CN100334215C - Antidisense immune and bactericidal function fusion protein hcml gene and its protein - Google Patents

Abstract

The present invention relates to a fusion protein hcml gene with the functions of disease resistant immunity and sterilization, and a protein thereof, which belongs to the biologic technical field. The fusion protein hcml gene is especially used for developing biological pesticide by a gene engineering technology and improving plant disease resistance and other beneficial production traits; the alpha helical structure of Hpal and CecropinA(CA) of streak disease bacteria of paddy rice and the alpha helical structure of Meltin(ME) are connected by polylinker and a free ring to be constructed into a fusion gene hcml, the magnitude of the gene hcml is 516 bp for encoding proteins of 171 amino acids, the isoelectric point of the gene hcml is pI5.4, and the gene hcml is stable for heat. A protein product encoded by the gene hcml has the functions of disease resistant immunity and sterilization and can be used for commercial use, such as the development of biological pesticide, the technical seed breeding of transgenic plants and agricultural biology, etc. A transgenic plant of the gene Hcml has damaging effect to pathogenic bacteria, and can strengthen the broad-spectrum disease resistance of plants and improve other production traits of plants.

Description

A kind of anti pathologic immunity and bactericidal function fusion protein hcml gene and protein thereof

One, technical field

The present invention's " a kind of anti pathologic immunity and bactericidal function fusion protein hcm1 gene and protein thereof " belongs to biological technical field, is exclusively used in by genetic engineering technique to develop biological pesticide and improve disease resistance of plant and other useful production traitss.

Two, background technology

The hpa1 gene is anaphylaxis and pathogenic (hypersensitive response and pathogenicity, hrp) gene cluster member from paddy rice cecospora spot bacterium (Xanthomonas oryzae pv.oryzicola) RS105 bacterial strain.This bacterial strain is public bacterial strain.The protein of hpa1 genetic expression has useful effects such as inducing anti-disease and promotion plant-growth.The protein Harpin of hpa1 genes encoding _XoocCommon trait with Harpin protein family: the protein wetting ability, to thermally-stabilised, iso-electric point is an acidity about 4.5, with the Harpins protein family member Harpin of report _EaAnd Harpin _PssDifference is Haprin _XoocContain a halfcystine, but 1～3 μ g/ml inducing plant produces anaphylactoid a kind of similar cell programming dead (PCD).Previous invention of the present invention " gene expression product of a kind of coding plant growth regulator and uses thereof " (patent No. ZL00135403.5) has been carried out patent protection to the Harpin albumen that obtains and in external direct application from the pathogenic Xanthomonas campestris.

Proteinaceous have an antibacterial or/and germicidal action mostly be antibacterial peptide class (antimicrobial peptide).The main member of Cecropin family mostly is the Cecropin A of 37 amino acid sizes.Linear Cecropin A is made up of 2 α spirals (helix), can be combined on the cytolemma of pathogenic micro-organism, causes the death of microorganism by forming ionic channel or ionic pump.The α spiral of N-end is active required basic structure (KLFKKIEKV).Antibacterial peptide extensively is present in plant, animal and the microorganism.

Another kind of antibacterial peptide also causes extensive concern, promptly melittin (bee venom toxin, melittin).Melittin also is cationic antibacterial peptide, and about 26 amino acid sizes have splitting action to bacterium, and eukaryotic cell is had hemolytic and toxic action.Also be made up of two α spirals, the α spiral of C-end is active required basic structure (AVLKVLTTGL).

Plant genetic engineering be with foreign gene with clear and definite function as goal gene, transform plant by certain technological line after random integration in Plant Genome, make it at plant interior expression, improve the disease-resistant, pest-resistant of plant and other useful proterties.As Bacillus thuringiensis (Bacillus thuringiensis, Bt) insecticidal proteins can be used as biotechnological formulation and sprays plant and carry out pest control, behind the gene transformation cotton, transgenic cotton also has resistance (A 01N63/02 CN95113498.1A) to bollworm; Behind antibacterial peptide (Cecropin) gene transformation tobacco, potato and the paddy rice, the transgenic plant performance has resistance (Jia Shirong, 1993) to the Micobial Disease on these plants.Can also improve crop quality such as storage tolerance, salt tolerance, amino acid quality and vegetable lipid etc. by transgenic technology.

Three, summary of the invention

Technical problem the purpose of this invention is to provide plant disease-resistant immunity and the sterilizing function fusion gene hcm1 that uses as biological pesticide and transgenic plant purpose, and its gene product has in the inducing plant body generation anti pathologic immunity function and has a sterilizing function external.Utilize this goal gene, can directly use on plant for biological pesticide by gene engineering method orientation expression fusion rotein and with its development, thereby also can obtain transgenic plant or obtain the genetic recombination body to obtain kind, strain or on producing, directly use or carry out agro-ecology breeding and transgenic plant by transgenic method to improve crop disease-resistant, pest-resistant proterties and to improve other production traitss of farm crop etc. as genetic resources as commercial use by microorganism reorganization.

Technical scheme

A kind of from the disease-resistant activator harpin of paddy rice cecospora spot bacterium encoding gene hpa1 by polylinker (DPGGGFGGKW), be connected with the α spiral KLFKKIEKV of antibacterial peptide (Cecropin) N-end, be connected by the α spiral AVLKVLTTGL of free ring (GQGIG) again, be built into fusion rotein encoding gene hcm1 (hpa1-cecropin-melittin) with melittin (Melittin) C-end.

A kind of anti pathologic immunity of the present invention and bactericidal function fusion protein hcm1 gene, its sequence are SEQ ID NO.1.The fused protein of hcm1 genes encoding, its sequence are SEQ ID NO.2.

Above-mentioned a kind of anti pathologic immunity and bactericidal function fusion protein hcm1 gene are to be made up by following method to form:

According to Cecropin A and Melittin peptide sequence, with the αLuo Xuanjiegou of Hpal with Cecropin A (CA): the αLuo Xuanjiegou of KLFKKIEKV and Melittin (ME): AVLKVLTTGL is by polylinker:DPGGGFGGKW and free ring: GQGIG is connected, and is configured to fusion gene hcm1:Hpa1-Cecropin-Melittin;

Utilize primer hpa1-F1 (SEQ ID NO.3): 5 '-CATATGAACTCTTTGAACACACAATTC-3 ' and hpa1-R1 (SEQ ID NO.4):

5 '-CTTACCGCCGAACCCGCCGCCCGGATCCTGCATCGATCCGCTGTCGTTC-3 ' increases from paddy rice cecospora spot bacterium by PCR and obtains hpa1 gene and multi-joint device part polylinker;

With first round PCR product is template, utilizes primer hpa1-F1 and primer Ca-R1 (SEQ ID NO.5): 5 '-CAATCTTCTTAAAGAGTTTCCACTTACCGCCGAACCCGCCGCCCGGATC-3 ' amplification to obtain hpa1 ∷ polylinker-Ca part;

Taking turns the PCR product with second is template, with hpa1-F1 and Ca-R2 (SEQ ID NO.6):

5 '-CTTAAGCACAGCGCCAATGCCTTGACCCACTTTTTCAATCTTCTTAAAGAGTTTC-3 ' is a primer, and pcr amplification obtains hpa1 ∷ polylinker ∷ Ca-hinge part;

With third round PCR product is template, with hpa1-F1 and Me-F1 (SEQ ID NO.7): 5 '-CTCGAGCTAGAGACCCGTGGTGAGCACCTTAAGCACAGCGCCAATGC-3 ' is a primer, pcr amplification obtains hpa1 ∷ polylinker ∷ Ca ∷ hinge ∷ Me fusion gene, called after hcm1 gene.

The goal gene hcm1 that is used for biological pesticide and transgenic plant provided by the present invention is characterized in that:

1.hcm1 the source of gene is connected Hpa1 and the αLuo Xuanjiegou (KLFKKIEKV) of Cecropin A (CA) and the αLuo Xuanjiegou (AVLKVLTTGL) of Melittin (ME) of paddy rice cecospora spot bacterium by polylinker (DPGGGFGGKW) and free ring (GQGIG), be configured to fusion gene hcm1 (Hpa1-Cecropin-Melittin).The N end of fused protein is the Hpa1 polypeptide, the C end is the αLuo Xuanjiegou (AVLKVLTTGL) of Melittin (ME), the αLuo Xuanjiegou (KLFKKIEKV) of Cecropin A (CA) and the αLuo Xuanjiegou (AVLKVLTTGL) of Melittin (ME) connect by GQGIG, and polylinker is positioned at the centre of fused protein.

2.hcm1 the physicochemical characteristics hcm1 gene of gene product size is 516bp, 171 the amino acid whose protein of encoding, and iso-electric point is pI5.4, to thermally-stabilised.Hcm1 is gene constructed can to excite the generation anaphylaxis at the protein that obtains on the expression vector on tobacco, the growth of phytopathogen is had restraining effect, uses can promote plant-growth, improve output and inducing plant produces the anti pathologic immunity effect on plant.Fused protein Hcm1 makes preparation, and the biological pesticide that can be used as controlling plant diseases uses.

3.hcm1 the transgenic plant technical tactic is cut gene constructed 35S promoter and the multienzyme between the marker gene uid at double base plasmid pBI121 of hcm1 on (XbaI and BamHI) cloning site, conversion or triparental mating method are transferred among the Agrobacterium tumefaciens EHA105.Infect rice cell with the embryo conversion method, plant transformed is organized in the regeneration plant and the acquisition T that grows in the substratum that contains Km in illumination box ₁For seed.

4.hcm1 can be used as goal gene, the feature hcm1 gene of transgenic plant carries out transgenic plant.The genetically modified crops that contain the hcm1 gene can obtain the resistance of wide spectrum of different disease and pests and improve the useful production traits.In transgenic plant, can detect the existence of hcm1 gene, and available RT-PCR method detects its expression on transcriptional level.Simultaneously, can detect the expression of defense response marker gene with methods such as RT-PCR and Northern blot hybridization equimolecular biology, as GST1, PR-1a and PR-1b etc.

Hcm1 gene provided by the present invention, its protein has anti pathologic immunity and sterilizing function, can be used as the goal gene of plant genetic engineering, is used for commercial uses such as biological pesticide development, transgenic plant and biotechnology breeding.

Beneficial effect

The present invention is according to Cecropin A and Melittin peptide sequence, the αLuo Xuanjiegou (KLFKKIEKV) of Hpa1 and Cecropin A (CA) and the αLuo Xuanjiegou (AVLKVLTTGL) of Melittin (ME) are connected by polylinker (DPGGGFGGKW) and free ring (GQGIG), are configured to fusion gene hcm1 (Hpa1-Cecropin-Melittin).The fusion rotein Hcm1 that this fusion gene obtains after expressing can give plant and produce the anti pathologic immunity function, can suppress the growth of phytopathogen.Gene product can be developed the control that is used for Plant diseases for biological pesticide.

Fusion rotein Hcm1 preparation is mixed with the aqueous solution, and final concentration is 15-30 μ g/ml.Growth to phytopathogen when giving birth to survey has restraining effect; The injection tobacco leaf has the anaphylactoid ability of generation; In seedling stage of crop growing season, tillering phase, initial bloom stage, just fruit phase or filling stage spray and use 2-3 time, can improve crop growth amount 10-10%, raising output is more than 6%, induces that to make the deposits yields disease resistance suitable with the conventional chemical sterilant.

As biological pesticide goal gene hcm1, its gene product has and excites anaphylaxis, inducing plant to produce broad spectrum resistance on non-host plant and phytopathogen is had sterilizing function, can by gene engineering method be structured in any can the expression system of expressing gene product on, obtain the hcm1 gene product with engineered method, and with its development for biological pesticide, be used for controlling plant diseases.

The hcm1 transgenic plant are that hcm1 is gene constructed on the Ti-of expression of plants plasmid binary vector pBI121.With the method for Agrobacterium tumefaciens (Agrobacterium tumefaciens) mediation, rice transformation has obtained transfer-gen plant, in the disease resistance of greenhouse and land for growing field crops evaluation transfer-gen plant.The hcm1 transgenic plant all have obvious enhancing to the disease resistance of fungi, bacterium and virus disease.The commentaries on classics hcm1 gene plant of paddy rice also shows low light tolerance, low temperature resistant, the good production traits such as plant type is downgraded, tiller more.

As transgenosis goal gene hcm1, can be incorporated in the plant gene, and in plant, express, the improvement of giving the resistance of wide spectrum of plant to disease, pathogenic bacteria being had sterilizing function and other production traitss is expressed in the transgenic paddy rice of hcm1 gene and can be detected hcm1 gene and defense response genes involved GST1, PR-1a, PR-1b and EF-1a etc.Can directly cultivate plants new variety or carry out routine and biotechnology breeding of the transgenic plant strain of hcm1 gene as genetic resources.

Four, description of drawings

The proteic aminoacid sequence feature of Fig. 1, hcm1 fusion gene nucleotide sequence and Hcm1

1-411 bp is the hpa1 gene, and gene product is harpin _XoocProtein; DPGGGFGGKW is multi-joint element (polylinker); KLFKKIEKV is the α-Luo Xuanjiegou of antibacterial peptide (Cecropin) N end; GQGIG is free ring (flexible hinge); AVLKVLTTGL is the α-Luo Xuanjiegou of melittin (Melittin) C end

Fig. 2, hcm1 gene make up the feature of the recombinant plasmid pBI121 ∷ hcm1 that transforms plant as goal gene

H：Hind?III；S：Sph?I；P：Pst?I；X：Xba?I；B：BamHI；Sm：Sma?I；Sa：Sac?I；?E：EcoR?I

Five, embodiment

The immunity of example 1 plant disease-resistant and sterilizing function hcm1 expression of gene product and directly use

Paddy rice cecospora spot bacterium (Xanthomonas oryzae pv.oryzicola) hrp gene cluster obtains (accession No.AY875714) by laboratory, place of the present invention.The present invention is according to Cecropin A and Melittin peptide sequence, the αLuo Xuanjiegou (KLFKKIEKV) of Hpa1 and Cecropin A (CA) and the αLuo Xuanjiegou (AVLKVLTTGL) of Melittin (ME) are connected by polylinker (DPGGGFGGKW) and free ring (GQGIG), are configured to fusion gene hcm1 (Hpal-Cecropin-Melittin).

Utilize primer hpa1-F1 (SEQ ID NO.3): 5 '-CATATGAACTCTTTGAACACACAATTC-3 ' and hpa1-R1 (SEQ ID NO.4):

5 '-CTTACCGCCGAACCCGCCGCCCGGATCCTGCATCGATCCGCTGTCGTTC-3 ' increases from paddy rice cecospora spot bacterium by PCR and obtains hpa1 gene and multi-joint componentry (polylinker);

With first round PCR product is template, utilizes primer hpa1-F1 and primer Ca-R1 (SEQ ID NO.5):

5 '-CAATCTTCTTAAAGAGTTTCCACTTACCGCCGAACCCGCCGCCCGGATC-3 ' amplification obtains hpa1 ∷ polylinker-Ca part;

Taking turns the PCR product with second is template, with hpa1-F1 and Ca-R2 (SEQ ID NO.6):

5 '-CTTAAGCACAGCGCCAATGCCTTGACCCACTTTTTCAATCTTCTTAAAGAGTTTC-3 ' is a primer, and pcr amplification obtains hpa1 ∷ polylinker ∷ Ca-hinge part;

With third round PCR product is template, with hpa1-F1 and Me-F1 (SEQ ID NO.7): 5 '-CTCGAGCTAGAGACCCGTGGTGAGCACCTTAAGCACAGCGCCAATGC-3 ' is a primer, pcr amplification obtains hpa1 ∷ polylinker ∷ Ca ∷ hinge ∷ Me fusion gene, called after hcm1 gene.

The pcr amplification condition is: 94 ℃/4min+ (94 ℃/30 sec+60 ℃/min+72 ℃/2min) * 35+72 ℃/7min of circulation.Final PCR product purification is connected in expression vector pET21 after NdeI and XhoI enzyme are cut ^aOn, the acquisition recombinant plasmid is pEHCM1 (pET30a ∷ hcm1).

With pEHCM1 transformed into escherichia coli BL21 (BL21/pET30 ^a∷ hcm1) the single colony inoculation of the recombinant microorganism BLHCM1 that obtains obtains to cultivate 6h under 37 ℃ among the recombinant microorganism BLHCM10 μ g/ml in the LB+Km 2 of 20ml, wherein make final concentration reach 1mM the IPTG adding of 1M, centrifugal collection thalline behind the cultivation 3h under 37 ℃, the back is in 100 ℃ of following water-bath 10min, the centrifugal 10min of cooling back 12000rpm, get supernatant and inject the tobacco leaf mesophyll cell, and generation anaphylaxis in the 24h (hypersensitive response, HR).Get 3 μ l supernatant liquors and carry out the 12%SDS-PAGE electrophoresis, calculate that according to the molecular weight of standard protein the molecular weight of this expressing protein is about 17kDa, this proteic expression amount accounts for 15% of total protein.The iso-electric point pI that disk isoelectric focusing electrophoresis (IEF) is measured fused protein is 5.4.Fusion gene hcm1 total length 516bp, 171 the amino acid whose protein of encoding.The aminoacid sequence of the nucleotide sequence of hcm1 fusion gene and Hcm1 fusion rotein as shown in Figure 1.

The single colony inoculation of genetic recombination microorganism BLHCM1 (BL21/pEHCM1) is cultivated 16～20h under 37 ℃ in LB+Km20 μ g/ml+IPTG1mM/L, oxygen-supply quantity is the 0.5L/ branch, and rotating speed is 110 rev/mins.Centrifugal (5,000rpm) 10min collects thalline, the 20mM Tris damping fluid (pH9.0) that adds 1/40 fermentation volume fully suspends, the N,O-Diacetylmuramidase (100mg/ml) that adds 10 μ l/25ml, behind the ice bath 10min, add 0.5ml 25ml PMSF (20mg/ml), the broken thalline 10min of ultrasonic wave (20KHz), the PMSF that adds 0.5ml/25ml again, ultrasonic disruption 10min again behind the mixing.Centrifugal 10 minutes of 10000rpm gets supernatant 10min under 100 ℃ of water-baths, cooling back 12, and the centrifugal 10min of 000rpm collects supernatant liquor.Proteinaceous concentration reaches 600 μ g/ml in the supernatant liquor.LB be substratum (contain 5g yeast powder, 10g polyprotein peptone among the 1L, 10g NaCl, pH7.0).Km is a kantlex, and IPTG is isopropylthio-beta galactose glycosides, and PMSF is a phenylmethylsulfonyl fluoride, and Tris is a Tutofusin tris.Down together.

The bacterial strain BLHCMl of high yield fusion rotein Hcm1 is rule 37 ℃ of following overnight incubation on the inclined-plane of LB+Km 20 μ g/ml.Single bacterium colony BLHCM1 is inoculated among the 100ml LB+Km 20 μ g/ml, and in 37 ℃, 110 change shaking culture 12h under the condition of branch.Add 20L LB+Km25 μ g/ml nutrient solution in the 30L fermentor tank, 1% amount inserts female the kind, and 37 ℃, 110 rev/mins and 0.5L/ divide under the condition of air flow and cultivate 12h.The LB that adds 700L in 1 ton of fermentor tank sterilizes (120 ℃, 2kg pressure, 30 minutes) afterwards add Km 40 μ g/ml+IPTG 1mM/L as fermented liquid by millipore filtration, 1% volume inserts cultivar, 32 ℃, 110 commentaries on classics divide and 1L/ divides air flow condition bottom fermentation to cultivate 20h, the back is 4500 rev/mins of centrifugal 10 minutes collection thalline in big whizzer, add 1/40 volume 20mMTris damping fluid (pH9.0) suspension thalline, the PMSF (20mg/ml) that adds 1/50 volume, the broken thalline of 100 ℃ of steam 10 minutes, the centrifugal collection product in cooling back, add the stopping composition drying and make the particulate state microcapsule, make the effective content of fusion rotein reach 1% (W/W).

Fusion rotein Hcm1 preparation is mixed with the aqueous solution, and final concentration is 15-30 μ g/ml.Growth to phytopathogen when giving birth to survey has restraining effect; The injection tobacco leaf has the anaphylactoid ability of generation; In seedling stage of crop growing season, tillering phase, initial bloom stage, just fruit phase or filling stage spray and use 2-3 time, can improve crop growth amount 10-10%, raising output is more than 6%, induces that to make the deposits yields disease resistance suitable with the conventional chemical sterilant.

Paddy rice slice pinta bacterium (X.o.pv.oryzicola) is public bacterial strain.PET21a carrier and e. coli strain bl21 are seen document Sambrook J., Fritsch EF., and and Maniatis is Cloning:A LaboratoryManual.2 T.1989.Molecular ^NdEd.Cold Spring Harbor Laboratory Press.

The structure of example 2 transgenic plant expression vector pBIHCM1 (pBI121 ∷ hcm1)

Be used to make up double base conversion-expression vector plasmid be to transform by T-DNA, wherein remove T-DNA25bp tumor-necrosis factor glycoproteins (LB and RB), outside promotor of rouge alkali synthetase gene (P-nos) and the terminator (T-nos), also have nptI that uses as selective marker in prokaryotic organism (bacterium) and the nptII that uses as selective marker in eukaryote (plant), the microbiotic that is used to select is Km and G-418.The promotor 35S (p35S) and beta-Glucuronidase (Gus) the gene uidA that in pBI121, also have the cauliflower mosaic virus of expressing in addition eukaryote.The hcm1 gene that uses as goal gene is inserted in the p35S and the multienzyme between the uid of pBI121 plasmid and cuts (Fig. 2) on the cloning site, obtains transgenic plant expression vector pBIHCM1 (pBI121 ∷ hcm1).The used restriction endonuclease of the present invention is XbaI and BamHI.Binary vector pBI121 is public carrier.

Example 3 expression of hcm1 gene in transgenic plant

The double base conversion carrier plasmid pBI121 ∷ hcm1 that makes up can directly transform and contains vir district helper plasmid and do not derive among competence Agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105 of plasmid with the pTiBo542 of Km resistance.EHA105 is the strain of deriving of the Km sensitivity of EHA101 (C58/pTiBo542).Also can helping plasmid pRK2013 to exist under the situation, transfer among the EHA105 with the bacillus coli DH 5 alpha that contains pBI121 ∷ hcm1 by triparental mating.Contain helper plasmid pTiBo542 (Km simultaneously ^s) and the Agrobacterium tumefaciens EHA105 that transforms plasmid (pBI121 ∷ hcm1) be used for the conversion of vegetable cell.Method leaf dish method, embryo conversion method or the pollen tube introduction method of reorganization Agrobacterium tumefaciens transformed plant cells.Explant screens the regeneration plant (T that is transformed in containing the plant regeneration substratum of Km ₀Generation).T ₁Screened transformed plant in 12 hours with Km (150 μ g/ml) immersion during for presprouting of seeds.

To T ₁Detect hcm1 expression of gene in transformation frequency and the plant for plant by strain system.PCR and Southern blot hybridization method can detect hcm1 gene and P-35S promotor in the transformed plant blade, and quantitative RT-PCR method can detect the RNA accumulation of hcm1 gene in the plant leaf.

Agrobacterium tumefaciens EHA105 is public bacterial strain, and the plant regeneration method of gene transformation and transformant is the ordinary method of transgenic plant.

The phenotypic characteristic of example 4 hcm1 gene transgenic paddy rice

The T of hcm1 gene transgenic paddy rice ₁Sow respectively in greenhouse and the popular district of disease isolation field piece for seed, with artificial inoculation with bring out capable infection method and measure the disease-resistant type of hcm1 transgenic paddy rice strain system rice blast (Magnaporthe grisea) and bacterial blight of rice (Xanthomonas oryzae pv.oryzae).Artificial inoculation method conventional spraying (Pyricularia oryzae) and leaf-cutting inoculation method (bacterial leaf spot pathogenic bacteria).Bring out capable infection method and be with the artificial spray inoculation susceptible variety of Pyricularia oryzae Xian excellent 63, be transplanted to after the morbidity disease identify the garden around and between furrow.1.8 meters of furrow face widths, seeding row spacing are 15 centimetres.The spore that brings out on the capable paddy rice scab can be propagated with the even STOCHASTIC DIFFUSION of wind and rain.The susceptible strain system and the kind 100% infected morbidity of setting out.Hcm1 transgenic paddy rice T1 all shows stronger resistance for individual plant to rice blast with to bacterial blight of rice.Being used for genetically modified parent's rice varieties has high quality japonica force to educate round-grained rice No. 7.Except that disease-resistant phenotype had clear improvement, transgenic line also showed characteristics such as tangible low light tolerance, low temperature resistant and plant type dwarfing.Hcm1 transgenic paddy rice strain called after Hcm transgenosis resists excellent rice.

The rice leaf spot bacteria and the Pyricularia oryzae that carry out the disease resistance evaluation are public.It is for No. 7 to produce to go up the kind of using that transgenosis parent's rice varieties force is educated round-grained rice, public.

Example 5 changes the feature that hcm1 trans-genetic hybrid rice strain starts plant defense

Can be by PCR and Southern blot hybridization method from paddy rice T ₁For total length insertion sequence that detects the hrfA gene in the plant and external source p35S promotor, account for 70%.Hcm1 transgenic paddy rice T ₁All do not show the phenomenon that causes producing anaphylactoid similar cell programming dead (PCD) for plant.Measure hcm1 gene and disease-resistant defence related gene expression with quantitative RT-PCR method, in transgenic plant strain system, can detect defence Expression of Related Genes such as GST1, PR-1a, PR-1b, and in parental line, not express.PCR, Southern blot hybridization and RT-PCR method are molecular biology conventional study methods.GST1, PR-1a and PR-1b are the significant gene that plant produces the disease resistance reaction, and be known and public.

About RS105: Chen Gongyou, Pan Xiaomei, Wang Jinsheng, the research of rice leaf spot bacteria (Xanthomonas oryzae pv.oryzae) chemomorphosis hrp gene mutation body and correlated character, Plant Pathology, 2001,31 (3): 199～207; About the plant genetic engineering molecule manipulation: Clark MS, Plant Molecular Biology, A Laboratory Manual, 1997, Springer, Berlin Germany;

Operate about recombinant cdna molecule: Sambrook J, Russell D.W., Molelcular cloning, A LaboratoryManual (3 ^RdEd), 2001, Spring Harbor Laboratory Press

PCR method about clone hpa1 dna homolog gene: Weiguang Zhu, Mark M.Magbanua and Fran F.White identification of Two Novel hrp-Associated Genes in the hrp Gene Cluster ofXanthomonas oryzae pv.oryzae, Journal of Bacteriology, 2000,182 (7): 1844-1853; About binary vector pBI121:Jefferson, R.A., Kavanash, T.A.﹠amp; Bevan, M.W. EMBO J.1987,6,3901-3907;

About cecropin and Melinttin:Moerman L, Bosteels S, Noppe W, et al.2002.Antibacterialand antifungal properties of a-helical, cationic peptides in the venom of scorpions fromsouthern Africa.Eur.J.Biochem.269,4799-4810

Sequence table

＜110〉Agricultural University Of Nanjing

＜120〉a kind of anti pathologic immunity and bactericidal function fusion protein hcml gene and protein thereof

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<221>primer

<222>(1)..(49)

<223>

<400>4

cttaccgccg?aacccgccgc?ccggatcctg?catcgatccg?ctgtcgttc 49

<210>5

<211>49

<212>DNA

＜213〉combination biosynthesizing

<220>

<221>primer

<222>(1)..(49)

<223>

<400>5

caatcttctt?aaagagtttc?cacttaccgc?cgaacccgcc?gcccggatc 49

<210>6

<211>55

<212>DNA

＜213〉combination biosynthesizing

<220>

<221>primer

<222>(1)..(55)

<223>

<400>6

cttaagcaca?gcgccaatgc?cttgacccac?tttttcaatc?ttcttaaaga?gtttc 55

<210>7

<211>47

<212>DNA

＜213〉combination biosynthesizing

<220>

<221>primer

<222>(1)..(47)

<223>

<400>7

ctcgagctag?agacccgtgg?tgagcacctt?aagcacagcg?ccaatgc 47

Claims (3)

1, a kind of anti pathologic immunity and bactericidal function fusion protein hcml gene, its sequence is SEQ ID NO.1.

2, the fused protein of described a kind of anti pathologic immunity of claim 1 and bactericidal function fusion protein hcml genes encoding, its sequence is SEQ ID NO.2.

3, according to described a kind of anti pathologic immunity of claim 1 and bactericidal function fusion protein hcml gene, it is characterized in that, is to be made up by following method to form:

According to Cecropin A and Melittin peptide sequence, with the αLuo Xuanjiegou of Hpal with Cecropin A (CA): the αLuo Xuanjiegou of KLFKKIEKV and Melittin (ME): AVLKVLTTGL is by polylinker:DPGGGFGGKW and free ring: GQGIG is connected, and is configured to fusion gene hcml:Hpal-Cecropin-Melittin;

Utilize primer hpal-F1 (SEQ ID NO.3): 5 '-CATATGAACTCTTTGAACACACAATTC-3 ' and hpal-R1 (SEQ ID NO.4):

5 '-CTTACCGCCGAACCCGCCGCCCGGATCCTGCATCGATCCGCTGTCGTTC-3 ' increases from paddy rice cecospora spot bacterium by PCR and obtains hpal gene and multi-joint componentry polylinker;

With first round PCR product is template, utilizes primer hpal-F1 and primer Ca-R1 (SEQ ID NO.5):

5 '-CAATCTTCTTAAAGAGTTTCCACTTACCGCCGAACCCGCCGCCCGGATC-3 ' amplification obtains the hpal::polylinker-Ca part;

Taking turns the PCR product with second is template, with hpal-F1 and Ca-R2 (SEQ ID NO.6):

5 '-CTTAAGCACAGCGCCAATGCCTIGACCCACTTTTTCAATCTTCTTAAAGAGTTTC-3 ' is a primer, and pcr amplification obtains the hpal::polylinker::Ca-hinge part;

With third round PCR product is template, with hpal-F1 and Me-F1 (SEQ ID NO.7): 5 '-CTCGAGCTAGAGACCCGTGGTGAGCACCTTAAG-CACAGCGCCAATGC-3 ' is a primer, pcr amplification obtains the hpal::polylinker::Ca::hinge::Me fusion gene, called after hcml gene.