CN102517308A - Construction and application of recombinant plasmid containing chitinase gene of apple leaf miner - Google Patents
Construction and application of recombinant plasmid containing chitinase gene of apple leaf miner Download PDFInfo
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Abstract
The invention relates to a new chitinase gene (LrCHI) of an apple leaf miner, in particular to the construction and the application of a recombinant plasmid carrier containing the chitinase gene of an insect. The LrCHI can be used for inhibiting funguses; and pRSET-LrCHI can be used for degrading chitin to fulfill the physiologic functions of digesting, modification, infection and the like.
Description
Technical field:
The invention belongs to biology field, relate to a kind of new plutella xylostella chitinase gene LrCHI, relate to the structure and the application of the reorganization prokaryotic vector of chitinase genes of insects.And relate to cloning process, construction of recombinant plasmid process and the LrCHI of plutella xylostella chitinase gene inhibition research to fungal growth.
Background technology:
Regitex FA (chitin) claim chitin, chitin again; Be through β-1; The 4-glycosidic link connects into the homopolymer of unbranched N-second phthalein GS amine, and it is the structural component of the many biologies of occurring in nature, mainly is present in the organisms such as invertebrates, algae, fungi.Insect chitin is insect cuticle and peritrophic important composition composition, all needs a certain amount of Regitex FA in each period of insect growth, growth.Insect chitinase is the metabolic important endo type protease of a kind of involved in insect Regitex FA, and it is through destroying chitinous β-1, and the 4-glycosidic link tentatively becomes low-molecular-weight oligomer with chitin degrading.The chitinase of insect is present in the poison gland of midgut, exuvial gland and some insect (like honeybee), in digestion, the metamorphosis of insect, playing an important role in the physiological activity such as infect.
In the whole life of insect, often not only express a kind of chitinase gene, the nearly 18 kinds of similar genes of chitinase that have.Different chitinase genes is being brought into play function corresponding at the different life stage of insect.Utilize technology such as southern hybridization, RT-PCR that the spatial and temporal expression of various insects chitinase gene is carried out systematic study; The result shows that I type chitinase gene all has the expression of certain level in the insect whole growth etap, is most important in the insect body, the most a kind of chitinase.I type chitinase gene also be study the most at present, one type of chitinase gene widely, up to now, from lepidopteran, Diptera, coleopteron, found and reported 50 surplus kind of I type chitinase gene.The molecular weight of insect I type chitinase is between 45～85kD; The field of activity of enzyme is between pH 4.0～8.0; Iso-electric point belongs to 18 family's chitinases mostly between 5.0～7.0, its protein is made up of 4 structural domains: N end signal peptide, catalytic domain, joining region and Regitex FA land; At catalytic domain and land one section conserved amino acid sequence is arranged respectively, the important evidence of design primer when being clone gene.The constructional feature of its I type chitinase gene of different types of insect is basic identical; With the maduca sexta I type chitinase gene of finding at first is the example explanation: this full length gene 2452bp; The ORFs that comprises a 1662bp; 554 amino acid of encoding are transcribed by 11 exons and 9 introns in the maduca sexta genome.
Because the vital role of insect chitinase in insect each item physiological activity, it is brought into becomes various countries scholar's research focus in recent years in the pest control.Tribactur (Bacillus thuringiensis Bt) is the microbial pesticide that a kind of quilt is extensively approved and used, reports such as Arora, and the adding of chitinase Chi36 makes Bt reduce by 30% to the toxic limit medium dose of Spodoptera litura larvae; Baculovirus is the narrow spectrum pathogenic micro-organism of insect; Very big application prospect is arranged in agricultural insect management; The reorganization of usefulness such as Fan Xiaojun has the two valency reorganization autographa california baculoviruss and the wild-type baculovirus difference infection of Chinese bollworm of Scorpio insect specific neurotoxin gene and maduca sexta chitinase gene, and the result shows that the toxic limit medium dose of recombinant virus reduces than wild-type virus is obvious; The constitutive expression of chitinase genes of insects in transgenic plant also obtained better prevention insect effect, and Gatehous etc. change chitinase genes of insects in the yam over to and express, and the transgenic Rhizoma Solani tuber osi of acquisition has strengthened the resistance to aphid.Ding etc. make an experiment to 4 kinds of chitinase transgene tobaccos that derive from maduca sexta, bacterium, actinomycetes and plant; All join in the newborn mealworm food with 1%～2% concentration; The chitinase transgene tobacco that the result finds to derive from bacterium and plant does not produce significantly influence to surviving rate and the growing state of larva, has eaten death in larva several days after the ovum hatching that contain insect chitinase transgene tobacco food and get.This explanation insect chitinase is more effective to the pest-resistant effect of enhancement of plant.People such as Zhang Hongbin extract chitinase respectively in silkworm and cotton boll polypide, under its optimum condition and yeast, penicillium spp on the LB solid medium, cultivate together, inhibition zone experiment shows that chitinase has obvious restraining effect to this fungi growth.These researchs show the truly have certain effect of insect chitinase to the inhibition of insect and fungi.
The shell of insect and some pathogenic fungies is resisted poor environment and predator as the physical chemistry barrier; It mainly is made up of Regitex FA and protein; Insect chitinase is applied in pest control and the fungi control; Utilizing shell, fungal cell wall that chitinase decomposes insect etc. to contain chitinous tissue and suppress its normal physiological activity, is a kind of safe, effective, feasible novel biocontrol strategy.
Summary of the invention:
The present invention provides the construction process of a kind of LrCHI gene clone with reorganization prokaryotic expression plasmid pRSET-LrCHI, and LrCHI is the plutella xylostella chitinase gene.
The plutella xylostella chitinase gene LrCHI that the present invention clone obtains is a kind of unknown gene, and its ORFs is made up of 1737bp, 578 amino acid of encoding; Predicted molecular weight is 64.4kDa; Iso-electric point pI is 5.49, and its sequence is SEQ ID NO:1, and the Blast that uses on the NCBI carries out homology analysis; Homology is higher between result's demonstration and the lepidopterous insects chitinase gene sequence, all above 75%.This gene is found first for the inventor and is reported, also is the gene of the first report of plutella xylostella simultaneously.
The present invention provides the recombinant plasmid vector pRSET-LrCHI of a kind of plutella xylostella chitinase gene (LrCHI), and it comprises pRSET carrier segments and plutella xylostella chitinase gene (LrCHI) SEQ ID NO:1.
Plasmid fragment pRSET in the described recombinant plasmid is a kind of prokaryotic expression carrier, is transformed by the PUC plasmid, and its main element that comprises is: the MCS of LrCHI and pRSET reorganization; But the T7 promotor of efficiently expressing exogenous gene LrCHI under the inducing of IPTG; Or be convenient to 6 * His of follow-up protein purification; Or help the ampicillin resistance gene of the screening of recon.Among the recombinant plasmid pRSET-LrCHI of the present invention, plutella xylostella chitinase gene LrCHI and plasmid fragment pRSET have xho I and hindIII restriction enzyme site.
The present invention provides the recombinant plasmid vector pRSET-LrCHI of a kind of plutella xylostella chitinase gene (LrCHI), after its foreign gene plutella xylostella chitinase gene (LrCHI) is inserted into the MCS of carrier pRSET, places under its T7 promotor.
The present invention provides the recombinant plasmid vector pRSET-LrCHI of a kind of plutella xylostella chitinase gene (LrCHI), and its recombinant plasmid contains ampicillin resistance gene and 6 * His albumen sequence label.
The present invention provides the method for a kind of construction of recombinant plasmid vector pRSET-LrCHI, may further comprise the steps:
(1), gets prepupal period plutella xylostella midgut, the total RNA of extraction midgut;
(2), the clone of plutella xylostella chitinase gene (LrCHI) SEQ ID NO:1;
(3), after cutting, the PCR product enzyme that contains SEQ ID NO:1 that plasmid vector pRSET and step 2 is obtained is connected.
The present invention provides the method for a kind of construction of recombinant plasmid vector pRSET-LrCHI; Utilize the terminal rapid amplifying technology of cDNA (RACE) amplification in the plutella xylostella body to obtain plutella xylostella chitinase gene (LrCHI) SEQ ID NO:1; And be connected on the plasmid pRSET, make up the prokaryotic expression carrier that contains plutella xylostella chitinase gene (LrCHI).
Above-mentioned steps also comprise step 4 promptly to recombinant plasmid carry out that enzyme is cut, PCR, order-checking identify.
The present invention provides in the method for construction of recombinant plasmid vector pRSET-LrCHI, and the plutella xylostella of step 1 is the laboratory artificial breeding, and feed formulation is: Semen Maydis powder 300g; Soybean cake powder 100g, yeast powder 100g, vitamins C 10g; Vitamin B complexes 1.5g, Sorbic Acid 1.5g, Hydrocerol A 2.5g; Propionic acid 5ml, agar powder 25g, water 1300ml.Raise that to get the larva midgut to the plutella xylostella of prepupal period subsequent use.
The process for extracting of the total RNA of midgut of step 1 comprises: with flesh tissue grind to form homogenate, centrifugal, leave standstill, separate with solvent.Be specially and add behind the chloroform centrifugally in the centrifuge tube respectively, get supernatant and add Virahol more respectively, leave standstill centrifugal, abandoning supernatant; Add ethanol again, centrifugal, supernatant discarded night, add 50 μ 1DEPC aqua sterilisas in the deposition; Inhale with the rifle head and to beat mixing, 65 ℃ of heating in water bath 10 minutes are stored in the liquid nitrogen.
The method of a unknown gene of clone has much at present; Delete choosing, the terminal Rapid Expansion technology of cDNA (RACE) etc. such as RNA fingerprint, cDNA library; What the present invention adopted is that RACE clones LrCHI, and the RACE technology mainly is made up of following several steps: cDNA is synthesized in the extraction of RNA and reverse transcription; According to the fragment between the conservative relatively aminoacid sequence design degenerate primer clone conserved regions; According to the conserved sequence design many 3 ' and 5 ' the RACE primer that obtain, utilize 3 ' and 5 ' RACE method to clone the 3 ' end and the 5 ' end of gene respectively.
The present invention provides in the method for construction of recombinant plasmid vector pRSET-LrCHI, step 2) concrete grammar be: prepupal period is got the plutella xylostella midgut, extracts the total RNA of midgut; With mRNA is substrate, synthetic cDNA one chain; With cDNA one chain is template, with round pcr amplification plutella xylostella chitinase gene (LrCHI); With primer amplification plutella xylostella chitinase gene (LrCHI) total length that has Xho I and HindIII restriction enzyme site.
The present invention provides in the method for construction of recombinant plasmid vector pRSET-LrCHI, according to the fragment between the conservative relatively aminoacid sequence design degenerate primer clone conserved regions; According to the conserved sequence design many 3 ' and 5 ' the RACE primer that obtain, utilize 3 ' and 5 ' RACE method to clone the 3 ' end and the 5 ' end of gene respectively.The clone of plutella xylostella chitinase gene (LrCHI) the SEQID NO:1 of step 2 is divided into conservative territory, 3 ' end and 5 ' and holds three parts to be taken up in order of priority the clone; The degenerate primer that the amplification of conservative territory is used is SEQ ID NO:2 and SEQ ID NO:3; The reverse transcription primer that 3 ' end clone uses is SEQ ID NO:4; The pcr amplification primer that 3 ' end clone uses is SEQ ID NO:5 and SEQ ID NO:6; The reverse transcription primer that 5 ' end clone uses is SEQ ID NO:7, and the pcr amplification primer that 5 ' end clone uses is SEQ ID NO:8 and SEQ ID NO:9.
The present invention provides in the method for construction of recombinant plasmid vector pRSET-LrCHI, and step 3 adopts Xho I, HindIII that plasmid and PCR product fragment are carried out double digestion.
The present invention makes up and recombinant expression pRSET-LrCHI through following step:
(1), the raising of plutella xylostella;
(2), prepupal period is got the plutella xylostella midgut, the total RNA of extraction midgut;
(3), be substrate with mRNA, synthetic cDNA one chain;
(4), be template with cDNA one chain, with round pcr amplification plutella xylostella chitinase gene;
(5), with the primer amplification LrCHI total length that has Xho I and HindIII restriction enzyme site;
(6), with Xho I and HindIII double digestion plasmid pRSET and LrCHI total length, connect two endonuclease bamhis;
(7), to recombinant plasmid carry out that enzyme is cut, PCR, order-checking identify;
(8), prokaryotic expression pRSET-LrCHI.
Plutella xylostella chitinase gene provided by the invention (LrCHI) can be applicable to degrade chitin and accomplishes insect digestion, metamorphosis, infects in the physiological function.
Plutella xylostella chitinase gene provided by the invention (LrCHI) can be applicable to suppress fungi.
Plutella xylostella chitinase gene provided by the invention (LrCHI) can be applicable to suppress penicillium spp.
But the recombinant plasmid pRSET-LrCHI successful expression that the present invention makes up, and be applied to degrade chitin accomplish insect digestion, abnormal, infect in the physiological function.
The recombinant plasmid pRSET-LrCHI that the present invention makes up can be applicable to suppress fungi.
The recombinant plasmid pRSET-LrCHI that the present invention makes up can be applicable to suppress penicillium spp.
Recombinant plasmid pRSET-LrCHI according to the invention is mainly used in the inhibition of fungi, and pRSET-LrCHI carries out the experiment of penicillium spp growth-inhibiting with it after expression in escherichia coli goes out chitinase, the research fungistatic effect.
For realizing above-mentioned purpose, the present invention adopts following technical proposals:
1, transformed into escherichia coli B21.
2, IPTG induces down and expresses LrCHI.
3, detect the inhibition effect of protein crude extract to penicillium spp.
Description of drawings:
Fig. 1 is a recombinant plasmid pRSET-LrCHI collection of illustrative plates
Fig. 2 is a prepupal period plutella xylostella larva worm attitude
Fig. 3 is PCR product agarose gel electrophoresis figure
Fig. 4 cuts for enzyme and detects agarose gel electrophoresis figure
Fig. 5 IPTG induces the expression of results of pRSET-LrCHI
Fig. 6 expression product is to the growth inhibitory effect figure of penicillium spp
Embodiment:
Following content is of the present invention further specifying, and should not be construed as limitation of the present invention.Do not running counter under the technological method essence involved in the present invention, the method among the present invention, condition and step are simply changed all belonging to the present invention and require the scope protected.
The present invention is the total length of template clone LrCHI gene through extracting total RNA of prepupal period plutella xylostella midgut with the cDNA after the reverse transcription.Be connected on the plasmid fragment pRSET that recombinates with terminal through double digestion, make up the prokaryotic expression carrier that contains LrCHI.
The ENZYMES that is adopted in the case study on implementation, molecule purification kit, recipient bacterium DH5a, plasmid pRSET and relevant biochemical reagents are market and buy, and relevant experimental implementation technology such as involved pcr amplification, reverse transcription, enzyme are cut, connected, conversion are with reference to Wang Xin " guidance of gene molecule biological experiment ".
The raising of embodiment 1 plutella xylostella larva:
For best experiment polypide material is provided to gene clone, the present invention carries out artificial breeding to the worm's ovum of plutella xylostella.
1) feed formulation
The specific configuration process is: 1. with Semen Maydis powder, soybean cake powder, yeast powder, agar and zero(ppm) water mixing, about 121 ℃ of autoclaving 20min.2. with vitamins C, vitamin B complexes, Sorbic Acid, Hydrocerol A, Oxacyclotetradecane,erythromycin deriv, propionic acid is added in the culture medium after sterilization after mixing.3. the liquid nutrient medium of mixing is poured in the porcelain dish and (just is paved with basically can), just can use after the cooled and solidified.
2) raising condition
The plutella xylostella worm's ovum was hatched under 28 ℃ two days, and larva hatches the back brushes in the ready plate with writing brush, and in plate, puts into feed prepared.With preservative film Pan Kou is sealed, and prick a little apertures in the above with syringe needle.Put it into 28 ℃ of constant temperature culture in the biochemical incubator, illumination every day 12 hours.
3) prepupal period polypide form
Cultivation polypide through in a few days begins to become black by transparent color; About 2 centimetres of most of polypide length, most polypide figure obviously increases after 6 days, and the back begins to grow the black speckle; Belly also also can obviously be found out its feeler for cyan; The velocity ratio of insect feed is very fast, baits mutually for preventing adult, it is changed over to raise in the box separately raise.Most of insect gets into prepupal period about 10 days; Showing as of prepupal period: be slow in action, motionless basically, belly is prone to up turn over, towards day, from afterbody begin to pupate, foot begins to shrink; Color is begun rubescent from afterbody by cyan one ring ring, belly is got fat, colour of skin gloss is glossy shape.Prepupal period plutella xylostella larva worm attitude is seen Fig. 2, the adult that gets into prepupal period is carried midgut get RNA, and all the other continue to raise.
The extraction of embodiment 2 total RNA:
1) gets midgut
The expression of chitinase genes of insects has space-time and tissue specificity, and research shows that the midgut of prepupal period is the righttest tissue of this gene expression amount.Midgut is gastral stage casing, and generally in a tubular form, front end connects glandular stomach, and the rear end is with Malpighian tube attachment region and hindgut boundary.The insect that hunger was handled four hours is dissected, because large intestine, midfield, small intestine are excessively not obvious, for preventing loss; So the whole piece intestines all extract; And then use microscope to confirm the midgut position, and begin then to dissect and extract, steep to saline water temporarily extracting good midgut.
2) extracting RNA
Because the RNA enzyme is ubiquitous, therefore in leaching process, to especially note preventing the pollution of RNA enzyme, the pre-treatment particularly important of experimental article.Concrete RNA extraction step:
The DEPC water of 1 usefulness 0.1% carries out pre-treatment to experiment material.
2 get and extract good flesh tissue and in liquid nitrogen, put into the glass grinding device and grind to form homogenate.
3 add ground flesh tissue respectively in the 1.5ml centrifuge tube, respectively add 1mlTrizol reagent again, firmly shake 10 seconds, and room temperature left standstill 5 minutes.
4 add the 0.2ml chloroform respectively in centrifuge tube, shook 15 seconds, leave standstill 2 minutes.Solution produces layering, divides three layers, and the upper strata is transparent, and there is a small amount of milky white precipitate in the middle level, and lower floor is red.
5 put into whizzer with centrifuge tube, at 4 ℃, and under the 12000rpm condition centrifugal 20 minutes.
6 get supernatant adds in the other EP pipe, adds the 0.5ml Virahol more respectively, liquid mixing gently in will managing, and room temperature left standstill 10 minutes.
74 ℃, under the 12000rpm condition centrifugal 20 minutes, abandon supernatant.
8 add 1ml 75% ethanol, washing precipitation gently.4 ℃, under the 7500rpm condition centrifugal 5 minutes, abandon supernatant.
9 add 50 μ l DEPC aqua sterilisas in deposition, inhale with the rifle head and beat mixing.65 ℃ of heating in water bath 10 minutes.
10 are stored in the liquid nitrogen.
RNA to extract is a template, uses Oligo dT to be synthetic cDNA one chain of primer.The reverse transcription system of 20ul:
Reaction conditions is 42 ℃ and hatches behind the 45min 85 ℃ and make protein denaturation 5min.
The clone and the structural analysis of embodiment 4 chitinase genes:
The method of a unknown gene of clone has much at present; Delete choosing, the terminal Rapid Expansion technology of cDNA (RACE) etc. such as RNA fingerprint, cDNA library; What the present invention adopted is that RACE clones LrCHI, and the RACE technology mainly is made up of following several steps: cDNA is synthesized in the extraction of RNA and reverse transcription; According to the fragment between the conservative relatively aminoacid sequence design degenerate primer clone conserved regions; According to the conserved sequence design many 3 ' and 5 ' the RACE primer that obtain, utilize 3 ' and 5 ' RACE method to clone the 3 ' end and the 5 ' end of gene respectively.
1) amplification in conservative territory
Through tens kinds of insect chitin enzyme amino acid sequences delivering announcement are carried out homology analysis; The result is illustrated in the inner sequence that has two sections high conservatives of its sequence; YDFDGLDLDWEYP and GAMTWAIDMD; Design degenerate primer 5 '-GGMTGGGARCTGACTGCTGC-3 ' (SEQ ID NO:2) and 5 '-CCTTGTARGCGTAGGGGCAYTTGCC-3 ' (SEQ ID NO:3) according to this; And be the conservative territory of substrate pcr amplification plutella xylostella chitinase with the cDNA that reverse transcription forms, electrophoresis result has obtained the fragment of about 450bp shown in 1 swimming lane of Fig. 3.
2) 3 ' end amplification
According to the peculiar poly of eukaryote mRNA (A) structure, design contains joint the primer 5 '-TGGAGACCAGCGTTTCTGAGATTC (T) of one section known array
18-3 ' (SEQ ID NO:4) is as the reverse transcription primer; According to the upstream primer CCACGTACCTGATCTTTGCCAGGAGC (SEQ ID NO:5) of acquired conservative territory sequences Design 3 ' race, utilize 3 ends of downstream joint primer 5 '-TGGAGACCAGCGTTTCTGAGATTC-3 ' (SEQ ID NO:6) pcr amplification chitinase gene again.Amplification is seen 2 swimming lanes of Fig. 3.
3) 5 ' end amplification
Because obtained the 3 ' terminal sequence of LrCHI; Can design the reverse transcription primer of special primer 5 '-TCCTTGATCCAGTTCATCTTG (SEQ ID NO:7) ATCT-3 ' according to this as 5 ' end amplification; Be not difficult to find that the A/T content of this primer is higher, make it under the temperature condition of reverse transcription, be easy to combine with template.Because 5 ' end of eukaryote mRNA does not have the oligomerization tail, therefore the amplification of 5 ' race need add the preceding paragraph homopolynucleotide tail artificially at the 3 ' end of its cDNA, its 5 ' end amplification of increasing on this basis.Be that cDNA to plutella xylostella behind the purifying carries out with gathering the cytosine(Cyt) tailing, again with upstream joints primer 5 '-TGGAGACCAGCGTTTCTGAGATTC (G) in the implementation process of the present invention
10Pcr amplification is carried out according to the special primer 5 '-GGTGTACGGAGCAGGATCGCCACC-3 ' (SEQ ID NO:9) of conserved sequence design in-3 ' (SEQ ID NO:8) and downstream, obtains 5 ' end of plutella xylostella chitinase gene.Amplification is seen 3 swimming lanes of Fig. 3.
4) structural analysis
To the sequence total length of the splicing back acquisition SEQ ID NO:1 of the gene order among the embodiment 4 (LrCHI), to form by 1737bp, 578 amino acid of encoding predict that its proteinic molecular weight and iso-electric point are respectively 64.2kDa, 5.42.Utilize the blast program among the NCBI that the LrCHI that finds among the present invention is carried out the homology similarity analysis; The result shows the homology of its nucleotide sequence and other insect chitinases higher (all more than 75%); With the homology of bollworm chitinase gene especially up to 98%, this explanation clone really of the present invention has obtained a kind of new insect chitin enzyme sequence.
The structure of embodiment 5 recombinant plasmid pRSET-LrCHI:
1, PCR expansion total length
With cDNA is substrate, with the primer amplification LrCHI total length that has restriction enzyme site Xho I, HindIII.The PCR condition is:
2, double digestion, connection and conversion
Sanprep plasmid a small amount of extraction agent box with buying in giving birth to worker's biotechnology ltd extracts plasmid pRSET, and the concrete operations step is produced specification sheets referring to it.With restriction enzyme Xho I, HindIII digested plasmid pRSET and pcr amplification product LrCHI total length, the enzyme tangent condition is 25 ℃ of water-bath 30min.With the DNA purification kit enzyme is cut product and carry out purifying, concrete steps are referring to the Microelute cycle-pure kit of Omega company test kit specification sheets.
With enzyme cut, plasmid behind the purifying is connected with the T4 ligase enzyme with the PCR fragment, linked system is 20ul: plasmid fragment 1ul, PCR product fragment 3ul, buffer 2ul, T4 ligase enzyme 1ul, dH
2O 13ul.22 ℃ connect 1 hour.
Get the linked system of 5ul, be added in the competence T cell of 50ul, ice bath 2min immediately behind 42 ℃ of heat shock 30s adds after the LB nutritive medium 37 ℃, 200rpm and hatched one hour.The bacterium liquid of getting 200ul is equably on the way to containing X-gal, IPTG, Amp
rSolid medium on, overnight cultures.Picking white recon is identified.
3, the evaluation of positive recombinant
Recombinant plasmid is carried out Xho I, the evaluation of HindIII double digestion, and the result sees Fig. 4
Recombinant plasmid is carried out PCR identify that the result sees 4 swimming lanes of Fig. 3
Recombinant plasmid is delivered to Beijing order-checking portion of living worker's biotechnology ltd check order, be inserted into correct, complete sequence direction below the T7 promotor of pRSET to guarantee LrCHI.
Embodiment 6 expression of recombinant plasmid pRSET-LrCHI in BL21:
1, pRSET-LrCHI transforms BL21
From-70 ℃, take out 50ulBL21 competence (the competent preparation of BL21 is operated referring to classical molecular biology),, flick mixing the recombinant plasmid pRSET-LrCHI adding of 5ul; Ice bath 25min, 42 ℃ of heat shock 30s place 2min on ice immediately; The LB liquid nutrient medium that adds 250 μ L balance to room temperatures, 200 commentaries on classics/min were hatched 1 hour for 37 ℃; Get 100ul bacterium liquid and be applied to equably on the flat board that contains 50ug/ulAmp, be inverted incubated overnight after just putting 30min for 37 ℃.
2, IPTG induces down and expresses LrCHI
Picking list bacterium colony is to LB under above-mentioned steps, hatches to OD600=0.4-0.6 under 37 ℃ of the 200 commentaries on classics/min.The bacterium liquid of getting 1ml is stored in-20 ℃ as blank, and adding final concentration in the remaining bacterium liquid is the IPTG of 1mM, abduction delivering 4 hours.With control group and the centrifugal 5min under 4000rpm of the bacterium liquid after IPTG induces; Discard supernatant; The Tris-HCL buffer that in bacterial sediment, adds 100ul uses the ultrasonic disruption appearance thalline to be carried out fragmentation, the centrifugal 10min of 13000rpm/min; Cleer and peaceful deposition in the separation, the urea that in deposition, adds 500ul makes its dissolving.Get the upward cleer and peaceful urea dissolved deposition of 25ul respectively and mix with 6 * SDS of 5ul, boiling water bath sex change 10min, moment is centrifugal, and the sample of getting 15ul carries out the SDS-PAGE gel electrophoresis.Electrophoresis result is seen Fig. 5, the target protein of about 65kDa of can having found out escherichia coli expression from figure.
The detection of embodiment 7 fungistatic effects
Get the hollow cylinder of three 8 * 6 * 10mm; Be fixed on the solid medium gently; Add the 100ul sterilized water in three right cylinders respectively, 100ul contains the supernatant of LrCHI prokaryotic expression, the broken liquid of intestinal bacteria of 100ul; The last penicillium spp (OD=0.3) that adds 100ul again in the right cylinder respectively, 37 ℃ of constant temperature culture 20 hours.Experimental result shows that the diameter of the growth bacterium circle of penicillium spp in the supernatant that contains the LrCHI prokaryotic expression is about 0.8cm; And the bacterium loop diameter that in bacterium liquid, only adds the sterilized water control group reaches 1.5cm; The bacterium loop diameter that adds the broken liquid control group of intestinal bacteria also is about 1.5cm; The growth phase of penicillium spp in the supernatant that contains the LrCHI prokaryotic expression obviously is suppressed for other two kinds of environment; This result shows that colibacillary broken liquid does not have remarkable influence to the growth of penicillium spp, and the LrCHI of prokaryotic expression then can suppress the growth of penicillium spp, the Regitex FA on reason possibly be the LrCHI catalytic hydrolysis penicillium spp cell walls; Destroyed its normal physiological structure, the result sees Fig. 6.
Claims (10)
1. a plutella xylostella chitinase gene (LrCHI), its sequence is SEQ ID NO:1.
2. plutella xylostella chitinase gene as claimed in claim 1 (LrCHI) is characterized in that plutella xylostella chitinase gene total length is made up of 578 amino acid of encoding 1737bp.
3. the recombinant plasmid vector pRSET-LrCHI of a plutella xylostella chitinase gene (LrCHI) is characterized in that comprising pRSET carrier segments and SEQ ID NO:1.
4. recombinant plasmid vector pRSET-LrCHI as claimed in claim 3 is characterized in that: after foreign gene plutella xylostella chitinase gene (LrCHI) is inserted into the MCS of carrier pRSET, place under its T7 promotor.
5. reorganization prokaryotic expression plasmid vector pRSET-LrCHI as claimed in claim 4 is characterized in that: recombinant plasmid contains ampicillin resistance gene and 6 * His albumen sequence label.
6. LrCHI method that makes up claim 3 recombinant plasmid vector pRSET-comprises following steps:
(1), gets prepupal period plutella xylostella midgut, the total RNA of extraction midgut;
(2), the clone of plutella xylostella chitinase gene (LrCHI) SEQ ID NO:1;
(3), after cutting, the PCR product enzyme that contains SEQ ID NO:1 that plasmid vector pRSET and step 2 is obtained is connected.
7. claim 1 or 2 described plutella xylostella chitinase genes (LrCHI) are accomplished its digestion, metamorphosis, are infected the application in the physiological function at degrade chitin.
8. claim 1 or 2 described plutella xylostella chitinase genes (LrCHI) application in suppressing fungi.
9. each described recombinant plasmid pRSET-LrCHI of claim 3-5 accomplishes its digestion, metamorphosis, infects the application in the physiological function at degrade chitin.
10. the application of each described recombinant plasmid pRSET-LrCHI of claim 3-5 in suppressing fungi.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110460067XA CN102517308A (en) | 2011-12-31 | 2011-12-31 | Construction and application of recombinant plasmid containing chitinase gene of apple leaf miner |
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| CN201110460067XA CN102517308A (en) | 2011-12-31 | 2011-12-31 | Construction and application of recombinant plasmid containing chitinase gene of apple leaf miner |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105274074A (en) * | 2014-06-19 | 2016-01-27 | 中国农业科学院棉花研究所 | Applications of Apolygus lucorum sialoprotein PG20 |
| CN105274073A (en) * | 2014-06-19 | 2016-01-27 | 中国农业科学院棉花研究所 | Applications of Apolygus lucorum sialoprotein PG10 |
| CN105296449A (en) * | 2014-06-19 | 2016-02-03 | 中国农业科学院棉花研究所 | Application of lygus lucorum sialoprotein PG2 |
| CN109258693A (en) * | 2018-09-30 | 2019-01-25 | 大连理工大学 | Application of the recombinant chitinase at desinsection or antibacterial aspect |
| CN113388597A (en) * | 2021-06-04 | 2021-09-14 | 福州大学 | Chitinase with antifungal activity and gene thereof |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105274074A (en) * | 2014-06-19 | 2016-01-27 | 中国农业科学院棉花研究所 | Applications of Apolygus lucorum sialoprotein PG20 |
| CN105274073A (en) * | 2014-06-19 | 2016-01-27 | 中国农业科学院棉花研究所 | Applications of Apolygus lucorum sialoprotein PG10 |
| CN105296449A (en) * | 2014-06-19 | 2016-02-03 | 中国农业科学院棉花研究所 | Application of lygus lucorum sialoprotein PG2 |
| CN109258693A (en) * | 2018-09-30 | 2019-01-25 | 大连理工大学 | Application of the recombinant chitinase at desinsection or antibacterial aspect |
| CN109258693B (en) * | 2018-09-30 | 2021-03-16 | 大连理工大学 | Application of recombinant chitinase in insecticidal or bacteriostatic aspects |
| CN113388597A (en) * | 2021-06-04 | 2021-09-14 | 福州大学 | Chitinase with antifungal activity and gene thereof |
| CN113388597B (en) * | 2021-06-04 | 2022-06-03 | 福州大学 | Chitinase with antifungal activity and gene thereof |
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