CN101985624A - Freesia 1-aminocyclopropane-1-carboxylate (ACC) synzyme FhACS1 protein coding sequence - Google Patents
Freesia 1-aminocyclopropane-1-carboxylate (ACC) synzyme FhACS1 protein coding sequence Download PDFInfo
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Abstract
The invention provides a freesia 1-aminocyclopropane-1-carboxylate (ACC) synzyme FhACS1 protein coding sequence, which comprises a nucleotide sequence nucleotide from the site of 1st to 1371st shown in SEQ ID NO.2 and polypeptide of amino acid sequence shown in SEQ ID NO.3, or conservative variation polypeptide, active fragments, or active derivatives of the amino acid sequence. The freesia ACC synzyme FhACS1 protein coding sequence of the invention provides science foundation for using the anti-sense ribose nucleic acid (RNA) technology to introduce anti-sense FhACS1 gene into the freesia in the future and inhibit systemization of ethylene in the freesia so as to prolong ornamental life of the freesia, and has great application value.
Description
Technical field
What the present invention relates to is a kind of albumen coded sequence of gene engineering technology field, particularly a kind of freesia ACC synthetic enzyme FhACS1 albumen coded sequence.
Background technology
As fresh cutting flower, not only require to have good ornamental value, also to keep long viewing period (shelf-lives, bottle are inserted the phase) simultaneously.From the mechanism angle analysis, one of initial reaction of petal aging is autocatalytically and produce ethene, on the one hand being that aging course produces ethene, be that the expression that the ethene that produces activates again with senescence-associated gene further promotes its aging on the other hand, and it is rotten to cause flower finally to wither.In the higher plant body, the ethene biosynthetic pathway is followed successively by: methionine(Met), S-ademetionine, ACC, ethene.Methionine(Met) forms the S-ademetionine under the effect of S-adenomethionine synthase, the S-ademetionine is at ACC synthetic enzyme (1-aminocyclopropane-1-carboxylate synthase, ACS) catalysis forms the precursor ACC of ethene down, and ACC forms ethene, CO under acc oxidase catalysis
2And HCN.Wherein catalysis SAM is the rate-limiting reaction of this approach to the ACC conversion, and ACC synthetic enzyme (ACS) is a plant Ethylene Biosynthesis rate-limiting enzyme.Utilize Antisense RNA Technique that antisense ACS gene is imported flowers, synthesizing of ethene suppressing can make and view and admire life-span prolongation (Halevy A H.1995).
Find by prior art documents, in a lot of plants, found the ACS multigene family.As Have A Ten, WelteringE J is at " Ethylene biosynthetic genes are differentially expressed during carnation flowersenescence (difference of Ethylene Biosynthesis genetic expression in the carnation aging course) " (Plant-Mol-Biol molecular biology of plants, 1997,34:89-97.) ethene that studies show that of carnation ACS gene is regulated and control fresh flower by change genetic expression and opened, and this gene expression amount of floral organ each several part there are differences.(Freesia * hybrida), the clone of ACS gene, expression pattern and ACS albumen coded sequence it be not immediately clear but for the flower bulbs freesia.
Further do not seeing the freesia ACS protein sequence pertinent literature report that theme any and of the present invention is relevant in the retrieval.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, a kind of freesia ACC synthetic enzyme FhACS1 albumen coded sequence is provided.The invention discloses with the closely-related protein gene of freesia FhACS1 and freesia FhACS1 protein sequence and nucleotide sequence thereof and at the expression pattern of freesia Different Organs, different developmental phases, for utilizing Antisense RNA Technique that antisense ACS gene is imported freesia from now on, thereby suppress synthesizing of the interior ethene of freesia body to prolong viewing and admiring the life-span of freesia flower, great application value is arranged.
The present invention is achieved by the following technical solutions:
The present invention includes have shown in the SEQ ID NO.2 from the nucleotide sequence of Nucleotide 1-1371 position and have the polypeptide of the aminoacid sequence shown in the SEQ ID NO.3, or its conservative property variation polypeptide or its active fragments, or its reactive derivative.
Described encoding sequence refer to the encode nucleotide sequence of polypeptide with freesia polypeptide FhACS1 protein-active is as 1-1371 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.2.This degenerate sequence is meant, is arranged in the 1-1371 position Nucleotide of SEQ ID NO.2, and having one or more codons to be encoded, the degenerate codon of same amino acid replaces the back and the sequence that produces.Because the degeneracy of codon, thus with SEQ ID NO.2 in 1-1371 position nucleotide sequence homology be low to moderate about 70% the degenerate sequence described sequence of SEQ IDNO.2 of also encoding out.This term also comprise with SEQ ID NO.2 in from the nucleotide sequence of the homology of nucleotide sequence at least 70% of Nucleotide 1-1371 position.
Polypeptide separated FhACS1 of the present invention, it comprises: the polypeptide with SEQ ID NO.3 aminoacid sequence.This polypeptide is the polypeptide with SEQID NO.3, this polypeptide is at the different growth and development stages of cut-flower, having that it's too late active size exist than big-difference in the Different Organs, and can be under environment-stress, plant hormone, chemical substance treatment the raising or the reduction of induced activity, ahead of time or the process of delay senility.
A kind of isolated dna molecular involved in the present invention, this molecule comprises the nucleotide sequence of the polypeptide with freesia FhACS1 protein active, and shows at least 70% homology from the nucleotides sequence of Nucleotide 1-1371 position among described nucleotide sequence and the SEQ ID NO.2; Perhaps described nucleotide sequence can with SEQ ID NO.2 in from the nucleotide sequence hybridization of Nucleotide 1-1371 position.
In the present invention, " isolating ", " purifying " DNA are meant, this DNA or fragment have been arranged in the sequence of its both sides under native state separates, and refers to that also this DNA or fragment with under the native state follow the component of nucleic acid to separate, and separates with follow its protein in cell.
The present invention also comprises encoding to have the variant form of sequence among the proteic SEQ ID NO.3 with natural freesia FhACS1 identical function.These variant forms comprise (but being not limited to): be generally disappearance, insertion and/or the replacement of 1-90 Nucleotide, and be added to 60 with inner nucleotide at 5 ' and/or 3 ' end.
" freesia ACC synthetic enzyme FhACS1 albumen " of the present invention refers to have the polypeptide of the SEQ ID NO.3 of freesia FhACS1 protein-active.This term also comprises having and the variant form relevant identical function of natural freesia FhACS1, SEQ ID NO.2 sequence.These variant forms comprise (but being not limited to): be generally 1-50 amino acid whose disappearance, insertion and/or replacement, and C-terminal and/or N-terminal add one or be 20 with interior amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises proteic active fragments of freesia FhACS1 and reactive derivative.
The variant form of freesia FhACS1 polypeptide of the present invention comprises: the albumen that homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, DNA that can relevant DNA hybridization with freesia FhACS1 under high or low rigorous condition are coded and the polypeptide or the albumen that utilize the antiserum(antisera) of freesia FhACS1 polypeptide to obtain.
In the present invention, " freesia FhACS1 conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID NO.3, has at the most 10 amino acid be replaced by similar performance or close amino acid and forms polypeptide.These conservative property variation polypeptide are preferably replaced according to table 1 and are produced.
Table 1
| Initial residue | Representational replacement | The preferred replacement |
| Ala(A) | Val；Leu；Ile | Val |
| Arg(R) | Lys；Gln；Asn | Lys |
| Asn(N) | Gln；His；Lys；Arg | Gln |
| Asp(D) | Glu | Glu |
| Cys(C) | Ser | Ser |
| Gln(Q) | Asn | Asn |
| Glu(E) | Asp | Asp |
| Gly(G) | Pro；Ala | Ala |
| His(H) | Asn；Gln；Lys；Arg | Arg |
| Ile(I) | Leu；Val；Met；Ala；Phe | Leu |
| Leu(L) | Ile；Val；Met；Ala；Phe | Ile |
| Lys(K) | Arg；Gln；Asn | Arg |
| Met(M) | Leu；Phe；Ile | Leu |
| Phe(F) | Leu；Val；Ile；Ala；Tyr | Leu |
| Pro(P) | Ala | Ala |
| Ser(S) | Thr | Thr |
| Thr(T) | Ser | Ser |
| Trp(W) | Tyr；Phe | Tyr |
| Tyr(Y) | Trp；Phe；Thr；Ser | Phe |
| Val(V) | Ile；Leu；Met；Phe；Ala | Leu |
Table 2
73% consistence is arranged in 1070 nt are overlapping
Query 136?gagaaccagctctcgttcaatcttctcgagtcttggcttgagcgtcaccctcacgctgcc 195
||||||||||||| || |||| ||||?||?||||||||| ||?|| ||?|?||
Sbjct 13 gagaaccagctctgctttgatctcatcgaatcgtggcttgagaaccatcccgacccagct 72
Query 196?gctttcaagacggaaggcacgtcgaagtcagttttccgggagttggctctcttccgggat 255
||?||||||| ||?|| | |?|?|?|?|||||||||?|?|||?|?||||?|||
Sbjct 73 gcattcaagaaagatggagc-----act-actattccgggagctcgctttgttccaggac 126
Query 256?taccatggcctccctgcttttaaacaagcattgacagaattcatgggtgaattgagagga 315
|||||||||?|?||?||?||?||?| |||||||| |||?||||||?|||?|?||||||
Sbjct 127?taccatggcttgccagccttcaagcgtgcattgactaaatacatgggagaagtaagagga 186
Query 316?aacaaggttgattttgaacctaacaaactggtcctcacagctggtgcaacctcggctaac 375
|||||?||?|?|||?||?||?|||| ||?||||||||?||||||||?||?||?||?||
Sbjct 187?aacaaagtagctttcgatcccaacaggctcgtcctcacggctggtgctacttctgccaat 246
Query 376?gagaccctcatgttctgccttgccgacccaggagaagcattcttgctccctactccatac 435
|||||?||||||||?||?||||||||?||?||||||||||||?|?||||||||||||||
Sbjct 247?gagactctcatgttttgtcttgccgaacctggagaagcattccttctgcctactccatac 306
Query 436?tatccagggtttgatcgtgatctcaagtggagaacaggagtggagatcgttccgatacac 495
||?|||||?||?|| |?||?|||||?|||?||||?||||?||||||||||||?||?||
Sbjct 307?tacccaggattcgacagagacctcaaatggcgaaccggagcggagatcgttcccatccat 366
Query 496?tgctcgagttcgaacagcttccggatcaccaagggcgccctcgaacaagcctatcggcat 555
||?|||||?||?|||?|||||?|||||||||| |||||?||| ||?||?| ||
Sbjct 367?tgttcgagctctaacggcttcaggatcaccaaaccggccctggaagctgcataccaagat 426
Query 556?gccggaaagtgcaatttgcgagttaagggagtgctgataaccaatccctccaatccactc 615
|| |||?|?| | ||||?||?||?||||||?|?|||||?||?|||||?|||?|
Sbjct 427?gcgcagaagcgtagcctcagagtgaaaggtgtgctggtgaccaacccgtccaacccattg 486
Query 616?ggcacgacgataaacaaagccgagctcgacatcctcgccgactttgtcgaagccaaagac 675
||?||||||?|?|?| | |||?||||||||?||?|?||||||||||| ||||?|||
Sbjct 487?gggacgacgctgacccgacacgaactcgacattcttgtcgactttgtcgtctccaaggac 546
Query 676?atccatttagtcggtgacgaaatctacgccggaacgaacttcgacatgccaggcttcatc 735
||||||?| ||?|||||||?||||||?|?||?||||||||||| |||?||?||||||
Sbjct 547?atccatctcatcagtgacgagatctactcagggaccaacttcgactcgccggggttcatc 606
Query 736?agtgttgcagaggcaataaaggagagaccccagatatccgaccgtgtccacattgtctat 795
|| ||||||||||?|?||||||?|| |?| |?|||?|?|| |?|||||?||?|
Sbjct 607?agcattgcagaggccacaaaggacaggaacaacgtctcccatcggattcacatcgtgtgc 666
Query 796?agcctctcgaaagacttcgggctaccgggtttccgagtcggtgcaatttattccaacaat 855
||||||||?||||| ||||?||?||?|||||?||?|||||||||||?||||| |?|||
Sbjct 667?agcctctctaaagatctcggcctcccaggttttcgtgtcggtgcaatctattcggagaat 726
Query 856?gatttggtcgtcgcggcggctacgaagatgtcgagcttcgggctgatatcgtcgcagact 915
|| ||||| |?||?|||||?||||||||?|||||?|||?||?|?||?||?||?||
Sbjct 727?gaagcagtcgtgtctgctgctactaagatgtcaagctttgggatggtctcttctcaaacc 786
Query 916?cagtatcttctttcggcaatgctagctgacaaggagtttacgaggaactacttggtggag 975
|||||?||?|| |||| ||?||?|?|||||?||?||?|| ||?|||?| |?|||
Sbjct 787?cagtacctcctcgcggcgttgttatccgacaaagaattcaccgataagtaccttctcgag 846
Query?976 aacaagaagaggctcaggaagaggcatggcgatcttgtcgagggtctccaaagggccggg 1035
|| |||||||?|||| ||?||||?|?| ||||||||?||?||?| ||| ||||
Sbjct?847 aatcagaagagactcaaagagcggcacgacatgcttgtcgaaggactgcgcaggatcggg 906
Query?1036?atcggttgcttggagagcaatgcgggcttgttctgttgggtagacatgaggcatctgctc 1095
|||||?||||||?| |?|?|||||?|||||||||?|||||?|||?||||?||?|||||
Sbjct?907 atcgggtgcttgaaaggaagtgcggccttgttctgctgggtggacgtgagacacctgctg 966
Query?1096?aagtctaatagtccccaaggagaaatggagctatggaagaagatgttgtacaatgtggga 1155
|||||?||?| |?|||||| ||||||||?||||||||||| |||| |?||||||
Sbjct?967 aagtccaacaccttcaaaggagcgatggagctgtggaagaagatagtgtatcaggtggga 1026
Query?1156?ctaaacatttctgcgggctcgtcgtgtcactccgacgaaccgggttggtt 1205
||||||||?|| ||||?||?|||||?||||?||||||||||||?|||||
Sbjct?1027?ctaaacatctcgccgggttcttcgtgccactgcgacgaaccggggtggtt 1076
Query: the encoding sequence of freesia ACC synthetic enzyme FhACS1
Sbjct: fruitlet bajiao banana ACC synthetic enzyme mRNA sequence
Table 2 is that the homology of the nucleotide sequence of freesia ACC synthetic enzyme FhACS1 of the present invention and fruitlet bajiao banana ACC synthase gene mRNA compares (GAP) table.
Table 3
73% consistence and 84% similarity are arranged in overlapping
Query?1 MNLSAKATCNSHGQDSSYFLGWEEYEKNPYHPTLNRSGIIQMGLAENQLSFNLLESWLER?180
M?LS?KA?CN?HGQDSSYFLGW+EYEKNPY?P N?+GIIQMGLAENQL?F+L+ESWLE
Sbjct?4 MLLSRKAACNIHGQDSSYFLGWQEYEKNPYDPITNPTGIIQMGLAENQLCFDLIESWLEN?63
Query?181?HPHAAAFKTEGTSKSVFRELALFRDYHGLPAFKQALTEFMGELRGNKVDFEPNKLVLTAG?360
HP AAFK+G +FRELALF+DYHGLPAFK+AL?++MGE+RGNKV?F+PN+LVLTAG
Sbjct?64 HPDPAAFKKDGAL—LFRELALFQDYHGLPAFKRALAKYMGEVRGNKVAFDPNRLVLTAG?121
Query?361?ATSANETLMFCLADPGEAFLLPTPYYPGFDRDLKWRTGVEIVPIHCSSSNSFRITKGALE?540
ATSANETLMFCLA+PGEAFLLPTPYYPGFDRD?KWRTG?EIVPIHCSSSN?FRITK?ALE
Sbjct 122?ATSANETLMFCLAEPGEAFLLPTPYYPGFDRDRKWRTGAEIVPIHCSSSNGFRITKPALE?181
Query 541?QAYRHAGKCNLRVKGVLITNPSNPLGTTINKAELDILADFVEAKDIHLVGDEIYAGTNFD?720
AY+?A?K?+LRVKGVL+TNPSNPLGTT+?+?ELDIL?DFV?+KDIHL+?DEIY?+GTNFD
Sbjct 182?AAYQDAQKRSLRVKGVLVTNPSNPLGTTLTRHELDILVDFVVSKDIHLISDEIYSGTNFD?241
Query 721?MPGFISVAEAIKERPQISDRVHIVYSLSKDFGLPGFRVGAIYSNNDLVVAAATKMSSFGL?900
PGFIS+AEA?K+R +S?R+HIV?SLSKD?GLPGFRVGAIYS?N+?VV+AATKMSSFG+
Sbjct 242?WPGFISIAEATKDRNNVSHRIHIVCSLSKDLGLPGFRVGAIYSENEAVVSAATKMSSFGM?301
Query 901?ISSQTQYLLSAMLADKEFTRNYLVENKKRLRKRHGDLVEGLQRAGIGCLESNAGLFCWVD?1080
+SSQTQYLL+A+L+DKEFT?YL+EN+KRL++RH?LVEGL+R?GIGCL?+?+A?LFCWVD
Sbjct 302?VSSQTQYLLAALLSDKEFTDKYLLENQKRLKERHDMLVEGLRRIGIGCLKGSAALFCWVD?361
Query?1081?MRHLLKSNSPQGEMELWKKMLYNVGLNISAGSSCHSDEPGWFRMCFANSQQETLNLAMDR?1260
MRHLLKSN+?+GEMELWKK++Y?VGLNIS?GSSCH?DEPGWFR+CFANMS++TL?LAM?R
Sbjct?362 MRHLLKSNTFKGEMELWKKIVYQVGLNISPGSSCHCDEPGWFRVCFANMSEDTLTLAMQR?421
Query?1261?LTSFVEL-------NYGVQRRRLPSSIVKWVLKLSPSTDRKAER 1371
L?SFV+ +G?QR?R?P?+?KWVL+LS?STDRK+ER
Sbjct?422 LKSFVDSGDCGSNHDSGHQRPRKP-FLTKWVLRLS-STDRKSER?463
Query: the aminoacid sequence of freesia ACC synthetic enzyme FhACS1
Sbjct: the aminoacid sequence (AAQ13435.1) of fruitlet bajiao banana ACC synthetic enzyme
Table 3 is that the homology of the aminoacid sequence of freesia ACC synthetic enzyme FhACS1 of the present invention and fruitlet bajiao banana ACC synthetic enzyme compares (FASTA) table.Wherein, identical amino acid marks with the amino acid monocase between two sequences.
Invention also comprises the analogue of freesia ACC synthetic enzyme FhACS1 albumen or polypeptide.The difference of these analogues and freesia ACC synthetic enzyme FhACS1 related polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide of enumerating.
(the not changing primary structure usually) form of modification comprises: the chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modification also comprises glycosylation, carries out glycosylation modified and polypeptide that produce in the procedure of processing as those in the synthetic and processing of polypeptide or further.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and finishes by polypeptide is exposed to.Modified forms also comprises have the phosphorylated amino acid residue sequence of (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Thereby also comprise the polypeptide that has been improved its anti-proteolysis performance or optimized solubility property by modifying.
In the present invention, the expression pattern of the methods analyst freesia ACC synthetic enzyme FhACS1 gene product of available real-time fluorescence quantitative PCR, whether and quantity the existence of mRNA transcript in cell of promptly analyzing the FhACS1 gene.
In addition, the nucleic acid molecule that the present invention can be used as probe has 8-100 continuous nucleotide of freesia FhACS1 nucleotide coding sequence usually, and this probe can be used for whether existing in the test sample the relevant nucleic acid molecule of coding freesia FhACS1.
The detection method that whether has freesia FhACS1 related nucleotide sequences in the test sample of the present invention comprises with above-mentioned probe and sample and hybridizing whether detection probes combination has taken place then.This sample is the product behind the pcr amplification, and wherein the pcr amplification primer is corresponding to freesia FhACS1 associated nucleotide encoding sequence, and can be positioned at the both sides or the centre of this encoding sequence.Primer length is generally 15-50 Nucleotide.
In addition, according to freesia FhACS1 nucleotide sequence of the present invention and aminoacid sequence, can be on the homology basis of nucleic acid homology or marking protein, relevant homologous gene of screening freesia FhACS1 or homologous protein.
In order to obtain the dot matrix with freesia FhACS1 genes involved, can screen freesia cDNA library with dna probe, these probes are under low rigorous condition, use
32P relevant all or part of of freesia FhACS1 cooked the radioactivity mark and.The cDNA library that is suitable for screening is the library from freesia.Structure is that biology field is well-known from the method in the cDNA library of interested cell or tissue.In addition, many such cDNA libraries also can buy, for example available from Clontech, and Stratagene, PaloAlto, Cal..This screening method can be discerned the nucleotide sequence of the gene family relevant with freesia FhACS1.
Freesia FhACS1 associated nucleotide full length sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.
In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.
In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.
Except producing with recombination method, the also available solid phase technique of the proteic fragment of the present invention, produced by direct peptide synthesis (people such as Stewart, (1969) solid-phase polypeptide is synthetic, WH Freeman Co., San Francisco; Merrifield J. (1963) J.Am Chem.Soc 85:2149-2154).Can carry out by hand or automatically at external synthetic protein.For example, can (Foster City CA) synthesizes peptide automatically with the 431A type peptide synthesizer of AppliedBiosystems.Can distinguish proteic each fragment of chemosynthesis the present invention, be connected to produce the molecule of total length with chemical process then.
Utilize freesia FhACS1 albumen of the present invention,, can filter out the interactional material of relevant generation with freesia FhACS1, perhaps acceptor, inhibitor or antagonist etc. by various conventional screening methods.
The present invention clones the synthetic proteic encoding sequence of rate-limiting enzyme ACC synthetic enzyme FhACS1 of the interior ethene of freesia body first and adopts the methods analyst FhACS1 expression of gene pattern of fluorescence real-time quantitative PCR, for utilizing Antisense RNA Technique that antisense ACS gene is imported freesia from now on, thereby the research and development of viewing and admiring the life-span that suppresses the synthetic prolongation freesia flower of ethene in the freesia body provides the foundation, freesia is one of the world's ten big cut-flowers, ornamental value is high, because flower takes place easily wilts, phenomenons such as petal variable color and influence it and view and admire the life-span, so the present invention has great application value.
Embodiment
Below in conjunction with test data in lab embodiments of the invention are elaborated: following examples are being to implement under the prerequisite with the technical solution of the present invention; provided detailed embodiment and process, but protection scope of the present invention is not limited to following embodiment.
The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, Sambrook equimolecular clone for example: the condition described in the laboratory manual (New York: press of cold spring harbor laboratory, 1989), or the condition of advising according to manufacturer.These embodiment only are used to the present invention is described and are not used in and limit the scope of the invention.
Embodiment 1
The clone of freesia ACC synthetic enzyme FhACS1 gene
1. the acquisition of vegetable material
Freesia is cultivated with biological institute oneself by Shanghai Communications University's agricultural.With health, Common Freesia Corm kind of the same size in matrix (perlite: vermiculite: peat 1: 1: 1), when treating that over-ground part is higher than 15cm, prepare DNA extraction or RNA.
2. the extracting of the total DNA of blade
With the total DNA in the CTAB method extracting blade.Get the spire sheet about 50mg, grind to form with grinding rod and add 400 μ L behind the powdery and extract damping fluid, in 65 ℃ of water-bath 45min; The cooling back adds the equal-volume chloroform: primary isoamyl alcohol (24: 1), and softly put upside down mixing and make its emulsification 10min; The centrifugal 10min of 12000rpm (18-20 ℃) draws supernatant in another clean 1.5mL centrifuge tube; Use chloroform: primary isoamyl alcohol (24: 1) as above method repeats extracting once, draws supernatant in another clean 1.5mL centrifuge tube; Add 2 times of isopyknic dehydrated alcohols of supernatant liquor, put upside down mixing; The centrifugal 10min of 12000rpm (18-20 ℃) carefully removes supernatant liquor; With 200 μ L75% ethanol rinsings, this moment is the flaky precipitate of white as seen.With 75% ethanol inhale make a call to two to three times after, carefully the ethanol sucking-off; After several minutes, blot ethanol with the Tip head; Be deposited in 55 ℃ of thermostat containers dry a moment to just occurring adding 50 μ L deionized water dissolvings when translucent, put-20 ℃ of storages.
3.RNA separation
With " the total RNA of leaf extracts test kit on a small quantity " extracted total RNA (RNA prep pure Plant Kit: precious biotechnology (Dalian) company limited product).Identify the RNA quality with the denaturing formaldehyde gel electrophoresis, on spectrophotometer, measure rna content then.
4. the full length sequence of gene is cloned
Utilize homologous genes clone principle, employing Genome Walking method (Genome Walking Kit: carry out the dna sequence dna clone precious biotechnology (Dalian) company limited), divide three phases to carry out:
(1) obtains the gene intermediate segment by PCR
With DNA is template, utilize primers F 1 (CA (A/G) ATGGGCCTCGC (C/A) GAGAA (C/T) CA) and R1 (CCA (G/A) CA (G/A) AAGAGCCCCGC (G/A) TTGC) to carry out PCR, amplification obtains about 1300bp fragment, reclaim and be connected on the pMD18-T Simple vector carrier, with RV-M and M13-47 as universal primer, adopt and stop thing fluorescent mark (Big-Dye, Perkin-Elmer, USA) method, (Perkin-Elmer checks order on USA) at ABI 377 sequenators.Sequencing result GCG software package (Wisconsin group, USA) the existing database of BLAST software search (Genebank) in, the homology of knowing its nucleotide sequence and proteins encoded and known fruitlet Musa ACC synthase gene is very high, so think that tentatively it is an ACC synthase gene.
(2) the method amplification 3 ' end sequence of usefulness Genome Walking
The three-wheel nest-type PRC is finished the amplification of 3 ' end sequence.
The first round: AP+3R3 (5 '-ACTCGGCACGACGATAAACA-3 ')
Second takes turns: AP+3R2 (5 '-AGCCAAAGACATCCATTTAGTCG-3 ')
Third round: AP+3R1 (5 '-CGGCGGCTACCAAGATGTCA-3 ')
Obtain FhACS1 3 ' end sequence, reclaim, be connected on the pMD18-T Simple vector carrier, with RV-M and M13-47 as universal primer, adopt thing fluorescent mark (Big-Dye, Perkin-Elmer, method USA) of stopping, (Perkin-Elmer checks order on USA) at ABI 377 sequenators.(Wisconsin group, USA) the existing database of BLAST software search (Genebank) in know that the homology of its nucleotide sequence and proteins encoded and fruitlet bajiao banana ACC synthase gene is higher to sequencing result with the GCG software package.
(3) the method amplification 5 ' end sequence of usefulness Genome Walking
The three-wheel nest-type PRC is finished the amplification of 5 ' end sequence.
The first round: AP+5F3 (5 '-CAAATTATTTATGTGCATGATGCATGC-3 ')
Second takes turns: AP+5F2 (5 '-CGAGGCAGAACATGAGGGTCTCGTG-3 ')
Third round: AP+5F1 (5 '-CACCCATGAATTTTGCCAATGCCTG-3 ')
Obtain FhACS1 5 ' end sequence, use with top the same method after recovery connects and check order.
The sequencing result that above-mentioned 3 kinds of methods are obtained carries out the overlap splicing, obtains splicing sequence and submits to BLAST to analyze.Result's proof FhACS1 gene that new clone obtains from freesia really is a gene relevant with the ACC synthetic enzyme.With ORFFingding (http://www.ncbi.nlm.nih.gov/gorf) the webpage prediction of sequencing result, found the initiator codon and the terminator codon of freesia FhACS1 gene in conjunction with NCBI.The FhACS1 full length gene 2576bp that is obtained (SEQ ID NO.1), 5 ' non-coding region has 433bp, and 3 ' non-coding region has 373bp; Its initiator codon is positioned at 434nt, and terminator codon is positioned at 2201nt; This gene has 3 introns, 4 exons.The position of intron is: 578-667nt, 805-1009nt, 1174-1277nt.
(4) acquisition of FhACS1 gene C DS opening code-reading frame sequence
With freesia petal RNA is template, and it is standby to obtain cDNA with " PrimeScript II 1st Strand cDNA Synthesis Kit " (precious biotechnology (Dalian) company limited) reverse transcription.According to the above gene order that obtains, begin to design upstream Auele Specific Primer ORF-F (5 '-ATGAATCTCTCTGCAAAAGCTAC-3 ') from initiator codon, the downstream primer ORF-R (5 '-CCTCTCGGCTTTCCGATCAGTCG-3 ') that comprises terminator codon in 3 ' end design, with freesia cDNA is that template is carried out RT-PCR, amplification obtains the long CDS opening code-reading frame sequence of 1371bp, is the proteic encoding sequence of FhACS1 (SEQ ID NO.2).
Embodiment 2
The sequence information of freesia FhACS1 gene and homology analysis
The new freesia FhACS1 total length CDS open reading frame sequence of the present invention is 1371bp, and detailed sequence is seen SEQ ID NO.2.Derive the aminoacid sequence of freesia FhACS1, totally 457 amino-acid residues according to CDS opening code-reading frame sequence.Detailed sequence is seen SEQ ID NO.3.
The CDS open reading frame sequence of freesia FhACS1 and the aminoacid sequence of proteins encoded thereof are carried out Nucleotide and protein homology retrieval with blast program in Non-redundantGenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS translations+PDB+SwissProt+Superdate+PIR database, found that it and fruitlet bajiao banana ACS gene (AB021907.1) have 73% homogeny (subordinate list 2) on nucleotide level; On amino acid levels, it and fruitlet bajiao banana ACS gene (AAQ13435.1) also have 73% consistence and 84% similarity (subordinate list 3).This shows that all there are higher homology in freesia FhACS1 gene and fruitlet bajiao banana ACS gene on nucleic acid still is protein level.
Embodiment 3
Freesia FhACS1 gene in each etap flower expression amount and flower in differential expression in the petal, stamen, gynoecium.
1. the acquisition of material: when the bud or flower that occurs each etap on the freesia inflorescence piece, take its whole flower (the flower compound sample of each etap) and isolating colored portion organ stamen, gynoecium, petal (compound sample of each etap) in the field, drop at once in the liquid nitrogen after sample wrapped with aluminium platinum paper respectively, then change stored for future use in-80 ℃ of Ultralow Temperature Freezers over to.
2.RNA extraction: utilize RNAprep pure Plant Kit (precious biotechnology (Dalian) company limited product) to extract the RNA in petal, stamen, the gynoecium in freesia whole flower and the flower.
3.RNA integrity and the determining of concentration: with plain agar sugar gel electrophoresis (gum concentration 1.2%; 0.5 * TBE electrophoretic buffer; 150v 15min) detects integrity.The formaldehyde electrophoresis of complete RNA can be observed 28S and 18S two bands significantly, and 28S approximately is that the 1.5-2.0 of 18S is doubly wide.If two bands are not obvious, illustrate that then RNA partly degrades.Purity is RNA preferably, A
260/ A
280About=2.0, A
260/ A
230About=2.0.With spectrophotometric determination OD value and calculate rna content: 1 OD
260=40 μ g/mL.
4.cDNA acquisition: the total RNA with 500ng is a template, according to the precious TaKaRa PrimeScript of biotech firm
TMIt is standby that RT reagentKit Perfect Real Time test kit operation instructions is carried out reverse transcription acquisition cDNA.
5. the design Auele Specific Primer is to carry out the expression amount of real-time fluorescence quantitative PCR analyzing gene in each organ and tissue.According to the freesia FhACS1 gene order that has obtained, utilize primer-design software primer premier 5.0 to be designed for the Auele Specific Primer that the FhACS1 gene quantification is analyzed among the Real-time PCR, the primer name is called Q-F (5 '-CCCAA GGAGA AATGGAGCTA-3 '), Q-R (5 '-ATTCG ACGAA CGACG TAAGC-3 ').Internal control gene is Actin, and its Auele Specific Primer is A-F (5 '-CATGA AGATC CTGACGGAGA-3 '), A-R (5 '-GAGTT GTAGG TGGTC TCATG-3 ').
6. make the typical curve of goal gene and internal control gene.With EASY Dilution (test kit provides) standard substance cDNA solution is carried out gradient dilution, be template with the cDNA solution after the dilution respectively then, Auele Specific Primer with goal gene and internal control gene carries out the Real-time pcr amplification, and reaction finishes the back and draws solubility curve and typical curve.Analyze solubility curve, judge whether the solubility curve of goal gene and internal control gene obtains simple spike, use this primer can obtain single pcr amplification product to judge.
7. the real time fluorescent quantitative analysis of goal gene in the testing sample.With synthetic cDNA article one chain is template, carries out quantitative fluorescence analysis with the primer amplified of goal gene and internal reference gene respectively, and Real-time PCR is reflected on the BIO-RAD Chromo 4 real-time quantitative instrument and carries out, and reaction system is 20 μ L.Three-step approach is adopted in reaction, 95 ℃ of sex change 20s, then 41 circulations: 95 ℃ of 15s; 60 ℃ of 15s; 72 ℃ of 25s.Whether after each amplification is finished, all do solubility curve, be special generation with the check amplified production.
8. adopt two calibration curve methods to make relative quantitative assay.The result shows that the expression amount of FhACS1 gene in each etap flower presents the significance difference opposite sex: wherein, FhACS1 expression of gene amount is higher when flower is opened with most of the opening fully, the highest when open fully, be 3.29 times of green small bud, for minimum expression phase (bud tapel colour developing stage) expression amount 32.6 times illustrate that the aging of this expression of gene and flower has tangible dependency.The FhACS1 gene is respectively stamen＞gynoecium＞petal from more to less at the expression amount of stamen, gynoecium, petal; FhACS1 expression of gene amount is respectively 2.62,0.46 times of expression amount in the petal in stamen, the gynoecium, and there were significant differences with expression amount in the petal, illustrates that this expression of gene is the closest with the aging relation of flower in the stamen.
Claims (4)
1. freesia ACC synthetic enzyme FhACS1 albumen coded sequence, it is characterized in that, comprise and have the nucleotide sequence and polypeptide shown in the SEQ ID NO.2 with the aminoacid sequence shown in the SEQ ID NO.3 from Nucleotide 1-1371 position, or its conservative property variation polypeptide or its active fragments, or its reactive derivative.
2. freesia ACC synthetic enzyme FhACS1 albumen coded sequence according to claim 1, it is characterized in that, described encoding sequence has the nucleotide sequence of the polypeptide of freesia polypeptide FhACS1 protein-active, as 1-1371 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.2.
3. freesia ACC synthetic enzyme FhACS1 albumen coded sequence according to claim 2, it is characterized in that, described degenerate sequence is meant, be arranged in the 1-1371 position Nucleotide of SEQ ID NO.2, having one or more codons to be encoded, the degenerate codon of same amino acid replaces the back and the sequence that produces.
4. the nucleic acid molecule as hybridization according to claim 2 is characterized in that, described polypeptide FhACS1, and it comprises: the polypeptide with SEQ ID NO.3 aminoacid sequence.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114107268A (en) * | 2021-11-09 | 2022-03-01 | 南开大学 | Single-enzyme activity mutant prepared by ACS double-enzyme activity key site mutation of higher plants and application thereof |
| CN114940995A (en) * | 2022-05-10 | 2022-08-26 | 浙江大学 | Persimmon RNA binding protein DkRBM24-1 and application thereof |
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2010
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114107268A (en) * | 2021-11-09 | 2022-03-01 | 南开大学 | Single-enzyme activity mutant prepared by ACS double-enzyme activity key site mutation of higher plants and application thereof |
| CN114107268B (en) * | 2021-11-09 | 2023-11-14 | 南开大学 | Single enzyme activity mutant prepared by mutation of ACS double enzyme activity key sites of higher plants |
| CN114940995A (en) * | 2022-05-10 | 2022-08-26 | 浙江大学 | Persimmon RNA binding protein DkRBM24-1 and application thereof |
| CN114940995B (en) * | 2022-05-10 | 2023-11-14 | 浙江大学 | Persimmon RNA binding protein DkRBM24-1 and application thereof |
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