CN100360664C - Nanjing bass 3-hydroxyl-3-methyl glutaryl coenzyme A reductase protein encoding sequence - Google Patents
Nanjing bass 3-hydroxyl-3-methyl glutaryl coenzyme A reductase protein encoding sequence Download PDFInfo
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Abstract
The present invention relates to a Nanjing linden tm-Hmgr protein coding sequence which belongs to the field of gene engineering. The separated DNA molecule comprises a nucleotide sequence coding a polypeptide with the protein activity of the Nanjing linden tm-Hmgr; the nucleotide sequence has at least 70% homology with the nucleotide sequence from 131th to 1888th nucleotides in SEQ ID No. 3, or the nucleotide sequence can be hybridized with the nucleotide sequence from 131th to 1888th nucleotides in the SEQ ID No. 3, under the condition of 40 to 55DEG C. The present invention is a reductase of a 3-hydroxy-3-methyl glutaryl coenzyme A, which is helpful to improve the content of terpenoid secondary metabolites or precursors thereof in the Nanjing linden, is capable of developing a novel protein resource and is helpful to protect the healthy growth of people; simultaneously, the present invention has important significance for the research and development of agriculture and medicine in China.
Description
Technical field
The present invention relates to molecular biology, gene engineering technology field.Particularly, the present invention relates to a kind of tm-Hmgr albumen of in tilia miqueliana, expressing (tilia miqueliana 3-hydroxy-3-methylglutaryl-coenzyme A reductase enzyme protein, Tilia miqucliana 3-hydroxy-3-methylglutaryl-CoA Reductase, TMHMGR) and nucleotide sequence.
Background technology
Tilia miqueliana (Tilia miqueliana Maxim) is commonly called as linden, belongs to the Tiliaceae lime tree and belongs to, and originates in each province, East China, east, Hunan, Guangdong, is born in hillside, the gully woods.Tilia miqueliana belongs to deciduous tree, and trunk is perfectly straight, and material is good, and growth is slow, and the life-span is long.Its leaf beauty, tree performance is quiet and beautiful, and summer, chrysanthemum was completely set, and is full of fragrance, is famous green shade seeds and good nectariferous plant.The lime tree plant is very famous herbal medicine in the west, drinks as medicinal herb tea among the people, has diuresis, is good for the stomach, treats neurodynia and function such as calm, contains the various active material.We find also that under study for action tilia miqueliana contains multiple active skull cap components, and comprising terpenoid, many terpenoids have good pharmacologically active, are the main components of Chinese medicine.The present resource of tilia miqueliana is more and more poorer, but its potential economy and pharmaceutical use are huge.
In recent years, the terpenoid secondary metabolism of many vegetable materials be studies have shown that HMGR is key enzyme in the mevalonate pathway.3-hydroxy-3-methylglutaric acid list acyl CoA (HMGCoA) generates mevalonic acid (MVA) under the effect of HMGR in mevalonate pathway.Because this reaction is a non-reversible process, so HMGR is considered to first rate-limiting enzyme in this approach.By improving the activity or the content of 3-hydroxy-3-methylglutaryl-coenzyme A reductase enzyme, the content of terpenoid secondary metabolite or its precursor in the raising tilia miqueliana that can be indirect.Modern science discovers that the terpenoid secondary metabolite is a natural active matter, is that to solve the Western medicine toxic side effect that the present world faces big, and difficult diseases such as cancer, acquired immune deficiency syndrome (AIDS) such as can't cure at a new way of a difficult problem.
In analysis to existing document, " Plant Cell (vegetable cell); 1992; 4 (10): 1333-1344 " reported and cloned the 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene from potato, announced the sequence of the 3-hydroxy-3-methyl glutaryl coenzyme reductase gene of rubber tree etc. on the NCBI website.But tilia miqueliana tm-Hmgr protein sequence and nucleotide sequence thereof report is not arranged as yet so far.
Before the present invention comes forth, any tilia miqueliana tm-Hmgr protein sequence and nucleotide sequence of mentioning in the present patent application thereof that disclose or reported do not arranged as yet.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, a kind of tilia miqueliana tm-Hmgr albumen coded sequence is provided.Make it comprise said gene Fusion gene construct, carry the new recombinant expression vector of this construct, by said expression vector transformed plant cells, and the transgenic plant and the offspring thereof of the said gene that produces by transformant, comprise plant seed and plant tissue, the transgenic plant that obtained will have the terpenoid secondary metabolite content that significantly improves.
The present invention is achieved by the following technical solutions, the present invention isolated dna molecular comprise: coding has the nucleotide sequence of polypeptide of tilia miqueliana tm-Hmgr protein-active, shows at least 70% homology from the nucleotides sequence of Nucleotide 131-1888 position among described nucleotide sequence and the SEQ IDNO.3; Perhaps described nucleotide sequence can be under 40-55 ℃ of condition with SEQ ID NO.3 in from the nucleotide sequence hybridization of Nucleotide 131-1888 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 131-1888 position.
The isolated tilia miqueliana tm-Hmgr of the present invention 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.
Vector dna molecule transformed host cells provided by the present invention, it is an eukaryotic cell.It comprises 8-100 continuous nucleotide in the described dna molecular.
The above-mentioned carrier of the present invention.This host cell is a tobacco 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 " tilia miqueliana tm-Hmgr albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with tilia miqueliana tm-Hmgr protein-active is as 131-1888 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.3.This degenerate sequence is meant, is arranged in the encoder block 131-1888 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 131-1888 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 131-1888 position.This term also comprise with SEQ ID NO.3 in from the homology of nucleotide sequence at least 70% of Nucleotide 131-1888 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 tilia miqueliana tm-Hmgr albumen 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 " tilia miqueliana tm-Hmgr albumen or polypeptide " refers to the SEQ ID NO.3 polypeptide of sequence that it has tilia miqueliana tm-Hmgr protein-active.This term also comprises the variant form that has with the SEQ ID NO.4 sequence of natural tilia miqueliana tm-Hmgr albumen identical function.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 tilia miqueliana tm-Hmgr and reactive derivative.
The variant form of tilia miqueliana tm-Hmgr protein polypeptide of the present invention comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low stringent condition can with the coded albumen of the DNA of tilia miqueliana tm-Hmgr protein D NA hybridization and the polypeptide or the albumen that utilize the serum of tilia miqueliana tm-Hmgr protein polypeptide to obtain.
In the present invention, " tilia miqueliana tm-Hmgr albumen 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 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
85％ identity in 910 nt over I ap
Query：207 attccaccaaagcctccgacgcactacctcctcctttgtatataatgaatgcggtgttct 266
||||||||||||| || |||||| | || | |||||||||| ||| |||||| |||||||
Sbjct： 825 attccaccaaagcttctgacgcattgccacttcctttgtatttaacgaatgctgtgttct 884
Query： 267 tcacgctcttcttctcggtggtttattttcttctttcccgttggcgtgaaaagatccgaa 326
|||| ||||| ||||||| ||||||||| ||||||| ||||||||| ||||||||||||
Sbjct： 885 tcactctctttttctcggcggtttatttccttctttgccgttggcgggaaaagatccgat 944
Query： 327 tctccacgcctctccacatcgtcaccttttccgagatcgttgcggttctcgctttagttg 386
||| ||||||||||| |||||||||||||||||||||||||| |||| |||| ||||
Sbjct： 945 cctctacgcctctccatgtcgtcaccttttccgagatcgttgcgattcttgcttccgttg 1004
Query： 387 cctcgtttatatatcttttgggattcttcgggattgactttgttcagtctttgattctcc 446
| |||||||| || |||||||| ||||| || || || || ||||| |||||| ||||||
Sbjct： 1005 cgtcgtttatttaccttttggggttctttggtatcgatttcgttcaatctttggttctcc 1064
Query： 447 gaccatcaactgacatttggaattccgaggaagaagaagaggaaaatgaagttttgctcc 506
| ||||| ||||| ||||| | | || || || |||| ||||| |||||||||||| |
Sbjct： 1065 ggccatcggctgacgtttgggctactgaagatgatgaagtggaaagtgaagttttgcttc 1124
Query： 507 gtaaagaagattcgcgtaaagtcccttgcggccaagctctcgattgctcgtttcctcctt 566
|||| |||||| | ||| | || ||||| || ||||| || ||| | ||| ||| |||
Sbjct： 1125 gtaatgaagatgctcgtcacgttccttgtggacaagcacttgatcggtcgattcgatctt 1184
Query： 567 tgcctccttcggcaccgattgtaactgcccagaaagttttcgatgaaaagcttgtggaag 626
||| ||| ||| ||| ||||||||||| ||||||| |||||||||| || |||| ||
Sbjct： 1185 tgcaacctccggaacctattgtaactgctgagaaagtgttcgatgaaatgcctgtgacag 1244
Query： 627 ttacaaccgaggaagacgaagaaataattaaatccgtagtggctggaacaaccccttcat 686
||| || ||||||||||||||||||||||| |||||| || ||||| || |||||||
Sbjct： 1245 ttatgactgaggaagacgaagaaataattagatccgttgtttgtggaatgactccttcat 1304
Query： 687 attctttggaatcgaaattaggtgattgcaagagagcgactgcgatcaggcgtgaggcgc 746
||||||||||||| ||||||| ||||||||||||||| |||||||||||||||||||
Sbjct： 1305 attctttggaatctaaattagatgattgcaagagagcagctgcgatcaggcgtgaggctt 1364
Query： 747 tgcagagattaacgggaaagtcattatcaggattgcccttggatggatttgattatgcgt 806
|||||||| |||||| ||||||||||||||||||||||||||||| |||||||||| ||
Sbjct： 1365 tgcagagaataacagggaagtcattatcaggattgcccttggatggttttgattatgagt 1424
Query： 807 cgattttagggcagtgttgtgagatgccggttgggtacgtgcaaattcccgtgggaattg 866
|||| || || ||||||||||||||||||||||| |||| ||| ||||||||||| ||||
Sbjct： 1425 cgatattgggacagtgttgtgagatgccggttggttacgagcagattcccgtgggcattg 1484
Query： 867 ctgggccgttgttgcttaatggaagagaatacacggttcctatggcaaccacggagggga 926
||||||| |||||||||||||||||||||||| ||||||| ||||||||||| || ||
Sbjct： 1485 ctgggcctttgttgcttaatggaagagaatactcggttcccatggcaaccaccgaaggat 1544
Query： 927 ccttggtggctagcactaataggggatgtaaggctattcatttgtctggtggagctacaa 986
|||||| |||||||||||||| || ||||||||||||||||||||||||||||||||||
Sbjct： 1545 gcttggtagctagcactaatagaggctgtaaggctattcatttgtctggtggagctacaa 1604
Query： 987 gtgttcttttgaaagatgggatgactagagctcctgtggttaggttcagtactgcgaaaa 1046
||||||| |||| |||||||||||||||||||||||| || |||||| |||| || ||||
Sbjct： 1605 gtgttctattgagagatgggatgactagagctcctgttgtgaggttcggtaccgcaaaaa 1664
Query： 1047 gagcagctgatctgaagttttatttggaggatcctgaaaatttcgagaccttggctgttg 1106
| || |||||||||||||| |||||||| ||||||||||||||||||||||||||| ||
Sbjct： 1665 gggcggctgatctgaagttgtatttggaagatcctgaaaatttcgagaccttggcttgtg 1724
Query： 1107 tttttaacag 1116
||||||||||
Sbjct： 1725 tttttaacag 1734
90％identity in 350 nt over I ap
Query： 1298 ggaaacttctgttcggacaaaaagccagctgcagtaaattggattgaaggacgtggcaaa 1357
|||||||||||||| ||||||||||||||||||||||||||||||||||| || ||||||
Sbjct： 2364 ggaaacttctgttccgacaaaaagccagctgcagtaaattggattgaaggccgaggcaaa 2423
Query： 1358 tctgttgtctgcgaggccatcattaaggatgatgtggtgaggaaggtcttgaagactggt 1417
||||| ||||| |||||||||||||| | |||||| |||| ||||||||||||||| ||
Sbjct： 2424 tctgtcgtctgtgaggccatcattaatggtgatgtagtgacgaaggtcttgaagacaagt 2483
Query： 1418 gtggaatctctcgtggagcttaacatgcttaagaaccttactggatctgctatggctgga 1477
|| ||||||||||||||||||||||||||||||||||||||||| ||||| |||||||||
Sbjct： 2484 gtagaatctctcgtggagcttaacatgcttaagaaccttactggttctgccatggctgga 2543
Query： 1478 gctctgggtggatttaatgcctatgccggtaacattgtgtctgctgtctacatagctacc 1537
|||||||||||||||||||| ||||| |||||||||| |||||||||| ||||| ||
Sbjct： 2544 gctctgggtggatttaatgctcatgccagtaacattgtcactgctgtctatatagccact 2603
Query： 1538 ggtcaagatccggctcaaaatgtcgagagctctcattgcataacgatgatggaagctgtt 1597
|| |||||||||| |||||||||||||||||||||||||| || |||||||||||||||
Sbjct： 2604 ggccaagatcctgcccaaaatgtcgagagctctcattgcatcaccatgatggaagctgtt 2663
Query： 1598 aatgatggcaaggaccttcacatctctgttacaatgccttccatcgaggt 1647
|||| |||||||||||||| ||||||| |||||||||||||| |||||
Sbjct： 2664 aatggcggcaaggaccttcatgtctctgtcacaatgccttccattgaggt 2713
88％identity in 244 nt over I ap
Query： 1644 aggttggcactgttggtggtggaactcagcttgcatctcagtcagcgtgtttgaacctgc 1703
||||||||||||||||||||||||||||||||||||| || ||||||||||||||||| |
Sbjct：3553 aggttggcactgttggtggtggaactcagcttgcatcacaatcagcttgtttgaaccttc 3612
Query：1704 tcggtgtcaagggtgcaagcaaagaatcacctggagaaaactctagaatgcttgcaacca 1763
| |||||||||||||||||||||||||| ||||||| |||||| | | | ||||| || |
Sbjct：3613 ttggtgtcaagggtgcaagcaaagaatcccctggagcaaactcaatactccttgcgacta 3672
Query：1764 ttgtagccggtgctgtccttgctggggagctgtcactcatgtctgcacttgcagctgggc 1823
| |||||||||||||||||||| || ||||||||||| ||||| |||||||| || ||||
Sbjct：3673 tcgtagccggtgctgtccttgccggcgagctgtcactgatgtcagcacttgctgccgggc 3732
Query：1824 aactaattaacagccatatgaagtacaataggtcaaataaggacgtgtccaaggcttctt 1883
||||| | || ||||||||||||||||||||||| | ||||| || ||||||| |||||
Sbjct：3733 aactagtgaaaagccatatgaagtacaataggtctagcaaggatgtttccaaggtttctt 3792
Query：1884 ccta 1887
||||
Sbjct：3793 ccta 3796
91％identity in 183 nt overIap
Query：1114 cagatcaagtagatttgctaggcttcaaggtatcaaatgtgcaattgctgggaagaatct 1173
||||||||| |||||||||||||||||| | ||||||||||||||||| |||||||||||
Sbjct：1829 cagatcaagcagatttgctaggcttcaaagcatcaaatgtgcaattgcagggaagaatct 1888
Query：1174 ctatttgagattcacttgcagtactggtgatgctatggggatgaacatggtttccaaggg 1233
||||| ||||||| ||| |||||||||||||||||||||||||||||||||||||||
Sbjct：1889 gtatttaagattctcatgctttactggtgatgctatggggatgaacatggtttccaaggg 1948
Query：1234 agtccaaaacgtattggatttccttcaaactgatttctctgacatggatgtcattggcat 1293
||| |||||||| || |||||||||||||| |||||| ||||||||||||||||||||||
Sbjct：1949 agttcaaaacgttttagatttccttcaaaccgatttccctgacatggatgtcattggcat 2008
Query：1294 ctc 1296
|||
Sbjct：2009 ctc 2011
96％i dentity in 30nt over I ap
Query：1983 catcatctctttgtaataagctaagtagac 2012
||||||||||| ||||||||||||||||||
Sbjct：3877 catcatctcttagtaataagctaagtagac 3906
Query: the nucleotide sequence of tilia miqueliana tm-Hmgr
Sbjct: the nucleotide sequence of upland cotton gh-Hmgr1 (AF038045.1)
Table 2 is that the homology of the nucleotide sequence of tilia miqueliana tm-Hmgr of the present invention and upland cotton (upland cotton) gh-Hmgr1 compares (GAP) table.
Table 3
83％ identity in 585 aa over I ap，87％similarity in 585 aa over I ap
Query：1 MEARRRSSTKPIQSLKTTKTVSLEENSTKASDALPPPLYIMNAVFFTLFFSVVYFLLSRW 60
ME RRSST I+S K + ++LE++STKASDALP PLY+NAVFFTLFFS VYFLL RW
Sbjct：1 METHRRSSTNSIRSHKPARPIALEDDSTKASDALPLPLYLTNAVFFTLFFSAVYFLLCRW 60
Query：61 REKIRISTPLHIVTFSEIVAVLALVASFIYLLGFFGIDFVQSLILRPSTDIWNSXXXXXX 120
REKIR STPLH+VTFSEIVA+LA VASFIYLLGFFGIDFVQSL+LRPS D+W+
Sbjct：61 REKIRSSTPLHVVTFSEIVAILASVASFIYLLGFFGIDFVQSLVLRPSADVWATEDDEVE 120
Query：121 XXVLLRKEDSRKVPCGQALDCSFPPLPPSAPIVTAQKVFXXXXXXXXXXXXXXXXXSVVA 180
VLLR ED+R VPCGQALD S L P P I VTA+KVF SVV
Sbjct：121 SEVLLRNEDARHVPCGQALDRSIRSLQPPEPIVTAEKVFDEMPVTVMTEEDEEIIRSVVC 180
Query：181 GTTPSYSLESKLGDCKRATAIRREALQRLTGKSLSGLPLDGFDYASILGQCCEMPVGYVQ 240
G TPSYSLESKL DCKRA AIRREALQR+TGKSLSGLPLDGFDY SILGQCCEMPVGY Q
Sbjct：181 GMTPSYSLESKLDDCKRAAAIRREALQRITGKSLSGLPLDGFDYESILGQCCEMPVGYEQ 240
Query：241 IPVGIAGPLLLNGREYTVPMATTEGTLVASTNRGCKAIHLSGGATSVLLKDGMTRAPVVR 300
IPVGIAGPLLLNGREY+VPMATTEG LVASTNRGCKAIHLSGGATSVLL+DGMTRAPVVR
Sbjct：241 IPVGIAGPLLLNGREYSVPMATTEGCLVASTNRGCKAIHLSGGATSVLLRDGMTRAPVVR 300
Query：301 FSTAKRAADLKFYLEDPENFETLAVVFNRSSRFARLQGIKCAIAGKNLYLRFTCSTGDAM 360
F TAKRAADLK YLEDPENFETLA VFNRSSRFARLQ IKCAIAGKNLYLRF+C TGDAM
Sbjct：301 FGTAKRAADLKLYLEDPENFETLACVFNRSSRFARLQSIKCAIAGKNLYLRFSCFTGDAM 360
Query：361 GMNMVSKGVQNVLDFLQTDFSDMDVIGISGNFCSDKKPAAVNWIEGRGKSVVCEAIIKDD 420
GMNMVSKGVQNVLDFLQTDF DMDVIGISGNFCSDKKPAAVNWIEGRGKSVVCEAII D
Sbjct：361 GMNMVSKGVQNVLDFLQTDFPDMDVIGISGNFCSDKKPAAVNWIEGRGKSVVCEAIINGD 420
Query：421 VVRKVLKTGVESLVELNMLKNLTGSAMAGALGGFNAYAGNIVSAVYIATGQDPAQNVESS 480
VV KVLKT VESLVELNMLKNLTGSAMAGALGGFNA+A NIV+AVYIATGQDPAQNVESS
Sbjct：421 VVTKVLKTSVESLVELNMLKNLTGSAMAGALGGFNAHASNIVTAVYIATGQDPAQNVESS 480
Query：481 HCITMMEAVNDGKDLHISVTMPSIEVGTVGGGTQLASQSACLNLLGVKGASKESPGENSR 540
HCITMMEAVN GKDLH+SVTMPSIEVGTVGGGTQLASQSACLNLLGVKGASKESPG NS
Sbjct：481 HCITMMEAVNGGKDLHVSVTMPSIEVGTVGGGTQLASQSACLNLLGVKGASKESPGANSI 540
Query：541 MLATIVAGAVLAGELSLMSALAAGQLINSHMKYNRSNKDVSKASS 585
+LATIVAGAVLAGELSLMSALAAGQL+SHMKYNRS+KDVSK SS
Sbjct：541 LLATIVAGAVLAGELSLMSALAAGQLVKSHMKYNRSSKDVSKVSS 585
Query: tilia miqueliana tm-Hmgr aminoacid sequence
Sbjct: upland cotton gh-Hmgr1 aminoacid sequence (GenBank Accession No.AAC05088.1)
Table 3 is that the homology of the aminoacid sequence of proteic aminoacid sequence of tilia miqueliana tm-Hmgr of the present invention and upland cotton gh-Hmgr1 compares (FASTA) table.Wherein, identical amino acid marks with the amino acid monocase between two sequences.
Invention also comprises the analogue of tilia miqueliana tm-Hmgr albumen or polypeptide.The difference of these analogues and natural 3-hydroxy-3-methylglutaryl-coenzyme A reductase enzyme 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, Y-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 tilia miqueliana tm-Hmgr protein polypeptide of the present invention, tilia miqueliana tm-Hmgr albumen coded sequence operationally can be connected in expression regulation sequence, thereby form tilia miqueliana tm-Hmgr 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 tilia miqueliana tm-Hmgr protein gene product promptly analyzes the existence of the proteic rna transcription thing of tilia miqueliana tm-Hmgr in cell.
In addition, can be used as the nucleic acid molecule of probe among the present invention, this molecule has 8-100 continuous nucleotide of tilia miqueliana tm-Hmgr albumen nucleotide coding sequence usually, preferably has 15-50 continuous nucleotide.This probe can be used for whether existing in the test sample the proteic nucleic acid molecule of coding tilia miqueliana tm-Hmgr.
The present invention relates to whether exist in the test sample method of tilia miqueliana tm-Hmgr pyrenoids 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 tilia miqueliana tm-Hmgr albumen nucleotide coding 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 tilia miqueliana tm-Hmgr pyrenoids nucleotide sequence of the present invention and aminoacid sequence, can be on the homology basis of nucleic acid homology or marking protein, screening tilia miqueliana tm-Hmgr albumen homology gene or homologous protein.
In order to obtain the dot matrix of the bark of eucommia cDNAs relevant with tilia miqueliana tm-Hmgr protein gene, can screen tilia miqueliana cDNA library with dna probe, these probes are under low stringent condition, use
32P to tilia miqueliana tm-Hmgr proteic all or part of do the radioactivity mark and.The cDNA library that most is suitable for screening is the library from tilia miqueliana.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 the proteic gene family of tilia miqueliana tm-Hmgr.
Tilia miqueliana tm-Hmgr pyrenoids thuja acid 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, WHFreeman Co., 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 AppliedBiosystems.Can distinguish proteic each fragment of chemosynthesis the present invention, be connected to produce the molecule of total length with chemical process then.
Utilize tilia miqueliana tm-Hmgr albumen of the present invention,, can filter out with tilia miqueliana tm-Hmgr albumen interactional material takes place, perhaps acceptor, inhibitor or short of money dose etc. by various conventional screening methods.Tilia miqueliana tm-Hmgr protein gene of the present invention can be used for improving the content of terpenoid secondary metabolite in the tilia miqueliana or its precursor by genetic engineering technique; and these secondary metabolites have huge using value clinically, and are helpful to the protection people's healthy growth.Thereby the present invention has great application prospect.
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 tilia miqueliana tm-Hmgr protein gene
1. separate tissue (isolation)
The tilia miqueliana young leaflet tablet derives from Medicinal Plant Biological Technology Key Laboratory, Jiangsu, take material after, place the freezing preservation of liquid nitrogen immediately.
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, GIBCO BRL, 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 some plant Hmgr, the design degenerate primer utilizes homologous genes clone principle, adopts Smart-RACE method (Clonetech test kit) to carry out the cDNA full-length clone, divides three phases to carry out:
(1)3’-RACE
PCR (UPM+F2) obtains TMF2 ' (936bp), 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 Hmgr gene of knowing its nucleotide sequence and proteins encoded and known upland cotton (Gossypiumhirsutum) etc. is very high, so think that tentatively it is a Hmgr gene.
(2)5’-RACE
According to 3 ' RACE result, design reverse special primer R2, obtain TMR2 ' (1358bp) (process is with (1)) through PCR (UPM+R2).Reclaim, be connected on the T-Easy carrier, with SP6 or T7 as universal primer, adopt stop the thing fluorescent mark (Big-Dye, Perkin-Elmer, method USA), (Perkin-Elmer checks order on USA) at the ABI377 sequenator.
(3) with 5 ' RACE sequencing result and 3 ' RACE sequencing result than preface and splice, obtain the full length fragment sequence information, and design a pair of special primer and carry out pcr amplification tm-Hmgr coding region (DF1+DR1) and obtain tm-Hmgr coding region (1758bp) (process with (1)).
The gene that result's proof of BLAST newly obtains from tilia miqueliana really is a plant Hmgr gene.By being used in combination above-mentioned 3 kinds of methods, obtained the complete encoding sequence of candidate's tilia miqueliana TmHMGR.Obtain on the total length basis of (comprising complete open reading frame at least) in splicing, further TmHMGRF1:5 '-ACTCAACCCCCGAAAACCG-3 ' (SEQ ID NO.1) is a forward primer to the design primer, oligonucleotide TmHMGRR1:5 '-ACAATTCTGTCATTTTACC-3 ' (SEQ ID NO.2) is a reverse primer, with total RNA is template, carry out the RT-PCR amplification, the PCR condition of TmHMGRF1/TmHMGRR2 be 94 ℃ 5 minutes, carried out 35 circulations in 2 minutes with 94 ℃ 1 minute, 55 ℃ 1 minute and 72 ℃ thereupon, extended 10 minutes with 72 ℃ at last.The electrophoresis detection pcr amplification product, the acquisition expanding fragment length is 2107bp.Clone, check order with pcr amplification product according to a conventional method then, obtain the sequence shown in the SEQ ID NO.3.
Embodiment 2
The sequence information and the homology analysis of tilia miqueliana tm-Hmgr protein gene
The length of the tilia miqueliana tm-Hmgr albumen full-length cDNA that the present invention is new is 2160 bp, and detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 131-1888 position Nucleotide.Derive the proteic aminoacid sequence of tilia miqueliana tm-Hmgr according to full-length cDNA, totally 585 amino-acid residues, molecular weight 62956.49, pI are 6.11.Detailed sequence is seen SEQ ID NO.4.
Proteic full length cDNA sequence of tilia miqueliana tm-Hmgr and coded protein thereof are carried out Nucleotide and protein homology retrieval with blast program in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-reduncant GenBank CDStranslations+PDB+SwissProt+Superdate+PIR database, found that it and upland cotton 3-hydroxy-3-methylglutaryl-coenzyme A reductase enzyme 1 gene (AF038045.1) have higher homology (subordinate list 2) on nucleotide level; On amino acid levels, it and upland cotton 3-hydroxy-3-methylglutaryl-coenzyme A reductase enzyme 1 (GenBank Accession No.AAC05088.1) have 78% homogeny and 86% similarity (seeing Table 3).This shows that all there are higher homology in tilia miqueliana tm-Hmgr albumen and upland cotton 3-hydroxy-3-methylglutaryl-coenzyme A reductase enzyme 1 on nucleic acid still is protein level, so can think that tilia miqueliana tm-Hmgr albumen is also similar on function.
Embodiment 3
Tilia miqueliana tm-Hmgr albumen carries out prokaryotic expression and purification in intestinal bacteria
In this embodiment, the tilia miqueliana tm-Hmgr albumen coded sequence of total length or fragment are built into commercial protein merge among the expression vector, to express and purification of recombinant proteins.
Tilia miqueliana tm-Hmgr protein polypeptide is carried out prokaryotic expression with the form of fusion rotein in intestinal bacteria.
Construction of prokaryotic expression vector, and transformed into escherichia coli
According to the proteic aminoacid sequence of tilia miqueliana tm-Hmgr, the primer of design protein-coding region, and on positive anti-primer, introduce restriction endonuclease sites (this decides according to pET32a (+) 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, tilia miqueliana tm-Hmgr protein gene is being guaranteed to be cloned into pET32a (+) carrier (Novagen) under the correct prerequisite of reading frame.Identify that good expression vector utilizes CaCl
2Method changes e. coli bl21 over to, and Screening and Identification obtains containing engineering bacteria BL21-pET32a (+)-3-hydroxy-3-methylglutaryl-coenzyme A reductase enzyme of pET32a (+)-3-hydroxy-3-methylglutaryl-coenzyme A reductase enzyme expression vector.
Express the isolation identification of the engineering bacteria of Trx-tm-Hmgr recombinant protein
The BL21-pET32a (+) of picking list bacterium colony-eu-Hmgr engineering bacteria contains jolting overnight incubation in the LB substratum of 100 μ g/ml penbritins in 3ml, draw nutrient solution by 1: 100 concentration and in new LB substratum (containing 100 μ g/ml penbritins), cultivated about 3 hours, to OD
600After reaching 0.5, adding IPTG continues at 37 ℃ to final concentration 1mmol/L and cultivated respectively 0,1,2,3 hours.It is centrifugal to get the different 1ml bacterium liquid of incubation time, in the bacterial precipitation thing, add lysate (2 * SDS sample-loading buffer, 50 μ l, distilled water 45 μ l, 3-mercaptoethanol 5 μ l), the suspendible bacterial precipitation, boiled in the boiling water bath 5 minutes, centrifugal 1 minute of 10000rpm, supernatant adds electrophoresis in the 12%SDS-PAGE glue.The bacterial strain that the protein content of dyeing back observation expection molecular weight size increases with the IPTG induction time is the engineering bacteria of expressing the Trx-3-tm-Hmgr fusion rotein.
The extraction purifying of Trx-3-tm-Hmgr fusion rotein
The proteic engineering bacteria BL21-pET32a of abduction delivering Trx-tm-Hmgr amalgamation and expression (+)-tm-Hmgr as stated above, collect thalline through centrifugation, and come the purifying inclusion body with BugBuster reagent and Benzonase nuclease according to the specification sheets of producer (Novagen).Inclusion body can with the dissolving damping fluid (50mM CAPS, pH 11.0,0.3%N-lauroylsarcosine) dissolve, (200mM Tris-HCl pH8.5) dialyses to use dialysis buffer liquid again.Use Histidine to carry out affinity chromatography then, and collect the Trx-tm-Hmgr fusion rotein through elution buffer (1M imidazole, 500mM NaCl, 20mM Tris-HCl pH 7.9) wash-out in conjunction with (HisBind) resin.Fusion rotein is the expressing protein of separable acquisition tm-Hmgr after 20 ℃ of enzymes of enteropeptidase are cut 16 hours.
The vitality test of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase enzyme of purifying
Press (Mol.Cell.Biol such as Thorsness, 1989,9:5702～5712) method is carried out the mensuration of enzyme activity to the 3-hydroxy-3-methylglutaryl-coenzyme A reductase enzyme of expression and purification, and research 3-hydroxy-3-methylglutaryl-coenzyme A under its effect generates the ability of mevalonic acid.Reaction system contains 0.05MTris-HCL (pH=7.5), 5mM DTT, 200mM NADPH, 300mM
14Acetyl-CoA of C mark (1mCi/mmol) and 20mM glucose 1-phosphate1-, every milliliter of 9.75mU glucose phosphate dehydrogenase is used for the regeneration of NADPH, cumulative volume is that the 100ul. reaction process is as follows: at first add 37 ℃ of water-baths of 400mg protein 5 minutes. add the HCL termination reaction of 20ml 6N then, mixture 37 ℃ of water-baths 15 minutes again make 3-hydroxy-3-methylglutaryl-coenzyme A change into mevalonic acid.Reacting final product separates on Bio-Rex 5 pillars with substrate material, and reacting final product can be washed from pillar.Available liquid scintillation register is measured
14The content of the reacting final product of C mark.The result shows that expressed proteins has the enzymic activity that the catalysis 3-hydroxy-3-methylglutaryl-coenzyme A generates mevalonic acid really.
Embodiment 4
Tilia miqueliana tm-Hmgr albumen carries out eukaryotic cell expression in tobacco
The structure that will contain the expression vector of goal gene (tilia miqueliana tm-Hmgr protein gene), according to the proteic full length sequence of tilia miqueliana tm-Hmgr (SEQ ID NO.3), design amplifies the primer that complete coding is read frame, and on the upstream and downstream primer, introduce restriction endonuclease sites (this is fixed by the carrier side 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, cDNA is cloned into intermediate carrier (as pBluescript) with tilia miqueliana tm-Hmgr protein gene, 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 tobacco.
Utilize leaf dish method transformation of tobacco
1. with the positive bacterium colony on the aseptic toothpick picking YEB selection flat board, be inoculated in 2ml YEB liquid (Sm
+, Kan
+), 28 degree, 200rpm shaking culture 24-36 hour;
2. under the room temperature 4, the centrifugal 10min of 000g;
3. abandon supernatant, thalline suspends with the 1/2MS liquid nutrient medium, is diluted to 5-20 times of original volume, makes about the OD600=0.5 of bacterium liquid;
4. get the aseptic blade of the tobacco about two weeks of growth, remove its main lobe arteries and veins, it is cut into about 1 square centimeter of square vanelets;
5. blade is put into the bacterium liquid for preparing, soaked 2-5min, on aseptic filter paper, blot bacterium liquid;
6. the blade through infecting is put on the MS substratum 28 ℃ of dark cultivations 48 hours;
7. blade is forwarded on the callus substratum (MS+6-BA 1.0mg/L+NAA 0.1mg/L+Kan 50mg/L+cb250mg/L), the formation of 7-15 days visible callus is cultivated in 25-28 ℃ of illumination down;
8. visible differentiation bud grows after about 20 days, treat that bud is grown up after, downcut, place on the root media (1/2MS+NAA0.5mg/L+Kan 25mg/L) and carry out root culture, take root about 2-7 days;
9. after waiting 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.
Utilize Northern blotting to detect the expression of tm-Hmgr albumen in transgenic tobacco plant
1.RNA extraction: treat the long RNA that extracts tobacco leaf during to 2-3 sheet leaf of transgene tobacco blade.With the plant of normal growth (condition is the same) in contrast, (GIBCO BRL USA) extracts and with reference to the preparation chapters and sections (Sambrook etc., 1989) of " molecular cloning " relevant RNA to utilize the TRIzol test kit.
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 labelling module 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 tm-HMGR transcriptional level of transgene tobacco is obviously more much higher than the expression level of genetically modified control material not.
The tm-Hmgr protein-active that contains in the transgenic tobacco plant of tilia miqueliana 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene (tmhmgr) is measured
1. proteic extraction: a) get the 500mg blade, add 1000ul 1*PBS (KH
2PO
40.2g/l, Na
2HPO
41.15g/l, KCl 0.2g/l, NaCl 8g/l) grind in 50ml eppondorf pipe; B) 13000,4 ℃ centrifugal 10 minutes; C) get supernatant, standby.Annotate: above process is in carrying out on ice.
2. proteic quantitative: with reference to Bradford method (Bradford, 1976).Get the 2ul protein sample, add 1mlBradford reagent, behind the mixing, spectrophotometric instrumentation OD
595Protein content calculates: 1 OD
595=28.57 μ g.
3.tm-Hmgr protein-active is measured the method (the detail operations process is with example 3) with reference to (Mol.Cell.Biol, 1989,9:5702～5712) such as Thorsness.The result shows that the tm-Hmgr protease activity ratio in the transgenic tobacco plant does not have genetically modified contrast obviously much higher (P＜0.05).Thereby the proof tilia miqueliana 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene of being cloned has the enzymic activity that the catalysis 3-hydroxy-3-methylglutaryl-coenzyme A generates mevalonic acid really once more, will can be used for utilizing transgenic technology to improve in the research and industrialization of terpenoid secondary metabolite of plant.
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
ACTCAACCCCCGAAAACCG
(2) information of SEQ ID NO.2
(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.2
ACAATTCTGTCATTTTACC
(3) information of SEQ ID NO.3
(i) sequence signature:
(A) length: 2160bp
(B) type: Nucleotide
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: Nucleotide
(iii) sequence description: SEQ ID NO.3
<210>1
<211>2160
<212>DNA
＜213〉tilia miqueliana (Tilia miqueliana Maxim)
<220>
<221>CDS
<222>(131)..(1888)
<223>
<400>1
acgcggggac tcaacccccg aaaaccgttt ttcccgaaac gaacctgaat cgtcttttca 60
ttcttgtact cttctcctcc accgctctat tcgccgacgg cttcctctgc catcgccacc 120
aacatacaaa atg gag gcc cgg cgg cga tca tcg act aaa cct att caa 169
tct ctc aag aca aca aaa act gtt tct tta gag gaa aat tcc acc aaa 217
gcc tcc gac gca cta cct cct cct ttg tat ata atg aat gcg gtg ttc 265
ttc acg ctc ttc ttc tcg gtg gtt tat ttt ctt ctt tcc cgt tgg cgt 313
gaa aag atc cga atc tcc acg cct ctc cac atc gtc acc ttt tcc gag 361
atc gtt gcg gtt ctc gct tta gtt gcc tcg ttt ata tat ctt ttg gga 409
ttc ttc ggg att gac ttt gtt cag tct ttg att ctc cga cca tca act 457
gac att tgg aat tcc gag gaa gaa gaa gag gaa aat gaa gtt ttg ctc 505
cgt aaa gaa gat tcg cgt aaa gtc cct tgc ggc caa gct ctc gat tgc 553
tcg ttt cct cct ttg cct cct tcg gca ccg att gta act gcc cag aaa 601
gtt ttc gat gaa aag ctt gtg gaa gtt aca acc gag gaa gac gaa gaa 649
ata att aaa tcc gta gtg gct gga aca acc cct tca tat tct ttg gaa 697
tcg aaa tta ggt gat tgc aag aga gcg act gcg atc agg cgt gag gcg 745
ctg cag aga tta acg gga aag tca tta tca gga ttg ccc ttg gat gga 793
ttt gat tat gcg tcg att tta ggg cag tgt tgt gag atg ccg gtt ggg 841
tac gtg caa att ccc gtg gga att gct ggg ccg ttg ttg ctt aat gga 889
aga gaa tac acg gtt cct atg gca acc acg gag ggg acc ttg gtg gct 937
agc act aat agg gga tgt aag gct att cat ttg tct ggt gga gct aca 985
agt gtt ctt ttg aaa gat ggg atg act aga gct cct gtg gtt agg ttc 1033
agt act gcg aaa aga gca gct gat ctg aag ttt tat ttg gag gat cct 1081
gaa aat ttc gag acc ttg gct gtt gtt ttt aac aga tca agt aga ttt 1129
gct agg ctt caa ggt atc aaa tgt gca att gct ggg aag aat ctc tat 1177
ttg aga ttc act tgc agt act ggt gat gct atg ggg atg aac atg gtt 1225
tcc aag gga gtc caa aac gta ttg gat ttc ctt caa act gat ttc tct 1273
gac atg gat gtc att ggc atc tct gga aac ttc tgt tcg gac aaa aag 1321
cca gct gca gta aat tgg att gaa gga cgt ggc aaa tct gtt gtc tgc 1369
gag gcc atc att aag gat gat gtg gtg agg aag gtc ttg aag act ggt 1417
gtg gaa tct ctc gtg gag ctt aac atg ctt aag aac ctt act gga tct 1465
gct atg gct gga gct ctg ggt gga ttt aat gcc tat gcc ggt aac att 1513
gtg tct gct gtc tac ata gct acc ggt caa gat ccg gct caa aat gtc 1561
gag agc tct cat tgc ata acg atg atg gaa gct gtt aat gat ggc aag 1609
gac ctt cac atc tct gtt aca atg cct tcc atc gag gtt ggc act gtt 1657
ggt ggt gga act cag ctt gca tct cag tca gcg tgt ttg aac ctg ctc 1705
ggt gtc aag ggt gca agc aaa gaa tca cct gga gaa aac tct aga atg 1753
ctt gca acc att gta gcc ggt gct gtc ctt gct ggg gag ctg tca ctc 1801
atg tct gca ctt gca gct ggg caa cta att aac agc cat atg aag tac 1849
aat agg tca aat aag gac gtg tcc aag gct tct tcc tag agaacacgta 1898
atgatattga tgaggctttg tcaagcctgg aatttgtgta aacttgcaag ttccacggta 1958
taaatgtatc gaatcttaag gagacatcat ctctttgtaa taagctaagt agacgtaatc 2018
tctttgtatt ttgttctttt ctttcttact tttcatattt aaggaaaaaa aaaaacaagc 2078
catgtaatct gagaattggg taaaatgaca gaattgtaaa ttaacctctt ttaaaaaaaa 2138
aaaaaaaaaa aaaaaaaaaa aa 2160
(4) information of SEQ ID NO.4
(i) sequence signature:
(A) length: 585 amino acid
(B) type: amino acid
(C) chain: strand
(D) topological framework: linearity
(ii). molecule type: polypeptide
(iii). sequence description: SEQ ID NO.4
<210>2
<211>585
<212>PRT
＜2 13〉tilia miqueliana (Tilia miqueliana Maxim)
<400>2
Met Glu Ala Arg Arg Arg Ser Ser Thu Lys Pro Ile Glu Ser Leu Lys
1 5 10 15
Thr Thr Lys Thr Val Ser Leu Glu Glu Asn Ser Thr Lys Ala Ser Asp
20 25 30
Ala Leu Pro Pro Pro Leu Tyr Ile Met Asn Ala Val Phe Phe Thr Leu
35 40 45
Phe Phe Ser Val Val Tyr Phe Leu Leu Ser Arg Trp Arg Glu Lys Ile
50 55 60
Arg Ile Ser Thr Pro Leu His Ile Val Thr Phe Ser Glu Ile Val Ala
65 70 75 80
Val Leu Ala Leu Val Ala Ser Phe Ile Tyr Leu Leu Gly Phe Phe Gly
85 90 95
Ile Asp Phe Val Glu Ser Leu Ile Leu Arg Pro Ser Thr Arg Ile Trp
100 105 110
Asn Ser Glu Glu Glu Glu Glu Glu Asn Glu Val Leu Leu Leu Lys Glu
115 120 125
Asp Ser Arg Lys Val Pro Cys Gly Glu Ala Leu Asp Cys Ser Phe Pro
130 135 140
Pro Leu Pro Pro Ser Ala Pro Ile Val Thr Ala Arg Lys Val Phe Asp
145 150 155 160
Glu Lys Leu Val Glu Val Thr Thr Glu Glu Asp Glu Glu Ile Ile Lys
165 170 175
Ser Val Val Ala Gly Thr Thr Pro Ser Tyr Ser Leu Glu Ser Lys Leu
180 185 190
Gly Asp Cys Lys Arg Ala Thr Ala Ile Arg Arg Glu Ala Leu Glu Arg
195 200 205
Leu Thr Gly Lys Ser Leu Ser Gly Leu Pro Leu Asp Gly Phe Asp Tyr
210 215 220
Ala Ser Ile Leu Gly Glu Cys Cys Glu Met Pro Val Gly Tyr Val Glu
225 230 235 240
Ile Pro Val Gly Ile Ala Gly Pro Leu Leu Leu Asn Gly Arg Glu Tyr
245 250 255
Thr Val Pro Met Ala Thr Thr Glu Gly Thr Leu Val Ala Ser Thr Asn
260 265 270
Arg Gly Cys Lys Ala Ile His Leu Ser Gly Gly Ala Thr Ser Val Leu
275 280 285
Leu Lys Asp Gly Met Thr Arg Ala Pro Val Val Arg Phe Ser Thr Ala
290 295 300
Lys Arg Ala Ala Asp Leu Lys Phe Tyr Leu Glu Asp Pro Glu Asp Phe
305 310 315 320
Glu Thr Leu Ala Val Val Phe Asn Arg Ser Ser Arg Phe Ala Arg Leu
325 330 335
Glu Gly Ile Lys Cys Ala Ile Ala Gly Lys Asn Leu Tyr Leu Arg Phe
340 345 350
Thr Cys Ser Thr Gly Asp Ala Met Gly Met Asn Met Val Ser Lys Gly
355 360 365
Val Glu Asn Val Leu Asp Phe Leu Glu Thr Asp Phe Ser Asp Met Asp
370 375 380
Val Ile Gly Ile Ser Gly Asn Phe Cys Ser Asp Lys Lys Pro Ala Ala
385 390 395 400
Val Asn Trp Ile Glu Gly Arg Gly Lys Ser Val Val Cys Glu Ala Ile
405 410 415
Ile Lys Asp Asp Val Val Arg Arg Val Leu Lys Thr Gly Val Glu Ser
420 425 430
Leu Val Glu Leu Asn Met Leu Lys Asn Leu Thr Gly Ser Ala Met Ala
435 440 445
Gly Ala Leu Gly Gly Phe Asn Ala Tyr Ala Gly Asn Ile Val Ser Ala
450 455 460
Val Tyr Ile Ala Thr Gly Glu Asp Pro Ala Glu Asn Val Glu Ser Ser
465 470 475 480
His Cys Ile Thr Met Met Glu Ala Val Asn Asp Gly Lys Asp Lys His
485 490 495
Ile Ser Val Thr Met Pro Ser Ile Glu Val Gly Thr Val Gly Gly Gly
500 505 510
Thr Glu Leu Ala Ser Glu Ser Ala Cys Leu Asn Leu Leu Gly Val Lys
515 520 525
Gly Ala Ser Lys Glu Ser Pro Gly Glu Asn Ser Arg Met Leu Ala Thr
530 535 540
Ile Val Ala Gly Ala Val Leu Ala Gly Glu Leu Ser Leu Met Ser Ala
545 550 555 560
Leu Ala Ala Gly Glu Leu Ile Asn Ser His Met Lys Tyr Asn Arg Ser
565 570 575
Asn Lys Asp Val Ser Lys Ala Ser Ser *
580 585
Claims (3)
1, the nucleotide sequence of tilia miqueliana 3-hydroxy-3-methylglutaryl-coenzyme A reductase enzyme protein is characterized in that described nucleotide sequence is made of the Nucleotide of 131-1888 position among the SEQ ID NO.3.
2, the aminoacid sequence of tilia miqueliana 3-hydroxy-3-methylglutaryl-coenzyme A reductase enzyme protein according to claim 1 is characterized in that described aminoacid sequence is the aminoacid sequence shown in the SEQ ID NO.4.
3, the nucleotide sequence transformed host cells of tilia miqueliana 3-hydroxy-3-methylglutaryl-coenzyme A reductase enzyme protein according to claim 1 is characterized in that described host cell is an eukaryotic cell.
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| CN1936004B (en) * | 2006-08-28 | 2010-09-08 | 徐州师范大学 | Medicinal plant radix euphorbiae pekinensis 3-hydroxy-3-methyl glutaryl coenzyme A reductase protein coding sequence |
| CN101250544B (en) * | 2008-04-08 | 2010-07-28 | 上海师范大学 | Salvia 3-hydroxy-3-methylglutaryl A reductase gene and its coding protein and application |
| CN101250540B (en) * | 2008-04-08 | 2010-06-09 | 上海师范大学 | Camptotheca acuminata 3-hydroxy-3-methylglutaryl A synthase gene and its coding protein and application |
| CN105112430B (en) * | 2015-09-11 | 2019-03-19 | 山东省农业科学院作物研究所 | The detection method of wheat 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene TaHMGR and its separation clone, rite-directed mutagenesis and enzyme function |
| CN107828804B (en) * | 2017-11-16 | 2021-02-02 | 广西壮族自治区农业科学院农产品加工研究所 | Mango HMGR gene, primer for cloning mango HMGR gene and cloning method thereof |
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| CN1381566A (en) * | 2001-04-18 | 2002-11-27 | 上海博德基因开发有限公司 | Polypeptide-hydroxymethyl glutaryl CoA reductase-9.35 and polynucleotide for coding it |
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Non-Patent Citations (1)
| Title |
|---|
| 高等植物的3-羟基-3-甲基戊二酰辅酶A 还原酶. 鞠世杰，阎秀峰.植物生理学通讯，第40卷第1期. 2004 * |
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