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Acacia cathepsin b and its encoding gene and probe and its application

A technology of cathepsin and azalea, which is applied in the application, genetic engineering, plant genetic improvement and other directions to achieve the effect of reducing the rate of cell proliferation and accelerating death

Inactive Publication Date: 2020-07-14
SHANGHAI JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no literature report related to Agapanthus cathepsin B protein and its coding gene sequence

Method used

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  • Acacia cathepsin b and its encoding gene and probe and its application
  • Acacia cathepsin b and its encoding gene and probe and its application
  • Acacia cathepsin b and its encoding gene and probe and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1, the cloning of Agapanthus Abaciens ApCathB gene

[0053] 1. Acquisition of plant material

[0054] Take the leaf tissue of Agapanthus adult seedlings for RNA extraction;

[0055] 2. Extraction of RNA

[0056] Total RNA (Trizol: Invitrogen) was extracted with "RNA prep pure Plant Total RNA Extraction Kit", and the integrity of RNA was identified by formaldehyde denaturing gel electrophoresis, and then the purity and concentration;

[0057] 3. Full-length cloning of genes

[0058] According to the protein function annotation results of Agapanthus japonica transcriptome sequencing (RNA-seq), the core fragment of ApCathB gene of Agapanthus japonica was obtained. Using the RACE method (SMARTer TM RACE cDNA Amplification Kit: Clonetech) for full-length cDNA cloning in three stages:

[0059] (1) PCR to obtain the middle fragment of the gene

[0060] The extracted RNA was reverse-transcribed (Prime Script Ⅱ 1st Strand cDNA Synthesis Kit: Treasure Bioengi...

Embodiment 2

[0071] Example 2, Sequence Information and Homology Analysis of Agapanthus Abaciens ApCathB Gene

[0072] The full-length open reading frame sequence of the new Agapanthus agapanthus ApCathB gene of the present invention is 1071bp, and the detailed sequence is shown in the sequence shown in SEQID NO.3. According to the sequence of the open reading frame, the amino acid sequence of the Agapanthus Abaciens ApCathB protein was deduced, with a total of 356 amino acid residues, a molecular weight of 39.51kDa, and an isoelectric point (pI) of 5.64. For the detailed sequence, see the sequence shown in SEQ ID NO.4;

[0073] The open reading frame sequence of Agapanthus ApCathB and the amino acid sequence of its encoded protein were nucleated in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS translations+PDB+SwissProt+Superdate+PIR databases using BLAST program Nucleotide and protein homology search, it was found that it has 81% identity with the oil palm Cathepsin...

Embodiment 3

[0074] Embodiment 3, Agapanthus ApCathB gene transformation model plant Arabidopsis thaliana

[0075] 1. Construction of the expression vector containing the gene of interest (ApCathB)

[0076] According to the full-length coding sequence of Agapanthus ApCathB gene (SEQ ID NO.3), design primers to amplify the complete coding reading frame, and introduce restriction endonuclease sites on the upstream and downstream primers respectively (this can be determined by the selected vector Depends), in order to construct the expression vector. Using the amplified product obtained in Example 1 as a template, after PCR amplification, the coding region sequence of the ApCathB gene was connected to an intermediate vector (such as pMD19-T) for sequencing, and then the coding region of the sequenced correct ApCathB gene The sequence is further cloned into an expression vector (such as pHB), and transformed into Agrobacterium tumefaciens (such as GV3101) under the premise that the reading ...

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Abstract

The invention discloses Agapanthus africanus cathepsin B, an encoding gene thereof, a probe and application. The Agapanthus africanus cathepsin B comprises protein (a) or protein (b), wherein the protein (a) is protein formed by an amino acid sequence as shown in SEQ ID No. 4; the protein (b) is protein which is obtained by subjecting the amino acid sequence as shown in SEQ ID No. 4 to substitution or deletion or by adding one or more amino acid to the amino acid sequence as shown in SEQ ID No. 4 and is derived from the protein (a), and the protein (b) has Agapanthus africanus ApCathB protein activity. The invention further provides a nucleotide sequence for encoding the protein and the probe for detecting the nucleotide sequence. The Agapanthus africanus cathepsin B, the encoding gene and the probe have the advantages that a foundation is laid for the further researches of enzyme properties and the expression level of the gene and the protein in various physiological and pathological states, the cathepsin B protein is obtained by the protein engineering technology, and the potential application prospect of the cathepsin B protein om tumor diagnosing and treating, agricultural disease and pest control, food and meat tendering and fragrance increasing, cell line separation and the like.

Description

technical field [0001] The present invention relates to Agapanthus cathepsin B (ApCathB) and its encoding gene and probe, in particular to Agapanthus cathepsin B (ApCathB) and its encoding gene, probe and application. Background technique [0002] Cathepsin B is a class of cysteine ​​proteolytic enzymes that cleave peptide bonds, and its catalytic action is realized by Cys and His, belonging to the papain family. It is active at pH 3.0-7.0, and will be irreversibly inactivated under alkaline conditions. Its hydrolysis site is -Arg-Arg- / -Xaa, and it can hydrolyze dipeptides in sequence at the carboxyl terminal, so it is also called carboxy-dipeptidase. [0003] Cathepsin B has broad-spectrum proteolytic activity, and has hydrolytic activity on various animal proteins (hemoglobin, serum albumin, vitellin, gelatin, etc.) and vegetable proteins (soybean, corn, cottonseed protein, etc.). Cathepsin B plays an essential role in the lysosomal protein degradation pathway. When extr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/50C12N15/57C12Q1/6895C12N15/11
CPCC12N9/63C12Q1/6895C12Q2600/158C12Y304/22001
Inventor 陈冠群申晓辉杨舟张荻
Owner SHANGHAI JIAOTONG UNIV
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