阅读说明：本技术 一种梨几丁质酶、其编码基因及其在提高植物抗病中的应用 (Chitinase of pear, encoding gene thereof and application of chitinase in improving disease resistance of plants ) 是由 张绍铃 陈启明 黄小三 董慧珍 齐开杰 殷豪 乔鑫 谢智华 乔清海 于 2021-08-17 设计创作，主要内容包括：本发明提供了一种梨几丁质酶、其编码基因及其在提高植物抗病中的应用,属于分子生物学技术领域。梨几丁质酶氨基酸序列如SEQIDNO:1所示。研究表明,梨几丁质酶表达量与梨品种抗病性呈正相关,高抗病品种中的表达量是高感病品种中表达量的4.5倍。同时梨几丁质酶基因经过原核表达系统重组表达后具有典型的几丁质酶活性,纯化的几丁质酶的比活力在最适条件下为14.85U/mg。该梨几丁质酶基因可以通过转基因技术获得过表达的转基因植株,从而提高植物的抗病能力,也可将其用于利用基因工程技术制备该几丁质酶应用到产业化中。(The invention provides chitinase of pear, a coding gene thereof and application thereof in improving disease resistance of plants, belonging to the technical field of molecular biology. The amino acid sequence of the chitinase of pear is shown as SEQIDNO: 1. Research shows that the expression level of chitinase in pear is positively correlated with the disease resistance of pear variety, and the expression level in high disease resistant variety is 4.5 times that in high disease susceptible variety. Meanwhile, the chitinase gene of pear 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 chitinase gene of pear can obtain an over-expressed transgenic plant through a transgenic technology, thereby improving the disease resistance of the plant, and can also be used for preparing the chitinase by utilizing a genetic engineering technology to be applied to industrialization.)

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

2. A gene PbrChiA encoding the chitinase according to claim 1, having a nucleotide sequence 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 multi-cloning site of the gene PbrChiA is XbaI/XhoI.

5. Use of the chitinase of pear as defined in claim 1, the gene PbrChiA as defined in claim 2 or the recombinant expression vector of plants as defined in claim 3 or 4 for improving disease resistance of plants.

6. The use of claim 5, wherein the disease in plant disease resistance comprises plant disease caused by pathogenic bacteria of the genus Staphylococcus (Botryosphaeria).

7. The use according to claim 6, wherein the plant disease caused by pathogens of the genus gluconobacter includes ring rot of fruit trees.

8. The use of claim 7, wherein the ring rot of fruit trees comprises pear ring rot and apple ring rot.

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

10. The use according to claim 9, wherein the chitinase produced has a specific activity of not less than 14.85U/mg at 35 ℃ and pH 8.

Technical Field

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

Background

The pear is an important economic fruit tree in the world, and as a popular healthy fruit, the demand of people on the yield and the quality of the pear is also increased year by year. However, the pears are often threatened by various plant diseases and insect pests in the planting and storage processes, wherein the pear black spot, anthracnose and ring spot are the most serious, and the pear black spot, the anthracnose and the ring spot are difficult to eradicate thoroughly due to wide transmission ways. The infected pear leaf sheets can generate gradually expanded disease spots until the whole leaves are necrotic and fall off; the branches and trunks of the diseased pear fruits can generate annular necrotic areas until phloem necrosis is stripped, and the appearance and quality of the fruits are seriously affected. Although the production of agricultural comprehensive control mainly using antifungal agents can achieve certain effects, the chemical agents cause pollution to the environment to different degrees. At present, no pear varieties immune to the diseases are found, and unknown genetic risks exist in a method for introducing exogenous resistance genes, so that the method for researching and utilizing the disease-resistant genes in pears to carry out molecular design breeding is the most economic and effective way for preventing and treating the diseases of pear trees.

When plants are infected by pathogenic fungi, the plants can form a complex and unique defense mechanism by themselves, and the complex and unique defense mechanism comprises the synthesis of phytochemicals, the expression of disease course related proteins and the like, so that the plants can obtain systemic resistance. However, no report about the control 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 chitinase of pear and a coding gene thereof, wherein the protein and gene expression level of the chitinase of pear are positively correlated with disease resistance, so that the chitinase of pear can be used for improving the disease resistance of plants.

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

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

The invention provides a gene PbrChiA for coding the chitinase of pear, and the nucleotide sequence 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 skeleton vector;

the multi-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 the disease resistance of plants.

Preferably, the disease in the disease resistance of the plant comprises a plant disease caused by a pathogenic bacterium of the genus staphylococcus (Botryosphaeria).

Preferably, the plant disease caused by a plasmodiophora pathogen includes ring rot of fruit trees.

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 produced chitinase has a specific activity of not less than 14.85U/mg at 35 ℃ and pH of 8.

The amino acid sequence of the chitinase is shown as SEQ ID NO. 1. According to the invention, the chitinase gene is obtained by screening through comparative analysis of transcriptome data of disease-resistant and disease-susceptible varieties before and after the infection of the ring spot, the gene is induced by the infection of the ring spot to be obviously up-regulated and expressed, and the up-regulation multiple of the gene in the disease-resistant varieties is higher. The pear chitinase is transferred into fruit trees by using a gene engineering mode, so that the disease resistance of plants can be improved, and the pear chitinase can also be used for preparing the chitinase by using a gene engineering technology and applied to industrialization.

Drawings

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

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

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

FIG. 4 is a comparison graph of the expression amounts of the chitinase gene PbrChiA in the resistant pear variety Dangshan pear and the susceptible pear variety Fengshui pear in example 5 of the present invention.

Detailed Description

The invention provides a chitinase of pear, the amino acid sequence is shown as SEQ ID NO 1. The biological source of chitinase is Tangshan crisp 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: MAMEASEFMASKTQTLALTLSLLILISSCKSSQA).

The invention also provides a gene PbrChiA for coding the chitinase of pear, and the nucleotide sequence 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 takes pCold-His as a skeleton vector. The inserted multiple cloning site of the gene PbrChiA is preferably XbaI/XhoI. The source of pCold-His in the present invention is not particularly limited, and any pCold-His known in the art may be used. In the present example, the pCold-His was purchased from Hu Nanfeng Hui Biotech Ltd.

In the present invention, the method for constructing the plant recombinant expression vector preferably comprises the following steps:

taking Dangshan pear leaf cDNA as a template, and carrying out PCR amplification by adopting a primer pair P1 and a primer pair P2 to obtain a PCR product from which signal peptides are removed;

performing double enzyme digestion on the PCR product and the skeleton vector with the signal peptide removed by adopting restriction enzymes XbaI and XhoI to obtain a PCR product and a linear skeleton vector after enzyme digestion;

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

In the 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 lower case letters indicate the sequence of the homology arms used to construct the expression vector. The reaction system for PCR amplification is preferably 50 μ L: phusion super fidelity PCR MasterMix 25. mu.L, Primer 1-P11.5. mu.L concentration 10. mu.M, Primer 1-P21.5. mu.L concentration 10. mu.M, Dangshan pear leaf cDNA template 1. mu.g, ddH₂O was supplemented to 50. mu.L. The reaction procedure of the PCR amplification is preferably as follows: : 3min at 94 ℃; 15s at 94 ℃; at 58 ℃ for 15 s; 72 ℃ for 1 min; 72 ℃ for 5 min; circulating for 29 times; keeping at 4 ℃. After obtaining the signal peptide-removed PCR product, it is preferably purified. The purification method is not particularly limited in the present invention, and may be carried out by a purification method known in the art, for example, by using a DNA product purification kit.

In the present invention, the reaction system of the double enzyme digestion is preferably as follows: two restriction enzymes XbaI and XhoI each 1. mu.L, digestion reaction buffer mix 5. mu.L, vector plasmid 1. mu.g, ddH₂O was supplemented to 50. mu.L. The reaction conditions of the double enzyme digestion are excellentThe following are selected: at 37 ℃ for 3 hours. The double cleavage is preferably carried out in a thermostated metal bath. The ligation is preferably performed using a recombinase. The present invention is not particularly limited in kind of the recombinase, and any kind of recombinase known in the art may be used. In the present example, the recombinase is purchased from noprazan-homologous recombinase. The reaction system for connecting the recombinase 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 for the recombinase ligation are preferably as follows: at 37 ℃ for 30 minutes. The joining is preferably carried out in a thermostatted metal bath.

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

In the present invention, the disease in the disease resistance of plants preferably includes plant diseases caused by pathogenic bacteria of the genus staphylococcus (Botryosphaeria). The plant disease caused by a plasmodiophora pathogen preferably comprises ring rot of fruit trees. The fruit tree ring rot preferably comprises pear ring rot and apple ring rot. The pathogenic bacteria of the pyricularia piricola disease are Botryosphaeria berengiana. The etiology of the apple ring rot disease is botrytis (Botryosphaeria dothidea).

In the invention, the method for improving the disease resistance of the plant preferably introduces the gene PbrChiA into the plant in the form of a plant recombinant expression vector, so that the chitinase of the pear is overexpressed in the plant. Because the expression quantity of the pear chitinase gene PbrChiA is positively correlated with the disease resistance of the pear variety ring spot, the transgenic plant over-expressed by the 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 to be exuded, thereby inhibiting the growth and the reproduction of fungi; the hydrolysate oligosaccharide can be used as an induction factor to start further defense reaction of the plant, and the antifungal capability of the plant is further improved.

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 a recombinant bacterium;

screening and culturing the recombinant bacteria and carrying out amplification culture to obtain a bacterial liquid;

and (3) carrying out induction culture on the bacterial liquid, separating supernatant, and purifying to obtain the recombinant expression chitinase.

The method for introducing into the prokaryotic expression system is not particularly limited in the present invention, and a transformation method well known in the art, for example, calcium chloride heat shock method, may be used.

In the present invention, the selection 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 180-220 rpm, and more preferably 200 rpm. The temperature of the shaking culture is preferably 36-38 ℃, and more preferably 37 ℃. The amplification culture preferably adopts shaking culture after bacterial liquid is diluted by times. The shaking culture is preferably carried out until the OD of the bacterial liquid is reached₆₀₀0.6 to 0.8, more preferably 0.7. The induction culture is preferably carried out at a concentration of 1mmol/L IPTG. The time for induction culture is preferably 18-22 h, and more preferably 20 h. The temperature of the induction culture is preferably 18-22 ℃, and more preferably 20 ℃. The rotation speed of the induction culture is preferably 180-220 rpm, and more preferably 200 rpm.

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

The method for separating the thalli is preferably to centrifuge for 8-12 min at the temperature of 3-5 ℃ and the rpm of 5500-6500, and is more preferably to centrifuge for 10min at the temperature of 4 ℃ and the rpm of 6000. The method for disrupting the cells of the present invention is not particularly limited, and any cell disruption method known in the art may be used. In the embodiment of the invention, the method for disrupting the bacteria by using ultrasonic is adopted, and the power of the ultrasonic is preferably 280-320W, and more preferably 300W. The interval time of the ultrasound is preferably 4s per 6s interval of the ultrasound treatment. The ultrasound is preferably continued for 20 min. The supernatant is preferably separated by centrifugation at 12000rpm for 20min at 4 ℃.

In the present invention, the supernatant is preferably used for protein purification of recombinant proteins using an AKTA protein purification system and a HIS-TAG column. The results 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 groved, and the result shows that the specific activity of the produced chitinase is preferably not lower than 14.85U/mg under the conditions of 35 ℃ and pH of 8. Therefore, the recombinant expression chitinase can be applied to industries such as food, cells and the like.

The chitinase, the gene encoding the same and the application thereof in improving plant disease resistance provided by the invention are described in detail in the following embodiments, but the chitinase and the gene cannot be understood as limiting the protection scope of the invention.

The embodiments described below are some, but not all embodiments of the invention. All other embodiments obtained by a person of ordinary skill in the art without any inventive step in connection with the embodiments of the present invention shall fall within the scope of protection of the present invention. Those whose specific conditions are not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer; the reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The molecular biology experimental methods which are not specifically described in the examples of the present invention are performed by referring to the specific methods listed in molecular cloning experimental manual (third edition) J. SammBruk, or according to the kit and product instructions; the technical means in the practice of the invention are, unless otherwise indicated, techniques commonly employed in the research field, and the starting materials used are commercially available.

Example 1

Preparation of Pear cDNA

(1) Extraction of Total RNA from Pear

Grinding 100mg of pear leaves with liquid nitrogen, extracting according to the method of the Tiangen RNA extraction kit specification, detecting the integrity of the pear leaves by 1.0% agarose gel electrophoresis, and measuring the RNA content and purity by a microspectrophotometer.

(2) Preparation of first Strand of chitinase cDNA

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

The reaction conditions are as follows: bathing at 42 deg.C for 30 min; the reaction was terminated by heating at 85 ℃ for 5 seconds, and the reaction mixture was stored under refrigeration.

By comparing and analyzing transcriptome data of disease-resistant and disease-susceptible varieties before and after the infection of the ring spot, a chitinase gene sequence which is induced by the infection of the ring spot and has obvious up-regulated expression and higher up-regulated times in the disease-resistant varieties is obtained by screening, and the gene is named as PbrChiA.

The nucleotide sequence of the chitinase gene PbrChiA (SEQ ID NO:2) of pear is marked by underlining that the signal peptide region corresponds to the nucleotide sequence:

ATGGCCATGGAGGCCAGTGAATTCATGGCATCCAAAACACAAACCCTAGCCCTAACTCTGTCCCTCTT GATCCTCATTTCTTCATGCAAGTCCTCCCAAGCCGCCGGAATTGCGATCTATTGGGGCCAAAACGGTAACGAAGGAACCTTAGCGGATGCTTGCAACTCAGGCAACTACCAGTTTGTTAACATAGCTTTCCTCATTACTTTCGGAAACAACCAAACCCCTGTCCTAAACCTCGCCGGCCACTGCGACCCCGCCAGTGGTACTTGCACGGGGCTGAGTGCCGACATCAGAACCTGCCAATCAAAAAACATAAAAGTCCTCCTCTCGATTGGAGGGGCCTCCGGAAGTTACTCTCTCACTTCAGCTGATGATGCAAGGCAAGTTGCTGATTACATCTGGAACAACTTCTTAGGTGGTCAGTCAGCTTCGCGCCCGCTTGGGGACGCGGTTTTGGACGGCGTTGATTTCGACATTGAGGCGGGTGGTGGGCAATTCTATGATGAGCTCGCCAGGTCACTCAACGGACACAACGGACAGGCAAAAACGGTCTATTTAGCCGCAGCTCCACAATGTCCGATCCCGGATGCTCACCTAGACGGCGCTATCCAAACCGGTTTATTTGACTACGTTTGGGTTCAGTTCTACAACAACCCCCCATGCCAGTATGCTGACGGTAATGCCAACGCTCTTTTGAACAGTTGGAGCCAATGGGCCTCGGTTCCGGCCACCCAGGTATTCATGGGGTTACCGGCGTCCACTGATGCCGCGGGCAGCGGATTTATTCCTGCTGATGCTCTCAAGTCACAAGTCCTTCCAACAATTAAGAATTCGGCGAAGTATGGAGGAGTTATGCTTTGGAGCAGGTGGTATGACATTAACAGCGGTTATAGTGCATCCATTAAGGACAGCAGGATCCATCGAGCTCGAGCTGCAGATGAATCGTAG。

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

MAMEASEFMASKTQTLALTLSLLILISSCKSSQAAGIAIYWGQNGNEGTLADACNSGNYQFVNIAFLITFGNNQTPVLNLAGHCDPASGTCTGLSADIRTCQSKNIKVLLSIGGASGSYSLTSADDARQVADYIWNNFLGGQSASRPLGDAVLDGVDFDIEAGGGQFYDELARSLNGHNGQAKTVYLAAAPQCPIPDAHLDGAIQTGLFDYVWVQFYNNPPCQYADGNANALLNSWSQWASVPATQVFMGLPASTDAAGSGFIPADALKSQVLPTIKNSAKYGGVMLWSRWYDINSGYSASIKDSRIHRARAADES。

example 2

Primer design of pear chitinase gene PbrChiA

Designing a Primer by using software Primer 5.0, and designing the Primer according to a chitinase gene PbrChiA sequence screened by a transcriptome library, wherein the Primer1 is used for amplifying the full length of a gene except a signal peptide corresponding region and constructing a protein expression vector, and lower case letters represent a homologous arm sequence used for constructing the expression vector; the Primer2 is used for real-time fluorescent quantitative PCR analysis to detect the relative expression quantity 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 Bioengineering Co., Ltd.

Example 3

Cloning and construction of protein expression vector for chitinase gene PbrChiA of pear

(1) PCR amplification of pear chitinase gene PbrChiA

The reaction system for PCR amplification is 50 μ L: phusion super fidelity PCR Master Mix 25. mu.L, 10. mu.M concentration Primer 1-P11.5. mu.L, 10. mu.M concentration Primer 1-P21.5. mu.L, Dangshan pear leaf cDNA template 1. mu.L, ddH₂O was supplemented to 50. mu.L.

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

(2) Amplification product recovery

The PCR product was recovered using a common DNA product purification kit.

(3) Double digestion and recovery of vector

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

(4) Construction of protein expression vectors

And (3) connecting the cloning product in the step (2) with the vector framework in the step (3) by utilizing Novozam homologous recombinase to obtain the recombinant plasmid pCold-PbrChiA.

Example 4

Expression and purification of chitinase PbrChiA of pear

Firstly, introducing the recombinant plasmid pCold-PbrChiA into escherichia coli BL21(DE3) to obtain the escherichia coli containing the recombinant plasmid pCold-PbrChiA, and naming the escherichia coli as a recombinant bacterium;

secondly, selecting a recombinant bacterium (expressing a single clone containing a His label pCold-PbrChiA), inoculating the recombinant bacterium to a kanamycin 5mL LB culture medium containing 100 mu g/mL, and culturing overnight in a shaking table at 37 ℃ and 200 rpm;

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

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

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

sixthly, adding 10mL of 1X PBS buffer solution into the bacterial sediment to resuspend the bacteria;

seventhly, ultrasonically breaking bacteria on ice, wherein the ultrasonic power is 300W, and ultrasonic treatment is carried out for 20min at intervals of 6s every time for 4 s;

eighthly, centrifuging at 4 ℃ and 12000rpm for 20min, collecting bacterial lysate supernatant, and placing on ice;

and a ninth step, performing protein purification on the recombinant protein by using an AKTA protein purification system and a HIS-TAG column of the company, and performing SDS-PAGE on the purified recombinant protein solution to obtain a protein band of 85.7kDa (the result is shown in figure 2).

Example 5

Research on enzymatic activity and properties of chitinase PbrChiA of pear

(1) Protein content determination

And (3) drawing a standard curve by taking Bovine Serum Albumin (BSA) as a standard substance, and determining the protein content by adopting a Coomassie brilliant blue method.

(2) Determination of enzyme activity and optimum reaction temperature of chitinase PbrChiA of pear

And (2) performing enzymatic activity determination on the purified pear chitinase PbrChiA according to the standard steps of the chitinase activity determination kit specification produced by Sigma company, wherein the reaction time is 1 hour, and the reaction temperature is set as the following gradient: 15 ℃, 25 ℃, 35 ℃,45 ℃ and 55 ℃. As shown in FIG. 3 (a), the optimum reaction temperature for the chitinase PbrChiA was 35 ℃.

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

And (3) under the same conditions as other conditions in the step (2), setting the reaction temperature to be 35 ℃, adjusting the pH value of the reaction system to be an integer value of 4-12, and carrying out inactivation determination on the pear chitinase PbrChiA. As a result, as shown in FIG. 3 (b), the optimum reaction pH of the chitinase PbrChiA was 8.

Example 6

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

Experimental materials: disease-resistant variety: dangshan pear; the susceptible variety is Fengshui pear^[1](ii) a RNA was extracted from the sample material by the method of example 1, and cDNA was obtained by reverse transcription.

Tubulin is selected as an internal reference and a Primer is Primer2 designed in example 2 by real-time fluorescent quantitative PCR; biological and technical repetition 3 each, reaction solutions were prepared according to the SYBR Premix Plus instructions of Takara,strictly controlling the sample application, use 2^-ΔΔCтThe relative expression amount is calculated.

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

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

The expression quantity of the chitinase gene PbrChiA of the pear is positively correlated with the disease resistance of the pear variety ring spot, and the expression quantity of the resistant variety Dangshan pear is 4.5 times of the expression quantity of the susceptible variety Fengshui 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 utilizing a genetic engineering technology and applied to industrialization.

Reference to the literature

1 piece of xylonite, Liu Qizhi, Zhang Guozhen, resistance difference of 6 kinds of pear fruits to ring rot and bacteriostasis of 4 bactericides to ring rot pathogen [ J ] plant protection, 2019,45(04): 224-.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Nanjing university of agriculture

<120> chitinase of pear, coding gene and application thereof in improving plant disease resistance

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 316

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Met Ala Met Glu Ala Ser Glu Phe Met Ala Ser Lys Thr Gln Thr Leu

1 5 10 15

Ala Leu Thr Leu Ser Leu Leu Ile Leu Ile Ser Ser Cys Lys Ser Ser

20 25 30

Gln Ala Ala Gly Ile Ala Ile Tyr Trp Gly Gln Asn Gly Asn Glu Gly

35 40 45

Thr Leu Ala Asp Ala Cys Asn Ser Gly Asn Tyr Gln Phe Val Asn Ile

50 55 60

Ala Phe Leu Ile Thr Phe Gly Asn Asn Gln Thr Pro Val Leu Asn Leu

65 70 75 80

Ala Gly His Cys Asp Pro Ala Ser Gly Thr Cys Thr Gly Leu Ser Ala

85 90 95

Asp Ile Arg Thr Cys Gln Ser Lys Asn Ile Lys Val Leu Leu Ser Ile

100 105 110

Gly Gly Ala Ser Gly Ser Tyr Ser Leu Thr Ser Ala Asp Asp Ala Arg

115 120 125

Gln Val Ala Asp Tyr Ile Trp Asn Asn Phe Leu Gly Gly Gln Ser Ala

130 135 140

Ser Arg Pro Leu Gly Asp Ala Val Leu Asp Gly Val Asp Phe Asp Ile

145 150 155 160

Glu Ala Gly Gly Gly Gln Phe Tyr Asp Glu Leu Ala Arg Ser Leu Asn

165 170 175

Gly His Asn Gly Gln Ala Lys Thr Val Tyr Leu Ala Ala Ala Pro Gln

180 185 190

Cys Pro Ile Pro Asp Ala His Leu Asp Gly Ala Ile Gln Thr Gly Leu

195 200 205

Phe Asp Tyr Val Trp Val Gln Phe Tyr Asn Asn Pro Pro Cys Gln Tyr

210 215 220

Ala Asp Gly Asn Ala Asn Ala Leu Leu Asn Ser Trp Ser Gln Trp Ala

225 230 235 240

Ser Val Pro Ala Thr Gln Val Phe Met Gly Leu Pro Ala Ser Thr Asp

245 250 255

Ala Ala Gly Ser Gly Phe Ile Pro Ala Asp Ala Leu Lys Ser Gln Val

260 265 270

Leu Pro Thr Ile Lys Asn Ser Ala Lys Tyr Gly Gly Val Met Leu Trp

275 280 285

Ser Arg Trp Tyr Asp Ile Asn Ser Gly Tyr Ser Ala Ser Ile Lys Asp

290 295 300

Ser Arg Ile His Arg Ala Arg Ala Ala Asp Glu Ser

305 310 315

<210> 2

<211> 951

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atggccatgg aggccagtga attcatggca tccaaaacac aaaccctagc cctaactctg 60

tccctcttga tcctcatttc ttcatgcaag tcctcccaag ccgccggaat tgcgatctat 120

tggggccaaa acggtaacga aggaacctta gcggatgctt gcaactcagg caactaccag 180

tttgttaaca tagctttcct cattactttc ggaaacaacc aaacccctgt cctaaacctc 240

gccggccact gcgaccccgc cagtggtact tgcacggggc tgagtgccga catcagaacc 300

tgccaatcaa aaaacataaa agtcctcctc tcgattggag gggcctccgg aagttactct 360

ctcacttcag ctgatgatgc aaggcaagtt gctgattaca tctggaacaa cttcttaggt 420

ggtcagtcag cttcgcgccc gcttggggac gcggttttgg acggcgttga tttcgacatt 480

gaggcgggtg gtgggcaatt ctatgatgag ctcgccaggt cactcaacgg acacaacgga 540

caggcaaaaa cggtctattt agccgcagct ccacaatgtc cgatcccgga tgctcaccta 600

gacggcgcta tccaaaccgg tttatttgac tacgtttggg ttcagttcta caacaacccc 660

ccatgccagt atgctgacgg taatgccaac gctcttttga acagttggag ccaatgggcc 720

tcggttccgg ccacccaggt attcatgggg ttaccggcgt ccactgatgc cgcgggcagc 780

ggatttattc ctgctgatgc tctcaagtca caagtccttc caacaattaa gaattcggcg 840

aagtatggag gagttatgct ttggagcagg tggtatgaca ttaacagcgg ttatagtgca 900

tccattaagg acagcaggat ccatcgagct cgagctgcag atgaatcgta g 951

<210> 3

<211> 34

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Ala Met Glu Ala Ser Glu Phe Met Ala Ser Lys Thr Gln Thr Leu

1 5 10 15

Ala Leu Thr Leu Ser Leu Leu Ile Leu Ile Ser Ser Cys Lys Ser Ser

20 25 30

Gln Ala

<210> 4

<211> 102

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atggccatgg aggccagtga attcatggca tccaaaacac aaaccctagc cctaactctg 60

tccctcttga tcctcatttc ttcatgcaag tcctcccaag cc 102

<210> 5

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atggagctcg gtaccctcga ggccggaatt gcgatc 36

<210> 6

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

agcagagatt acctatctag actacgattc atctgc 36

<210> 7

<211> 18

<212> DNA

<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