CN105624173B - The unknown membrane protein gene OsMP1 of one rice and its application in resistant gene engineering - Google Patents
The unknown membrane protein gene OsMP1 of one rice and its application in resistant gene engineering Download PDFInfo
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- CN105624173B CN105624173B CN201610151567.8A CN201610151567A CN105624173B CN 105624173 B CN105624173 B CN 105624173B CN 201610151567 A CN201610151567 A CN 201610151567A CN 105624173 B CN105624173 B CN 105624173B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
- C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
Abstract
The invention belongs to genetic engineering fields, are related to the unknown membrane protein gene OsMP1 of a rice and its application in resistant gene engineering.Paddy gene OsMP1, the cDNA sequence of the gene is as shown in SEQ ID NO.1, and the rice memebrane protein of coding, amino acid sequence is as shown in SEQ ID NO.2.The invention discloses rice membrane protein gene OsMP1 and its encoded protein.Rice membrane protein gene OsMP1 is to report for the first time in rice, is according to genetic chip and iTRAQ protein science before and after analysis Heikezijing inoculation Pyricularia oryzae as a result, one screened is obviously by the unknown membrane protein gene of rice blast inoculation inducing expression.MRNA expression analysis shows that the gene is inoculated with by Pyricularia oryzae and induces, and the seedling pest resistance of transgenic rice plant is enhanced.Therefore it is expected to can be used as target gene importing plant, disease resistance of plant is improved, to carry out plant species improvement.
Description
Technical field
The invention belongs to genetic engineering field, it is related to a kind of unknown membrane protein gene OsMP1 of rice and its in disease-resistant gene
Application in engineering.
Background technique
Plasmalemma of plant albumen is located at cell and extraneous intersection, is capable of the signal transduction of mediated cell and the external world, executes
The substantially important biological function of cell.Plasmalemma protein is resisted pathogen and is infected in defense response signal transduction path and rises in plant
Very important effect is arrived.
The OsMP1 that the present invention clones is the unknown plasmalemma protein of rice, and expression is obviously inoculated with by Pyricularia oryzae and is induced, turn
The anti-pest of gene plant are remarkably reinforced.It studies the function of OsMP1 and it is applied in resistant gene engineering, to raising
Rice anti-rice blast resistance has great significance.
Summary of the invention
It is an object of the invention to the open unknown membrane protein gene OsMP1 of rice.
It is a further object of the present invention to provide the disease resistence gene engineer applications of gene OsMP1.
The purpose of the present invention can be achieved through the following technical solutions:
Paddy gene OsMP1, the cDNA sequence of the gene is as shown in SEQ ID NO.1.
The rice memebrane protein of paddy gene OsMP1 coding of the present invention, amino acid sequence such as SEQ ID NO.2 institute
Show.
Expression vector containing the paddy gene OsMP1.
The expression vector is preferably by I enzyme of Spe of the paddy gene OsMP1 insertion expression vector Ubi-pBA002
Obtained by enzyme site.
The genetic engineering application of paddy gene OsMP1 of the present invention.
Rice membrane protein gene OsMP1 of the present invention is cultivated in rice blast resistance kind by genetic engineering means
Application.
Rice blast resistance kind is being cultivated by genetic engineering means using the rice membrane protein gene OsMP1, is being led to
Often first with OsMP1 gene of the present invention gene constructed plant expression vector as a purpose, then the recombinant expression containing the gene carried
Body converts target plant.
Using OsMP1 gene of the present invention gene constructed plant expression vector as a purpose, wherein any starting can be used
Sub such as cauliflower mosaic virus (CAMV) 35S promoter, Ubiquitin promoter or other promoters, in the expression vector
It when necessary may include enhancer, whether transcriptional enhancer or translational enhancer.It can be used to simplify the identification of transformed cells
Selected marker includes the enzyme with antibiotic resistance, can also be used color change (such as β-glucuronidase GUS) or
(such as luciferase) is shone come the enzyme of the compound identified, it is also possible to unmarked selection.Expression vector used can be used
Ti-plasmids, Ri plasmid, plant viral vector etc..Method for transformation can with agrobacterium-mediated transformation, particle bombardment, pollen tube passage method or
Other methods convert plant.
The genetic engineering application of rice membrane protein gene OsMP1, preferably includes following steps:
1) rice anti-rice blast bacterium kind " Heikezijing " total serum IgE is extracted
2) clone of paddy gene OsMP1
The total serum IgE reverse transcription that step 1) is obtained synthesizes the first chain of cDNA, as template, P1:SEQ ID NO.3 and
P2:SEQ ID NO.4 is primer, and pEASY-T1Blunt carrier is connected to after carrying out PCR amplification with high fidelity enzyme, and sequencing obtains
The cDNA sequence SEQ ID NO.1 of paddy gene OsMP1；
3) building of plant expression vector
According to the cDNA sequence SEQ ID NO.1 of rice Os MP1 gene, design amplifies drawing for complete coding reading frame
Object, and same introducing restriction endonuclease sites Spe I, P3 and P4 primer sequence are distinguished on upstream primer P3 and downstream primer P4
Column are respectively as follows: SEQ ID NO.5 and SEQ ID NO.6；Using the pcr amplification product obtained in step 2) as template, through PCR amplification
Afterwards, OsMP1 is cloned into expression by recombination method and carried by the cDNA of OsMP1 using the restriction endonuclease sites Spe I introduced
Body Ubi-pBA002, sequencing identification ensure that code area reading frame is correct in expression vector；
4) acquisition of transgenic plant
The expression vector Ubi-pBA002-OsMP1 that step 3) obtains is transferred to Agrobacterium, is further transferred to rice sense rice blast
Germ kind " Suyunuo ", the disease resistance for carry out after real time PCR is verified rice to the transgenic plant of acquisition are commented
Valence obtains Varieties Resistant To Rice Blast.
Beneficial effect
1, the invention discloses rice membrane protein gene OsMP1 and its encoded protein.Rice membrane protein gene
OsMP1 is to report for the first time in rice, and Heikezijing is inoculated with gene microarray analysis, iTRAQ protein science result before and after Pyricularia oryzae
Show that the gene is inoculated with obvious induction by Pyricularia oryzae with mRNA expression analysis.Therefore being expected to, which can be used as target gene and import, plants
Object improves disease resistance of plant, to carry out plant species improvement.
2, OsMP1 gene of the invention comes from rice, has the optimization password for being suitable for the expression of the monocotyledons such as rice
Son, genetic engineering recipient plant are more suitable for rice, jade except dicotyledon, such as soybean, cotton, tobacco
The monocotyledons such as rice, wheat.
3, construction of expression vector obtains transgenic plant, and the transgenic plant resistance for obtaining overexpression is remarkably reinforced, and
The resistance that point mutation knocks out the transgenic plant of expression obviously weakens, and illustrates that the gene is related with Rice Resistance To Rice Blast, has
Genetic engineering application value.
4, using DUALmembrane system, the interaction egg of an OsMP1 has been screened in Heikezijing cDNA library
White MP-P1 has SNARE_assoc structural domain, belongs to the relevant Golgi protein family of SNARE, contains SNARE structural domain
Albumen can play an important role in plant defense response.
Detailed description of the invention
The subcellular localization of Fig. 1, OsMP1 albumen the result shows that, be primarily targeted for rice plasma membrane.
The yeast two-hybrid result of Fig. 2, OsMP1 albumen.
Specific embodiment
Embodiment 1
1) it (is blast resisting for Taihu Lake basin japonica rice local varieties that rice varieties " Heikezijing " is selected in the extraction of total serum IgE
Bacterium kind), after rice seedling length to 3-4 leaf phase, inoculation processing is carried out with magnaporthe grisea spore, takes 0h, 4h after processing respectively,
8h, 12h, for 24 hours, the blade of 48h uses liquid nitrogen frozen immediately, is stored in -80 DEG C of refrigerators.Partial blade is taken respectively, is ground with mortar
It is broken, it is transferred to the 1.5mL EP pipe (TRIzol Reagents is purchased from Invitrogen, USA) for filling Trizol lysate, is filled
After dividing oscillation, extracted total RNA, electroresis appraisal total serum IgE quality.
2) the proteomics result before and after the clonal analysis Heikezijing inoculation rice blast of rice membrane protein gene OsMP1
It was found that the obvious induction that the memebrane protein is inoculated with by Pyricularia oryzae.Using OryzasativaLcv.Nipponbare gene OsMP1 sequence as probe sequence, water is searched for
Rice genome sequence and est sequence database, splicing obtains the full length sequence of 1408bp rice membrane protein gene OsMP1, and sets
Count both ends primer P1 and P2:
P1:5-CACGCCTTGCTATTCCTGTA-3 (SEQ ID NO.3),
P2:5-GATTACTCTAACCCGCCCATA-3 (SEQ ID NO.4),
The first chain of cDNA of the total serum IgE reverse transcription synthesis obtained using step 1) is template, using P1 and P2 as primer, is protected with height
True enzyme (being purchased from Roche) carries out PCR amplification, and PCR program is as follows: 95 DEG C of initial denaturation 5min, 95 DEG C of denaturation 40sec, 56 DEG C of renaturation
40sec, 72 DEG C of extension 90sec are connected to pEASY- after 72 DEG C of extensions 5min, last 72 DEG C of extensions 10min after 30 recycle
T1Blunt carrier, the sequencing of commission Nanjing Si Pu King Company obtain the cDNA sequence SEQ ID NO.1 of membrane protein gene OsMP1.
Analyze the cDNA sequence SEQ ID NO.1 and its coding Protein S EQ of the rice membrane protein gene OsMP1 of above-mentioned acquisition
ID NO.2。
Embodiment 2
The rice 3-4 leaf phase after carrying out real-time quantitative RT-PCR analysis inoculation processing with primer P1, the P2 designed in embodiment 1
The expression of overground part seedling, the results showed that, the expression of OsMP1 is remarkably reinforced after inoculation magnaporthe grisea spore 4 hours, shows
The expression of OsMP1 gene is related with Pyricularia oryzae processing.
Embodiment 3
The cDNA full length sequence (see SEQ ID NO.1) of the OsMP1 obtained according to embodiment 1, design amplify complete volume
The primer of code reading frame, and restriction endonuclease sites Spe I, P3 and P4 are introduced on upstream primer P3 and downstream primer P4
Primer sequence are as follows:
P3:5-CTACGCGTTTAATTAACTAGTCACGCCTTGCTATTCCTGTA-3 (SEQ ID NO.5),
P4:5‐GGGAAATTCGAGCTCACTAGTGATTACTCTAACCCGCCCATA‐3(SEQ ID NO.6),
Using the amplified production obtained in embodiment 1 as template, using P3 and P4 as primer, after PCR amplification, recombination method is utilized
The cDNA of OsMP1 is connected in expression vector Ubi-pBA002 (purchase is in Wuhan Miao Ling Biotechnology Co., Ltd), sequencing
Identification ensures that code area reading frame is correct in expression vector, then is transferred to Agrobacterium, is further transferred to rice sense rice blast
Bacterium kind " Suyunuo " (Taihu Lake basin japonica rice local varieties).Real time PCR verifying is carried out to the transgenic plant of acquisition
The disease resistance evaluation of rice is carried out afterwards.The transgenosis T1 for growing to the 3-4 leaf phase is carried out at magnaporthe grisea spore inoculation for plant
Reason, the average sick series that plant is investigated after 7 days is 2.7 grades, and incidence of leaf is averaged scab number as 6.7 and scab average length
For 0.86mm, and the average sick series of adjoining tree is 7 grades and incidence of leaf be averaged scab number is 38 and scab is averaged
Length is 4.60mm, the results showed that, transgenic plant shows the obvious resistance to Pyricularia oryzae compared with the control.
Embodiment 4
Point mutation carrier is constructed according to using CH system (purchase is in Wuhan Miao Ling Biotechnology Co., Ltd), according to
CRISPR experimental design normal process analyzes target gene sequence, design primer P5 and P6, primer sequence are as follows:
P5:5-TGGCCAAGTTCACCTCTGCAACGG-3 (SEQ ID NO.7),
P6:5‐AAACCCGTTGCAGAGGTGAACTTG‐3(SEQ ID NO.8)
The Target sequence that clip size is 20bp is obtained through PCR amplification, target fragment (Target) is connected to CH and is carried
On body (Cas9n+chimeric guide RNA containing+85nt of tracrRNA), CH-OsMP1 carrier is constructed.
It after EcoRI and HindIII difference digestion CH-Target carrier and pCAMBIA1300 carrier, connects, obtains through T4DNA ligase
P1300-CH-OsMP1 carrier.Sequencing identification ensures that code area reading frame is correct in expression vector, then is transferred to Agrobacterium,
Further it is transferred to rice anti-rice blast bacterium kind " Heikezijing " (Taihu Lake basin japonica rice local varieties).The transgenosis of acquisition is planted
Strain carries out the disease resistance evaluation of progress rice after real time PCR verifying.The transgenosis T1 of 3-4 leaf phase will be grown to for plant
Magnaporthe grisea spore inoculation processing is carried out, the average sick series that plant is investigated after 7 days is 3.8 grades, and incidence of leaf is averaged scab number
Mesh is 5 and scab average length is 1.2mm, and the average sick series of adjoining tree is 0 grade of disease-free spot, the results showed that, turn base
Because plant shows to weaken Pyricularia oryzae resistance compared with the control.
In conclusion the OsMP1 gene that the present inventor provides is the new gene separated in rice, function and Rice Resistance
Characteristic of disease is related, can be used as target gene and imports plant, disease resistance of plant is improved, to carry out plant species improvement.Using this hair
Bright OsMP1 gene gene constructed plant expression vector as a purpose, wherein any promoter such as Cauliflower Mosaic can be used
Viral (CAMV) 35S promoter, Ubiquitin promoter or other promoters may include enhancing in the expression vector when necessary
Son, whether transcriptional enhancer or translational enhancer.It include pair to simplify the identification of transformed cells selected marker can be used
The enzyme of antibiotic resistance can also be used color change (such as β-glucuronidase GUS) or shine (such as luciferase)
Enzyme come the compound identified, it is also possible to unmarked selection.Ti-plasmids, Ri plasmid, plant can be used in expression vector used
Viral vectors etc..Method for transformation can be planted with converting through agrobacterium-mediated transformation, particle bombardment, pollen tube passage method or other methods
Object.
Claims (2)
1. paddy gene OsMP1 is cultivating the application in rice blast resistance kind, the rice base by genetic engineering means
Because the cDNA sequence of OsMP1 is as shown in SEQ ID NO. 1.
2. the application according to claim 1, it is characterised in that include the following steps:
1) rice anti-rice blast bacterium kind " Heikezijing " total serum IgE is extracted
2) clone of paddy gene OsMP1
The total serum IgE reverse transcription that step 1) is obtained synthesizes the first chain of cDNA, as template, P1: SEQ ID NO.3 and P2:
SEQ ID NO.4 is primer, and pEASY-T1 Blunt carrier is connected to after carrying out PCR amplification with high fidelity enzyme, and sequencing obtains water
The cDNA sequence SEQ ID NO. 1 of rice gene OsMP1；
3) building of plant expression vector
According to the cDNA sequence SEQ ID NO. 1 of rice Os MP1 gene, design amplifies the primer of complete coding reading frame,
And same introducing restriction endonuclease sites Spe I, P3 and P4 primer sequence are distinguished on upstream primer P3 and downstream primer P4
Column are respectively as follows: SEQ ID NO.5 and SEQ ID NO.6；Using the PCR amplified production obtained in step 2 as template, through PCR
After amplification, by the cDNA of OsMP1 using the restriction endonuclease sites Spe I introduced, OsMP1 is cloned by table by recombination method
Up to carrier Ubi-pBA002, sequencing identification ensures that code area reading frame is correct in expression vector；
4) acquisition of transgenic plant
The expression vector Ubi-pBA002-OsMP1 that step 3) obtains is transferred to Agrobacterium, is further transferred to rice sense Pyricularia oryzae
Kind " Suyunuo " obtains Varieties Resistant To Rice Blast.
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Non-Patent Citations (7)
| Title |
|---|
| Identification of quantitative trait loci conferring blast resistance in Bodao, a japonica rice landrace;J Huan et al.;《Genetics and Molecular Research》;20141127;第13卷(第4期);第9756-9765页 |
| Oryza sativa Japonica Group cDNA clone:J023073J15, full insert sequence，Accession number：AK070844.1;Kikuchi S et al.;《Genbank》;20081204;第1-2页 |
| OsNPSN11转基因水稻抗稻瘟病蛋白质组学分析及其产物互作蛋白的鉴定;党亮亮;《中国优秀硕士学位论文全文数据库 农业科技辑》;20150315(第3期);第D046-57页 |
| Recent progress and understanding of the molecular mechanisms of the rice–Magnaporthe oryzae interaction;JINLING LIU et al.;《Molecular plant pathology》;20101231;第11卷(第3期);第419-427页 |
| unnamed protein product [Oryza sativa Japonica Group]，Accession number：BAG92168.1;Kikuchi S et al.;《Genbank》;20081204;第1-2页 |
| 利用三个抗性鉴定指标对黑壳子粳和苏御糯RIL群体进行抗稻瘟病性QTL定位;曾国应;《中国优秀硕士学位论文全文数据库 农业科技辑》;20150315(第3期);第D047-23页 |
| 水稻与稻瘟病菌互作机制研究进展;张红生等;《南京农业大学学报》;20121231;第35卷(第5期);第1-8页 |
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