CN1284853C - Gene encoding protease inhibitor and encoded protein thereby - Google Patents
Gene encoding protease inhibitor and encoded protein thereby Download PDFInfo
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- CN1284853C CN1284853C CN 200510040452 CN200510040452A CN1284853C CN 1284853 C CN1284853 C CN 1284853C CN 200510040452 CN200510040452 CN 200510040452 CN 200510040452 A CN200510040452 A CN 200510040452A CN 1284853 C CN1284853 C CN 1284853C
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- dsti
- protease inhibitor
- descurainia
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Abstract
The present invention discloses a protease inhibitor gene cDNA sequence and an encoding protein sequence thereof, which belongs to the fields of gene engineering and protein engineering. The present invention relates to a cDNA sequence SEQID No. 1 of a descurainia protease inhibitor gene DsTI-1 and an encoding amino acid sequence SEQID No. 2. The gene of the present invention is reported for the first time in dicotyledonous plant descurainia and participates in an insect resistance reaction of descurainia. The mRNA expression analysis indicates that the gene is expressed by insect pest and harm induction. The transgene experiment proves that the overexpression of the gene can obviously enhance the insect resistance of rapes. The DsTI-1 can be used as a purpose gene to be led into plants so as to enhance insect resistance of the plants. The yeast expression products have the action of inhibiting the activity of proteinase, and the gene and the encoding protein can be applied to the fields of medicine and biochemistry.
Description
Technical field
The invention discloses a kind of protease inhibitor gene dna sequence dna and coded protein sequence thereof, belong to genetically engineered field and protein engineering field.Specifically, relate to plant and press down the relevant insect-resistance of gene, the key gene and the enzyme of resistance, also relate to the proteinase inhibitor production application in medical science, the biological chemistry with proteolytic enzyme.
Technical background
Protease inhibitor gene is generally accepted for people in the vital role of aspects such as plant resistance to insect, field of medicaments.It can be used to improve the insect-resistance of plant, makes farm crop reduce production loss more than 15%; Also can be used for the treatment of pancreatitis, antitumor, prevent and treat aspects such as cerebrovascular disease, cardiac surgery.
In the classification, proteolytic enzyme suppressor gene family can be divided into 4 classes, serpin gene, inhibitors of metalloproteinase gene, cystatin gene, asparaginic acid protease inhibitors gene (Ryan, 1992), mainly be distributed in 4 (Graminaceae of section, Leguminosae, Solanaceae, Crucifera) in.The proteolytic enzyme suppressor gene of Cruciferae belongs to the Serine type, has the different structure of proteolytic enzyme inhibition of gene expression proteolytic enzyme with non-Cruciferae species.The protease inhibitor gene of descurainia sophia (l.) webb ex prantl belongs to Cruciferae serpin gene.The proteinase inhibitor of Cruciferae can either suppress trypsinase, can suppress Chymotrypsin again.Retrieve authentic data storehouse NCBI (www.ncbi.nlm.nih.gov) as can be known: at present in the Cruciferae species, 9 protease inhibitor genes have been had been found that, they are respectively ATTI1-7, RTI, MTI-2, the patent that the gene of these similar sequences has been declared is (referring to document Clauss M.J., and Mitchell-Olds T., 2004.Functinal divergence in tandemly duplicated Arabodopsis thaliana trypsin inhibitorgenes.Genetics 166:1419-1436).
We have at first found protease inhibitor gene in this broadleaf weed plant descurainia sophia (l.) webb ex prantl.
Summary of the invention
Technical problem the objective of the invention is to disclose a kind of protease inhibitor gene and proteins encoded thereof, and this gene can be used as goal gene and imports plant from descurainia sophia (l.) webb ex prantl, improves insect-resistance, carries out plant species improvement; Also can be applied to protein engineering, produce the proteinase inhibitor goods.
Technical scheme
Protease inhibitor gene of the present invention (DsTI-1), its nucleotide sequence SEQ ID NO.1 is:
GAGAGAAGATGGTCATGGCAATGAAGTCTGTTTCTACCTTCGCCATCTTTTGCAT
TCTCTTTTTGGTTATCTTTGAAACGTCTGAGATAGAAGCGCAGCTGCAGGAAAG
ACAATGCCTCAAAGAATATGGCGGTGATGTTGGTTTCAGCTTTTGTGCACCTCG
AATATTCCCATCGTTCTGTGATCGGAACTGCCGTAACAACAAGGGGGCGAAAGG
TGGAATATGCCGTTGGGAACAGAACAACGCTATTGGTGTTAGATGCTTATGTGA
CTTTTGCGGAGAAGAGCCTTCTAATCTGATTCTAAGTCGTATTTGAATCTTGCAT
GTGTCATGGTTTATGTGATAAGAAAGGTAAAATGGTCCACGTAATGTTATAATAA
TGAAGTTAAATTAAGAATAACGAAACGCAAGACTTTGCTTGTGTGAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAA
Above-mentioned protease inhibitor gene (DsTI-1) is from descurainia sophia (l.) webb ex prantl, and its encoded protein matter from descurainia sophia (l.) webb ex prantl, has avtive spot ring sequence C APRIFP, also has N end secreting signal peptide, belongs to the precursor protein of proteinase inhibitor.
It has aminoacid sequence SEQ ID NO.2 as follows:
MVMAMKSVSTFAIFCILFLVIFETSEIEAQLQERQCLKEYGGDVGFSFCAPRIFPSFC
DRNCRNNKGAKGGICRWEQNNAIGVRCLCDFCGEEPSNLILSRI
Beneficial effect
1, the invention discloses a kind of protease inhibitor gene (DSTI-1 gene) and coded protein thereof.Protease inhibitor gene is the reported first in the dicotyledons descurainia sophia (l.) webb ex prantl, is expected to be applied to biological approach and produces proteinase inhibitor.This gene can be used as goal gene and imports plant from descurainia sophia (l.) webb ex prantl (Descurainia sophia web.), improves plant resistance to insect, to carry out plant species improvement.Coded protein has pest-resistant function.
2, the DSTI-1 gene function that provides of the inventor is the effect with arrestin enzymic activity.No matter the mRNA expression analysis shows the DSTI-1 gene the descurainia sophia (l.) webb ex prantl seedling, still growth, ripening stage, induced by insect pest, injury, adverse circumstance and expresses enhancing.
3, DSTI-1 gene of the present invention is from descurainia sophia (l.) webb ex prantl, has the optimizing codon that monocotyledonss such as being suitable for descurainia sophia (l.) webb ex prantl, rape, Arabidopis thaliana is expressed, its genetically engineered recipient plant is except monocotyledons, outside paddy rice, corn, wheat etc., be more suitable for waiting soybean, cotton, tobacco dicotyledons.
4, utilize DSTI-1 gene of the present invention to make up plant expression vector as goal gene, wherein available any promotor, for example cauliflower mosaic virus (CAMV) 35S promoter, Ubiquitin promotor or other promotor, can comprise enhanser in case of necessity in this expression vector, no matter be transcriptional enhancer or translational enhancer.Can use selected marker for the evaluation of simplifying transformant and comprise enzyme antibiotics resistance, also can utilize the enzyme of the compound that colour-change (for example B-glucuronidase GUS) or luminous (for example luciferase) discern, also available unmarked selection.Used expression vector can use Ti-plasmids, Ri plasmid, plant viral vector etc.Method for transformation can be used through agrobacterium-mediated transformation, particle bombardment, pollen tube passage method or other method and transform plant.
5, DSTI-1 gene coded protein not only can suppress tryptic activity, and can suppress the activity of Chymotrypsin, therefore also may use the method for protein engineering, produces the proteins encoded of DSTI-1 gene, is applied to aspects such as medical science, Biochemical Research.
Embodiment
Embodiment 1
Select ruderal plant descurainia sophia (l.) webb ex prantl " tx-1 " (weeds in field is collected in rural area, Taixing City, Jiangsu, called after tx-1) for use.Select ripening stage angle fruit for use, adopt SMART technique construction cDNA total length library.Building process is: extract the total RNA of descurainia sophia (l.) webb ex prantl angle fruit, obtain mRNA through Oligotex test kit purifying.Utilization contains the synthetic cDNA article one chain of CDS/3 ' PCR primer of Sfi B restriction enzyme site, and contains the template that the SMART oligonucleotide of Sfi A restriction enzyme site extends out at mRNA 5 ' end as cDNA article one chain.By the LD-PCR synthetic double chain cDNA, through protease K digesting, Sfi (A﹠amp; B) enzyme is cut, is crossed CHROMA PIN 400 post fractional separation and removes the following small molecules of 400bp, is connected on the λ TriplEx2 carrier, packs, and promptly obtains the original library of fruit cDNA, descurainia sophia (l.) webb ex prantl angle.Intestinal bacteria XL1-Blue plate is measured and the PCR Rapid identification shows: the original titre in this library is 1.49 * 10
6Pfu/ml, recombination fraction are 91.24%, and library, amplification back titre is 1.15 * 10
9Pfu/ml.10 plaques of picking at random, pcr amplification inserts fragment, and its length illustrate that the library quality that makes up is higher between 800bp～2kb, can satisfy the needs that the ESTs library construction reaches research work such as from library separation screening important gene.So far successfully made up fruit cDNA library, first descurainia sophia (l.) webb ex prantl angle, for the excellent gene clone of descurainia sophia (l.) webb ex prantl and the degeneration-resistant molecule mechanism of systematic study descurainia sophia (l.) webb ex prantl are laid a good foundation.
The a large amount of positive plaques of picking through being converted to plasmid, adopt T7 promotor primer to check order at random.From a large amount of cDNA full length sequence sequencing results, found protease inhibitor gene.The sequence of complete of this gene comprises coding region and non-coding region.The gene that the result of BLAST and molecule modeling result proof newly obtain from descurainia sophia (l.) webb ex prantl really is a protease inhibitor gene.This gene encoding production has arrestin enzyme active sites ring, has pest-resistant function.
Embodiment 2
The cDNA sequence SEQ ID NO.1 of above-mentioned acquisition descurainia sophia (l.) webb ex prantl protease inhibitor gene DsTI-1, its encoded protein matter, from descurainia sophia (l.) webb ex prantl (Descurainia sophia), its aminoacid sequence is SEQ ID NO.2, through in database relatively its proteins encoded and protease inhibitor gene Arabidopis thaliana ATTI2, sinapsis alba MTI-2 in the database, the amino acid similarity of rape RTI be respectively 63%, 59%, 61%, with the similarity of ATTI1, AATI3-7 less than 60%.(seeing SEQ ID NO.2).The signal peptide sequence of the N end of DsTI-1 shows that DsTI-1 is positioned on the endoplasmic reticulum, is consistent with sinapsis alba, rape, Arabidopis thaliana conclusion.
Embodiment 3
Different tissues by descurainia sophia (l.) webb ex prantl extracts RND (RNApure kit), and plate, pcr amplification are touched in conduct through reverse transcription cDNA.With DsTI-1 cDNA sequence in the example 1, design primer P1, P2:
P1：5’-TCTTTCTACCTTCGCCATCT-3’
P2：5’-CAAAGTCTTGCGTTTCGTTA-3’
PCR reaction conditions: 10 * PCR buffer, 5 μ l; 2mM dNTP 1.5 μ l; Primer 1 μ l; Touch plate, 2 μ l; The TaqDNA polysaccharase, 1 μ l; The ddH that adds sterilization at last
2O is to the 50 μ l that reach of cumulative volume; PCR response procedures: 94 ℃ and pre-sex change 5min; Denaturation temperature is 94 ℃, time 50s, 53 ℃ of renaturation temperature, time 50s, 72 ℃ of elongating temperatures, time 50s, 33 circulations; 72 ℃ are extended 10min again.
Sxemiquantitative RT-PCR analyzes the expression of descurainia sophia (l.) webb ex prantl overground part, and the result shows that the descurainia sophia (l.) webb ex prantl overground part is under insect pest, injury, adverse circumstance (arid) inductive condition, and 6h significantly strengthens expression, and through the gray scale scanning analysis, its mRNA expression amount is more than 3 times of contrast.The expression that DsTI-1 is described and insect pest, environment stress are relevant.In angle fruit and seed, express under this gene normal circumstances.
Experimental example 4
The full length sequence of the DsTI-1 that obtains according to embodiment 1 uses constitutive promoter 35S, makes up the carrier that transforms the DsTI-1 gene, and the gene here is to have removed N end signal peptide and 7 amino acid whose purpose fragments of C end.
Have Sac1 below the design respectively, a pair of primer of BamH1 restriction enzyme site is used for removing N end signal peptide and 8 amino acid of C end of DsTI-1 gene.Downstream primer sports TAA with TCT and produces a terminator.
Upstream primer 5---GAgagctcATGGTCATGGCAATGAAGT---3
Downstream primer 5---ATggatccTTAAGGCTCTTTCTAAGCAA---3
Utilize above-mentioned primer to carry out pcr amplification, PCR product electrophoresis is reclaimed, use Sac1, two kinds of restriction enzymes of BamH1 are the gene fragment and the carrier Pcam-BIA1301 that cut back to close of enzyme respectively, the target gene fragment of waving and linear carrier are connected linear carrier with the T4 dna ligase with target gene fragment under 16 ℃; Transformed into escherichia coli JM109 competent cell, on the substratum of LB native land, (contain the kam microbiotic) and be coated with lithographic plate, the resistance bacterium colony that obtains, the several bacterium colonies of picking are through liquid LB culture medium culturing, do the PCR preliminary evaluation respectively, extract plasmid, use Sac1, the BamH1 double digestion, carry out enzyme and cut evaluation, obtain positive colony.Transforming Agrobacterium LBA4404, and carrying out enzyme and cut evaluation, the positive colony of acquisition is the Agrobacterium of carrying the foreign gene plasmid and is used for transforming.Agrobacterium-mediated transformation transforms rape (two low kinds, two No. 3 of China), and the result shows: in spire, proteinase inhibitor content has improved more than 2 times, and the content in the old blade improves more.
Experimental example 5:
According to the gene order of experiment 4, transformed yeast DS115 carries out expression study.Through experiments such as vector construction, transformant screening, SDS-PAGE, proteic purification, determinations of activity, prove that this gene not only can suppress the activity of bovine trypsin, and can suppress the activity of Chymotrypsin that the dissociation equilibrium constant is 2 * 10
-10With 4.3 * 10
-7, similar with the RTI and the sinapsis alba MTI-2 of rape.Therefore, according to the mechanism of action of protease inhibitor gene, can think that the DsTI-1 gene has good insect-resistance, its encoded protein also may be used at aspects such as medical science as proteinase inhibitor.
The inventor has cloned a cDNA from dicotyledons descurainia sophia (l.) webb ex prantl (Descurainia sophia), proteins encoded enzyme inhibitor gene, called after DsTI-1.The aminoacid sequence of proteins encoded through compare it in database and Arabidopis thaliana, sinapsis alba, rape has similarity, is respectively 63%, 59%, 61%.DsTI-1 cDNA total length 456bp contains the ORF of 309bp, and coding contains 102 amino acid whose protein, and 5 '-UTR is 8bp, and 3 '-UTR is 139bp.The N end signal peptide sequence of DsTI-1 proteins encoded shows that it is positioned on the endoplasmic reticulum.
The mRNA expression analysis shows that descurainia sophia (l.) webb ex prantl is being subjected to DsTI-1 genetic expression enhancing under insect pest, injury, the adverse environmental factor, and the overground part expression amount is more than 3 times of contrast.Transgenic research shows, changes the DsTI-1 gene over to rape, the similar contrast of transfer-gen plant growing way under normal operation, and also protease inhibitory activity is high more than 2 times, shows to have significantly improved insect-resistance.This gene can be used as goal gene and imports plant, improves plant resistance to insect, to carry out plant species improvement.
In sum, the DsTI-1 gene that the inventor provides is an isolating new gene in the dicotyledons descurainia sophia (l.) webb ex prantl first, and its function is to produce proteinase inhibitor, improves plant resistance to insect.Can utilize DsTI-1 gene of the present invention to make up plant expression vector as goal gene, wherein available any promotor is cauliflower mosaic virus (CAMV) 35S promoter, Ubiquitin promotor or other promotor for example, can comprise enhanser in case of necessity in this expression vector, no matter be transcriptional enhancer or translational enhancer.Can use selected marker for the evaluation of simplifying transformant and comprise enzyme antibiotics resistance, also can utilize the enzyme of the compound that colour-change (for example B-glucuronidase GUS) or luminous (for example luciferase) discern, also available unmarked selection.Used expression vector can use Ti-plasmids, Ri plasmid, plant viral vector etc.Method for transformation can be used through agrobacterium-mediated transformation, particle bombardment, pollen tube passage method or other method and transform plant.
DsTI-1 gene of the present invention is from descurainia sophia (l.) webb ex prantl, have and be suitable for the optimizing codon that cress is expressed, its genetically engineered recipient plant outside soybean, cotton, tobacco etc., also is suitable for monocotyledonss such as paddy rice, corn, wheat except dicotyledons.The proteins encoded of a kind of plant protease inhibitor gene of the present invention also is suitable for producing proteinase inhibitor.
Sequence that the present invention relates to and mark apportion are as follows:
(1) information of SEQ ID NO.1
(i) sequence signature:
(A) length: 456bp
(B) type: Nucleotide
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: Nucleotide
(iii) sequence description: SEQ ID NO.1
(2) information of SEQ IDNO.2
(i) sequence signature:
(A) length: 102a.a
(B) type: amino acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: protein
(iii) sequence description: SEQ ID NO.2
Sequence table
＜110〉Agricultural University Of Nanjing
＜120〉a kind of protease inhibitor gene and coded protein thereof
<140>2005100404523
<141>2005-06-09
<160>2
<170>PatentIn?version?3.1
<210>SEQ?ID?NO.1
<211>456
<212>DNA
＜213〉descurainia sophia (l.) webb ex prantl (Descurainia sophia)
<221>CDS
<222>(9)..(317)
<221>polyA_site
<222>(427)..(456)
<221>5’UTR
<222>(1)..(8)
<221>mRNA
<222>(1)..(456)
<221>3’UTR
<222>(318)..(456)
<400>1
gagagaag?atg?gtc?atg?gca?atg?aag?tct?gtt?tct?acc?ttc?gcc?atc?ttt 50
Met?Val?Met?Ala?Met?Lys?Ser?Val?Ser?Thr?Phe?Ala?Ile?Phe
1 5 10
tgc?att?ctc?ttt?ttg?gtt?atc?ttt?gaa?acg?tct?gag?ata?gaa?gcg?cag 98
Cys?Ile?Leu?Phe?Leu?Val?Ile?Phe?Glu?Thr?Ser?Glu?Ile?Glu?Ala?Gln
15 20 25 30
ctg?cag?gaa?aga?caa?tgc?ctc?aaa?gaa?tat?ggc?ggt?gat?gtt?ggt?ttc 146
Leu?Gln?Glu?Arg?Gln?Cys?Leu?Lys?Glu?Tyr?Gly?Gly?Asp?Val?Gly?Phe
35 40 45
agc?ttt?tgt?gca?cct?cga?ata?ttc?cca?tcg?ttc?tgt?gat?cgg?aac?tgc 194
Ser?Phe?Cys?Ala?Pro?Arg?Ile?Phe?Pro?Ser?Phe?Cys?Asp?Arg?Asn?Cys
50 55 60
cgt?aac?aac?aag?ggg?gcg?aaa?ggt?gga?ata?tgc?cgt?tgg?gaa?cag?aac 242
Arg?Asn?Asn?Lys?Gly?Ala?Lys?Gly?Gly?Ile?Cys?Arg?Trp?Glu?Gln?Asn
65 70 75
aac?gct?att?ggt?gtt?aga?tgc?tta?tgt?gac?ttt?tgc?gga?gaa?gag?cct 290
Asn?Ala?Ile?Gly?Val?Arg?Cys?Leu?Cys?Asp?Phe?Cys?Gly?Glu?Glu?Pro
80 85 90
tct?aat?ctg?att?cta?agt?cgt?att?tgaatcttgc?atgtgtcatg?gtttatgtga 344
Ser?Ash?Leu?Ile?Leu?Ser?Arg?Ile
95 100
taagaaaggt?aaaatggtcc?acgtaatgtt?ataataatga?agttaaatta?agaataacga 404
aacgcaagac?tttgcttgtg?tgaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa?aa 456
<210>SEQ?ID?NO.2
<211>102
<212>PRT
＜213〉descurainia sophia (l.) webb ex prantl (Descurainia Sophia)
<400>2
Met?Val?Met?Ala?Met?Lys?Ser?Val?Ser?Thr?Phe?Ala?Ile?Phe?Cys?Ile
1 5 10 15
Leu?Phe?Leu?Val?Ile?Phe?Glu?Thr?Ser?Glu?Ile?Glu?Ala?Gln?Leu?Gln
20 25 30
Glu?Arg?Gln?Cys?Leu?Lys?Glu?Tyr?Gly?Gly?Asp?Val?Gly?Phe?Ser?Phe
35 40 45
Cys?Ala?Pro?Arg?Ile?Phe?Pro?Ser?Phe?Cys?Asp?Arg?Asn?Cys?Arg?Ash
50 55 60
Asn?Lys?Gly?Ala?Lys?Gly?Gly?Ile?Cys?Arg?Trp?Glu?Gln?Asn?Asn?Ala
65 70 75 80
Ile?Gly?Val?Arg?Cys?Leu?Cys?Asp?Phe?Cys?Gly?Glu?Glu?Pro?Ser?Asn
85 90 95
Leu?Ile?Leu?Ser?Arg?Ile
100
Claims (2)
1. protease inhibitor gene, from descurainia sophia (l.) webb ex prantl (Descurainia sophia), called after DsTI-1 gene, its nucleotide sequence SEQ ID NO.1 is:
GAGAGAAGATGGTCATGGCAATGAAGTCTGTTTCTACCTTCGCCATCTTTTGCATTCTCTTTTTGGTTATCTTTGAAACGTCTGAGATAGAAGCGCAGCTGCAGGAAAGACAATGCCTCAAAGAATATGGCGGTGATGTTGGTTTCAGCTTTTGTGCACCTCGAATATTCCCATCGTTCTGTGATCGGAACTGCCGTAACAACAAGGGGGCGAAAGGTGGAATATGCCGTTGGGAACAGAACAACGCTATTGGTGTTAGATGCTTATGTGACTTTTGCGGAGAAGAGCCTTCTAATCTGATTCTAAGTCGTATTTGAATCTTGCATGTGTCATGGTTTATGTGATAAGAAAGGTAAAATGGTCCACGTAATGTTATAATAATGAAGTTAAATTAAGAATAACGAAACGCAAGACTTTGCTTGTGTGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA。
2. the described a kind of protease inhibitor gene of claim 1, from descurainia sophia (l.) webb ex prantl (Descurainia sophia), called after DsTI-1 gene, its coded protein sequence SEQ ID NO.2 is:
MVMAMKSVSTFAIFCILFLVIFETSEIEAQLQERQCLKEYGGDVGFSFCAPRIFPSFCDRNCRNNKGAKGGICRWEQNNAIGVRCLCDFCGEEPSNLILSRI。
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|---|---|---|---|
| CN 200510040452 CN1284853C (en) | 2005-06-09 | 2005-06-09 | Gene encoding protease inhibitor and encoded protein thereby |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510040452 CN1284853C (en) | 2005-06-09 | 2005-06-09 | Gene encoding protease inhibitor and encoded protein thereby |
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| Publication Number | Publication Date |
|---|---|
| CN1721537A CN1721537A (en) | 2006-01-18 |
| CN1284853C true CN1284853C (en) | 2006-11-15 |
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ID=35912144
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| CN 200510040452 Expired - Fee Related CN1284853C (en) | 2005-06-09 | 2005-06-09 | Gene encoding protease inhibitor and encoded protein thereby |
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| Country | Link |
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Granted publication date: 20061115 Termination date: 20100609 |