Application of psasy1 gene in regulating abnormal behavior of pollen mother cell meiosis and regulating plant growth rate

Abstract

The present application relates to the technical field of genetic engineering, in particular to the application of PsASY1 gene in regulating abnormal behavior of plant pollen mother cell meiosis and regulating plant growth speed. The present application establishes a system of overexpressing PsASY1 gene in tobacco, and the results show that overexpression of PsASY1 gene can cause abnormal pollen mother cell meiosis of tree peony, and also can improve the growth speed of plants, which is embodied in improving the crown width, leaf size and plant height of plants. It can be seen that the PsASY1 protein, PsASY1 gene or biological material overexpressing PsASY1 gene provided by the present application can be used in regulating plant pollen mother cell meiosis, cultivating polyploid tree peony, cultivating sterile tree peony and the like. The present application provides new genetic material for tree peony molecular breeding, and also provides theoretical basis and technical reference for creating tree peony polyploid breeding by using sexual polyploidization approach.

Description

Technical Field

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

[0002] Peony (Paeonia suffruticosa), originating in China, is a perennial deciduous flowering shrub belonging to the genus Paeonia in the family Paeoniaceae. It is an important ornamental, medicinal, and emerging oilseed crop in my country. Peonies are widely distributed in my country and are divided into different cultivar groups based on cultivation regions and wild origins: the Central Plains group, the Jiangnan group, the Northwest group, and the Southwest group. Long-standing cultivation evolution and modern breeding techniques have led to an increasingly rich variety of peony varieties; currently, there are over 3,000 varieties worldwide, of which nearly 2,000 are found in China.

[0003] In the breeding of ornamental plant varieties, polyploids have attracted widespread attention from breeders due to their advantages such as larger floral organs, more and thicker petals, more vibrant colors, delayed flowering in some species, and stronger resistance to adverse conditions. Peonies have large and few chromosomes, making them suitable as material for research on chromosomal abnormalities or mutations. Wild species of the peony section are diploid with a basic chromosome number of 5. All peony species have the same karyotype, with a chromosome number of 2n = 2x = 10. Current research on peony meiosis mainly focuses on observing abnormal meiotic behavior. Meanwhile, there is little research on peony growth traits. Therefore, it is necessary to explore the relationship between genes in peonies and meiotic behavior and its growth traits. Summary of the Invention

[0004] The purpose of this invention is to provide the 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 prior art.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] The present invention provides a PsASY1 protein, the amino acid sequence of which 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 which is shown in SEQ ID NO.1.

[0008] This invention provides a biological material containing the aforementioned PsASY1 gene.

[0009] This invention provides the use of the above-described PsASY1 protein, the above-described PsASY1 gene, or the above-described biological material in any one or more of the following:

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

[0011] (2) Regulates 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 the following: crown width, leaves, and plant height;

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

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

[0017] Preferably, by overexpressing the PsASY1 gene in plants, the aim is to promote the formation of 2n pollen grains, cultivate polyploid plants, cultivate sterile plants, improve plant growth traits, increase the content of anthocyanins and chlorophyll in plants.

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

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

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

[0021] This invention provides a method for cultivating polyploid plants, including 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] More preferably, the plant includes tobacco or peony.

[0023] The present invention provides a method for cultivating sterile plants, including 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] More preferably, the plant includes tobacco or peony.

[0025] This invention provides a method for improving plant growth traits, the method 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; the growth traits include one or more of crown width, leaf height and plant height.

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

[0027] This invention provides a method for cultivating plants with large canopies, large leaves, and tall stems, the method 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] More preferably, the plant includes tobacco or peony.

[0029] As an additional option, the present invention provides the application of the above-mentioned PsASY1 protein, the above-mentioned PsASY1 gene, or the above-mentioned biological materials in regulating plant crown width, thereby increasing the plant crown width by overexpressing the expression level of the PsASY1 gene in plants.

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

[0031] As an optional addition, the present invention provides a method for cultivating large-canopy plants, 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.

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

[0033] As an additional option, the present invention provides the application of the above-mentioned PsASY1 protein, the above-mentioned PsASY1 gene, or the above-mentioned biological materials in regulating plant leaf size, thereby increasing the plant leaf size by overexpressing the expression level of the PsASY1 gene in the plant.

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

[0035] As an optional addition, the present invention provides a method for cultivating large-leaved plants, 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.

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

[0037] As an additional option, the present invention provides the application of the above-mentioned PsASY1 protein, the above-mentioned PsASY1 gene, or the above-mentioned biological materials in regulating plant height, thereby increasing plant height by overexpressing the expression level of the PsASY1 gene in plants.

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

[0039] As an optional addition, the present invention provides a method for cultivating tall plants, 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.

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

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

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

[0043] As an optional addition, 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] More preferably, the plant includes tobacco or peony.

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

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

[0047] As an optional addition, the present invention provides a method for cultivating plants with high chlorophyll 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.

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

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

[0050] This invention established a system for overexpressing the PsASY1 gene in tobacco. Results showed that overexpression of the PsASY1 gene led to abnormal meiosis in peony pollen mother cells and increased plant growth rate, specifically manifested in increased crown width, leaf size, and plant height. Therefore, the PsASY1 protein, PsASY1 gene, or biomaterials overexpressing the PsASY1 gene provided by this invention can be used to regulate meiosis in plant pollen mother cells, cultivate polyploid peonies, cultivate sterile peonies, and improve plant growth rate, anthocyanin content, and chlorophyll content. This invention provides new gene materials for peony molecular breeding and also provides a theoretical basis and technical reference for breeding work to create polyploid peonies using sexual polyploidization. Attached Figure Description

[0051] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

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

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

[0054] Figure 3 Alignment and phylogenetic analysis of the conserved motifs of PsASY1 gene with those of known ASY1 in other species;

[0055] Figure 4 Analysis of the HORMADs family; where a is the analysis of conserved motifs of the HORMADs family in Paeonia lactiflora, Arabidopsis thaliana and tobacco; b is the phylogenetic tree of the HORMADs family in Paeonia lactiflora, Arabidopsis thaliana and tobacco; c is the localization analysis of the HORMADs family on the chromosome of Paeonia lactiflora.

[0056] Figure 5 pSuper1300-PsASY1 genetically transformed tobacco (Nicotiana tabacum); where a represents the co-culture stage; bc represents the Hyg selection culture stage; de ​​represents the rooting induction stage; and f represents the regenerated plant.

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

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

[0059] Figure 8 Phenotypic changes in different transgenic tobacco lines; among them, WT is wild-type large-leaf tobacco, and OE-3, OE-4 and OE-10 are transgenic tobaccos positive for the PsASY1 gene;

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

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

[0062] Figure 11 The results of the survey on the number of leaves, crown width, plant height, and total anthocyanin content of transgenic tobacco are shown below. In this paper, a represents the number of leaves; b represents the crown width; c represents the plant height; and d represents the total anthocyanin content (*p<0.05, **p<0.01, ***p<0.001).

[0063] Figure 12 The values ​​of L*, a*, and b* for transgenic tobacco leaves are: a = L*, b = a*, and c = b*. Figure 13 The results of the survey on chlorophyll content in transgenic tobacco leaves; where a is chlorophyll a content; b is chlorophyll b content; c is total chlorophyll content (*p<0.05, **p<0.01, ***p<0.001);

[0064] Figure 14 The pSuper1300-PsASY1 recombinant vector spectrum;

[0065] Figure 15 PCR verification of the pSuper1300-PsASY1 recombinant vector bacterial culture; lanes 1-9 are bacterial cultures of the pSuper1300-PsASY1 overexpression gene vector.

[0066] Figure 16 This describes the meiotic process of pollen mother cells in 'Fengdanbai' cultivar transiently overexpressed with pSuper1300; where a represents interphase; b represents leptotene; c represents pachytene; de ​​represents diplotene; f represents diakinesis; g represents metaphase I (5 neatly arranged rod-shaped bivalents); h represents anaphase I; ik represents telophase I; l represents prophase II; m represents metaphase II; n represents anaphase II; o represents telophase II; and p represents tetrad stage.

[0067] Figure 17 Abnormal meiotic behavior of 'Fengdanbai' pollen mother cells overexpressing the PsASY1 gene; where ad represents chromosome bridge; eg represents chromosome fragments in telophase I; h represents lagging chromosomes and fragments; il represents two chromosome fragments in anaphase I; mn represents multiple chromosome fragments in anaphase I; o represents the simultaneous presence of chromosome bridges and fragments; p represents uneven chromosome segregation; q represents triterpenoids; r represents 4 nuclei and 1 micronucleus; st represents 4 nuclei and 2 micronuclei.

[0068] Figure 18 TTC staining and germination status of pollen grains (a) and 2n pollen grains (b) overexpressing PsASY1'Fengdanbai' were determined. Detailed Implementation

[0069] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of 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 terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0071] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0072] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

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

[0074] Example 1: Cloning of the target gene

[0075] 1. Materials and Methods

[0076] 1.1 Plant materials

[0077] The peony variety is 'Fengdanbai'; the wild-type tobacco seeds were a gift from Professor Shu Qingyan of the Peony Group at the Institute of Botany, Chinese Academy of Sciences.

[0078] 1.2 Cloning of the target gene

[0079] (1) cDNA synthesis

[0080] Total RNA was extracted from the stamens of 'Fengdanbai' variegated plant before and after colchicine treatment using a plant total RNA extraction kit. RNA concentration was determined using a Nanodrop 2000 (Thermo) ultraviolet spectrophotometer, and RNA quality was ensured by 1% agarose gel electrophoresis. (Refer to...) cDNA synthesis was performed according to the RTSuperMix for qPCR (+gDNAwiper) kit instructions. The resulting cDNA was diluted 3-5 times and used as a template for quantitative real-time PCR. 1 μg of P1 RNA was used as a template (the specific treatment procedure for P1 was as follows: during the meiotic stage of 'Fengdanbai' peony, colchicine was injected into peony buds using an injection method (colchicine concentration of 0.25%). Buds between the small bell stage and the large bell stage were selected for treatment. A disposable syringe was used to puncture the petals, and the colchicine solution was injected into the center of the bud until the sepals exuded fluid). Refer to [the instructions for the kit]. The reverse transcription of cDNA was performed according to the instructions of the III1st Strand cDNA Synthesis Kit (+gDNAwiper). The specific operation procedure was as follows: a 16 μL reaction system (4×gDNAwiperMix 4 μL; RNA 1 μg; ddH2O to 16 μL) was prepared on ice, and the mixture was gently pipetted to mix well. The mixture was then incubated at 42°C for 2 min. Next, 5×HiScriptIIIqRTSuperMix was added, and the mixture was gently pipetted up and down to mix well. After a brief centrifugation, the droplets collected from the tube wall were collected at the bottom of the tube and then placed in a 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℃ 5 seconds

[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. The target gene was amplified according to the instructions of the 2×Taq Plus Master MixⅡ (DyePlus), and the program steps in Table 3 were run in the PCR instrument (30 cycles).

[0088] Table 3 Procedure Steps

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

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

[0091] After the reaction, the obtained PCR products were detected by agarose gel electrophoresis to check the specificity of the PCR products. The prepared gel products were placed under a UV gel imaging system, and bands of the same length and size as expected were observed. The gel was then cut under UV light and stored in a 1.5 mL centrifuge tube. The DNA gel was then extracted according to the FastPure DNA gel extraction kit. The instructions for using the Gel DNA Extraction Mini Kit (Novizan) describe the purification and recovery of target DNA.

[0092] (5) Connecting the T-carrier

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

[0094] (6) Transform the colonic competent state

[0095] The ligation product was transferred to E. coli competent cells Fast-T1 Competent Cells. The target DNA was added to the competent cells according to the instructions. The cells were incubated on ice for 30 min, then heat-shocked at 42°C for 30 s and then quickly placed on ice for 2 min.

[0096] Add 900 μL of antibiotic-free LB liquid medium to a centrifuge tube, mix well, and incubate at 37°C and 200 rpm for 1 hour. Centrifuge at 5000 rpm for 5 minutes and remove 800 μL of supernatant. Resuspend the bacteria in the remaining medium and gently spread evenly onto agar plates containing LB solid medium (100 μg / mL) containing Amp antibiotic. After the bacterial suspension has been completely absorbed by the medium, invert the sealed plates and incubate overnight at 37°C.

[0097] (7) Bacterial culture sequencing verification

[0098] After the dispersed and relatively large single colonies grew on the plate, single colonies were picked and placed into liquid medium containing Amp (100 mg / mL) lysate broth (Luria-Balani Medium, LB). After shaking at 200 rpm and 37°C for 5 hours, the colonies were sequenced (Qingke). The sequencing results were compared with PsASY1 using DNAMAN 8.0 software.

[0099] (8) Plasmid extraction

[0100] After sequencing, the bacterial culture was incubated overnight at 37°C with vigorous shaking at 200 rpm. Plasmids were extracted using a high-purity plasmid DNA mini-scale kit (Novizan), and after confirming their quality and concentration, they were stored at -20°C for later use.

[0101] 1.3 Bioinformatics Analysis

[0102] Bioinformatics analysis was conducted by examining the molecular characteristics of the coding regions of target genes and their functional relationships. This included predicting the physicochemical properties of amino acids, their hydrophilicity / hydrophobicity, signal peptides, conserved domains, and the secondary and tertiary structures of the encoded products. This revealed the relationship between the molecular biological characteristics of the gene coding regions and their functions. Based on this, software such as DNAMAN and MEGA were used to compare homologous sequences of amino acids from various plants, constructing phylogenetic trees for different species. The websites used for analysis are shown in Table 4.

[0103] Table 4. Online Analysis Software Links

[0104] database Function Website ProtParam Physicochemical properties of proteins http: / / web.expasy.org / protparam / ProtScale Hydrophobicity analysis of proteins https: / / web.expasy.org / protscale / TMHMM protein transmembrane domains http: / / www.cbs.dtu.dk / services / TMHMM / SignalP Predicting signal peptides 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 Conservative domain analysis http: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi

[0105] 1.4 Construction of overexpression vectors

[0106] (1) Preparation of linearized carriers

[0107] Find suitable restriction enzyme sites on the Super1300 vector and digest the Super1300 vector with restriction endonucleases ApaⅠ and KpnI. Prepare the reaction system according to Table 5.

[0108] Table 5 Reaction System

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

[0110] The enzyme was digested at 37°C for 5 min, and then immediately placed on ice to obtain the linearized Super1300 vector. The digestion was verified by 1% agarose gel electrophoresis, and the DNA was purified and recovered using a DNA gel recovery kit.

[0111] (2) Target gene ligation homologous arm

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

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

[0114] The homologous recombination reaction system was prepared on ice, as shown in Table 6.

[0115] Table 6 Homologous recombination reaction system

[0116] reagents volume Linearized carrier 2μL Insert fragment (diluted) 1μL 2×ClonExpressMix 5μL <![CDATA[ddH2O]]> 2μL

[0117] Fragment recombination reaction, 50°C, 10 min; cool to 4°C or immediately on ice.

[0118] Using the ligation product of the recombinant reaction as a template, efficient transformation was performed using the Fast-T1 chemicompetent cell kit (Novizan). The specific culture method was as follows: the ligation product was transferred to *E. coli* competent cells (Fast-T1), and the competent cells were then plated onto LB solid medium containing Amp (100 mg / mL). The sealed plates were incubated upright at 37°C for 10 min until the bacterial culture was completely absorbed by the medium. The plates were then inverted and incubated overnight. After the growth of dispersed and relatively large single colonies, single colonies were picked and transferred to liquid medium containing Amp (100 μg / mL) lysate (Luria-Balani Medium, LB). After shaking for 5 h, sequencing was performed.

[0119] (4) Transformation with Agrobacterium tumefaciens GV3101

[0120] After overnight shaking, plasmids were extracted from correctly sequenced colonies. Agrobacterium tumefaciens GV3101 competent cells were transformed according to the GV3101 Chemically Competent Cell instructions. 0.01–1 μg of plasmid was added to every 100 μL of competent cells, and the mixture was incubated sequentially on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and on ice for 5 min.

[0121] Add 700 μL of antibiotic-free LB liquid medium, incubate at 28°C and 200 rpm for 2-3 hours, centrifuge at 6000 pm for 1 min, and remove 600 μL of supernatant. Resuspend the bacteria in the remaining medium by aspirating, and then spread them onto agar plates containing Kans (50 μg / mL) and Rif (20 μg / mL) antibiotics using a sterile spreader. Incubate upside down at 28°C for 2-3 days.

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

[0123] PCR detection of the pSuper1300-PsASY1 recombinant plasmid was performed. A single, dispersed, and relatively plump Agrobacterium colony was picked from a sterilized pipette tip and injected into LB liquid medium containing 100 μg / mL Kan and 50 μg / mL LRif. The medium was gently shaken and incubated at 28°C and 200 rpm for about 5 hours. After that, the bacterial culture was tested for positive recombinant plasmids by PCR. Plasmids were extracted from bacterial cultures with clear bands and stored at -20°C for later use. Correct bacterial cultures were stored at -80°C with 50% glycerol.

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

[0125] The specific steps of Agrobacterium tumefaciens-mediated leaf disc transformation of tobacco leaves are as follows:

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

[0127] (2) At room temperature, the bacterial precipitate was collected after centrifugation at 5000 rpm for 10 min. The bacterial cells were resuspended in MS0 liquid medium (MS medium + 30 g / L sucrose, pH = 5.8) to obtain MS0 resuspension for infecting leaves.

[0128] (3) Take leaves from well-grown, sterile tobacco seedlings and cut them into 1cm pieces. 2 Small pieces on the left and right;

[0129] (4) Place small pieces of tobacco leaves into MS0 resuspension solution with the underside of the leaves facing down and gently shake to ensure that the bacterial solution fully contacts the wounds on the leaf edges and immerse for 10-15 minutes.

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

[0131] (6) The leaves were inoculated onto 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 co-culture, add 500 mg / L cephalosporin (Cef) to double-distilled water (ddH2O) and wash off any remaining Agrobacterium on the leaf surface. After blotting the water with sterile filter paper, transfer the leaves to 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) for differentiation induction and selection. Place the leaves in an incubator and change the MS1 medium every 10 days.

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

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

[0135] (10) Four weeks after transplanting to the substrate, three young leaves were collected from each individual plant, and tobacco leaf DNA was extracted according to the instructions of the Plant Genomic DNA Extraction Kit (Novizan). PCR amplification was performed using the DNA as a template and pSuper1300-F / R (SEQ ID NO. 6 and SEQ ID NO. 7) as primers. Transgenic positive tobacco lines were identified by clear and correctly sized bands. Subsequently, the meiotic process of pollen mother cells and the determination of vegetative growth indicators were observed.

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

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

[0138] (2) Take 1 mL each of Agrobacterium GV3101 from pSuper1300 and pSuper1300-PsASY1 and inoculate 60 mL of LB liquid medium (containing 50 mg / L Rif, 100 mg / L Kan, 50 mg / L Gentamicin, 200 mmol / L Acetyleugenol, and 10 mmol / L LMES) and incubate at 28°C with shaking at 200 rpm until the OD of different treatments is reached. 600 value.

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

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

[0141] (5) Adjust the OD of the resuspended bacterial culture with the infection solution. 600 Value and the set bacterial culture OD 600 The values ​​were the same, and the OD values ​​of pSuper1300 and pSuper1300-PsASY1 bacterial cultures were... 600 Same value; dark treatment at room temperature for 4 hours;

[0142] The bacterial solution was injected into the peony bud stage using an injection method. A 1ml sterile syringe was used to puncture the sepals and petals from the side of the bud and inject the infection solution into the center of the bud until the sepals exuded liquid. The process of meiosis in pollen mother cells was observed by taking pictures and samples 3, 5, 7 and 12 days after injection.

[0143] 1.7 Cytological observation of pollen mother cells

[0144] Flower buds (1-1.2 cm in diameter) in the meiotic stage were collected. After removing the petals, the buds were fixed in Carnoy's fixative (anhydrous ethanol: glacial acetic acid = 3:1, v / v). After vacuuming for 30 minutes, the buds were placed in a 4°C refrigerator. After 24 hours, the buds were rinsed 2-3 times with 90% ethanol and once with 70% ethanol. The buds were then transferred to 70% ethanol and stored at 4°C for long-term use. For observation, 1-3 anthers were placed on a glass slide. The anthers were squeezed with a glass rod to release the pollen mother cells. 1-2 drops of carbofuran staining solution were added, and the slides were squeezed with a dissecting needle for 3-5 minutes. Impurities were removed, and a coverslip was placed on top. The coverslip was then pressed again with the thumb to disperse the cells, creating a temporary slide. The morphology of chromosomes at various stages of meiosis was observed under a microscope and photographed.

[0145] Statistical analysis and pollen viability testing of 1.82n pollen.

[0146] Peony buds were collected 2 days before opening, petals were removed, and the buds were placed on tracing paper and air-dried naturally indoors for 48 hours. Mature pollen was collected, dried with silica gel, and stored at -20°C. The pollen was stirred and swelled in 0.9M mannitol CPW isotonic solution for about 2 hours, and observed and photographed using an optical microscope (Leica DM500). Based on the fact that the diameter of 2n pollen grains is more than 1.3 times that of normal pollen grains, the ratio of 2n pollen grains was statistically analyzed.

[0147] (1) TTC staining method

[0148] A 0.5% TTC staining solution was prepared using phosphate buffer (0.83 g Na₂HPO₄·2H₂O and 0.27 g KH₂PO₄ dissolved in 100 ml distilled water, pH = 7.2). A small amount of pollen was placed on a glass slide, and 1-2 drops of the staining solution were added. The slide was stirred thoroughly with a dissecting needle, covered with a coverslip, and incubated at 35°C for at least 2 hours until the number of stained pollen grains no longer increased. Observation under an optical microscope revealed that pollen grains stained red were considered viable, while those not stained red were considered non-viable. Ten fields of view were randomly selected for photographing and recording the staining rate. Staining rate = (Number of red-stained pollen grains / Total number of pollen grains) × 100%.

[0149] (2) In vitro germination method

[0150] Prepare a 10% sucrose agar medium. Use a dropper to take a small amount of the medium and place it in the groove of a concave glass slide. Let it stand for about 15 minutes until it solidifies. Spread a small amount of pollen evenly on the medium. Place the prepared slide in a 22°C incubator and observe the germination process under a microscope. Calculate the germination rate.

[0151] 2. Results

[0152] 2.1 Cloning of the target gene

[0153] A 6282 bp nucleotide sequence was obtained from the transcriptome data Unigene (pos.gene61825). A known genome search revealed its CDS length to be 1971 bp (SEQ ID NO.1), which was 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 cDNA derived from the reversed RNA of 'Fengdanbai' stamens as a template. Electrophoresis of the PCR products showed a single specific band around 1971 bp. Figure 1The sequencing assembly results were compared with the transcriptome data using multiple sequence alignment. The results showed that the obtained gene sequence was consistent with the CDS region sequence of the PsASY1 gene in the transcriptome. Blast analysis of this sequence on NCBI revealed a high degree of similarity to 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 amino acid physicochemical properties, prediction analysis of amino acid hydrophobicity and hydrophilicity, and prediction analysis of the secondary and tertiary structures of the encoded product.

[0156] The physicochemical properties of the protein were predicted using the ProtParam online analysis tool. The CDS region of the PsASY1 gene is 1971 bp long, encoding 656 amino acids (PsASY1 protein, amino acid sequence: MHFVTHQCSLVSLNFKFQTKT *, SEQ ID NO. 8), mainly containing 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 C1. 5942 H 9915 N 1971 O 2476 S 355 It has a total of 20,659 atoms, a theoretical isoelectric point (PI) of 4.98, an instability index of 35.20, and is therefore classified as a stable protein. Its average hydrophobicity index is 0.757.

[0157] ProtScale was used to analyze the hydrophilicity and hydrophobicity of proteins. Figure 2 In the a) group, the maximum hydrophobicity value of the PsASY1 protein is 2.25, and the minimum is -0.65, indicating it is generally a slightly hydrophobic protein. SignalP was used to predict the signal peptide ( Figure 2 (b) The results showed that PsASY1 protein lacks a signal peptide and is a non-secretory protein. The transmembrane domains of the protein were predicted using the TMHMM online tool. Figure 2 c) The results showed that PsASY1 protein lacks a transmembrane domain and is not a membrane protein. Secondary and tertiary structure prediction was performed using PSIPRED and SWISS-MODEL; the results showed that PsASY1 protein structural elements included α-helices (29.11%), random coils (57.93%), and extended chains (12.96%). Figure 2 (de in the middle).

[0158] The ASY1 protein sequences of other plants with sequence homology of over 80% were downloaded from NCBI. Using TBtools, the amino acid sequence encoded by the PsASY1 gene was compared with the amino acid sequences of 26 species, including *Actinidia chinensis*, *Actinidia eriantha*, *Camellia sinensis*, *Camellia lanceoleosa*, *Camellia sinensis*, grape (*Vitis vinifera*), and peach (*Prunus persica*). MEME Suite was used to compare their conserved motif DNA binding sites, revealing that the PsASY1 protein is quite similar to the conserved motifs of other species. Figure 3 To investigate the homologous evolution of the PsASY1 protein with the amino acid sequences of other species, a phylogenetic tree was constructed using MEGA and PsASY1 protein sequences from other species to explore the homologous evolution of the PsASY1 protein's amino acid sequence with other species. The results showed that the PsASY1 protein has a high degree of affinity with species such as *Actinidia chinensis*, *Ipomoea nil*, and *Camellia sinensis*, and is more closely clustered in the phylogenetic tree, forming a single group.

[0159] Existing research indicates that the ASY1 gene actually originates from the HORMA domain-containing protein family. Based on genomic data, this study analyzed all genes from this family in three species: peony, Arabidopsis thaliana, and tobacco. Figure 4In the phylogenetic tree, the number of genes was found to be relatively small. The key gene PsASY1 (Pos.gene61825) selected in this embodiment is closely related to Arabidopsis thaliana AT1G67370.1, and tobacco Nta08g04630, Nta03g10930, Nta05g24160, and Nta06g29970. Correspondingly, their conserved Motif DNA binding site sequences show similar patterns. Chromosomal localization analysis of peony HORMADs family genes revealed that the 15 genes belonging to the HORMADs family screened from the peony genome are distributed on different chromosomes, with PsASY1 located on chromosome 03. Figure 4 (c)

[0160] 2.3 Identification of target gene overexpression vectors

[0161] The CDS region fragment of the PsASY1 gene was ligated into the plant expression vector pSuper1300 to construct the pSuper1300-PsASY1 recombinant vector. Figure 14 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, PCR verification was performed. Electrophoresis of the PCR products showed a single correct band at approximately 1971 bp. Figure 15 Sequencing results of the positive plasmid showed that the sequence identity was over 99%, and the pSuper1300-PsASY1 overexpression recombinant vector was successfully constructed.

[0162] 2.4 Tobacco Conversion

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

[0164] RNA was extracted from the transgenic lines and identified by real-time quantitative PCR to verify the relative expression levels of the genes. Figure 6 The results showed that the relative expression level of the PsASY1 gene in the transgenic lines was higher than that in wild-type tobacco, which further verified 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 Quantitative real-time PCR detection, pollen mother cell observation, and phenotypic observation of transgenic tobacco

[0166] DNA was extracted from the obtained PsASY1 transgenic tobacco and subjected to PCR detection. Wild-type tobacco DNA was used as a negative control (CK), and pSuper1300-PsASY1 recombinant vector was used as a positive control. In all PsASY1 transgenic tobacco samples, specific bands of the expected size were amplified. Figure 7 While no fragment was amplified in WT, this preliminarily indicates that the exogenous PsASY1 gene has been normally expressed in the transgenic tobacco lines. RNA was extracted from the transgenic tobacco lines and analyzed by real-time quantitative PCR to verify the relative expression level of the gene. The results showed that the relative expression level of the PsASY1 gene was significantly higher than that in wild-type tobacco, further confirming that the tobacco plants were positive transgenic plants. Figure 7 The results of normal meiosis in tobacco pollen mother cells are as follows: Figure 9 As shown in the figure. The results of observing abnormal meiotic behavior in tobacco pollen mother cells overexpressing the PsASY1 gene are as follows. Figure 10 As shown. The results indicated that in tobacco pollen mother cells overexpressing the PsASY1 gene, interphase lag of chromosomes occurred, haploids and lagging chromosomes coexisted, trigonosomes and lagging chromosomes were observed, telophase II chromosomes were lagging, and chromosome segregation was uneven, ultimately leading to the formation of 2n pollen grains. Therefore, overexpression of the PsASY1 gene can promote the formation of 2n pollen grains. Based on this, the formation of 2n pollen grains can be used to cultivate polyploid plants and sterile plants. The L*a*b* values ​​of the obtained PsASY1 transgenic tobacco were measured. The results showed that the L* value of the first line of transgenic tobacco plants was decreased, significantly different from the control group; the a* and b* values ​​of the first and third lines of transgenic tobacco plants were also decreased, 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 was increased, and the difference from the control group was significant. Figure 13 The anthocyanin content of the obtained PsASY1 transgenic tobacco was determined. The anthocyanin content of the transgenic tobacco plants in lines 1, 2, and 3 was significantly increased compared with the control group. Figure 11 In the D-means expression of PsASY1 in the recombinant vector, increased expression levels and meiotic disorder further suggest that PsASY1 may be a gene affecting abnormal meiosis in peony, and its enhanced function may lead to the formation of 2n pollen. The phenotypic growth rate of tobacco and wild-type plants was significantly faster than that of wild-type plants. After 15 days of transplantation, although the number of leaves in transgenic tobacco plants was similar to that of wild-type tobacco plants, the crown width, number of leaves, and plant height of transgenic tobacco plants were significantly greater than those of wild-type plants. Figure 8 and Figure 11AC in the middle.

[0167] 2.6 Effects of transient overexpression of PsASY1 on meiosis in 'Fengdanbai' pollen mother cells

[0168] The anthers collected from flower buds within 3-12 days after injection were pressed into slides to observe the meiotic process of pollen mother cells. The results of normal meiosis observation are as follows: Figure 16 The results show abnormal behaviors compared to the normal meiotic process, such as chromosome bridges, uneven chromosome separation, and chromosome fragmentation. Figure 17 This ultimately leads to the formation of 2n pollen grains. Therefore, overexpression of the PsASY1 gene can promote the formation of 2n pollen grains in 'Fengdanbai'. The pollen viability of the control group and the PsASY1-expressing group was statistically analyzed. The results showed that the total staining rate of pollen overexpressing PsASY1 was approximately 84.67%, while that of the control group was approximately 81.98%. In 'Fengdanbai' overexpressing PsASY1, the proportion of 2n pollen was approximately 6.23%, the 2n staining rate was approximately 71.2%, and the 1n staining rate was approximately 87.14%. This indicates that there was no significant difference in pollen viability between the PsASY1-expressing and control groups. Figure 18 ).

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

Claims

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

8.

2. A protein encoding the PsASY1 protein of claim 1 PsASY1 Genes, characterized by, The PsASY1 The nucleotide sequence of the gene is shown as SEQ ID NO.

1.

3. A biological material comprising the gene of claim 2. PsASY1 3. A biological material comprising the gene of claim 2.

4. The PsASY1 protein of claim 1, and the protein of claim 2 PsASY1 The use of the gene or the biomaterial of claim 3 in any one or more of the following: (1) Cultivating polyploid plants; (2) Regulating plant growth traits; the growth traits include one or more of the following: crown width, leaf height and plant height; (3) Regulate the anthocyanin content in plants; (4) Regulate the chlorophyll content of plants; Through overexpression of plant PsASY1 The expression level of genes can be adjusted to achieve the goals of cultivating polyploid plants, improving plant growth traits, increasing anthocyanin content, and increasing chlorophyll content. The plants mentioned are tobacco and peony.

5. A method of promoting the formation of 2n pollen grains in a plant, characterized in that, Including overexpression in plants PsASY1 The steps for determining gene expression levels; PsASY1 The nucleotide sequence of the gene is shown in SEQ ID NO.1; the plants are tobacco and peony.

6. A method of breeding a polyploid plant, comprising the steps of, Including overexpression in plants PsASY1 The steps for determining gene expression levels; PsASY1 The nucleotide sequence of the gene is shown in SEQ ID NO.1; the plants are tobacco and peony.

7. A method of improving a growth trait in a plant, comprising: The method includes overexpression of plant... PsASY1 The steps for determining gene expression levels; PsASY1 The nucleotide sequence of the gene is shown in SEQ ID NO.1; the growth traits include one or more of the following: crown width, leaf height, and plant height; the plant is tobacco.

8. A method for cultivating plants with large crowns, large leaves, and tall stems, characterized in that, The method includes overexpression of plant... PsASY1 The steps for determining gene expression levels; PsASY1 The nucleotide sequence of the gene is shown in SEQ ID NO.1; the plant is tobacco.

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