CN102464710A - Helicoverpa armigera juvenile hormone binding protein (Ha-JHBP) and encoding gene and application thereof - Google Patents
Helicoverpa armigera juvenile hormone binding protein (Ha-JHBP) and encoding gene and application thereof Download PDFInfo
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Abstract
The invention discloses a helicoverpa armigera juvenile hormone binding protein (Ha-JHBP) and an encoding gene and application thereof. The helicoverpa armigera juvenile hormone binding protein provided by the invention is a protein shown as (a) or (b), wherein (a) is constituted by an amino acid sequence shown as a sequence 1 in a sequence table, and (b) is obtained by performing substitution and/or deletion and/or addition of one or more amino acid residues on an amino acid residue sequence shown as a sequence 1, is relevant to helicoverpa armigera growth and is derived the sequence 1. The invention further provides an encoding gene of the protein and a functional fragment of the encoding gene. By establishing a dsRNA (double-stranded Ribonucleic Acid) plant expression vector in a way of taking a Ha-JHBP gene as a target gene and expressing a large quantity of dsRNAs of the Ha-JHBP in plants, the insect resistance of transgenic plants is enhanced. The Ha-JHBP has important application values in the fields of biological prevention and control of phytophagous insects, plant insect resistance gene engineering and molecular biology.
Description
Technical field
The present invention relates to a kind of bollworm JH binding protein and encoding sox and application.
Background technology
Insect pest is the important factor of restriction higher yield of crops.According to statistics, the financial loss average out to 20%～30% that various crops suffer because of insect pest, hundreds billion of approximately dollars of annual loss.Spray chemical pesticide, biotic pesticide and be the main method of preventing and treating at present.Because factors such as human enhancing to environment protection and self health perception, pest resistance, pesticide residue make a large amount of conventional pesticides needs of incompatibility modern agricultural development.
Utilize genetic engineering means to cultivate advantages such as pest-resistant new variety have provide protection continuity, aboundresources, toxicity is single-minded, environmental pollution is little, the cycle is short, cost is low, and purpose is strong.Therefore, the appearance of transgenic anti-insect plants can solve pest-resistant problem to a great extent.As derive from the mikrobe bacillus thuringiensis (Bacillusthuringiensis, Bt) toxoprotein gene successfully changes various crops and fruit tree forest over to, the phytohemagglutinin gene that derives from higher plant also is used to obtain transgenic anti-insect plants.
Though insect is slow to the resistance development of transgenic plant, also be a problem that can not be ignored.The field resistance evidence of transgenic plant, insect just produces resistance to the Bt toxalbumin after 12 breedings, and (insecticidal crystal protein, ICP) plant of gene generally can only be used 8～10 years on producing to change single crystallin.In view of this, the resistance of insect is potential problems comparatively serious in the insect-resistant transgenic engineering, presses for to seek new method or the new effectively next or special control plant pest of gene.
RNA disturbs (RNA interfefence; RNAi) phenomenon is meant and utilizes endogenous or exogenous double-stranded RNA (double stranded RNA; DsRNA) the specificity degraded takes place in mRNA in the mediated cell, causes target gene expression reticent, produces function corresponding phenotype disappearance.The RNAi phenomenon of dsRNA mediation is found in the multiple biologies such as fungi, fruit bat, plant, trypanosome, hydra, turbellarian worm, zebra fish, big mouse successively.Also there is the systemic propagation phenomenon of RNAi signal in most of insects.There are some researches show ovum, haemocoele or the local organization of the dsRNA direct injection being advanced insect, the specificity that can cause remote target gene is reticent.In addition, except direct injection dsRNA, can also induce insect to produce the RNAi phenomenon through the method for feeding dsRNA.Existing at present research shows that the dsRNA of the corresponding target genes of feeding successfully reduces rapamycin target protein (targetof rapamycin, expression level amTOR) and bollworm (Helicoverpa armigera) the P450 expression of gene level of shallow brown volume moth (Epiphyas postvittana) the larva Procaine esterase expression level of apple, honeybee (Apismellifera).Thereby, be the new insect pest control method of foundational development with the RNAi technology, the New Policy of high specific and environmental safety is provided for agricultural insect management.
Insect hormone plays an important role in the growing of insect.Regulate the insect hormone that insect grows; It is synthetic, related key gene majority has very strong species specificity in transportation and the pathways metabolism; Promptly the dna homolog property with other species is low; Especially to compare with Human genome almost do not have homology, and this has reduced " effect of missing the target " of RNAi technology to a great extent, thereby guarantee is provided for the application security aspect.Therefore; In plant, express the dsRNA of insect hormone genes involved; Its advantage only is that the insect of phytophagy is had restraining effect; Can also through expressing not homotactic dsRNA a certain or a certain growing of insect of genus be exerted an influence simultaneously, thereby reach the purpose of accurate, safe control insect pest according to the species specificity of insect hormone genes involved.
Summary of the invention
The purpose of this invention is to provide a kind of bollworm JH binding protein and encoding sox and application.
Protein provided by the invention, name is called Ha-JHBP albumen, available from lepidopteran noctuid bollworm (Helicoverpa armigera), is (a) or (b) as follows:
(a) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 1;
(b) with the aminoacid sequence of sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant with the bollworm growth by sequence 1 deutero-protein.
In order to make the protein in (a) be convenient to purifying, proteinic N-terminal or C-terminal that can the aminoacid sequence shown in the sequence 1 is formed in by sequence table connect label as shown in table 1.
The sequence of table 1 label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (being generally 5) | RRRRR |
| Poly-His | 2-10 (being generally 6) | |
| FLAG | ||
| 8 | DYKDDDDK | |
| Strep-tag?II | 8 | WSHPQFEK |
| c- |
10 | EQKLISEEDL |
Above-mentioned (b) but in the protein synthetic, also can synthesize its encoding sox earlier, carry out biology again and express and to obtain.Proteinic encoding sox in above-mentioned (b) can be through the codon with one or several amino-acid residue of disappearance in the dna sequence dna shown in the sequence in the sequence table 2; And/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table 1.
The gene of encoding said proteins (Ha-JHBP gene) also belongs to protection scope of the present invention.
Said gene can be following 1) or 2) or 3) or 4) dna molecular:
1) sequence 2 is held the dna molecular shown in the 12nd to 740 Nucleotide from 5 ' in the sequence table;
2) dna molecular shown in the sequence 2 in the sequence table;
3) under the rigorous condition of height with 1) or 2) the dna sequence dna hybridization that limits and the dna molecular of coding bollworm growth associated protein;
4) with 1) or 2) dna sequence dna that limits has 90% above homology and the dna molecular of the bollworm growth associated protein of encoding.
The rigorous condition of said height be 2 * SSPE (or 2 * SSC), in the solution of 0.1%SDS, under 68 ℃, hybridize and wash film.
The recombinant expression vector, expression cassette, transgenic cell line or the reorganization bacterium that contain said gene all belong to protection scope of the present invention.
The present invention also protects the sequence 2 that the contains ordered list dna fragmentation from 5 ' terminal 207-689 position Nucleotide.
Said dna fragmentation can be dna fragmentation first, dna fragmentation second or dna fragmentation third;
Said dna fragmentation first can be like the sequence 2 of sequence table from shown in 5 ' the terminal 207-689 position Nucleotide;
Said dna fragmentation second can be like the sequence 2 of sequence table from shown in 5 ' the terminal 204-691 position Nucleotide;
Said dna fragmentation third can be the DNA that contains forward sequence and reverse sequence; Said forward sequence like the sequence 4 of sequence table from shown in 5 ' terminal the 15th to 497 Nucleotide, said reverse sequence like the sequence 4 of sequence table from shown in 5 ' terminal the 711st to 1193 Nucleotide.Said dna fragmentation third is preferably DNA shown in the sequence 4 of sequence table.
The recombinant expression vector, expression cassette, transgenic cell line or the reorganization bacterium that contain said dna fragmentation all belong to protection scope of the present invention.
Available existing plant expression vector construction contain said gene or said dna fragmentation recombinant expression vector.Said plant expression vector comprises double base agrobacterium vector and the carrier etc. that can be used for the plant micropellet bombardment.Said plant expression vector also can comprise 3 ' end untranslated zone of foreign gene, promptly comprises the dna fragmentation of polyadenylic acid signal and any other participation mRNA processing or genetic expression.Said polyadenylic acid signal can guide polyadenylic acid to join 3 ' end of mRNA precursor.When using said gene or said dna fragmentation to make up the recombinant plant expression vector, before its transcription initiation Nucleotide, can add any enhancement type promotor or constitutive promoter, they can use separately or be used in combination with other plant promoter; In addition; When using gene of the present invention or dna fragmentation to make up plant expression vector; Also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser zones can be ATG initiator codon or neighboring region initiator codon etc.; But must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of said translation wave and initiator codon is widely, can be natural, also can be synthetic.Translation initiation region can be from transcription initiation zone or structure gene.For the ease of transgenic plant cells or plant being identified and screening; Can process used plant expression vector, can produce enzyme or the gene of luminophor, antibiotic marker thing or the anti-chemical reagent marker gene etc. of colour-change with resistance as adding the coding that in plant, to express.
Said recombinant expression vector specifically can be as follows (I) or (II):
(I) recombinant plasmid that the MCS that said dna fragmentation third is inserted recombinant plasmid pC2300PN obtains; Said recombinant plasmid pC2300PN is for being skeleton carrier with plant expression vector pCAMBIA2300; The sequence 8 of insertion sequence table is from the DNA shown in 5 ' terminal the 12nd to 295 Nucleotide between PstI and EcoRI restriction enzyme site; The sequence 9 of insertion sequence table is from the DNA shown in 5 ' terminal the 14th to 194 Nucleotide, the recombinant plasmid that obtains between PstI and HindIII restriction enzyme site;
(II) said dna fragmentation first or said dna fragmentation second are inserted the recombinant plasmid that the MCS of L4440 plasmid obtains.
Said reorganization bacterium specifically can be (II) described recombinant plasmid importing intestinal bacteria HT115 (DE3) is obtained the bacterium of recombinating.
Above-mentioned (I) described recombinant plasmid is a kind of interference carrier; Under the driving of cotton curve leaf disease virus (CLCuV) complementary strand promotor (PRP) in plant overexpression to the RNA interfering of Ha-JHBP gene; Plant-feed insect has reduced the Ha-JHBP gene at the intravital expression level of insect after getting this plant of food; Thereby the hormonal equilibrium of upsetting insect influences it and grows, and reaches pest-resistant purpose.
The present invention also protects a kind of method of cultivating transgenic plant, is described dna fragmentation third is imported in the purpose plant, obtains the transgenic plant that the bollworm resisting ability is higher than said purpose plant.Said dna fragmentation specifically can import in the said purpose plant through said recombinant expression vector.Carry the expression vector of said dna fragmentation can be through using conventional biological method transformed plant cells or tissues such as Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity led, agriculture bacillus mediated, particle gun, and the plant transformed tissue cultivating become plant.Said dna fragmentation third specifically can import said purpose plant through (I) described recombinant plasmid.Said purpose plant can be monocotyledons or dicotyledons.Said dicotyledons is preferably tobacco (like tobacco bred Nicotiana tabacum cv.Xanthi).
The present invention also protects a kind of double-stranded RNA, and one of which bar chain is held (its complementary strand is held shown in the 29th to 516 Nucleotide from 5 ' like sequence in the sequence table 7) shown in the 16th to 503 Nucleotide like sequence in the sequence table 6 from 5 '.Said double-stranded RNA can be used for preparing the preparation of killing heliothis armigera.So the present invention protects a kind of preparation that is used for killing heliothis armigera simultaneously, its activeconstituents is said double-stranded RNA.
The level of neotonin plays a crucial role at the metamorphosis process that the regulation and control insect larvae becomes adult through each phase in the insect body, and as the insect of one type of locust, neotonin also is its important factor that is changed to the migration phase by stationary phase of a control.Many insects have the different adult form with difference in functionality, and hormone is determining them will become the adult of which kind of form.Albumen provided by the invention combines with the neotonin (JH) that is secreted into hemolymph from corpus allatum, makes it avoid the degraded of nonspecific esterase or neotonin cyclooxygenase (JHEH).
With present pest-resistant compared with techniques; The present invention has following beneficial effect: at first; Ha-JHBP gene provided by the present invention is an isolating gene in the lepidopteran bollworm; Its coded JH binding protein transports the degraded of avoiding nonspecific esterase or neotonin cyclooxygenase (JHEH) in vivo for the secreted neotonin of bollworm corpus allatum and plays an important role, the Ha-JHBP gene in insect body changes of expression level to the generation great influence of growing of insect; Secondly, utilize Ha-JHBP gene of the present invention to make up the dsRNA plant expression vector as goal gene, under the driving of cotton curve leaf disease virus PRP promotor in plant the dsRNA of great expression Ha-JHBP, improved the insect resistance capacity of transfer-gen plant.The present invention is for the biological control of plant-feed insect, engineering of insect-resistant plant, and biology field performance important use is worth.
Description of drawings
Fig. 1 is the structural representation of recombinant plasmid pLJP.
Fig. 2 is two groups of mortality analysis histograms of handling bollworms in the feeding experiment of embodiment 2.
Fig. 3 is the structural representation of interference fragment.
Fig. 4 is the synoptic diagram of plant interior expression dsRNA.
Fig. 5 is the structural representation of interference carrier PRP:dsJHBP.
Fig. 6 is that the PCR of transgene tobacco detects figure; M is DL2000plus Marker; CK is a wild-type tobacco; 1-10 is the positive tobacco of PCR.
Fig. 7 is that transgene tobacco Northern blot detects figure; CK is a wild-type tobacco; 1-6 is the positive tobacco of PCR.
Fig. 8 is the mortality analysis of bollworm; The X axle is represented the fate of cotton bollworm larvae feed transgene tobacco blade; The Y axle is represented the mortality ratio of bollworm.
Fig. 9 is the body weight analysis of inoculation survival bollworm after 8 days.
After Figure 10 was transgene tobacco feeding bollworm, real-time PCR detected Ha-JHBP mrna expression in the insect body.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment like no specified otherwise, is ordinary method.Used test materials among the following embodiment like no specified otherwise, is to buy from routine biochemistry reagent shop and obtains.Quantitative test in following examples all is provided with repeated experiments three times, results averaged.
Bacterial expression vector L4440: the public can obtain with developmental biology institute from Chinese Academy of Sciences's heredity; Reference: Timmons, L.and Fire, A. (1998) Specific interference by ingested dsRNA.Nature 395:854-854.); The details of this carrier can inquire on the internet, is concrete network address http://www.addgene.org/pgvecl? Cmd=findpl&identifier=1654.
Intestinal bacteria HT115 (DE3): the public can obtain with developmental biology institute from Chinese Academy of Sciences's heredity; Reference: Timmons; L.; Court; D.L.and Fire, A. (2001) Ingestion of bacteriallyexpressed dsRNAs can produce specific and potent genetic interference inCaenorhabditis elegans.Gene 263:103-112.).
Agrobacterium tumefaciens EHA105: the public can obtain with developmental biology institute from Chinese Academy of Sciences's heredity; Reference: Hood EE, Gelvin SB, Melchers S, Hoekema A (1993) New Agrobacterium helperplasmids for gene transfer to plants (EHA105) .Trans Res 2:208-218.
Used aseptic tobacco bred is Nicotiana tabacum cv.Xanthi among the embodiment 4, and the public can obtain with developmental biology institute from Chinese Academy of Sciences's heredity; Reference: A.NATO; S.BAZETOUX; Y.MATHIEUPhotosynthetic Capacities and Growth Characteristics of Nicotiana tabacum (cv.Xanthi) Cell Suspension Cultures Physiologia Plantarum Volume 41; Issue 2, pages 116-123, and October 1977.
Bollworm (Helicoverpa armigera): the public can obtain with developmental biology institute from Chinese Academy of Sciences's heredity; Reference: Wu KM; Lu YH; Feng HQ, Jiang YY, Zhao JZ.Suppression ofcotton bollworm in multiple crops in China in areas with Bt toxin-containingcotton.Science.2008 Sep 19; 321 (5896): 1676-8.
The LB substratum: solvent is a water, contains yeast extract paste 5g/L, peptone 10g/L, NaCl 10g/L, regulates pH to 7.0 with the 10M NaOH aqueous solution.
The YEB substratum: solvent is a water, contains beef extract 5g/L, yeast extract paste 1g/L, peptone 5g/L, sucrose 5g/L, MgSO47H
2O 0.04g/L, agar 15g/L regulate pH to 7.2 with the 10M NaOH aqueous solution.
Be total to culture medium: with the MS substratum is basic medium, and other contains IAA (indolylacetic acid) 0.1mg/L, 6-BA (6-benzyl aminopurine) 1mg/L, AS (Syringylethanone) 100uM.
Screening culture medium: with the MS substratum is basic medium, and other contains IAA (indolylacetic acid) 0.1mg/L, 6-BA (6-benzyl aminopurine) 1mg/L, Cef (cephamycin) 400mg/L, Kan (kantlex) 100mg/L.
Regeneration culture medium: with the MS substratum is basic medium, and other contains 6-BA (6-benzyl aminopurine) 1mg/L, Cef (cephamycin) 400mg/L, Kan (kantlex) 100mg/L.
Root media: with the MS substratum is basic medium, and other contains Cef (cephamycin) 200mg/L, Kan (kantlex) 100mg/L.
The discovery of embodiment 1, bollworm JH binding protein and encoding sox thereof
1, the JHBP gene order according to known insect designs degenerate primer (P1/P2) as follows:
P1：5’-AGTGA(C/T)ATA(A/G)AATGC(T/A)T(G/A)AGCAA-3’；
P2：5’-GG(C/T)TC(T/A)CCAA(T/A)GATTTC(A/G)CAAG-3’。
2, extract total RNA of 3 instar bollworm grubs, obtain cDNA through the external reverse transcription of RT-PCR.
3, the cDNA with step 2 is a template, carries out pcr amplification with step 1 designed primer, obtains pcr amplification product.
4, the pcr amplification product of step 3 is cloned on the pEASY-Blunt sequencing vector checks order, sequencing result shows that the nucleotide sequence of pcr amplification product is shown in the sequence 5 of sequence table.
5, extract the total RNA of 3 bollworms in age and make primer extension assay, hold at 5 ' of mRNA to add RNA joint RAP1; The synthetic cDNA of RNA reverse transcription that 5 ' end is added joint; With this cDNA is template; According to sequence 5 designs, 5 ' end amplimer (P3) and 3 ' end amplimer (P4), adopt the method for 5 ' RACE and 3 ' RACE (the primer UP5-I/UP5-II/P3 and UP3/P4) to obtain 5 ' end and 3 ' end unknown nucleotide sequence then; Splicing obtains full length sequence.
RAP1：5’-GCUGAUGGCGAUGAAUGAACACUGCGUUUGCUGGCUUUGAUGAAA-3’；
UP5-I：5’-GCTGATGGCGATGAATGAACACTG-3’；
UP5-II：5’-CGCGGATCCGAACACTGCGTTTGCTGGCTTTGATG-3’；
UP3：5’-oligo(dT)25-3’；
P3：5’-GCAGTTGTTGAAGCTGTATAAGCGCC-3’；
P4：5’-GGAGAACCGAACGTGGATATTGG-3’。
6, it is following that the full length sequence that obtains according to splicing designs primer P5/P6:
P5：5’-ATGGCAGTTTATAGGAGCTTG-3’；
P6：5’-TTAAATGTCAGTGAAAAAGGCTT-3’。
7, the cDNA with step 2 is a template, carries out pcr amplification with step 6 designed primer, obtains pcr amplification product.
8, the pcr amplification product of step 7 is cloned on the pEASY-Blunt sequencing vector, obtains recombinant plasmid.
9, recombinant plasmid is checked order, sequencing result shows, the nucleotide sequence of pcr amplification product like the sequence 2 of sequence table from shown in 5 ' terminal the 12nd to 740 Nucleotide.
With the protein called after Ha-JHBP albumen shown in the sequence 1 of sequence table.The corresponding protein sequence of Ha-JHBP albumen and present known lepidopteran noctuid is compared; Similarity is about 80%; Analyze through SMART (The SimpleModular Architecture Research Tool), Ha-JHBP albumen possesses and neotonin bonded structural domain.The proteic unnamed gene of Ha-JHBP of will encoding is the Ha-JHBP gene, its ORFs like the sequence 2 of sequence table from shown in 5 ' terminal the 12nd to 740 Nucleotide.
The sequence 2 of preparation sequence table is from DNA shown in 5 ' terminal the 12nd to 740 Nucleotide.The dna clone for preparing to pEASY-Blunt sequencing vector (available from the Beijing Quanshijin Biotechnology Co., Ltd), is obtained recombinant plasmid pJHBP.
One, the structure of recombinant plasmid pLJP
1, is template with recombinant plasmid pJHBP, carries out pcr amplification, reclaim pcr amplification product (the Ha-JHBP gene is guarded section JHBP1-BX) with primer P7-BglII/P8-XhoI.
P7-BglII：5’-GAAGATCTGATGTTGTGTATGA-3’；
P8-XhoI：5’-CCGCTCGAGTTAAAAGTCGTGGT-3’。
2,, reclaim enzyme and cut product with the pcr amplification product of restriction enzyme BglII and XhoI double digestion step 3.
3,, reclaim carrier framework with restriction enzyme BglII and XhoI double digestion bacterial expression vector L4440.
4, the carrier framework of the enzyme of step 2 being cut product and step 3 is connected, and obtains recombinant plasmid pLJP.The carrier that sets out of recombinant plasmid pLJP is L4440, and the sequence 2 of between the BglII of the carrier that sets out and XhoI restriction enzyme site, having inserted sequence table is from the DNA shown in 5 ' the terminal 204-691 position Nucleotide.Recombinant plasmid pLJP structural representation is seen Fig. 1, comprises the Ha-JHBP gene between two T7 promotors and the T7 promotor.Through two T7 promotors, recombinant plasmid pLJP can the sequence 6 of expressed sequence table shown in the RNA fragment shown in the sequence 7 of RNA fragment and sequence table, two RNA fragments can form double-stranded RNA.
Two, the preparation of reorganization bacterium and contrast bacterium
With recombinant plasmid pLJP transformed into escherichia coli HT115 (DE3), obtain the bacterium of recombinating.
With bacterial expression vector L4440 transformed into escherichia coli HT115 (DE3), obtain contrasting bacterium.
Three, cotton bollworm larvae feeding experiment
1, preparation RNA sample
Bacterium 37 ℃, 200rpm shaking culture 8-10h in the LB substratum will recombinate; Be transferred in the fresh LB substratum in 2% (volumn concentration) ratio then, shaking culture is to OD
600Be about 0.6; Add IPTG, shaking culture 4 hours is collected thalline; From thalline, extract total RNA (RNA sample first).
To contrast bacterium 37 ℃, 200rpm shaking culture 8-10h in the LB substratum; Be transferred in the fresh LB substratum in 2% (volumn concentration) ratio then, shaking culture is to OD
600Be about 0.6; Add IPTG, shaking culture 4 hours is collected thalline; From thalline, extract total RNA (RNA sample second).
2, artificial diet
Bollworm is raised artificial diet prescriptions (1L): analysis for soybean powder 80g, wheat-flour 80g, yeast powder 16g, agar 14g, 20 of vitamins Cs; 15 of vitamins Bs, Sorbic Acid 1g, Sodium Benzoate 1g, glacial acetic acid 5ml; Nepal gold ethyl ester 2g, F-1991 (Benlate) 0,05-0.1g, adding water to volume is 1L; Boil the cooling back for solid, carry out feeding experiment.
3, cotton bollworm larvae feeding experiment
2 instar bollworm grubs are divided into two groups, establish three repetitions for every group, each repeats 24 larvas, carries out feeding experiment respectively, and is specific as follows:
First group: the surface (every gram feed spraying 10ul) that the RNA sample first of 100ng/ul evenly is sprayed on artificial diet; 2 instar bollworm grubs are placed on the artificial diet after the spraying, change new treated artificial diet every day;
Second group: the surface (every gram feed spraying 10ul) that the RNA sample second of 100ng/ul evenly is sprayed on artificial diet; 2 instar bollworm grubs are placed on the artificial diet after the spraying, change new treated artificial diet every day;
After 10 days, the mortality ratio of statistics bollworm is seen Fig. 2.The mortality ratio of first group of cotton bollworm larvae is higher than second group.
The structure of embodiment 3, interference carrier
One, synthetic interference fragment
1, DNA (the intron intervening sequence shown in the sequence 3 of composition sequence table; 5 ' end has the PstI enzyme and cuts recognition site, and 3 ' end has the PstI enzyme and cuts recognition site), be inserted into carrier pUC19 (available from Takara company; Catalog number is D3219) the PstI restriction enzyme site between; Obtain recombinant plasmid pUC19i,, obtain enzyme and cut the product first with restriction enzyme BglII and XhoI double digestion plasmid pUC19i.
2, be template with recombinant plasmid pJHBP, carry out pcr amplification, reclaim pcr amplification product with primer P7-BglII/P8-XhoI.
P7-BglII：5’-GAAGATCTGATGTTGTGTATGA-3’；
P8-Xho?I：5’-CCGCTCGAGTTAAAAGTCGTGGT-3’。
With restriction enzyme BglII and XhoI double digestion pcr amplification product, obtain enzyme and cut product second.
3, enzyme is cut the product first and cut product second with enzyme and be connected, obtain recombinant plasmid pUC19i-JHBP (S),, obtain enzyme and cut product third with restriction enzyme SalI and XbaI double digestion plasmid pUC19i-JHBP (S).
4, be template with recombinant plasmid pJHBP, carry out pcr amplification, reclaim pcr amplification product with primer P7-BglII/P9-XbaI.
P7-BglII：5’-GAAGATCTGATGTTGTGTATGA-3’；
P9-XbaI：5’-GCTCTAGAGTTAAAAGTCGTGGT-3’。
With restriction enzyme BglII and XbaI double digestion pcr amplification product, obtain enzyme and cut the product fourth.
5, enzyme is cut the product fourth and cut product third with enzyme and be connected, obtain connecting product, promptly have the recombinant plasmid pUC19i-dsJHBP of interference fragment.Among the recombinant plasmid pUC19i-dsJHBP, inserted interference fragment at the PstI of pUC19 restriction enzyme site.
Interference fragment is a Ha-JHBP gene forward sequence from 5 ' terminal the 15th to 497 Nucleotide shown in the sequence 4 of sequence table, and the 711st to 1193 Nucleotide is the reverse sequence of Ha-JHBP gene.Fig. 3 is the structural representation of interference fragment; " Seq S " is Ha-JHBP gene forward sequence, and " Seq A " is the reverse sequence of Ha-JHBP gene, and " intron " is intervening sequence.Fig. 4 is the synoptic diagram of plant interior expression RNA interfering; " Seq S " is Ha-JHBP gene forward sequence, and " Seq A " is the reverse sequence of Ha-JHBP gene, and " | " represented hydrogen bond.
Two, the structure of interference carrier
Plant expression vector pCAMBIA2300 is (available from CAMBIA company; Detailed sequence is seen GenBank:AF234315.1, network address: http://www.ncbi.nlm.nih.gov/nuccore/7638145) go up and make up connection cotton curve leaf disease virus (CLCuV) complementary strand promotor PRP and T-NOS terminator, obtain plasmid pC2300PN.
1, the DNA shown in the sequence 8 of composition sequence table (sequence 8 of sequence table is the PRP fragment from 5 ' terminal the 12nd to 295 Nucleotide).
2, through with restriction enzyme PstI/EcoR I double digestion, the DNA shown in the sequence 8 is inserted between the Pst I/EcoR I site of pCAMBIA2300, obtain recombinant plasmid pCAMBIA2300-PRP.
3, the DNA shown in the sequence 9 of composition sequence table (sequence 9 of sequence table is the T-NOS fragment from 5 ' terminal the 14th to 194 Nucleotide).
4, through with restriction enzyme PstI/HindIII double digestion, the DNA shown in the sequence 9 is inserted between the PstI/HindIII site of pCAMBIA2300-PRP, obtain recombinant plasmid pC2300PN.
5, cut recombinant plasmid pUC19i-dsJHBP with restriction enzyme PstI enzyme, reclaim the interference fragment shown in the sequence 4, be inserted into the PstI restriction enzyme site of pC2300PN, obtain interference carrier PRP:dsJHBP.The structural representation of interference carrier PRP:dsJHBP is seen Fig. 5, comprises cotton curve leaf disease virus (CLCuV) promotor PRP, interference fragment and T-NOS terminator and border, the left and right sides.
The acquisition of embodiment 4, transgene tobacco and resistance are identified
One, the acquisition of transgene tobacco
1, changes interference carrier PRP:dsJHBP electric shock over to agrobacterium tumefaciens EHA105, obtain the Agrobacterium of recombinating.
2, the Agrobacterium of will recombinating change to be drawn a YEB solid medium (containing kantlex 50mg/L, Rifampin 50mg/L), carries out activation; Picking mono-clonal bacterial plaque is in YEB liquid nutrient medium (containing kantlex 50mg/L, Rifampin 50mg/L), and 28 ℃ of 200rpm cultivated 16～20 hours; Ratio in 4% (volumn concentration) is inoculated in YEB liquid nutrient medium (containing Syringylethanone 100uM) then, and 28 ℃, 200rpm shaking culture are to OD
600Value reaches 0.5～0.6 (about 4 hours); 4 ℃, 4000rpm are centrifugal, collect thalline; Thalline is suspended with MS liquid nutrient medium (containing Syringylethanone 200uM), and regulating the OD value is 0.15～0.20, is reorganization bacterium bacteria suspension.
3, aseptic tobacco leaf is cut into the leaflet dish, puts into reorganization bacterium bacteria suspension, infected 10 minutes.
4, the leaflet dish after taking-up is infected, filter paper is inhaled unnecessary bacterium liquid a little, and the leaf dish back side is laid on the common culture medium up, and 25 ℃ of dark conditions were cultivated 72 hours; To transfer on the screening culture medium through the leaf dish of cultivating altogether, 28 ℃ of 16/8h photoperiods cultivated 15～20 days down; Have the leaf of callus dish to be transferred on the regeneration culture medium by screening culture medium with growing, 28 ℃ of 16/8h are cultured to little regeneration bud and occur under the photoperiod; Downcut budlet and move on to strong plantlets and rootage in the root media, treat to move in the vermiculite nutrition earth mixtures behind the long root.
5, PCR identifies
The seedling of will taking root carries out PCR and identifies (P10/P11), shows that the plant of 430bp left and right sides band is the PCR positive plant.
P10：5’-GACGAAGGCTTGGGACTGGT-3’；
P11：5’-TGGCATAGCGTCTTCTTCCG-3’。
The PCR reaction conditions: 95 ℃, 1 circulation in 5 minutes; 95 ℃ 40 seconds, 56 ℃ 40 seconds, 72 ℃ 40 seconds, 35 circulations; 72 ℃ were extended 10 minutes.
The electrophorogram of the product of PCR is seen Fig. 6.
6, Northern blot identifies
PCR male plant is carried out Northern blot identifies.
Get total RNA that the 50ug blade extracts, carry out 15% polyacrylamide gel electrophoresis, RNA is transferred on the Hybond-NX Hybond membrane, with 37 ℃ of hybridization of probe 12-16 hour (probe is that the sequence 2 of sequence table is from the DNA shown in 5 ' the terminal 207-689 position Nucleotide).
Northern blot qualification result is seen Fig. 7, and the plant of swimming lane 1,2,4,5 has stronger hybridization signal.Transfer-gen plant hybridizes corresponding band.Northern blot identifies that the male plant is transfer-gen plant (T
0Generation).Plant called after T with swimming lane 1
0-1, with the plant called after T of swimming lane 2
0-2, with the plant called after T of swimming lane 5
0-5.
Two, the acquisition of contrast tobacco
PC2300PN is replaced interference carrier PRP:dsJHBP, adopt the step 1 identical operations, obtain changeing the empty carrier tobacco, as the contrast of transgene tobacco.
Three, pest-resistant evaluation
Respectively with transgene tobacco (T
0-1, T
0-2, T
0-5), 3 strain T
0In generation, changes the empty carrier tobacco and carries out feeding experiment, and is specific as follows: plant is placed hot-house culture (growth temperature is 25 ℃, and periodicity of illumination is 16/8h), during plant strain growth to 7～10 leaf ages, inoculate 3 instar bollworm grubs, 10 larvas of every strain tobacco inoculation; Add up the mortality ratio of larva every day.
Duration of test, the mortality ratio of cotton bollworm larvae are seen Fig. 8 (getting the MV and the MV that changes the empty carrier tobacco of transgene tobacco).From beginning in the 4th day of inoculation, the mortality ratio of being tried worm on the transgenic tobacco plant is apparently higher than changeing the empty carrier tobacco.
Inoculate after 8 days, 10 survivals are still arranged on the 3 strain transgene tobaccos, 3 strains are changeed in the empty carrier tobacco still has 23 survivals.Get the bollworm alive that remains on the plant, weigh and with the worm tuple according to carrying out variance analysis.See Fig. 9.The larva body weight of feeding transgene tobacco is significantly less than the larva body weight that feeding changes the empty carrier tobacco, P＜0.05 (0.013).
Inoculate after 8 days, get the bollworm alive that remains on the plant, extract total RNA, cDNA is synthesized in reverse transcription.Adopt the method for QRT-PCR to detect Ha-JHBP genetic expression in the cotton boll polypide as template cDNA.
The Ha-JHBP gene primer was during QRT-PCR detected:
P12：5’-ATTGCTTGCATTTGCGAGTTGTGT-3’；
P13：5’-TCCGGGAAACCATTGCTTGT-3’。
Confidential reference items ACTIN gene primer is:
P14：5’-CCTGGTATTGCTGACCGTATGC-3’；
P15：5’-CTGTTGGAAGGTGGAGAGGGAA-3’。
All individual average results of surviving are shown in figure 10.The mRNA level that is inoculated in the interior Ha-JHBP gene of cotton boll polypide of transgene tobacco significantly is lower than the bollworm that is inoculated in commentaries on classics empty carrier tobacco, explains that getting food transgenic tobacco plant blade can reduce Ha-JHBP expression of gene amount in the cotton boll polypide.
Claims (10)
1. protein is (a) or (b) as follows:
(a) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 1;
(b) with the aminoacid sequence of sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant with the bollworm growth by sequence 1 deutero-protein.
2. coding claim 1 said proteic gene is following 1) or 2) or 3) or 4) dna molecular:
1) sequence 2 is held the dna molecular shown in the 12nd to 740 Nucleotide from 5 ' in the sequence table;
2) dna molecular shown in the sequence 2 in the sequence table;
3) under the rigorous condition of height with 1) or 2) the dna sequence dna hybridization that limits and the dna molecular of coding bollworm growth associated protein;
4) with 1) or 2) dna sequence dna that limits has 90% above homology and the dna molecular of the bollworm growth associated protein of encoding.
3. the recombinant expression vector, expression cassette, transgenic cell line or the reorganization bacterium that contain claim 2 or 3 said genes.
4. the sequence 2 that contains ordered list is from the RNA shown in the sequence 6 of the dna fragmentation of 5 ' terminal the 207th to 689 Nucleotide or sequence table.
5. dna fragmentation as claimed in claim 4 is characterized in that: said dna fragmentation is dna fragmentation first, dna fragmentation second or dna fragmentation third;
Said dna fragmentation first like the sequence 2 of sequence table from shown in 5 ' terminal the 207th to 689 Nucleotide;
Said dna fragmentation second like the sequence 2 of sequence table from shown in 5 ' terminal the 204th to 691 Nucleotide;
Said dna fragmentation third is for containing the DNA of forward sequence and reverse sequence; Said forward sequence like the sequence 4 of sequence table from shown in 5 ' terminal the 15th to 497 Nucleotide, said reverse sequence like the sequence 4 of sequence table from shown in 5 ' terminal the 711st to 1193 Nucleotide; Said dna fragmentation third is preferably DNA shown in the sequence 4 of sequence table.
6. the recombinant expression vector, expression cassette, transgenic cell line or the reorganization bacterium that contain claim 4 or 5 said dna fragmentations.
7. recombinant expression vector as claimed in claim 6 is characterized in that: said recombinant expression vector for (I) as follows or (II):
(I) recombinant plasmid that the MCS that said dna fragmentation third is inserted recombinant plasmid pC2300PN obtains; Said recombinant plasmid pC2300PN is for being skeleton carrier with plant expression vector pCAMBIA2300; The sequence 8 of insertion sequence table is from the DNA shown in 5 ' terminal the 12nd to 295 Nucleotide between Pst I and EcoRI restriction enzyme site; The sequence 9 of insertion sequence table is from the DNA shown in 5 ' terminal the 14th to 194 Nucleotide, the recombinant plasmid that obtains between PstI and HindIII restriction enzyme site;
(II) said dna fragmentation first or said dna fragmentation second are inserted the recombinant plasmid that the MCS of bacterial expression vector L4440 obtains;
Said reorganization bacterium obtains the bacterium of recombinating for (II) described recombinant plasmid is imported intestinal bacteria HT115 (DE3).
8. a method of cultivating transgenic plant is that the described dna fragmentation third of claim 5 is imported in the purpose plant, obtains the transgenic plant that the bollworm resisting ability is higher than said purpose plant.
9. method as claimed in claim 8 is characterized in that: said dna fragmentation third imports said purpose plant through (I) described recombinant plasmid of claim 7; Said purpose plant is monocotyledons or dicotyledons; Said dicotyledons is preferably tobacco.
10. preparation that is used for killing heliothis armigera, its activeconstituents is a double-stranded RNA, a chain of said double-stranded RNA like the sequence 6 of sequence table from shown in the 16th to 503 Nucleotide of 5 ' end.
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