CN103789323A - Protein capable of inducing defense response of rice, as well as encoding gene, preparation method and use thereof - Google Patents
Protein capable of inducing defense response of rice, as well as encoding gene, preparation method and use thereof Download PDFInfo
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- CN103789323A CN103789323A CN201410010747.5A CN201410010747A CN103789323A CN 103789323 A CN103789323 A CN 103789323A CN 201410010747 A CN201410010747 A CN 201410010747A CN 103789323 A CN103789323 A CN 103789323A
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Abstract
The invention provides an encoding gene of a plant immunologic active substance capable of inducing the defense response of rice. The encoding gene has a nucleotide sequence shown as SEQ ID NO.1. The invention also provides a protein for inducing the defense response of rice. The protein has a nucleotide sequence shown as SEQ ID NO.7. The protein expressed by the gene sequence SEQ ID NO.1 is discovered to remarkably induce the immune response of rice, is a novel rice immunologically active substance, and has huge application potential to the prevention and control of rice diseases, and huge economic benefits and social benefits are further created.
Description
Technical field
The present invention relates to a kind of albumen and encoding gene thereof that can inducing paddy rice defense response.Particularly, the present invention relates to bacterial origin immunologic active material, it can cause paddy rice immune response.
Background technology
Paddy rice is one of China's staple food crop, and cultivated area accounts for 1/4 of national cultivated area, and annual production accounts for 1/2 of national total grain output nearly.The harm of rice disease has a strong impact on the production of paddy rice always, if effectively do not prevented and not treated, and 30,000,000,000 kilograms of average annual lost units, even prevent and treat under condition existing, average annual loss may reach 20,000,000,000 kilograms.Therefore, studying and prevent and treat rice disease tool is of great significance.
The kind of rice disease is a lot, and the whole world has nearly hundred kinds, and wherein bacterial leaf-blight generation area is large, popular by force, harm is serious, is one of " three large important diseases " on paddy rice.Because after bacterial leaf spot morbidity, focus is arranged in Rice Vascular Bundle, so the control of bacterial leaf spot is still more difficult.Recent result of study shows, transforms after microbial source gene in paddy rice, and this transgenic rice plant can show the resistance to relevant disease, has proved that paddy rice can excite the lower generation disease resistance of being correlated with at external antigenicity substance.
Along with Production requirement and the market thriving demand to Organic food of agriculture high added value in recent years, market is needed badly and can effectively be prevented the corps diseases anti-product of agriculture of non agricultural chemical residuum again.Therefore exploitation has immunocompetent biological activity protein, is a kind of irreplaceable mode for Guarantee Grain Production and the quality that improves People's livelihood.
Summary of the invention
Therefore, the object of this invention is to provide a kind of gene and albumen thereof of bacterial origin immunologic active material that can inducing paddy rice defense response.
As follows for realizing the technical scheme of above-mentioned purpose:
On the one hand, the invention provides a kind of encoding gene of plant immunizing activities material, described encoding gene has the nucleotide sequence as shown in SEQ ID NO.1.Or described encoding gene has the nucleotide sequence as shown in SEQID NO.2.
Preferably, the nucleotide sequence of described encoding gene is as shown in SEQ ID NO.1 or SEQ ID NO.2.
Above-mentioned encoding gene provided by the invention can derive from oat acidophilic bacteria Acidovorax avenae(and be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC on December 30th, 2013, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), culture presevation CGMCC No.8661; Further be accredited as oat acidophilic bacteria Acidovorax avenae subsp.Avenae (Aaa)) genomic dna, be the gene of Novel rice immunologic active material provided by the invention.Especially, herein by nucleotide sequence as the encoding gene called after " fliC " of SEQID NO.1.
On the other hand, the invention provides a kind of albumen for inducing paddy rice defense response, described albumen has the aminoacid sequence as shown in SEQ ID NO.7;
Preferably, the aminoacid sequence of described albumen is as shown in SEQ ID NO.7.
Above-mentioned albumen provided by the invention can be obtained by the encoding gene coding with nucleotide sequence as shown in SEQ ID NO.1.According to the specific embodiment of the present invention, the albumen of aminoacid sequence as shown in SEQ ID NO.7 has the molecular weight of 51kD, herein by its called after albumen " FliC ".
Another aspect, the invention provides the preparation method of above-mentioned albumen, and described preparation method comprises the following steps:
(1) obtain its encoding gene;
(2) use the encoding gene that step (1) obtains to build protein expression vector, and transformed intestinal bacteria;
(3) albumen described in the escherichia coli expression through transforming that induction step (2) obtains.
Preferably, described step (1) comprising:
Take the genomic dna of oat acidophilic bacteria Acidovorax avenae (CGMCC No.8661) as template, adopt the increase encoding gene of described albumen of PCR method.
Wherein, can directly obtain the nucleotide sequence as shown in SEQ ID NO.1 from the genomic dna amplification of oat acidophilic bacteria Acidovorax avenae CGMCC No.8661 with upstream primer SEQ ID NO.5, downstream primer SEQ ID NO.6, it is the encoding gene of albumen FliC.
Or, according to specific embodiment of the invention scheme, can obtain the nucleotide sequence as shown in SEQ ID NO.2 from the genomic dna amplification of oat acidophilic bacteria Acidovorax avenae CGMCC No.8661 (Aaa) with upstream primer SEQ ID NO.3, downstream primer SEQ ID NO.4, it comprises the nucleotide sequence shown in SEQ ID NO.1.Using this nucleotide sequence and carrier pMD-18T construction recombination plasmid as template, then with upstream primer SEQ ID NO.5, downstream primer SEQ ID NO.6 this recombinant plasmid that increases, obtain the nucleotide sequence as shown in SEQ ID NO.1.
Again or, can obtain the nucleotide sequence as shown in SEQ ID NO.2 from the genomic dna amplification of oat acidophilic bacteria Acidovorax avenae CGMCC No.8661 (Aaa) with upstream primer SEQ ID NO.3, downstream primer SEQ ID NO.4, be directly used in step (2) and build protein expression vector.
Preferably, described step (2) comprising:
Use restriction enzyme BamH I and Hind III double digestion step (1) obtains respectively encoding gene and carrier pET-28a, connect with T4 ligase enzyme the fragment obtaining afterwards, thereby build expression of recombinant proteins plasmid, be then transformed in competence coli strain.According to specific embodiment of the invention scheme, coli strain is BL21(DE3).
Preferably, described step (3) comprising:
With the IPTG of 0.5mM, the e. coli bl21 through transforming that inducing culture step (2) obtains, obtains the albumen of 51kD; Preferably, the condition of described inducing culture is 10 ℃, rotating speed 150rpm.
On the one hand, experiment showed, that albumen provided by the invention can cause the immune response of plant, particularly paddy rice again.Therefore, the present invention also provides described encoding gene or albumen in the purposes of preparing in plant immunization preparation.Preferably, described immunological reagent is paddy rice immunological reagent.
Particularly, the method for obtaining specific gene of the present invention and albumen comprises the following steps:
(1) obtaining oat acidophilic bacteria Acidovorax avenae CGMCC No.8661 (Aaa) sequence is the flagellin gene of SEQ ID NO.1;
(2) the gene constructed immunoreactive protein expression vector obtaining with step (1).
Preferably, aforesaid method is further comprising the steps of:
(3) immunoreactive protein expression vector step (2) being built is converted in e. coli bl21 (DE3) competence and carries out abduction delivering.
In aforesaid method, preferably, step (1) comprises the following steps:
Take oat acidophilic bacteria Acidovorax avenae CGMCC No.8661 (Aaa) genomic dna as template, employing PCR method amplification of nucleotide acid sequence is that the gene order of SEQ ID NO.2 builds up in T carrier pMD-18T, the recombinant plasmid called after pMD-18T-fliC obtaining; Wherein, the nucleotides sequence of upstream primer is classified SEQ ID NO:3 as, and the nucleotides sequence of downstream primer is classified SEQ ID NO.4 as;
In the same subspecies of oat acidophilic bacteria that the gene order of SEQ ID NO.2 and genome sequence are checked order completely, strains A cidovorax avenae subsp.avenae ATCC19860 carries out the compare of analysis of gene, and definite kernel nucleotide sequence is the gene of SEQ ID NO.1.
Afterwards, in step (2), take pMD-18T-fliC as template, the nucleotides sequence of upstream primer is classified SEQ ID NO.5 as, the nucleotides sequence of downstream primer is classified SEQ ID NO.6 as, the gene order of pcr amplification SEQ ID NO.1, is cloned in prokaryotic expression carrier pET-28a, the recombinant expression vector called after pET-28a-fliC obtaining.
Then, in step (3), transform the intestinal bacteria that obtain through IPTG inducing culture step (2), obtaining protein sequence is the albumen that SEQ ID NO.7, size are about 51KD.
The present invention utilizes oat acidophilic bacteria Acidovorax avenae CGMCC No.8661 and the interior bacterial strain oat acidophilic bacteria Acidovorax avenae subsp.avenae ATCC19860 flagellin of same subspecies (in GenBank, its mRNA sequence Locus:YP_004236748, CDS sequence is 1, 479bp, Gene ID:10309252, complete genome sequence Locus:NC_015138, gene annotation is " flagellin domain-containing protein ") gene has this feature of higher homology, design degenerated primer, clone obtains large gene fragment, and through comparing the gene of having determined a kind of Novel rice immunologic active material, called after fliC, again so the albumen that obtains its coding be the flagellin FliC of oat acidophilic bacteria.Experiment showed, the flagellin prokaryotic expression of this oat acidophilic bacteria purifying is concentrated after, process rice leaf, detect and on paddy rice, produced H
2o
2, this shows that albumen has excited the immune response of paddy rice inherence.
The immune response of plant has certain effect to the invasion that stops pathogen.FliC has outstanding biology function characteristic, can produce anaphylaxis (hypersensitive response, HR) by inducing paddy rice, causes the immune response of paddy rice.This characteristic is that a kind of novel paddy rice immunologic active material of exploitation and broad spectrum plant immunization induction preparation provide the foundation, and by determining the active function territory of oat acidophilic bacteria flagellin, can make a kind of plant immunization preparation, for strengthening the disease resistance of plant.
Accompanying drawing explanation
Below, describe by reference to the accompanying drawings embodiment of the present invention in detail, wherein:
Fig. 1 shows the clone of the SEQ ID NO.2 sequence of utilizing the conserved sequence amplified fliC of comprising full-length gene.Wherein Figure 1A is clone's collection of illustrative plates of SEQ ID NO.2; Figure 1B is the electrophoresis result figure of pcr amplification SEQ ID NO.2, and wherein M is marker (1kb ladder) (day root code:MD113).
Fig. 2 shows the structure of fliC total length SEQ ID NO.1 expression vector.Wherein, Fig. 2 A is clone's collection of illustrative plates of pET-28a-fliC expression vector; Fig. 2 B is the electrophoresis result figure of pcr amplification fliC full length sequence, and wherein M is marker (1kb ladder); Fig. 2 C is expression vector pET-28a-fliC double digestion checking electrophoresis result figure, and wherein M is marker (1kb ladder).
Fig. 3 shows the prokaryotic expression situation of FliC albumen.Fig. 3 A is the polyacrylamide gel electrophoresis result figure of FliC albumen, and M is albumen marker (94KD) (day root code:MP102); Fig. 3 B is the Western blot checking of FliC albumen, and M is albumen marker (245KD) (day root code:MP206).
Fig. 4 shows FliC albumen processing rice leaf situation.PBS is the damping fluid that dissolves FliC albumen.
Embodiment
Below in conjunction with embodiment, the present invention is further described in detail, the embodiment providing is only in order to illustrate the present invention, rather than in order to limit the scope of the invention.
In following embodiment, the various test materialss that use, reagent, carrier etc., all can obtain by commercial sources; Related various experimental working techniques, comprise that structure, the enzyme of preparation, clone or the expression vector of extraction, the cDNA of RNA cut, connection, bacterial strain transform, screening etc., are the research technique of routine this area in.
Utilize the method design degenerated primer amplification oat acidophilic bacteria flagellin gene of homology comparison.
1, clone oat acidophilic bacteria Acidovorax avenae CGMCC No.8661 (Aaa) flagellin gene
According to the homology of flagellin gene between oat acidophilic bacteria subspecies, we are according to all primers of other subspecies of oat acidophilic bacteria Acidovorax avenae subsp.avenae ATCC19860 design amplification Aaa flagellin gene (fliC) total length of order-checking of genome, take Aaa complete genome DNA as template, with high-fidelity enzymatic amplification PCR.Then amplified fragments is connected to carrier pMD-18T (TaKaRa code:D101A) upper (ligation is with reference to pMD18-T vector test kit specification sheets), the recombinant plasmid called after pMD-18T-fliC obtaining.Order-checking has obtained the gene fragment that size is 2,328bp, is SEQ ID NO.2.Construction of recombinant plasmid process is shown in Fig. 1.
Primer sequence:
Upstream primer (SEQ ID NO.3): 5'-GCTGGGAACAAACATTACTGCC-3';
Downstream primer (SEQ ID NO.4): 5'-GGTTCTGCACCTGCACGTTG-3'.
2, build FLIC expression vector
Utilize primer amplified fliC gene, take pMD-18T-fliC as template, primer adds respectively restriction enzyme site, and sequence is as follows:
Upstream primer SEQ ID NO.5:
5'-CGGGATCCATGGCATCCACCATCAACAC-3'；
Downstream primer SEQ ID NO.6:
5'-CCAAGCTTTCAACGCAGCAGGGACAACA-3'。
Order-checking has obtained the gene fragment of size for Isosorbide-5-Nitrae 79bp, is SEQ ID NO.1.
Use respectively specific limited enzyme BamH I (TaKaRa code:D1010A) and Hind III (TaKaRa code:D1060A) enzyme to cut this gene fragment and expression vector pET-28a, then use T4 ligase enzyme (TaKaRa code:D2050) to connect, be built into protein expression vector, called after pET-28a-fliC(ligation is with reference to T4DNA ligase enzyme specification sheets).This expression vector is transformed to e. coli bl21 (DE3), and screening, the positive colony that authentication sequence information is correct, be fliC expression vector.Building process is shown in Fig. 2.
3, FLIC protein expression
Recombinant expression plasmid pET-28a-flic is gone in expression strain BL21 (DE3), the IPTG that is 0.5mM through final concentration is at 10 ℃, rotating speed is under the condition of 150rpm, inducing culture 24h, obtained the big or small fusion rotein that is about 51kD, solubility detects finds that albumen has expression in supernatant.Protein electrophoresis and Western Blot detailed process: the albumen of purifying is carried out to SDS-PAGE protein electrophoresis, transfer on pvdf membrane by half dry type, add 5% skimmed milk to seal non-specific site as coating buffer, at room temperature steadily shake 2h, add mouse monoclonal antibody primary antibodie Anti-His(1:1000), at 4 ℃, spend the night, abandon the anti-IgG-HRP(1:5000 of sheep anti mouse two that primary antibodie adds horseradish peroxidase) (Beijing Tian Gen biochemical technology company limited), at room temperature steadily shake 2h, abandoning two resists, add the super quick luminous nitrite ion of Super ECL Plus, by chemoluminescence imager observations.The results are shown in Figure 3.
The albumen of Prokaryotic expression, purification is processed paddy rice, detects the generation of defense response
Be about the rice leaf of 2 weeks sizes of purifying FliC injection of 0.3mg/ml by the concentration that is dissolved in PBS.After 24h, the blade of clip treatment sites, with DAB(3,3 '-diaminobenzidine) dyeing processing, then with alcohol decolouring, take pictures under the microscope afterwards.As can be seen from Figure 4, detect and on paddy rice, produced H
2o
2, this shows that albumen energy inducing paddy rice generation anaphylaxis has excited the immune response of paddy rice inherence.
Claims (8)
1. an encoding gene for plant immunizing activities material, is characterized in that, described encoding gene has the nucleotide sequence as shown in SEQ ID NO.1.
2. encoding gene according to claim 1, is characterized in that, described encoding gene has the nucleotide sequence as shown in SEQ ID NO.2;
Preferably, the nucleotide sequence of described encoding gene is as shown in SEQ ID NO.1 or SEQ ID NO.2.
3. for an albumen for inducing paddy rice defense response, it is characterized in that, described albumen has the aminoacid sequence as shown in SEQ ID NO.7;
Preferably, the aminoacid sequence of described albumen is as shown in SEQ ID NO.7.
4. the preparation method of albumen according to claim 3, is characterized in that, described preparation method comprises the following steps:
(1) obtain encoding gene according to claim 1 and 2;
(2) use the encoding gene that step (1) obtains to build protein expression vector, and transformed intestinal bacteria;
(3) albumen described in the escherichia coli expression through transforming that induction step (2) obtains.
5. preparation method according to claim 4, is characterized in that, described step (1) comprising:
Take the genomic dna of oat acidophilic bacteria Acidovorax avenae CGMCC No.8661 as template, adopt the PCR method encoding gene according to claim 1 and 2 that increases.
6. according to the preparation method described in claim 4 or 5, it is characterized in that, described step (2) comprising:
Use restriction enzyme BamH I and Hind III double digestion step (1) obtains respectively encoding gene and carrier pET-28a, connect with T4 ligase enzyme the fragment obtaining afterwards, thereby build expression of recombinant proteins plasmid, be then transformed in competence coli strain.
7. according to the preparation method described in any one in claim 4 to 6, it is characterized in that, described step (3) comprising:
The e. coli bl21 through transforming obtaining with the IPTG inducing culture step (2) of 0.5mM, obtains the albumen of 51kD;
Preferably, the condition of described inducing culture is 10 ℃, rotating speed 150rpm.
Encoding gene according to claim 1 and 2 or according to claim 3 albumen in the purposes of preparing in plant immunization preparation.
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| KR20220066618A (en) * | 2020-11-16 | 2022-05-24 | 아주대학교산학협력단 | Toll-like receptor 1/2 and/or 4 Activating Peptides and Uses Thereof |
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Non-Patent Citations (4)
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| CHE,F.S.等: "GenBank Accession Number:AB040139.1", 《GENBANK》 * |
| FANG-SIK CHE等: "Flagellin from an Incompatible Strain of Pseudomonas avenae Induces a Resistance Response in Cultured Rice Cells", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
| 伍甜等: "燕麦嗜酸菌鞭毛素的提纯及其活性检测", 《生物技术通报》 * |
| 刘莹等: "鞭毛素蛋白云造蚤悦葬葬葬诱导水稻免疫反应的活性测定", 《植物保护学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20220066618A (en) * | 2020-11-16 | 2022-05-24 | 아주대학교산학협력단 | Toll-like receptor 1/2 and/or 4 Activating Peptides and Uses Thereof |
| KR102543157B1 (en) | 2020-11-16 | 2023-06-14 | 아주대학교산학협력단 | Toll-like receptor 1/2 and/or 4 Activating Peptides and Uses Thereof |
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Application publication date: 20140514 |