CN113637661B - Pear chitinase, coding gene thereof and application thereof in improving plant disease resistance - Google Patents

Abstract

The invention provides pear chitinase, a coding gene thereof and application thereof in improving plant disease resistance, and belongs to the technical field of molecular biology. The amino acid sequence of the pear chitinase is shown as SEQ ID NO. 1. Studies show that the expression level of the chitinase of the pear is positively correlated with the disease resistance of the pear variety, and the expression level in the high disease resistance variety is 4.5 times of that in the high disease resistance variety. Meanwhile, the pear chitinase gene has typical chitinase activity after being recombined and expressed by a prokaryotic expression system, and the specific activity of the purified chitinase is 14.85U/mg under the optimal condition. The pear chitinase gene can obtain an over-expressed transgenic plant through a transgenic technology, so that the disease resistance of the plant is improved, and the pear chitinase gene can be used for preparing the chitinase by utilizing a genetic engineering technology and applied to industrialization.

Description

Pear chitinase, coding gene thereof and application thereof in improving plant disease resistance

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to pear chitinase, a coding gene thereof and application thereof in improving plant disease resistance.

Background

Pears are important economic fruit trees in the world, and as a popular healthy fruit, the requirements of people on the yield and quality of pears are also increasing year by year. However, pears are often threatened by various diseases and insect pests in the planting and storage processes, wherein the damage of pear black spot, anthracnose and ring spot is the most serious, and the pear black spot, anthracnose and ring spot are difficult to thoroughly eradicate due to wide transmission paths. The leaves of the infected pear tree can generate gradually expanded disease spots until the whole leaf is necrotic and falls off; the fruit branches and trunks of the infected pears can have a ring necrosis area until the phloem is necrotic and stripped, which seriously affects the appearance and quality properties of the fruits. Although agricultural comprehensive control mainly using antifungal agents can play a certain role in production, the chemical agents cause pollution to the environment to different degrees. At present, pear varieties which are immune to the diseases are not found, and the method for introducing exogenous resistance genes has unknown genetic risks, so that molecular design breeding is researched and carried out by utilizing the disease resistance genes in the pears, and the method is the most economical and effective way for preventing and treating pear diseases.

When a plant is infected by pathogenic fungi, complex and unique defense mechanisms are formed, including synthesis of plant protection elements, expression of proteins related to the disease course and the like, so that the plant obtains systemic resistance. However, no report on prevention and treatment of fungal pathogens by pear-derived chitinase exists at present.

Disclosure of Invention

In view of the above, the present invention aims to provide a pear chitinase and a gene encoding the same, which can be used for improving the disease resistance of plants because the protein and gene expression levels of the pear chitinase are positively correlated with the disease resistance.

The invention also aims at providing application of the pear chitinase and the coding gene thereof in improving plant disease resistance.

The invention provides pear chitinase, and the amino acid sequence of the pear chitinase is shown as SEQ ID NO. 1.

The invention provides a gene PbrChiA for encoding pear chitinase, and the nucleotide sequence of the gene is shown as SEQ ID NO. 2.

The invention provides a plant recombinant expression vector, which comprises the gene PbrChiA.

Preferably, pCold-His is used as a framework vector;

the insertion multiple cloning site of the gene PbrChiA is XbaI/XhoI.

The invention provides application of the pear chitinase, the gene PbrChiA or the plant recombinant expression vector in improving plant disease resistance.

Preferably, the disease in plant disease resistance includes a plant disease caused by a staphylococcus (Botryosphaeria) pathogen.

Preferably, the plant disease caused by the pathogenic bacteria of the genus Phytocassum comprises fruit tree ring rot.

Preferably, the fruit tree ring rot comprises pear ring rot and apple ring rot.

The invention provides application of the gene PbrChiA or the plant recombinant expression vector in producing chitinase.

Preferably, the specific activity of the produced chitinase is not lower than 14.85U/mg at 35 ℃ and pH of 8.

The amino acid sequence of the pear chitinase provided by the invention is shown as SEQ ID NO. 1. The invention obtains chitinase genes through comparing and analyzing transcriptome data of disease-resistant and disease-sensitive varieties before and after ring spot infection, the genes are obviously up-regulated and expressed under the induction of ring spot infection, and the up-regulating multiple is higher in the disease-resistant varieties. The pear chitinase is transferred into fruit trees in a genetic engineering mode, so that the disease resistance of plants can be improved, and the pear chitinase can be used for preparing the chitinase by using a genetic engineering technology and applied to industrialization.

Drawings

FIG. 1 is a PCR electrophoresis chart of the chitinase gene PbrChiADNA of the present invention in example 3, wherein lane 1 is a DNA standard molecule, lanes 2, 3, 4 are the resistant pear variety Dangshan pear, lanes 5, 6, 7 are the susceptible pear variety Fengshan pear ^[1] ；

FIG. 2 shows the results of SDS-PAGE detection of pear chitinase PbrChiA in example 4 of the present invention;

FIG. 3 shows the effect of temperature and pH on the activity of the pear chitinase PbrChiA in the present invention, wherein (a) is the effect of reaction temperature on the activity of the pear chitinase PbrChiA, and (b) is the effect of reaction pH on the activity of the pear chitinase PbrChiA;

FIG. 4 is a graph showing comparison of the expression level of the chitinase gene PbrChiA of the pear in the resistance pear variety Dangshan pear and the easy pear variety Fengshan pear in example 5.

Detailed Description

The invention provides pear chitinase, and the amino acid sequence of the pear chitinase is shown as SEQ ID NO. 1. The biological source of chitinase is Shang Shansu pear (Pyrus bretshneider Rehd). The chitinase has a length of 316 amino acid sequences, wherein the N-terminal comprises a signal peptide consisting of 34 amino acids (SEQ ID NO:3: MAMEASEFMASTKTQTLALTLSLLILISSCKSSQA).

The invention also provides a gene PbrChiA for encoding the pear chitinase, and the nucleotide sequence of the gene PbrChiA is shown as SEQ ID NO. 2. The nucleotide sequence of the signal is shown in SEQ ID NO. 4 (ATGGCCATGGAGGCCAGTGAATTCATGGCATCCAAAACACAAACCCTAGCCCTAACTCTGTCCCTCTTGATCCTCATTTCTTCATGCAAGTCCTCCCAAGCC).

The invention provides a plant recombinant expression vector, which comprises the gene PbrChiA. The plant recombinant expression vector preferably uses pCold-His as a framework vector. The insertion multiple cloning site of the gene PbrChiA is preferably XbaI/XhoI. The source of pCold-His is not particularly limited in the present invention, and sources of pCold-His known in the art may be used. In an embodiment of the invention, the pCold-His is purchased from Hunan Fenghui Biotechnology Co.

In the present invention, the construction method of the plant recombinant expression vector preferably comprises the steps of:

performing PCR amplification by using the Dangshan pear leaf cDNA as a template and adopting a P1 and P2 primer pair to obtain a PCR product from which the signal peptide is removed;

double enzyme digestion is carried out on the PCR product with the signal peptide removed and the skeleton carrier by adopting double enzyme digestion of restriction enzymes XbaI and XhoI, so as to obtain the digested PCR product and the linear skeleton carrier;

and connecting the PCR product after the enzyme digestion with a linear framework vector to obtain a plant recombinant expression vector.

In the present invention,the nucleotide sequence of the P1 is shown as SEQ ID NO 5 (5' -atggagctcggtacc)ctcgagGCCGGAATTGCGATC-3'); the nucleotide sequence of the P2 is shown as SEQ ID NO. 6 (5' -agcagagattaccta)tct agaCTACGATTCATCTGC-3'); wherein the lowercase letters indicate homologous arm sequences used to construct the expression vector. The reaction system for PCR amplification is preferably 50. Mu.L: phusion super-fidelity PCR MasterMix 25 mu L,10 mu M concentration Primer 1-P1.5 mu L,10 mu M concentration Primer 1-P2.5 mu L, dangshan pear leaf cDNA template 1 mu g, ddH ₂ O was replenished to 50. Mu.L. The reaction procedure for PCR amplification is preferably: :94 ℃ for 3min;94 ℃ for 15s;58 ℃ for 15s;72 ℃ for 1min;72 ℃ for 5min; cycling 29 times; maintained at 4 ℃. After obtaining the PCR product from which the signal peptide is removed, purification is preferably performed. The method of purification is not particularly limited in the present invention, and may be accomplished by purification methods well known in the art, for example, by using a DNA product purification kit.

In the present invention, the reaction system for double cleavage is preferably as follows: two restriction enzymes XbaI and XhoI each 1. Mu.L, cleavage reaction buffer mix 5. Mu.L, vector plasmid 1. Mu.g, ddH ₂ O was replenished to 50. Mu.L. The reaction conditions for the double cleavage are preferably as follows: at 37℃for 3 hours. The double cleavage is preferably carried out in a constant temperature metal bath. The ligation is preferably accomplished using a recombinase. The type of the recombinant enzyme is not particularly limited, and any type of recombinant enzyme known in the art may be used. In an embodiment of the invention, the recombinase is purchased from a noni-endoribonuclease. The reaction system for the ligation of the recombinant enzyme is preferably as follows: cloning product 4. Mu.L, vector fragment 3. Mu.L, homologous recombination reaction buffer mix 2. Mu.L, recombinase 1. Mu.L. The reaction conditions under which the recombinase is linked are preferably as follows: at 37℃for 30 minutes. The connection is preferably carried out in a constant temperature metal bath.

The invention provides application of the pear chitinase, the gene PbrChiA or the plant recombinant expression vector in improving plant disease resistance.

In the present invention, the disease in plant disease resistance preferably includes a plant disease caused by a staphylococcus (Botryosphaeria) pathogen. The plant disease caused by the pathogenic bacteria of the genus Botrytis preferably comprises fruit tree ring rot. The fruit tree ring rot preferably comprises pear ring rot and apple ring rot. The pathogenic bacteria of the pear ring rot is Botryosphaeria berengeriana. The apple ring rot pathogen is a viticola (Botryosphaeria dothidea).

In the method for improving plant disease resistance, the gene PbrChiA is preferably introduced into plants in the form of a plant recombinant expression vector, so that pear chitinase is overexpressed in the plants. Because the expression quantity of the pear chitinase gene PbrChiA is positively correlated with the leaf scald disease resistance of the pear variety, the transgenic plant with the over-expressed pear chitinase gene PbrChiA is obtained by a transgenic technology, and the disease resistance of the plant can be improved. In the invention, chitinase catalyzes the hydrolysis of fungal cell wall chitin to enable protoplasm of the fungal cell wall chitin to extravasate, so that the growth and propagation of fungi are inhibited; the hydrolysate oligosaccharide can be used as an inducer to start further defense reaction of the plant, and further improve the antifungal capability of the plant.

The invention provides application of the gene PbrChiA or the plant recombinant expression vector in producing chitinase.

In the present invention, the method for producing chitinase preferably comprises the steps of:

introducing the prepared plant recombinant expression vector into a prokaryotic expression system to obtain recombinant bacteria;

screening, culturing and expanding culturing the recombinant bacteria to obtain bacterial liquid;

and (3) performing induction culture on the bacterial liquid, separating a supernatant, and purifying to obtain the recombinant expressed chitinase.

The method of introducing the prokaryotic expression system is not particularly limited in the present invention, and transformation methods well known in the art, such as a calcium chloride heat shock method, may be employed.

In the present invention, the screening culture was performed by shaking culture using 5mL of LB medium containing 100. Mu.g/mL of kanamycin. The rotation speed of the shaking culture is preferably 180 to 220rpm, more preferably 200rpm. The temperature of the shaking culture is preferably 36 to 38℃and more preferablyIs 37 ℃. The expansion culture is preferably performed by shaking culture after dilution of the bacterial liquid by a multiple ratio. The shaking culture is preferably carried out until the OD of the bacterial liquid is reached ₆₀₀ And 0.6 to 0.8, more preferably 0.7. The induction culture is preferably carried out at a concentration of 1mmol/L IPTG. The time of the induction culture is preferably 18 to 22 hours, more preferably 20 hours. The temperature of the induction culture is preferably 18 to 22℃and more preferably 20 ℃. The rotation speed of the induction culture is preferably 180 to 220rpm, more preferably 200rpm.

In the present invention, the separation supernatant includes separation of cells, cell disruption, and separation of supernatant.

The method for separating the cells is preferably centrifugation at 5500 to 6500rpm at 3 to 5℃for 8 to 12 minutes, more preferably centrifugation at 6000rpm at 4℃for 10 minutes. The method for disrupting the cells of the present invention is not particularly limited, and methods for disrupting cells known in the art may be used. In the embodiment of the invention, the method of ultrasonic cell disruption is adopted, and the power of ultrasonic waves is preferably 280-320W, more preferably 300W. The interval time of the ultrasound is preferably 4 s/6 s of the ultrasound treatment. The time of the ultrasound preferably lasts 20min. The separation method of the supernatant is preferably centrifugation at 12000rpm at 4℃for 20min.

In the present invention, the supernatant is preferably subjected to protein purification of recombinant proteins using an AKTA protein purification system and a HIS-TAG column. The result showed that the purified recombinant protein solution was subjected to SDS-PAGE to obtain a protein band of 85.7 kDa.

In the invention, the enzymolysis conditions of the produced chitinase are searched, and the result shows that the specific activity of the produced chitinase is preferably not lower than 14.85U/mg at 35 ℃ and pH of 8. Therefore, the recombinant expressed chitinase can be applied to the industries of foods, cells and the like.

The pear chitinase, the coding gene and the application thereof in improving plant disease resistance provided by the invention are described in detail below with reference to examples, but are not to be construed as limiting the scope of the invention.

The embodiments described below are some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art without the benefit of the teachings of this invention, are intended to be within the scope of the invention. The specific conditions are not noted in the examples, and are carried out according to conventional conditions or conditions suggested by the manufacturer; the reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The molecular biological experimental methods which are not specifically described in the embodiment of the invention are all carried out by referring to the specific methods listed in the J. Sam Brookfield of the guidelines for molecular cloning experiments (third edition), or according to the specifications of the kit and the product; the technical means used in the practice of the present invention are conventional in the art, and raw materials are commercially available, unless otherwise specified.

Example 1

Preparation of pear cDNA

(1) Extraction of total RNA from pear

100mg of pear leaves are ground by liquid nitrogen, extracted according to the method on a specification of a Tiangen RNA extraction kit, the integrity of the pear leaves is detected by agarose gel electrophoresis of 1.0%, and the RNA content and purity are measured by a micro-spectrophotometer.

(2) Preparation of first strand of pear chitinase cDNA

Reverse transcription adopts reverse transcriptase produced by the whole gold company, and the reaction system is as follows: mu.g of RNA, 1. Mu.l Oligo (dT), 10. Mu.l 2xTS Reaction Mix,1. Mu.l RT/RI Enzyme Mix, 1. Mu.l gDNA reverse, and the reaction system was made up to 20. Mu.l with RNase-free Water.

The reaction conditions are as follows: warm bath at 42 ℃ for 30min; heating at 85deg.C for 5s to terminate the reaction, and freeze preserving.

The transcriptome data of disease-resistant and disease-sensitive varieties before and after the infection of the ring spot are compared and analyzed, and a chitinase gene sequence which is obviously up-regulated and expressed by the induction of the infection of the ring spot and has higher up-regulation multiple in the disease-resistant varieties is obtained by screening and is named as a gene PbrChiA.

The nucleotide sequence of the pear chitinase gene PbrChiA (SEQ ID NO: 2) is underlined to mark the nucleotide sequence corresponding to the signal peptide region:

ATGGCCATGGAGGCCAGTGAATTCATGGCATCCAAAACACAAACCCTAGCCCTAACTCTGTCCCTCTT GATCCTCATTTCTTCATGCAAGTCCTCCCAAGCCGCCGGAATTGCGATCTATTGGGGCCAAAACGGTAACGAAGGAACCTTAGCGGATGCTTGCAACTCAGGCAACTACCAGTTTGTTAACATAGCTTTCCTCATTACTTTCGGAAACAACCAAACCCCTGTCCTAAACCTCGCCGGCCACTGCGACCCCGCCAGTGGTACTTGCACGGGGCTGAGTGCCGACATCAGAACCTGCCAATCAAAAAACATAAAAGTCCTCCTCTCGATTGGAGGGGCCTCCGGAAGTTACTCTCTCACTTCAGCTGATGATGCAAGGCAAGTTGCTGATTACATCTGGAACAACTTCTTAGGTGGTCAGTCAGCTTCGCGCCCGCTTGGGGACGCGGTTTTGGACGGCGTTGATTTCGACATTGAGGCGGGTGGTGGGCAATTCTATGATGAGCTCGCCAGGTCACTCAACGGACACAACGGACAGGCAAAAACGGTCTATTTAGCCGCAGCTCCACAATGTCCGATCCCGGATGCTCACCTAGACGGCGCTATCCAAACCGGTTTATTTGACTACGTTTGGGTTCAGTTCTACAACAACCCCCCATGCCAGTATGCTGACGGTAATGCCAACGCTCTTTTGAACAGTTGGAGCCAATGGGCCTCGGTTCCGGCCACCCAGGTATTCATGGGGTTACCGGCGTCCACTGATGCCGCGGGCAGCGGATTTATTCCTGCTGATGCTCTCAAGTCACAAGTCCTTCCAACAATTAAGAATTCGGCGAAGTATGGAGGAGTTATGCTTTGGAGCAGGTGGTATGACATTAACAGCGGTTATAGTGCATCCATTAAGGACAGCAGGATCCATCGAGCTCGAGCTGCAGATGAATCGTAG。

the amino acid sequence of pear chitinase PbrChiA (SEQ ID NO: 1) is underlined to mark the amino acid sequence corresponding to the signal peptide region:

MAMEASEFMASKTQTLALTLSLLILISSCKSSQAAGIAIYWGQNGNEGTLADACNSGNYQFVNIAFLITFGNNQTPVLNLAGHCDPASGTCTGLSADIRTCQSKNIKVLLSIGGASGSYSLTSADDARQVADYIWNNFLGGQSASRPLGDAVLDGVDFDIEAGGGQFYDELARSLNGHNGQAKTVYLAAAPQCPIPDAHLDGAIQTGLFDYVWVQFYNNPPCQYADGNANALLNSWSQWASVPATQVFMGLPASTDAAGSGFIPADALKSQVLPTIKNSAKYGGVMLWSRWYDINSGYSASIKDSRIHRARAADES。

example 2

Primer design of pear chitinase gene PbrChiA

Primer design is carried out by using software Primer 5.0, and primers are designed according to a chitinase gene PbrChiA sequence screened by a transcriptome library, wherein Primer1 is used for amplifying the whole length of genes except a corresponding region of a signal peptide and constructing a protein expression vector, and lowercase letters represent homologous arm sequences used for constructing the expression vector; primer2 was used for real-time fluorescent quantitative PCR analysis to detect the relative expression of the pear chitinase gene PbrChiA in different resistant pear varieties:

Primer 1

P1:5’-atggagctcggtaccctcgagGCCGGAATTGCGATC-3’(SEQ ID NO:5)；

P2:5’-agcagagattacctatctagaCTACGATTCATCTGC-3’(SEQ ID NO:6)；

Primer 2

P1:5’-TGGGGCCAAAACGGTAAC-3’(SEQ ID NO:7)

P2:5’-TTTATGTTTTTTGATTGG-3’(SEQ ID NO:8)。

the above primers were synthesized by Shanghai Biotechnology Co.

Example 3

Cloning and constructing protein expression vector of pear chitinase gene PbrChiA

(1) PCR amplification of pear chitinase gene PbrChiA

The reaction system for PCR amplification was 50. Mu.L: phusion super-fidelity PCR Master Mix 25. Mu.L, primer 1-P1.5. Mu.L with concentration of 10. Mu.M, primer 1-P2.5. Mu.L with concentration of 10. Mu.M, dangshan pear leaf cDNA template 1. Mu.L, ddH ₂ O was replenished to 50. Mu.L.

The reaction conditions are as follows: 94 ℃ for 3min;94 ℃ for 15s;58 ℃ for 15s;72 ℃ for 1min;72 ℃ for 5min; cycling 29 times; the temperature was kept at 4℃and the result of the electrophoresis was examined (see FIG. 1).

(2) Amplification product recovery

The PCR products were recovered using a common DNA product purification kit.

(3) Double enzyme cutting and recovering of carrier

The vector pCold-His was digested with the restriction enzymes XbaI and XhoI to recover the vector backbone (about 5700 bp).

(4) Protein expression vector construction

And (3) connecting the cloning product in the step (2) and the vector skeleton in the step (3) by using the noni-endoribonuclease to obtain a recombinant plasmid pCold-PbrChiA.

Example 4

Expression and purification of pear chitinase PbrChiA

Firstly, introducing a recombinant plasmid pCold-PbrChiA into escherichia coli BL21 (DE 3) to obtain escherichia coli containing the recombinant plasmid pCold-PbrChiA, which is named as recombinant bacterium;

secondly, selecting a monoclonal of recombinant bacteria (expressing pCold-PbrChiA containing His tag), inoculating to 5mL LB medium containing 100 mug/mL kanamycin, and culturing overnight in a shaking table at 37 ℃ and 200 rpm;

thirdly, inoculating the bacterial liquid into 100mL of LB liquid medium containing 100 mug/mL kanamycin according to the volume ratio of 1:100, and culturing at 37 ℃ with shaking at 200rpm until the OD600 reaches 0.6-0.8;

fourthly, adding IPTG (inducer) to the concentration of 1mmol/L, and carrying out shaking culture at 20 ℃ and 200rpm for 20 hours;

fifthly, collecting the bacterial liquid into a centrifuge tube, centrifuging at the temperature of 4 ℃ and at the speed of 6000rpm for 10min, discarding the supernatant, and collecting the precipitate;

sixthly, adding 10mL of 1 XPBS buffer solution to the bacterial sediment to resuspend the bacterial cells;

seventh, crushing bacteria on ice by ultrasonic waves, wherein the ultrasonic power is 300W, and carrying out ultrasonic treatment for 20min at intervals of 6s for 4s each time;

eighth step, centrifugation is carried out at 12000rpm for 20min at 4 ℃, bacterial lysate supernatant is collected and placed on ice;

ninth, the collected supernatant was subjected to protein purification using an AKTA protein purification system and HIS-TAG column of the company, and SDS-PAGE was performed on the purified recombinant protein solution to obtain a protein band of 85.7kDa (results are shown in FIG. 2).

Example 5

Studies on enzymatic activity and properties of pear chitinase PbrChiA

(1) Protein content determination

A standard curve is drawn by taking Bovine Serum Albumin (BSA) as a standard substance, and the content of protein is measured by adopting a Coomassie brilliant blue method.

(2) Enzyme activity and optimal reaction temperature determination of pear chitinase PbrChiA

The purified pear chitinase PbrChiA was subjected to enzymatic activity assay according to standard procedures of the chitinase activity assay kit instruction sheet produced by sigma company, wherein the reaction time was 1 hour, and the reaction temperature was set as the following gradient: 15 ℃, 25 ℃, 35 ℃,45 ℃ and 55 ℃. As a result, as shown in FIG. 3 (a), the optimal reaction temperature of the pear chitinase PbrChiA was 35 ℃.

(3) Determination of optimal reaction pH value of pear chitinase PbrChiA

And (3) under the same conditions as the other conditions in the step (2), setting the reaction temperature to 35 ℃, adjusting the pH value of a reaction system to an integer value of 4-12, and carrying out no-activity measurement on the pear chitinase PbrChiA. As a result, the optimal reaction pH of the pear chitinase PbrChiA was 8, as shown in FIG. 3 (b).

Example 6

Expression analysis of pear chitinase gene PbrChiA in resistant and susceptible varieties

Experimental materials: disease-resistant variety: dangshan pear; the disease-sensitive variety is Fengshui pear ^[1] The method comprises the steps of carrying out a first treatment on the surface of the RNA of the sample material was extracted by the method of example 1, and subjected to reverse transcription to obtain cDNA.

The real-time fluorescent quantitative PCR selects Tubulin as an internal reference, and the Primer is Primer2 designed in the example 2; 3 biological replicates and technical replicates, each of which was prepared according to the SYBR Premix Plus specification of Takara, and the sample was applied under stringent control, using 2 ^-ΔΔCт The relative expression level was calculated by the method.

20 mu L of a reaction system, wherein the reaction conditions are as follows: 94 ℃ for 5min; the cyclic reaction was 94℃for 3 seconds; 60 ℃ for 10 seconds; 72 ℃,30 seconds; for 45 cycles.

As shown in FIG. 4, the experimental result shows that the pear chitinase PbrChiA has typical chitinase activity, and the specific activity of the pear chitinase is 14.85U/mg under the conditions that the reaction temperature is 35 ℃ and the pH is 8.

The expression level of the pear chitinase gene PbrChiA is positively correlated with the disease resistance of pear variety ring rot, and the expression level of the resistance variety Dangshan pear is 4.5 times of that of the disease-sensitive variety Fengshan pear. Therefore, the transgenic plant with the pear chitinase gene PbrChiA over-expressed is obtained through a transgenic technology, the disease resistance of the plant can be improved, and the transgenic plant can also be used for preparing the chitinase by using a genetic engineering technology and applied to industrialization.

Reference to the literature

1 piece of celluloid, liu Jizhi, zhang Guozhen.6 pear fruit has resistance difference to ring spot and bacteriostasis of 4 bactericides to ring spot bacteria [ J ]. Plant protection, 2019,45 (04): 224-228.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Sequence listing

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<213> Artificial sequence (Artificial Sequence)

<400> 7

tggggccaaa acggtaac 18

<210> 8

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

tttatgtttt ttgattgg 18

Claims (6)

1. The Dangshan pear chitinase is characterized in that the amino acid sequence is shown as SEQ ID NO. 1.

2. A gene PbrChiA encoding the pear chitinase of claim 1, wherein the nucleotide sequence is shown in SEQ ID No. 2.

3. A plant recombinant expression vector comprising the gene PbrChiA of claim 2.

4. The plant recombinant expression vector according to claim 3, wherein pCold-His is used as a backbone vector;

the insertion multiple cloning site of the gene PbrChiA is XbaI/XhoI.

5. Use of the gene PbrChiA of claim 2 or the plant recombinant expression vector of claim 3 or 4 for the production of chitinase.

6. The use according to claim 5, wherein the chitinase produced has a specific activity of not less than 14.85U/mg at 35℃and pH 8.