CN1995355B - Southernwood 1-hydroxy-2-methyl-2-(E)- cyclobutenyl 4-diphosphoric acid reductase protein encoding sequence - Google Patents

Abstract

The invention discloses an arteannuin 1-hydroxy-2-methyl-2-(E)-cyclobutenyl 4-pyrophosphoric acid reductase protein coded sequence in the gene engineering domain, which is characterized by the following: the ribonucleotide sequence possesses 70% consanguinity with 36-1403th in the SEQ ID NO. 1, which an cross the 36-1403th in the SEQ ID NO. 1 under 45-55 deg. c.

Description

Southernwood 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium coded sequence of reductase enzyme protein

Technical field

What the present invention relates to is a kind of albumen coded sequence, and particularly a kind of southernwood 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium coded sequence of reductase enzyme protein belongs to the genetically engineered field.

Background technology

In recent years statistical information shows: the world has more than 20 hundred million people of more than 100 countries and regions to live in the popular district of malaria, has every year 2.7 hundred million people to suffer from this disease approximately, wherein has nearly 3,000,000 people to die from malaria.Just because of the harm of malaria is so serious, the World Health Organization has just come into effect one " the malaria plan is eliminated in the whole world " nineteen fifty-seven, but effect does not show so far.Its reason be from the sixties plasmodium main antimalarial drug such as chloroquine is produced resistance, make these medicines lose the antimalarial drug effect.Many in the world for this reason countries have all dropped into a large amount of manpower and materials and have removed to research and develop new antimalarial agent.Artemisine natural product and derivative thereof are the class new antimalarial agents that China scientific worker excavates motherland's medicine and pharmacology treasure-house research and development, have independent intellectual property right, are widely used in the treatment of malaria at present.The demand of the annual Artemisinin in the whole world is about 150 tons, and output only is about 15 tons, and obviously supply falls short of demand, and price has reached about 225 dollars/g at present.Carry out the Artemisinin correlative study and have good economic benefits and social benefit.

Improve the scientific and technological content of China's sweet wormwood product at present, it is imperative to promote the sweet wormwood industrial status, and this depends on the innovation in the source of science and technology, and modern biotechnology can play decisive role at production high-quality Artemisinin medicine source side face.The serious scarcity in high-quality medicine source is the greatest problem that faces in the present Artemisinin production.In recent years, seeking and enlarging natural drug Artemisinin medicine source was a very active research field, had obtained very big progress, and mainly comprise following three aspects: (1) utilizes traditional breeding technology to cultivate the sweet wormwood Cultivar of high content arteannuin; (2) Artemisinin is chemical complete synthesis or semi-synthetic; (3) utilize modern biotechnology to improve artemislnin content in the sweet wormwood.In these research fields, the required cycle of sweet wormwood Cultivar of cultivating high content arteannuin is oversize, efficient is very low, the content of Artemisinin can not be greatly improved, in addition, extract from natural sweet wormwood, output is subjected to the restriction in resource, environment and season, and the production cost height, yields poorly, is difficult to meet the need of market; Though Artemisinin can manually synthesize, cost height, difficulty are big, also fail to put into production.

By contrast, utilize genetic engineering technique genetic improvement sweet wormwood, carry out the research of Artemisinin metabolic engineering, produce Artemisinin and just have very big advantage: (1) is in the biotechnology research field, the researchist has established good theory and practice basis to the success of sweet wormwood isolated culture and sweet wormwood genetic transformation for utilizing biotechnology to produce Artemisinin; (2) utilize genetic engineering technique genetic improvement sweet wormwood fast and efficiently on gene level, obtain that the target pharmaceutical ingredient content greatly improves and the transgenosis medicinal plant (comprising seedling, hairly root, clone etc.) of tool genetic stability has had much successfully report; (3) owing at present illustrated the mechanism of some important steps in the Artemisinin biosynthetic metabolism approach and relevant rate-limiting enzyme gene is cloned, make the metabolic engineering of realizing Artemisinin become possibility.

Artemisinin belongs to sesquiterpene derivative, and the biosynthetic pathway of Artemisinin belongs to Plant Isoprenoid Metabolic Pathway.There are two approach at least in the biosynthesizing of plant isoprenoid, promptly is positioned at the mevalonate pathway and the pyruvic acid/phosphoglyceraldehyde approach that is positioned at plastid of kytoplasm.The final step that is positioned at the pyruvic acid/phosphoglyceraldehyde approach of plastid is methylol butenyl-4-phosphoric acid (hydroxymethylbutenyl 4-diphosphate, HMBPP) (1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase HDR) is converted into isopentenyl pyrophosphate (IPP) and dimethylallylpyrophosphate (DMAPP) (5: 1) under the effect at 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase enzyme.They are two basic general precursors of 5 carbon of all natural terpenes.Yet the research about plant hdr gene only has seldom report, what the expression of lytB gene in intestinal bacteria of adonis amurensis (Adonisaestivalis) proved its coding is the gene with HDR function, and the document is only studied the clone of this gene and the functional verification of coded protein; 2005, people such as Guevara-Garc í a reported clone, the checking of HDR protein function and the hdr of the Arabidopis thaliana hdr gene expression at Arabidopis thaliana at The PlantCell, make that research is goed deep into about plant hdr; In order to investigate the effect of hdr in the diterpene biosynthesizing, Botella-Pav í a etc. are composition overexpression Japanese yew diene synthetic enzyme (TXS) gene in Arabidopis thaliana at first, finds that the content of Japanese yew diene in the Arabidopis thaliana is not high; Yet at the same time in the Arabidopis thaliana of overexpression HDR and TXS gene, the Japanese yew diene content improves 13 times than the Arabidopis thaliana of single TXS of commentaries on classics, this studies show that hdr is a rate-limiting enzyme gene in the terpene substances biosynthesizing, be the critical function gene of carrying out the terpene metabolic engineering.Yet regrettably, up to the present, yet there are no the report of clone HDR gene from sweet wormwood.Nucleotide sequence according to 1-hydroxy-2-methyl-2-(the E)-butenyl 4-tetra-sodium reductase enzyme of part plant among the GeneBank, we have cloned correlated series from sweet wormwood in conjunction with the RACE technology, according to search and the functional verification result of GeneBank, be southernwood 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase gene with this unnamed gene.

In analysis to existing document, do not see the clone of the HDR gene on pyruvic acid in the sweet wormwood/phosphoglyceraldehyde biosynthetic pathway, 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 southernwood 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium coded sequence of reductase enzyme protein is provided.The invention discloses the aminoacid sequence of southernwood 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase enzyme and the nucleotide sequence of southernwood 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase gene, for finally breaking the rate-limiting reaction step on pyruvic acid/phosphoglyceraldehyde approach, for the metabolic engineering of Artemisinin provides a functional gene, and utilizing transgenic technology to improve the application of artemislnin content.

The present invention is achieved by the following technical solutions: at the isolated dna molecular of the present invention, this molecule comprises: coding has southernwood 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase enzyme nucleotide sequence, and shows 70% homology from the nucleotides sequence of Nucleotide 36-1403 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 36-1403 position.

The present invention isolated southernwood 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium coded sequence of reductase enzyme protein coded 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.

Dna molecular of the present invention comprises 8-100 continuous nucleotide in the described dna molecular.

Dna molecular transformed host cells of the present invention is an eukaryotic cell.This host cell is a sweet wormwood 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, refer to encode has the nucleotide sequence of active polypeptide to term " southernwood 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium coded sequence of reductase enzyme protein ", as 36-1403 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.3 1.This degenerate sequence is meant, is arranged in the encoder block 36-1403 position Nucleotide of SEQ ID NO.1 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.1 in 36-1403 position nucleotide sequence homology be low to moderate about 70% the degenerate sequence described sequence of SEQ ID NO.1 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.1 in from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 36-1403 position.This term also comprise with SEQ ID NO.3 in from the homology of nucleotide sequence at least 70% of Nucleotide 36-1403 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.1 with southernwood 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase enzyme 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 " southernwood 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase enzyme " refers to have the SEQ ID NO.1 polypeptide of sequence of protein-active.This term also comprises having variant form relevant identical function, SEQ ID NO.3 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 its active fragments and reactive derivative.

The variant form of southernwood 1-hydroxy-2-methyl-2-of the present invention (E)-butenyl 4-tetra-sodium reductase gene comprises: homologous sequence, the conservative property varient, allelic variant, natural mutation, the induced mutation body, under high or low stringent condition can with the coded albumen of DNA of southernwood 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase gene DNA hybridization, and the polypeptide protein that utilizes the serum acquisition of southernwood 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase enzyme coded polypeptide.

In the present invention, " southernwood 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase enzyme conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID NO.1, 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

Initial residue	Representational replacement	The preferred replacement
			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

82％identity in 458aa overlap
			Query SbjctQuery Sbjct Query Sbjct Query Sbjct Query Sbjct Query Sbjct Query Sbjct Query Sbjct	1 1 56 59 116 119 176 179 236 239 296 299 356 359 416 419	MASLQLTPLSTRTDYLSLPADIKVFRCRKPLTVRCSGGDTSSST-----QFDAKVFRHNL MA+L+ +P ST T+ LSLP D+K+FRCRKPL+VRCSGGD+SS + FDAKVFRHNL MATLRFSPFSTCTE-LSLP-DVKLFRCRKPLSVRCSGGDSSSPSVASGSDFDAKVFRHNL TRSENYNRKGFGHKKETLELMSQEYFSDIIKTLKENNYEYTWGNVTVKLAEAFGFCWGVE TRSENYNRKGFGHKKETLELM+QEY SDIIKTLKENN EYTWGNVTVKLAEA+GFCWGVE TRSENYNRKGFGHKKETLELMNQEYTSDIIKTLKENNNEYTWGNVTVKLAEAYGFCWGVE RAVQIAYEARKQFPDDKIWITNQIIHNPTVNKRLEEMEVTDIPIDGGEKQFDVVDKGDVV RAVQIAYEARKQFPD+KIWITN+IIHNPTVNKRLEEMEV DIP+ GEKQFDV+DKGDVV RAVQIAYEARKQFPDEKIWITNEIIHNPTVNKRLEEMEVKDIPVKDGEKQFDVIDKGDVV ILPAFGAAVDEMRILSNKEVQIVDTTCPWVTKVWNVVEKHKKGDYTSVIHGKHNHEETVA ILPAFGAAV+EM LS+K+VQIVDTTCPWV+KVW VEKHKKG YTS+IHGK++HEETVA ILPAFGAAVNEMLTLSDKQVQIVDTTCPWVSKVWTSVEKHKKGAYTSIIHGKYSHEETVA TASFAGKFIVVKNIDEATYVCDYILGGKLNGSSSTKEAFMEKFKSAVSEGFDPDKDLVKA TASFAGK+++VKN+DEATYVCDYILGGKLNGSSSTKEAF+EKFK AVS GFDPD DLVK TASFAGKYVIVKNMDEATYVCDYILGGKLNGSSSTKEAFLEKFKYAVSNGFDPDTDLVKT GIANQTTMLKGETEEIGKLLERTMMQKYGVENVNNHFLSFNTICDATQERQDAMYKLVDD G+ANQTTMLKGETEEIGKL+ERTMM KYGVEN HF+SFNTICDATQERQDAMYKLVD+ GVANQTTMLKGETEEIGKLVERTMMSKYGVENATEHFISFNTICDATQERQDAMYKLVDE KVDLMLVIGGFNSSNTSHLQEIAEERKIPSYWIDSEKRIGPGNRIAYKLLHGELVEQENW K+DLMLV+GG+NSSNTSHLQEI EER IPSYWID+ +R+GPGNRIAYK +HGELVE+ENW KMDLMLVVGGWNSSNTSHLQEIPEERGIPSYWIDTVERVGPGNRIAYKTMHGELVEKENW LPKGPITIGVTSGASTPDKVVEDALLKVFEIKREEALQL LPKGP+TIG+TSGASTPDK VED L+VFE IKREE+LQ LPKGPLTIGITSGASTPDKAVEDVLKRVFEIKREESLQF

The aminoacid sequence of Query: southernwood 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase enzyme

The aminoacid sequence (ABB88836.2) of Sbjct: stevia rebaudianum (Stevia rebaudiana) 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase enzyme

Table 2 is that the homology of the aminoacid sequence of southernwood 1-hydroxy-2-methyl-2-of the present invention (E)-butenyl 4-tetra-sodium reductase enzyme and Vinca 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase enzyme compares (FASTA) table.Wherein, identical amino acid marks with the amino acid monocase between two sequences.

Invention also comprises the analogue of southernwood 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase enzyme protein or polypeptide.The difference of these analogues and southernwood 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase enzyme protein 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 southernwood 1-hydroxy-2-methyl-2-of the present invention (E)-butenyl 4-tetra-sodium reductase enzyme protein, the encoding sequence of southernwood 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase gene operationally can be connected in expression regulation sequence, thereby form southernwood 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase gene 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 plastid transit peptides DNA as precursor expression and participate in the plastid location of polypeptide, plastid transit peptides 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 plastid transit peptide 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.

In addition, can be used as the nucleic acid molecule of probe among the present invention, this molecule has 8-100 continuous nucleotide of southernwood 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase gene encoding sequence usually, preferably has 15-50 continuous nucleotide.This probe can be used for whether existing in the test sample nucleic acid molecule of coding southernwood 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase enzyme.

The present invention relates to whether exist in the test sample method of southernwood 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase gene 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 southernwood 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase gene 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.

Southernwood 1-hydroxy-2-methyl-2-of the present invention (E)-butenyl 4-tetra-sodium reductase gene 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 relevant nucleotide sequence design 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 be about 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 Freeman 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 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 southernwood 1-hydroxy-2-methyl-2-of the present invention (E)-butenyl 4-tetra-sodium reductase enzyme, by various conventional screening methods, can filter out and southernwood 1-hydroxy-2-methyl-2-(E)-interactional material of the relevant generation of butenyl 4-tetra-sodium reductase enzyme, perhaps acceptor, inhibitor or short of money dose etc.

The present invention has tangible effect on the raising artemislnin content.Improve the output of Artemisinin, to satisfy the great demand of the whole world to anti-malaria medicaments.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 HarborLaboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1

The clone of southernwood 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase gene

1. separate tissue (Isolation)

Sweet wormwood from Chongqing tenth of the twelve Earthly Branches sun gather, planting seed in the greenhouse, is treated that the sweet wormwood seedling grows to 10cm when high, 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, 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)

Nucleotide conserved sequence according to 1-hydroxy-2-methyl-2-(the E)-butenyl 4-tetra-sodium reductase gene of each kind of plant, utilize homologous genes clone principle, adopt RACE method (Clontech test kit) to carry out the cDNA full-length clone, divide three phases to carry out:

(1) the pulsating clone of core

PCR[BP001 (5 '-GTYGAGCGYGCHGTBCAGATYGC-3 ')+BP002 (5 '-CATCYTCMACDRCCTTRTCNGG-3 ')] obtain the core segment of 2002BP (976bp), be connected to after the recovery on the pGEMT-Easy carrier, with SP6 or T7 as universal primer, adopt and stop thing fluorescent mark (Big-Dye, Perkin-Elmer, USA) method, (Perkin-Elmer checks order on USA) at ABI 377 sequenators.Sequencing result GCG software package (Wisconsin group, USA) BLAST in and the existing database of FASTA software search (Genebank+EMBL), the homology of HDR gene of knowing its nucleotide sequence and proteins encoded and known stevia rebaudianum is higher, so think that tentatively it is the fragment of a 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase gene.

(2)3’-RACE

PCR[AP+BP003 (5 '-GTTACATCTGGTGCTTCTAC-3 ')] obtain 2002BP 3 ' terminal sequence (389bp), 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 HDR gene of knowing its nucleotide sequence and proteins encoded and stevia rebaudianum is very high, so think that tentatively it is a 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase gene.

(3).5’-RACE

First round PCR[AAP+BP004 (5 '-ATCTTGTCATCAGGGAACTG-3 ')]

Second takes turns PCR[(AUAP+BP005 (5 '-CTAGCTTCATAAGCAATCTGC-3 ')] obtain 2002BP5 ' terminal sequence (406bp), (process is with (1)).

Overlap splicing with sequencing result, obtain the full length fragment sequence information, and design a pair of special primer and carry out pcr amplification sweet wormwood HDR coding region (BP006F1 (5 '-ATGGCGTCTTTGCAGCTAAC-3 ')+BP007R1 (5 '-CTACACCAATTGCAGGGC-3 ')) obtain the coding region (1368bp) of HDR.

The gene that result's proof of BLAST newly obtains from sweet wormwood really is a 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase gene.Because known homology 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase gene has the function that HMBPP is reduced to IPP and DMAPP, so infer that this gene has identical functions.

By being used in combination above-mentioned 3 kinds of methods, obtained the complete encoding sequence of southernwood 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase enzyme of candidate.Obtain on the total length basis of (comprising complete open reading frame) in splicing, further F1:5 '-CGCGGGACACACAAAAACACA-3 ' is a forward primer to design primer BP008, BP009 R1:5 '-GTAACAAGTCGTTTATAGCAACC-3 ' is a reverse primer, with total RNA is template, carry out the RT-PCR amplification, the PCR condition of F1/R2 is: 94 ℃ of pre-sex change 2min; Then 94 ℃ of sex change 45s, 50 ℃ of annealing 45s, 72 ℃ extend 2min; 30 circulations; Last 72 ℃ are extended 8min; Electrophoresis detection PCR product, obtaining the amplification fragment length is 1617bp.Clone with pcr amplification product according to a conventional method then, order-checking obtains the sequence shown in the SEQ ID NO.1.

Embodiment 2

Southernwood 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase gene sequence information and homology analysis

The length of 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase gene sequence full-length cDNA that the present invention is new is 1696bp, and detailed sequence is seen SEQ ID NO.1, and wherein open reading frame is positioned at 36-1403 position Nucleotide (1428 Nucleotide).Derive southernwood 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase enzyme aminoacid sequence according to full-length cDNA, totally 455 amino-acid residues, molecular weight is 51.44kDa, iso-electric point (pI) is 5.63.Detailed sequence is seen SEQIDNO.1.

The full length cDNA sequence and the coded protein sequence thereof of southernwood 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase gene are carried out Nucleotide and protein homology retrieval with blast program in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS translations+PDB+SwissProt+Superdate+PIR database, found that it and stevia rebaudianum 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase gene sequence have 83% homogeny (subordinate list 2) on nucleotide level; On amino acid levels, it and stevia rebaudianum HDR have 82% homogeny on protein level, (subordinate list 3).This shows, all there is higher homology in southernwood 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase gene and stevia rebaudianum 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase gene on nucleic acid still is protein level, so can think that southernwood 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase enzyme also has similar effect on pyruvic acid/phosphoglyceraldehyde approach.

Embodiment 3

Southernwood 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase enzyme protein or polypeptide carry out functional verification in intestinal bacteria

In this embodiment, the sweet wormwood HDR encoding sequence of total length or segment are built among the commercial protein expression carrier,, make the host bacterium obtain the ability of on auxotrophic substratum, growing to express this albumen.

According to the aminoacid sequence of sweet wormwood HDR, design amplifies the primer of the protein-coding region of HDR, and introduces restriction endonuclease sites (this decides according to the pQE30 carrier of selecting for use) on positive anti-primer respectively, so that construction of expression vector.With sweet wormwood RNA is template, carry out RT-PCR amplification after, sweet wormwood HDR gene is cloned into pQE30 carrier (Novagen) guaranteeing to read under the correct prerequisite of frame.Identify that good expression vector utilizes CaCl ₂Method change over to intestinal bacteria MG1655ara＜ispH, Screening and Identification obtains containing the engineering bacteria of pQE30-HDR expression vector.Simultaneously pQE30 is changed in the same way MG1655ara＜ispH, Screening and Identification obtains containing the engineering bacteria of pQE30 expression vector, in contrast the experiment.

Intestinal bacteria MG1655ara＜〉ispH is the bacterial strain of HDR transgenation, on the LB of no pectinose substratum, can't grow.The engineering bacteria that contains the pQE30-HDR expression vector can be grown on the LB substratum of IPTG (sec.-propyl-b-d-thiogalactoside) and GLc (glucose), and the engineering bacteria that contains the pQE30 expression vector can not be grown on the LB of IPTG and GLu substratum.Illustrating that the gene of implementing to obtain in the example 1 has the function of 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase enzyme, is southernwood 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase gene really.

Embodiment 4

Southernwood 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase enzyme protein or polypeptide carry out eukaryotic cell expression and transfer-gen plant in sweet wormwood artemislnin content detects

The structure that contains the expression vector of goal gene, according to southernwood 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase gene encoding sequence (SEQ ID NO.1), 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, southernwood 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase gene cDNA is cloned into intermediate carrier (as pGEM T-ease), further be cloned into binary expression vector (as improved pCAMBIA1304), guaranteeing to identify expression vector under the correct prerequisite of reading frame, again it is changed in the Agrobacterium rhizogenes, utilize leaf dish law technology to transform sweet wormwood.

1. send out seedling: take the sophisticated seed of sweet wormwood, with 0.1% (M/V) mercuric chloride (HgCl ₂) solution soaking 5 minutes, with aseptic water washing 6 times; Seeds of southernwood is seeded in (substratum was contained in the triangular flask of 150ml, in 121 ℃ of sterilizations 20 minutes) on the seed germination substratum, and this culture medium prescription is: the MS minimum medium, add 30g/L sucrose, and regulating the medium pH value is 5.8, adds 5% agar powder again.Cultivate the young shoot of sweet wormwood in illumination box, culture condition is: 25 ℃, dark condition is cultivated down.After treating that seed is sprouted, the change culture condition is: 25 ℃, and illumination in 12 hours, intensity of illumination is 25 μ mol.m ^-2.s ^-1By the time the plant length of a film can be used for genetic transformation constantly to the 3cm height.

2. transform altogether and cultivate: the young leaflet tablet of sweet wormwood aseptic seedling is whittled into about 1cm with small blade ²Put into prior cultured bacterium liquid (OD ₆₀₀Value is between 0.4-0.6) infected 3-5 minute.Then take out and put into the dark cultivation of common substratum 2 days.

3. degerming is cultivated: after cultivating end altogether, explant is put into the degerming culture medium culturing.4 all subcultures once.After treating that end is cultivated in degerming, change the resistance screening substratum over to and carry out transgenosis hairly root preliminary screening.

Remove bacterium culture medium: MS+cb (cynnematin) (250mg/l)

Screening culture medium: MS+hygr (Totomycin) (30mg/l)

4. the isolated culture of hairly root: downcut when long when hairly root grows to about 4cm, do not containing on the MS substratum of any plant hormone succeeding transfer culture in the constant incubator of 25 ± 1.0 ℃ of unglazed photographs.Per three all subcultures once, through carrying out liquid fermentation and culture behind the succeeding transfer culture several times, further to carry out Molecular Detection.

5. the Molecular Detection of transgene abrotanum

The extraction of hairly root DNA and purifying all with reference to molecular cloning experiment guide (Sambrook, J.et al 1993) according to the Ri plasmid gene sequential analysis design rolB of Furner etc., the Auele Specific Primer of rolC gene.The a pair of primer of rolB gene is: 5 '-GCTCTTGCAGTGCTAGATTT-3 ' and 5 '-GAAGGTGCAAGCTACCTCTC-3 '.The a pair of primer of rolC gene is 5 '-CTCCTGACATCAAACTCGTC-3 ' and 5 '-TGCTTCGAGTTATGGGTACA-3 '.The clip size of rolB, rolC gene is 423bp and 626bp respectively.

Owing to contain 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase gene in the sweet wormwood, so when the hairly root that changes 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase gene is cloned, can not detect direct DXR gene, and the Totomycin on the detection plant expression vector (hygr) resistant gene, the detection use primer fhygr of hygromycin gene (812bp) (5 '-CGATTTGTGTACGCCCGACAGTC-3 ') and rhygr

(5 '-CGATGTAGGAGGGCGTGGATATG-3 ', annealing temperature, 58 ℃).

PCR result shows: the root that is obtained is a hairly root, and the detection of hygromycin gene is shown that southernwood 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase gene successfully has been incorporated into the sweet wormwood genome.

6.1-the northern blot of hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase gene in the sweet wormwood hairly root analyzes

1) extraction of .RNA: (GIBCO BRL USA) extracts and the preparation chapters and sections (Sambrook etc., 1989) of the relevant RNA of reference " molecular cloning " extract each monoclonal RNA of sweet wormwood transgenosis hairly root to utilize the TRIzol test kit.

2) .RNA's is quantitative: with reference to " molecular cloning " (Sambrook etc., 1989), and spectrophotometric instrumentation OD ₂₆₀Rna content calculates: 1 OD ₂₆₀=40 μ g/ml.

3). total RNA agarose gel electrophoresis separates: 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) shift on the .RNA nylon membrane: before 1) shifting, 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 blot result shows: 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase gene transcriptional level is obviously more higher than blank hairly root in the hairly root of commentaries on classics southernwood 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase gene.

7. the HPLC of Artemisinin analyzes in the hairly root of commentaries on classics 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase gene

Artemisinin extracts: the sweet wormwood transgenosis hairly root of oven dry is ground into fine powder, accurately take by weighing the 0.125g fine powder and place extraction flask, add 30～60 ℃ of sherwood oils of 10ml, in ultrasonic bath, extract 30min, filter, decompression and solvent recovery dissolves residue again with 1ml methyl alcohol, centrifugal 10min is to precipitate insoluble part under the 12000r/min, and supernatant liquor is used to measure artemislnin content.

The preparation of high-pressure liquid phase test sample and standard substance: get Artemisinin methanol solution 200 μ l in the 10ml test tube, add 800 μ l methyl alcohol, 4ml 0.2%NaOH solution, mixing, in 50 ℃ of water-baths, react 30min, be cooled to room temperature, get the 0.5ml reaction solution in the 1.5ml plastic centrifuge tube, add 100 μ l methyl alcohol, 400 μ l 0.05mol/L acetic acid, mixing is crossed the little column purification of C18 pre-separation.According to the Artemisinin standard solution that is used for high pressure liquid phase analysis with the quadrat method preparation, standard substance concentration is respectively and contains Artemisinin standard substance 0,40,80,120,160,200,240 μ g in every 10ml solution.

The high-pressure liquid phase testing conditions: chromatographic column is the C18 post, 150mm * 416mm, and moving phase is the 0.01mol/L phosphoric acid buffer: methyl alcohol (55: 45), pH7.0, flow velocity 1ml/min, UV-detector detects wavelength and is located at 258nm, volume injected 10 μ l.Under these conditions, the Artemisinin appearance time is greatly about 4min.

Measurement result shows: artemislnin content is obviously than artemislnin content height in the wild-type sweet wormwood in the transgene abrotanum.

Sequence table

＜110〉Southwestern University

＜120〉southernwood 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase gene sequence

<140>

<141>

<160>2

<170>PatentIn version 3.1

<210>1

<211>1696

<212>DNA

＜213〉sweet wormwood (Artemisia annua L.)

CGCGGGACAC ACAAAAACAC ACGCAGCATA ATTCA ATG GCG TCT TTG CAG CTA ACA 56

Met Ala Ser Leu Gln Leu Thr

1 5

CCT CTG TCA ACT CGC ACA GAC TAC CTC TCC TTA CCT GCA GAC ATA AAA 104

Pro Leu Ser Thr Arg Thr Asp Tyr Leu Ser Leu Pro Ala Asp Ile Lys

10 15 20

GTA TTC CGG TGC CGG AAG CCG TTA ACA GTC CGA TGC TCC GGC GGT GAC 152

Val Phe Arg Cys Arg Lys Pro Leu Thr Val Arg Cys Ser Gly Gly Asp

25 30 35

ACG TCA TCG TCA ACG CAA TTT GAT GCG AAG GTG TTC AGG CAT AAT TTG 200

Thr Ser Ser Ser Thr Gln Phe Asp Ala Lys Val Phe Arg His Asn Leu

40 45 50 55

ACA AGG AGC GAG AAT TAT AAT AGG AAA GGA TTT GGT CAT AAG AAG GAG 248

Thr Arg Ser Glu Asn Tyr Asn Arg Lys Gly Phe Gly His Lys Lys Glu

60 65 70

ACT CTT GAG CTC ATG AGT CAG GAG TAT TTT AGC GAC ATT ATA AAG ACT 296

Thr Leu Glu Leu Met Ser Gln Glu Tyr Phe Ser Asp Ile Ile Lys Thr

75 80 85

TTG AAG GAG AAT AAC TAC GAA TAT ACA TGG GGA AAT GTC ACT GTA AAG 344

Leu Lys Glu Asn Asn Tyr Glu Tyr Thr Trp Gly Asn Val Thr Val Lys

90 95 100

CTT GCA GAA GCT TTT GGT TTT TGT TGG GGT GTT GAG CGT GCC GTC CAG 392

Leu Ala Glu Ala Phe Gly Phe Cys Trp Gly Val Glu Arg Ala Val Gln

105 110 115

ATT GCT TAT GAA GCT AGG AAA CAG TTC CCT GAT GAC AAG ATA TGG ATC 440

Ile Ala Tyr Glu Ala Arg Lys Gln Phe Pro Asp Asp Lys Ile Trp Ile

120 125 130 135

ACA AAT CAA ATT ATT CAC AAC CCT ACT GTT AAC AAG AGG CTA GAA GAG 488

Thr Asn Gln Ile Ile His Asn Pro Thr Val Asn Lys Arg Leu Glu Glu

140 145 150

ATG GAA GTT ACG GAT ATC CCC ATT GAC GGC GGA GAG AAA CAG TTT GAT 536

Met Glu Val Thr Asp Ile Pro Ile Asp Gly Gly Glu Lys Gln Phe Asp

155 160 165

GTT GTT GAC AAG GGC GAT GTT GTA ATT CTG CCT GCC TTT GGA GCT GCA 584

Val Val Asp Lys Gly Asp Val Val Ile Leu Pro Ala Phe Gly Ala Ala

170 175 180

GTA GAC GAG ATG CGG ATT TTG AGT AAC AAA GAA GTA CAA ATA GTC GAT 632

Val Asp Glu Met Arg Ile Leu Ser Asn Lys Glu Val Gln Ile Val Asp

185 190 195

ACA ACA TGC CCA TGG GTG ACT AAG GTG TGG AAT GTT GTC GAA AAG CAT 680

Thr Thr Cys Pro Trp Val Thr Lys Val Trp Asn Val Val Glu Lys His

200 205 210 215

AAG AAG GGG GAC TAT ACG TCA GTA ATT CAT GGT AAA CAT AAC CAT GAG 728

Lys Lys Gly Asp Tyr Thr Ser Val Ile His Gly Lys His Asn His Glu

220 225 230

GAG ACT GTA GCA ACA GCA TCT TTT GCA GGA AAG TTT ATC GTT GTG AAG 776

Glu Thr Val Ala Thr Ala Ser Phe Ala Gly Lys Phe Ile Val Val Lys

235 240 245

AAC ATT GAT GAG GCG ACA TAT GTG TGT GAT TAC ATT CTT GGT GGT AAA 824

Asn Ile Asp Glu Ala Thr Tyr Val Cys Asp Tyr Ile Leu Gly Gly Lys

250 255 260

CTT AAT GGA TCT AGC TCA ACC AAA GAA GCT TTC ATG GAG AAA TTC AAA 872

Leu Asn Gly Ser Ser Ser Thr Lys Glu Ala Phe Met Glu Lys Phe Lys

265 270 275

TCT GCA GTT TCT GAG GGT TTT GAT CCT GAC AAG GAT CTT GTC AAA GCT 920

Ser Ala Val Ser Glu Gly Phe Asp Pro Asp Lys Asp Leu Val Lys Ala

280 285 290 295

GGT ATC GCA AAC CAA ACT ACA ATG TTG AAG GGA GAA ACA GAG GAG ATA 968

Gly Ile Ala Asn Gln Thr Thr Met Leu Lys Gly Glu Thr Glu Glu Ile

300 305 310

GGA AAA TTA TTA GAA AGA ACT ATG ATG CAA AAG TAT GGA GTA GAA AAT 1016

Gly Lys Leu Leu Glu Arg Thr Met Met Gln Lys Tyr Gly Val Glu Asn

315 320 325

GTC AAC AAT CAT TTC CTA AGT TTT AAC ACC ATC TGT GAC GCC ACT CAG 1064

Val Asn Asn His Phe Leu Ser Phe Asn Thr Ile Cys Asp Ala Thr Gln

330 335 340

GAG CGA CAA GAT GCT ATG TAT AAG TTG GTA GAT GAC AAA GTT GAT CTT 1112

Glu Arg Gln Asp Ala Met Tyr Lys Leu Val Asp Asp Lys Val Asp Leu

345 350 355

ATG TTG GTA ATT GGT GGG TTC AAC TCG AGC AAC ACC TCA CAC TTA CAA 1160

Met Leu Val Ile Gly Gly Phe Asn Ser Ser Asn Thr Ser His Leu Gln

360 365 370 375

GAG ATT GCT GAG GAG CGT AAA ATT CCA TCT TAT TGG ATT GAC AGT GAG 1208

Glu Ile Ala Glu Glu Arg Lys Ile Pro Ser Tyr Trp Ile Asp Ser Glu

380 385 390

AAA AGA ATA GGC CCT GGA AAC CGA ATA GCT TAC AAG TTA TTG CAC GGT 1256

Lys Arg Ile Gly Pro Gly Asn Arg Ile Ala Tyr Lys Leu Leu His Gly

395 400 405

GAA TTG GTT GAG CAA GAA AAC TGG CTA CCA AAG GGG CCT ATC ACA ATT 1304

Glu Leu Val Glu Gln Glu Asn Trp Leu Pro Lys Gly Pro Ile Thr Ile

410 415 420

GGT GTT ACA TCT GGT GCT TCT ACC CCT GAC AAG GTT GTT GAA GAT GCT 1352

Gly Val Thr Ser Gly Ala Ser Thr Pro Asp Lys Val Val Glu Asp Ala

425 430 435

CTT CTT AAG GTA TTT GAG ATC AAA CGT GAG GAA GCC CTG CAA TTG GTG 1400

Leu Leu Lys Val Phe Glu Ile Lys Arg Glu Glu Ala Leu Gln Leu Val

400 445 450 455

TAG ATTAGGCATG TACCTTGGCA AGAGAATCGA ACAGTATAGA TAAATCAAGT 1453

***

CTGAGCACCG AAACATGATG TCATCCATTG CAACTTGCAG CTGCATGTAC CTTTATATCT 1513

GAAATGTATT GTATGCATAC TACAAAACCA TGGGTTCAGT ATCGACATTG ATACACACCA 1573

CCCTTTAATA TGAAGTATCA TGGCTCTGGT TGCTATAAAC GACTTGTTAC AAAAAAAAAA 1633

AAAAAACAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAG AAAAAAAAAA 1693

AAA 1696

<210>2

<211>456

<212>PRT

＜213〉sweet wormwood (Artemisia annua L.)

<400>2

Met Ala Ser Leu Gln Leu Thr Pro Leu Ser Thr Arg Thr Asp Tyr Leu

1 5 10 15

Ser Leu Pro Ala Asp Ile Lys Val Phe Arg Cys Arg Lys Pro Leu Thr

20 25 30

Val Arg Cys Ser Gly Gly Asp Thr Ser Ser Ser Thr Gln Phe Asp Ala

35 40 45

Lys Val Phe Arg His Asn Leu Thr Arg Ser Glu Asn Tyr Asn Arg Lys

50 55 60

Gly Phe Gly His Lys Lys Glu Thr Leu Glu Leu Met Ser Gln Glu Tyr

65 70 75 80

Phe Ser Asp Ile Ile Lys Thr Leu Lys Glu Asn Asn Tyr Glu Tyr Thr

85 90 95

Trp Gly Asn Val Thr Val Lys Leu Ala Glu Ala Phe Gly Phe Cys Trp

100 105 110

Gly Val Glu Arg Ala Val Gln Ile Ala Tyr Glu Ala Arg Lys Gln Phe

115 120 125

Pro Asp Asp Lys Ile Trp Ile Thr Asn Gln Ile Ile His Asn Pro Thr

130 135 140

Val Asn Lys Arg Leu Glu Glu Met Glu Val Thr Asp Ile Pro Ile Asp

145 150 155 160

Gly Gly Glu Lys Gln Phe Asp Val Val Asp Lys Gly Asp Val Val Ile

165 170 175

Leu Pro Ala Phe Gly Ala Ala Val Asp Glu Met Arg Ile Leu Ser Asn

180 185 190

Lys Glu Val Gln Ile Val Asp Thr Thr Cys Pro Trp Val Thr Lys Val

195 200 205

Trp Asn Val Val Glu Lys His Lys Lys Gly Asp Tyr Thr Ser Val Ile

210 215 220

His Gly Lys His Asn His Glu Glu Thr Val Ala Thr Ala Ser Phe Ala

225 230 235 240

Gly Lys Phe Ile Val Val Lys Asn Ile Asp Glu Ala Thr Tyr Val Cys

245 250 255

Asp Tyr Ile Leu Gly Gly Lys Leu Asn Gly Ser Ser Ser Thr Lys Glu

260 265 270

Ala Phe Met Glu Lys Phe Lys Ser Ala Val Ser Glu Gly Phe Asp Pro

275 280 285

Asp Lys Asp Leu Val Lys Ala Gly Ile Ala Asn Gln Thr Thr Met Leu

290 295 300

Lys Gly Glu Thr Glu Glu Ile Gly Lys Leu Leu Glu Arg Thr Met Met

305 310 315 320

Gln Lys Tyr Gly Val Glu Asn Val Asn Asn His Phe Leu Ser Phe Asn

325 330 335

Thr Ile Cys Asp Ala Thr Gln Glu Arg Gln Asp Ala Met Tyr Lys Leu

340 345 350

Val Asp Asp Lys Val Asp Leu Met Leu Val Ile Gly Gly Phe Asn Ser

355 360 365

Ser Asn Thr Ser His Leu Gln Glu Ile Ala Glu Glu Arg Lys Ile Pro

370 375 380

Ser Tyr Trp Ile Asp Ser Glu Lys Arg Ile Gly Pro Gly Asn Arg Ile

385 390 395 400

Ala Tyr Lys Leu Leu His Gly Glu Leu Val Glu Gln Glu Asn Trp Leu

405 410 415

Pro Lys Gly Pro Ile Thr Ile Gly Val Thr Ser Gly Ala Ser Thr Pro

420 425 430

Asp Lys Val Val Glu Asp Ala Leu Leu Lys Val Phe Glu Ile Lys Arg

435 400 445

Glu Glu Ala Leu Gln Leu Val TAG ***

450 455 456

Claims (3)

1. the nucleic acid of coding southernwood 1-hydroxy-2-methyl-2-(E)-butenyl 4-tetra-sodium reductase enzyme is characterized in that described nucleotide sequence is made of the nucleotide sequence from nucleic acid 36-1403 position among the SEQ ID NO.1.

2. southernwood 1-hydroxy-2-methyl-the 2-of nucleic acid encoding according to claim 1 (E)-butenyl 4-tetra-sodium reductase enzyme is characterized in that by the aminoacid sequence shown in the SEQ ID NO.2.

3. nucleic acid transformed host cells according to claim 1 is characterized in that described host cell is an eukaryotic cell.