阅读说明：本技术 梨PbrSTONE基因及其应用 (Pear PbrSTONE gene and application thereof ) 是由 吴俊� 薛程 薛雍松 张明月 李甲明 张绍铃 秦梦帆 赵渴姣 汪润泽 于 2021-02-05 设计创作，主要内容包括：本发明公开了梨PbrSTONE基因及其应用。一种分离自‘砀山酥梨’具有调控梨果实石细胞发育的PbrSTONE基因,其核苷酸序列为SEQ ID No.1所示,其编码的氨基酸序列为序列表SEQ ID No.2所示。通过在梨果实中瞬时过量表达以及基因沉默技术,证实了PbrSTONE可以改变果实石细胞和木质素的积累过程；过表达PbrSTONE的转基因拟南芥花序茎中,木质素含量及其G型单体显著增加,同时木质化束间纤维细胞层数增多,导管细胞的次生细胞壁显著加厚。由此证明PbrSTONE基因参与调控梨果实石细胞木质素的形成。(The invention discloses a pear PbrSTONE gene and application thereof. A PbrSTONE gene which is separated from a Dangshan pear and has the function of regulating and controlling the development of pear stone cells has the nucleotide sequence shown in SEQ ID No.1, and the coded amino acid sequence is shown in a sequence table SEQ ID No. 2. The transient over-expression and gene silencing technology in pear fruits prove that PbrSTONE can change the accumulation process of stone cells and lignin of fruits; in the transgenic arabidopsis inflorescence stem over expressing PbrSTONE, the lignin content and G type monomers thereof are obviously increased, the number of the fibrous cell layers among lignification bundles is increased, and the secondary cell wall of ductal cells is obviously thickened. Thus, the PbrSTONE gene is proved to be involved in the regulation of the formation of the lignin of the stone cells of the pear fruits.)

1. A PbrSTONE gene separated from the Dangshan pear and used for regulating the development of pear stone cells has a CDS sequence shown in SEQ ID No.1 and a coded protein sequence shown in SEQ ID No. 2.

2. The PbrSTONE gene-encoded protein of claim 1, wherein the amino acid sequence is as shown in SEQ ID No. 2.

3. A recombinant expression vector comprising the gene of claim 1.

4. The recombinant expression vector of claim 4, wherein pCAMBIA1301 is used as a starting vector, and the insertion site of the gene of claim 1 is between Xba I and BamH I.

5. A host bacterium comprising the gene of claim 1.

6. The primer pair for cloning the gene sequence of claim 1, characterized in that the sequence of the upstream primer PbrSTONE-F1 is shown as SEQ ID No.3, and the sequence of the downstream primer PbrSTONE-R1 is shown as SEQ ID No. 4.

7. Use of the gene of claim 1 for regulating stone cell formation and/or lignin synthesis.

8. Use of the recombinant expression vector of claim 3 or 4 for regulating lignin synthesis in a pyritum cell and/or an arabidopsis inflorescence stem.

Technical Field

The invention belongs to the field of plant genetic engineering, relates to a pear PbrSTONE gene and application thereof, and particularly relates to a PbrSTONE gene for regulating and controlling pear stone cell development, which is obtained by separating and cloning from a Dangshan pear.

Background

The pear is a perennial woody plant of the genus Pyrus (Pyrus L.) of the subfamily Persicae (Rosaceae), is the third largest fruit tree species in China, has long cultivation history and wide planting area (Tengyuan, 2017). China is used as a large export country of pears, and the cultivation area and the yield of pears are the first world in recent years. However, some traditional main cultivars in China, such as Dangshan pears, have high stone cell content, poor fruit quality and weak market competitiveness. The stone cells are the special characters in the pulp of the pear and are also important factors influencing the quality of the fruit. Therefore, the method is especially important for determining the content difference of stone cells and analyzing the forming mechanism of the stone cells and improving the quality of the pear fruits.

Pear stone cells are a type of short stone cells, and a plurality of stone cells are aggregated into stone cell masses in the pear fruit development process (Nii et al, 2008; Zhang et al, 2017). The size and number of stone cell mass in the pulp affects the fruit quality and thus the eating mouthfeel and processing quality (Choi et al, 2007; Wu et al, 2013; Brahem et al, 2017; Zhang et al, 2017). The sclerite cells developed from parenchyma cells mainly contain cellulose and lignin, and lignin deposition is an important factor causing hardening of the cell walls of the sclerite cells, so that the relationship between lithocyte synthesis and lignin metabolism is close. The lignin of stone cells is mainly G-lignin and contains a small amount of S-lignin, but the G-lignin and S-lignin content of different pear varieties can be different (Cai et al, 2010; Jin et al, 2013).

Lignin, a polyphenol polymer, is deposited in the secondary cell walls of vascular plants, not only provides mechanical strength and hydrophobicity to plant vascular bundles, but also has the effect of preventing pathogenic bacteria from invading. In lignin-deficient Arabidopsis mutants, plants show cell wall collapse and lodging, which fully embodies the importance of lignin in the secondary wall (Zhong et al, 1998; Ruel et al, 2009). However, in the case of wood for papermaking, lignin is an important factor affecting paper quality and papermaking process, and a large amount of strong acid and alkali chemicals are required to remove lignin, which not only increases production cost, but also causes serious environmental pollution. In view of this, researchers have been expecting plants with less lignin content but without affecting their primary function by altering the synthetic pathway of lignin (Vanhole et al, 2012; ZHao, 2016).

Disclosure of Invention

The invention aims to provide a PbrSTONE gene for regulating and controlling the stone cell development of pear fruits.

Another purpose of the invention is to provide the application of the gene.

The purpose of the invention can be realized by the following technical scheme:

a PbrSTONE gene which is separated from Dangshan pear and has the function of promoting the synthesis of lignin in stone cells has the nucleotide sequence shown as SEQ ID No.1 and comprises an open reading frame of 819 bp; 272 amino acids are coded, and the coded amino acid sequence is shown in a sequence table SEQ ID No. 2.

The invention also provides a recombinant expression vector containing the PbrSTONE gene.

The recombinant expression vector takes pCAMBIA1301 as a starting vector, and the insertion site of the PbrSTONE gene is between Xba I and BamH I.

Host bacteria containing the PbrSTONE gene of the invention.

The primer pair of the PbrSTONE gene cDNA sequence is cloned, the sequence of an upstream primer PbrSTONE-F1 is shown as SEQ ID No.3, and the sequence of a downstream primer PbrSTONE-R1 is shown as SEQ ID No. 4.

The PbrSTONE gene disclosed by the invention is applied to promotion of lignin synthesis in stone cells.

The recombinant expression vector disclosed by the invention is applied to promotion of lignin synthesis in stone cells.

Advantageous effects

In combination with the data of the whole gene association analysis, the invention discovers a novel gene (PbrSTONE). The transient over-expression and gene silencing technology in pear fruits prove that PbrSTONE can change the accumulation process of stone cells and lignin of fruits; in the transgenic arabidopsis inflorescence stem over expressing PbrSTONE, the lignin content and G type monomers thereof are obviously increased, the number of the fibrous cell layers among lignification bundles is increased, and the secondary cell wall of ductal cells is obviously thickened. Thus, it was demonstrated that the PbrSTONE gene is involved in the regulation of stone cell formation in pear fruits. The discovery of the gene complements and perfects a mechanism for regulating and controlling the metabolism of the lignin of the stone cells in the pear fruits, provides a theoretical basis for improving the content of the stone cells of the pear fruits, and provides gene resources for reducing the content of the lignin by gene editing in the paper industry and the bioenergy industry.

Compared with the prior art, the invention has the following advantages and effects:

the discovery of PbrSTONE gene provides new gene resource for molecular breeding for reducing organism lignin synthesis, provides new genetic resource for implementing green agriculture, and the development and utilization of the genetic resource are beneficial to reducing agricultural cost and realizing environmental protection.

2. Through an agrobacterium-mediated transformation method, the function verification of the PbrSTONE gene is carried out in young pear fruits and arabidopsis thaliana, and the result shows that the PbrSTONE gene has the advantage of simultaneously regulating and controlling a plurality of lignin synthesis pathway enzyme genes, so that a more efficient way is provided for molecular breeding.

Drawings

FIG. 1 is a schematic view of a vector of example 1 of the present invention.

FIG. 2 is the functional analysis of the PbrSTONE gene transient injection pear young fruit of the invention.

Wherein: a, the relative expression quantity of the PbrSTONE gene in different tissues and different development periods of the Dangshan pear. b, staining the pear fruit section of the transient expression PbrSTONE gene by phloroglucinol. c and d, lignin content of pear pulp. e and f, expression quantity of genes related to pear fruit lignin synthesis.

FIG. 3 shows the functional analysis of PbrSTONE gene in transgenic Arabidopsis plants. Wherein: WT: a wild type; the rest numbers are as follows: and (4) transgenic lines. a, the overexpression of the PbrSTONE gene influences the expression of lignin synthetase related genes. b-d, lignin content of inflorescence stems of wild type and transgenic Arabidopsis plants after eight weeks of growth (b), lignin monomer composition (c) and the number of interbeam lignified cells (d). (e-p) histological analysis of inflorescence stems of wild type and transgenic Arabidopsis plants eight weeks after growth, with wild type on the left and transgenic Arabidopsis on the right: e-f, toluidine blue staining indicates cell morphology; g-l, Wiesner,and UV light detecting the spatial distribution of lignin deposition; m-p, detecting the thickness of the secondary cell wall by a transmission electron microscope.

Detailed Description

The present invention will be described in detail with reference to specific examples. From the following description and these examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

Example 1 isolation cloning of PbrSTONE Gene and construction of overexpression vector

Mu.g of Dangshan pear early pulp RNA was extracted and reverse transcribed using one-step gDNA removal and cDNA synthesis kit (Transgen, China), according to the instructions. Xba I and BamH I were selected as endonucleases based on the multiple cloning site of pCAMBIA-1301 vector and the cleavage site analysis on the coding region sequence of PbrSTONE gene. Primers SEQ ID NO.3 and SEQ ID NO.4 with restriction sites were designed using Snapgene software according to the general principle of primer design. A50. mu.L reaction system contained 200ng of cDNA, 1 Xbuffer (TransStart FastPfu Buffer), 10mM dNTP, 1U of Taq Polymerase (TransStart FastPfu DNA Polymerase) (the Buffer and Taq Polymerase were purchased from TRANS corporation), and 500nM of the above primer. The PCR reaction was performed on an eppendorf amplification machine according to the following procedure: pre-denaturation at 95 ℃ for 2min, denaturation at 95 ℃ for 20 sec, annealing at 60 ℃ for 20 sec, extension at 72 ℃ for 1min, 35 thermal cycles, extension at 72 ℃ for 10 min, and storage at 4 ℃. One single PCR band product was generated.

After the PCR product was detected by 1% agarose gel electrophoresis, DNA fragments were recovered using a small gel recovery kit (purchased from Haokang century, according to the instructions provided in the kit). The total volume of the double enzyme digestion system of the pCAMBIA1301 vector is 50. mu.L, which contains 10. mu.L of pCAMBIA1301 vector plasmid obtained by plasmid extraction, 5. mu.L of 10 XBuffer (purchased from NEB company), 1. mu.L of Xba I, 1. mu.L of BamH I and 33. mu.L of water. The resulting mixture was digested at 37 ℃ for 3 hours and then recovered. The restriction enzyme digested expression vector pCAMBIA1301 and PbrSTONE gene were ligated at 37 ℃ for 30 minutes using recombinase Exnase II (available from Vazyme). The total reaction volume was 20. mu.L, containing 5 XCE II Buffer 4. mu.L, Exnase II 2. mu.L, PCR-recovered product of PbrSTONE gene 2. mu.L, double digestion-recovered product of pCAMBIA1301 vector 6. mu.L and water 6. mu.L. Taking 10 mu L of the ligation product to transform escherichia coli competent DH5 alpha, screening positive clones in an LB solid plate containing 50mg/L kanamycin, extracting plasmids to perform enzyme digestion and PCR identification, and sending recombinant plasmid samples to a biological company for sequencing. Sequencing results show that the total length of the PbrSTONE gene is 819bp, the nucleotide sequence of the PbrSTONE gene is shown in SEQ ID NO.1, the PbrSTONE gene can encode protein with 272 amino acid residues, and the sequence of the PbrSTONE gene is shown in SEQ ID NO. 2. Phylogenetic tree analysis shows that homologous genes exist in PbrSTONE in other species, but the functions of the genes are not reported in relevant documents. We named the recombinant vector 35S-PbrSTONE-GFP and the map of the constructed vector is shown in FIG. 1. The recombinant vector was introduced into Agrobacterium GV3101 using a freeze-thaw method.

Example 2 analysis of spatiotemporal expression patterns of PbrSTONE Gene

'Dangshan crisp pear' different tissue samples were collected from the Kogyou orchard of Jiangsu province (2015). Total RNA was extracted by CTAB method (Porebski et al, 1997) and the quality of the extracted samples was checked by spectrophotometer and agarose gel. Mu.g of the total RNA extracted was used for reverse transcription using one-step gDNA removal and cDNA synthesis kit (Transgen, China), the method was as described in the specification. The primers used in the fluorescent quantitative PCR are gene specific primer pairs: SEQ ID No.5 and SEQ ID No. 6; GAPDH was used as an internal reference gene, and a fluorescent quantitation kit was purchased from Roche. The used instrument of Real-timesEPCR is Roche 480 quantitative PCR instrument, and the reaction system is as follows: 2 × SYBR GreenI Master Mix 10 μ L, upstream and downstream primers (10 μ M)0.4 μ L, 2 μ LcDNA, 7.2 μ LPCR grade water. The reaction condition is 95 ℃ denaturation for 5 min; pre-denaturation at 95 ℃ for 5s, annealing at 60 ℃ for 5s, and extension at 72 ℃ for 10s, and repeating 45 cycles; melting curves were analyzed at 65 ℃ to 95 ℃ with 1 ℃ increase every 5 s.

Studies have shown that there is a large amount of lignin synthesis in the early stages of pear fruit development and vascular tissues (Xue et al, 2019). qRT-PCR results of different pear tissues show that the expression level of PbrSTONE in the early stage of fruit development is obviously higher than that in the later stage; the expression level in the stem and anther was also significantly higher than in the leaf (FIG. 2 a). In conclusion, PbrSTONE was highly expressed in fruits, stems and anthers 21 to 49 days after flowering, suggesting that PbrSTONE expression, consistent with the timing and space of lignin deposition, is likely to be involved in lignin synthesis in stem and pulp stone cells and in anther dehiscence.

Example 3 transient transformation of Pear fruit and detection of Lignin content and Gene expression

(1) Instantaneous transformation of pear fruit

The method for injecting the agrobacterium containing the PbrSTONE over-expression vector into the Dangshan pear' about 35 days after flowers by an agrobacterium-mediated method comprises the following steps:

1. activating agrobacterium containing the correct plasmid on a solid culture medium, and growing for 48 hours in an incubator at the temperature of 28 ℃;

2. to a 100mL Erlenmeyer flask was added 30mL of a solution containing R⁺And K⁺The liquid LB culture medium of (1) is prepared by picking activated agrobacterium tumefaciens with a gun head, and growing for 12 hours in a shaker at 28 ℃ and 200 rpm;

3. pouring the bacterial liquid into a 50mL centrifuge tube at 6000rpm, centrifuging for 15 minutes, and collecting all thalli;

4. resuspending the pellet in appropriate amount of induction medium (10mM MgCl2, 10mM MES, 200mM acetosyringone, pH 5.6), adjusting OD to 0.8-1.2, and inducing with decolorizing shaker at room temperature for 4 hr;

5. injecting the staining solution into pear fruits 35 days after blossom, injecting at least 10 fruits each time, and performing biological repetition for three times;

6. culturing in dark for 24 hr, placing in an incubator with 16 hr illumination/8 hr darkness photoperiod, maintaining at 22 deg.C, and culturing for 7-10 days;

(2) histological analysis

Cutting off fruits by a blade, and dyeing by phloroglucinol-hydrochloric acid, and specifically comprises the following steps:

1. firstly, treating the mixture with 30% hydrochloric acid solution (V/V) for 1 minute;

2. dissolving 10% phloroglucinol in 80% ethanol (W/V);

3. dripping the solution on the slice treated by hydrochloric acid, and dyeing for 5 minutes;

4. finally, the reaction is stopped by washing with sufficient water.

Phloroglucinol-hydrochloric acid staining shows that the staining effect of lignin of a part excessively expressing PbrSTONE is obviously enhanced, and the part injected with pCAMBIA1301 vector has no obvious difference with the part not injected (figure 2 b).

(3) Injection site lignin content detection

The content of lignin in stone cells is expressed as a percentage of the measured value/weight of the sample used, with reference to the bromoacetyl method (Acker et al, 2013). There was no significant difference in the lignin content between the injected pCAMBIA1301 vector and the non-injected part, while the lignin content was significantly increased in the part overexpressing PbrSTONE (FIGS. 2 c-d).

(4) Detection of relative expression quantity of injection site PbrSTONE and lignin synthesis related gene

The system and procedure for RNA extraction, cDNA synthesis, and fluorescent quantitative PCR were as described in example 2. The site overexpressing PbrSTONE not only increased the expression level of PbrSTONE, but also significantly increased the expression levels of Pbr4CL1, Pbr4CL2, PbrC3H1, PbrCAD, PbrCSE, PbrLAC2, PbrLAC15 and PbrLAC18 (FIGS. 2 e-f). Taken together, these results indicate that overexpression of PbrSTONE promotes lignin synthesis in stone cells of pome fruits.

TABLE 1 Pear real-time fluorescent quantitative primers

Example 4 genetic transformation of Arabidopsis thaliana and identification of transformed plant molecules

Col-0 Arabidopsis thaliana was infected by the floral dip method with Agrobacterium containing a PbrSTONE overexpression vector (Clough & Bent, 1998). The specific method comprises the following steps:

1. using a solution containing 50mg/L K⁺And 100mg/L R⁺The solid LB culture medium is used for marking and activating agrobacterium, and is cultured for 36 hours in an incubator at the temperature of 28 ℃;

2. the single clone on the thread was picked up with a sterilized toothpick or tip, placed in a 100mL Erlenmeyer flask, and 30mL of 50mg/L K solution was added⁺And 100mg/L R⁺The liquid LB medium of (1) was cultured in a shaker at 28 ℃ for 12 hours at 200 rpm;

3. centrifuging the mixture for 20 minutes at 5000rpm by using a 50mL centrifuge tube to collect thalli;

4. resuspending the cells in equal volume of transformation medium [ 1/2 MS; 5% sucrose (W/V); 10 mug/L of 6-BA; adjusting the pH value to 5.7 by using KOH; 0.025% surfactant (V/V);

5. cutting off siliques and opened flowers of Arabidopsis thaliana to be transformed;

6. soaking the arabidopsis inflorescence in a transformation medium containing thalli, vacuumizing to 380 mm mercury by using a vacuum pump, and soaking for 5 minutes;

7. the cells were placed in a 22 ℃ culture room protected from light for 24 hours, and then cultured under long-day conditions (16 hours light/8 hours dark) at 22 ℃.

Taking arabidopsis T1 generation positive plants with two weeks of hygromycin resistance, taking inflorescence stems to extract RNA, and detecting the expression quantity of PbrSTONE by fluorescent quantitative PCR, wherein primers are SEQ ID NO.5 and SEQ ID NO. 6. Of these, 3 strains (OE-3, OE-6, OE-8) were present in higher expression and their homozygotes were used in subsequent experiments. The T3 generation homozygous seeds and wild type seeds were sown to germination medium [ MS; 3% sucrose (W/V); 0.75% agar (W/V). After the seeds germinate, the seedlings are moved to nutrient soil and cultured under the conditions of 22 ℃ and long day (16 hours of light/8 hours of dark).

Example 5 measurement of Lignin-related physiological indices of transgenic plants

(1) Detection of relative expression quantity of genes related to synthesis of transgenic arabidopsis inflorescence stem lignin

The method comprises the following steps of taking the primary inflorescence stems of 4-week-old plants of wild type and transgenic lines, extracting RNA, synthesizing cDNA, and carrying out fluorescent quantitative PCR (polymerase chain reaction) according to example 2.

In the over-expression PbrSTONE Arabidopsis thaliana plant, the expression levels of the lignin synthesis genes AtC3H1 and AtLAC17 are both increased remarkably (FIG. 3a), which indicates that PbrSTONE causes the excessive accumulation of lignin in the stem by activating the expression of the lignin synthesis related genes.

TABLE 2 Arabidopsis thaliana real-time fluorescent quantitative primers

(2) Determination of lignin content and monomer content of transgenic arabidopsis

5 strains of each of the wild type and T3 generation transgenic lines were selected, 10cm long stems were collected from the lower part of the inflorescence stem, cut into 2mm long pieces, and the mixed samples were dried to constant weight. 5mg of the dried sample was taken to extract CWR for lignin analysis, the procedure was as follows: the samples were placed in 2mL centrifuge tubes and treated for 30 minutes each step, passed through water (98 ℃ C.), ethanol (76 ℃ C.), chloroform (59 ℃ C.), acetone (54 ℃ C.), and finally dried to constant weight. Bromoacetyl-soluble lignin content determination, reference (Acker et al, 2013). The lignin composition was determined by the thioacid analysis method, method reference (Lapierre et al, 1995). A derivative formed by the action of thioacid lysis of lignin is derivatized by trimethyl silane and then detected by GC-MS. The test was repeated four times, each time with the same amount of sample.

The lignin content in the inflorescence stems of the transgenic lines was determined to be significantly increased (fig. 3 b); and the content of the transgenic line G type monomer is obviously increased, but the content of the S type monomer is not obviously different (figure 3 c); at the same time, the number of cell layers between transgenic lines was significantly increased (fig. 3 d).

(3) Observation of Stem tissue Structure of transgenic Arabidopsis inflorescence

1 tissue section staining

The inflorescence stems of the wild type and transgenic Arabidopsis plants grown for eight weeks were taken 1cm from the bottom, embedded in 5% agarose gel and 100 μm thick sections were cut using a Leica VT1000S shaker.

Toluidine blue staining: staining the slices in 0.3% toluidine blue reagent for 30s, washing with deionized water, and observing;

and (3) Wiesner dyeing: the sections were placed in a Wiesner reagent [ 37% HCl: 3% phloroglucinol alcoholic solution ═ 1: 2 (v: v) for 1min, washing with deionized water and observing;

dyeing: and (3) placing the slices in 0.5% potassium permanganate aqueous solution for 2min, washing with deionized water, adding 3.7% HCl for washing twice, sucking the supernatant, adding concentrated ammonia water for 1min, directly taking out the slices, and observing.

The stained sections were observed using a Nikon Ni-U microscope.

2 Lignin autofluorescence Observation

The unstained section is observed by a Nikon Ti-E inverted fluorescence microscope, a diode laser emits 355/25nm laser, and the laser intensity, the amplification factor and the gain setting of a photomultiplier are kept consistent among different samples.

3 Transmission electron microscope

Fixing a sample which is the same as the paraffin section for 12 hours at 4 ℃ by using 2.5 percent glutaraldehyde stationary liquid, and quickly putting the sample into fresh 2.5 percent glutaraldehyde stationary liquid after taking the sample; the material is obtained by operating at low temperature of 0-4 deg.C, and the instruments and fixative solution for obtaining material are cooled; since the fixed solution has poor permeability, glutaraldehyde has a penetration depth of 0.5mm, and osmic acid has a penetration depth of 0.25mm, the sample size is about 1.5mm × 3mm, and the thickness is not more than 2 mm; for a material floating on the stationary liquid, it is necessary to completely immerse the material under the stationary liquid by air suction to sufficiently fix the material. Method reference (Whitehill et al, 2016). The thickness of the secondary cell wall of xylem cells was measured with ImageJ software.

Through paraffin section, lignin autofluorescence detection and transmission electron microscope detection, the morphology of xylem cells of the stem of transgenic arabidopsis is normal, but lignin is obviously accumulated, autofluorescence is strong, and secondary cell walls are thicker (fig. 3 e-p).

Primary references

Tengwen 2017, development of phylogeny of Pyria plants and origin research of Oriental pear varieties, academic journal of fruit tree 34(3):370-378.

Acker RV,Vanholme R,Storme V,Mortimer JC,Dupree P,Boerjan W.2013.Lignin biosynthesis perturbations affect secondary cell wall composition and saccharification yield in Arabidopsis thaliana.Biotechnology for Biofuels 6(1):1-17.

Brahem M,Renard CMGC,Gouble B,Bureau S,Le Bourvellec C.2017.Characterization of tissue specific differences in cell wall polysaccharides of ripe and overripe pear fruit.Carbohydrate polymers 156:152-164.

Cai Y,Li G,Nie J,Lin Y,Nie F,Zhang J,Xu Y.2010.Study of the structure and biosynthetic pathway of lignin in stone cells of pear.Scientia Horticulturae 125(3):374-379.

Choi J-H,Choi J-J,Hong K-H,Kim W-S,Lee S-H.2007.Cultivar differences of stone cells in pear flesh and their effects on fruit quality.Horticulture Environment and Biotechnology 48(1):27-31.

Clough SJ,Bent AF.1998.Floral dip:a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana.Plant Journal for Cell&Molecular Biology 16(6):735.

Jin Q,Yan C,Qiu J,Zhang N,Lin Y,Cai Y.2013.Structural characterization and deposition of stone cell lignin in Dangshan Su pear.Scientia Horticulturae 155:123-130.

Lapierre C,Pollet B,Rolando C.1995.New insights into the molecular architecture of hardwood lignins by chemical degradative methods.Research on Chemical Intermediates 21(3-5):397.

Nii N,Kawahara T,Nakao Y.2008.The development of stone cells in Japanese pear fruit.The Journal of Horticultural Science and Biotechnology 83(2):148-153.

Porebski S,Bailey LG,Baum BR.1997.Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components.Plant Molecular Biology Reporter 15(1):8-15.

Ruel K,Berrio-Sierra J,Derikvand MM,Pollet B,Thévenin J,Lapierre C,Jouanin L,Joseleau JP.2009.Impact of CCR1 silencing on the assembly of lignified secondary walls in Arabidopsis thaliana.New Phytologist 184(1):99-113.

Vanholme R,Morreel K,Darrah C,Oyarce P,Grabber JH,Ralph J,Boerjan W.2012.Metabolic engineering of novel lignin in biomass crops.New Phytologist 196(4):978-1000.

Whitehill JG,Henderson H,Schuetz M,Skyba O,Yuen MM,King J,Samuels AL,Mansfield SD,Bohlmann J.2016. Histology and cell wall biochemistry of stone cells in the physical defence of conifers against insects.Plant Cell Environ 39(8):1646-1661.

Wu J,Wang Z,Shi Z,Zhang S,Ming R,Zhu S,Khan MA,Tao S,Korban SS,Wang H.2013.The genome of the pear(Pyrus bretschneideri Rehd.).Genome research 23(2):396-408.

Xue C,Yao JL,Qin MF,Zhang MY,Allan AC,Wang DF,Wu J.2019.PbrmiR397a regulates lignification during stone cell development in pear fruit.Plant Biotechnol J 17(1):103-117.

Zhang J,Cheng X,Jin Q,Su X,Li M,Yan C,Jiao X,Li D,Lin Y,Cai Y.2017.Comparison of the transcriptomic analysis between two Chinese white pear(Pyrus bretschneideri Rehd.)genotypes of different stone cells contents.PLoS One 12(10).

Zhao Q.2016.Lignification:flexibility,biosynthesis and regulation.Trends in plant science 21(8):713-721.

Zhong R,Morrison WH,Negrel J,Ye Z-H.1998.Dual methylation pathways in lignin biosynthesis.The plant cell 10(12): 2033-2045.

Sequence listing

<110> Nanjing university of agriculture

<120> pear PbrSTONE gene and application thereof

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 819

<212> DNA

<213> Dangshan pear' (Dangshan Pirum)

<400> 1

atggcgcgtg gtctcctcgc ttgctttgga tcaaaaggag gatcccgcac gtcccatgaa 60

aagcatgctg cgggggcaac agcgcatgtc tccgctgagg agcagcagcg gagcgggccg 120

gtgctggtgg agctgttctc gtcgcagggc tgcagcacct cgccggtggc agagctgctg 180

gtgtcgaggc ttgggagagg ggattttgat gggacggatc tgcctcctgt ggtggtgctg 240

gcgttccacg tggactactg ggactacatg ggctggaagg acccatatgg gtcaagccag 300

tggactgtac ggcagaaggc ctatgtcgag gccttgggtc tggacaccat gttcaccccc 360

cagattgtcc ttcaaggtag ggcccaatgt gttggcaatg atgagagtgc cttgctaacg 420

tcgatcaagg atgcacctag atatcctgca cccgcattcc aggcaacatt ccaaaggcca 480

tcgccggact ccctgcaagt atccctaaca gggtcattga ggtcaaaggt ggacaactac 540

ggtgtcaatg tgatggtggc gctgtacgag aacgggctga tcaccgactg ccccaaggga 600

gagaaccaag gacgtgtcct gtccaacgac tttgtcgtcc gacgacttga aaagctctgc 660

actgtcaaag acattgctgc aaagaagacc atttctggaa ccatcaactt ctccttgtgg 720

gaaggtttca atccagccaa atgtggcatg gccctcttcg ttcagaaccc ttcccatcaa 780

atttttggtt tgcagaactt tcagctgccg gacaattag 819

<210> 2

<211> 272

<212> PRT

<213> Dangshan pear' (Dangshan Pirum)

<400> 2

Met Ala Arg Gly Leu Leu Ala Cys Phe Gly Ser Lys Gly Gly Ser Arg

1 5 10 15

Thr Ser His Glu Lys His Ala Ala Gly Ala Thr Ala His Val Ser Ala

20 25 30

Glu Glu Gln Gln Arg Ser Gly Pro Val Leu Val Glu Leu Phe Ser Ser

35 40 45

Gln Gly Cys Ser Thr Ser Pro Val Ala Glu Leu Leu Val Ser Arg Leu

50 55 60

Gly Arg Gly Asp Phe Asp Gly Thr Asp Leu Pro Pro Val Val Val Leu

65 70 75 80

Ala Phe His Val Asp Tyr Trp Asp Tyr Met Gly Trp Lys Asp Pro Tyr

85 90 95

Gly Ser Ser Gln Trp Thr Val Arg Gln Lys Ala Tyr Val Glu Ala Leu

100 105 110

Gly Leu Asp Thr Met Phe Thr Pro Gln Ile Val Leu Gln Gly Arg Ala

115 120 125

Gln Cys Val Gly Asn Asp Glu Ser Ala Leu Leu Thr Ser Ile Lys Asp

130 135 140

Ala Pro Arg Tyr Pro Ala Pro Ala Phe Gln Ala Thr Phe Gln Arg Pro

145 150 155 160

Ser Pro Asp Ser Leu Gln Val Ser Leu Thr Gly Ser Leu Arg Ser Lys

165 170 175

Val Asp Asn Tyr Gly Val Asn Val Met Val Ala Leu Tyr Glu Asn Gly

180 185 190

Leu Ile Thr Asp Cys Pro Lys Gly Glu Asn Gln Gly Arg Val Leu Ser

195 200 205

Asn Asp Phe Val Val Arg Arg Leu Glu Lys Leu Cys Thr Val Lys Asp

210 215 220

Ile Ala Ala Lys Lys Thr Ile Ser Gly Thr Ile Asn Phe Ser Leu Trp

225 230 235 240

Glu Gly Phe Asn Pro Ala Lys Cys Gly Met Ala Leu Phe Val Gln Asn

245 250 255

Pro Ser His Gln Ile Phe Gly Leu Gln Asn Phe Gln Leu Pro Asp Asn

260 265 270

<210> 3

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

acacggggga ctctagaatg gcgcgtggtc 30

<210> 4

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ttgctcacca tggatccatt gtccggcagc tga 33

<210> 5

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ccagattgtc cttcaaggta gg 22

<210> 6

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ttgcctggaa tgcgggtg 18