Application of PsASY1 gene in regulation and control of meiosis abnormality behaviors of plant pollen mother cells and regulation and control of plant growth speed

Abstract

The invention relates to the technical field of gene engineering, in particular to application of a PsASY1 gene to regulation and control of meiosis abnormality behaviors of plant pollen mother cells and regulation and control of plant growth speed. A system for overexpressing the PsASY1 gene is established in tobacco, and the result shows that the overexpressing of the PsASY1 gene can cause meiosis abnormality of peony pollen mother cells and can also improve the growth speed of plants, which is specifically reflected in improvement of the crown breadth, the leaf size and the plant height of the plants. Therefore, the PsASY1 protein, the PsASY1 gene or a biological material for overexpressing the PsASY1 gene provided by the invention can be used for regulating and controlling meiosis of plant pollen mother cells, cultivating polyploidy peony, cultivating sterile peony and the like. The invention provides a new gene material for peony molecular breeding, and also provides a theoretical basis and a technical reference for breeding work of creating peony polyploidy by using a sexual polyploidy approach.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to the application of the PsASY1 gene in regulating abnormal meiotic behavior of plant pollen mother cells and regulating plant growth rate. Background Art

[0002] The peony (Paeonia suffruticosa) originates in China and is a perennial deciduous flowering shrub of the genus Paeonia in the family Paeoniaceae. It is an important ornamental and medicinal plant and an emerging oilseed crop in my country. Peonies are widely distributed in my country and are divided into different cultivars based on their cultivation regions and wild origins: the Central Plains, Jiangnan, Northwest, and Southwest groups. Thanks to a long history of cultivation and modern breeding techniques, peony varieties are increasingly diverse. Currently, there are over 3,000 varieties worldwide, of which nearly 2,000 are in China.

[0003] In the breeding of ornamental plants, polyploids have attracted widespread attention due to their advantages, including large floral organs, numerous and thick petals, vibrant colors, delayed flowering in some species, and strong stress resistance. Peonies, with their large and small chromosome counts, are suitable for studying chromosomal abnormalities and mutations. Wild species of the peony group are diploid, with a base chromosome number of 5. All peony species share a common karyotype, with a chromosome number of 2n = 2x = 10. Current research on peony meiosis focuses primarily on observing abnormal meiotic behavior. Furthermore, little research has been conducted on peony growth traits. Therefore, it is necessary to investigate the relationship between genes in the peony and meiotic behavior and growth traits. Summary of the Invention

[0004] The purpose of the present invention is to provide an application of the PsASY1 gene in regulating abnormal meiotic behavior of plant pollen mother cells and regulating plant growth rate, so as to solve the problems existing in the above-mentioned prior art.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a PsASY1 protein, the amino acid sequence of the PsASY1 protein is shown in SEQ ID NO.8.

[0007] The present invention provides a PsASY1 gene encoding the above-mentioned PsASY1 protein. The nucleotide sequence of the PsASY1 gene is shown in SEQ ID NO.1.

[0008] The present invention provides a biological material containing the PsASY1 gene.

[0009] The present invention provides the use of the aforementioned PsASY1 protein, the aforementioned PsASY1 gene or the aforementioned biological material in any one or more of the following:

[0010] (1) Regulating meiosis of plant pollen mother cells

[0011] (2) regulating the formation of 2n pollen grains in plants;

[0012] (3) Cultivating polyploid plants;

[0013] (4) Cultivating sterile plants;

[0014] (5) regulating plant growth traits; the growth traits include one or more of crown width, leaf length, and plant height;

[0015] (6) Regulating the content of anthocyanins in plants;

[0016] (7) Regulate the chlorophyll content of plants.

[0017] Preferably, by overexpressing the PsASY1 gene in plants, the purpose of promoting the formation of plant 2n pollen grains, cultivating polyploid plants, cultivating sterile plants, improving plant growth traits, increasing plant anthocyanin content and increasing plant chlorophyll content is achieved.

[0018] Further preferably, the plant includes tobacco or peony.

[0019] The present invention provides a method for promoting the formation of 2n pollen grains in plants, comprising the step of overexpressing the expression level of the PsASY1 gene in the plant; the nucleotide sequence of the PsASY1 gene is shown in SEQ ID NO.1.

[0020] Further preferably, the plant includes tobacco or peony.

[0021] The present invention provides a method for cultivating polyploid plants, comprising the step of overexpressing the expression level of the PsASY1 gene in the plant; the nucleotide sequence of the PASY1 gene is shown in SEQ ID NO.1.

[0022] Further preferably, the plant includes tobacco or peony.

[0023] The present invention provides a method for cultivating sterile plants, comprising the step of overexpressing the expression level of the PsASY1 gene in the plant; the nucleotide sequence of the PsASY1 gene is shown in SEQ ID NO.1.

[0024] Further preferably, the plant includes tobacco or peony.

[0025] The present invention provides a method for improving plant growth traits, comprising the step of overexpressing the expression level of the PsASY1 gene in the plant; the nucleotide sequence of the PsASY1 gene is shown in SEQ ID NO.1; and the growth traits comprise one or more of crown width, leaf width, and plant height.

[0026] Further preferably, the plant includes tobacco or peony.

[0027] The present invention provides a method for cultivating plants with large crowns, large leaves and tall stalks, comprising the step of overexpressing the expression level of the PsASY1 gene in the plant; the nucleotide sequence of the PsASY1 gene is shown in SEQ ID NO.1.

[0028] Further preferably, the plant includes tobacco or peony.

[0029] As an additional solution, the present invention provides the use of the above-mentioned PsASY1 protein, the above-mentioned PsASY1 gene or the above-mentioned biological material in regulating the crown width of plants, thereby achieving the purpose of increasing the crown width of plants by overexpressing the expression level of the PsASY1 gene in plants.

[0030] Further preferably, the plant includes tobacco or peony.

[0031] As an additional solution, the present invention provides a method for cultivating plants with large crown width, comprising the step of overexpressing the expression level of the PsASY1 gene in the plant, wherein the nucleotide sequence of the PsASY1 gene is shown in SEQ ID NO.1.

[0032] Further preferably, the plant includes tobacco or peony.

[0033] As an additional solution, the present invention provides the use of the above-mentioned PsASY1 protein, the above-mentioned PsASY1 gene or the above-mentioned biological material in regulating the size of plant leaves, thereby achieving the purpose of enlarging plant leaves by overexpressing the expression level of the PsASY1 gene in plants.

[0034] Further preferably, the plant includes tobacco or peony.

[0035] As an additional solution, the present invention provides a method for cultivating large-leaf plants, comprising the step of overexpressing the expression level of the PsASY1 gene in the plant, wherein the nucleotide sequence of the PsASY1 gene is shown in SEQ ID NO.1.

[0036] Further preferably, the plant includes tobacco or peony.

[0037] As an additional solution, the present invention provides the use of the above-mentioned PsASY1 protein, the above-mentioned PsASY1 gene or the above-mentioned biological material in regulating plant height, thereby achieving the purpose of increasing plant height by overexpressing the expression level of the PsASY1 gene in the plant.

[0038] Further preferably, the plant includes tobacco or peony.

[0039] As an additional solution, the present invention provides a method for cultivating tall plants, comprising the step of overexpressing the expression level of the PsASY1 gene in the plant, wherein the nucleotide sequence of the PsASY1 gene is shown in SEQ ID NO.1.

[0040] Further preferably, the plant includes tobacco or peony.

[0041] As an additional solution, the present invention provides a method for increasing the anthocyanin content in plants, comprising the step of overexpressing the expression level of the PsASY1 gene in the plant; the nucleotide sequence of the PsASY1 gene is shown in SEQ ID NO.1.

[0042] Further preferably, the plant includes tobacco or peony.

[0043] As an additional solution, the present invention provides a method for cultivating plants with high anthocyanin content, the method comprising the step of overexpressing the expression level of the PsASY1 gene in the plant, the nucleotide sequence of the PsASY1 gene being shown in SEQ ID NO.1.

[0044] Further preferably, the plant includes tobacco or peony.

[0045] As an additional solution, the present invention provides a method for increasing the chlorophyll content of a plant, comprising the step of overexpressing the expression level of the PsASY1 gene in the plant; the nucleotide sequence of the PsASY1 gene is shown in SEQ ID NO.1.

[0046] Further preferably, the plant includes tobacco or peony.

[0047] As an additional solution, the present invention provides a method for cultivating plants with high chlorophyll content, which includes the step of overexpressing the expression level of the PsASY1 gene in the plant, and the nucleotide sequence of the PsASY1 gene is shown in SEQ ID NO.1.

[0048] Further preferably, the plant includes tobacco or peony.

[0049] The present invention discloses the following technical effects:

[0050] The present invention establishes a system for overexpressing the PsASY1 gene in tobacco. The results show that overexpressing the PsASY1 gene can cause abnormal meiosis in peony pollen mother cells and also increase the growth rate of the plant, which is specifically reflected in increasing the plant's crown width, leaf size, and plant height. It can be seen that the PsASY1 protein, PsASY1 gene, or biological materials overexpressing the PsASY1 gene provided by the present invention can be used to regulate plant pollen mother cell meiosis, cultivate polyploid peonies, cultivate sterile peonies, and other aspects such as increasing plant growth rate, increasing plant anthocyanin content, and increasing plant chlorophyll content. The present invention provides new genetic materials for peony molecular breeding, and also provides a theoretical basis and technical reference for breeding work using sexual polyploidization to create peony polyploids. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0052] Figure 1 Agarose gel electrophoresis of the PCR clone of PsASY1 gene;

[0053] Figure 2 Prediction of the hydrophilicity / hydrophobicity (a), signal peptide (b), transmembrane structure (c), secondary (d) and tertiary structure (e) of the PsASY1 gene;

[0054] Figure 3 To compare the PsASY1 gene with the known ASY1 conserved motifs in other species and conduct phylogenetic tree analysis;

[0055] Figure 4 HORMADs family analysis; a is the analysis of conserved motifs of HORMADs family in peony, Arabidopsis and tobacco; b is the phylogenetic tree of HORMADs family in peony, Arabidopsis and tobacco; c is the location analysis of HORMADs family on peony chromosome;

[0056] Figure 5 Figure 1 shows the genetic transformation of tobacco (Nicotiana tabacum) with pSuper1300-PsASY1. a is the co-cultivation stage; bc are the Hyg screening and cultivation stages; de is the rooting induction stage; f is the regenerated plant.

[0057] Figure 6PCR detection of PsASY1 transgenic tobacco; Maeker is a standard protein, Positive is the pSuper1300-PsASY1 recombinant vector, WT is wild-type tobacco, and OE-3, OE-4, and OE-10 are PsASY1 transgenic tobacco.

[0058] Figure 7 is the expression level of PsASY1 gene in transgenic tobacco lines; asterisks indicate significant differences (*p<0.05, **p<0.01, ***p<0.001);

[0059] Figure 8 The phenotypic changes of different transgenic tobacco lines are shown in Figure 2. WT is the wild-type big-leaf tobacco, and OE-3, OE-4, and OE-10 are PsASY1 gene transgenic tobacco lines.

[0060] Figure 9 This is the observation of the normal meiotic process of wild-type tobacco pollen mother cells; a is interphase; b is leptotene; c is pachytene; d is diplotene; e is diakinesis; f is metaphase I; gh is anaphase I; i is telophase I; j is diakinesis II; k is pachytene II; l is metaphase II; m is anaphase II; n is telophase II; o is tetrad; p is mature pollen;

[0061] Figure 10 Abnormal meiotic behavior of tobacco pollen mother cells overexpressing the PsASY1 gene; ac indicates interphase chromosome lagging; d indicates chromosome bridge; ef indicates micronuclei in interphase; g indicates lagging chromosome; h indicates telophase chromosome bridge; ij indicates multiple lagging chromosomes; k indicates vertical spindle; l indicates micronuclei in telophase II; mo indicates triploids and lagging chromosomes; p indicates tetrads and lagging chromosomes; q indicates uneven chromosome segregation; r indicates triploids; s indicates quinads; and t indicates triploids and a micronucleus.

[0062] Figure 11 The results of the investigation on leaf number, crown width, plant height and total anthocyanin content of transgenic tobacco are shown in Figure 2, where a is the number of leaves, b is the crown width, c is the plant height, and d is the total anthocyanin content (*p<0.05, **p<0.01, ***p<0.001).

[0063] Figure 12 are the L* value, a* value and b* value of transgenic tobacco leaves; where a is the L* value; b is the a* value; c is the b* value; Figure 13 The results of the investigation on chlorophyll content in transgenic tobacco leaves are shown in Table 1. a represents chlorophyll a content; b represents chlorophyll b content; and c represents total chlorophyll content (*p<0.05, **p<0.01, ***p<0.001).

[0064] Figure 14 This is the map of the pSuper1300-PsASY1 recombinant vector;

[0065] Figure 15 This is the PCR verification of the pSuper1300-PsASY1 recombinant vector bacterial solution; among them, lanes 1-9 are the bacterial solutions of the pSuper1300-PsASY1 overexpression gene vector;

[0066] Figure 16 Meiotic process of pollen mother cells in 'Fengdanbai' transiently overexpressing pSuper1300; a is interphase; b is leptotene; c is pachytene; de ​​is diplotene; f is diakinesis; g is metaphase I (five neatly arranged rod-shaped bivalents); h is anaphase I; ik is telophase I; l is prophase II; m is metaphase II; n is anaphase II; o is telophase II; p is tetrad stage

[0067] Figure 17 Abnormal meiotic behavior in pollen mother cells of 'Fengdanbai' overexpressing the PsASY1 gene; ad represents a chromosome bridge; eg represents a chromosome fragment in telophase I; h represents a lagging chromosome and a fragment; il represents two chromosome fragments in anaphase I; mn represents multiple chromosome fragments in anaphase I; o represents the coexistence of a chromosome bridge and a fragment; p represents uneven chromosome segregation; q represents triplospores; r represents four nuclei and one micronucleus; and st represents four nuclei and two micronuclei.

[0068] Figure 18 Pollen TTC staining and germination determination of control pollen grains (a) and 2n pollen grains overexpressing PsASY1'Fengdanbai' (b). DETAILED DESCRIPTION

[0069] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0070] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0071] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0072] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0073] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0074] Example 1 Cloning of target gene

[0075] 1. Materials and Methods

[0076] 1.1 Plant materials

[0077] The peony variety is 'Fengdanbai'; the wild tobacco seeds were donated by Shu Qingyan, Professor of the Peony Group, Institute of Botany, Chinese Academy of Sciences.

[0078] 1.2 Cloning of target genes

[0079] (1) cDNA synthesis

[0080] Plant total RNA was extracted using a plant total RNA extraction kit to extract total RNA from stamens of 'Fengdanbai' before and after colchicine treatment. RNA concentration was determined using a Nanodrop 2000 (Thermo) ultraviolet spectrophotometer and RNA quality was ensured by 1% agarose gel electrophoresis. cDNA synthesis was performed according to the instructions of the RTSuperMixforqPCR (+gDNAwiper) kit. The cDNA obtained by the reaction was diluted 3-5 times and used as a template for fluorescent quantitative PCR. 1 μg of P1 RNA was used as a template (the specific treatment process of P1 was: during the meiotic stage of 'Fengdanbai', the peony buds were treated with colchicine by injection (the concentration of colchicine was 0.25%). The buds between the small bell stage and the large bell stage were selected for treatment, and the petals were pierced with a disposable syringe, and the colchicine solution was injected into the center of the bud until the sepals exuded liquid). cDNA was synthesized by reverse transcription according to the instructions of the III1st Strand cDNA Synthesis Kit (+ gDNAwiper). The specific operation process is as follows: prepare 16 μL of reaction system (4× gDNAwiper Mix 4 μL; RNA 1 μg; ddH2O to 16 μL) on ice, mix evenly by pipetting, and incubate at 42°C for 2 min; then add 5× HiScript III qRT Super Mix and gently pipette up and down to mix. After a brief centrifugation, collect the droplets on the tube wall at the bottom of the tube and place it in the PCR instrument. The reaction temperature and time are shown in Table 1.

[0081] Table 1 Reaction temperature and time

[0082] temperature time 37℃ 15min 85℃ 5sec

[0083] (2) Cloning of gene coding regions and PCR amplification of target genes

[0084]

[0085] Table 2 Primer sequences used for gene cloning

[0086]

[0087] Using cDNA as a template, PCR amplification was performed. PCR amplification of the target gene was performed according to the instructions of 2×Taq Plus Master Mix II (DyePlus), and the program steps in Table 3 were run in a PCR instrument (the number of cycles was 30).

[0088] Table 3 Procedure steps

[0089] temperature time 95℃ 3min 95℃ 15sec 60℃ 20sec 72℃ 60sec / kb 72℃ 5min

[0090] (4) Purification and recovery of DNA gel products

[0091] After the reaction, the obtained PCR products were subjected to agarose gel electrophoresis to detect the specificity of the PCR products. The obtained gel products were placed under a UV gel imager. Bands of the expected length were observed. The gel was cut under UV light and stored in a 1.5 mL centrifuge tube. The DNA was recovered according to the DNA gel recovery kit FastPure Gel DNA Extraction Mini Kit (Novozymes) instructions for purification and recovery of target DNA.

[0092] (5) Connecting T vector

[0093] According to the instructions of the 5-min TA / Blunt-Zero Cloning Kit, a 5 μL reaction system was prepared on ice, and the recovered product was ligated to the vector by a blunt-end Blunt-Zero cloning reaction for 5 min.

[0094] (6) Transformation into the competent colon

[0095] The ligation product was transferred to the E. coli Fast-T1 Competent Cell. According to the instructions, the target DNA was added to the competent cell, and it was placed on ice for 30 minutes. After that, it was heat-shocked at 42℃ for 30 seconds and then quickly placed on ice for 2 minutes.

[0096] Add 900 μL of antibiotic-free LB medium to the centrifuge tube, mix thoroughly, and shake at 37°C at 200 rpm for 1 hour. Centrifuge at 5000 rpm for 5 minutes, and remove 800 μL of the supernatant. Resuspend the cells in the remaining medium by pipetting, and gently spread the suspension evenly on a plate containing LB solid medium supplemented with the antibiotic Amp (100 μg / mL). Once the suspension has been completely absorbed by the medium, seal the plate and place it upside down in a 37°C incubator for overnight incubation.

[0097] (7) Bacterial liquid sequencing verification

[0098] After dispersed and large single colonies grew on the plate, single clones were picked and transferred to Luria-Balani Medium (LB) liquid culture medium containing Amp (100 mg / mL). The culture was shaken at 200 rpm and 37°C for 5 h before sequencing (Qingke). The sequencing results were aligned with PsASY1 using DNAMAN8.0 software.

[0099] (8) Plasmid extraction

[0100] The correct sequencing bacteria were shaken at 200 rpm and 37°C overnight. Plasmids were extracted using a high-purity plasmid DNA miniprep kit (Novagen), and after confirming the quality and concentration, they were stored at -20°C until use.

[0101] 1.3 Bioinformatics analysis

[0102] Bioinformatics analysis was performed to link the molecular characteristics of the target gene's coding region sequence with its function. This included predictions of amino acid physicochemical properties, hydrophilicity, signal peptide prediction, conserved domain analysis, and secondary and tertiary structure predictions of the encoded product. This revealed the relationship between the molecular biological characteristics of the gene's coding region and its function. Based on this, homologous amino acid sequences from various plants were compared using software such as DNAMAN and MEGA, and phylogenetic trees were constructed for different species. The analysis sites are shown in Table 4.

[0103] Table 4 Online analysis software analysis link

[0104] database Function Website ProtParam Protein physicochemical properties http: / / web.expasy.org / protparam / ProtScale Protein hydrophobicity analysis https: / / web.expasy.org / protscale / TMHMM Protein transmembrane domain http: / / www.cbs.dtu.dk / services / TMHMM / SignalP Predicted signal peptide http: / / www.cbs.dtu.dk / services / SignalP SOPMA Protein secondary structure prediction http: / / pbil.ibcp.fr SWISS-MODEL Protein tertiary structure prediction http: / / swissmodel.expasy.org / NCBI Conserved domain analysis http: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi

[0105] 1.4 Construction of overexpression vector

[0106] (1) Preparation of linearized vector

[0107] Find the appropriate restriction enzyme sites on the Super1300 vector and double-digest the Super1300 vector with restriction enzymes Apa I and Kpn I. Set up the reaction system according to Table 5.

[0108] Table 5 Reaction system

[0109] Reagents volume Super1300 plasmid 10 μL ApaⅠ 1 μL KpN 1 μL 10×Mbuffer 5μL <![CDATA[ddH2O]]> 33μL

[0110] Digest the enzyme in a 37°C thermostat for 5 minutes and immediately place on ice to obtain the linearized Super1300 vector. Verify the success of the digestion by 1% agarose gel electrophoresis and purify and recover the DNA with a DNA gel recovery kit.

[0111] (2) Target gene connects homology arms

[0112] Primers were designed online using the (CE) Primer-BLAST website (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ). Conditions were set to automatically generate amplification primers for the insert fragment (SEQ ID NO. 2 and SEQ ID NO. 3 in Table 2). The insert fragment was amplified by PCR using a high-fidelity enzyme. After amplification and electrophoresis, the fragment was recovered using a DNA gel recovery kit.

[0113] (3) Connection and transformation of target gene and linear vector

[0114] Prepare the homologous recombination reaction system on ice as shown in Table 6.

[0115] Table 6 Homologous recombination reaction system

[0116] Reagents volume Linearized vector 2μL Insert fragment (after dilution) 1 μL 2×ClonExpressMix 5μL <![CDATA[ddH2O]]> 2μL

[0117] Fragment recombination reaction, 50℃, 10min; then reduce to 4℃ or immediately cool on ice.

[0118] The ligated product from the recombination reaction was used as a template for efficient transformation using the Fast-T1 chemically competent cell kit (Novozymes). The specific culture method was as follows: the ligated product was transferred to the competent E. coli Fast-T1, and then the competent cells were plated on LB solid medium containing Amp (100 mg / mL). The sealed plate was placed upright in a 37°C incubator for 10 minutes. After the bacterial solution was completely absorbed by the medium, the plate was inverted and incubated overnight. After the plate grew dispersed and large single colonies, single clones were picked and transferred to Luria-Balani medium (LB) liquid medium containing Amp (100 μg / mL) and shaken gently for 5 hours before sequencing.

[0119] (4) Transformation of Agrobacterium tumefaciens GV3101

[0120] After overnight shaking of the correctly sequenced colonies, plasmids were extracted. Transform Agrobacterium tumefaciens GV3101 competent cells according to the GV3101 Chemically Competent Cell instructions. Add 0.01-1 μg of plasmid per 100 μL of competent cells, shake thoroughly, and incubate on ice for 5 minutes, in liquid nitrogen for 5 minutes, in a 37°C water bath for 5 minutes, and in an ice bath for 5 minutes.

[0121] Add 700 μL of antibiotic-free LB medium and shake at 200 rpm at 28°C for 2-3 hours. Centrifuge at 6000 rpm for 1 minute, and remove 600 μL of supernatant. Resuspend the cells in the remaining medium by pipetting. Then, use a sterile spreader to spread onto a plate containing Kan (50 μg / mL) and Rif (20 μg / mL) antibiotics. Incubate the plate in an inverted position at 28°C for 2-3 days.

[0122] (5) Identification of recombinant vector plasmid.

[0123] The pSuper1300-PsASY1 recombinant plasmid was tested by PCR. A dispersed and relatively full single Agrobacterium colony was picked up with a sterilized pipette tip, and the pipette tip was injected into LB liquid culture medium containing 100 μg / mL Kan and 50 μg / mL Rif, and gently shaken to mix. After being cultured at 28°C and 200 rpm for about 5 hours, a PCR test of the bacterial solution was performed to detect positive recombinant plasmids. The bacterial solution with clear bands was selected to extract the plasmid, and the solution was stored at -20°C for later use. The correct bacterial solution was preserved at -80°C with 50% glycerol.

[0124] 1.5 Agrobacterium tumefaciens-mediated transformation of tobacco and screening of transgenic lines

[0125] The Agrobacterium tumefaciens-mediated leaf disc method for tobacco leaf transformation is as follows:

[0126] (1) Take the activated bacterial solution of positive Agrobacterium tumefaciens at a ratio of 1:100 and add it to 25 mL of LB liquid medium containing 100 μg / mL Kan and 50 μg / mL R-Lif. Cultivate at 28°C and 180 rpm with shaking until the OD 600 =0.6;

[0127] (2) After centrifugation at 5000 rpm for 10 min at room temperature, the bacterial pellet was collected and resuspended in MS0 liquid medium (MS medium + 30 g / L sucrose, pH = 5.8) to obtain MS0 resuspension for leaf infection;

[0128] (3) Take the leaves of sterile tobacco seedlings that have grown well and cut them into 1cm 2 Small pieces on the left and right;

[0129] (4) Place a small piece of tobacco leaf in the MS0 resuspension solution, with the back of the leaf facing down, and gently shake it to allow the bacterial solution to completely contact the leaf edge wound. Inoculate for 10-15 minutes.

[0130] (5) Place the infected leaves on sterile filter paper to absorb any bacterial liquid remaining on the surface;

[0131] (6) The leaves were inoculated onto a solid co-culture medium (MS medium + 20 g / L sucrose + 7.5 g / L agar, pH = 5.8) and co-cultured at 28°C in the dark for 3 days;

[0132] (7) After the co-cultivation, 500 mg / L cefotaxime (Cef) was added to double-distilled H2O (ddH2O) to clean the Agrobacterium remaining on the leaf surface. After drying with sterile filter paper, the leaves were transferred to the differentiation induction and screening medium MS1 (MS medium + 1.0 mg / L 6-BA + 0.2 mg / L NAA + 20 g / L sucrose + 7.5 g / L agar + 20 mg / L Hyg + 200 mg / L Cef, pH = 5.8) containing hygromycin (Hyg), and cultured in an artificial incubator. The MS1 medium was replaced every 10 days.

[0133] (8) When the differentiated seedlings grew to 2-3 cm, they were separated from the callus and transferred to MS2 rooting medium (1 / 2 MS medium + 20 g / L sucrose + 7.5 g / L agar + 100 mg / L Kan + 200 mg / L Cef, pH = 5.8) to induce rooting.

[0134] (9) After the root system is well developed, wash the agar on the roots of the tobacco tissue culture seedlings with sterile water. Plant them in a cultivation medium with a ratio of peat soil to vermiculite of 3:1 (V / V). After culturing for 1 week, remove the film and culture normally.

[0135] (10) After four weeks of transplantation, three young leaves were collected from each individual plant and DNA was extracted from tobacco leaves using a plant genomic DNA extraction kit (Novagen). PCR amplification was performed using DNA as a template and pSuper1300-F / R (SEQ ID NO. 6 and SEQ ID NO. 7) as primers. Transgenic tobacco lines with clear bands of the correct size were identified. Meiosis of pollen mother cells and vegetative growth indicators were then observed.

[0136] 1.6pSuper1300-PsASY1 transient overexpression in 'Fengdanbai'

[0137] (1) Set pSuper1300-PsASY1 as the target gene and the corresponding pSuper1300 empty vector as the control;

[0138] (2) 1 mL each of Agrobacterium GV3101 containing pSuper1300 and pSuper1300-PsASY1 was inoculated into 60 mL of LB liquid medium (containing 50 mg / L Rif, 100 mg / L Kan, 50 mg / L gentamicin (Gen), 200 mmol / L acetosyringone (As), and 10 mol / L LMES) and cultured at 200 rpm / min and 28°C until the OD values ​​of different treatments were reached. 600 value.

[0139] (3) Determine the bacterial solution concentration OD 600 =0.8-1.0, centrifuge at 4000 rpm at room temperature for 10 min, collect the bacteria, and discard the supernatant.

[0140] (4) Resuspend in infection buffer (10 mM MgCl2, 20 mM AAS, 10 mM MES (pH = 5.6);

[0141] (5) Use infection solution to adjust the OD of the resuspended bacterial solution 600 The value is consistent with the set bacterial solution OD 600 The values ​​are the same, and the OD values ​​of pSuper1300 and pSuper1300-PsASY1 bacterial solutions are 600 The values ​​were the same; dark treatment at room temperature for 4 h;

[0142] The bacterial liquid was infected by injection during the bud stage of peony. A 1 ml sterile syringe was used to pierce the sepals and petals from the side of the bud and inject the infection liquid into the center of the bud until the sepals exuded liquid. Photos were taken and samples were taken 3d, 5d, 7d, and 12d after injection to observe the meiotic process of pollen mother cells.

[0143] 1.7 Cytological Observation of Pollen Mother Cells

[0144] Take flower buds (1-1.2 cm in diameter) in the meiotic stage, remove the petals, and fix them in Carnoy's fixative (anhydrous ethanol: glacial acetic acid = 3:1, v / v). After vacuuming for 30 minutes, place them in a 4°C refrigerator. After 24 hours, rinse the material with 90% ethanol 2-3 times and 70% ethanol once. Then transfer it to 70% ethanol and store it in a 4°C refrigerator for long-term use. During observation, remove 1-3 anthers and place them on a glass slide. Use a glass rod to squeeze the anthers to release pollen mother cells. After adding 1-2 drops of Carbofuchsin dye, squeeze them with a dissecting needle. After 3-5 minutes, pick out impurities, cover with a coverslip, and press the coverslip again with your thumb to disperse the cells. Make a temporary press slide. Observe the morphology of chromosomes at various stages of meiosis under a microscope and take photos.

[0145] 1.82n pollen statistics and pollen viability test

[0146] Peony buds were collected 2 days before opening, petals removed, and placed on sulphite paper. The buds were then naturally dried in the shade for 48 hours. Mature pollen was collected, dried on silica gel, and stored at -20°C. The pollen was stirred and imbibed in an isotonic solution of 0.9 M mannitol (CPW) for approximately 2 hours. The pollen was observed and photographed using a light microscope (Leica DM500). The ratio of 2n pollen was calculated based on the diameter of the 2n pollen grain being at least 1.3 times the diameter of a normal pollen grain.

[0147] (1) TTC staining

[0148] Prepare a 0.5% TTC staining solution using phosphate buffer (0.83g Na₂HPO₄·2H₂O and 0.27g KH₂PO₄ dissolved in 100ml distilled water, pH = 7.2). Place a small amount of pollen on a glass slide and add 1-2 drops of the staining solution. Stir thoroughly with a dissecting needle. Cover with a coverslip and incubate at 35°C for at least 2 hours until the number of pollen staining stops increasing. Observe under a light microscope. Pollen that stains red is considered viable, while pollen that does not stain red is considered dead. Randomly select 10 fields of view and photograph them to record the staining rate. Staining rate = number of pollen staining red / total number of pollen counted × 100%.

[0149] (2) In vitro germination method

[0150] Prepare an agar medium with a 10% sucrose concentration. Use a pipette to draw a small amount of the medium into the groove of a concave glass slide and let it sit for about 15 minutes until it solidifies. Evenly spread a small amount of pollen on the medium. Place the prepared slide in a 22°C incubator. Observe germination under a microscope and calculate the germination rate.

[0151] 2. Results

[0152] 2.1 Cloning of target genes

[0153] A 6282bp nucleotide sequence was obtained from the transcriptome data Unigene (pos.gene61825). A known genome query revealed that its CDS length was 1971bp (SEQ ID NO.1), named PsASY1, encoding 656 amino acids. Primers PsASY1-F / R (SEQ ID NO.2 and SEQ ID NO.3) were designed at both ends of the sequence. PCR amplification was performed using the cDNA of the reversed RNA of the stamens of 'Fengdanbai' as a template. Electrophoresis of the PCR product revealed a single specific band near 1971bp ( Figure 1A multiple sequence alignment of the sequencing results with the transcriptome data showed that the obtained gene sequence was consistent with the CDS region of the PsASY1 gene in the transcriptome. A blast analysis of this sequence on NCBI revealed high similarity with other ASY1 plant proteins registered in GeneBank.

[0154] 2.2 Bioinformatics analysis of target genes

[0155] Bioinformatics analysis was performed on the coding region sequence of the PsASY1 gene, including prediction analysis of the physicochemical properties of amino acids, prediction analysis of the hydrophobicity and hydrophilicity of amino acids, and prediction analysis of the secondary and tertiary structures of the encoded product.

[0156] The protein physicochemical properties were predicted using the ProtParam online analysis tool. The CDS region of the PsASY1 gene is 1971 bp long and encodes 656 amino acids (PsASY1 protein, amino acid sequence: MHFVTHQCSLVSLNFKFQTKT *, SEQ ID NO. 8), mainly composed of alanine (32.3%), glycine (24.1%), threonine (25.6%), and cysteine ​​(18.0%). The relative molecular mass of the encoded protein is 159966.11 Da, and the molecular formula is C 5942 H 9915 N 1971 O 2476 S 355 The total number of atoms is 20,659, the theoretical isoelectric point PI value is 4.98; the instability index is 35.20, so it is a stable protein; the average hydrophobicity index is 0.757.

[0157] ProtScale was used to analyze the hydrophilicity and hydrophobicity of proteins ( Figure 2 In a), the maximum hydrophobicity of PsASY1 protein is 2.25, and the minimum is -0.65, which indicates that it is a hydrophobic protein. SignalP was used to predict the signal peptide ( Figure 2 The results show that PsASY1 protein has no signal peptide and is a non-secretory protein. The protein transmembrane domain was predicted using the TMHMM online tool ( Figure 2 c), the results showed that PsASY1 protein did not have a transmembrane domain and was not a membrane protein. PSIPRED and SWISS-MODEL were used to predict the secondary and tertiary structures of the protein; the results showed that the structural elements of PsASY1 protein included α-helix (29.11%), random coil (57.93%), extended chain (12.96%) ( Figure 2 in de).

[0158] Other plant ASY1 protein sequences with sequence homology of more than 80% were downloaded from NCBI. The amino acid sequence encoded by the PsASY1 gene was aligned with the amino acid sequences of 26 species, including Actinidia chinensis, Actinidia eriantha, Camellia sinensis, Camellia lanceoleosa, Camellia sinensis, Vitis vinifera, and Prunus persica, using TBtools. The conserved Motif DNA binding sites were compared using MEME Suite. It was found that the conserved motifs of PsASY1 protein were similar to those of other species ( Figure 3 To investigate the homologous evolutionary relationship between the PsASY1 protein and the amino acid sequences of other species, the ASY1 protein sequences of other species were combined with the PsASY1 protein using MEGA to construct a phylogenetic tree. The results showed that the PsASY1 protein is highly related to species such as Actinidia chinensis, Ipomoea nil, and Camellia sinensis, with the proteins clustering closer together in the phylogenetic tree.

[0159] Existing studies have shown that the ASY1 gene actually originates from the HORMADs protein family (HORMA domain-containing). Combined with genomic data, all genes of this family in three species, peony, Arabidopsis, and tobacco, were analyzed ( Figure 4The number of genes was small. The key gene PsASY1 (Pos.gene61825) selected in this example was closely related to Arabidopsis AT1G67370.1, tobacco Nta08g04630, Nta03g10930, Nta05g24160, and Nta06g29970 in the phylogenetic tree. Correspondingly, their conserved Motif DNA binding site sequences showed a similar pattern. The location analysis of the peony HORMADs family genes on chromosomes found that the 15 genes belonging to the HORMADs family screened from the peony genome were arranged on different chromosomes, among which PsASY1 was located on chromosome 03 ( Figure 4 c).

[0160] 2.3 Identification of target gene overexpression vector

[0161] The CDS region fragment of the PsASY1 gene was ligated with the plant expression vector pSuper1300 to construct the pSuper1300-PsASY1 recombinant vector ( Figure 14 ). PCR verification was performed using the pSuper1300-PsASY1 recombinant vector as a template and pSuper1300-PsASY1-F and pSuper1300-PsASY1-R (SEQ ID NO.4 and SEQ ID NO.5) as primers. The electrophoresis results of the PCR product showed that there was a single correct band at approximately 1971 bp ( Figure 15 Sequencing analysis of the positive plasmids showed that the sequence identity was over 99% with the original sequence, and the pSuper1300-PsASY1 overexpression recombinant vector was successfully constructed.

[0162] 2.4 Transformation of tobacco

[0163] The PsASY1 gene was transferred into wild-type tobacco using the leaf disc method mediated by Agrobacterium tumefaciens. After co-cultivation, screening culture, bud induction, rooting, and transplanting, Hyg-resistant regenerated seedlings were obtained. Figure 5 ).

[0164] RNA of transgenic lines was extracted and identified by real-time fluorescence quantitative PCR to verify the relative expression of genes ( Figure 6 The results showed that the relative expression of the PsASY1 gene in the transgenic lines was higher than that in the wild-type tobacco, further verifying that the tobacco plants were positive transgenic plants. The relative expression levels of the third line (OE-3), the fourth line (OE-4), and the tenth line (OE-10) were 717 times, 1768 times, and 708 times that of the wild-type plants, respectively.

[0165] 2.5 Fluorescence quantitative PCR detection, pollen mother cell observation and phenotypic observation of transgenic tobacco

[0166] The DNA of the PsASY1 transgenic tobacco was extracted and tested by PCR. The wild-type tobacco DNA was used as the negative control (CK) and the pSuper1300-PsASY1 recombinant vector was used as the positive control. The PsASY1 transgenic tobacco could amplify specific bands with the expected size ( Figure 7 ), while no fragment was amplified in WT, which preliminarily indicated that the exogenous PsASY1 gene was normally expressed in the transgenic tobacco line. RNA was extracted from the transgenic tobacco line for real-time fluorescence quantitative PCR detection to verify the relative expression of the gene. The results showed that the relative expression of the PsASY1 gene was significantly higher than that in wild-type tobacco, which further verified that the tobacco plant was a positive transgenic plant ( Figure 7 The results of normal meiosis observation of tobacco pollen mother cells are as follows. Figure 9 The abnormal meiotic behavior of tobacco pollen mother cells overexpressing PsASY1 gene is shown in Figure 10 As shown. The results showed that the interphase of tobacco pollen mother cells that overexpressed the PsASY1 gene was delayed, the univalents and chromosomes were lagging, the triplets and lagging chromosomes coexisted, the telophase II chromosomes were lagging, and the chromosomes were unevenly separated, which eventually led to the formation of 2n pollen grains. It can be seen that overexpression of the PsASY1 gene can promote the formation of 2n pollen grains. Based on this, the formation of 2n pollen grains can cultivate polyploid plants and sterile plants. The L*a*b* values ​​of the obtained PsASY1 transgenic tobacco were measured, and the results showed that the L* value of the first line of transgenic tobacco plants was reduced, which was significantly different from the control group; the a* and b* values ​​of the first and third lines of transgenic tobacco plants were reduced, which were significantly different from the control group ( Figure 12 The chlorophyll content of the obtained PsASY1 transgenic tobacco was measured. The chlorophyll a content of the first and second lines of transgenic tobacco plants increased, which was significantly different from the control group ( Figure 13 ) The anthocyanin content of the obtained PsASY1 transgenic tobacco was determined. The anthocyanin content of the first, second and third lines of transgenic tobacco plants increased, and the difference was significant compared with the control group ( Figure 11 D) in the figure, the expression level of the PsASY1 overexpression recombinant vector increased, and meiosis was disrupted, which further indicates that PsASY1 may be the gene that affects meiotic abnormalities in peony, and its enhanced function may lead to the formation of 2n pollen. The phenotypic growth rate of tobacco plants is faster than that of wild-type plants, with a significant difference. 15 days after transplantation, it was observed that although the number of leaves of transgenic tobacco plants was similar to that of wild-type tobacco plants, the crown width, leaves, and plant height of transgenic tobacco plants were significantly larger than those of wild-type plants ( Figure 8 and Figure 11AC in).

[0167] 2.6 Effects of transient overexpression of PsASY1 on the meiotic process of pollen mother cells in ‘Fengdanbai’

[0168] The anthers in the flower buds collected within 3-12 days of injection were pressed to observe the meiotic process of pollen mother cells. The results of normal meiosis observation were as follows: Figure 16 Compared with the normal meiotic process, abnormal behaviors such as chromosome bridges, uneven chromosome separation and chromosome fragmentation are shown ( Figure 17 ), which eventually leads to the formation of 2n pollen grains. This shows that overexpression of the PsASY1 gene can promote the formation of 2n pollen grains in 'Fengdanbai'. The pollen viability of the control group and those expressing PsASY1 was statistically analyzed, and the results showed that the total staining rate of pollen overexpressing PsASY1 was about 84.67%, and the total staining rate of the control group was about 81.98%; in the 'Fengdanbai' that overexpressed PsASY1, the proportion of 2n pollen was about 6.23%, the 2n staining rate was about 71.2%, and the 1n staining rate was about 87.14%. This shows that there is no significant difference in pollen vitality between the overexpression of PsASY1 and the control group ( Figure 18 ).

[0169] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A PsASY1 protein, characterized in that The amino acid sequence of the PsASY1 protein is shown in SEQ ID NO.

8.

2. A PsASY1 gene encoding the PsASY1 protein according to claim 1, characterized in that: The nucleotide sequence of the PsASY1 gene is shown in SEQ ID NO.

1.

3. A biological material containing the PsASY1 gene according to claim 2.

4. Use of the PsASY1 protein according to claim 1, the PsASY1 gene according to claim 2, or the biomaterial according to claim 3 in any one or more of the following: (1) Regulating meiosis of plant pollen mother cells (2) regulating the formation of 2n pollen grains in plants; (3) Cultivating polyploid plants; (4) Cultivating sterile plants; (5) regulating plant growth traits; the growth traits include one or more of crown width, leaf length, and plant height; (6) Regulating the content of anthocyanins in plants; (7) Regulate the chlorophyll content of plants.

5. The use according to claim 4, characterized in that By overexpressing the PsASY1 gene in plants, the purpose of promoting abnormal meiotic behavior in plant pollen mother cells, promoting the formation of plant 2n pollen grains, cultivating polyploid plants, cultivating sterile plants, improving plant growth traits, increasing plant anthocyanin content and increasing plant chlorophyll content is achieved.

6. A method for promoting the formation of 2n pollen grains in plants, characterized in that: The method comprises the step of overexpressing the expression level of the PsASY1 gene in the plant; the nucleotide sequence of the PsASY1 gene is shown in SEQ ID NO.

1.

7. A method for cultivating polyploid plants, characterized in that: The method comprises the step of overexpressing the expression level of the PsASY1 gene in the plant; the nucleotide sequence of the PsASY1 gene is shown in SEQ ID NO.

1.

8. A method for cultivating sterile plants, characterized in that: The method comprises the step of overexpressing the expression level of the PsASY1 gene in the plant; the nucleotide sequence of the PsASY1 gene is shown in SEQ ID NO.

1.

9. A method for improving plant growth traits, characterized in that: The method comprises the step of overexpressing the expression level of the PsASY1 gene in the plant; the nucleotide sequence of the PsASY1 gene is shown in SEQ ID NO.1; and the growth traits comprise one or more of crown width, leaf length and plant height.

10. A method for cultivating plants with large crowns, large leaves and tall stems, characterized in that: The method comprises the step of overexpressing the expression level of the PsASY1 gene in the plant; the nucleotide sequence of the PsASY1 gene is shown in SEQ ID NO.1.

Images