CN100532552C

CN100532552C - Gene sequence of coding perinereis albuhitensis grube cysteine protease inhibitor and its amino acid sequence and application

Info

Publication number: CN100532552C
Application number: CNB2007101150097A
Authority: CN
Inventors: 李荣贵; 杜桂彩
Original assignee: Qingdao University
Current assignee: Qingdao University
Priority date: 2007-11-20
Filing date: 2007-11-20
Publication date: 2009-08-26
Anticipated expiration: 2027-11-20
Also published as: CN101186916A

Abstract

The invention relates to gene sequence and amino acid sequence of coding perinereis aibuhitensis grube cysteine protease inhibitor and insect-resistant activity of recombinant protein produced by the gene. The gene sequence and the amino acid sequence of the code protein are respectively shown like sequence 1 and sequence 2 in the chart, wherein the length of nucleotide sequence of the gene of coding perinereis aibuhitensis grube cysteine protease inhibitor is 333bp, the total length of the amino acid sequence of coding perinereis aibuhitensis grube cysteine protease inhibitor includes 110 amino acid residues, and recombinant cysteine protease inhibitor is clearly characterized by insect-resistant activity, which belongs to the field of agricultural biotechnology. Based on the gene of clamworm cysteine protease inhibitor of the invention, the coding gene of the cysteine protease inhibitor can be cloned from the cDNA library of the perinereis aibuhitensis grube. The gene of the cysteine protease inhibitor can be introduced into the plant to cultivate anti-insect plants by gene recombination technique. The gene production has recombinant clamworm cysteine protease inhibitor with insect-resistant activity, which can also be showed in other cells. The recombinant protein can be considered as novel biological pesticide to protect the agriculture from pest hazard.

Description

A kind of gene order of coding perinereis albuhitensis grube cystatin and aminoacid sequence thereof and application

Technical field:

The present invention relates to a kind of gene and aminoacid sequence thereof of coding perinereis albuhitensis grube cystatin and the anti-insect activity of using the recombinant protein of this genes produce, belong to agricultural biological technical field.

Background technology:

Proteinase inhibitor is that a class can the active material of arrestin enzymic hydrolysis, extensively is present in animal, plant, microorganism and the human body.They are by combining with protease activities position and allosteric site, and the catalytic activity of inhibitory enzyme or prevention proenzyme are converted into activated enzyme.This proteinoid plays an important role at the aspects such as degraded, immune response and metastases of modulin in the body.The proteinase inhibitor of a lot of plants is also by suppressing the activity of insect body endoproteinase, the performance defense function.Difference according to its effect substrate and character thereof, proteinase inhibitor can be divided into serpin, cystatin, inhibitors of metalloproteinase and acid protease inhibitor, wherein, can to suppress in the digestive tube with the serine protease be the growth of the insect of major protein enzyme to serpin; Cystatin is then to being that the control of insect of Coleoptera of main digestive ferment is valuable with L-Cysteine HCL Anhydrous.This two proteinoids enzyme inhibitors has complementarity on pest-resistant spectrum.

The gene of the cystatin of the multiple biology of current encoder is cloned.(2002) such as white persons of outstanding talent have reported the encoding gene of having cloned a cystatin from mandarin sturgeon and acipenser schrencki respectively, mandarin sturgeon cystatin full length gene 635bp wherein, the full length gene sequence of acipenser schrencki is 629bp, two cystatins that gene is all encoded and is made up of 112 amino acid.Abrahamson etc. (1987) have reported the gene order of people's cysteine proteinase inhibitor C, Colella etc. (1989) have cloned the encoding gene of cystatin in the Ovum Gallus domesticus album, and the distribution in different tissues is studied to this albumen.Yamashita etc. (1996) have cloned the gene that L-Cysteine HCL Anhydrous suppresses from Oncorhynchi, and have studied the expression in different tissues, have inquired into its possible biologic activity.Xu Anlong etc. are in its patent (patent No.: disclose a kind of jellyfish cystatin gene and expression thereof and application 03126567.7), this full length gene 396bp, encoded protein enzyme inhibitors total length contains 131 amino-acid residues, comprise 18 amino acid whose signal peptides and 118 amino acid whose maturation proteins, recombinant protein has the activity of typical cystatin, and papoid is had the obvious suppression effect.

Perinereis aibuhitensis is a kind of ocean annelid that China's seashore extensively distributes, and a kind of little peptide (nereistoxin) wherein successfully is used as biological pesticide, but at home and abroad there is no the research report of clam worm cystatin and encoding gene thereof at present.

Summary of the invention:

The object of the present invention is to provide a kind of gene order of new perinereis aibuhitensis cystatin and the aminoacid sequence of its proteins encoded, and the anti-insect activity of using the recombinant protein of this gene preparation.This technology is for cultivating zoophobous by transgenic technology, and perhaps by make up engineering bacteria production reorganization clam worm cystatin in gene recombination technology, preparing novel biological pesticide aspect has good prospects for application.

In order to realize the foregoing invention purpose, the present invention has made up the cDNA library of perinereis aibuhitensis: at first extracted the mRNA of perinereis aibuhitensis, reverse transcription becomes the cDNA rear clone to plasmid pAP3neo, and Transformed E .coli DH10B has made up the cDNA library.

The present invention measures by the sequence to the cDNA library of perinereis aibuhitensis, has therefrom screened the cDNA of cystatin, and this cDNA contains the open reading frame of 333bp, the 110 amino acid constitutive proteins of encoding.

To expression vector pET-15b, recombinant plasmid transformed E.coliBL21 (DE3) has made up engineering bacteria with the gene clone of perinereis aibuhitensis cystatin in the present invention, and IPTG induces this expression of gene, and the recombinant protein of expression is through Ni ²⁺The resin affinity chromatography has obtained electrophoretically pure reorganization cystatin.

The recombinant protein that the present invention obtains has restraining effect to the papoid that belongs to L-Cysteine HCL Anhydrous, and the insect holotrichia oblita that belongs to Coleoptera is had tangible anti-insect activity, can prepare the novel biopesticide with anti-insect activity.

A kind of gene order with perinereis aibihitensis Grube cystatin of thrombolysis activity, this sequence is as follows: atggagtcggcacttcaacagaaatctgtcatgatgtctggtggtttaggcccaac tcttcctgctgatccggagacgcaagttatttgtgatgaggtaaaaaaccagctgg agggcgaagttggacgtaactttgctgagtacaatgccatcctttttaggagtcaa gtagttgctggaaccgtcctcttcatcaaagtgcacgttggccatgctgattacat tcatattcgcatcttcataccactgccaggtccaggaaaaaccagtcttggagggt accaactacacaagactaaggatgatccaatcaaatattttaataataattag

A kind of gene order with perinereis aibuhitensis cystatin of thrombolysis activity, its amino acid sequence coded is as follows:

MESALQQKSVMMSGGLGPTLPADPETQVICDEVKNQLEGEVGRNFAEYNAILFRSQVVAGTVLFIKVHVGHADYIHIRIF

IPLPGPGKTSLGGYQLHKTKDDPIKYFNNN

Gene according to clam worm cystatin of the present invention, can from the cDNA library of perinereis aibuhitensis, clone the encoding gene of cystatin, can be by gene recombination technology with the cystatin gene transfered plant to cultivate zoophobous, also can in other cell, express the reorganization clam worm cystatin that this genes produce has anti-insect activity, recombinant protein can be used as novel biological pesticide, control agricultural insect pest.

Description of drawings:

Fig. 1 cuts the agarose gel electrophoresis figure of evaluation for the enzyme of expression plasmid pET-15bI.

Fig. 2 is the SDS-PAGE analysis chart of the purifying of reorganization cystatin.

Fig. 3 is the restraining effect activation analysis figure of reorganization cystatin to papoid.

Fig. 4 measures figure for the anti-insect activity of reorganization cystatin.

Embodiment:

The present invention is further illustrated below by specific embodiment.

Embodiment 1: the structure in perinereis aibuhitensis cDNA library and the clone of encoding cysteine protease inhibitor gene

1, is material with 2g perinereis aibuhitensis biological tissue, uses RNAiso Reagent (TaKaRa Code No.D312) to extract total RNA.

2, use Oligotex-dT30mRNA purification kit (TaKaRa Code No.9086) to extract mRNA.

3, be template with 5 μ g mRNA, use TaKaRa cDNA library construction test kit construction cDNA library, specifically may further comprise the steps:

(1) utilizes ThermoScript II M-MLV and Oligo (dT) anchor primer to synthesize article one chain of cDNA, use 5-methyl dCTP when synthetic;

(2) utilize RNaseH, e. coli dna polymerase and dna ligase to synthesize the second chain of cDNA;

(3) utilize the T4DNA polysaccharase to make the terminal smoothing of double-stranded cDNA;

(4) be connected with the joint that contains the NotI recognition sequence, carry out enzyme with NotI again and cut;

(5) use DNA to reclaim test kit (TaKaRa Code No.9092) and remove short chain cDNA;

(6) be connected with plasmid pAP3neo, get 20 μ l and connect product;

(7) get 2 μ l and connect product Transformed E .coli DH10B, the construction cDNA library, storage capacity is: 7.8 * 10 ⁵

(8) utilize ABI dna sequence analysis instrument, sequential analysis is carried out in the cDNA library, therefrom screen the cDNA of encoding cysteine protease inhibitor, obtain 2 of positive colonies altogether, two sequences are identical.

The result is as follows:

IPLPGPGKTSLGGYQLHKTKDDPIKYFNNN

Embodiment 2: the expression of cystatin gene in intestinal bacteria and the purifying of expression product

1, according to the cDNA sequence that obtains, design a pair of primer,

Primer 1:5 ' ACATGACTACATATGGAGTCGGCAC 3 '; Primer 2: 5 ' ACCCTCGAGCTAATTATTATTA 3 ' is a template with the cDNA of perinereis albuhitensis grube protease, utilize Taq archaeal dna polymerase amplification cystatin gene, amplification condition: 94 ℃ of sex change 45sec, 50 ℃ of annealing 45sec, 72 ℃ are extended 45sec.Obtain the 330bp fragment (among Fig. 1: 1.pET-15bI/XbaI, XhoI; 2. λ DNA/EcoRI, HindIII).

2, cut rear clone to expression vector pET-15b with NdeI and XhoI, construction of expression vector pET-15bI with expression vector Transformed E .coliBL21 (DE3), makes up engineering bacteria.

3, picking engineering bacteria list bacterium colony, being inoculated in 20ml contains in the LB substratum of 100 μ g/ml penbritins, 30 ℃ of shaking culture 12h, be transferred to and continue to cultivate 4h in the same substratum of 2L under the similarity condition, add IPTG (isopropyl-) to 1.0mM, continue to cultivate 8h, the centrifugal collection thalline of 8000 * g.

4, thalline resuspended with 100ml Tris-Cl damping fluid (20mM, pH8.0, the 5mM imidazoles, 0.5MNaCl), the ultrasonic disruption cell.

5, the lysate of the engineering bacteria of abduction delivering behind the ultrasonic disruption cell, 4 ℃, the centrifugal 30min of 15000 * g gets supernatant liquor.

6, supernatant liquor flows through Ni ²⁺Resin column (2.6 * 8cm), and usefulness 100ml washings (20mM, pH8.0, the 50mM imidazoles, 0.5MNaCl).

7, with elution buffer (20mM, pH8.0, the 500mM imidazoles, 0.5MNaCl) elution of bound albumen, the perinereis aibuhitensis cystatin of must recombinating, the molecular weight of recombinant protein are 14kDa.Among Fig. 2: 1. cystatin; 2. middle molecular weight standard albumen (is followed successively by 14,25,45,67,94kDa) from top to bottom.

Embodiment 3: the reorganization cystatin is to the restraining effect of papoid

Papoid is mixed in buffered soln with the recombinant protein of different mol ratio, measures the activity of papoid behind 25 ℃ of insulation 15min, used buffered soln be Tris-Cl buffered soln (50mM, pH7.0, the 2mM dithiothreitol (DTT), 50mMNaCl).During mensuration at the Tris-Cl of 4.5ml buffered soln (50mM, pH7.0, the 2mM dithiothreitol (DTT), 50mMNaCl) add that 50 μ l are untreated or the papoid (0.1mg/ml) handled with recombinant protein and the L-BAPNA (chromogenic substrate of L-Cysteine HCL Anhydrous of 100 μ l, 2.0mg/ml), add 10% hydrochloric acid soln, 350 μ l with termination reaction behind 37 ℃ of insulation 10min, measure the relative reactivity that absorbance is determined papoid in 405nm wavelength place.Relative reactivity (%) with papoid is an ordinate zou, the mol ratio of recombinant protein and papoid is that X-coordinate is drawn, the result as shown in Figure 3, increase along with the recombinant protein mol ratio, the activity of papoid reduces gradually, when the mol ratio of papoid and recombinant protein was 1:2, the activity of papoid only remained 6%.The recombinant protein that purifying is described is cystatin really.

Embodiment 4: the anti-insect activity experiment of reorganization cystatin

Lamp lures gathers holotrichia oblita in rearging cage, and 50 every group, 25 ℃, lucifuge is raised, and feeds with the fresh apple leaf.(20mM's recombinant protein pH7.0) dialyses, and sprays certain density reorganization cystatin then on the surface of feed to phosphate buffer solution, to spray phosphate buffer solution (20mM, pH7.0) for contrast, raised 3 days, statistics is respectively organized the mortality ratio of holotrichia oblita.With mortality ratio (%) is ordinate zou, reorganization cystatin (mg/ml) is drawn for X-coordinate, the result as shown in Figure 4, raising along with reorganization cystatin concentration, insect mortality improves gradually, impose a group of 2mg/ml recombinant protein, mortality ratio reaches 90%.

Sequence table

＜110〉University Of Qingdao

＜120〉a kind of gene order of coding perinereis albuhitensis grube cystatin and aminoacid sequence thereof and application

<160>2

<170>PatentIn?3.1

<210>1

<211>333

<212>cDNA

＜213〉enclose Nereis (Perinereis)

<400>1

<210>2

<211>110

<212>PRT

＜213〉enclose Nereis (Perinereis)

<400>2

Claims

1, a kind of gene order of coding perinereis albuhitensis grube cystatin is characterized in that it being the nucleotide sequence shown in the sequence 1 in the sequence table.

2, a kind of aminoacid sequence of perinereis aibuhitensis cystatin is characterized in that it being the aminoacid sequence shown in the sequence 2 in the sequence table.

3, a kind of application of gene order of coding perinereis albuhitensis grube cystatin is characterized in that utilizing the gene order of the described perinereis aibuhitensis cystatin of claim 1 to prepare the biological pesticide with anti-insect activity.