CN106811453B

CN106811453B - African agapanthus cathepsin B, coding gene and probe thereof, and application of African agapanthus cathepsin B

Info

Publication number: CN106811453B
Application number: CN201710008419.5A
Authority: CN
Inventors: 陈冠群; 申晓辉; 杨舟; 张荻
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2017-01-05
Filing date: 2017-01-05
Publication date: 2020-07-14
Anticipated expiration: 2037-01-05
Also published as: CN106811453A

Abstract

The invention discloses a agapanthus cathepsin B, a coding gene and a probe thereof, and application thereof, wherein the protein is the protein of the following (a) or (B): (a) a protein consisting of an amino acid sequence shown as SEQ ID No. 4; (b) and (b) a protein which is derived from the protein (a) and has the agapanthus ApCathB protein activity, wherein the amino acid sequence shown in SEQ ID NO.4 is subjected to substitution, deletion or addition of one or more amino acids. The invention also provides a nucleic acid sequence for coding the protein and a probe for detecting the nucleic acid sequence; the invention lays a foundation for further researching the property of the enzyme and the expression level of the gene and the protein in various physiological and pathological states, and further utilizes the protein engineering technology to obtain the cathepsin B protein, and develops the potential application prospect in the aspects of diagnosing and treating tumors, preventing and treating agricultural diseases and insect pests, tenderizing and enhancing the meat quality of food, separating cell strains and the like.

Description

African agapanthus cathepsin B, coding gene and probe thereof, and application of African agapanthus cathepsin B

Technical Field

The invention relates to agapanthus cathepsin B (ApCathB) and an encoding gene and a probe thereof, in particular to agapanthus cathepsin B (ApCathB) and an encoding gene and a probe thereof and application thereof.

Background

Cathepsin B is a cysteine proteolytic enzyme for cracking peptide bonds, the catalytic action of the cathepsin B is realized by Cys and His, and the cathepsin B belongs to papain family. Has activity at pH3.0-7.0, and can be irreversibly inactivated under alkaline condition. The hydrolysis site is-Arg-Arg-/-Xaa and is capable of sequentially hydrolyzing dipeptides at the carboxy terminus, and is therefore also called carboxy-dipeptidase (carboxy-dipeptidase).

Cathepsin B has a broad spectrum of proteolytic activity, and has hydrolytic activity against various animal proteins (hemoglobin, serum protein, vitellin, gelatin, etc.) and plant proteins (soybean, corn, cottonseed protein, etc.). Cathepsin B plays an essential role in the pathway of lysosomal degradation of proteins, and when extracellular proteins, plasma proteins, hormones, phagocytized bacteria and the like enter cells, they are hydrolyzed by proteolytic enzymes in lysosomes and undergo intracellular digestion, thereby maintaining the precise balance between protein synthesis and degradation. Meanwhile, cathepsin B also participates in the ways of plant defense against exogenous bacteria, plant senescence, apoptosis and the like. Wherein cathepsin B can directly or indirectly activate Caspase to trigger apoptosis, thereby accelerating tissue autolysis.

With the gradual and intensive research on cathepsin B, the research on the properties, genes and protein levels of the cathepsin B in various physiological and pathological states shows potential application prospects in the aspects of diagnosis and treatment of tumors, prevention and treatment of agricultural diseases and insect pests, tenderization and aroma enhancement of food meat quality, separation of cell strains and the like. The gene encoding cathepsin B has been cloned from a variety of plants including: arabidopsis, rice, maize, soybean, etc. However, the cloning, expression pattern and protein sequence of cathepsin B in ornamental plants, particularly bulbous flowers, are not known. At present, no literature report related to agapanthus cathepsin B protein and coding gene sequences thereof exists.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to fill the blank of the cloning and expression pattern analysis of agapanthus cathepsin B (ApCathB) gene and agapanthus ApCathB protein, provides an agapanthus ApCathB protein, and also provides a nucleic acid sequence for coding the protein and a probe for detecting the nucleic acid sequence; the invention discloses a physiological effect and an expression mode of a agapanthus ApCathB gene transformed into arabidopsis thaliana, thereby providing a theoretical basis for regulating and controlling the time-space characteristics of ApCathB gene expression by using a genetic engineering technology in the future and having great application value.

The purpose of the invention is realized by the following technical scheme:

in a first aspect, the present invention provides agapanthus cathepsin B comprising the following protein (a) or (B):

(a) a protein consisting of an amino acid sequence shown as SEQ ID No. 4;

(b) and (b) a protein which is derived from the protein (a) and has the agapanthus ApCathB protein activity, wherein the amino acid sequence shown in SEQ ID NO.4 is subjected to substitution, deletion or addition of one or more amino acids.

Preferably, the protein is a sequence obtained by deleting, inserting and/or substituting 1-50 amino acids of an amino acid sequence shown in SEQ ID NO.4, or adding 1-20 amino acids at the C terminal and/or the N terminal.

Preferably, the protein is a sequence formed by replacing 1-10 amino acids in an amino acid sequence shown in SEQ ID NO.4 by amino acids with similar or similar properties.

In a second aspect, the invention provides a nucleic acid sequence encoding agapanthus cathepsin B.

Preferably, the nucleic acid sequence is in particular:

(a) the base sequence is shown as 1st to 1071 st sites of SEQ ID NO. 3;

or (b) a sequence having at least 70% homology with the nucleic acid shown in SEQ ID NO.3 at positions 1-1071;

or (c) a sequence capable of hybridizing with the nucleic acid shown in the 1st to 1071 st positions of SEQ ID NO. 3.

Preferably, the nucleic acid sequence is specifically deletion, insertion and/or substitution of 1-90 nucleotides in the nucleic acid sequence shown in 1 st-1071 st position of SEQ ID NO.3, or a sequence formed by adding 60 nucleotides to the 5 'and/or 3' end.

In a third aspect, the invention provides a probe for detecting a nucleic acid sequence encoding the agapanthus cathepsin B, wherein the probe is a nucleic acid molecule comprising 8-100 continuous nucleotides of the nucleic acid sequence. The probe can be used for detecting whether nucleic acid molecules related to the agapanthus cathepsin B (ApCathB) exist in a sample.

In a fourth aspect, the invention provides an application of a agapanthus cathepsin B protein coding gene, wherein the base sequence of the gene is shown in SEQ ID No.3, 1st to 1071 st sites, and the application comprises the following steps: the gene is used for preventing and controlling agricultural diseases and insect pests, tenderizing and flavoring food meat quality and separating cell strains by adopting a genetic engineering means.

In the present invention, the term "agapanthus cathepsin B (ApCathB) protein coding sequence" refers to a nucleotide sequence encoding a polypeptide having agapanthus ApCathB protein activity, such as nucleotide sequences 1 to 1071 shown in SEQ ID NO.3 and degenerate sequences thereof. The degenerate sequence is a sequence generated by replacing one or more codons with degenerate codons for coding the same amino acid in nucleotides 1 to 1071 shown in SEQ ID NO. 3. Due to the degeneracy of codons, a degenerate sequence with homology as low as about 70% with the 1st to 1071 st nucleotide sequences shown in SEQ ID NO.3 can also encode the sequence shown in SEQ ID NO. 4. The term also includes nucleotide sequences having at least 70% homology with the nucleotide sequence shown in SEQ ID NO. 3.

The term also includes variants of the sequence shown in SEQ ID NO.3 that encode the same function of the native agapanthus cathepsin B (ApCathB) protein. These variants include (but are not limited to): usually 1-90 nucleotides, and within 60 nucleotides at the 5 'and/or 3' end.

In the present invention, the term "agapanthus cathepsin b (ApCathB) refers to a polypeptide having the sequence shown in SEQ ID No.4 having an agapanthus ApCathB protein activity. The term also includes variants of the sequence of SEQ ID No.4 that have the same function as the native agapanthus ApCathB protein. These variants include (but are not limited to): usually 1 to 50 amino acid deletions, insertions and/or substitutions, and C-terminal and/or N-terminal addition of one or less than 20 amino acids. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, the addition of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the function of the protein. The term also includes active fragments and active derivatives of the agapanthus ApCathB protein.

The variant form of agapanthus cathepsin b (apcathb) of the present invention comprises: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridizes under high or low stringency conditions with agapanthus ApCathB-associated DNA, and polypeptides or proteins obtained using antisera to agapanthus ApCathB proteins.

In the present invention, the "agapanthus ApCathB conservative variant polypeptide" refers to a polypeptide formed by replacing at most 10 amino acids with amino acids having similar or similar properties, as compared with the amino acid sequence shown in SEQ ID No. 4. These conservative variant polypeptides are preferably generated by substitution according to table 1.

TABLE 1

The analogs also include analogs having residues other than the naturally occurring L-amino acids (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., β, gamma-amino acids), it being understood that the polypeptides of the invention are not limited to the representative polypeptides listed above.

Modified (generally without altering primary structure) forms include: chemically derivatized forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications in the synthesis and processing of the polypeptide or in further processing steps. Such modification may be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylase. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides modified to increase their resistance to proteolysis or to optimize solubility.

In the invention, the physiological effect and the expression mode of the agapanthus ApCathB gene in arabidopsis can be analyzed by a real-time fluorescent quantitative PCR method, namely the function of the protein coded by the agapanthus ApCathB gene is analyzed.

The detection method for detecting whether the agapanthus ApCathB related nucleotide sequence exists in the sample comprises the steps of hybridizing the probe and the sample, and then detecting whether the probe is combined. The sample is a product after PCR amplification, wherein PCR amplification primers correspond to a nucleotide coding sequence related to agapanthus ApCathB and can be positioned at two sides or in the middle of the coding sequence. The length of the primer is generally 15 to 50 nucleotides.

In addition, the agapanthus ApCathB nucleotide sequence and the agapanthus ApCathB amino acid sequence can screen related homologous genes or homologous proteins of the agapanthus ApCathB on the basis of nucleic acid homology or homology of expressed proteins.

To obtain arrays of agapanthus ApCathB related genes, agapanthus cDNA libraries can be screened with DNA probes under low stringency conditions³²P is obtained by radioactive labeling of all or part of related agapanthus ApCathB. A suitable cDNA library for screening is a library from agapanthus. Methods for constructing cDNA libraries from cells or tissues of interest are well known in the field of molecular biology. In addition, many such cDNA libraries are also commercially available, for example, from Clontech, Stratagene, Palo Alto, Calif. The screening method can identify the nucleotide sequence of the gene family related to the agapanthus ApCathB.

The agapanthus ApCathB related nucleotide full-length sequence or the fragment thereof can be obtained by a PCR amplification method, a recombination method or an artificial synthesis method. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed herein, particularly open reading frame sequences, and the sequences can be amplified using commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art as templates. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order.

When the sequence of interest is obtained, the sequence of interest can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

Furthermore, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

In addition to being produced recombinantly, fragments of the proteins of the invention can be produced by direct peptide synthesis using solid phase techniques (Stewart et al, (1969) solid phase peptide synthesis, WH Freeman Co., San Francisco; Merrifield J. (1963) J.am chem.Soc 85: 2149-. In vitro synthesis of proteins can be performed manually or automatically. For example, peptides can be synthesized automatically using a model 431A peptide synthesizer from Applied Biosystems (Foster City, Calif.). Fragments of the proteins of the invention can be chemically synthesized separately and then chemically linked to produce full-length molecules.

The agapanthus cathepsin B (ApCathB) can be used for screening substances related to the agapanthus cathepsin B (ApCathB) protein and having interaction, or inhibitors, antagonists and the like by various conventional screening methods.

The agapanthus has extremely high ornamental value and wide application, and has very high application prospect in garden planning. Therefore, research on adaptability to different environments and resistance characteristics to diseases and insect pests is a prerequisite for promoting popularization and application of the pesticide.

Compared with the prior art, the invention has the following beneficial effects:

1) the invention clones the coding sequence of cathepsin B (ApCathB) in the plant body of the African agapanthus for the first time, transforms the coding sequence into the model plant Arabidopsis thaliana, adopts a fluorescence real-time quantitative PCR method to analyze the physiological effect and the expression mode of the ApCathB gene in the Arabidopsis thaliana, and provides a theoretical basis for regulating and controlling the space-time expression of the ApCathB gene by utilizing a gene engineering technology in the future, thereby providing a theoretical basis for the popularization and the application of the African agapanthus in the landscape.

2) The obtained in-vivo cathepsin B of the agapanthus plant can accelerate cell death and obviously reduce cell proliferation rate under the extreme stress environment. The method is favorable for the characteristic, and can be applied to the aspects of diagnosing and treating tumors, preventing and treating agricultural diseases and insect pests, tendering and increasing flavor of food meat quality, separating cell strains and the like.

Drawings

Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:

FIG. 1 shows the results of homology comparison (GAP) of the nucleotide sequences of mRNA of the African agapanthus ApCathB gene and the oil palm Cathepsin B-like gene according to the present invention;

FIG. 2 is the results of a homology comparison (FASTA) of the amino acid sequences of the African agapanthus ApCathB protein and the oil palm Cathepsin B-like protein of the present invention, wherein the same amino acids are indicated by amino acid one-letter symbols between the two sequences;

FIG. 3 shows wild type and ApCathB transgenic Arabidopsis thaliana at H₂O₂Phenotypic observation of growth status under stress;

FIG. 4 shows wild type and ApCathB transgenic Arabidopsis thaliana at H₂O₂Single seedlings growing under stress bear fruit weight;

FIG. 5 shows the recombinant E.coli Transetta: the growth condition of PET-30a-ApCathB in the stress culture medium;

FIG. 6 shows quantitative analysis of wild type and ApCathB transgenic Arabidopsis ApCathB gene expression.

Detailed Description

The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.

The experimental procedures, for which specific conditions are not indicated in the following examples, are generally carried out according to conventional conditions, for example, those described in molecular cloning, for example, Sambrook et al, a laboratory Manual (New York: Cold Spring Harbor L laboratory Press,1989), or according to the conditions recommended by the manufacturer.

Example 1 cloning of African agapanthus ApCathB Gene

1. Obtaining plant material

Taking leaf tissues of adult seedlings of the agapanthus, and extracting RNA;

extraction of RNA

Extracting total RNA (Trizol: Invitrogen) by using an RNA prep pure plant total RNA extraction kit, identifying the integrity of the RNA by using formaldehyde denaturing gel electrophoresis, and then determining the purity and concentration of the RNA on a Spectrophotometer (Thermo Scientific NanODROP 1000 Spectrophotometer);

3. full Length cloning of Gene

And obtaining the core fragment of the agapanthus ApCathB gene according to the protein function annotation result of agapanthus transcriptome sequencing (RNA-seq). By RACE (SMARTer)^TMRACE cDNA Amplification Kit: Clonetech) was performed in three stages:

(1) PCR to obtain intermediate fragments of the Gene

Reverse transcription of the extracted RNA (Prime Script II 1st Strand cDNA Synthesis Kit: Bao bioengineering (Dazong) Co., Ltd.), PCR using primers ApCathB F (SEQ ID NO.1) and ApCathB R (SEQ ID NO.2) with first Strand cDNA as a template, amplification to obtain a 1403bp fragment, recovery and ligation to pMD19-TSimplevector vector, sequencing using RV-M and M13-47 as universal primers using terminator fluorescent marker (Big-Dye, Perkin-Elmer, USA) on ABI377 sequencer (Perkin-Elmer, USA), sequencing by B L AST (http:// blast. NCBI. n. lm. nih. gov) on NCBI website, comparison of existing database (Bank) with known nucleic acid sequence and known homology of oil palm gene, and known homology of Haemain CathB gene, which is considered as a high AppsB gene;

(2)3′RACE

two rounds of nested PCR completed the amplification of the 3' end sequence.

A first round: UPM +3 ' -GSP1 (5'-GCTAACCTCACCTCGCTTGCCATCTA-3') (SEQ ID NO.5)

And a second round: NUP +3 ' -GSP2 (5'-GTTGTTACCGACGAGTGCGACCCATA-3') (SEQ ID NO.6)

3 ' RACE obtains a 3 ' terminal sequence (724bp) of African agapanthus Apcathb, the 3 ' terminal sequence is recovered and connected to a pMD19-T Simple vector, RV-M and M13-47 are used as universal primers, a method of terminator fluorescence labeling (Big-Dye, Perkin-Elmer, USA) is adopted for sequencing on an ABI377 sequencer (Perkin-Elmer, USA), and the sequencing result is compared with an existing database (GenBank) through B L AST (http:// blast.ncbi.nlm.nih.gov /) carried out on an NCBI website, and the homology of a nucleic acid sequence and a coding protein of the kit with known Cathepsin B genes of date and oil palm is known to be high;

(3)5′RACE

5 'RACE ready cDNA was used as a template to complete the amplification of 5' end sequence by two rounds of nested PCR.

A first round: UPM +5 ' -GSP1 (5'-CACCGTCCTCGCTAGTTCCCCAACC-3') (SEQ ID NO.7)

And a second round: NUP +5 ' -GSP2 (5'-GAAATATGGGTCGCACTCGTCGGTAA-3') (SEQ ID NO.8)

UPM and NUP provide a kit, 5 'RACE obtains the 5' terminal sequence (686bp) of the African agapanthus ApCathB gene, the same method is used for sequencing after recovery and connection, the sequencing results of the sequences obtained by the 3 methods are spliced, the spliced sequence is submitted to B L AST analysis, the result proves that the ApCathB gene newly obtained from African agapanthus is indeed a cathepsin B gene, the sequencing results are combined with ORF finishing (http:// www.ncbi.nlm.nih.gov/gorf) prediction of NCBI to discover the start codon and the stop codon of the African agapanthus ApCathB gene, specific primers ORF-F (5'-ATGATGATGATGAAGAAGGGGATCATGTAT-3') (SEQ ID NO.13) and ORF-R (5'-CTATATCCATGCGAGCGAAGCACC-3') (SEQ ID NO.14) are respectively designed from the start codon and the stop codon according to the obtained sequences, PCR is carried out by taking African agapanthus cDNA as a template to amplify the coding sequence (SEQ ID NO.3) of 1071bp African agapanthus ApCathB protein.

Example 2 sequence information and homology analysis of African agapanthus ApCathB Gene

The sequence of the full-length open reading frame of the novel agapanthus ApCathB gene is 1071bp, and the detailed sequence is shown as a sequence represented by SEQID NO. 3. Deducing an amino acid sequence of the agapanthus ApCathB protein according to the open reading frame sequence, wherein the amino acid sequence has 356 amino acid residues, the molecular weight is 39.51kDa, the isoelectric point (pI) is 5.64, and the detailed sequence is shown as a sequence shown in SEQ ID NO. 4;

nucleotide and protein homology searches using the B L AST program in the Non-redundant GenBank + EMB L + DDBJ + PDB and Non-redundant GenBank CDS translations + PDB + SwissProt + Superdate + PIR database revealed 81% identity at the nucleotide level with the oil palm Catherin B-like gene (accession # XM-010909454.1), as shown in FIG. 1 (Sbcoding sequence of Query: Lotus ApthB; jct: mRNA sequence of oil palm Catherin B-like), and at the amino acid level, it also showed 81% identity and 90% similarity with the oil palm Catherin B-like protein (accession # XP-010907756.1), as shown in FIG. 2 (the levels of the amino acid sequences of Query: Cathay B protein; the amino acid sequences of the protein Aphyllin B-like protein, and thus the amino acid sequences of the other Cathay B proteins are known to be present in the Non-redundant GenBank, EMBL.: Aphyllin B-protein sequences, and thus the amino acid sequence of the protein Aphyllin the Non-redundant Aphyllin Aphyllum protein.

Example 3 African agapanthus ApCathB Gene transformation model plant Arabidopsis thaliana

1. Construction of expression vector containing target Gene (ApCathB)

According to the full-length coding sequence (SEQ ID NO.3) of the agapanthus ApCathB gene, primers for amplifying a complete coding reading frame are designed, and restriction endonuclease sites (which can be determined according to the selected vector) are respectively introduced on the upstream primer and the downstream primer so as to construct an expression vector. The amplification product obtained in the embodiment 1 is used as a template, after PCR amplification, the coding region sequence of the ApCathB gene is connected to an intermediate vector (such as pMD19-T) for sequencing, the coding region sequence of the ApCathB gene with correct sequencing is further cloned into an expression vector (such as pHB), the ApCathB gene is transferred into Agrobacterium tumefaciens (such as GV3101) on the premise of correct reading frame identification, and PCR identification is carried out on the transformed Agrobacterium tumefaciens, so that the plant expression vector containing the ApCathB gene is successfully transferred into the Agrobacterium tumefaciens.

2. Agrobacterium tumefaciens mediated transformation of arabidopsis thaliana

(1) Pre-shaking Agrobacterium, namely selecting a positive monoclonal to be 25ml of YEP liquid culture medium containing 50 mg/L Kan, 50 mg/L gentamicin and 25 mg/L Rif, shaking the bacteria at the speed of 200rpm at the temperature of 28 ℃ for 24 h;

(2) expanding and culturing agrobacterium, namely expanding and culturing the pre-shaken agrobacterium liquid into a YEP culture medium containing 400m L Kan resistance at the ratio of 1:100, culturing for 13-16h at the temperature of 28 ℃ and the rpm of 200, and culturing until the absorbance OD600 reaches 1.5-2.0 to collect the agrobacterium under the conditions of 23 ℃, 5000rpm and 8 min;

(3) and (3) cutting off all the siliques and full and white flowers on the plants one day before or on the day of transformation to prepare 500m L1/2 MS solution containing 5% of cane sugar, suspending the collected agrobacterium tumefaciens precipitates by using a small amount of MS solution, shaking uniformly, adding 0.04% (v/v) Silwet L-77 and 10 mu L6-BA (mother solution is 1mg/m L) into the rest cane sugar solution, stirring uniformly, mixing uniformly before transformation, soaking the stems and inflorescences of the plants in bacterial liquid for 30s, taking out the drained bacterial liquid, putting the drained bacterial liquid into a disposable plastic bag, sealing and moisturizing, covering a black box after all the plants are transformed, culturing in the dark for 24h, taking out the plants, standing the plants vertically, watering and culturing to ensure that the plants have sufficient water.

3. Screening of transgenic Positive lines

And (3) harvesting seeds of the transformed plants after the siliques are completely mature, placing the transformed plants in a drying culture dish filled with filter paper at room temperature for a week to completely dry the seeds, filtering the seeds by using a 50-mesh stainless steel sieve, removing the siliques, collecting transgenic T1 generation seeds, sowing the seeds in a plug tray, screening seedling resistance by using 0.05% (v/v) glufosinate-ammonium to obtain T2 generation transgenic plants, and continuously screening until T3 generation homozygotic transgenic plants are obtained.

4. ApCathB gene expression difference of transgenic arabidopsis plant

Cutting leaves of 0.2g of arabidopsis wild type and ApCathB transgenic arabidopsis plants, extracting RNA, preparing cDNA and carrying out real-time quantitative PCR analysis. The specific primer for quantitative analysis of ApCathB gene in Real-time PCR is qApCathB-F (5'-TTTGATGCTAGAACAGCTTGG-3') (SEQ ID NO.9), the primer qApCathB-R (5'-GGCCATTATAGGATATCCCC-3') (SEQ ID NO.10), the reference gene is Arabidopsis thaliana UBQ5 gene, and the primer is UBQ5-F (5'-GACGCTTCATCTCGTCC-3' (SEQ ID NO.11)), UBQ5-R (5'-CCACAGGTTGCGTTAG-3') (SEQ ID NO. 12). By using

The relative quantitative analysis of the method shows that the expression level of ApCathB in the transgenic arabidopsis is higher, which is 2.6 times of that of the reference gene UBQ5 and is 1894 times of that of the wild plant (figure 6). Indicating that ApCathB is not expressed in wild plants.

Example 4 functional verification of African agapanthus ApCathB Gene transfer to Arabidopsis thaliana

H at different concentrations₂O₂Treatment of simulated oxidative stress, wild type and ApCathB transgenic Arabidopsis plants in H₂O₂The phenotype under stress was significantly different (FIG. 3), in FIG. 3, ① represents wild type Arabidopsis thaliana, ② and ③ represent ApCathB transgenic Arabidopsis thaliana, ApCathB transgenic seedlings vs. wild type seedlings H₂O₂The induced oxidative stress is more sensitive, the growth of seedlings is obviously inhibited, and ApCathB transgenic seedlings grow in H₂O₂The weight of a single seedling under stress was only around 50% of that of the wild type (FIG. 4). And with H₂O₂The increase of the concentration also gradually reduces the growth amount of seedlings, and the seedlings tend to die. The results show that ApCathB can make seedlings more sensitive to abiotic stress and accelerate cell death.

Example 5, recombinant escherichia coli Transetta: stress resistance functional verification of PET-30a-ApCathB

1. Construction of prokaryotic expression vector containing target gene (ApCathB)

The full-length coding sequence (SEQ ID NO.3) of the agapanthus ApCathB gene was ligated to a prokaryotic expression vector pET-30a according to the method of example 3, and transferred into E.coli (DN5 α) for mass amplification, and plasmids were extracted.

2. Expression of recombinant proteins

The constructed vector pET-30a-ApCathB is transferred into an escherichia coli Transetta strain for recombinant protein expression, firstly, positive single clones are picked to 5ml of L B liquid culture medium containing 50 mg/L kanamycin and 15 mg/L chloramphenicol, bacteria are shaken overnight at 37 ℃ and 200rpm, secondly, the pre-shaken Transetta bacterial liquid is expanded to a culture medium containing 50m L kanamycin and 15 mg/L chloramphenicol resistance L B at a ratio of 1:100, the bacteria liquid is cultured at 37 ℃ and 200rpm for about 3 hours to enable the OD600 value of the bacterial liquid to be 0.6-0.8, and then IPTG with the final concentration of 0.8mM is added into the cultured bacterial liquid for protein expression induction, and the expression is shown to be carried out for 2-8 hours at 25 ℃.

3. Identification of stress resistance of recombinant protein

Single colonies of E.coli Transetta containing pET-30a and pET-30a-ApCathB vectors were inoculated into 5m L liquid medium L B containing 50 mg/L kanamycin and 15 mg/L chloramphenicol, respectively, and shaken overnight at 37 ℃ and 200rpm, the next day was inoculated into NaCl (200, 400, 600, 800mM), KCl (200, 400, 600, 800mM), PEG6000(2.5, 5.0, 10, 15%) and different pHs (3.0, 5.0, 7.0, 9.0, 11.0) at a ratio of 1:100, respectively, and simultaneously 0.8mM TG was added to induce expression, and OD600 values were measured after 12h, as shown in FIG. 5, which indicates that the recombinant proteins pET-30 a-ApthB were less resistant to pET-30a under different stress conditions, indicating that AptB proteins can inhibit the proliferation rate of E.coli and are not favorable to the environment where stress is present.

The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.

SEQUENCE LISTING

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gtggaagtcg ccttcacagt ttatgaggat tttgctcact ataaatcagg gatctacaag 840

catgttacag gtggtgtaat gggtggccat gctgtgaaat taattggttg gggaactagc 900

gaggacggtg aagattattg gcttcttgca aatcagtgga acagaggttg gggcgatgat 960

ggttacttca agattgtgag gggaagcaat gaatgcggta ttgaagagga tgtggttgca 1020

ggattgcctt ctaccaagaa tttagcaggt gcttcgctcg catggatata g 1071

<210>4

<211>356

<212>PRT

<213>Artificial

<220>

<223>artificial sequence

<400>4

Met Met Met Met Lys Lys Gly Ile Met Tyr Ala Leu Pro Leu Pro Leu

1 5 10 15

Leu Leu Leu Leu Val Leu Thr Ala Glu Ile Gln Phe Leu Gln Val Val

20 25 30

Ala Val Lys Pro Ile Pro Gln Ala Lys Gln Asp Ala Lys Ile Leu Gln

35 40 45

Asp Ser Ile Val Glu Lys Val Asn Ser Asn Pro Asn Ala Gly Trp Arg

50 55 60

Ala Ser Ile Asn Ser Arg Phe Ser Asn Tyr Thr Arg Gly Gln Phe Gln

65 70 75 80

Tyr Ile Leu Gly Val Lys Gln Leu Leu Gln Thr Ala Leu Glu Gly Phe

85 90 95

Pro Val Lys Thr Tyr Pro Lys Leu Leu Lys Leu Pro Lys Gln Phe Asp

100 105 110

Ala Arg Thr Ala Trp Pro Gln Cys Ser Thr Ile Gly Lys Ile Leu Asp

115 120 125

Gln Gly His Cys Gly Ser Cys Trp Ala Phe Gly Ala Val Glu Ser Leu

130 135 140

Ser Asp Arg Phe Cys Ile Asn Phe Gly Met Asn Ile Ser Leu Ser Val

145 150 155 160

Asn Asp Leu Leu Ser Cys Cys Gly Phe Met Cys Gly Asp Gly Cys Asp

165 170 175

Gly Gly Tyr Pro Ile Met Ala Trp Arg Tyr Phe Val Gln Asn Gly Val

180 185 190

Val Thr Asp Glu Cys Asp Pro Tyr Phe Asp Ala Glu Gly Cys Ala His

195 200 205

Pro Gly Cys Glu Pro Leu Tyr Pro Thr Pro Gln Cys Glu Lys Lys Cys

210 215 220

Lys Ala Asn Asn Leu Val Trp Met Glu Thr Lys His Phe Ser Val Asp

225 230 235 240

Ala Tyr Arg Val Ser Ser Asp Pro His Asp Ile Met Gln Glu Val Tyr

245 250 255

Thr His Gly Pro Val Glu Val Ala Phe Thr Val Tyr Glu Asp Phe Ala

260 265 270

His Tyr Lys Ser Gly Ile Tyr Lys His Val Thr Gly Gly Val Met Gly

275 280 285

Gly His Ala Val Lys Leu Ile Gly Trp Gly Thr Ser Glu Asp Gly Glu

290 295 300

Asp Tyr Trp Leu Leu Ala Asn Gln Trp Asn Arg Gly Trp Gly Asp Asp

305 310 315 320

Gly Tyr Phe Lys Ile Val Arg Gly Ser Asn Glu Cys Gly Ile Glu Glu

325 330 335

Asp Val Val Ala Gly Leu Pro Ser Thr Lys Asn Leu Ala Gly Ala Ser

340 345 350

Leu Ala Trp Ile

355

<210>5

<211>26

<212>DNA

<213>Artificial

<220>

<223>Artificial Sequence

<400>5

gctaacctca cctcgcttgc catcta 26

<210>6

<211>26

<212>DNA

<213>Artificial

<220>

<223>Artificial Sequence

<400>6

gttgttaccg acgagtgcga cccata 26

<210>7

<211>25

<212>DNA

<213>Artificial

<220>

<223>Artificial Sequence

<400>7

caccgtcctc gctagttccc caacc 25

<210>8

<211>26

<212>DNA

<213>Artificial

<220>

<223>Artificial Sequence

<400>8

gaaatatggg tcgcactcgt cggtaa 26

<210>9

<211>21

<212>DNA

<213>Artificial

<220>

<223>Artificial Sequence

<400>9

tttgatgcta gaacagcttg g 21

<210>10

<211>20

<212>DNA

<213>Artificial

<220>

<223>Artificial Sequence

<400>10

ggccattata ggatatcccc 20

<210>11

<211>17

<212>DNA

<213>Artificial

<220>

<223>Artificial Sequence

<400>11

gacgcttcat ctcgtcc 17

<210>12

<211>16

<212>DNA

<213>Artificial

<220>

<223>Artificial sequence

<400>12

ccacaggttg cgttag 16

<210>13

<211>30

<212>DNA

<213>Artificial

<220>

<223>Artificial Sequence

<400>13

atgatgatga tgaagaaggg gatcatgtat 30

<210>14

<211>24

<212>DNA

<213>Artificial

<220>

<223>Artificial sequence

<400>14

ctatatccat gcgagcgaag cacc 24

Claims

1. A agapanthus cathepsin B, which is characterized by comprising a protein of the following (a):

(a) a protein consisting of an amino acid sequence shown as SEQ ID No. 4.

2. A nucleic acid sequence encoding the agapanthus cathepsin B of claim 1.

3. The nucleic acid sequence encoding African agapanthus cathepsin B according to claim 2, which is in particular:

(a) the base sequence is shown in SEQ ID NO.3 1-1071.