CN102094026B

CN102094026B - Protein HlpB gene capable of stimulating plant to perform allergic reaction

Info

Publication number: CN102094026B
Application number: CN201010575259A
Authority: CN
Inventors: 陈功友; 邹华松; 邹丽芳; 车亦舟; 李玉蓉
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2010-12-07
Filing date: 2010-12-07
Publication date: 2012-09-05
Anticipated expiration: 2030-12-07
Also published as: CN102094026A

Abstract

The invention relates to an hlpB gene which belongs to the technical field of biology, comes from plant pathogenic xanthomonas campestris and can stimulate the plant to perform allergic reaction. The sequence of the gene is shown in SEQ ID NO.1 and is 552bp in length; the hlpB gene is coded by 182 amino acids; the sequence of the amino acids is shown in SEQ ID NO.2; the hlpB protein does not contain cysteine and contains rich glycine, the isoelectric point is 5.28, the molecular weight is 19.5kDa, the protein has thermal instability, is sensitive to protease and can stimulate tobacco to perform allergic reaction. The hlpB gene obtained in the invention can be used as the gene resource for the breeding for disease resistance of plant and the hlpB protein can be used as the protein drug to stimulate the plant to have disease resistance.

Description

Excite plant to produce anaphylactoid protein HlpB gene

Technical field

The present invention relates to a kind of gene of biological technical field, specifically is to excite plant to produce anaphylactoid protein HlpB gene in a kind of pathogenic Xanthomonas campestris.

Background technology

The resistance that plant is infected pathogen shows non-host and two aspects of host-resistance.The Gram-negative plant pathogenetic bacteria produces anaphylaxis on non-host plant (Hypersensitive response is to be determined by some exciton in the pathogenic bacteria HR).This HR is a kind of anti pathologic immunity reaction, and the corresponding protein that produces HR that excites is called the harpin proteinoid.The harpin albumen kind of from the Gram-negative plant pathogenetic bacteria, having identified at present has HrpZ, HrpN, HrpW; PopA1; Hpa1, HopAK1 and HopP1, and only exist HrpW and these two of Hpa1 to excite non-host plant to produce the harpin proteinoid of HR in the clear and definite pathogenic Xanthomonas campestris.These harpin albumen have common characteristic: the tool wetting ability, be rich in glycocoll, to thermally-stabilised, responsive, acidic protein to proteolytic enzyme.

The Harpin proteinoid has important biological function.The HR that the harpin proteinoid produces on plant is a kind of cell dead form of programming, and is accompanied by in this process that active oxygen is seted out, the Whitfield's ointment accumulation, pathogenesis-related proteins produces and callose increase etc., thereby increases the anti pathologic immunity property of plant.The Harpin proteinoid is applied on the plant, can promote plant-growth, increase crop yield and inducing plant generation disease resistance etc.Pathogenic Xanthomonas campestris harpin proteinoid and encoding sox thereof have Resistant breeding value and biological and ecological methods to prevent plant disease, pests, and erosion is worth; And having a function of separating with differential plant anti pathologic immunity gene, harpin proteinoid and encoding sox thereof that excavation can make plant produce in the pathogenic Xanthomonas campestris that anti pathologic immunity reacts have important scientific value and more practical value.

Literature search through to prior art is found, does not find can excite in the pathogenic Xanthomonas campestris plant to produce the HlpB albumen of anti pathologic immunity reaction and the relevant report of corresponding encoded gene hlpB.

Summary of the invention

The objective of the invention is to overcome deficiency of the prior art, provide a kind of and excite plant to produce anaphylactoid protein HlpB encoding sox.HlpB albumen of the present invention can make the non-host plant of cause of disease Xanthomonas campestris produce anaphylaxis.

The present invention realizes through following technical scheme:

The plant that excites that the present invention relates to produces anaphylactoid protein HlpB gene, and its sequence is SEQ ID NO.1, and sequence length is 552bp; 183 amino acid of described HlpB genes encoding, aminoacid sequence are SEQ ID NO.2; Do not contain halfcystine in the described aminoacid sequence, isoelectric points of proteins is 5.28, is acid, is rich in glycocoll, molecular weight 19.5kD, and glycocoll content reaches 20%.Described HlpB gene product HlpB can make tobacco produce anaphylaxis.Anaphylaxis is the result that disease resistance of plant is activated.

HlpB gene of the present invention is to be made up by following method to form:

Utilize primer hlpB-F (SEQ ID NO.3): 5 '-TAGAATTCATGGCCCGCGG-3 ' and hlpB-R (SEQ ID NO.4): 5 '-ATTGTCGACTCAGAACGGGATATCGT-3 ' can be from paddy rice streak germ RS105 bacterial strain, rice leaf spot bacteria PXO99 through pcr amplification ^AAmplification obtains the hlpB gene in bacterial strain, cabbage black rot bacterium Xcv 85-10 bacterial strain, c itrus canker germ Xac 306 bacterial strains and the capsicum spot germ Xcc8004 strain gene group DNA.Be cloned on the coli expression carrier pET41a (+) through EcoR I and Sal I restriction enzyme site, be transformed among the expressive host bacterium BL21 (DE3), and screen the positive transformant with kalamycin resistance, this transformant is that HlpB expresses engineering strain.Engineering bacillus strain process abduction delivering, ultrasonication, ultracentrifugal method obtains the thick leach protein of solubility HlpB.After cutting the GST label, thick leach protein process GSTtrap column purification and enteropeptidase enzyme obtain complete HlpB pure protein.HlpB pure protein injection three lives cigarette, discovery can make tobacco produce anaphylaxis.

Compared with prior art, the present invention has following beneficial effect:

The exciton albumen HlpB that the present invention obtains has the proteic characteristic of harpin; Can excite tobacco to produce HR reaction and system's acquisition disease resistance; This provides the important function of gene resource for genetically engineered medicament and the transgenic plant that design inducing plant generation system obtains disease resistance, has important more practical value.

Related paddy rice cecospora spot bacterial strain RS105 and rice leaf spot bacteria PXO99 among the present invention ^ABacterial strain is at document " Wu Xiaomin; Li Yurong; Zou Lifang; And Chen Gongyou.2007.Gene-for-gene relationships between rice and diverse avrBs3/pthA avirulence genes in Xanthomonas oryzae pv.oryzae.Plant Pathology, 56 (1): 26-34 " open in; C itrus canker germ 306 bacterial strains that relate to are at document " da Silva; A.C.R.; Ferro, J.A., Reinach; F.C., et al.2002.Comparison of the genomes of two Xanthomonas pathogens with differing host specificities.Nature 417 (6887): 459-463 " in open; The oranges and tangerines cabbage black rot bacterium 85-10 bacterial strain of design is at document " Thieme; F.; Koebnik, R., Bekel; T., et al.2005.Insights into Genome Plasticity and Pathogenicity of the Plant Pathogenic Bacterium Xanthomonas campestris pv.vesicatoria Revealed by the Complete Genome Sequence.J.Bacteriol.187 (21): 7254-7266 " in open; Capsicum spot germ 8004 bacterial strains that relate to are at " Qian; W.; Jia, Y.-T., Ren; S.-X., et al.2005.Comparative and functional genomic analyses of the pathogenicity of phytopathogen Xanthomonas campestris pv.campestris.Genome Res.15 (6): 757-767 " in open.

Four, description of drawings

Fig. 1, HlpB protein expression and purifying electrophorogram

Fig. 2, HlpB excite anaphylactoid minimum concentration test on tobacco

The HlpB albumen in Fig. 3, different pathogenic Xanthomonas campestris source excites anaphylaxis figure on tobacco

Five, specific examples embodiment

Below in conjunction with specific embodiment, further set forth the present invention.These embodiment only be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions in the following example; Usually according to normal condition; For example the Sambrook equimolecular is cloned: laboratory manual (New York:Cold Spring Harbor Laboratory Press; 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment

1, pathogenic Xanthomonas campestris hlpB gene prokaryotic vector construction

Present embodiment is from paddy rice streak germ RS105 bacterial strain, rice leaf spot bacteria PXO99 ^AThe hlpB gene is a masterplate in bacterial strain, cabbage black rot bacterium 85-10 bacterial strain, c itrus canker germ 306 bacterial strains and capsicum spot germ 8004 strain gene group DNAs; Design primer hlpB-F (SEQ ID NO.3) and hlpB-R (SEQ ID NO.4); Carry out pcr amplification, the pcr amplification condition is: 94 ℃/4min+ (94 ℃/30sec+55 ℃/30sec+72 ℃/30sec) * 35+72 ℃/10min of circulation.Final PCR product is connected the pMDl8-T carrier, connects product transformed competence colibacillus cell DH5 α, the sub-pThlpB of recombinant conversion that screening has the Amp resistance.Recombinant conversion reclaims the hlpB fragment through EcoR I and Sal I double digestion; Connect into through same enzyme and cut the expression vector pET41a (+) of processing; Connect product and transform DH5 α; Obtain correct recombinant plasmid pEhlpB,, obtain recombinant bacterial strain BL21pEhlpB pEhlpB transformed into escherichia coli BL21 (DE3).

Paddy rice streak germ RS105 bacterial strain, rice leaf spot bacteria PXO99 ^ABacterial strain, cabbage black rot bacterium 85-10 bacterial strain, c itrus canker germ 306 bacterial strains and capsicum spot germ 8004 bacterial strains are public bacterial strain.PMD18-T and pET41a (+) carrier are public bacterial strain, and the carrier characteristic is seen document: TAKARA Company products specification sheets and Nerk Company products specification sheets.E. coli bl21 (DE3) bacterial strain is seen document Sambrook J., Fritsch EF., and and Maniatis is Cloning:A Laboratory Manual.2 T.1989.Molecular ^NdThe used restriction endonuclease of ed.Cold Spring Harbor Laboratory Press. the present invention is EcoR I and Sal I.

2, proteic prokaryotic expression of HlpB and purifying

Present embodiment expression strain BL21pEHlpB is from mono-clonal; The seed liquor of 37 ℃ of incubated overnight is forwarded in the fresh LB+Km substratum by 1%, and thalli growth gets into logarithmic phase behind the enlarged culturing 3h, adds the IPTG of final concentration 1mM; After continuing inducing culture 4h under 37 ℃, collect thalline.With PBS damping fluid suspension thalline, through behind the ultrasonic cell-break, the residual body of centrifugal removal bacterium, supernatant are the thick leach protein (Fig. 1 total protein) that contains GST-HlpB.Behind the thick leach protein process GSTrap protein purification column purification; Obtain the albumen (Fig. 1 purifying protein) behind the purifying; Handle 16h excision GST albumen label with 22 ℃ of enteropeptidase again; HlpB that obtains and GST mixed protein liquid are removed GST through GSTrap protein purification post, and flowing out is HlpB pure protein (Fig. 1 removes label protein) mutually.And with BCA Kit protein quantification kit measurement HlpB pure protein concentration.Proteic prokaryotic expression is conventional molecular genetic operation, and GSTrap protein purification post is a GE Health Company products, and BCA Kit is the albumen accurate quantification test kit that green skies company produces.Present embodiment three lives cigarette is that HR measures the most frequently used tobacco bred Nicotiana tabacum cv Xanthi, and is public.

3, HlpB excites the anaphylactoid mensuration of tobacco

Present embodiment HlpB is after BCA kit measures protein concentration; Adjustment final concentration 1mg/ml; And to use PBS buffer to carry out gradient dilution be 9 kinds of different concentration; Inoculation three lives cigarette confirms that minimum HR excites concentration, and the Hpa1 with same concentrations injects tobacco as positive control simultaneously, observes the tobacco leaf anaphylaxis behind the 24h.Find that through measuring it is 0.1mg/ml (Fig. 2) that HlpB excites the Cmin of HR.

4, the HlpB albumen of pathogenic Xanthomonas campestris excites the anaphylactoid mensuration of tobacco

Present embodiment is from rice leaf spot bacteria PXO99 ^AClone the hlpB gene in the erwinia amylovora of Xanthomonas campestris such as bacterial strain, cabbage black rot bacterium 85-10 bacterial strain, c itrus canker germ 306 bacterial strains and capsicum spot germ 8004 bacterial strains and non-xanthomonas; Be structured on pET41a (+) carrier; Extract protein liquid; Adjustment final concentration 1mg/ml, injection inoculation three lives cigarette is with the HlpB of same concentrations _XocThe tobacco leaf anaphylaxis is observed in the positive contrast of albumen behind the 24h.Discovery is from rice leaf spot bacteria PXO99 ^AThe HlpB albumen of bacterial strain, cabbage black rot bacterium 85-10 bacterial strain, c itrus canker germ 306 bacterial strains and capsicum spot germ 8004 bacterial strains all can excite tobacco to produce anaphylaxis, but the HlpB of erwinia amylovora can not excite anaphylaxis (Fig. 3) on tobacco.

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;

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

Method about clone harpin genes involved: Wen W.G, Wang J.S, Defense response in plants induced by Harpin _Xoo, an elicitor of Hypersensitive response from Xanthomonas oryzae pv.oryzae, 2003, Journal of Agricultural Biotechnology;

About cloning vector pMD18-T and expression vector pET41a (+): TAKARA carrier specification sheets and the explanation of Nerk expression vector;

About albumen pronucleus expression and purifying: Nerk gene escherichia expression system specification sheets.

Claims

1. one kind excites plant to produce anaphylactoid protein HlpB gene, it is characterized in that its sequence is SEQ ID NO.1, and sequence length is 552bp, and 183 amino acid of described HlpB genes encoding, aminoacid sequence are SEQ ID NO.2.

2. the purposes that excites plant to produce anaphylactoid protein HlpB gene according to claim 1 is characterized in that, described HlpB gene product HlpB makes tobacco produce anaphylaxis, and anaphylaxis is the result that disease resistance of plant is activated.