOX5.2 gene can promote proliferation of cells. Further transfer of the dark cultured callus pieces for 40 days to the screening differentiation medium for about 2 months, the occurrence of green leaf-like secondary buds in the callus pieces transformed with PtrWOX5.2 gene (c of FIG. 3) was found, whereas no formation of green cluster buds was observed in the control vector-transformed callus (d of FIG. 3), and this result indicated that the overexpression of PtrWOX5.2 gene promoted not only cell proliferation but also bud regeneration ability.

Aiming at the problem that poplar haploid cells are difficult to regenerate buds, the invention takes the callus induced by LM50 anther culture as a receptor material, and can promote the growth rate of the callus and improve the bud regeneration efficiency through the overexpression of PtrWOX5.2 genes. In order to further verify the effect, the haploid cell line 1588 of the aspen which is identified as an anther culture source is taken as a receptor material, and through over-expression of PtrWOX5.2 genes, the growth of callus formed by induction of the haploid cell line can be accelerated, and the regeneration of buds can be effectively promoted. These results indicate that overexpression of the PtrWOX5.2 gene can improve the regeneration efficiency of poplar haploid plants.

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Claims (9)

1. A method for promoting plant regeneration efficiency of poplar haploids by improving expression of PtrWOX5.2 gene in poplar is characterized in that PtrWOX5.2 gene is a gene encoding PtrWOX5.2 protein, and PtrWOX5.2 protein is protein of A1, A2 or A3:

a1, the amino acid sequence is the protein of SEQ ID No.2 in the sequence table;

A2, substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID No.2 in the sequence table to obtain a protein which has more than 80% of identity with the protein shown in A1 and has similar functions;

A3, a fusion protein obtained by linking protein tags at the N terminal or/and the C terminal of A1 or A2.

2. The method according to claim 1, wherein the coding sequence of the PtrWOX5.2 gene is the DNA molecule of SEQ ID No.1 of the sequence Listing.

3. The method of claim 2, wherein the promotion of plant regeneration efficiency of poplar haploids is expressed as a combination of any one or more of Y1-Y3:

y1 promotes proliferation of cells;

Y2 accelerates the growth of callus;

Y3 promotes differentiation of clumped buds.

4. The method of claim 3, wherein the PtrWOX5.2 gene is introduced into a haploid cell of a receptor poplar to obtain a poplar with high haploid plant regeneration efficiency, and the haploid plant regeneration efficiency of the poplar with high haploid plant regeneration efficiency is higher than that of the haploid plant regeneration efficiency of the haploid of the receptor poplar.

5. The ptrwox5.2 protein as set forth in claim 1.

6. The PtrWOX5.2 protein related biomaterial according to claim 5, wherein the PtrWOX5.2 protein related biomaterial is any one of the following B1 to B5:

b1, a nucleic acid molecule encoding PtrWOX5.2 protein;

b2, an expression cassette comprising a nucleic acid molecule as described in B1;

B3, a recombinant vector comprising the nucleic acid molecule of B1, or a recombinant vector comprising the expression cassette of B2;

b4, a recombinant microorganism comprising a nucleic acid molecule according to B1, or a recombinant microorganism comprising an expression cassette according to B2, or a recombinant microorganism comprising a recombinant vector according to B3;

b5, a transgenic plant cell line comprising a nucleic acid molecule according to B1, or a transgenic plant cell line comprising an expression cassette according to B2, or a transgenic plant cell line comprising a recombinant vector according to B3.

7. The biological material according to claim 6, wherein the coding sequence of the nucleic acid molecule B1 is the nucleic acid molecule of SEQ ID No.1 of the sequence Listing.

8. A plant reagent comprising, as an active ingredient, a substance that promotes or increases the PtrWOX5.2 gene according to claim 1 or 2 or increases the abundance of the PtrWOX5.2 protein according to claim 1 or 2.

9. Use of the method according to any one of claims 1 to 4, or the ptrwox5.2 protein according to claim 5, or the biological material according to claim 6 or 7, or the plant agent according to claim 8 in poplar breeding.