A gene PbrbHLH195 regulating lignin synthesis in pear fruit

Abstract

The invention discloses a gene PbrbHLH195 for regulating the synthesis of lignin in pear fruit ， The CDS sequence of the gene is shown in SEQ ID NO.1. The gene was constructed and overexpressed by injecting the vector into the fruit of Dangshan Pear. Compared with the uninjected part and the part injected with empty control GFP, the stone cell and lignin content increased significantly. The gene was constructed and overexpressed by introducing the vector into Arabidopsis. PbrbHLH195 The secondary cell walls of the vessel cells and intervascular fiber cells of the transgenic Arabidopsis thaliana were significantly thickened, the accumulation of lignin in the stems increased, and the expression of genes related to lignin biosynthesis increased significantly. The discovery of this gene provides a new gene resource for fruit quality breeding and is an important candidate gene for future genetic engineering to improve fruit quality breeding.

Description

Technical Field

[0001] The invention belongs to the field of plant genetic engineering, and particularly relates to a gene PbrbHLH195 for regulating the biosynthesis of lignin in pear fruit. Background Art

[0002] Pear (Pyrus spp.) is one of the most important fruit trees in the world. It is grown in all temperate countries and is an effective supply of important agricultural products. The increase in stone cells is one of the main reasons for the decline in pear fruit quality, which directly affects the economic benefits of the pear industry. Stone cells are a type of cell unique to pear fruit. They cause problems such as rough flesh and less juice, and are an important factor affecting fruit quality. The formation of stone cells is closely related to the thickening of the secondary cell wall (SCW) and the deposition of lignin.

[0003] Pear (Pyrus spp.), belonging to the Rosaceae family, has a long history of cultivation, strong adaptability, and is widely distributed in temperate and subtropical regions. It is one of the most important fruit trees in the world. Stone cells are a type of cell unique to pears, which are formed mainly through the thickening of secondary cell walls and the accumulation of lignin (Tao et al., 2009). Based on the development of high-throughput sequencing technology, some key genes that regulate the formation of stone cells in pear fruits have been identified. However, due to the complexity of the stone cell formation mechanism, the regulatory network behind it has not been fully resolved. Therefore, further exploring the key factors of stone cell formation is of great significance for accelerating the improvement of pear fruit quality.

[0004] The bHLH transcription factor family is the second largest eukaryotic transcription factor family in plants, and plays an important regulatory role in plant growth and development and abiotic stress. bHLH has a highly conserved domain, consisting of about 60 amino acids, with two functionally different regions, one is the basic region containing 13-17 basic amino acids at the N-terminus, which can bind to the cis-acting element E-BOX (5'-CACGTG-3') on the target gene, and the other is the HLH (helix-loop-helix) region at the C-terminus, which is composed of two α-helices connected by a loop of variable length. Proteins containing the HLH motif can form homodimers or heteromultimers with other proteins and contribute to the specific binding of DNA (Feller et al, 2011). At present, bHLH transcription factors have been reported in crops such as rice (Li et al, 2006), peanut (Gao et al, 2017), corn (Zhang et al, 2018), poplar (Lorenzo et al, 2010), and tomato (Wang et al, 2015). Research on bHLH transcription factors involved in the regulation of secondary metabolism has also achieved certain results.

[0005] bHLH transcription factors are widely present in various tissues of higher plants and participate in various signal transductions, anabolism, and responses to adverse stresses. Some members are involved in secondary cell wall development and lignin accumulation. For example, in chrysanthemum, the bHLH protein CmHLB regulates lignification by interacting with CmKNAT7 (Zhao et al., 2022). The 'EjbHLH14-EjHB1-EjPRX12' cascade has been shown to be involved in low temperature-induced lignin deposition in loquat fruit (Zhang et al., 2022). In rice, overexpression of OsbHLH034 increased lignin accumulation (Onohata and Gomi, 2020). The above is the research on bHLH transcription factors in lignin deposition, but the research on this transcription factor in pear in regulating cell lignification has not been reported. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention aims to provide a key gene for the formation of stone cells in pear fruit, which is isolated and cloned from the variety 'Dangshan Pear' (Pyrus bretschneideri) with high stone cell content. The applicant named it PbrbHLH195, and its CDS sequence is shown in SEQ ID NO.1, and its corresponding protein sequence is shown in the sequence table SEQ ID NO.2. The discovery of this gene may provide new insights into lignin synthesis and quality improvement of pear fruit.

[0007] Another object of the present invention is to provide an application of the gene PbrbHLH195 involved in the synthesis of pear lignin. The gene was constructed into an overexpression vector and introduced into Arabidopsis thaliana by Agrobacterium-mediated genetic transformation. The obtained transgenic material was verified by biological function, indicating that the cloned PbrbHLH195 gene of the present invention has the function of promoting lignin accumulation and secondary cell wall thickening.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] In a first aspect, the present invention provides the use of gene PbrbHLH195 in the following (A1)-(A3):

[0010] (A1) Application of increasing lignin content in plants;

[0011] (A2) Use in the preparation of products for increasing the lignin content in plants;

[0012] (A3) Application in breeding for increasing the lignin content in plants;

[0013] The CDS sequence of the gene PbrbHLH195 is shown in SEQ ID NO.1.

[0014] In a second aspect, the present invention also provides the use of the protein encoded by the gene PbrbHLH195 in the following (A1)-(A3):

[0015] (A1) Application of increasing lignin content in plants;

[0016] (A2) Use in the preparation of products for increasing the lignin content in plants;

[0017] (A3) Application in breeding for increasing the lignin content in plants;

[0018] The amino acid sequence of the protein is shown in SEQ ID NO.2.

[0019] The * indicates a stop codon. The secondary structure of the protein is mainly irregular coil and α-helix, which is a stable protein. The number of amino acids in the protein is 688.

[0020] The third invention, the present invention also provides the use of a recombinant expression vector and a transient expression vector containing the gene PbrbHLH195 in the following (A1)-(A3):

[0021] (A1) Application of increasing lignin content in plants;

[0022] (A2) Use in the preparation of products for increasing the lignin content in plants;

[0023] (A3) Application in breeding for increasing the lignin content in plants.

[0024] The present invention can use existing plant expression vectors to construct a recombinant expression vector containing the gene PbrbHLH195.

[0025] When the gene PbrbHLH195 is used to construct a recombinant plant overexpression vector, a cauliflower mosaic virus (CAMV) 35S strong promoter can be added before its transcription start nucleotide; when the gene of the present invention is used to construct a plant expression vector, an ATG start codon is used, but it must be the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence.

[0026] In order to facilitate the identification and screening of transgenic plants, the plant expression vectors used are processed and genes (luciferase genes) that encode luminescent compounds expressed in plants and antibiotic markers with resistance (kanamycin markers) are added. Considering the safety of transgenic plants, no selective marker genes are added and transformed plants can be directly screened with hygromycin.

[0027] In a specific embodiment, the backbone vector of the recombinant expression vector is pCAMBIA1300-GFP.

[0028] In a fourth aspect, the present invention also protects the use of a recombinant bacterium containing the gene PbrbHLH195 described above in the following (A1) to (A3):

[0029] (A1) Application of increasing lignin content in plants;

[0030] (A2) Use in the preparation of products for increasing the lignin content in plants;

[0031] (A3) Application in breeding for increasing the lignin content in plants.

[0032] In a specific embodiment, the use is achieved by transferring the gene PbrbHLH195 into the target plant for overexpression.

[0033] In one embodiment of the present invention, the gene encoding the protein is introduced into Arabidopsis thaliana using pCAMBIA1300-GFP, a vector for directing the expression of foreign genes in plants, to obtain transgenic Arabidopsis thaliana plants. The expression vector carrying the gene can be transformed into Arabidopsis thaliana by using the Agrobacterium-mediated method (flower infection method), and the transformed Arabidopsis thaliana seeds are collected.

[0034] In one embodiment of the present invention, the gene encoding the protein was transiently introduced into the young fruit of 'Dangshan Pear' 35 days after flowering using pCAMBIA1300-GFP, and the transiently injected fruit was obtained to measure its related indicators.

[0035] The plant described in the present invention can be either a monocot or a dicot, such as Arabidopsis thaliana, pear, etc.

[0036] The present invention also relates to the application of the gene in the genetic improvement of fruit quality. The gene is overexpressed in Arabidopsis, and the obtained transgenic strains are verified by biological functions. The lignin content is significantly increased, the secondary cell walls of stem vascular cells are significantly thickened, and the expression level of genes related to lignin synthesis is also significantly increased.

[0037] In a fifth aspect, the present invention protects a method for increasing the lignin content of pear fruit, which is achieved by increasing the expression level of the gene PbrbHLH195 in pear.

[0038] In a specific embodiment, the method is achieved by transferring the gene PbrbHLH195 into the target plant for overexpression.

[0039] In a sixth aspect, the present invention protects a method for increasing the lignin content in Arabidopsis thaliana, wherein the method is achieved by increasing the expression level of the gene PbrbHLH195 in Arabidopsis thaliana.

[0040] In a specific embodiment, the method is achieved by transferring the gene PbrbHLH195 into the target plant for overexpression.

[0041] Beneficial Effects

[0042] The gene PbrbHLH195 for regulating the synthesis of lignin in pear fruit provided by the present invention has the following beneficial effects compared with the prior art:

[0043] (1) This paper first discovered that PbrbHLH195 can positively regulate the content of lignin in pear fruit.

[0044] (2) The gene PbrbHLH195 provided by the present invention was overexpressed in Arabidopsis thaliana, and the obtained transgenic strains were verified by biological functions. The lignin content was significantly increased, the secondary cell walls of stem vascular cells were significantly thickened, and the expression levels of genes related to lignin synthesis were also significantly increased.

[0045] (3) The discovery of the gene PbrbHLH195 in the present invention provides a new gene resource for fruit quality breeding and is an important candidate gene for future genetic engineering to improve fruit quality breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1-Figure 5 This is the transient overexpression analysis of PbrbHLH195 in pear fruit.

[0047] Figure 1 This is the phloroglucinol-hydrochloric acid staining image of 'Dangshan Pear' after PbrbHLH195 was overexpressed for 7 days. 35S represents the empty control, 35S-PbbHLH195 (also labeled as 35S::PbbHLH195) represents the overexpression of PbrbHLH195 mediated by the 35S strong promoter, and the scale bar = 1 cm.

[0048] Figure 2 is the expression level of overexpressed PbrbHLH195 in pear fruit.

[0049] Figure 3 Lignin content of overexpressed PbrbHLH195 in pear fruit.

[0050] Figure 4 is the stone cell content of overexpressed PbrbHLH195 in pear fruit.

[0051] Figure 5 is the expression level of lignin-related genes.

[0052] Figure 6-Figure 10 This is the overexpression analysis of PbrbHLH195 in Arabidopsis.

[0053] Figure 6 The phenotypes of wild-type and PbrbHLH195 overexpressing plants after 55 days of growth. Scale bar = 5 cm.

[0054] Figure 7 is qRT-PCR analysis, indicating that PbrbHLH195 was successfully overexpressed in the transgenic lines.

[0055] Figure 8 Lignin content analysis of wild-type and PbrbHLH195 overexpressing plants.

[0056] Fig. 9 Toluidine blue and phloroglucinol-HCl staining and UV-excited lignin autofluorescence observation of paraffin sections of stems of wild-type and PbrbHLH195-overexpressing Arabidopsis plants. Scale bar = 50 μm.

[0057] Fig.10 Figure 2 Transmission electron microscopy analysis of interfascicular fibers and vessel cells in wild-type and PbrbHLH195 transgenic plants (scale bar = 10 μm) and statistical analysis of secondary cell wall (SCW) thickness. More than 20 cells were counted in each line (*p < 0.05, **p < 0.01). DETAILED DESCRIPTION

[0058] The present invention is described in detail below in conjunction with specific embodiments. Based on the following description and embodiments, those skilled in the art can determine the essential features of the present invention, and can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention to make it suitable for various uses and conditions.

[0059] The experimental methods in the following examples without specifying specific conditions are generally based on the known methods in the art. The experimental materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores.

[0060] Example 1 Obtaining the Pear PbrbHLH195 Gene

[0061] According to the PbrbHLH195 gene sequence, a specific primer pair for amplifying the sequence was designed using Primer Premier 5.0.

[0062] The specific steps are as follows:

[0063] Using Dangshan Pear cDNA as a template, Phanta Max Super-Fidelity DNA Polymerase (Vazyme, China) was used for amplification. The amplification system is shown in Table 1, the amplification program is shown in Table 2, and the amplification primer sequences are:

[0064] PbrbHLH195-F: gagaacacgggggactctagaATGGCTGCTTTAGTGTATATTGTGATC;

[0065] PbrbHLH195-R: gcccttgctcaccatggatccCATCAGTAAAACCGCTGTGAAGC.

[0066]

[0067]

[0068] The amplified product was purified and recovered using the FastPure Gel DNA Extraction Mini Kit (Vazyme, China). The pCAMBIA1300-GFP overexpression vector (TransGen, China) was digested with Xba I and BamH I restriction endonucleases (Themo Scientific, China). The digestion system is shown in Table 3. After reacting at 37°C for 2 hours, the digested vector was purified and recovered using the FastPure Gel DNA Extraction Mini Kit (Vazyme, China). The purified product and the double-digested vector were ligated using the ClonExpress II One Step Cloning Kit (Vazyme, China) to construct the expression vector 35s-PbrbHLH195. The ligation system is shown in Table 4. After incubation at 37°C for 30 minutes, the competent E. coli DH5α (Tsingke, China) was transformed. The E. coli transformation method is as follows:

[0069] (1) Add 20 μL of the ligation product to 50 μL of E. coli competent DH5α (Tsingke, China) cells melted in an ice bath, mix gently, and place on ice for 30 min;

[0070] (2) After heat shock in a 42°C water bath for 45 seconds, place in ice for 2 minutes. During this process, the centrifuge tube should not be shaken;

[0071] (3) Add 600 μL of LB liquid medium without antibiotics and culture at 37°C and 200 rpm for 1-2 h to allow the bacteria to recover;

[0072] (4) After centrifugation at 4000 rpm for 3 min, discard 500 μL of the supernatant, resuspend and take 100 μL of the revived competent cells and evenly apply them to LB solid culture medium containing the corresponding antibiotics. Place the culture dish upside down in a 37°C constant temperature incubator and culture overnight;

[0073]

[0074]

[0075]

[0076] 12-16h after transformation, single clones on the plate were picked and placed in 1mL centrifuge tubes, LB liquid culture medium containing corresponding antibiotics was added, and the culture was shaken at 37℃ until the bacterial solution became turbid, and then positive identification was performed. The reagent used was 2 × Rapid Taq MasterMix (Vazyme, China), the reaction system is shown in Table 5, and the PCR program is shown in Table 6. After obtaining the positive clone, the positive clone was sent to Shanghai Bioengineering Company for sequencing, and the gene sequence of PbrbHLH195 was obtained based on the sequencing results.

[0077]

[0078] The gene sequence of the gene in pear was cloned by the expression vector PbrbHLH195-F. After sequencing, it was found that the GFP vector separated a 2064bp CDS sequence, whose sequence was SEQ ID NO.1 and the length was 2064bp; the gene encodes a protein of 688 amino acids, whose sequence is SEQ ID NO.2.

[0079] The correct bacterial solution was expanded and used for bacterial strain and plasmid extraction. The bacterial solution was kept in a ratio of bacterial solution: glycerol = 7:3 (V:V), mixed, quick-frozen in liquid nitrogen, and stored at -80°C for later use. Plasmid extraction used the FastPurePlasmid Mini Kit (Vazyme, China) to extract plasmids.

[0080] The competent Agrobacterium GV3101 (Weidi Biotechnology Co., Ltd., Shanghai, China) was thawed on ice, and then the recombinant vector plasmid containing the target gene was added. After standing on ice for 5 minutes, it was quickly frozen in liquid nitrogen for 5 minutes, then heat-shocked at 37℃ for 5 minutes, and on ice for 5 minutes. Then, 700μL of antibiotic-free liquid LB was added and shaken for 3 hours (shaker setting 28℃, 250rpm / min). Centrifuged at low speed for 5 minutes, discarded part of the supernatant, and 100μL was resuspended and evenly spread on LB solid medium containing antibiotics (kana 50μg / mL; rifampicin 50μg / mL) with a coating rod. Inverted in a 28℃ incubator, after 48 hours, a single clone point was picked with a sterile toothpick for PCR verification. The PCR amplification system is shown in Table 6. The Agrobacterium bacterial liquid was stored in the ratio of bacterial liquid: glycerol = 7:3 (V:V), and mixed, quickly frozen in liquid nitrogen, and stored at -80℃ for later use.

[0081] Example 2 Analysis of stone cell and lignin content in pulp

[0082] The stone cell content in the fruit pulp was determined by the freezing separation method. Three fruits of the same size (young fruits were mostly taken), the peel was removed, and the edible part of the fruit was taken according to the quartering method. 100 g was weighed and placed in a -20℃ refrigerator for 24 h. After thawing at room temperature, 200 ml of distilled water was added and the pulp was thawed with a tissue masher (1000-1500 r·min -1 ) and crush for 5 min. Then transfer the homogenate to a 1000ml beaker, stir with a glass rod for 1 min, let it stand for 5 min, let the stone cells fully settle at the bottom of the beaker, pour out the upper suspension, suspend the precipitate in 0.5 M hydrochloric acid solution for 30 min, stir every 5 min, remove the floating matter, rinse with distilled water 5-6 times, collect the first few suspensions and rinse. Combine the obtained stone cells, filter with coarse filter paper, and finally separate the pure stone cells, dry to constant weight, and weigh.

[0083] Use a ten-thousandth balance to accurately weigh 0.01g of pulp powder sample, grind it into a homogenate with 95% ethanol, dilute it to 5ml, centrifuge it at 12000g for 2min, discard the supernatant, wash it with 95% ethanol 3 times, and then wash it with alcohol: n-hexane = 1:2 (V / V) 3 times, and blow it dry in a fume hood. Then add 2ml of 25% bromoacetyl acetate solution, warm it in a 70℃ water bath for 30min, add 0.9ml of 2M NaOH solution to terminate the reaction, then add 5ml of acetic acid and 0.1ml of 7.5M hydroxylamine chloride solution, dilute it to 10mL with glacial acetic acid, measure the absorbance at 280nm, and finally check the lignin content through the lignin standard sample (Sigma-Aldrich, USA) curve (Syros et al., 2004).

[0084] Example 3 Analysis of transient expression in pear fruit

[0085] The culture conditions of Agrobacterium strain were the same as those in Example 1. After discarding the supernatant, the bacterial precipitate was resuspended in the infiltration medium (10 mM MgCl2, 10 mM MES, 200 μM AS, pH 5.6, OD600 = 1.0). After 2-4 h of induction in the dark at room temperature, the infiltration was injected into the equatorial part of the young fruit of 'Dangshan Suli' at 35 DAF.

[0086] The results showed that 7 days after injection, an increase in lignin staining was observed at the injection sites of PbrbHLH195 compared with the corresponding non-injection sites and empty vector annotations ( Figure 1 ). PbrbHLH195 was successfully overexpressed at the injection site ( Figure 2 ). Stone cells ( Figure 3 ) and lignin ( Figure 4 ) content increased significantly, and the expression of lignin synthesis-related genes at the injection site also increased significantly after overexpression ( Figure 5 ). Therefore, PbrbHLH195 positively regulates the lignin content in pear fruit.

[0087] Example 4 Overexpression of Arabidopsis

[0088] (1) The Agrobacterium strain containing the expression vector 35S-PbrbHLH195 (hereinafter referred to as the PbrbHLH195 Agrobacterium strain) verified by PCR in Example 1 was added with 100 μL of the PbrbHLH195 Agrobacterium strain into 20 mL of LB liquid medium (containing 50 μg / mL kanamycin and 50 μg / mL rifampicin), and cultured at 28°C in a shaking incubator for 16 h;

[0089] (2) After centrifugation at 4000 rpm for 10 min, discard the supernatant and resuspend the cells in an equal volume of transformation medium (2.25 g / L MS medium, 5 g / L sucrose, 10 μg / L 6-BA, pH adjusted to 5.7 with KOH), and add SILWETL-77 to a final concentration of 0.025%;

[0090] (3) Cut off the siliques and opened flowers of the wild-type Arabidopsis thaliana to be transformed (bolting 10-15 cm);

[0091] (4) Soak the preserved Arabidopsis flowers in the bacterial solution and evacuate to 0.6-0.8 KPa for 5 minutes;

[0092] (5) Incubate in dark at 22°C for 24 h, then remove the plants and culture them normally. Harvest the seeds for screening.

[0093] The harvested T0 generation seeds were planted in screening medium (containing MS medium, 30 g / L sucrose, 0.75% agar, 20 mg / L hygromycin, 100 mg / L timentin and 100 mg / L carboxymethyl) for screening, and the grown seedlings were moved into plastic pots with a mixture of vermiculite and soil (1:2) and cultured in a greenhouse with a photoperiod of 16 h light / 8 h dark and a relative humidity of 40%. The seeds were collected after they matured.

[0094] Example 5 Physiological index determination of transgenic Arabidopsis

[0095] The T2 seeds and wild-type seeds of Example 4 were planted in MS medium to form T3 transgenic plants for physiological quantity measurement. Wild-type and T3 transgenic Arabidopsis seeds were germinated in a petri dish for one week, and the root length was measured using a ruler (50 replicates), and transplanted into a plastic pot with a mixture of vermiculite and soil (1:2), and cultured in a greenhouse with a photoperiod of 16h light / 8h dark and a relative humidity of 40%. The height of the aboveground part of the plant was measured and photographed at the flowering stage (5 weeks old) (50 replicates) ( Figure 6 ). At maturity, the height of the above-ground part of the plant was measured and photographed, and the callose content of the primary inflorescence stems was measured; the primary inflorescence stems were dried to constant weight and ground into powder using a sample grinder to measure the lignin content.

[0096] After 8 weeks of Arabidopsis cultivation, three T3 transgenic lines and wild-type Arabidopsis were randomly selected, and the stems (about 5 cm) of Arabidopsis were cut and fixed in 0.1 M sodium bicarbonate buffer (pH 7.4) containing 2.5% glutaraldehyde and 2% paraformaldehyde at 4 °C (Kim et al., 2017). The stems were rinsed with 0.1 M dimethicone buffer (pH 7.4) and then 1% OsO4 at 4 °C. The sections were dehydrated using different concentrations of ethanol (10, 20, 30, 40, 50, 60, 70, 80, 90, and 100%) and embedded in butyric epoxy resin. After 2 days, thin (80–90 nm) sections were cut using an ultramicrotome (LKB-8800, Sweden) and stained with uranyl acetate and lead citrate on nickel grids. Finally, the sections were observed using a transmission electron microscope (Hitachi, Tokyo, Japan).

[0097] Prepare paraffin sections and stain with toluidine blue and phloroglucinol as follows:

[0098] Ethanol dehydration: Use 75%-100% ethanol, dehydrate in 5 levels for 2 hours.

[0099] Transparency: ethanol and xylene are gradually transparent according to the proportion, anhydrous ethanol: xylene = 3:1 elution for 40 minutes;

[0100] Treat with anhydrous ethanol: xylene = 1:1 for 40 min; treat with anhydrous ethanol: xylene = 1:3 for 4 min; soak in pure xylene for 1 h, repeat once.

[0101] Wax dipping: add half volume of xylene, add half volume of paraffin, heat to 75°C in an oven, infiltrate the melted paraffin for 2h, and repeat once.

[0102] Embedding: After the wax immersion is completed, use tweezers to pick up the material and place it in a paper box and embed the material with pure wax solution that has melted into liquid.

[0103] Trimming and sectioning: The embedded material was trimmed into a trapezoidal shape according to the position of the material and sectioned using a Leica RM 2015 hand-cranked microtome with a thickness of approximately 6 μm.

[0104] Spreading and sticking: Pick up the cut sample gently with tweezers, put it into a 35-45℃ water bath for spreading, pick it up with a glass slide after the wax sheet is spread, and put it into a 40℃ oven for drying.

[0105] Dewaxing: insert the slide with the sample into xylene, dewax for 15 minutes, wash for 2 minutes with xylene: anhydrous ethanol = 1:1, rinse with anhydrous ethanol in different levels (100%, 95%, 90%, 85%, 80%, 75%, 70%), and wash for 2 minutes from high concentration to low concentration.

[0106] Staining: Stain with toluidine blue staining solution for 24 hours, wash twice with 95% ethanol, anhydrous ethanol, xylene: anhydrous ethanol = 1:1, and pure xylene for 30 seconds respectively, cover the slides with neutral gum, and dry them in a 40℃ oven.

[0107] The images were observed and collected using an upright fluorescence microscope.

[0108] The results showed that qRT-PCR confirmed that PbrbHLH195 was successfully overexpressed in the transgenic lines ( Figure 7 ). The lignin content measurement showed that the content of G-type lignin and H-type lignin in the inflorescence stem increased significantly ( Figure 8 In addition, paraffin sections of stems of wild-type and PbrbHLH195 transgenic plants were stained with toluidine blue, phloroglucinol-hydrochloric acid, and observed for lignin autofluorescence under ultraviolet light excitation. It was found that the staining of lignin tissue in transgenic plants was stronger than that in wild-type plants, and the lignin autofluorescence signals of vascular cells and interfascicular fiber cells were stronger than those in wild-type plants ( Fig. 9 In addition, transmission electron microscopy was used to examine the anatomical characteristics of the secondary cell walls in the stems of PbrbHLH195 overexpressing plants. The results showed that the SCW thickness of interfascicular fiber cells and vascular cells in PbrbHLH195 transgenic plants was significantly higher than that in the wild type ( Fig.10), further confirming that PbrbHLH195 positively regulates lignin deposition. Collectively, these findings suggest that PbrbHLH195 promotes lignin deposition and SCW thickening during stone cell development.

[0109] SEQ ID NO.1 Biological source: Pear; Latin name: Pyrus bretschneideri

[0110]

[0111] SEQ ID NO.2 Biological source: Pear; Latin name: Pyrus bretschneideri

[0112] *

[0113] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the attached claims.

Claims

1. Genes PbBHLH195 Application in the following (A1)-(A3): (A1) Application of increasing the lignin content in pear fruit or Arabidopsis thaliana; (A2) Use in the preparation of a product for increasing the lignin content in pear fruit or Arabidopsis thaliana; (A3) Application in breeding for increasing the lignin content in pear fruit or Arabidopsis thaliana; The gene PbBHLH195 The CDS sequence is shown in SEQ ID NO.1; The application involves PbBHLH195 This can be achieved by overexpressing the gene in pear fruit or Arabidopsis thaliana.

2. The gene according to claim 1 PbBHLH195 The encoded protein is used in the following (A1)-(A3): (A1) Application of increasing the lignin content in pear fruit or Arabidopsis thaliana; (A2) Use in the preparation of a product for increasing the lignin content in pear fruit or Arabidopsis thaliana; (A3) Application in breeding for increasing the lignin content in pear fruit or Arabidopsis thaliana; The amino acid sequence of the protein is shown in SEQ ID NO.2; The application involves PbBHLH195 This can be achieved by overexpressing the gene in pear fruit or Arabidopsis thaliana.

3. Containing the gene according to claim 1 PbBHLH195 Use of the recombinant expression vector and / or transient expression vector in the following (A1)-(A3): (A1) Application of increasing the lignin content in pear fruit or Arabidopsis thaliana; (A2) Use in the preparation of a product for increasing the lignin content in pear fruit or Arabidopsis thaliana; (A3) Application in breeding for increasing the lignin content in pear fruit or Arabidopsis thaliana; The gene PbBHLH195 The CDS sequence is shown in SEQ ID NO.1; The application involves PbBHLH195 This can be achieved by overexpressing the gene in pear fruit or Arabidopsis thaliana.

4. The use according to claim 3, characterized in that: The backbone vector of the recombinant expression vector is pCAMBIA1300-GFP.

5. Containing the gene according to claim 1 PbBHLH195 The use of the recombinant bacteria in the following (A1)-(A3): (A1) Application of increasing the lignin content in pear fruit or Arabidopsis thaliana; (A2) Use in the preparation of a product for increasing the lignin content in pear fruit or Arabidopsis thaliana; (A3) Application in breeding for increasing the lignin content in pear fruit or Arabidopsis thaliana; The gene PbBHLH195 The CDS sequence is shown in SEQ ID NO.1; The application involves PbBHLH195 This can be achieved by overexpressing the gene in pear fruit or Arabidopsis thaliana.

6. A method for increasing the lignin content of pear fruit, characterized in that: The method involves PbBHLH195 The gene is transferred into pear fruit for overexpression. PbBHLH195 The CDS sequence is shown in SEQ ID NO.1 。 7. The method according to claim 6, characterized in that The method improves the gene expression in pear PbBHLH195 to achieve the expression level.

8. A method for increasing the lignin content in Arabidopsis thaliana, characterized in that: The method involves PbBHLH195 The gene is transferred into Arabidopsis thaliana and overexpressed. PbBHLH195 The CDS sequence is shown in SEQ ID NO.1 。 9. The method according to claim 8, characterized in that The method improves the gene expression in Arabidopsis thaliana PbBHLH195 to achieve the expression level.

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