CN1528895A - Cabbage type rape drought-resistant salinity-tolerant transcription factor BnHB6 protein coding seuquence - Google Patents
Cabbage type rape drought-resistant salinity-tolerant transcription factor BnHB6 protein coding seuquence Download PDFInfo
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Abstract
The invention is a kind of cabbage type rape drought resisting salt-enduring rerecord factor BnHB6 protein coding series, which belongs to gene project field. It includes: coding polypeptide nucleotide series which has cabbage type rape BnHB6 protein activity and at least 70% of the series has consanguinity to 207-1142 bit series of SEQ ID NO.3 nucleotide; or it can cross with 207-1142 bit nucleotide series of SEQ ID NO.3. the invention has prominent effect in drought resisting and salt-enduring, it has large application value. It can decrease the loss of plant production caused by drought or semiarid environment, alkaline.
Description
Technical field
What the present invention relates to is a kind of albumen coded sequence, and particularly a kind of cabbage type swede type rape drought resistance and salt tolerance transcription factor BnHB6 albumen coded sequence belongs to the genetically engineered field.
Background technology
Modern age the terrestrial climate environment variation and the activity of human beings desert, the face of land and the semidesertization that cause, and the existence on big area arid and semi-arid area and saline and alkaline beach ground requires to have the varieties of crops that are suitable for planting in these areas can constantly obtain popularization.To be exactly that the gene pairs farm crop that will show salt-tolerant drought-resistant carry out genetic engineering modified when previous important means, to obtain the drought-enduring new crop varieties of anti-salt.Plant transcription factor can start relevant salt-tolerant drought-resistant gene and proteic expression improves the salt-tolerant drought-resistant of plant.Comprise with source capsule district-leucine zipper (Homeodomain leucine-zipper) (HD-Zip) zone transcription factor be a protein families of finding in the plant.This gene contains one with source capsule zone and 6 zippers that leucine is formed, can be combined in DNA and go up the expression that starts downstream gene, be one roughly the same source capsule (Homeobox) zone be HB in conjunction with the albumen of (Binding) so get this gene of first letter designation.This type of transcription factor plays a significant role in growth, growth and the degeneration-resistant reaction of plant.Transcription factor HB6 gene all has tangible enhancing to express under the different adverse circumstance environment of plant, and starts the relevant salt-tolerant drought-resistant expression of gene in downstream, is important regulatory gene in the conduction of plant ABA signal.
Find in the retrieval of prior art document: magazine " Plant Molecular Biology (molecular biology of plants) " is 1999,40:1073-1083 has delivered article " The HD-Zip gene ATHB6 in Arabidopsisis expressed in developing leaves; roots and carpels and up-regulated by waterdeficit conditions ", and (Arabidopis thaliana HD-Zip gene A THB6 is the leaf of growing, express in root and the carpel and be subjected to moisture to lack just to regulate and control) "; though the document to gene A THB6 the expression under drought stress is handled affirm fully; be the positively related gene of dormin ABA abduction delivering; report up till now shows the salt tolerant of the transgenic arabidopsis plant of this gene; the effect of drought resisting and disease-resistant worm aspect does not obviously improve, find to have the report of close ties document in addition so far as yet with theme of the present invention.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, a kind of swede type rape drought resistance and salt tolerance transcription factor BnHB6 albumen coded sequence is provided, make it have tangible effect, can obviously be reduced in the loss of various farm crop on biological yield that arid and saline and alkaline half saline-alkali soil are planted on the ground at drought resistance and salt tolerance.
The present invention is achieved by the following technical solutions:
The invention provides a kind of isolated dna molecular, this molecule comprises: coding has the nucleotide sequence of the polypeptide of swede type rape BnHB6 protein active, and shows at least 70% homology from the nucleotides sequence of Nucleotide 207-1142 position among described nucleotide sequence and the SEQ ID NO.3; Perhaps described nucleotide sequence can with SEQ IDNO.3 in from the nucleotide sequence hybridization of Nucleotide 207-1142 position.Preferably, described encoding sequence has the polypeptide of the aminoacid sequence shown in the SEQID NO.3, or its conservative property variation polypeptide or its active fragments, or its reactive derivative.More preferably, described encoding sequence has among the SEQ ID NO.3 nucleotide sequence from Nucleotide 207-1142 position.
The isolated swede type rape HD-ZIP of the present invention class related protein polypeptide BnHB6, it comprises: the polypeptide with SEQID NO.3 aminoacid sequence, preferably, this polypeptide is to have SEQ ID NO.3 polypeptide of sequence, this polypeptide can all can play a role under salt, drought, waterflooding, wound, active oxygen and Whitfield's ointment are handled, and Whitfield's ointment is the signaling molecule of plant disease-resistant worm.
Carrier provided by the invention, it comprises above-mentioned dna molecular.A kind of nucleic acid molecule, it comprises 8-100 continuous nucleotide in the described dna molecular.
The present invention is with above-mentioned dna molecular transformed host cells, and it is an eukaryotic cell.This host cell is an Arabidopis thaliana in example.
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.
In the present invention, term " swede type rape drought resistance and salt tolerance albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with swede type rape BnHB6 protein-active is as 207-1142 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.3.This degenerate sequence is meant, is arranged in the encoder block 207-1142 position Nucleotide of SEQ ID NO.3 sequence, 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.3 in 207-1142 position nucleotide sequence homology be low to moderate about 70% the degenerate sequence described sequence of SEQ ID NO.3 of also encoding out.This term also comprises can be under the moderate stringent condition (70%-85% consistence), better under the height stringent condition among (85% above consistence) and the SEQ ID NO.3 from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 207-1142 position.This term also comprise with SEQ ID NO.3 in from the homology of nucleotide sequence at least 70% of Nucleotide 207-1142 position, preferably at least 80%, more preferably at least 90%, at least 95% nucleotide sequence best.
This term also comprises encoding to have the variant form of open reading frame sequence among the proteic SEQ IDNO.3 with natural swede type rape BnHB6 identical function.These variant forms comprise (but being not limited to): several (are generally 1-90, preferably 1-60, more preferably 1-20,1-10 best) disappearance, insertion and/or the replacement of Nucleotide, and several (are generally in 60 to hold interpolation 5 ' and/or 3 ', preferably being in 30, more preferably is in 10, is in 5 best) Nucleotide.
In the present invention, term " swede type rape drought resistance and salt tolerance albumen or polypeptide " refers to have the SEQ ID NO.3 polypeptide of sequence of swede type rape BnHB6 protein-active.This term also comprises having and the variant form relevant identical function of natural swede type rape BnHB6, SEQ ID NO.4 sequence.These variant forms comprise (but being not limited to): several (are generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) 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 swede type rape BnHB6 and reactive derivative.
The variant form of swede type rape BnHB6 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 swede type rape BnHB6 under high or low rigorous condition are coded and the polypeptide or the albumen that utilize the antiserum(antisera) of swede type rape BnHB6 polypeptide to obtain.
In the present invention, " swede type rape BnHB6 conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID NO.3, have 10 at the most, preferably at the most 8, more preferably 5 amino acid is replaced by similar performance or close amino acid and is formed polypeptide at the most.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
85％?identity?in?204-1142nt?overlap
Query：204??TTGATGATGAAGAGATTAAGCAGTTCAGATTCAGTGGGTGGTCTCATCTCTTTATGTCCC?263
||||||||||||||||||||?||||||||||||||||||||||||||||||||||||||
Sbjct：216??TTGATGATGAAGAGATTAAGTAGTTCAGATTCAGTGGGTGGTCTCATCTCTTTATGTCCT?275
Query：264??ACTACTTCCACAGATCAGCCGAGTCCAAGAAGATACGG---GAGAGAATTTCAGTCGATG?320
||?||||||||||||?|||?||||||?||?||||||||???||||||?||||||||||||
Sbjct：276??ACAACTTCCACAGATGAGCAGAGTCCGAGGAGATACGGTGGGAGAGAGTTTCAGTCGATG?335
Query：321??CTTGAAGGTTACGAGGAGGAAGAAGAAGAAGCCATAACCGAGGAAAGAGGACAAACCGGT?380
|||||||||||||||||???||||||||||||?|||???||?|||||||||||????||
Sbjct：336??CTTGAAGGATACGAGGA---AGAAGAAGAAGCTATAGTAGAAGAAAGAGGACACGTGGGC?392
Query：381??TTAGCCGAGAAGAAGAGACGGTTAAGCATTAACCAAGTTAAAGCCTTGGAGAAAAATTTC?440
||??|?||||||||||||?|||||||||||||||||||||||||?||||||||?|||||
Sbjct：393??TTGTCGGAGAAGAAGAGAAGGTTAAGCATTAACCAAGTTAAAGCTTTGGAGAAGAATTTT?452
Query：441??GAGTTAGAGAACAAGCTTGAGCCCGAGAGGAAAGTGAAGCTAGCTCAAGAACTTGGTCTC?500
|||||||||||?|||||||||||?|||||||||||?|||?|||||||||||||||||||
Sbjct：453??GAGTTAGAGAATAAGCTTGAGCCTGAGAGGAAAGTTAAGTTAGCTCAAGAACTTGGTCTT?512
Query：501??CAACCTCGTCAAGTAGCTGTTTGGTTTCAGAACCGCCGTGCGCGGTGGAAGACAAAACAG?560
||||||||||||||?||||||||||||||?|||||?|||||?||||||||||||||||||
Sbjct：513??CAACCTCGTCAAGTTGCTGTTTGGTTTCAAAACCGTCGTGCTCGGTGGAAGACAAAACAG?572
Query：561??CTCGAGAAAGATTACGGTGTTCTCAAAACGCAGTACGATTCTCTCCGCCATAACTTCGAT?620
||?||||||||||||||||||||?|||||?|||||||||||||||||?||||||||?|||
Sbjct：573??CTTGAGAAAGATTACGGTGTTCTTAAAACCCAGTACGATTCTCTCCGTCATAACTTTGAT?632
Query：621??TCCCTCCGCCGTGACAATGAATCTCTTCTTCAAGAGATCGGTAAACTAAAAGCTAAGCTA?680
||||||||||||||||||||||||||?|||||||||||??|||||||?|||?|?|||||
Sbjct：633??TCCCTCCGCCGTGACAATGAATCTCTCCTTCAAGAGATTAGTAAACTGAAAACGAAGCTT?692
Query：681??AACGGAGAAGAAGAAGTTGAAGAAGATGATGAAGATGAAGAGAACAAC---GCGGTCACG?737
||?||||?||?||?|???|||||||??????||||?||||||||||||??|||||||||
Sbjct：693??AATGGAGGAGGAGGA---GAAGAAG------AAGAAGAAGAGAACAACGCGGCGGTGACA?743
Query：738??ATGGAGAGTGATGTTTCCGTCAAGGAAGAAGAAGTTTCGTTGCCGGAGGAGCTTACAGAT?797
|?||||||||||?||||?|||||||?||||||||||||||||||||||?||?|||||||
Sbjct：744??ACGGAGAGTGATATTTCGGTCAAGGAGGAAGAAGTTTCGTTGCCGGAGAAGATTACAGAG?803
Query：798??CCGCCGTCTTCTCCTCCGCAGCTTCTAGAGCATTCCGACAGTTTCAATTACCGGAGTTTC?857
|?|||||?||||||||?|||?||||?||?|||||?||??||?|?|||||||||||||||
Sbjct：804??GCACCGTCGTCTCCTCCACAGTTTCTTGAACATTCTGATGGTCTTAATTACCGGAGTTTC?863
Query：858??ACCGACCTCCGCGACCTTCTTCCGTTAAAGGCCGCGGCTTCCTC---CGTCGCCGCCGCT?914
||?||?||?||?||?||||||||?||||||||?||||||||?||???||?||||||?|||
Sbjct：864??ACAGATCTACGTGATCTTCTTCCATTAAAGGCGGCGGCTTCTTCATTCGCCGCCGCAGCT?923
Query：915??GGATCGTCGGACAGTAGCGATTCGAGCGCCGTGTTGAACGAGGAAAGTAGCTCTAACGTT?974
|||||?||?||||||||||||||?|||||??||?||||?||?|||||?||||||||?||
Sbjct：924??GGATCTTCAGACAGTAGCGATTCAAGCGCTCTGCTGAATGAAGAAAGCAGCTCTAATGTC?983
Query：975??AC---GGCGGCTCCGGCGACGGTTCCCCGGCGGCAGTTTCTTGCAGTTTGTGAAAATGGA?1031
||???|||||||||||?|||||||?||?||?||?|?||||||?|||||||||||||||||
Sbjct：984??ACTGTGGCGGCTCCGGTGACGGTT-CCAGGAGGTAATTTCTTCCAGTTTGTGAAAATGGA?1042
Query：1032?GCAGACGGAGGATCACGACGACTTTCTGAGTGGAGAAGAAGCGTGCGGGTTTTTCTCCGA?1091
|||||||||||||||?||?|||||||||||||||||||||||?||?|??||?||?|||||
Sbjct：1043?GCAGACGGAGGATCATGAGGACTTTCTGAGTGGAGAAGAAGCTTGTGAATTCTTTTCCGA?1102
Query：1092?TGAACAGCCACCGTCTCTGCACTGGTATTCCACCGTTGATCAGTGGAA?1139
||||||?||?||||||||?||||||||?||||||||||||||?|||||
Sbjct：1103?TGAACAACCGCCGTCTCTACACTGGTACTCCACCGTTGATCATTGGAA?1150
Query：1321?TTTCGTTCAGTTCTTGTAATTAAGGATCAT?1350
||||||?||?||||||||||||||||||||
Sbjct：1346?TTTCGTGCACTTCTTGTAATTAAGGATCAT?1375
Query: the nucleotide sequence of swede type rape BnHB6
Sbjct: the nucleotide sequence of Arabidopis thaliana AtHB6 (AY063819)
Table 2 is that the homology of swede type rape BnHB6 albumen of the present invention and the proteic nucleotide sequence of Arabidopis thaliana AtHB6 compares (GAP) table.
Table 3
86％?identity?in?311aa?overlap，90％?similarity?in?311aa?overlap
Query：1???MMKRLSSSDSVGGLISLCPTTSTDQPSPRRY-GREFQSMLEGYEEEEEEAITEERGQTGL?59
MMKRLSSSDSVGGLISLCPTTSTD+?SPRRY?GREFQSMLEGY?EEEEEAI?EERG??GL
Sbjct：1???MMKRLSSSDSVGGLISLCPTTSTDEQSPRRYGGREFQSMLEGY-EEEEEAIVEERGHVGL?59
Query：60??AEKKRRLSINQVKALEKNFELENKLEPERKVKLAQELGLQPRQVAVWFQNRRARWKTKQL?119
+EKKRRLSINQVKALEKNFELENKLEPERKVKLAQELGLQPRQVAVWFQNRRARWKTKQL
Sbjct：60??SEKKRRLSINQVKALEKNFELENKLEPERKVKLAQELGLQPRQVAVWFQNRRARWKTKQL?119
Query：120?EKDYGVLKTQYDSLRHNFDSLRRDNESLLQEIGKLKAKLNGEEEVEEDDEDEENNAVTME?179
EKDYGVLKTQYDSLRHNFDSLRRDNESLLQEI?KLK?KLNG????EE++E+????AVT?E
Sbjct：120?EKDYGVLKTQYDSLRHNFDSLRRDNESLLQEISKLKTKLNGGGGEEEEEENNA--AVTTE?177
Query：180?SDVSVKEEEVSLPEELTDPPSSPPQLLEHSDSFNYRSFTDLRDLLPLK-AAASSVAAAGS?238
SD+SVKEEEVSLPE++T+?PSSPPQ?LEHSD??NYRSFTDLRDLLPLK?AA+S??AAAGS
Sbjct：178?SDISVKEEEVSLPEKITEAPSSPPQFLEHSDGLNYRSFTDLRDLLPLKAAASSFAAAAGS?237
Query：239?SDSSDSSAVLNEESSSNVT-AAPATVPRGSFLQFVKMEQTEDHDDFLSGEEACGFFSDEQ?297
SDSSDSSA+LNEESSSNVT?AAP?TVP?G+F?QFVKMEQTEDH+DFLSGEEAC?FFSDEQ
Sbjct：238?SDSSDSSALLNEESSSNVTVAAPVTVPGGNFFQFVKMEQTEDHEDFLSGEEACEFFSDEQ?297
Query：298?PPSLHWYSTVDQWN?311
PPSLHWYSTVD?WN
Sbjct：298?PPSLHWYSTVDHWN?311
Query: the aminoacid sequence of swede type rape BnHB6
Sbjct: the aminoacid sequence of Arabidopis thaliana AtHB6 (S47136)
Table 3 is that the homology of the aminoacid sequence of swede type rape BnHB6 albumen of the present invention and Arabidopis thaliana AtHB6 compares (FASTA) table.Wherein, identical amino acid marks with the amino acid monocase between two sequences.
The analogue of invention swede type rape BnHB6 albumen or polypeptide.The difference of these analogues and natural swede type rape BnHB6 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 that exemplifies.
(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, can select various carrier known in the art for use, the carrier as commercially available comprises plasmid, clay etc.When producing swede type rape BnHB6 polypeptide of the present invention, swede type rape BnHB6 encoding sequence operationally can be connected in expression regulation sequence, thereby form swede type rape BnHB6 protein expression vector.
As used herein, " operationally being connected in " refer to a kind of like this situation, and promptly some part of linear DNA sequence can influence the activity of same other parts of linear DNA sequence.For example, if signal peptide DNA as precursor expression and participate in the secretion of polypeptide, signal peptide (secretion leader sequence) DNA operationally is connected in polypeptid DNA so; If transcribing of promotor control sequence, it is operationally to be connected in encoding sequence so; When if ribosome bind site is placed in the position that can make its translation, it is operationally to be connected in encoding sequence so.Generally, " operationally being connected in " means adjacent, then means in reading frame adjacent for the secretion leader sequence.
In the present invention, term " host cell " is an eukaryotic cell.Eukaryotic host cell commonly used comprises yeast cell, tobacco cell and other vegetable cell.
Whether and quantity the expression of also available Northern blotting technical Analysis swede type rape BnHB6 gene product, the existence of rna transcription thing in cell of promptly analyzing swede type rape BnHB6.
In addition, the nucleic acid molecule that can be used as probe provided by the invention, this molecule have 8-100 continuous nucleotide of swede type rape BnHB6 nucleotide coding sequence usually, preferably have 15-50 continuous nucleotide.This probe can be used for whether existing in the test sample the relevant nucleic acid molecule of coding swede type rape BnHB6.
The method that whether has swede type rape BnHB6 related nucleotide sequences in the test sample of the present invention, it comprises with above-mentioned probe and sample and hybridizing whether detection probes combination has taken place then.Preferably, this sample is the product behind the pcr amplification, and wherein the pcr amplification primer is corresponding to swede type rape BnHB6 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 swede type rape BnHB6 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 swede type rape BnHB6 or homologous protein.
In order to obtain the dot matrix of the swede type rape cDNAs relevant with swede type rape BnHB6 genes involved, can screen swede type rape cDNA library with dna probe, these probes are under low rigorous condition, use
32P relevant all or part of of swede type rape BnHB6 cooked the radioactivity mark and.The cDNA library that most is suitable for screening is the library from swede type rape.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, Palo Alto, Cal..This screening method can be discerned the nucleotide sequence of the gene family relevant with swede type rape BnHB6.
Swede type rape BnHB6 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 is produced (people such as Stewart, (1969) Solid-Phase Peptide Synthesis, WH FreemanCo., San Francisco by direct peptide synthesis; 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 Applied Biosystems.Can distinguish proteic each fragment of chemosynthesis the present invention, be connected to produce the molecule of total length with chemical process then.
Utilize swede type rape BnHB6 albumen of the present invention,, can filter out the interactional material of relevant generation with swede type rape BnHB6, perhaps acceptor, inhibitor or antagonist etc. by various conventional screening methods.
The present invention has substantive distinguishing features and marked improvement, and the present invention has tangible effect at drought resistance and salt tolerance, can obviously be reduced in the loss of various farm crop on biological yield that arid and saline and alkaline half saline-alkali soil are planted on the ground.Have large-area arid and semi-arid, saline and alkaline half saline and alkaline area on the earth, therefore, the present invention has very big using value.
Embodiment
Below in conjunction with the concrete data of laboratory test, further set forth the present invention with specific embodiment:
The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, for example the Sambrook equimolecular is cloned: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, 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 swede type rape BnHB6 gene
1. separate tissue (isolation)
Swede type rape (kind is " a Shanghai oil 15 ") is buied from the market, places 28 ℃ to germinate 24 hours swede type rape, is seeded in then in the greenhouse, when treating that the swede type rape blade is the 3-5 sheet, prepares DNA extraction or RNA.
2.RNA separation (RNA isolation)
Get portion of tissue, grind, add the 1.5mL EP pipe that fills lysate, fully after the vibration, move in the glass homogenizer again with mortar.Move to after the homogenate in the 1.5mL EP pipe, and extracted total RNA (TRIzol Reagents, GIBCOBRL, USA).Identify total RNA quality with the denaturing formaldehyde gel electrophoresis, on spectrophotometer, measure rna content then.
3. the full-length clone of gene (Cloning of Full-length cDNA)
According to the amino acid conserved sequence of Arabidopis thaliana AtHB6 gene, utilize homologous genes clone principle, adopt RACE method (GibcoBRL test kit) to carry out the cDNA full-length clone, divide three phases to carry out:
(1)RT-PCR
PCR[HB001 (SEQ ID NO.1)+HB002 (SEQ ID NO.2)] obtain 2003BP (761bp), reclaim, be connected on the pGEMT-Easy carrier, with SP6 or T7 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.Sequencing result GCG software package (Wisconsin group, USA) BLAST in and the existing database of FASTA software search (Genebank+EMBL), the homology of knowing its nucleotide sequence and proteins encoded and known cress such as Arabidopis thaliana (Arabidopsis thaliana) AtHB6 gene is very high, so think that tentatively it is a HD-ZIP gene.
(2)3’-RACE
PCR[AP+HB301 (5 '-CGCCGCTGGATCGTCGGACA-3 ')] obtain HB3 ' (474bp), reclaim, be connected on the T-Easy carrier, with SP6 or T7 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.Sequencing result GCG software package (Wisconsin group, USA) BLAST in and the existing database of FASTA software search (Genebank+EMBL), the homology of knowing its nucleotide sequence and proteins encoded and known cress such as Arabidopis thaliana (Arabidopsis thaliana) AtHB6 gene is very high, so think that tentatively it is one and HD-ZIP genes involved.
(3).5’-RACE
First round PCR[AAP+RD501 (5 '-GTGATCCTCCGTCTGCTCCA-3 ')]
Second takes turns PCR[(AUAP+RD502 (5 '-TCCGGCAACGAA (A/T) CTTCTTC-3 ')) obtain HB5 ' (about 233bp) (process with (1))
With the overlap splicing of sequencing result, the fragment that discovery procedure (1) obtains is the complete coding region of this gene.
The gene that result's proof of BLAST newly obtains from swede type rape really is a gene relevant with Arabidopis thaliana AtHB6.
By being used in combination above-mentioned 3 kinds of methods, obtained candidate's the proteic complete encoding sequence of swede type rape BnHB6 (SEQ ID NO.3).
Embodiment 2
The sequence information and the homology analysis of swede type rape BnHB6 gene
The length of the swede type rape BnHB6 full-length cDNA that the present invention is new is 1572bp, and detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 207-1142 position Nucleotide (935 Nucleotide).Derive the aminoacid sequence of swede type rape BnHB6 according to full-length cDNA, totally 311 amino-acid residues, molecular weight is 35176.49 dalton, iso-electric point (pI) is 4.57.Detailed sequence is seen SEQ ID NO.3.
Full length cDNA sequence and the coded protein thereof of swede type rape BnHB6 are carried out Nucleotide and protein homology retrieval with blast program in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDStranslations+PDB+SwissProt+Superdate+PIR database, found that it and arabidopsis gene AtHB6 have 85% homogeny on nucleotide level, (subordinate list 2); On amino acid levels, it and arabidopsis gene AtHB6 (S47136) also have 86% homogeny (subordinate list 3).This shows that there are higher homology in swede type rape gene BnHB6 and arabidopsis gene AtHB6 on nucleic acid still is protein level.Arabidopsis gene AtHB6 (S47136) has been proved to be obviously to strengthen under drought condition and has expressed, and brings into play the effect of wanting emphatically, can think that swede type rape gene BnHB6 also has similar effect aspect drought resistance and salt tolerance.
Embodiment 3
Swede type rape gene BnHB6 albumen or polypeptide carry out the salt tolerance of drought of eukaryotic cell expression and transfer-gen plant in Arabidopis thaliana identifies
The structure that contains the expression vector of goal gene (swede type rape gene BnHB6)
Full length sequence (SEQ ID NO.3) according to swede type rape gene BnHB6, design amplifies the primer that complete coding is read frame, and on the upstream and downstream primer, introduce restriction endonuclease sites (this is decided by the carrier of selecting for use) respectively, so that construction of expression vector.Amplified production with acquisition among the embodiment 1 is a template, behind pcr amplification, with swede type rape gene BnBDCl gene cDNA clone to intermediate carrier (as pBluescript), further be cloned into binary expression vector (as pBI121 and improved pCAMBIA2300), guaranteeing to identify good expression vector under the correct prerequisite of reading frame, again it is changed in the Agrobacterium, utilize leaf dish law technology transformation mode plant Arabidopis thaliana.
1. send out seedling: seed is with rinsed with sterile water 15-20 minute, uses 70% ethanol disinfection again 1 minute, sterilizes 10-12 minute with 0.1% mercuric chloride then.Use aseptic water washing 5 times at last again.Washed seed is blotted with thieving paper, put into the MS substratum.Illumination cultivation 5 days is treated that seedling is long just can cut seedling to 4-5 centimetre.
2. cut seedling: clip 0.5-1 centimetre hypocotyl small segment is put into pre-culture medium, cultivates 2 days.
Pre-culture medium: MS+6BA (0.2mg/l)+2.4D (1.2mg/l)
3. transform altogether and cultivate: the hypocotyl that will cultivate in advance 2 days is put into prior cultured bacterium liquid (OD value 0.4-0.6) and infected 3-5 minute.Then take out and put into the dark cultivation of pre-culture medium 2 days.
4. screening and culturing: cultivate altogether finish after, explant is put into screening culture medium.2 all subcultures once.The differentiation of callus formation and bud is arranged in 2-4 week.After treating green bud length to 2 centimetre, cutting-out is taken root.
Screening culture medium: MS+6BA (4.5mg/l)+NAA (0.1mg/l)+AgNO3 (6mg/l)+cb (250mg/l)+Kan (20mg/l)
Root media: MS+NAA (0.5mg/l)+cb (250mg/l)+Kan (5mg/l)
5. transformed plant is cultivated; After treating well developed root system, plant is taken out, clean the solid medium that adheres to, move in the soil, just begun to treat to take off lens again behind the robust plant, cultivate in the greenhouse with lens cover several days with sterilized water.
The salt tolerance of drought that contains the transgenic arabidopsis plant of swede type rape gene BnHB6 is identified
In view of coding BnHB6, be proved to be under the drought resistance and salt tolerance condition as the AtHB6 gene of Arabidopis thaliana and played a role, and the AtHB6 of swede type rape gene BnHB6 transcriptional level and Arabidopis thaliana has higher homology, can further carry out drought resistance and salt tolerance to the transgenic arabidopsis plant that contains swede type rape gene BnHB6 and identify.Seed (2 days, 7 days, 14 days, 21 days, 28 days) the back research BnHB6 gene of handling transfer-gen plant and transfer-gen plant with 300mM sodium-chlor and 300mM treatment with mannitol in transfer-gen plant expression and various processing to the growing state of plant.Northern blot analytical results proves that transfer-gen plant BnHB6 transcriptional level its expression amount after salt and treatment with mannitol significantly strengthens, though and the raising of contrast non-transgenic plant expression amount is starkly lower than transfer-gen plant.The non-transgenic plant strain growth is slow in addition, and is withered and dead under salt and treatment with mannitol at last, and transgenic plant still can normal growth, and are just slow slightly than undressed plant strain growth.In anti-anaerobism, anti-oxidant and disease-resistant test, transfer-gen plant also obviously shows comparison according to (not transfer-gen plant) growth normally.This proof swede type rape gene BnHB6 gene has more effective and the function in degeneration-resistant border widely than Arabidopis thaliana gene A tHB6, will can be used for utilizing in the research and industrialization production of transgenic technology improvement plant drought salt tolerant.
Embodiment 4
The copy number analysis of swede type rape gene BnHB6 in swede type rape
Adopt ordinary method from the swede type rape blade, to extract DNA (with reference to " molecular cloning ", Sambrook etc., 1989), cut DNA[13 μ g (microgram)/sample with HindIII Xba I BamH and EcoRI enzyme respectively] after, DNA is gone to after Hybond membrane (nylon membrane) goes up.Use the Amersham Pharmacia Gene Images of company
TMContents CDP-Star
TMLabelling module (PRN3540), we are labeled as probe with the BnBDC1 gene coding region, hybridize (in 60 ℃ of hybridization 16 hours) then.Take out film, place film washing liquid I (1*SSC, 1%SDS) in, in 60 ℃ of rinsings 3 times, each 15 minutes.Change over to film washing liquid II (0.1*SSC, 1%SDS) in 60 ℃ of rinsings 3 times, each same 15 minutes.With X-ray sheet compressing tablet 60-90 minute, develop then, photographic fixing (method is with reference to Roche DIG labeled test kit specification sheets).Result (Southern blot) finds to occur many hybridization bands on Hybond membrane, illustrate that swede type rape gene BnHB6 copy number in swede type rape is not less than 2.
Embodiment 5
The expression pattern analysis of swede type rape gene BnHB6 in the swede type rape of different treatment
1.RNA extraction: the swede type rape seedling of the 3-5 sheet leaf of will having grown is earlier after 200mM N.F,USP MANNITOL 0.5 hour, 100mMNaCl3 hour, 4 ℃ of 48 hours, 1500 μ J/m2 uviolizings 0.5 hour, submerging treatment two days, after the scissors wound 0.5 hour, 200uM H2023 hour, 100 μ M dormins 4 hours and 500uM Whitfield's ointment were handled in 12 hours, use TRIzol test kit (GIBCO BRL then, USA) extract also with reference to " molecular cloning " preparation chapters and sections (Sambrook etc., 1989) about RNA.
2.RNA quantitatively: with reference to " molecular cloning " (Sambrook etc., 1989), spectrophotometric instrumentation OD
260Rna content calculates: 1 OD
260=40 μ g/ml.
3 total RNA agarose gel electrophoresis separate: 1) get 6ml 25* (doubly) electrophoretic buffer, add the 117ml sterilized water, mixing.2) take by weighing the 1.5g agarose, join in the above-mentioned solution, heating and melting in microwave oven changes in 55 ℃ of water-baths.3) in stink cupboard, get 26.8ml formaldehyde, join in 55 ℃ the gelating soln mixing.4) pour into rapidly in the glue plate, room temperature water placing flat 30 minutes treats that gelling is solid.5) RNA (20 μ g) that extracts is dissolved in the RNA denaturing soln, heated 10 minutes down, be placed on ice immediately then at 65 ℃.6) in sample, add 2ul 10* sample-loading buffer, mixing.7) do not cover point sample under the condition of glue in electrophoresis liquid, 5V/cm voltage electrophoresis is about 5 hours.
4.RNA shift on the nylon membrane: 1) before the transfer, nylon membrane is soaked with 10*SSC.2) moistening film is covered exactly on film, two filter paper identical with film size are put in the 2*SSC solution moistening, cover on film, get rid of bubble.3) put one on the filter paper and fold and the identical thieving paper of film size, put a sheet glass and a weight on thieving paper, horizontal positioned shifted 12-20 hour.4) after the transfer, film was toasted 2 hours in 80 ℃.
5. the detecting of hybridization signal on the film: 1) film is immersed in 5 * Dendart ' s, 0.1%SDS, 0.1mg/ml salmon sperm dna], 65 ℃ of following prehybridizations 2 hours.2) will use Gene Images
TMContents CDP-Star
TMThe sex change 5 minutes in boiling water of the probe of labellingmodule mark directly adds 1) hybridization solution in, in 65 ℃ of hybridization 16-24 hour.3) take out film, place film washing liquid I (1*SSC, 1%SDS) in, in 65 ℃ of rinsings 3 times, each 15 minutes.Change over to film washing liquid II (0.1*SSC, 1%SDS) in 65 ℃ of rinsings 3 times, each 15 minutes.4) use X-ray sheet compressing tablet 60-90 minute, development, photographic fixing (method is with reference to Roche DIG labeled test kit specification sheets) then.Northern hybridization shows: except deepfreeze, different chemical substances, environment and HORMONE TREATMENT, all raising in various degree swede type rape BnHB6 transcriptional level.Illustrate that swede type rape BnHB6 transcriptional level under arid, salt, wound and active oxygen are coerced, all can play a role.Whitfield's ointment is handled and is also obviously improved this expression of gene level, illustrates that this gene also can play a role under disease and pest is coerced.
The expression pattern analysis that swede type rape BnHB6 gene is handled at the different time of N.F,USP MANNITOL and dormin
1.RNA extraction: the swede type rape seedling of the 3-5 sheet leaf of will having grown, then according to method extracting RNA above-mentioned and carries out quantitatively through 200mM treatment with mannitol 2 minutes, 5 minutes, 20 minutes, 1 hour, 3 hours, 6 hours and 12 hours; The swede type rape seedling of 3-5 sheet leaf of will having grown is simultaneously handled through the 100mM dormin, and treatment time and method for extracting are the same.
2.Northern hybridizing method is with embodiment 5.Test-results proves that along with the N.F,USP MANNITOL and the prolongation in dormin treatment time, the expression of BnHB6 progressively strengthens, and illustrates that BnHB6 is arid and adverse circumstance signal hormone dormin abduction delivering, plays the part of important role in rape resistance.
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: 21bp
(B) type: Nucleotide
(C) chain: strand
(D) topological framework: linearity
(ii). molecule type: oligonucleotide
(iii). sequence description: SEQ ID NO.1
GC?ATA?ACT?T(A/T/C/G)G?ATT?CCC?TCC
(2) information of SEQ ID NO.2
(i) sequence signature:
(A) length: 20bp
(B) type: Nucleotide
(C) chain: strand
(D) topological framework: linearity
(ii). molecule type: oligonucleotide
(iii). sequence description: SEQ ID NO.2
(A/T)TT?CCC?GCC?CA(A/G)A(A/T/C/G)T?TAA?CC
(2) information of SEQ ID NO.3
＜110〉Shanghai Communications University
＜120〉swede type rape drought resistance and salt tolerance transcription factor BnHB6 albumen coded sequence
<140>
<160>2
<170>PatentIn?version?3.1
<210>1
<211>1572
<212>DNA
＜213〉swede type rape (Brassica napus)
<400>1
ttcatatgga?aaacaaagaa?ctctgtctct?ctctctcctc?tcttactgaa?tagaatctcc???60
ccattgttct?aatttatctc?ttccttcaaa?gtattgatca?tcaagtacta?gatgcatttt??120
aaactgagag?ttaaaacaaa?caaaaaaatc?attattttga?agtaactaaa?aggtttttga??180
gaagaccctt?tttgtagtgc?tgtttgatga?tgaagagatt?aagcagttca?gattcagtgg??240
gtggtctcat?ctctttatgt?cccactactt?ccacagatca?gccgagtcca?agaagatacg??300
ggagagaatt?tcagtcgatg?cttgaaggtt?acgaggagga?agaagaagaa?gccataaccg??360
aggaaagagg?acaaaccggt?ttagccgaga?agaagagacg?gttaagcatt?aaccaagtta??420
aagccttgga?gaaaaatttc?gagttagaga?acaagcttga?gcccgagagg?aaagtgaagc??480
tagctcaaga?acttggtctc?caacctcgtc?aagtagctgt?ttggtttcag?aaccgccgtg??540
cgcggtggaa?gacaaaacag?ctcgagaaag?attacggtgt?tctcaaaacg?cagtacgatt??600
ctctccgcca?taacttcgat?tccctccgcc?gtgacaatga?atctcttctt?caagagatcg??660
gtaaactaaa?agctaagcta?aacggagaag?aagaagttga?agaagatgat?gaagatgaag??720
agaacaacgc?ggtcacgatg?gagagtgatg?tttccgtcaa?ggaagaagaa?gtttcgttgc??780
cggaggagct?tacagatccg?ccgtcttctc?ctccgcagct?tctagagcat?tccgacagtt??840
tcaattaccg?gagtttcacc?gacctccgcg?accttcttcc?gttaaaggcc?gcggcttcct??900
ccgtcgccgc?cgctggatcg?tcggacagta?gcgattcgag?cgccgtgttg?aacgaggaaa??960
gtagctctaa?cgttacggcg?gctccggcga?cggttccccg?gggcagtttc?ttgcagtttg?1020
tgaaaatgga?gcagacggag?gatcacgacg?actttctgag?tggagaagaa?gcgtgcgggt?1080
ttttctccga?tgaacagcca?ccgtctctgc?actggtattc?caccgttgat?cagtggaact?1140
gaggcttttg?atcaccggga?gagggttttt?gaattggatt?aaaatgttct?gttatgattc?1200
gccggagaag?attgaaaaag?cggagggcgg?aatgggaata?atggtgaaat?tgcaagggtt?1260
cattctgggc?gggaatataa?ttaattaggg?tgatttttgt?aattatgcgc?tctcttgcgt?1320
ttcgttcagt?tcttgtaatt?aaggatcatg?cgaatttgag?aaaggggtgg?aaaattctaa?1380
agggcgaaaa?ctagaaatgg?aatctttgcg?ggattgaatt?caagaatttg?aaagaattca?1440
cgtgtaataa?attttgcagt?attttaaatg?attttataag?attttaatgc?aattgtataa?1500
tttttatgtt?tatcttttgc?catagaattt?aagtgaatgt?aaattaattt?gatggaaaaa?1560
aaaaaaaaaa?aa?????????????????????????????????????????????????????1572
<210>2
<211>311
<212>PRT
＜213〉swede type rape (Brassica napus)
<400>2
MMKRLSSSDS?VGGLISLCPT?TSTDQPSPRR?YGREFQSMLE?GYEEEEEEAI?TEERGQTGLA???60
EKKRRLSINQ?VKALEKNFEL?ENKLEPERKV?KLAQELGLQP?RQVAVWFQNR?RARWKTKQLE??120
KDYGVLKTQY?DSLRHNFDSL?RRDNESLLQE?IGKLKAKLNG?EEEVEEDDED?EENNAVTMES??180
DVSVKEEEVS?LPEELTDPPS?SPPQLLEHSD?SFNYRSFTDL?RDLLPLKAAA?SSVAAAGSSD??240
SSDSSAVLNE?ESSSNVTAAP?ATVPRGSFLQ?FVKMEQTEDH?DDFLSGEEAC?GFFSDEQPPS??300
LHWYSTVDQW?N???????????????????????????????????????????????????????311
Claims (5)
1, a kind of swede type rape drought resistance and salt tolerance transcription factor BnHB6 albumen coded sequence, it is characterized in that, comprise: coding has the nucleotide sequence of the polypeptide of swede type rape BnHB6 protein active, and shows at least 70% homology from the nucleotides sequence of Nucleotide 207-1142 position among described nucleotide sequence and the SEQ ID NO.3; Perhaps described nucleotide sequence can with SEQ ID NO.3 in from the nucleotide sequence hybridization of Nucleotide 207-1142 position.
2, swede type rape drought resistance and salt tolerance transcription factor BnHB6 albumen coded sequence according to claim 1, it is characterized in that, described encoding sequence has 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.
3, swede type rape drought resistance and salt tolerance transcription factor BnHB6 albumen coded sequence according to claim 1 and 2 is characterized in that, described encoding sequence has among the SEQ ID NO.3 nucleotide sequence from Nucleotide 207-1142 position.
4, swede type rape drought resistance and salt tolerance transcription factor BnHB6 albumen coded sequence according to claim 1 is characterized in that, comprises: 8-100 continuous nucleotide in the dna molecular.
5, swede type rape drought resistance and salt tolerance transcription factor BnHB6 albumen coded sequence according to claim 4 is characterized in that, the dna molecular transformed host cells, and it is an eukaryotic cell.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101386645B (en) * | 2008-10-29 | 2011-10-19 | 中国农业科学院作物科学研究所 | Plant salt tolerant protein and encoding gene and application thereof |
| CN102417911A (en) * | 2010-09-28 | 2012-04-18 | 华中农业大学 | Method for over-expressing brassica napus BnLAS gene for improving plant drought resistance |
| EP2487246A3 (en) * | 2007-08-02 | 2012-09-19 | BASF Plant Science GmbH | Transgenic plants with increased stress tolerance and yield |
| CN102094006B (en) * | 2009-12-11 | 2013-03-06 | 上海市农业科学院 | Brassica napus ERF-B3 family transcription factor gene and preparation method thereof |
-
2003
- 2003-10-16 CN CNA2003101079671A patent/CN1528895A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2487246A3 (en) * | 2007-08-02 | 2012-09-19 | BASF Plant Science GmbH | Transgenic plants with increased stress tolerance and yield |
| CN101386645B (en) * | 2008-10-29 | 2011-10-19 | 中国农业科学院作物科学研究所 | Plant salt tolerant protein and encoding gene and application thereof |
| CN102094006B (en) * | 2009-12-11 | 2013-03-06 | 上海市农业科学院 | Brassica napus ERF-B3 family transcription factor gene and preparation method thereof |
| CN102417911A (en) * | 2010-09-28 | 2012-04-18 | 华中农业大学 | Method for over-expressing brassica napus BnLAS gene for improving plant drought resistance |
| CN102417911B (en) * | 2010-09-28 | 2013-05-08 | 华中农业大学 | Method for over-expressing brassica napus BnLAS gene for improving plant drought resistance |
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