CN1528889A - Chinese cabbage 9-cis-carotene epoxide dioxygen zymoprotein coding sequence - Google Patents
Chinese cabbage 9-cis-carotene epoxide dioxygen zymoprotein coding sequence Download PDFInfo
- Publication number
- CN1528889A CN1528889A CNA2003101079722A CN200310107972A CN1528889A CN 1528889 A CN1528889 A CN 1528889A CN A2003101079722 A CNA2003101079722 A CN A2003101079722A CN 200310107972 A CN200310107972 A CN 200310107972A CN 1528889 A CN1528889 A CN 1528889A
- Authority
- CN
- China
- Prior art keywords
- cis
- sequence
- chinese cabbage
- nucleotide
- epoxies carotene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
The invention is a coding series of celery cabbage 9- series- epoxy caritinoid oxydol enzyme protein, which belongs to gene project field. It includes: codes the nucleotide series of polypeptide which has celery cabbage 9- series- epoxy caritinoid oxydol enzyme protein activity, and at least 70% of the series has consanguinity with SEQ ID NO.3 nucleotide from 89 to 1774; or the series can cross with the 89-1774 bit nucleotide series of SEQ ID NO.3. the product in the invention has key effect to the ABA synthsis, it can upgrades the inversion resisting property of plant effectively.
Description
Technical field
What the present invention relates to is a kind of albumen coded sequence, and particularly a kind of Chinese cabbage 9-cis-epoxies carotene dioxygenase albumen coded sequence belongs to the genetically engineered field.
Background technology
A lot of experiments show that dormin (being called for short ABA) is a kind of stress hormone, play an important role when plant opposing external environment is coerced, and can both influence ABA in the intravital level of plant as heat, salt, arid, envrionment conditions such as cold and weedicide etc.In some growth courses of plant such as seed germination, fetal development and the fruit maturation, certain variation all can take place in ABA content.ABA has vital role in plant growth and development process.In the ABA biosynthesizing, except that extraneous envrionment conditions rise induce, the more intravital factors of plant also play even more important regulating effect as enzyme and cofactors.Aspect enzyme adjusting biosynthesizing, 9-cis-epoxies carotene dioxygenase (NCED) is playing a crucial role aspect the biosynthesizing of ABA.
In analysis to existing document, though " The Plant Journal; 2001; 27:325-333 " affirms fully the proteic function of NCED, point out that this gene can increase the expression of ABA under drought stress, but do not point out that this expression of gene is relevant with adverse circumstances such as salt and ultraviolets, do not find to have the report of close ties document 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 Chinese cabbage 9-cis-epoxies carotene dioxygenase albumen coded sequence is provided.Disclose and Chinese cabbage 9-cis-epoxies carotene peroxidase (NCED, nine-cisepoxycarotenoid dioxygenase) nucleotide sequence of closely-related proteic aminoacid sequence and Chinese cabbage 9-cis-epoxies carotene dioxygenase gene, and, comprise the improvement of the adverse circumstances such as drought resisting, water logging and anti-salt of plant in the application that utilizes on transgenic technology improvement plant 9-cis-epoxies carotene peroxidase.
The present invention is achieved by the following technical solutions: at the isolated dna molecular of the present invention, this molecule comprises: coding has the nucleotide sequence of the polypeptide of Chinese cabbage 9-cis-epoxies carotene dioxygenase protein active, and shows at least 70% homology from the nucleotides sequence of Nucleotide 89-1774 position among described nucleotide sequence and the SEQ ID NO.3; Perhaps described nucleotide sequence can be under 45-55 ℃ of condition with SEQ ID NO.3 in from the nucleotide sequence hybridization of Nucleotide 89-1774 position.Preferably, described sequence encoding has the polypeptide of the aminoacid sequence shown in the SEQ ID NO.3.More preferably, described sequence has among the SEQ ID NO.3 nucleotide sequence from Nucleotide 89-1774 position.
The isolated Chinese cabbage 9-cis of the present invention-epoxies carotene peroxidase related protein polypeptide, it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQ ID NO.3 aminoacid sequence, or its reactive derivative.Preferably, this polypeptide is to have SEQ ID NO.3 polypeptide of sequence.
Carrier provided by the present 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 carrier 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 and have separated under native state, refer to that also this DNA or fragment with under the native state follow the component of nucleic acid to separate, and separate with the protein of in cell, following it.
In the present invention, term " Chinese cabbage 9-cis-epoxies carotene peroxidase albumen (or polypeptide) encoding sequence " refers to encode and has Chinese cabbage 9-cis-nucleotide sequence of the polypeptide of epoxies carotene peroxidase protein-active, as 89-1774 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.3.This degenerate sequence is meant, is arranged in the encoder block 89-1774 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 89-1774 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, better under the height stringent condition with SEQ ID NO.3 in from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 89-1774 position.This term also comprise with SEQ ID NO.3 in from the homology of nucleotide sequence at least 70% of Nucleotide 89-1774 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 ID NO.3 with natural Chinese cabbage 9-cis-epoxies carotene peroxidase 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 " Chinese cabbage 9-cis-epoxies carotene peroxidase albumen or polypeptide " refers to have Chinese cabbage 9-cis-the SEQ ID NO.3 polypeptide of sequence of epoxies carotene peroxidase protein-active.This term also comprises having and variant form natural Chinese cabbage 9-cis-relevant identical function of epoxies carotene peroxidase, 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 the active fragments and the reactive derivative of Chinese cabbage 9-cis-epoxies carotene peroxidase associated protein.
The variant form of Chinese cabbage 9-cis of the present invention-epoxies carotene peroxidase polypeptide comprises: the albumen that homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, DNA that can relevant DNA hybridization with Chinese cabbage 9-cis-epoxies carotene peroxidase under high or low stringent condition are coded and the polypeptide or the albumen that utilize the serum of Chinese cabbage 9-cis-epoxies carotene peroxidase polypeptide to obtain.
In the present invention, " Chinese cabbage 9-cis-epoxies carotene peroxidase conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID NO.3, there are 10 at the most, preferably at the most 8, more preferably 5 amino acid similar performances or close amino acid are replaced and are 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
87％?identity?in?1636?nt?overlap
Query：6????ggagatgggtcggtggtaatcatactaaactaccattatcgtcttctcaaagctccgcct?65
||||||||?|?|||||?|||||||||?|?|??|||||||||||||||||||||||||?||
Sbjct：157??ggagatggcttggtggcaatcatactcagccgccattatcgtcttctcaaagctccgact?216
Query：66???tgaataattcttgctccgtacccatgacaaatcgttcccaacgaaagctcaatgtttctt?125
|||?|?|||?|?|||||?||||?|||?|?|?||||??|??|||?||||||||||||||?|
Sbjct：217??tgagttattgtagctccttacctatggccagtcgtgtcacacgtaagctcaatgtttcat?276
A??L??A??Q??E??Q??L??Q??E??E??A??B
Query：126??ctgcgcttcacactcatcctgctctccatttccccaagcaatcctccacctctcccgcca?185
|||||||||||||||?|||?|||||?||||||||?||||||||?||?|?|||||||||||
Sbjct：277??ctgcgcttcacactcctccagctcttcatttccctaagcaatcatcaaactctcccgcca?336
F??F??T???D??F??C??R??P?B??Y??D??P??L??E??B??B??T??B??P??E
Query：186??ttattgtcaacccaaaaaccaaagaatccgacaccaaacagatgagcttgttccagagag?245
||?||||?||?||?|||?|||||||||||?||||?||||||||||??|||||||||||||
Sbjct：337??ttgttgttaagcccaaagccaaagaatccaacactaaacagatgaatttgttccagagag?396
T??S??K??A??M??T??H??I??B?N??B??G??S??M??W??A??M??E??H???D
Query：?246??cggcggcggcggcgctggacgcggcggagggtttcctcgtgagccacgagcgacagcatc?305
||||||||||?|||?||||||||||||||||||||||?||?|||||||||???|??||?|
Sbjct：?397??cggcggcggcagcgttggacgcggcggagggtttccttgtcagccacgagaagctacacc?456
B??F??G??L??I??D??C??H??H??E?T??C??E??C??P??L??F??L??P???E
Query：?306??ccctccccaaaaccgccgatcctagcgttcagatcgccggaaacttcgctccggtgaacg?365
|?||?||?|||||?||?||||||||?|||||||||||||||||?||?|||||||||||?|
Sbjct：?457??cgcttcctaaaacggctgatcctagtgttcagatcgccggaaattttgctccggtgaatg?516
C??C??R??Y??H??H?C??R??L??D??M???M??B??E??C??A??T??L??H??B
Query：?366??aacagcctctccggcgtaacctcccggtggtcggaaaaataccggattccatcaaaggag?425
|||||||??||||||||||?||?|||||||||||||||?|?||?||||||||||||||||
Sbjct：?517??aacagcccgtccggcgtaatcttccggtggtcggaaaacttcccgattccatcaaaggag?576
M??T??F??C??S??B??M??P??W?P???E??T??L??T??L??R??T??F??M??A
Query：?426??tgtacgtgcgcaacggagccaacccgcttcacgagccggtgacaggtcaccacttcttcg?485
||||?||||||||||||||?|||||?||||||||||||||||||||||||||||||||||
Sbjct：?577??tgtatgtgcgcaacggagctaacccacttcacgagccggtgacaggtcaccacttcttcg?636
M??Y??C??L??K??N??Y??N??B??K??C??T??E??L??N??B??Q??Q??H??C
Query：?486??acggagacgggatggttcacgccgtgacattcgaggacggttcagctagctacgcgtgcc?545
||||||||||?||||||||||||||?|?||||||??|||||||||||||||||||?||||
Sbjct：?637??acggagacggtatggttcacgccgtcaaattcgaacacggttcagctagctacgcttgcc?696
H??S??I??I??P??Y??Y??A??S??E??C??A??Y??L??N??L??Y??I??E??S
Query：?546??ggtttactctgaccaaccggtttatccaggaacgtcaattaggtcgaccggttttcccca?605
|||||||||?|||?|||||||||?|?||||||||||||||?|||||||||||||||||||
Sbjct：?697??ggtttactcagactaaccggtttgttcaggaacgtcaattgggtcgaccggttttcccca?756
L??B??S??M??L??N??E??H?S??E???N??F??I??S??H??P??P??A??K??G
Query：?606??aggccatcggcgagcttcacggccacaccggtattgctcgactcatgctattctacgcca?665
|?||||||||?||||||||||||||||||||||||||?||||||||||||||||||||||
Sbjct：?757??aagccatcggtgagcttcacggccacaccggtattgcccgactcatgctattctacgcca?816
S??C??I??B??B??B??E??E?A??E???P??E??E??W??T??N??W??L??D??F
Query：?666??gagccgcagccggtttagtcgacccggctcacggaaccggagtagctaacgccggtttag?725
||||?|||||||||?|||||||||||||?|||||||||||?|||||||||||||||||?|
Sbjct：?817??gagctgcagccggtatagtcgacccggcacacggaaccggtgtagctaacgccggtttgg?876
G??F??G??H??P??T??E??I??W??Y??I??C??S??S??H??T??T??R??M??H
Query：?726??tctatttcaataaccggttattagccatgtcggaagacgatttgccttaccaagtccgga?785
|||||||||||??|||||||||?||?||||||||?||?|||||?|||||||||||?|?||
Sbjct：?877??tctatttcaatggccggttattggctatgtcggaggatgatttaccttaccaagttcaga?936
L??I??C??F??Y??T??B??M??E??M??H??R??I??B??P??S??C??C??Y??I
Query：?786??tcactccaggtggagacttgaaaaccgttggccgttacgatttcgacgggcagttagaat?845
|||||||???||||||?||?|||||||||||?||?|?||||||?||?||?||?|||||||
Sbjct：?937??tcactcccaatggagatttaaaaaccgttggtcggttcgattttgatggacaattagaat?996
K??E??Q??L??M??F??B??B??Q??Q??H??P??L??B??B??F??N??E??A??I
Query：?846??ccacaatgatcgcccacccgaaagtcgacccggaatccggcgagctattcgctctaagct?905
||||||||||?|||||||||||||||||||||||||||||?||?||?||||||?||||||
Sbjct：?997??ccacaatgattgcccacccgaaagtcgacccggaatccggtgaactcttcgctttaagct?1056
P??L??H??D??K??I??A??P??Q??H??L??Q??I??E??B??H??P??S??M??E
Query：?906??acgacgtcgtttcaaagccttacttaaagtacttccgcttctcaccggacggagaaaaat?965
|||||||||||||||||||||||?||||?||||||||?|||||||||||||||???||||
Sbjct：?1057?acgacgtcgtttcaaagccttacctaaaatacttccgattctcaccggacggaactaaat?1116
C??H??P??S??F??Y?F??R??M??L???R??P??M??S??B??B??H??R??S??L
Query：?966??cgccggacgtcgagatccagctcgatcagccgacgattatgcacgacttgccgataacag?1025
|?||||||||||||||?|||||?||||||||?|||||?||||||||?||??||||?||||
Sbjct：?1117?caccggacgtcgagattcagcttgatcagccaacgatgatgcacgatttcgcgattacag?1176
T??F??L?E??C??S??E??P??H??B???M???P??D??T??I??K??A??S??E??L
Query：?1026?agaacttcgtcgttttaccggaccagcaagtcgtgttcaagctccaggagatgatccgcg?1085
|||||||||||||??||||?||||||||||||||?||||||||?|?||||||||||||||
Sbjct：?1177?agaacttcgtcgtcgtacctgaccagcaagtcgttttcaagctgccggagatgatccgcg?1236
Q??C??H??F??H??R??Y?C??B??A???M??L??H??C??C??P??A??I??Y??B
Query：?1086?gcggctctccggtgatctacgacaaggagaaggttgcaagattcggaattttagacaaat?1145
|?||?|||||||||?|?|||||||||?|?|||||?|||||||||||?|||||||||||||
Sbjct：?1237?gtgggtctccggtggtttacgacaagaacaaggtcgcaagattcgggattttagacaaat?1296
Q??R??P??L??H??H??E??S??K??T??L??L??Y??N??N??F??R??B??L??C
Query：?1146?acgcggcggactcgtcgggaatcaggtggatcgaagctccggactgcttctgcttccacc?1205
||||?|??||?||?|||???||?|?||||||?||?|||||?||?||||||||||||||?|
Sbjct：?1297?acgccgaagattcatcgaacattaagtggattgatgctccagattgcttctgcttccatc?1356
T??N??T??B??C??A??C??D??T?P???H??B??L??C??T??T??T??Y??T??R
Query：?1206?tctggaacgcgtgggaggagccggaaacagaggaggtcgtcgtgatcgggtcgtgcatga?1265
||||||||||?|||||?|||||?||||||||?||?|||||||||||?|||||?||?||||
Sbjct：?1357?tctggaacgcttgggaagagccagaaacagatgaagtcgtcgtgatagggtcctgtatga?1416
F??L??K??L??Y?T??I??C??A??D???C??D??L??E??C??I??T??M??P??F
Query：?1266?ctccgccggactcaattttcaacgagtccgacgagaatctcaagagcgtcctctccgaga?1325
||||?||?||||||||||||||||||||?|||||||||||||||||?|||||?||?||?|
Sbjct：?1417?ctccaccagactcaattttcaacgagtctgacgagaatctcaagagtgtcctgtctgaaa?1476
B??P??Y?C??L??Q??Y??E??Q??I???F??Y??N??A??L??A??F??Y??G??T
Query：?1326?ttcggctcaacctcagaaccggagagtccactcgccgtccgatcatctccgacggagatc?1385
|?||?||?||?||||?||||||?||?||?|||||||||||||||||||||?|||?|||||
Sbjct：?1476?tccgcctgaatctcaaaaccggtgaatcaactcgccgtccgatcatctccaacgaagatc?1536
R??S??T??S??C??H??E??F?A??B???T??D??B??K??C??H??C??H??F??F
Query：?1386?aacaagtcaacctcgaagcagggatggtgaaccggaacatgctcggccgtaagaccaagt?1445
||||||||||||||||||||||||||||?|||?|?|||||||||||||||||?|||||?|
Sbjct：?1537?aacaagtcaacctcgaagcagggatggtcaacagaaacatgctcggccgtaaaaccaaat?1596
D??D??C??A??B?P??E??D??L??P???N??T??C??N??Y??B??R??B??C??M
Query：?1446?tcgcttacctcgctttagccgagccgtggcctaaagtctccggctttgcgaaagtcgatc?1505
||||||||?|?|||||||||||||||||||||||||||||?||?||?||?|||||?||||
Sbjct：?1597?tcgcttacttggctttagccgagccgtggcctaaagtctcaggattcgctaaagttgatc?1656
I??S??H?P??I??M??C??B??B??P???L??N??Y??Y??L??P??Q??E??N??B
Query：?1506?tcgccaccggagaggtcaagaagcatctctacggcgatgaccgttacggtggagagcctc?1565
||?|?||?|||||?||?|||||?|||||?|||||||||?||||||||||?||||||||||
Sbjct：?1657?tcactactggagaagttaagaaacatctttacggcgataaccgttacggaggagagcctc?1716
P??T??F??N??C??T??I??Q??B??S??P??H??T??A??M??P??L??P??D??Q
Query：?1566?tgtttctccccggtgagggagca---gaagacgatgggcacatcctctgttttgttcacg?1622
|||||||||||||?||?||||?|???||||||||?||?||||||||||||||?|||||||
Sbjct：?1717?tgtttctccccggagaaggaggagaggaagacgaaggatacatcctctgtttcgttcacg?1776
Y??E??D??S??B?L??F??C??E??T???T??H??L??T??T??T??T??T??Y??Q
Query：?1623?acgagaagacatggaaatc?1641
|||||||||||||||||||
Sbjct：?1777?acgagaagacatggaaatc?1795
F??L??S??K??Q??M
Query: the nucleotide sequence of Chinese cabbage 9-cis-epoxies carotene peroxidase associated protein 9-cis-epoxies carotene dioxygenase
Sbjct: Arabidopis thaliana 9-cis-epoxies carotene peroxidase II 33kDa oxygen evolution protein gene nucleotide sequence (NM_112304)
Table 2 is that the homology of the nucleotide sequence of Chinese cabbage 9-cis of the present invention-epoxies carotene peroxidase albumen (9-cis-epoxies carotene dioxygenase) and Arabidopis thaliana oxygen evolution albumen (OEC33) compares (GAP) table.
Table 3
87％?identity?in?632aa?overlap，?92％?similarity?in?632aa?overlap
Query：1???MTNRSQRKLNVSSALHTHPALHFPKQSSTSPAIIVNPKTKESDTKQMSLFQRAAAAALDA?60
M?+R??RKLNVSSALHT?PALHFPKQSS?SPAI+V?PK?KES+TKQM+LFQRAAAAALDA
Sbjct：40??MASRVTRKLNVSSALHTPPALHFPKQSSNSPAIVVKPKAKESNTKQMNLFQRAAAAALDA?99
Query：61??AEGFLVSHERQHPLPKTADPSVQIAGNFAPVNEQPLRRNLPVVGKIPDSIKGVYVRNGAN?120
AEGFLVSHE+?HPLPKTADPSVQIAGNFAPVNEQP+RRNLPVVGK+PDSIKGVYVRNGAN
Sbjct：100?AEGFLVSHEKLHPLPKTADPSVQIAGNFAPVNEQPVRRNLPVVGKLPDSIKGVYVRNGAN?159
Query：121?PLHEPVTGHHFFDGDGMVHAVTFEDGSASYACRFTLTNRFIQERQLGRPVFPKAIGELHG?180
PLHEPVTGHHFFDGDGMVHAV?FE?GSASYACRFT?TNRF+QERQLGRPVFPKAIGELHG
Sbjct：160?PLHEPVTGHHFFDGDGMVHAVKFEHGSASYACRFTQTNRFVQERQLGRPVFPKAIGELHG?219
Query：181?HTGIARLMLFYARAAAGLVDPAHGTGVANAGLVYFNNRLLAMSEDDLPYQVRITPGGDLK?240
HTGIARLMLFYARAAAG+VDPAHGTGVANAGLVYFN?RLLAMSEDDLPYQV+ITP?GDLK
Sbjct：?220?HTGIARLMLFYARAAAGIVDPAHGTGVANAGLVYFNGRLLAMSEDDLPYQVQITPNGDLK?279
Query：?241?TVGRYDFDGQLESTMIAHPKVDPESGELFALSYDVVSKPYLKYFRFSPDGEKSPDVEIQL?300
TVGR+DFDGQLESTMIAHPKVDPESGELFALSYDVVSKPYLKYFRFSPDG?KSPDVEIQL
Sbjct：?280?TVGRFDFDGQLESTMIAHPKVDPESGELFALSYDVVSKPYLKYFRFSPDGTKSPDVEIQL?339
Query：?301?DQPTIMHDLPITENFVVLPDQQVVFKLQEMIRGGSPVIYDKEKVARFGILDKYAADSSGI?360
DQPT+MHD??ITENFVV+PDQQVVFKL?EMIRGGSPV+YDK?KVARFGILDKYA?DSS?I
Sbjct：?340?DQPTMMHDFAITENFVVVPDQQVVFKLPEMIRGGSPVVYDKNKVARFGILDKYAEDSSNI?399
Query：?361?RWIEAPDCFCFHLWNAWEEPETEEVVVIGSCMTPPDSIFNESDENLKSVLSEIRLNLRTG?420
+WI+APDCFCFHLWNAWEEPET+EVVVIGSCMTPPDSIFNESDENLKSVLSEIRLNL+TG
Sbjct：?400?KWIDAPDCFCFHLWNAWEEPETDEVVVIGSCMTPPDSIFNESDENLKSVLSEIRLNLKTG?459
Query：?421?ESTRRPIISDGDQQVNLEAGMVNRNMLGRKTKFAYLALAEPWPKVSGFAKVDLATGEVKK?480
ESTRRPIIS+?DQQVNLEAGMVNRNMLGRKTKFAYLALAEPWPKVSGFAKVDL?TGEVKK
Sbjct：?460?ESTRRPIISNEDQQVNLEAGMVNRNMLGRKTKFAYLALAEPWPKVSGFAKVDLTTGEVKK?519
Query：?481?HLYGDDRYGGEPLFLPGEGA-EDDGHILCFVHDEKTWKSCVNVIDAKTMSAEPVAVVELP?539
HLYGD+RYGGEPLFLPGEG??ED+G+ILCFVHDEKTWKS?+?+++A?++??E??A?V+LP
Sbjct：?520?HLYGDNRYGGEPLFLPGEGGEEDEGYILCFVHDEKTWKSELQIVNAVSLEVE--ATVKLP?577
Query：?540?HRVPYGFHAFFVTEEQLQEQTL?561
RVPYGFH??F+??+?L?+Q?+
Sbjct：?578?SRVPYGFHGTFIGADDLAKQVV?599
Query: the aminoacid sequence of Chinese cabbage 9-cis-epoxies carotene peroxidase associated protein 9-cis-epoxies carotene dioxygenase
Sbjct: Arabidopis thaliana 9-cis-epoxies carotene peroxidase II 33kDa oxygen evolution Argine Monohydrochloride sequence (NP_188062)
Table 3 is that the homology of the aminoacid sequence of Chinese cabbage 9-cis of the present invention-epoxies carotene peroxidase albumen (9-cis-epoxies carotene dioxygenase) and 9-cis-epoxies carotene dioxygenase compares (FASTA) table.Wherein, identical amino acid marks with the amino acid monocase between two sequences.
Invention also comprises the analogue of Chinese cabbage 9-cis-epoxies carotene peroxidase albumen or polypeptide.The difference of these analogues and natural 9-cis-epoxies carotene peroxidase 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 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 Chinese cabbage 9-cis of the present invention-epoxies carotene peroxidase polypeptide, Chinese cabbage 9-cis-epoxies carotene peroxidase encoding sequence operationally can be connected in expression regulation sequence, thereby form Chinese cabbage 9-cis-epoxies carotene peroxidase 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 Chinese cabbage 9-cis-epoxies carotene peroxidase gene product, the existence of rna transcription thing in cell of promptly analyzing Chinese cabbage 9-cis-epoxies carotene peroxidase.
In addition, can be used as the nucleic acid molecule of probe among the present invention, this molecule has 8-100 continuous nucleotide of Chinese cabbage 9-cis-epoxies carotene peroxidase nucleotide coding sequence usually, preferably has 15-50 continuous nucleotide.This probe can be used for whether existing in the test sample coding Chinese cabbage 9-nucleic acid molecule that cis-epoxies carotene peroxidase is relevant.
The present invention relates to whether exist in the test sample method of Chinese cabbage 9-cis-epoxies carotene peroxidase nucleotide sequence, 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 Chinese cabbage 9-cis-epoxies carotene peroxidase 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 Chinese cabbage 9-cis of the present invention-epoxies carotene peroxidase nucleotide sequence and aminoacid sequence, can be on the homology basis of nucleic acid homology or marking protein, screening Chinese cabbage 9-cis-relevant homologous gene of epoxies carotene peroxidase or homologous protein.
In order to obtain the dot matrix with Chinese cabbage 9-cis-Chinese cabbage cDNAs that epoxies carotene peroxidase genes involved is relevant, can screen Chinese cabbage cDNA library with dna probe, these probes are under low stringent condition, use
32P relevant all or part of of Chinese cabbage 9-cis-epoxies carotene peroxidase cooked the radioactivity mark and.The cDNA library that most is suitable for screening is the library from Chinese cabbage.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 with Chinese cabbage 9-cis-gene family that epoxies carotene peroxidase is relevant.
Chinese cabbage 9-cis of the present invention-epoxies carotene peroxidase associated nucleotide full length sequence 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 Chinese cabbage 9-cis of the present invention-epoxies carotene peroxidase albumen, by various conventional screening methods, can filter out the interactional material of relevant generation with Chinese cabbage 9-cis-epoxies carotene peroxidase, perhaps acceptor, inhibitor or short of money dose etc.
Among the present invention, 9-cis-epoxies carotene peroxidase plays a crucial role in ABA is synthetic, and obviously body ground improves the resistance of crop.Plant can run into arid, water logging, adverse circumstance such as damage to plants caused by sudden drop in temperature in the process of growth, 9-cis of the present invention-epoxies carotene peroxidase II gene 9-cis-epoxies carotene dioxygenase is just possessing this function, it can strengthen the content of plant ABA under adverse circumstance, so that improve the resistance of plant to adverse circumstance.Therefore, the present invention has very big using value.
Embodiment
Below in conjunction with the concrete testing data in laboratory and in conjunction with specific embodiments, further set forth the present invention.These embodiment only are used to the present invention is described and are not used in and limit the scope of the invention.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.
Embodiment 1
The clone of Chinese cabbage 9-cis-epoxies carotene peroxidase gene
1. separate tissue (isolation)
Chinese cabbage (kind is " heat-resisting No. 3 ") is buied from the market, places 28 ℃ to germinate 24 hours Chinese cabbage, is seeded in then in the greenhouse, when treating that the Chinese cabbage blade is the 4-5 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 9-cis-epoxies carotene peroxidase II 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[BP001 (SEQ ID NO.1)+BP002 (SEQ ID NO.2)] obtain 2002BP (about 1.1kb), 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 the photosynthetic oxygen evolution gene of its nucleotide sequence and proteins encoded and known cress such as Arabidopis thaliana (Arabidopsis thaliana) is very high, so think that tentatively it is a 9-cis-epoxies carotene peroxidase gene.
(2)3’-RACE
PCR[AP+BP003 (5 '-GACGAGAAGACATGGAAATC-3 ')] obtain 2002BP3 ' (about 300bp), 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 the photosynthetic oxygen evolution gene of its nucleotide sequence and proteins encoded and known cress such as Arabidopis thaliana (Arabidopsis thaliana) is very high, so think that tentatively it is one and 9-cis-epoxies carotene peroxidase genes involved.
(3).5’-RACE
First round PCR [AAP+BP004 (5 '-CGTCTTCCGACATGGCTAAT--3 ')]
Second takes turns PCR[(AUAP+BPR005 (5 '-TACCGGTGTGGCCGTGAAGC-3 ')) obtain 2001BP 5 ' (about 500bp) (process is 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 the result of BLAST proof newly obtains from Chinese cabbage really be one with gene that plant 9-cis-epoxies carotene peroxidase is relevant.
By being used in combination above-mentioned 3 kinds of methods, obtained the complete encoding sequence (SEQ ID NO.3) of Chinese cabbage 9-cis-epoxies carotene peroxidase associated protein of candidate.
Embodiment 2
The sequence information and the homology analysis of Chinese cabbage 9-cis-epoxies carotene peroxidase genes involved
The length of Chinese cabbage 9-cis-epoxies carotene peroxidase full-length cDNA that the present invention is new is 1151bp, and detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 89-1774 position Nucleotide (699 Nucleotide).Derive the aminoacid sequence of Chinese cabbage 9-cis-epoxies carotene peroxidase according to full-length cDNA, totally 233 amino-acid residues, molecular weight is 167.63kDa, iso-electric point (pI) is 4.89.Detailed sequence is seen SEQ ID NO.3.
Full length cDNA sequence that Chinese cabbage 9-cis-epoxies carotene peroxidase is relevant and coded protein thereof carry out Nucleotide and protein homology retrieval with blast program in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundantGenBank CDS translations+PDB+SwissProt+Superdate+PIR database, found that it and Arabidopis thaliana 9-cis-epoxies carotene peroxidase gene (NM_112304) have 87% homogeny, (subordinate list 2) on nucleotide level; On amino acid levels, it and Arabidopis thaliana suppress 9-cis-epoxies carotene peroxidase albumen (NP_188062) also 87% homogeny (subordinate list 3).This shows that all there are higher homology in Chinese cabbage 9-cis-epoxies carotene peroxidase gene 9-cis-epoxies carotene dioxygenase and Arabidopis thaliana 9-cis-epoxies carotene peroxidase albumen on nucleic acid still is protein level.Arabidopis thaliana Arabidopis thaliana 9-cis-epoxies carotene peroxidase albumen be proved to be with 9-cis-epoxies carotene peroxidase in oxygen evolution have tangible effect, so can think that 9-cis-epoxies carotene dioxygenase is suppressing also to have similar effect on 9-cis-epoxies carotene peroxidase.
Embodiment 3
Chinese cabbage 9-cis-epoxies carotene peroxidase associated protein or polypeptide carry out the resistance of eukaryotic cell expression and transfer-gen plant in Arabidopis thaliana identifies
The structure that contains the expression vector of goal gene (Chinese cabbage 9-cis-epoxies carotene dioxygenase gene), the full length sequence (SEQ ID NO.3) relevant according to Chinese cabbage 9-cis-epoxies carotene peroxidase, 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 Chinese cabbage 9-cis-epoxies carotene peroxidase 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 resistance that contains the transgenic arabidopsis plant of Chinese cabbage 9-cis-epoxies carotene dioxygenase gene is identified, in view of coding 9-cis-epoxies carotene dioxygenase, AtNCED1 gene as Arabidopis thaliana has been proved to be ABA synthetic key gene, and the AtNCED1 of Chinese cabbage 9-cis-epoxies carotene dioxygenase transcriptional level and Arabidopis thaliana has higher homology, further to containing carrying out property of the transgenic arabidopsis plant evaluation of Chinese cabbage 9-cis-epoxies carotene dioxygenase.We utilize RT-PCR and Northern blot that transfer-gen plant is detected, and two kinds of methods prove that all goal gene has been incorporated in the genome of Arabidopis thaliana.With normal Arabidopis thaliana is contrast, the Arabidopis thaliana plant that changes 9-cis-epoxies carotene peroxidase gene is carried out ABA, arid, Whitfield's ointment, water logging, freezing, uviolizing and sodium-chlor coerce processing, treatment condition are: NaCL 200Mm handled 5 hours; SA (Whitfield's ointment) 500 μ M 24 hours; ABA 100 μ M handled 5 hours; N.F,USP MANNITOL 200mM 5 hours; On filter paper, place 2 hours as the arid processing.More than handled plant position all be the young leaflet tablet of plant.Transfer-gen plant with normal illumination is contrast, and the result proves that changeing 9-cis-epoxies carotene dioxygenase gene plant all is better than the plant that does not change this gene on aspect the anti-above-mentioned adverse circumstance.This proof 9-cis-general adverse circumstance of epoxies carotene dioxygenase gene pairs has resistance, and Chinese cabbage 9-cis-epoxies carotene dioxygenase gene will can be used for utilizing in the research and industrialization production of transgenic technology improvement plant stress-resistance.
The Chinese cabbage 9-cis-epoxies carotene peroxidase gene 9-cis-copy number analysis of epoxies carotene dioxygenase in Chinese cabbage
Adopt ordinary method from the Chinese cabbage blade, to extract DNA (with reference to " molecular cloning ", Sambrook etc., 1989), use BamH I respectively, EcoRI, Xba I and HindIII enzyme are cut DNA[13 μ g (microgram)/sample] 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 BcpFLC 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 RocheDIG labeled test kit specification sheets).Result (Southern blot) finds to occur many hybridization bands on Hybond membrane, illustrate that 9-cis-epoxies carotene dioxygenase gene belongs to multi-copy gene in Chinese cabbage.
The Chinese cabbage 9-cis-epoxies carotene peroxidase genes involved 9-cis-expression pattern analysis of epoxies carotene dioxygenase in Chinese cabbage
1.RNA extraction: with Chinese cabbage " heat-resisting No. 3 " (greenhouse sowing, temperature is set to 25-26 ℃, light application time is 16 hours).Extract the RNA of Chinese cabbage tender leaf when treating blade length to 2-3 sheet leaf.With the plant of normal growth in contrast, the plant of coercing processing with ABA, arid, Whitfield's ointment, water logging, freezing, uviolizing and sodium-chlor is an analytic target, utilize TRIzol test kit (GIBCO BRL, 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; The plant that the plant 9-cis of the Chinese cabbage of normal growth-epoxies carotene dioxygenase transcriptional level is handled than above-mentioned adverse circumstance is obviously on the low side, this explanation 9-cis-epoxies carotene dioxygenase transcriptional level and adverse circumstance utmost point significant correlation.
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: 19bp
(B) type: Nucleotide
(C) chain: strand
(D) topological framework: linearity
(ii). molecule type: oligonucleotide
(iii). sequence description: SEQ ID NO.1
CACCACTTCTTCGACGGAG
(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
GATTTCCATGTCTTCTCGTC
(2) information of SEQ ID NO.3
＜110〉Shanghai Communications University
＜120〉Chinese cabbage 9-cis-epoxies carotene dioxygenase albumen coded sequence
<140>
<141>
<160>2
<170>PatentIn?version?3.1
<210>1
<211>2020
<212>DNA
＜213〉Chinese cabbage (Brassica Campestris ssp.Pekinensis)
CTCTCGGAGA?TGGGTCGGTG?GTAATCATAC?TAAACTACCA?TTATCGTCTT?CTCAAAGCTC??60
CGCCTTGAAT?AATTCTTGCT?CCGTACCCAT?GACAAATCGT?TCCCAACGAA?AGCTCAATGT??120
TTCTTCTGCG?CTTCACACTC?ATCCTGCTCT?CCATTTCCCC?AAGCAATCCT?CCACCTCTCC??180
CGCCATTATT?GTCAACCCAA?AAACCAAAGA?ATCCGACACC?AAACAGATGA?GCTTGTTCCA??240
GAGAGCGGCG?GCGGCGGCGC?TGGACGCGGC?GGAGGGTTTC?CTCGTGAGCC?ACGAGCGACA??300
GCATCCCCTC?CCCAAAACCG?CCGATCCTAG?CGTTCAGATC?GCCGGAAACT?TCGCTCCGGT??360
GAACGAACAG?CCTCTCCGGC?GTAACCTCCC?GGTGGTCGGA?AAAATACCGG?ATTCCATCAA??420
AGGAGTGTAC?GTGCGCAACG?GAGCCAACCC?GCTTCACGAG?CCGGTGACAG?GTCACCACTT??480
CTTCGACGGA?GACGGGATGG?TTCACGCCGT?GACATTCGAG?GACGGTTCAG?CTAGCTACGC??540
GTGCCGGTTT?ACTCTGACCA?ACCGGTTTAT?CCAGGAACGT?CAATTAGGTC?GACCGGTTTT??600
CCCCAAGGCC?ATCGGCGAGC?TTCACGGCCA?CACCGGTATT?GCTCGACTCA?TGCTATTCTA??660
CGCCAGAGCC?GCAGCCGGTT?TAGTCGACCC?GGCTCACGGA?ACCGGAGTAG?CTAACGCCGG??720
TTTAGTCTAT?TTCAATAACC?GGTTATTAGC?CATGTCGGAA?GACGATTTGC?CTTACCAAGT??780
CCGGATCACT?CCAGGTGGAG?ACTTGAAAAC?CGTTGGCCGT?TACGATTTCG?ACGGGCAGTT??840
AGAATCCACA?ATGATCGCCC?ACCCGAAAGT?CGACCCGGAA?TCCGGCGAGC?TATTCGCTCT??900
AAGCTACGAC?GTCGTTTCAA?AGCCTTACTT?AAAGTACTTC?CGCTTCTCAC?CGGACGGAGA??960
AAAATCGCCG?GACGTCGAGA?TCCAGCTCGA?TCAGCCGACG?ATTATGCACG?ACTTGCCGAT??1020
AACAGAGAAC?TTCGTCGTTT?TACCGGACCA?GCAAGTCGTG?TTCAAGCTCC?AGGAGATGAT??1080
CCGCGGCGGC?TCTCCGGTGA?TCTACGACAA?GGAGAAGGTT?GCAAGATTCG?GAATTTTAGA??11401
CAAATACGCG?GCGGACTCGT?CGGGAATCAG?GTGGATCGAA?GCTCCGGACT?GCTTCTGCTT??1200
CCACCTCTGG?AACGCGTGGG?AGGAGCCGGA?AACAGAGGAG?GTCGTCGTGA?TCGGGTCGTG??1260
CATGACTCCG?CCGGACTCAA?TTTTCAACGA?GTCCGACGAG?AATCTCAAGA?GCGTCCTCTC??1320
CGAGATTCGG?CTCAACCTCA?GAACCGGAGA?GTCCACTCGC?CGTCCGATCA?TCTCCGACGG??1380
AGATCAACAA?GTCAACCTCG?AAGCAGGGAT?GGTGAACCGG?AACATGCTCG?GCCGTAAGAC?1440
CAAGTTCGCT?TACCTCGCTT?TAGCCGAGCC?GTGGCCTAAA?GTCTCCGGCT?TTGCGAAAGT?1500
CGATCTCGCC?ACCGGAGAGG?TCAAGAAGCA?TCTCTACGGC?GATGACCGTT?ACGGTGGAGA?1560
GCCTCTGTTT?CTCCCCGGTG?AGGGAGCAGA?AGACGATGGG?CACATCCTCT?GTTTTGTTCA?1620
CGACGAGAAG?ACATGGAAAT?CATGTGTGAA?TGTGATTGAC?GCAAAAACAA?TGTCTGCTGA?1680
GCCAGTGGCA?GTGGTGGAGC?TGCCGCATAG?GGTTCCATAT?GGCTTCCATG?CCTTCTTTGT?1740
TACAGAGGAA?CAACTCCAGG?AACAAACTCT?TACATAAGAC?AAGTTGTGTG?CATAATTATA?1800
TTATATGTTA?TATGCTACTA?TGAAGACGTT?GGAGAACCGA?GAAATTGGAT?GATTCAGACA?1860
TACGTATTAC?ATATACTACA?TGTATATTCA?TTTGAATGCC?TCGTTTCTGT?TTACATAAGA?1920
ATTTACATTT?TCATTTCAAT?AGTAAACTAG?GGTTCTTCTA?TCAAATAAAA?AAAAAAAGAA?1980
AAAAAAAAAA?AAAAAAAAAA?AAAAAAAAAA?AAAAAAAAAA???????????????????????2020
<210>2
<211>562
<212>PRT
＜213〉Chinese cabbage (Brassica Campestris ssp.Pekinensis)
<400>2
ALAQEQLQEE?ABFFTDFCRP?BYDPLEBBTB?PETSKAMTHI?BNBGSMWAME?HDBFGLIDCH?60
HETCECPLFL?PECCRYHHCR?LDMMBECATL?HBMTFCSBMP?WPETLTLRTF?MAMYCLKNYN?120
BKCTELNBQQ?HCHSIIPYYA?SECAYLNLYI?ESLBSMLNEH?SENFISHPPA?KGSCIBBBEE?180
AEPEEWTNWL?DFGFGHPTEI?WYICSSHTTR?MHLICFYTBM?EMHRIBPSCC?YIKEQLMFBB?240
QQHPLBBFNE?AIPLHDKIAP?QHLQIEBHPS?MECHPSFYFR?MLRPMSBBHR?SLTFLECSEP?300
HBMPDTIKAS?ELQCHFHRYC?BAMLHCCPAI?YBQRPLHHES?KTLLYNNFRB?LCTNTBCACD?360
TPHBLCTTTY?TRFLKLYTIC?ADCDLECITM?PFBPYCLQYE?QIFYNALAFY?GTRSTSCHEF?420
ABTDBKCHCH?FFDDCABPED?LPNTCNYBRB?CMISHPIMCB?BPLNYYLPQE?NBPTFNCTIQ?480
BSPHTAMPLP?DQYEDSBLFC?ETTHLTTTTT?YQFLSKQMAH?SEMAMPNBII?TPSASSQMPF?540
DLTPDADLTS?SBNLMYQSYN?AK??????????????????????????????????????????562
Claims (5)
1, a kind of Chinese cabbage 9-cis-epoxies carotene dioxygenase albumen coded sequence, it is characterized in that, comprise: coding has the nucleotide sequence of the polypeptide of Chinese cabbage 9-cis-epoxies carotene dioxygenase protein active, and shows at least 70% homology from the nucleotides sequence of Nucleotide 89-1774 position among described nucleotide sequence and the SEQ ID NO.3; Perhaps described nucleotide sequence can be under 45-55 ℃ of condition with SEQ ID NO.3 in from the nucleotide sequence hybridization of Nucleotide 89-1774 position.
2, Chinese cabbage 9-cis according to claim 1-epoxies carotene dioxygenase albumen coded sequence, it is characterized in that, described sequence encoding has polypeptide or its conservative property variation polypeptide or its active fragments of the aminoacid sequence shown in the SEQ ID NO.3, or its reactive derivative.
3, Chinese cabbage 9-cis according to claim 1 and 2-epoxies carotene dioxygenase albumen coded sequence is characterized in that, this sequence has among the SEQ ID NO.3 nucleotide sequence from Nucleotide 89-1774 position.
4, Chinese cabbage 9-cis according to claim 1-epoxies carotene dioxygenase albumen coded sequence is characterized in that, comprises: 8-100 continuous nucleotide in the dna molecular.
5, Chinese cabbage 9-cis according to claim 4-epoxies carotene dioxygenase albumen coded sequence is characterized in that, the dna molecular transformed host cells, and it is an eukaryotic cell.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2003101079722A CN1528889A (en) | 2003-10-16 | 2003-10-16 | Chinese cabbage 9-cis-carotene epoxide dioxygen zymoprotein coding sequence |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2003101079722A CN1528889A (en) | 2003-10-16 | 2003-10-16 | Chinese cabbage 9-cis-carotene epoxide dioxygen zymoprotein coding sequence |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1528889A true CN1528889A (en) | 2004-09-15 |
Family
ID=34304575
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2003101079722A Pending CN1528889A (en) | 2003-10-16 | 2003-10-16 | Chinese cabbage 9-cis-carotene epoxide dioxygen zymoprotein coding sequence |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1528889A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103732740A (en) * | 2011-08-10 | 2014-04-16 | 诺维信公司 | Polypeptides having peroxygenase activity and polynucleotides encoding same |
| CN113215180A (en) * | 2021-06-08 | 2021-08-06 | 吉林大学 | Corn 9-cis-epoxy carotenoid dioxygenase protein gene ZmVP14 and application |
-
2003
- 2003-10-16 CN CNA2003101079722A patent/CN1528889A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103732740A (en) * | 2011-08-10 | 2014-04-16 | 诺维信公司 | Polypeptides having peroxygenase activity and polynucleotides encoding same |
| US9487761B2 (en) | 2011-08-10 | 2016-11-08 | Novozymes A/S | Polypeptides having peroxygenase activity and polynucleotides encoding same |
| CN103732740B (en) * | 2011-08-10 | 2018-07-31 | 诺维信公司 | Polypeptide with peroxidase activity and the polynucleotides for encoding the polypeptide |
| US10465173B2 (en) | 2011-08-10 | 2019-11-05 | Novozymes A/S | Polypeptides having peroxygenase activity and polynucleotides encoding same |
| CN113215180A (en) * | 2021-06-08 | 2021-08-06 | 吉林大学 | Corn 9-cis-epoxy carotenoid dioxygenase protein gene ZmVP14 and application |
| CN113215180B (en) * | 2021-06-08 | 2023-03-28 | 吉林大学 | Corn 9-cis-epoxy carotenoid dioxygenase protein gene ZmVP14 and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1807453A (en) | Bacterial leaf spot resistance related protein and its coding gene and uses | |
| CN1854154A (en) | Rice blast resistant related protein, its coding gene and use | |
| CN1420932A (en) | Apomixis conferred by expression of SERK interacting proteins | |
| CN1840542A (en) | Rice tillering related protein, genes encoding same, and use thereof | |
| CN1807611A (en) | Alfalfa cysteine prolease and its coding gene and uses | |
| CN1297661C (en) | A rice blast resistance gene, its encoded protein and use thereof | |
| CN1528889A (en) | Chinese cabbage 9-cis-carotene epoxide dioxygen zymoprotein coding sequence | |
| CN1563386A (en) | Reducing enzyme protein coded sequence of sulfoxide methionine of cotton | |
| CN1600861A (en) | Protein coded sequence of binding factor in ethane response element of cotton | |
| CN1289664C (en) | EPSP synthase of variable halomonas high resistance glyphosate and its encoding sequence | |
| CN1295334C (en) | Wheat antidisense related gene TaEDR1 and its application | |
| CN1219060C (en) | Cabbage rape BnBDC1 protein coding sequence | |
| CN1273600C (en) | Coding sequence of Chinese yew tarad-ane C13-lateral chain-N-benzoyl transiting enzymic protein | |
| CN1252270C (en) | Taxaceae 3-hydroxy-3-methylpentadiacyl cozymase A synthetic zymoprotein coding sequence | |
| CN1566146A (en) | Paddy rice stalk extension gene, coded protein and application thereof | |
| CN1594570A (en) | Protein coded sequence of protein kinase 1 activated by Brassica napus L. cell mitogen | |
| CN1769436A (en) | Nanjing bass 3-hydroxyl-3-methyl glutaryl coenzyme A reductase protein encoding sequence | |
| CN1284855C (en) | Betaine aldehyde dehydrogenase gene and its encoded protein | |
| CN1232530C (en) | Ethane cyclic amp receptor protein of wheat and its coding sequence | |
| CN1191354C (en) | Method for producing transgene paddy with amount of spikelet being increased | |
| CN1760363A (en) | Coded sequence of reductase enzyme protein of eucommia 3-hydroxy-3-coenzyme of methyl glutaryl A | |
| CN1528895A (en) | Cabbage type rape drought-resistant salinity-tolerant transcription factor BnHB6 protein coding seuquence | |
| CN1179974C (en) | Reverse-corelation-resisting signal transfer gene of plant and protein coded thereof | |
| CN1557952A (en) | Solanum lycopersicoides dun. S1Ve1 receptor protein encoding sequence and use thereof | |
| CN1597959A (en) | Code sequence of anti-anaerobic enolase protein in rape of cabbage type |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |