CN117327710A - Intercellular continuous filament callose binding protein gene for regulating and controlling pear lignin synthesis - Google Patents

Abstract

The invention discloses an intercellular continuous filament callus binding protein gene PbPDCB16 for regulating and controlling pear lignin synthesis, wherein the CDS sequence of the gene is shown as SEQ ID NO. 1. Injecting the gene construction overexpression vector into the Dangshan pear fruits, wherein compared with the uninjected part and the injection of no-load contrast GFP, the lignin content is reduced, and the callose content is increased; the gene construction overexpression vector is introduced into Arabidopsis, the obtained PbPDCB16 transgenic Arabidopsis has obviously thinned duct, wood fiber and fiber cell wall among fiber bundles, the quantity of intercellular continuous filaments is reduced, the aperture of the intercellular continuous filaments is blocked, the accumulation of lignin in stems is reduced, the growth of plants is inhibited, and the expression level of genes related to lignin biosynthesis is reduced. Provides new gene resources for fruit quality breeding, and is an important candidate gene for future genetic engineering improvement of fruit quality breeding.

Description

Intercellular continuous filament callose binding protein gene for regulating and controlling pear lignin synthesis

Technical Field

The invention belongs to the field of plant genetic engineering, and particularly relates to an intercellular continuous filament binding protein gene PbPDCB16 for regulating and controlling pear lignin synthesis.

Background

Pears (Pyrus) are native to China, which is the country with the highest yield in the world (FAO, 2022), with yield up to 1610 tens of thousands of tons. Stone cells are a unique cell in pear fruits, whose number and size can directly affect the texture of the fruit (Zhang et al, 2017). Cells thicken by primary wall lignification and further thicken and harden on Secondary Cell Walls (SCW) to form stone cells. Lignin is the major component of stone cells (Tao et al 2009). Therefore, reducing the lignin content of the pear fruit can prevent the secondary wall thickening and lignin deposition of parenchyma cells, thereby inhibiting the development of stone cells.

It is important to find genes that can inhibit parenchyma thickening and lignin deposition in parenchyma cells, and thus inhibit the development of stone cells.

Disclosure of Invention

The invention aims to provide a pear fruit callose binding protein gene which is a gene of an plasmodesmata callose binding protein separated and cloned from Dangshan pear' (Pyrus bretschneideri) with high lignin content, and the gene is named as PbPDCB16 by the applicant, wherein the CDS sequence of the gene is shown as SEQ ID NO.1, and the corresponding protein sequence of the gene is shown as a sequence table SEQ ID NO.2. The discovery of the gene may provide new insight for the study of lignin synthesis.

It is still another object of the present invention to provide a gene PbPDCB16 involved in inhibiting pear lignin synthesis and its use in improving fruit quality. The gene is constructed into an overexpression vector, and the overexpression vector is introduced into arabidopsis through agrobacterium-mediated genetic transformation, and biological function verification shows that the cloned PbPDCB16 gene has the function of regulating callose accumulation and further inhibiting lignin synthesis.

The aim of the invention is achieved by the following technical scheme:

in a first aspect, the present invention provides a gene PbPDCB16, the CDS sequence of which is shown in SEQ ID NO. 1.

In a second aspect, the invention also provides a protein encoded by the gene PbPDCB16, and the amino acid sequence of the protein is shown as SEQ ID NO.2.

Wherein, the stop codon is represented, the secondary structure of the protein is mainly irregular curl and alpha-helix, and the protein is stable. The number of amino acids of the protein was 219.

In a third aspect, the invention also provides a recombinant expression vector, a transient expression vector, or a recombinant bacterium comprising the gene PbPDCB16.

The invention can construct a recombinant expression vector containing the gene PbPDCB16 by using the existing plant expression vector.

When the gene PbPDCB16 is used for constructing a recombinant plant overexpression vector, a cauliflower mosaic virus (CAMV) 35S strong promoter can be added before transcription initiation nucleotides; when the gene of the present invention is used to construct a plant expression vector, the ATG initiation codon is used, but must be the same as the reading frame of the coding sequence to ensure proper translation of the entire sequence.

To facilitate the identification and selection of transgenic plants, the plant expression vectors used are processed, and genes encoding compounds which produce luminescence (luciferase genes), antibiotic markers with resistance (kanamycin markers) expressed in plants are added. From the safety aspect of transgenic plants, the transformed plants can be directly screened by hygromycin without adding any selectable marker gene.

In a fourth aspect, the invention also provides a primer pair for amplifying the full length or any fragment of the gene PbPDCB16.

In one embodiment, applicants designed a pair of primers and cloned the full-length cDNA sequence of the gene PbPDCB16 using PCR techniques.

The nucleotide sequences of the PCR primer pairs are as follows:

forward primer 1: namely PbPDCB16-GFP-F, gagaacacgggggactctagaATGGCTGCTTTAGTGTATATTGTGATC;

reverse primer 1: namely PbPDCB16-GFP-R, gcccttgctcaccatggatccCATCAGTAAAACCGCTGTGAAGC.

In a fifth aspect, the invention also provides the use of the gene PbPDCB16, the protein, the recombinant expression vector, the transient expression vector or recombinant bacteria or the primer in plant breeding.

In some embodiments, the invention provides the use of the gene PbPDCB16, the protein, the recombinant expression vector, the transient expression vector, or the recombinant bacterium or the primer in the cultivation of plants with low lignin content.

In other embodiments, the invention provides the use of the gene PbPDCB16, the protein, the recombinant expression vector, the transient expression vector or the recombinant bacterium or the primer in genetic improvement of fruit quality. In particular to application in cultivating fruits with low lignin content.

In one embodiment of the present invention, a vector that directs the expression of a foreign gene in plants using pCAMBIA1300 (GFP), a gene encoding the protein is introduced into arabidopsis thaliana, and a transgenic arabidopsis thaliana plant can be obtained. The expression vector carrying the gene can be transformed into Arabidopsis thaliana by using an Agrobacterium-mediated method (floral dip method), and transformed Arabidopsis thaliana seeds can be harvested.

In one embodiment of the invention, pCAMBIA1300 (GFP) is used to guide the expression vector of exogenous gene in plant, the gene encoding the protein is transiently introduced into young fruit of Dangshan pear fruit 35DAF (DAF, days after flowering), and the transiently injected fruit is obtained to determine the relevant index.

The plants of the invention may be both monocotyledonous and dicotyledonous plants, such as: arabidopsis thaliana, pear, etc.

The invention also relates to application of the gene in genetic improvement of fruit quality, the gene is overexpressed in arabidopsis thaliana, and the obtained transgenic strain is verified by biological functions to have obviously increased callose content, obviously reduced quantity of plasmodesmata, inhibited expression quantity of genes related to lignin synthesis and obviously reduced lignin content.

According to the invention, the space-time expression of the PbPDCB16 gene in different development stages of pear fruits is analyzed by using qRT-PCR technology, and the lignin content in the pear fruits in different development stages is analyzed, so that the analysis result shows that the PbPDCB16 gene has high expression quantity in early development stages of the fruits and has an opposite trend to the lignin change trend. The cloned plasmodesmata callose binding protein gene PbPDCB16 has important significance for reducing the pear lignin content and breeding research.

Drawings

Fig. 1 shows the lignin content change in pulp during pear fruit development, and the difference is significant in different lower case letters on the column (P < 0.05).

FIG. 2 is a DNA identification of Arabidopsis positive seedlings transformed with the PbPDCB16 gene.

FIG. 3 is an identification of Arabidopsis thaliana transformed with the PbPDCB16 gene. (a) Relative expression pattern of PbPDCB16 in transgenic arabidopsis; (b, e) root length of WT and PbPDCB16 transgenic arabidopsis seedlings after 9 d; after 8 weeks, the stem diameter (c) and the plant height (d, f) of the WT and PbPDCB16 transgenic lines arabidopsis seedlings. The different lower case letters on the bars indicate significant differences (P < 0.05).

FIG. 4 shows the measurement of physiological indexes of Arabidopsis thaliana transformed with the PbPDCB16 gene. Lignin content (fig. 4 a) and callose content (fig. 4 b) of WT and transgenic lines. The different lower case letters on the bars indicate significant differences (P < 0.05).

Fig. 5 is that overexpression of PbPDCB16 affects PD and cell wall formation. PD status of WT (FIG. 5 a) and transgenic lines (FIGS. 5 b-d); morphology and number of PD in WT (FIG. 5 e) and overexpressed Arabidopsis (FIGS. 5 f-h) stems.

FIG. 6 is an analysis of the overexpression of PbPDCB16 in 35DAF pear fruit (FIG. 6 a), A indicates empty vector GFP injection, B indicates PbPDCB16-GFP injection site, and no injection site is labeled a, B. Fig. 6a is a photograph taken 7d after infestation. (FIGS. 6 b-d) expression level, lignin content and callose content of over-expressed PbPDCB16 in pear fruit. The different lower case letters on the bars indicate significant differences (P < 0.05).

Detailed Description

The present invention will be described in detail with reference to specific examples. From the following description and 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.

The following examples do not address the specific conditions of the experimental procedure, and are generally in accordance with means well known in the art. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.

EXAMPLE 1 cloning of full Length CDS of pear PbPDCB16 Gene

Based on the PbPDCB16 gene sequence, a specific Primer pair for amplifying the sequence is designed by utilizing a Primer Premier 5.0.

The method comprises the following specific steps:

the Dangshan pear cDNA is used as a template, phanta Max Super-Fidelity DNA Polymerase (Vazyme, china) is adopted for amplification, an amplification system is shown in a table 1, an amplification program is shown in a table 2, and an amplification primer sequence is as follows:

PbPDCB16-GFP-F：

gagaacacgggggactctagaATGGCTGCTTTAGTGTATATTGTGATC；

PbPDCB16-GFP-R：

gcccttgctcaccatggatccCATCAGTAAAACCGCTGTGAAGC；

TABLE 1 Gene amplification System

TABLE 2 Gene amplification PCR procedure

The amplified product was purified using FastPure Gel DNA Extraction Mini Kit gel recovery kit (Vazyme, china). GFP vector (TransGen, china) was double digested with Xba I and BamH I restriction enzymes (Themo Scientific, china), and the double digested system was shown in Table 3, and after 2 hours of reaction at 37℃the digested vector was purified and recovered using FastPure Gel DNA Extraction Mini Kit gel recovery kit (Vazyme, china). The purified product and the double digested vector were ligated using ClonExpress IIOne Step Cloning Kit ligase independent single fragment rapid cloning kit (Vazyme, china) to construct the expression vector PbPDCB16-GFP, the ligation system is shown in Table 4, and E.coli competent DH 5. Alpha. Chemically Competent Cell (Tsingke, china) was transformed after incubation at 37℃for 30 min. The method for transforming the escherichia coli comprises the following steps:

(1) mu.L of the ligation product was added to 50. Mu.L of ice-bath thawed E.coli competent DH 5. Alpha. Chemically Competent Cell (Tsingke, china) cells, gently mixed, and left on ice for 30min;

(2) Placing in ice for 2min after heat shock for 45s in a water bath at 42 ℃ and keeping the centrifuge tube from shaking;

(3) Adding 600 μl of LB liquid medium without antibiotics, shaking at 37deg.C, culturing at 200rpm for 1-2h to revive bacteria;

(4) Centrifuging at 4000rpm for 3min, discarding 500 μl of supernatant, re-suspending, collecting 100 μl of recovered competent cells, uniformly coating on LB solid medium containing corresponding antibiotics, placing the culture dish upside down in a constant temperature incubator at 37deg.C, and culturing overnight;

TABLE 3 double enzyme cleavage System for GFP vector

TABLE 4 GFP vector ligation System

TABLE 5 Positive identification reaction System

After transformation, monoclonal on a flat plate is picked up in a 1mL centrifuge tube, LB liquid culture medium containing corresponding antibiotics is added, shaking culture is carried out on the flat plate at a temperature of 37 ℃ until bacterial liquid is turbid, and then positive identification is carried out. The reagents were 2X Rapid Taq Master Mix (Vazyme, china), the reaction systems are shown in Table 5, and the PCR procedures are shown in Table 6. After the positive clone is obtained, the positive clone is sent to Shanghai Biotechnology company for sequencing, and the gene sequence of PbPDCB16 is obtained according to the sequencing result.

TABLE 6 Gene amplification PCR procedure

Cloning an expression vector PbPDCB16-GFP to obtain a gene sequence of the gene in pears, and sequencing to find that the GFP vector is separated to obtain a 660bp CDS sequence, wherein the sequence is SEQ ID NO.1, and the length is 660bp; the gene codes protein with 219 amino acids and the sequence is SEQ ID NO.2.

Expanding propagation of a bacterial liquid with correct sequencing, and extracting a strain and a plasmid, wherein the bacterial liquid is preserved according to the ratio: glycerol = 7:3 (V: V), mixing, quick freezing in liquid nitrogen, and preserving at-80deg.C. Plasmid extraction plasmids were extracted using FastPure Plasmid Mini Kit (Vazyme, china) kit.

Thawing Agrobacterium competent GV3101 (Biotechnology limited only, shanghai, china) on ice, adding recombinant vector plasmid containing target gene, standing on ice for 5min, quick-freezing with liquid nitrogen for 5min, heat-shock at 37deg.C for 5min, and ice for 5min, adding 700 μl of antibiotic-free liquid LB, and shake culturing with shaking table for 3h (shaking table set 28 deg.C, 250 rpm/min). The mixture was centrifuged at a low speed for 5min, part of the supernatant was discarded, 100. Mu.L of the suspension was left, and the suspension was spread evenly on LB solid medium containing antibiotics (kana 50. Mu.g/mL; rifampicin 50. Mu.g/mL) with a spreading bar. The mixture is placed in an incubator at 28 ℃ for 48 hours, and then a sterile toothpick is picked up to carry out PCR verification on a monoclonal spot, and a PCR amplification system is shown in Table 6. The agrobacterium strain retaining seed is prepared from the following bacterial strains in proportion: glycerol = 7:3 (V: V), mixing, quick freezing in liquid nitrogen, and preserving at-80deg.C.

EXAMPLE 2 analysis of lignin content in pulp during fruit development

0.01g of pulp powder sample was accurately weighed using a ten-thousandth balance, ground to homogenate using 95% ethanol, fixed to 5ml, centrifuged at 12000rpm for 2min, the supernatant was discarded, washed 3 times with 95% ethanol, and then with ethanol: n-hexane=1:2 (V/V) was washed 3 times and dried in a fume hood for further use. 2mL of 25% bromoacetoacetic acid solution was added, the reaction was stopped by heating in a 70℃water bath for 30min, 0.9mL of 2M NaOH solution was added, 5mL of acetic acid and 0.1mL of 7.5M hydroxylamine chloride solution were added, the volume was fixed to 10mL with glacial acetic acid, the absorbance was measured at 280nm, and finally the lignin content was determined by a lignin standard sample (Sigma-Aldrich, USA) curve (Syros et al 2004).

The variation of lignin content during fruit development was studied in this experiment and lignin content in pear fruits was determined at 15DAF-55DAF (fig. 1). During this period, the lignin content in the pear fruit increases rapidly.

EXAMPLE 3 Arabidopsis thaliana transformation of PbPDCB16 Gene

(1) The correct Agrobacterium strain containing the expression vector PbPDCB16-GFP (hereinafter referred to as PbPDCB16 Agrobacterium strain) was confirmed by PCR in example 1, 100. Mu.L of PbPDCB16 Agrobacterium strain was added to 20mL of LB liquid medium (containing 50. Mu.g/mL kanamycin and 50. Mu.g/mL rifampicin), and shake-cultured at 28℃for 16 hours;

(2) The cells were centrifuged at 4000rpm for 10min, the supernatant was discarded, resuspended in an equivalent volume of transformation medium (2.25 g/L MS medium, 5g/L sucrose, 10. Mu.g/L6-BA, KOH adjusted pH to 5.7) and SILWETL-77 was added to a final concentration of 0.025%;

(3) Cutting off the horned fruits and the flowers of the wild arabidopsis thaliana (10-15 cm for bolting) to be transformed;

(4) Soaking the flowers remained in Arabidopsis thaliana in the bacterial liquid, vacuumizing to 0.6-0.8KPa, and soaking for 5min;

(5) Dark culturing for 24 hours at 22 ℃, then taking out the plants for normal culturing, and harvesting seeds to be screened.

Harvested T0 generation seeds were screened in screening medium (containing MS medium, 30g/L sucrose, 0.75% agar, 20mg/L hygromycin, 100mg/L timentin and 100mg/L carboxylation), the grown seedlings were transferred into plastic pots with a mixture of vermiculite and soil (1:2), cultivated in a greenhouse with 16h light/8 h darkness and 40% relative humidity photoperiod, and seeds were harvested after maturation. The T1 generation transgenic system leaves are taken to extract DNA by using a CTAB method.

And (3) identifying the transgenic arabidopsis positive plant, detecting by using the extracted DNA as a template and using two pairs of primers, adding a gene reverse primer and a PbPDCB16 specific primer to a 35S promoter forward primer, and detecting by agarose gel electrophoresis. The primer sequences were as follows:

35S-GFP-F：5’-TCCTCGGATTCCATTGCCCAGC-3’；

PbPDCB16-GFP-R：5’-CATCAGTAAAACCGCTGTGAAGC-3’。

the results showed that in the T1 generation plants, 20 positive seedlings were identified in total by DNA extraction (fig. 2). RNA was extracted from the positive shoots detected and qRT-PCR detection was performed to detect the expression level findings, wherein lines OE-2, OE-13, OE-19, which exhibited high levels of PbPDCB16 expression, were selected for further analysis (FIG. 3 a).

Example 4 determination of physiological index of PbPDCB16 transgenic Arabidopsis thaliana

The T2 generation seed and wild type seed of example 3 were used to form T3 generation transgenic line plants in MS medium for physiological quantity determination. Wild-type and T3-generation transgenic arabidopsis seeds were germinated in petri dishes for one week, root length was measured using a ruler (50 replicates) and transplanted into plastic pots with a mixture of vermiculite and soil (1:2), and grown in a greenhouse with 16h light/8 h darkness and 40% relative humidity. The aerial parts of the plants were measured for plant height at anthesis (5 weeks of age) and photographed (50 replicates). Measuring the plant height of the overground part of the plant in the mature period, taking the stem of the primary inflorescence, and measuring the callose content; and (3) drying the stems of the primary inflorescences to constant weight, grinding into powder by a sample grinder, and measuring the lignin content.

After 8 weeks of arabidopsis culture, 3T 3-generation transgenic lines and wild type arabidopsis were randomly selected, the stems (about 5 cm) of arabidopsis were cut and fixed in 0.1M sodium dicarbonate buffer (pH 7.4) containing 2.5% glutaraldehyde and 2% paraformaldehyde at 4 ℃ (Kim et al, 2017). The stems were rinsed with 0.1M dimethoxy acid buffer (pH 7.4) and then with 1% OsO4 at 4 ℃. The sections were dehydrated using different concentrations of ethanol (10, 20, 30, 40, 50, 60, 70, 80, 90 and 100%) and embedded in butyl epoxy. After 2 days, thin (80-90 nm) sections were cut with an ultra microtome (LKB-8800, sweden) and stained with uranyl acetate and lead citrate on a nickel grid. Finally, the sections were observed using a transmission electron microscope (Hitachi, tokyo, japan).

Paraffin sections were prepared and safranin fast green stained as follows:

ethanol dehydration: sequentially dehydrating with 75% -100% ethanol for 2h according to 5 grades.

And (3) transparency: ethanol and dimethylbenzene are transparent step by step according to proportion, and absolute ethanol: xylene = 3:1 eluting for 40min;

absolute ethyl alcohol: xylene = 1:1, treating for 40min; absolute ethyl alcohol: xylene = 1:3, treating for 4min; soaking in pure xylene for 1 hr, and repeating once.

Wax dipping: half of the volume of xylene was added, half of the volume of paraffin was added, the temperature was raised to 75 ℃ in an oven, and the melted paraffin was immersed for 2 hours and repeated once.

Embedding: after the wax dipping is finished, the materials are clamped by forceps and placed in a paper box, and the materials are embedded by pure wax solution melted into a liquid state.

Trimming the slice: the embedded material was trimmed to a trapezoid depending on the location of the material, and sliced with a Leica RM 2015 hand slicer to a thickness of about 6 μm.

Spreading and sticking: slightly clamping the cut sample with small tweezers, placing into a water bath kettle at 35-45 ℃ for spreading, taking out the sample with a glass slide after spreading the wax sheet, and placing into a baking oven at 40 ℃ for baking.

Dewaxing: the slide with the attached sample was inserted into xylene, dewaxed for 15min, xylene: absolute ethanol = 1:1 for 2min, and rinsing with absolute ethanol (100%, 95%, 90%, 85%, 80%, 75%, 70%) in different grades, and washing sequentially from high concentration to low concentration for 2min.

Dyeing: safranin-fast green dyeing method. Firstly, the red dye liquor is used for dyeing for 24 hours, 70 percent, 75 percent, 80 percent and 85 percent of ethanol are used for respectively cleaning for 30 seconds, the solid green dye liquor is used for dyeing for 1min, and 95 percent of ethanol, absolute ethanol and xylene are used for dyeing: absolute ethanol = 1: 1. the chips were covered with neutral gum and dried in an oven at 40 c.

The pictures were collected by observation with a forward fluorescent microscope.

As a result, it was found that the transgenic lines showed shorter root lengths (FIGS. 3b, e). In the maturation stage (8 weeks), the stems of the WTs were about 30% thicker than those of the transgenic plants (fig. 3 c), the heights of the transgenic plants being on average 26% shorter than those of the WTs (fig. 3d, f). Thus, after PbPDCB16 is overexpressed, the development of the transgenic plants is delayed. In addition, the lignin content and the expression level of lignin biosynthesis genes of transgenic plants were significantly reduced (fig. 4 a). Furthermore, more callose was accumulated in the transgenic plants compared to the wild type (fig. 4 b). Meanwhile, transmission Electron Microscopy (TEM) showed that transgenic plants had fewer PDs, single morphology and blocking phenomena (fig. 5 a-d). The intersubular fibers and xylem cells are the main stem tissue supporting the growth of the standing inflorescences, stem segments are stained with safranin fast green staining, and the change of cells is observed. The cell wall thickness was significantly reduced compared to WT (fig. 5 e-h). These results indicate that PbPDCB16 inhibits lignin deposition and cell wall lignification.

Example 6 Pear fruit transient injection of PbPDCB16 Gene

The culture conditions of the agrobacterium strain are the sameExample 3 bacterial pellet after supernatant removal was resuspended in permeation medium (10 mM MgCl ₂ 、10mM MES、200μM AS，pH 5.6，OD ₆₀₀ =1.0). At room temperature, the young fruit of Dangshan pear is injected and permeated at the equatorial part of the young fruit of the Dangshan pear after being induced for 2-4 hours in a dark place.

The results showed that after injection for 7d, reduced lignin staining was observed at the injection point of PbPDCB16 compared to the corresponding non-injection point and GFP (fig. 6 a). The expression of PbPDCB16 was higher after overexpression, about 2.5-fold higher than the other parts (fig. 6 b). Lignin content was reduced (fig. 6 c) and callose content at the injection site was significantly higher after overexpression (fig. 6 d). Thus, pbPDCB16 can reduce lignin content in pear fruits.

Claims (10)

1. The CDS sequence of the gene PbPDCB16 is shown as SEQ ID NO. 1.

2. The protein encoded by the gene PbPDCB16 of claim 1, wherein the amino acid sequence of the protein is shown as SEQ ID NO.2.

3. A recombinant expression vector, transient expression vector or recombinant bacterium comprising the gene PbPDCB16 of claim 1.

4. A primer pair for amplifying the full length or any fragment of the gene PbPDCB16 of claim 1.

5. The primer pair of claim 4, wherein the primer pair is as follows:

forward primer 1: namely PbPDCB16-GFP-F,

gagaacacgggggactctagaATGGCTGCTTTAGTGTATATTGTGATC；

reverse primer 1: namely PbPDCB16-GFP-R,

gcccttgctcaccatggatccCATCAGTAAAACCGCTGTGAAGC。

6. use of the gene PbPDCB16 of claim 1, the protein of claim 2, the recombinant expression vector of claim 3, the transient expression vector or the recombinant bacterium or the primer pair of claim 4 or 5 in plant breeding; the application is that the gene PbPDCB16 is transferred into plants to be over-expressed.

7. Use of the gene PbPDCB16 of claim 1, the protein of claim 2, the recombinant expression vector of claim 3, the transient expression vector or the recombinant bacterium or the primer pair of claim 4 or 5 for cultivating plants with low lignin content; the application is that the gene PbPDCB16 is transferred into plants to be over-expressed.

8. Use of the gene PbPDCB16 of claim 1, the protein of claim 2, the recombinant expression vector of claim 3, the transient expression vector or the recombinant bacterium or the primer pair of claim 4 or 5 for genetic improvement of fruit quality; the application is that the gene PbPDCB16 is transferred into plants to be over-expressed.

9. Use of the gene PbPDCB16 of claim 1, the protein of claim 2, the recombinant expression vector of claim 3, the transient expression vector or the recombinant bacterium or the primer pair of claim 4 or 5 for cultivating low lignin fruits; the application is that the gene PbPDCB16 is transferred into plants to be over-expressed.

10. The use according to any one of claims 6 to 9, wherein the plant is arabidopsis thaliana or pyri.