CN105543341A - Detection and analysis method of hickory nut RCA gene clone - Google Patents
Detection and analysis method of hickory nut RCA gene clone Download PDFInfo
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Abstract
The invention discloses a detection and analysis method of hickory nut RCA gene clone. The method comprises the following steps: 1, sequence retrieval and primer designing; 2, RCA gene intermediate fragment amplification; 3, RCA gene 3' end and 5' end sequence amplification; 4, CcRCAalpha and CcRCAbeta cDNA sequence characteristic analysis; 5, RCA genome sequence analysis; 6, hickory nut RCA phylogenetic tree analysis; and 7, analysis of RCA expression in hickory nut blades in different growth stages. RACE and RT-PCR technologies are used to clone in order to obtain two full-length genomes of hickory nut, bioinformatics analysis is combined to analyze differences among a base sequence, an amino acid coding sequence and other characteristics, and the expression mode of the RCA genes is searched according to the RCA gene expression level in the blades in different growth stages, so the method lays foundation for increase of the photosynthetic rate and improvement of the stress resistance by regulating the RCA through a gene engineering means.
Description
[technical field]
The present invention relates to bionic technical field, the technical field of the determination method of particularly a kind of Semen Caryae Cathayensis RCA gene clone.
[background technology]
Hickory Juglandaceae hickory plant, about has 20 kinds, and wherein 4 kinds originate in China, originate in W. Asia and western part of Asia Han dynasty imports China into, breeds domestic variety with external complete difference through centuries.Basic subtropical zone has juglandaceae plant to distribute in the world, and kind is hundreds of.There is distribution domestic various places, and northeast is with autumn (wild) in the majority North China, and also there is distribution northwest.
Semen Caryae Cathayensis when carrying out photosynthesis, the catalysis of committed step fixed co2 process need Rubisco enzyme.The activity of Rubisco enzyme is regulated and controled by mechanism complicated in plant materials, and wherein large quantity research has confirmed that Rubiscoactivase (RCA) can activate and maintain the catalytic activity of Rubisco enzyme.Simultaneously RCA is also considered to plant and coerces in various varying environment lower to photosynthetic key regulatory factor, Mate etc. find that expression of plants low-level RCA can cause the minimizing of nitrogen accumulation and total biomass, and RCA expresses the individuality of seldom or not expressing and then cannot survive under atmospheric carbon dioxide concentration simultaneously.Therefore, correlative study improves CO2 specific absorption by RCA regulation and control finally to improve crop yield significant.
[summary of the invention]
Object of the present invention solves the problems of the prior art exactly, the determination method of a kind of Semen Caryae Cathayensis RCA gene clone is proposed, the present invention utilizes RACE and RT-PCR technology, clone obtains Semen Caryae Cathayensis 2 RCA full length genes, in conjunction with bioinformatic analysis, analyze at base sequence, encoding amino acid sequence and other property differences, and the expression pattern of RCA gene is sought by the RCA expression conditions of different development stage blade, regulate and control RCA for utilizing genetic engineering means and lay the first stone to increase photosynthetic rate and to improve resistance.
For achieving the above object, the determination method of a kind of Semen Caryae Cathayensis RCA of the present invention gene clone, is characterized in that, comprise the following steps:
A) sequence retrieval and design of primers: by clone and comparison confirms that Semen Caryae Cathayensis exists 2 RCA genes, utilize the sequence of the ORF of β type RCA to be marked in Semen Caryae Cathayensis genome as inquiry and carry out homology coupling, through Homologous gene sequences comparison and transcript profile sequential analysis, retrieve in conjunction with Semen Caryae Cathayensis genomic fragment, design primer is ActinFor:GCTGAACGGGAAATTGTCActin internal reference primer, ActinRev:AGAGATGGCTGGCTGGAAGAGGActin internal reference primer, Rca-abF:GACTTCGCCACTACGACCTGRca Gene Partial fragment expands gets, Rca-adR:TTCTCCATACGACCATCACGRca Gene Partial fragment expands gets, 3 ' RACE-a:GAACCACCCAATACACTGTCAACAACCAAa subunit 3 terminal specific amplification, 5 ' RACE-a:CCATCCATGTTGTTGTCCAGGTCGTAGTGa subunit 5 terminal specific amplification, 3 ' RACE-b:GAACCACCCAATACACAGTCAACAACCAAb subunit 3 terminal specific amplification, 5 ' RACE-b:CCATCCATGTTGTTGTCCAAGTTGTACTGb subunit 5 terminal specific amplification, qPCR-aF:GTGAAGAGGGTCCAACTTGCCGAa subunit is quantitative, qPCR-aR:GCTCCCATCATCACTCCTCGCAGa subunit is quantitative, qPCR-bF:GGCAAACAGACCGCCAAGGACAb subunit is quantitative, qPCR-bR:TGGAAGAGCGAGTCAACCATACCCb subunit is quantitative, Genome-aF:ACCTTCCATTAAAGTGCTGCGTGCa subunit gene group increases, Genome-aR:CCAAGGGCAGCCTCGCTCAAGTa subunit gene group increases, Genome-b1F:ACGAGGATGCTATTAAGAGCGGAACb subunit gene group increases, Genome-b1R:GAAGTTGGATCAAAGTTCTCTGGCAb subunit gene group increases,
B) RCA gene intermediate segment amplification: combine existing Semen Caryae Cathayensis transcript profile according to homologous sequence, utilize PrimerPremier5.0 software design intermediate segment primer, and using β-Actin as reference gene, carry out pcr amplification and obtain product, through 1% agarose gel electrophoresis visible 550bp object band;
C) amplification of RCA gene 3 ' end and 5 ' terminal sequence: design α and β RACE primer respectively according to intermediate segment sequencing result, with 3 ' end reverse transcription cDNA for template, 3 ' end primer and UPM (UniversalPrimerMix) once increase and obtain product, through 1.5% agarose gel electrophoresis visible 850bp object band, in like manner, 5 ' end primer and UPM once increase and obtain product, through 1.5% agarose gel electrophoresis visible 900bp object band, because α and β subunit intermediate segment sequence similarity is very high, so specificity amplification primer is close, and RACE amplified production size almost identical explanation α and β subunit 3 terminal sequence and 3 end untranslated regions and 5 terminal sequences and 5 end untranslated region similar length, all sequencing results obtain α and β subunit cDNA total length through splicing and comparison,
D) analysis of CcRCA α and CcRCA β cDNA sequence characteristic: the cDNA total length of CcRCA α and CcRCA β is close, all comprise complete open reading frame (ORF) and part 3 end not translation sequences, predictive coding 472, 435 amino acid, theoretical relative molecular mass 51.52kD and 47.52kD, BLAST analyzes both displays similar 85.44%, article two, protein sequence N holds front 55 and 57 amino acid to be predicted as chloroplast transit peptides (cTP), so CcRCA α and CcRCA β maturation protein should comprise 417 and 378 amino acid, corresponding molecular mass is 46.11kD and 42.11kD, the protein sequence that CcRCA α derives has more 37 amino acid at C end than CcRCA β, wherein comprise the critical sites that two cysteine residues are RCA redox modulating, two sections of conservative ATP-binding domain territory GGKGQGKS and LFIND lay respectively at 162 to 170 and 222 to 227 of aminoacid sequence, according to the research to spinach and tobacco RCA, in Semen Caryae Cathayensis RCA 169 Methionin and Rubisco activates and ATP enzyme live closely related, the aspartic acid of 227 is essential to the meticulous adjustment of γ-phosphate bond in subunit cohesive process, in addition, the amino acid that CcRCA α encodes holds many 37 than CcRCA β at C, also 26 amino acid classes differences are had in the sequence contrasted, although do not obtain the not translation sequences of whole CcRCA α and CcRCA β, but there is obviously difference in acquired sequence, SignalP4.1Server program is utilized to analyze two proteins, do not find signal peptide, the equal non-secreted protein of two proteins is described, utilize the chloroplast transit peptides (cTP) of ChloroP1.1Server program predicted protein matter sequence, result represents, two gene protein sequences are all containing chloroplast transit peptides, and cTP preamble sequence length is respectively 55 and 57, the PSORT program of ExPASy is utilized to predict protein subcellular location, result shows that RAC is positioned in chloroplast(id), by the membrane spaning domain of TMpred program predicted protein matter, result shows, two albumen have 2 transbilayer helixs, respectively in amino acid 88th ~ 107 and 143-162 region, illustrate that the albumen that CcRCA encodes may be transmembrane protein, comprehensive analysis is known, CcRCA is cell nucleus gene coding, the zymoprotein that chloroplast(id) cyst membrane plays a role,
E) RCA genomic sequence analysis: in order to confirm whether RCA and RCA shears from same Pre-mRNA different positions, Semen Caryae Cathayensis genomic dna is analyzed, gene-specific primer is utilized to expand the genomic dna sequence getting CcRCA α, CcRCA β, CcRCA α genomic dna comprises 6 introns and 7 exons, and length is respectively 1590bp and 1419bp; CcRCA β comprises 5 introns and 6 exons, length is respectively 817bp and 1308bp, utilize DNAMAN software carry out Multiple Sequence Alignment display CcRCA α and CcRCA β similarity higher, reach 73.23%, consistent with the similarity system design result of mRNA before, the analysis of RCA genomic dna confirms two kinds of RCAmRNA having found respectively by 2 independently genes encoding, i.e. 1 α type gene and 1 β type gene;
F) Phylogenetic analysis of Semen Caryae Cathayensis RCA: the blastx program using NCBI, albumen homology comparison is carried out to CcRCA α and CcRCA β gene, find that the RCA aminoacid sequence of Semen Caryae Cathayensis and many plants has higher homology, adopt the adjacent method (Neighbor-Joining of MEGA5.05 software, NJ), build the systematic evolution tree of the RCA aminoacid sequence of Semen Caryae Cathayensis CcRCA α and 22 kind of plant such as CcRCA β and pecan tree, systematic evolution tree shows, Semen Caryae Cathayensis CcRCA α and pecan tree (Carayaillinoensis), the sibship of American red-maple (Acerrubrum) apple (Malusdomestica) etc. is close, CcRCA β and grape (Vitisvinifera), soybean (Glycinemax) and cotton (Gossypiumhirsutum) the most close, mutually unisonly be respectively 88%, 89% and 88%, wherein its conserved structure domain analysis is found, its conserved regions has 2 ATP-binding site P-ring sequences and ATPasesensor2motif special site, all belong to ABC_ATPaseSuperfamilies superfamily gene, but all with dragon spruce (Piceasitchensis), the sibship of chlamydomonas etc. is far away,
G) RCA expression analysis in different growing stage Semen Caryae Cathayensis blade: week about in the afternoon getting after the different end of the branch outermost spire of homophyletic or blade be numbered, rapid liquid nitrogen freezing process, be placed in-70 DEG C of Refrigerator stores, improved method of CTAB and test kit (TaRaKaMiniBESTUniversalRNAExtractionKit) conbined usage is adopted to extract the total serum IgE of different growing stage blade, to extract gained total serum IgE for template, OligodT is primer, according to ReverseTranscriptaseM-MLV (RNaseH-) test kit (TaKaRa) process specifications synthesis cDNA first chain, detect the relative expression quantity of gene in order to RT-qPCR after diluting 5 times, the Hickory Leaves grown under natural condition is at the expression amount of different times RCA, along with the change of growth date, CcRCA α is close with CcRCA beta gene expression trend.
As preferably, the CcRCA α in described steps d, step e, step f, step g refers to the 3 ' end that this experiment employing 3 '/5 ' RACE and RT-PCR technology Successful amplification go out Semen Caryae Cathayensis 2 RCA genes, and sequence assembly obtains full length cDNA sequence respectively; CcRCA β in described steps d, step e, step f, step g refers to the 5 ' end that this experiment employing 3 '/5 ' RACE and RT-PCR technology Successful amplification go out Semen Caryae Cathayensis 2 RCA genes, and sequence assembly obtains full length cDNA sequence respectively.
Beneficial effect of the present invention: the present invention utilizes RACE and RT-PCR technology, clone obtains Semen Caryae Cathayensis 2 RCA full length genes, in conjunction with bioinformatic analysis, analyze at base sequence, encoding amino acid sequence and other property differences, and the expression pattern of RCA gene is sought by the RCA expression conditions of different development stage blade, regulate and control RCA for utilizing genetic engineering means and lay the first stone to increase photosynthetic rate and to improve resistance.
Feature of the present invention and advantage will be described in detail by reference to the accompanying drawings by embodiment.
[accompanying drawing explanation]
Fig. 1 is RCA gene intermediate segment agarose gel electrophoresis figure of the present invention;
Fig. 2 is RCA gene 3 of the present invention, end and 5, RLM-RACE result agarose gel electrophoresis figure;
Fig. 3 is that CcRCA α of the present invention and CcRCA beta amino acids alignment scheme;
Fig. 4 is RCA α genomic dna structural representation of the present invention;
Fig. 5 is RCA β genomic dna structural representation of the present invention;
Fig. 6 is the systematic evolution tree schematic diagram of the different plant α type RCA aminoacid sequences based on NJ method of the present invention;
Fig. 7 is the systematic evolution tree schematic diagram of the different plant beta type RCA aminoacid sequences based on NJ method of the present invention.
[embodiment]
Consult Fig. 1-7, the determination method of a kind of Semen Caryae Cathayensis RCA of the present invention gene clone, is characterized in that, comprise the following steps:
A) sequence retrieval and design of primers: by clone and comparison confirms that Semen Caryae Cathayensis exists 2 RCA genes, utilize the sequence of the ORF of β type RCA to be marked in Semen Caryae Cathayensis genome as inquiry and carry out homology coupling, through Homologous gene sequences comparison and transcript profile sequential analysis, retrieve in conjunction with Semen Caryae Cathayensis genomic fragment, design primer is ActinFor:GCTGAACGGGAAATTGTCActin internal reference primer, ActinRev:AGAGATGGCTGGCTGGAAGAGGActin internal reference primer, Rca-abF:GACTTCGCCACTACGACCTGRca Gene Partial fragment expands gets, Rca-adR:TTCTCCATACGACCATCACGRca Gene Partial fragment expands gets, 3 ' RACE-a:GAACCACCCAATACACTGTCAACAACCAAa subunit 3 terminal specific amplification, 5 ' RACE-a:CCATCCATGTTGTTGTCCAGGTCGTAGTGa subunit 5 terminal specific amplification, 3 ' RACE-b:GAACCACCCAATACACAGTCAACAACCAAb subunit 3 terminal specific amplification, 5 ' RACE-b:CCATCCATGTTGTTGTCCAAGTTGTACTGb subunit 5 terminal specific amplification, qPCR-aF:GTGAAGAGGGTCCAACTTGCCGAa subunit is quantitative, qPCR-aR:GCTCCCATCATCACTCCTCGCAGa subunit is quantitative, qPCR-bF:GGCAAACAGACCGCCAAGGACAb subunit is quantitative, qPCR-bR:TGGAAGAGCGAGTCAACCATACCCb subunit is quantitative, Genome-aF:ACCTTCCATTAAAGTGCTGCGTGCa subunit gene group increases, Genome-aR:CCAAGGGCAGCCTCGCTCAAGTa subunit gene group increases, Genome-b1F:ACGAGGATGCTATTAAGAGCGGAACb subunit gene group increases, Genome-b1R:GAAGTTGGATCAAAGTTCTCTGGCAb subunit gene group increases,
B) RCA gene intermediate segment amplification: combine existing Semen Caryae Cathayensis transcript profile according to homologous sequence, utilize PrimerPremier5.0 software design intermediate segment primer, and using β-Actin as reference gene, carry out pcr amplification and obtain product, through 1% agarose gel electrophoresis visible 550bp object band;
C) amplification of RCA gene 3 ' end and 5 ' terminal sequence: design α and β RACE primer respectively according to intermediate segment sequencing result, with 3 ' end reverse transcription cDNA for template, 3 ' end primer and UPM (UniversalPrimerMix) once increase and obtain product, through 1.5% agarose gel electrophoresis visible 850bp object band, in like manner, 5 ' end primer and UPM once increase and obtain product, through 1.5% agarose gel electrophoresis visible 900bp object band, because α and β subunit intermediate segment sequence similarity is very high, so specificity amplification primer is close, and RACE amplified production size almost identical explanation α and β subunit 3 terminal sequence and 3 end untranslated regions and 5 terminal sequences and 5 end untranslated region similar length, all sequencing results obtain α and β subunit cDNA total length through splicing and comparison,
D) analysis of CcRCA α and CcRCA β cDNA sequence characteristic: the cDNA total length of CcRCA α and CcRCA β is close, all comprise complete open reading frame (ORF) and part 3 end not translation sequences, predictive coding 472, 435 amino acid, theoretical relative molecular mass 51.52kD and 47.52kD, BLAST analyzes both displays similar 85.44%, article two, protein sequence N holds front 55 and 57 amino acid to be predicted as chloroplast transit peptides (cTP), so CcRCA α and CcRCA β maturation protein should comprise 417 and 378 amino acid, corresponding molecular mass is 46.11kD and 42.11kD, the protein sequence that CcRCA α derives has more 37 amino acid at C end than CcRCA β, wherein comprise the critical sites that two cysteine residues are RCA redox modulating, two sections of conservative ATP-binding domain territory GGKGQGKS and LFIND lay respectively at 162 to 170 and 222 to 227 of aminoacid sequence, according to the research to spinach and tobacco RCA, in Semen Caryae Cathayensis RCA 169 Methionin and Rubisco activates and ATP enzyme live closely related, the aspartic acid of 227 is essential to the meticulous adjustment of γ-phosphate bond in subunit cohesive process, in addition, the amino acid that CcRCA α encodes holds many 37 than CcRCA β at C, also 26 amino acid classes differences are had in the sequence contrasted, although do not obtain the not translation sequences of whole CcRCA α and CcRCA β, but there is obviously difference in acquired sequence, SignalP4.1Server program is utilized to analyze two proteins, do not find signal peptide, the equal non-secreted protein of two proteins is described, utilize the chloroplast transit peptides (cTP) of ChloroP1.1Server program predicted protein matter sequence, result represents, two gene protein sequences are all containing chloroplast transit peptides, and cTP preamble sequence length is respectively 55 and 57, the PSORT program of ExPASy is utilized to predict protein subcellular location, result shows that RAC is positioned in chloroplast(id), by the membrane spaning domain of TMpred program predicted protein matter, result shows, two albumen have 2 transbilayer helixs, respectively in amino acid 88th ~ 107 and 143-162 region, illustrate that the albumen that CcRCA encodes may be transmembrane protein, comprehensive analysis is known, CcRCA is cell nucleus gene coding, the zymoprotein that chloroplast(id) cyst membrane plays a role,
E) RCA genomic sequence analysis: in order to confirm whether RCA and RCA shears from same Pre-mRNA different positions, Semen Caryae Cathayensis genomic dna is analyzed, gene-specific primer is utilized to expand the genomic dna sequence getting CcRCA α, CcRCA β, CcRCA α genomic dna comprises 6 introns and 7 exons, and length is respectively 1590bp and 1419bp; CcRCA β comprises 5 introns and 6 exons, length is respectively 817bp and 1308bp, utilize DNAMAN software carry out Multiple Sequence Alignment display CcRCA α and CcRCA β similarity higher, reach 73.23%, consistent with the similarity system design result of mRNA before, the analysis of RCA genomic dna confirms two kinds of RCAmRNA having found respectively by 2 independently genes encoding, i.e. 1 α type gene and 1 β type gene;
F) Phylogenetic analysis of Semen Caryae Cathayensis RCA: the blastx program using NCBI, albumen homology comparison is carried out to CcRCA α and CcRCA β gene, find that the RCA aminoacid sequence of Semen Caryae Cathayensis and many plants has higher homology, adopt the adjacent method (Neighbor-Joining of MEGA5.05 software, NJ), build the systematic evolution tree of the RCA aminoacid sequence of Semen Caryae Cathayensis CcRCA α and 22 kind of plant such as CcRCA β and pecan tree, systematic evolution tree shows, Semen Caryae Cathayensis CcRCA α and pecan tree (Carayaillinoensis), the sibship of American red-maple (Acerrubrum) apple (Malusdomestica) etc. is close, CcRCA β and grape (Vitisvinifera), soybean (Glycinemax) and cotton (Gossypiumhirsutum) the most close, mutually unisonly be respectively 88%, 89% and 88%, wherein its conserved structure domain analysis is found, its conserved regions has 2 ATP-binding site P-ring sequences and ATPasesensor2motif special site, all belong to ABC_ATPaseSuperfamilies superfamily gene, but all with dragon spruce (Piceasitchensis), the sibship of chlamydomonas etc. is far away,
G) RCA expression analysis in different growing stage Semen Caryae Cathayensis blade: week about in the afternoon getting after the different end of the branch outermost spire of homophyletic or blade be numbered, rapid liquid nitrogen freezing process, be placed in-70 DEG C of Refrigerator stores, improved method of CTAB and test kit (TaRaKaMiniBESTUniversalRNAExtractionKit) conbined usage is adopted to extract the total serum IgE of different growing stage blade, to extract gained total serum IgE for template, OligodT is primer, according to ReverseTranscriptaseM-MLV (RNaseH-) test kit (TaKaRa) process specifications synthesis cDNA first chain, detect the relative expression quantity of gene in order to RT-qPCR after diluting 5 times, the Hickory Leaves grown under natural condition is at the expression amount of different times RCA, along with the change of growth date, CcRCA α is close with CcRCA beta gene expression trend, CcRCA α in described steps d, step e, step f, step g refers to the 3 ' end that this experiment employing 3 '/5 ' RACE and RT-PCR technology Successful amplification go out Semen Caryae Cathayensis 2 RCA genes, and sequence assembly obtains full length cDNA sequence respectively,
CcRCA β in described steps d, step e, step f, step g refers to the 5 ' end that this experiment employing 3 '/5 ' RACE and RT-PCR technology Successful amplification go out Semen Caryae Cathayensis 2 RCA genes, and sequence assembly obtains full length cDNA sequence respectively.
The present invention utilizes RACE and RT-PCR technology, clone obtains Semen Caryae Cathayensis 2 RCA full length genes, in conjunction with bioinformatic analysis, analyze at base sequence, encoding amino acid sequence and other property differences, and the expression pattern of RCA gene is sought by the RCA expression conditions of different development stage blade, regulate and control RCA for utilizing genetic engineering means and lay the first stone to increase photosynthetic rate and to improve resistance.
Above-described embodiment is to explanation of the present invention, is not limitation of the invention, anyly all belongs to protection scope of the present invention to the scheme after simple transformation of the present invention.
<110> parasol pine is permanent, Zheng Ping Song, Ji Guocun, Qiu Lingling
Zhejiang A & F University
The determination method of <120> Semen Caryae Cathayensis RCA gene clone
<160>1
<170>PrimerPremier5.0
<210>1
<211>1230
<212>DNA
<213> synthetic
<400>1
ActinForGCTGAACGGGAAATTGTC18
ActinRevAGAGATGGCTGGCTGGAAGAGG22
Rca-abFGACTTCGCCACTACGACCTG20
Rca-adRTTCTCCATACGACCATCACG20
3’RACE-aGAACCACCCAATACACTGTCAACAACCAA29
5’RACE-aCCATCCATGTTGTTGTCCAGGTCGTAGTG29
3’RACE-bGAACCACCCAATACACAGTCAACAACCAA29
5’RACE-bCCATCCATGTTGTTGTCCAAGTTGTACTG29
qPCR-aFGTGAAGAGGGTCCAACTTGCCGA23
qPCR-aRGCTCCCATCATCACTCCTCGCAG23
qPCR-bFGGCAAACAGACCGCCAAGGACA22
qPCR-bRTGGAAGAGCGAGTCAACCATACCC24
Genome-aFACCTTCCATTAAAGTGCTGCGTGC24
Genome-aRCCAAGGGCAGCCTCGCTCAAGT22
Genome-b1FACGAGGATGCTATTAAGAGCGGAAC25
Genome-b1RGAAGTTGGATCAAAGTTCTCTGGCA25
CcRcAαMAAAVSTIGAVNQAPLSLNSSGVGASVPSSAFLGSSLKKLTPRFT..KVS48
CcRcAβMAAAVSTIGTVNRvPLSLNSTGAGASVPSSAFLGSSLKKVTSPFNNSKVS50
CcRcAαTGSFKVVAEIEEDKQTDKDKWKGLAFDTSDDQQDITRGKGMVDSLFQAPT98
CcRcAβTGSFKVVAEVEEGKQTAKDKWKGLAFDESDDQQDITRGKGMVDSLFQAPT100
CcRcAαGAGTHYAVMSSYDYISTGLRHYDLDNNMDGFYIAPAFMDKLVVHITKNFM148
CcRcAβGSGTHYAVMSSYDYISTGLRQYNLDNNMDGFYIAPAFMDKLVVHISKNFM150
CcRcAαSLPNIKIPLILGIWGGKGQGKSFQCELVFAKMGISPIMMSAGELESGNAG198
CcRcAβSLPNIKIPLILGIWGGKGQGKSFQCELVFAKMGISPIMMSAGELESGNAG200
CcRcAαEPAKLIRQRYREAADIIRKGKMCCLFINDLDAGAGRMGGTTQYTVNNQMV248
CcRcAβEPAKLIRQRYREAADIIRKGKMCCLFINDLDAGAGRMGGTTQYTVNNQMV250
CcRcAαNATLMNIADNPTNVQLPGMYNKEENPRVPVIVTGNDSSTLYAPLIRDGRM298
CcRcAβNATLMNIADNPTNVQLPGMYNKEENPRVPIIVTGNDFSTLYAPLIRDGRM300
CcRcAαEKFYWAPTREDRIGVCQGIFRTDKVAADDIVKLVDTFPGQSIDFFGALRA348
CcRcAβEKFYWAPTREDRIGVCKGIFRTDKVADDDIVKLVDTFPGQSIDFFGALRA350
CcRcAαRVYDDEVRKWVSGVGVDGIGKRLVNSKEGPPTFEQPQMTLEKLLEYGNML398
CcRcAβRVYDDEVRKWISGVGVDGVGKRLVNSKEGPPSFEQPQMTLEKLLEYGNML400
CcRcAαVQEQENVKRVQLADKYLSEAALGEANEDSIKSGTFYGKAAQQVNTPVPEG448
CcRcAαVQEQENVKRVQLADKYLSEAALGDANEDAIKSGTF435
CcRcAαCTDPNAENFDPTARSDDGSCLYTF472
Claims (2)
1. a determination method for Semen Caryae Cathayensis RCA gene clone, is characterized in that, comprises the following steps:
A) sequence retrieval and design of primers: by clone and comparison confirms that Semen Caryae Cathayensis exists 2 RCA genes, utilize the sequence of the ORF of β type RCA to be marked in Semen Caryae Cathayensis genome as inquiry and carry out homology coupling, through Homologous gene sequences comparison and transcript profile sequential analysis, retrieve in conjunction with Semen Caryae Cathayensis genomic fragment, design primer is ActinFor:GCTGAACGGGAAATTGTCActin internal reference primer, ActinRev:AGAGATGGCTGGCTGGAAGAGGActin internal reference primer, Rca-abF:GACTTCGCCACTACGACCTGRca Gene Partial fragment expands gets, Rca-adR:TTCTCCATACGACCATCACGRca Gene Partial fragment expands gets, 3 ' RACE-a:GAACCACCCAATACACTGTCAACAACCAAa subunit 3 terminal specific amplification, 5 ' RACE-a:CCATCCATGTTGTTGTCCAGGTCGTAGTGa subunit 5 terminal specific amplification, 3 ' RACE-b:GAACCACCCAATACACAGTCAACAACCAAb subunit 3 terminal specific amplification, 5 ' RACE-b:CCATCCATGTTGTTGTCCAAGTTGTACTGb subunit 5 terminal specific amplification, qPCR-aF:GTGAAGAGGGTCCAACTTGCCGAa subunit is quantitative, qPCR-aR:GCTCCCATCATCACTCCTCGCAGa subunit is quantitative, qPCR-bF:GGCAAACAGACCGCCAAGGACAb subunit is quantitative, qPCR-bR:TGGAAGAGCGAGTCAACCATACCCb subunit is quantitative, Genome-aF:ACCTTCCATTAAAGTGCTGCGTGCa subunit gene group increases, Genome-aR:CCAAGGGCAGCCTCGCTCAAGTa subunit gene group increases, Genome-b1F:ACGAGGATGCTATTAAGAGCGGAACb subunit gene group increases, Genome-b1R:GAAGTTGGATCAAAGTTCTCTGGCAb subunit gene group increases,
B) RCA gene intermediate segment amplification: combine existing Semen Caryae Cathayensis transcript profile according to homologous sequence, utilize PrimerPremier5.0 software design intermediate segment primer, and using β-Actin as reference gene, carry out pcr amplification and obtain product, through 1% agarose gel electrophoresis visible 550bp object band;
C) amplification of RCA gene 3 ' end and 5 ' terminal sequence: design α and β RACE primer respectively according to intermediate segment sequencing result, with 3 ' end reverse transcription cDNA for template, 3 ' end primer and UPM (UniversalPrimerMix) once increase and obtain product, through 1.5% agarose gel electrophoresis visible 850bp object band, in like manner, 5 ' end primer and UPM once increase and obtain product, through 1.5% agarose gel electrophoresis visible 900bp object band, because α and β subunit intermediate segment sequence similarity is very high, so specificity amplification primer is close, and RACE amplified production size almost identical explanation α and β subunit 3 terminal sequence and 3 end untranslated regions and 5 terminal sequences and 5 end untranslated region similar length, all sequencing results obtain α and β subunit cDNA total length through splicing and comparison,
D) analysis of CcRCA α and CcRCA β cDNA sequence characteristic: the cDNA total length of CcRCA α and CcRCA β is close, all comprise complete open reading frame (ORF) and part 3 end not translation sequences, predictive coding 472, 435 amino acid, theoretical relative molecular mass 51.52kD and 47.52kD, BLAST analyzes both displays similar 85.44%, article two, protein sequence N holds front 55 and 57 amino acid to be predicted as chloroplast transit peptides (cTP), so CcRCA α and CcRCA β maturation protein should comprise 417 and 378 amino acid, corresponding molecular mass is 46.11kD and 42.11kD, the protein sequence that CcRCA α derives has more 37 amino acid at C end than CcRCA β, wherein comprise the critical sites that two cysteine residues are RCA redox modulating, two sections of conservative ATP-binding domain territory GGKGQGKS and LFIND lay respectively at 162 to 170 and 222 to 227 of aminoacid sequence, according to the research to spinach and tobacco RCA, in Semen Caryae Cathayensis RCA 169 Methionin and Rubisco activates and ATP enzyme live closely related, the aspartic acid of 227 is essential to the meticulous adjustment of γ-phosphate bond in subunit cohesive process, in addition, the amino acid that CcRCA α encodes holds many 37 than CcRCA β at C, also 26 amino acid classes differences are had in the sequence contrasted, although do not obtain the not translation sequences of whole CcRCA α and CcRCA β, but there is obviously difference in acquired sequence, SignalP4.1Server program is utilized to analyze two proteins, do not find signal peptide, the equal non-secreted protein of two proteins is described, utilize the chloroplast transit peptides (cTP) of ChloroP1.1Server program predicted protein matter sequence, result represents, two gene protein sequences are all containing chloroplast transit peptides, and cTP preamble sequence length is respectively 55 and 57, the PSORT program of ExPASy is utilized to predict protein subcellular location, result shows that RAC is positioned in chloroplast(id), by the membrane spaning domain of TMpred program predicted protein matter, result shows, two albumen have 2 transbilayer helixs, respectively in amino acid 88th ~ 107 and 143-162 region, illustrate that the albumen that CcRCA encodes may be transmembrane protein, comprehensive analysis is known, CcRCA is cell nucleus gene coding, the zymoprotein that chloroplast(id) cyst membrane plays a role,
E) RCA genomic sequence analysis: in order to confirm whether RCA and RCA shears from same Pre-mRNA different positions, Semen Caryae Cathayensis genomic dna is analyzed, gene-specific primer is utilized to expand the genomic dna sequence getting CcRCA α, CcRCA β, CcRCA α genomic dna comprises 6 introns and 7 exons, and length is respectively 1590bp and 1419bp; CcRCA β comprises 5 introns and 6 exons, length is respectively 817bp and 1308bp, utilize DNAMAN software carry out Multiple Sequence Alignment display CcRCA α and CcRCA β similarity higher, reach 73.23%, consistent with the similarity system design result of mRNA before, the analysis of RCA genomic dna confirms two kinds of RCAmRNA having found respectively by 2 independently genes encoding, i.e. 1 α type gene and 1 β type gene;
F) Phylogenetic analysis of Semen Caryae Cathayensis RCA: the blastx program using NCBI, albumen homology comparison is carried out to CcRCA α and CcRCA β gene, find that the RCA aminoacid sequence of Semen Caryae Cathayensis and many plants has higher homology, adopt the adjacent method (Neighbor-Joining of MEGA5.05 software, NJ), build the systematic evolution tree of the RCA aminoacid sequence of Semen Caryae Cathayensis CcRCA α and 22 kind of plant such as CcRCA β and pecan tree, systematic evolution tree shows, Semen Caryae Cathayensis CcRCA α and pecan tree (Carayaillinoensis), the sibship of American red-maple (Acerrubrum) apple (Malusdomestica) etc. is close, CcRCA β and grape (Vitisvinifera), soybean (Glycinemax) and cotton (Gossypiumhirsutum) the most close, mutually unisonly be respectively 88%, 89% and 88%, wherein its conserved structure domain analysis is found, its conserved regions has 2 ATP-binding site P-ring sequences and ATPasesensor2motif special site, all belong to ABC_ATPaseSuperfamilies superfamily gene, but all with dragon spruce (Piceasitchensis), the sibship of chlamydomonas etc. is far away,
G) RCA expression analysis in different growing stage Semen Caryae Cathayensis blade: week about in the afternoon getting after the different end of the branch outermost spire of homophyletic or blade be numbered, rapid liquid nitrogen freezing process, be placed in-70 DEG C of Refrigerator stores, improved method of CTAB and test kit (TaRaKaMiniBESTUniversalRNAExtractionKit) conbined usage is adopted to extract the total serum IgE of different growing stage blade, to extract gained total serum IgE for template, OligodT is primer, according to ReverseTranscriptaseM-MLV (RNaseH-) test kit (TaKaRa) process specifications synthesis cDNA first chain, detect the relative expression quantity of gene in order to RT-qPCR after diluting 5 times, the Hickory Leaves grown under natural condition is at the expression amount of different times RCA, along with the change of growth date, CcRCA α is close with CcRCA beta gene expression trend.
2. the determination method of a kind of Semen Caryae Cathayensis RCA gene clone as claimed in claim 1, it is characterized in that: the CcRCA α in described steps d, step e, step f, step g refers to the 3 ' end that this experiment employing 3 '/5 ' RACE and RT-PCR technology Successful amplification go out Semen Caryae Cathayensis 2 RCA genes, and sequence assembly obtains full length cDNA sequence respectively; CcRCA β in described steps d, step e, step f, step g refers to the 5 ' end that this experiment employing 3 '/5 ' RACE and RT-PCR technology Successful amplification go out Semen Caryae Cathayensis 2 RCA genes, and sequence assembly obtains full length cDNA sequence respectively.
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