CN102021197A - Plant bivalent expression vector and construction method thereof - Google Patents

Abstract

The present invention relates to the plant genetic engineering field, concretely a plant bivalent expression vector and construction method thereof. As shown by the basic group sequence in the plant bivalent expression vector sequence table SEQ ID No.1, a beta-carotene ketolase gene and a beta-carotene hydroxylase gene are inserted into the same expression vector to obtain the plant bivalent expression vector. The construction of the plant bivalent expression vector comprises the following steps: cloning the bkt and crtR-B genes from haematococcus pluvialis; ligating the bkt gene into a pBI1221 vector; enzyme digesting the pBI1221 vector with the bkt gene to obtain a 35S-bkt-NOS fragment and inserting the fragment into a pCAMBIA1301 vector; finally, inserting the crtR-B gene into the pCAMBIA1301 vector with the bkt fragment to obtain the plant bivalent expression vector containing the bkt and the crtR-B genes. During the transformation of higher plants by the vector, full use of the precursor substance of the beta-carotene in the plant can be made to efficiently synthesize astaxanthin.

Description

A kind of plant bivalent expression vector and construction process thereof

Technical field

The present invention relates to plant genetic engineering field, to a kind of β-Hu Luobusu ketonize enzyme gene (bkt) in the Haematocoocus Pluvialls and β-Hu Luobusu '-hydroxylase gene (crtR-B) are transformed plant bivalent expression vector and construction process thereof synchronously specifically.

Technical background

Astaxanthin is a kind of carotenoid with extremely strong anti-oxidant activity, is with a wide range of applications.The unicell green alga Haematocoocus Pluvialls can synthesize and accumulate astaxanthin under the environment-stress condition, content is that of other biology arrives several magnitude up to 4% of dry cell weight, becomes the natural astaxanthin synthetics of present first-selection.The astaxanthin of Haematocoocus Pluvialls is a synthetic under the effect of a series of enzymes, wherein β-Hu Luobusu ketonize enzyme and β-Hu Luobusu hydroxylase are two key enzymes, their actings in conjunction transform β-Hu Luobusu and form astaxanthin (Grunewald K etc., 2000, Plant Physiol, 122:1261-1268).These two enzymes all are bifunctional enzymes, and β-Hu Luobusu ketonize enzyme can be a substrate with β-Hu Luobusu and zeaxanthin respectively, on C4 and C4 ' position, introduce ketone group (Fraser PD etc., 1998, Eur J Biochem, 252:229-236); The β-Hu Luobusu hydroxylase can be a substrate with β-Hu Luobusu and angle xanthin respectively, introduce respectively in C3 and C3 ' position a hydroxyl (Linden H, 1999, BiochimBiophys Acta, 1446:203-212).General synthesizing astaxanthin not in plants such as corn, peanut, so will utilize precursor substance synthesizing astaxanthin in other plant, β-Hu Luobusu ketonize enzyme and β-Hu Luobusu hydroxylase are absolutely necessary.

Now (the GenBank sequence number: D45881) (the GenBank sequence number: AF162276) clone comes out with the crtR-B gene with the bkt gene in the Haematocoocus Pluvialls (Haematococcus pluvialis), this makes utilization metabolic engineering means a large amount of natural astaxanthins of synthetic production in higher plant become possibility, has had the scientific research personnel to attempt in this field now.Mann etc. with the β-Hu Luobusu ketonize enzyme gene of being cloned into combine with tomato pds gene promoter change tobacco over to after, carotenoid composition and content do not change in the plant leaf, but in the nectary chromoplastid of flower, accumulated the astaxanthin of high density, make colored color from red (the Mann V etc. of xanthochromia, 2000, Nat Biotechnol, 18:888-892).Structural analysis shows that the astaxanthin that transgene tobacco forms has identical chirality with the natural astaxanthin that Haematocoocus Pluvialls forms.Ralley etc. are with β-Hu Luobusu ketonize enzyme and β-Hu Luobusu '-hydroxylase gene and lead the peptide fusion, and under the driving of the CaMV35S of tobacco mosaic virus (TMV) promotor, change tomato and tobacco over to, also successfully in the blade of tomato and tobacco, expressed (Ralley L, 2004, The Plant Journal, 39:477-486).Yet these work mainly are that the term single gene among bkt or the crtR-B is changeed into plant, or two genes are connected under the same promotor start, and when two gene constructed content that whether can improve astaxanthin when on identical carrier and respectively starting regulation and control by two promotors are also unknown, therefore make up the plant bivalent expression vector that contains β-Hu Luobusu ketonize enzyme gene and β-Hu Luobusu '-hydroxylase gene, make two genes change plant simultaneously over to, significant to improve the research that transforms astaxanthin resultant quantity in the plant by genetic engineering means.

Summary of the invention

The object of the present invention is to provide β-Hu Luobusu ketonize enzyme gene (bkt) in the Haematocoocus Pluvialls and β-Hu Luobusu '-hydroxylase gene (crtR-B) are transformed plant bivalent expression vector and construction process thereof synchronously.

For achieving the above object, the technical solution used in the present invention is:

1. shown in the base sequence among the plant bivalent expression vector sequence table SEQ ID No.1, it is inserted on the pCAMBIA1301 expression vector jointly for β-Hu Luobusu ketonize enzyme gene (bkt) and β-Hu Luobusu '-hydroxylase gene (crtR-B), the plant bivalent expression vector that obtains.

2. described plant bivalent expression vector total length is 12727bp, and bkt and crtR-B expression of gene are by the CaMV35S promotor startup of upstream separately.

3. at first, clone's β-Hu Luobusu ketonize enzyme gene (bkt) and β-Hu Luobusu '-hydroxylase gene (crtR-B) from Haematocoocus Pluvialls; Secondly, the bkt that obtains and crtR-B are connected to obtain pET-28a (+)-bkt and pET-28a (+)-crtR-B carrier on two pET-28a (+) carrier; Once more, be template with pET-28a (+)-bkt, P ₁And P ₂Be the primer PCR amplification, product cuts back to close fragment and pBI221 carrier T through BamHI and SacI enzyme ₄Dna ligase connects, and obtains the pBI221-bkt intermediate carrier, uses HindIII, this intermediate carrier of EcoRI double digestion and pCAMBIA1301 carrier afterwards again, and uses T ₄The dna ligase connection obtains the pCAMBIA1301-bkt intermediate carrier; At last, be template with pET-28a (+)-crtR-B, P ₃And P ₄Be the primer PCR amplification, product and pCAMBIA1301-bkt intermediate carrier are cut through BglII and BstEII enzyme, and the fragment of recovery is by T ₄On the pCAMBIA1301-bkt carrier after dna ligase is connected to crtR-B gene forward enzyme and cuts, i.e. plant bivalent expression vector shown in the base sequence among the calling sequence table SEQ ID No.1.

4. described P ₁, P ₂, P ₃, P ₄Primer is respectively: P ₁: 5 ' AAA GGATCCATGCACGTCGCATC 3 ', P ₂: 5 ' AAC GAGCTCTCATGCCAAGGCAG 3 ', P ₃: 5 ' CCC AGATCTATGCTGTCGAAGCTGC 3 ', P ₄: 5 ' AAA GGTCACCCTACCGCTTGGACCA 3 '.

5. described bkt upstream region of gene has 35S promoter, and the downstream has the NOS termination signal, is built into the 35S-bkt-NOS fragment, and this fragment oppositely is inserted on the pCAMBIA1301 carrier; The crtR-B upstream region of gene has 35S promoter, and the downstream has the NOS termination signal, constitutes the 35S-crtR-B-NOS fragment, and this fragment forward is inserted on the pCAMBIA1301 carrier.

The advantage that the present invention had:

1. the present invention is building up on the identical carrier by will clone the β-Hu Luobusu ketonize enzyme gene (bkt) and the β-Hu Luobusu '-hydroxylase gene (crtR-B) that come out from Haematocoocus Pluvialls, so that two genes of bkt and crtR-B change in the plant for mediation by Agrobacterium, make and express β-Hu Luobusu ketonize enzyme and β-Hu Luobusu hydroxylase in the plant simultaneously, and then two kinds of problems that enzyme can not be expressed synchronously that two genes imported plant materials respectively and cause have been overcome, simultaneously can make full use of the efficient synthesizing astaxanthin of β-Hu Luobusu precursor substance in the plant, the present invention and two genes carrier construction respectively transform plant again to compare method for transformation more simple.

2. another advantage of the present invention is that two upstream region of gene all have corresponding 35S promoter to start, and starts two genes with a promotor and compares, and gene transcript expression efficient is higher; And the Haematocoocus Pluvialls hydroxylase of expressing on the bivalent expression carrier of the present invention is a bifunctional enzyme, can be that substrate can be a substrate with the angle xanthin again with the β-Hu Luobusu, introduce a hydroxyl respectively at C3 and C3 ' position, catalysis generates zeaxanthin and astaxanthin, and zeaxanthin can be used as the substrate of ketonize enzyme, the Haematocoocus Pluvialls hydroxylase of expressing on the bivalent expression carrier of the present invention is a bifunctional enzyme simultaneously, can be that substrate can be substrate with the zeaxanthin again with the β-Hu Luobusu, on C4 and C4 ' position, introduce a ketone group respectively, catalysis generates angle xanthin and astaxanthin, and the angle xanthin that generates can be used as the substrate of hydroxylase.

3. itself do not possess the involved enzyme gene in the higher plant and synthesizing astaxanthin in vivo, behind expression vector conversion plant of the present invention, thereby make it have hydroxylase in the algae and ketonize enzyme gene synthesizing astaxanthin in vivo, transformed the carotene pathways metabolism of plant, made it synthesize the product of high added value.

4. utilize carrier of the present invention to transform astaxanthin that higher plant produced and have identical sterie configuration, overcome the problem that the method for utilizing chemosynthesis can not generate the astaxanthin with native configurations with natural astaxanthin in Haematocoocus Pluvialls and other bacterium.

5. cloned genes is all from the eukaryote Haematocoocus Pluvialls on the bivalent expression carrier of the present invention, this with compare from some prokaryotic organism clone genes involveds such as bacteriums, when transforming the eucaryon higher plant, the probability of the foreign gene silence that is changed over to is lower.

Description of drawings

Fig. 1 makes up schema for the pBI221-bkt intermediate carrier.

Fig. 2 makes up schema for the pCAMBIA1301-bkt intermediate carrier.

Fig. 3 is pCMBIA1301-bkt intermediate carrier figure.

Fig. 4 is pCAM[BIA1301-bkt-crtR-B intermediate carrier figure.

Fig. 5 is the two valency vector construction schemas of pCAMBIA1301-bkt-crtR-B.

Fig. 6 is that the PCR of bkt gene among the expression vector pCAMBIA1301-bkt-crtR-B detects.

Fig. 7 is that the PCR of crtR-B gene among the expression vector pCAMBIA1301-bkt-crtR-B detects.

Embodiment

Further illustrate the present invention in the example below, this does not limit the scope of the invention.

Experiment material and test kit

All DNA restriction enzymes, T ₄Dna ligase, La Taq archaeal dna polymerase, the TaqDNA polysaccharase, all available from Dalian TakaRa company, plasmid extraction kit, sepharose reclaim test kit in a small amount, the total genome of plant extracts test kit available from Shanghai China Shun biotechnology company limited.

Embodiment 1

Shown in the base sequence among the plant bivalent expression vector sequence table SEQ ID No.1, it is inserted on the pCAMBIA1301 expression vector jointly for β-Hu Luobusu ketonize enzyme gene (bkt) and β-Hu Luobusu '-hydroxylase gene (crtR-B), the plant bivalent expression vector that obtains.

Sequence table SEQ ID No.1

ATGCTGTCGAAGCTGCAGTCAATCAGCGTCAAGGCCCGCCGCGTTGAACTAGCCCGCGACATCACGCGGCCCA

AAGTCTGCCTGCATGCTCAGCGGTGCTCGTTAGTTCGGCTGCGAGTGGCAGCACCACAGACAGAGGAGGCGCT

GGGAACCGTGCAGGCTGCCGGCGCGGGCGATGAGCACAGCGCCGATGTAGCACTCCAGCAGCTTGACCGGGC

TATCGCAGAGCGTCGTGCCCGGCGCAAACGGGAGCAGCTGTCATACCAGGCTGCCGCCATTGCAGCATCAATT

GGCGTGTCAGGCATTGCCATCTTCGCCACCTACCTGAGATTTGCCATGCACATGACCGTGGGCGGCGCAGTGCC

ATGGGGTGAAGTGGCTGGCACTCTCCTCTTGGTGGTTGGTGGCGCGCTCGGCATGGAGATGTATGCCCGCTATG

CACACAAAGCCATCTGGCATGAGTCGCCTCTGGGCTGGCTGCTGCACAAGAGCCACCACACACCTCGCACTGG

ACCCTTTGAAGCCAACGACTTGTTTGCAATCATCAATGGACTGCCCGCCATGCTCCTGTGTACCTTTGGCTTCTG

GCTGCCCAACGTCCTGGGGGCGGCCTGCTTTGGAGCGGGGCTGGGCATCACGCTATACGGCATGGCATATATG

TTTGTACACGATGGCCTGGTGCACAGGCGCTTTCCCACCGGGCCCATCGCTGGCCTGCCCTACATGAAGCGCCT

GACAGTGGCCCACCAGCTACACCACAGCGGCAAGTACGGTGGCGCGCCCTGGGGTATGTTCTTGGGTCCACAG

GAGCTGCAGCACATTCCAGGTGCGGCGGAGGAGGTGGAGCGACTGGTCCTGGAACTGGACTGGTCCAAGCGG

TAGGTGACCAGCTCGAATTTCCCCGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGC

CGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGA

CGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATA

TAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATCGGGAATTAAACTATCAGT

GTTTGACAGGATATATTGGCGGGTAAACCTAAGAGAAAAGAGCGTTTATTAGAATAACGGATATTTAAAAGGG

CGTGAAAAGGTTTATCCGTTCGTCCATTTGTATGTGCATGCCAACCACAGGGTTCCCCTCGGGATCAAAGTACT

TTGATCCAACCCCTCCGCTGCTATAGTGCAGTCGGCTTCTGACGTTCAGTGCAGCCGTCTTCTGAAAACGACAT

GTCGCACAAGTCCTAAGTTACGCGACAGGCTGCCGCCCTGCCCTTTTCCTGGCGTTTTCTTGTCGCGTGTTTTAG

TCGCATAAAGTAGAATACTTGCGACTAGAACCGGAGACATTACGCCATGAACAAGAGCGCCGCCGCTGGCCTG

CTGGGCTATGCCCGCGTCAGCACCGACGACCAGGACTTGACCAACCAACGGGCCGAACTGCACGCGGCCGGCT

GCACCAAGCTGTTTTCCGAGAAGATCACCGGCACCAGGCGCGACCGCCCGGAGCTGGCCAGGATGCTTGACCA

CCTACGCCCTGGCGACGTTGTGACAGTGACCAGGCTAGACCGCCTGGCCCGCAGCACCCGCGACCTACTGGAC

ATTGCCGAGCGCATCCAGGAGGCCGGCGCGGGCCTGCGTAGCCTGGCAGAGCCGTGGGCCGACACCACCACG

CCGGCCGGCCGCATGGTGTTGACCGTGTTCGCCGGCATTGCCGAGTTCGAGCGTTCCCTAATCATCGACCGCAC

CCGGAGCGGGCGCGAGGCCGCCAAGGCCCGAGGCGTGAAGTTTGGCCCCCGCCCTACCCTCACCCCGGCACAG

ATCGCGCACGCCCGCGAGCTGATCGACCAGGAAGGCCGCACCGTGAAAGAGGCGGCTGCACTGCTTGGCGTGC

ATCGCTCGACCCTGTACCGCGCACTTGAGCGCAGCGAGGAAGTGACGCCCACCGAGGCCAGGCGGCGCGGTGC

CTTCCGTGAGGACGCATTGACCGAGGCCGACGCCCTGGCGGCCGCCGAGAATGAACGCCAAGAGGAACAAGC

ATGAAACCGCACCAGGACGGCCAGGACGAACCGTTTTTCATTACCGAAGAGATCGAGGCGGAGATGATCGCG

GCCGGGTACGTGTTCGAGCCGCCCGCGCACGTCTCAACCGTGCGGCTGCATGAAATCCTGGCCGGTTTGTCTGA

TGCCAAGCTGGCGGCCTGGCCGGCCAGCTTGGCCGCTGAAGAAACCGAGCGCCGCCGTCTAAAAAGGTGATGT

GTATTTGAGTAAAACAGCTTGCGTCATGCGGTCGCTGCGTATATGATGCGATGAGTAAATAAACAAATACGCA

AGGGGAACGCATGAAGGTTATCGCTGTACTTAACCAGAAAGGCGGGTCAGGCAAGACGACCATCGCAACCCA

TCTAGCCCGCGCCCTGCAACTCGCCGGGGCCGATGTTCTGTTAGTCGATTCCGATCCCCAGGGCAGTGCCCGCG

ATTGGGCGGCCGTGCGGGAAGATCAACCGCTAACCGTTGTCGGCATCGACCGCCCGACGATTGACCGCGACGT

GAAGGCCATCGGCCGGCGCGACTTCGTAGTGATCGACGGAGCGCCCCAGGCGGCGGACTTGGCTGTGTCCGCG

ATCAAGGCAGCCGACTTCGTGCTGATTCCGGTGCAGCCAAGCCCTTACGACATATGGGCCACCGCCGACCTGG

TGGAGCTGGTTAAGCAGCGCATTGAGGTCACGGATGGAAGGCTACAAGCGGCCTTTGTCGTGTCGCGGGCGAT

CAAAGGCACGCGCATCGGCGGTGAGGTTGCCGAGGCGCTGGCCGGGTACGAGCTGCCCATTCTTGAGTCCCGT

ATCACGCAGCGCGTGAGCTACCCAGGCACTGCCGCCGCCGGCACAACCGTTCTTGAATCAGAACCCGAGGGCG

ACGCTGCCCGCGAGGTCCAGGCGCTGGCCGCTGAAATTAAATCAAAACTCATTTGAGTTAATGAGGTAAAGAG

AAAATGAGCAAAAGCACAAACACGCTAAGTGCCGGCCGTCCGAGCGCACGCAGCAGCAAGGCTGCAACGTTG

GCCAGCCTGGCAGACACGCCAGCCATGAAGCGGGTCAACTTTCAGTTGCCGGCGGAGGATCACACCAAGCTGA

AGATGTACGCGGTACGCCAAGGCAAGACCATTACCGAGCTGCTATCTGAATACATCGCGCAGCTACCAGAGTA

AATGAGCAAATGAATAAATGAGTAGATGAATTTTAGCGGCTAAAGGAGGCGGCATGGAAAATCAAGAACAAC

CAGGCACCGACGCCGTGGAATGCCCCATGTGTGGAGGAACGGGCGGTTGGCCAGGCGTAAGCGGCTGGGTTGT

CTGCCGGCCCTGCAATGGCACTGGAACCCCCAAGCCCGAGGAATCGGCGTGACGGTCGCAAACCATCCGGCCC

GGTACAAATCGGCGCGGCGCTGGGTGATGACCTGGTGGAGAAGTTGAAGGCCGCGCAGGCCGCCCAGCGGCA

ACGCATCGAGGCAGAAGCACGCCCCGGTGAATCGTGGCAAGCGGCCGCTGATCGAATCCGCAAAGAATCCCG

GCAACCGCCGGCAGCCGGTGCGCCGTCGATTAGGAAGCCGCCCAAGGGCGACGAGCAACCAGATTTTTTCGTT

CCGATGCTCTATGACGTGGGCACCCGCGATAGTCGCAGCATCATGGACGTGGCCGTTTTCCGTCTGTCGAAGCG

TGACCGACGAGCTGGCGAGGTGATCCGCTACGAGCTTCCAGACGGGCACGTAGAGGTTTCCGCAGGGCCGGCC

GGCATGGCCAGTGTGTGGGATTACGACCTGGTACTGATGGCGGTTTCCCATCTAACCGAATCCATGAACCGAT

ACCGGGAAGGGAAGGGAGACAAGCCCGGCCGCGTGTTCCGTCCACACGTTGCGGACGTACTCAAGTTCTGCCG

GCGAGCCGATGGCGGAAAGCAGAAAGACGACCTGGTAGAAACCTGCATTCGGTTAAACACCACGCACGTTGC

CATGCAGCGTACGAAGAAGGCCAAGAACGGCCGCCTGGTGACGGTATCCGAGGGTGAAGCCTTGATTAGCCG

CTACAAGATCGTAAAGAGCGAAACCGGGCGGCCGGAGTACATCGAGATCGAGCTAGCTGATTGGATGTACCG

CGAGATCACAGAAGGCAAGAACCCGGACGTGCTGACGGTTCACCCCGATTACTTTTTGATCGATCCCGGCATC

GGCCGTTTTCTCTACCGCCTGGCACGCCGCGCCGCAGGCAAGGCAGAAGCCAGATGGTTGTTCAAGACGATCT

ACGAACGCAGTGGCAGCGCCGGAGAGTTCAAGAAGTTCTGTTTCACCGTGCGCAAGCTGATCGGGTCAAATGA

CCTGCCGGAGTACGATTTGAAGGAGGAGGCGGGGCAGGCTGGCCCGATCCTAGTCATGCGCTACCGCAACCTG

ATCGAGGGCGAAGCATCCGCCGGTTCCTAATGTACGGAGCAGATGCTAGGGCAAATTGCCCTAGCAGGGGAA

AAAGGTCGAAAAGGTCTCTTTCCTGTGGATAGCACGTACATTGGGAACCCAAAGCCGTACATTGGGAACCGGA

ACCCGTACATTGGGAACCCAAAGCCGTACATTGGGAACCGGTCACACATGTAAGTGACTGATATAAAAGAGAA

AAAAGGCGATTTTTCCGCCTAAAACTCTTTAAAACTTATTAAAACTCTTAAAACCCGCCTGGCCTGTGCATAAC

TGTCTGGCCAGCGCACAGCCGAAGAGCTGCAAAAAGCGCCTACCCTTCGGTCGCTGCGCTCCCTACGCCCCGC

CGCTTCGCGTCGGCCTATCGCGGCCGCTGGCCGCTCAAAAATGGCTGGCCTACGGCCAGGCAATCTACCAGGG

CGCGGACAAGCCGCGCCGTCGCCACTCGACCGCCGGCGCCCACATCAAGGCACCCTGCCTCGCGCGTTTCGGT

GATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGA

GCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCCGGGCGCAGCCATGACCCAGTCACGTA

GCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGG

TGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTC

GCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAA

TCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGC

GTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGC

GAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACC

CTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGG

TATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTG

CGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTG

GTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTA

CACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTT

GATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAA

AGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGG

ATTTTGGTCATGCATTCTAGGTACTAAAACAATTCATCCAGTAAAATATAATATTTTATTTTCTCCCAATCAGGC

TTGATCCCCAGTAAGTCAAAAAATAGCTCGACATACTGTTCTTCCCCGATATCCTCCCTGATCGACCGGACGCA

GAAGGCAATGTCATACCACTTGTCCGCCCTGCCGCTTCTCCCAAGATCAATAAAGCCACTTACTTTGCCATCTT

TCACAAAGATGTTGCTGTCTCCCAGGTCGCCGTGGGAAAAGACAAGTTCCTCTTCGGGCTTTTCCGTCTTTAAA

AAATCATACAGCTCGCGCGGATCTTTAAATGGAGTGTCTTCTTCCCAGTTTTCGCAATCCACATCGGCCAGATC

GTTATTCAGTAAGTAATCCAATTCGGCTAAGCGGCTGTCTAAGCTATTCGTATAGGGACAATCCGATATGTCGA

TGGAGTGAAAGAGCCTGATGCACTCCGCATACAGCTCGATAATCTTTTCAGGGCTTTGTTCATCTTCATACTCTT

CCGAGCAAAGGACGCCATCGGCCTCACTCATGAGCAGATTGCTCCAGCCATCATGCCGTTCAAAGTGCAGGAC

CTTTGGAACAGGCAGCTTTCCTTCCAGCCATAGCATCATGTCCTTTTCCCGTTCCACATCATAGGTGGTCCCTTT

ATACCGGCTGTCCGTCATTTTTAAATATAGGTTTTCATTTTCTCCCACCAGCTTATATACCTTAGCAGGAGACAT

TCCTTCCGTATCTTTTACGCAGCGGTATTTTTCGATCAGTTTTTTCAATTCCGGTGATATTCTCATTTTAGCCATT

TATTATTTCCTTCCTCTTTTCTACAGTATTTAAAGATACCCCAAGAAGCTAATTATAACAAGACGAACTCCAATT

CACTGTTCCTTGCATTCTAAAACCTTAAATACCAGAAAACAGCTTTTTCAAAGTTGTTTTCAAAGTTGGCGTAT

AACATAGTATCGACGGAGCCGATTTTGAAACCGCGGTGATCACAGGCAGCAACGCTCTGTCATCGTTACAATC

AACATGCTACCCTCCGCGAGATCATCCGTGTTTCAAACCCGGCAGCTTAGTTGCCGTTCTTCCGAATAGCATCG

GTAACATGAGCAAAGTCTGCCGCCTTACAACGGCTCTCCCGCTGACGCCGTCCCGGACTGATGGGCTGCCTGTA

TCGAGTGGTGATTTTGTGCCGAGCTGCCGGTCGGGGAGCTGTTGGCTGGCTGGTGGCAGGATATATTGTGGTGT

AAACAAATTGACGCTTAGACAACTTAATAACACATTGCGGACGTTTTTAATGTACTGAATTAACGCCGAATTAA

TTCGGGGGATCTGGATTTTAGTACTGGATTTTGGTTTTAGGAATTAGAAATTTTATTGATAGAAGTATTTTACAA

ATACAAATACATACTAAGGGTTTCTTATATGCTCAACACATGAGCGAAACCCTATAGGAACCCTAATTCCCTTA

TCTGGGAACTACTCACACATTATTATGGAGAAACTCGAGCTTGTCGATCGACAGATCCGGTCGGCATCTACTCT

ATTTCTTTGCCCTCGGACGAGTGCTGGGGCGTCGGTTTCCACTATCGGCGAGTACTTCTACACAGCCATCGGTC

CAGACGGCCGCGCTTCTGCGGGCGATTTGTGTACGCCCGACAGTCCCGGCTCCGGATCGGACGATTGCGTCGC

ATCGACCCTGCGCCCAAGCTGCATCATCGAAATTGCCGTCAACCAAGCTCTGATAGAGTTGGTCAAGACCAAT

GCGGAGCATATACGCCCGGAGTCGTGGCGATCCTGCAAGCTCCGGATGCCTCCGCTCGAAGTAGCGCGTCTGC

TGCTCCATACAAGCCAACCACGGCCTCCAGAAGAAGATGTTGGCGACCTCGTATTGGGAATCCCCGAACATCG

CCTCGCTCCAGTCAATGACCGCTGTTATGCGGCCATTGTCCGTCAGGACATTGTTGGAGCCGAAATCCGCGTGC

ACGAGGTGCCGGACTTCGGGGCAGTCCTCGGCCCAAAGCATCAGCTCATCGAGAGCCTGCGCGACGGACGCAC

TGACGGTGTCGTCCATCACAGTTTGCCAGTGATACACATGGGGATCAGCAATCGCGCATATGAAATCACGCCA

TGTAGTGTATTGACCGATTCCTTGCGGTCCGAATGGGCCGAACCCGCTCGTCTGGCTAAGATCGGCCGCAGCGA

TCGCATCCATAGCCTCCGCGACCGGTTGTAGAACAGCGGGCAGTTCGGTTTCAGGCAGGTCTTGCAACGTGAC

ACCCTGTGCACGGCGGGAGATGCAATAGGTCAGGCTCTCGCTAAACTCCCCAATGTCAAGCACTTCCGGAATC

GGGAGCGCGGCCGATGCAAAGTGCCGATAAACATAACGATCTTTGTAGAAACCATCGGCGCAGCTATTTACCC

GCAGGACATATCCACGCCCTCCTACATCGAAGCTGAAAGCACGAGATTCTTCGCCCTCCGAGAGCTGCATCAG

GTCGGAGACGCTGTCGAACTTTTCGATCAGAAACTTCTCGACAGACGTCGCGGTGAGTTCAGGCTTTTTCATAT

CTCATTGCCCCCCGGGATCTGCGAAAGCTCGAGAGAGATAGATTTGTAGAGAGAGACTGGTGATTTCAGCGTG

TCCTCTCCAAATGAAATGAACTTCCTTATATAGAGGAAGGTCTTGCGAAGGATAGTGGGATTGTGCGTCATCCC

TTACGTCAGTGGAGATATCACATCAATCCACTTGCTTTGAAGACGTGGTTGGAACGTCTTCTTTTTCCACGATG

CTCCTCGTGGGTGGGGGTCCATCTTTGGGACCACTGTCGGCAGAGGCATCTTGAACGATAGCCTTTCCTTTATC

GCAATGATGGCATTTGTAGGTGCCACCTTCCTTTTCTACTGTCCTTTTGATGAAGTGACAGATAGCTGGGCAAT

GGAATCCGAGGAGGTTTCCCGATATTACCCTTTGTTGAAAAGTCTCAATAGCCCTTTGGTCTTCTGAGACTGTA

TCTTTGATATTCTTGGAGTAGACGAGAGTGTCGTGCTCCACCATGTTATCACATCAATCCACTTGCTTTGAAGA

CGTGGTTGGAACGTCTTCTTTTTCCACGATGCTCCTCGTGGGTGGGGGTCCATCTTTGGGACCACTGTCGGCAG

AGGCATCTTGAACGATAGCCTTTCCTTTATCGCAATGATGGCATTTGTAGGTGCCACCTTCCTTTTCTACTGTCC

TTTTGATGAAGTGACAGATAGCTGGGCAATGGAATCCGAGGAGGTTTCCCGATATTACCCTTTGTTGAAAAGTC

TCAATAGCCCTTTGGTCTTCTGAGACTGTATCTTTGATATTCTTGGAGTAGACGAGAGTGTCGTGCTCCACCATG

TTGGCAAGCTGCTCTAGCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCAC

GACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCAC

CCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGA

AACAGCTATGACCATGATTACGCCCGATCTAGTAACATAGATGACACCGCGCGCGATAATTTATCCTAGTTTGC

GCGCTATATTTTGTTTTCTATCGCGTATTAAATGTATAATTGCGGGACTCTAATCATAAAAACCCATCTCATAAA

TAACGTCATGCATTACATGTTAATTATTACATGCTTAACGTAATTCAACAGAAATTATATGATAATCATCGCAA

GACCGGCAACAGGATTCAATCTTAAGAAACTTTATTGCCAAATGTTTGAACGATCGGGGAAATTCGTCATGCC

AAGGCAGGCACCAGGCCACGCCCGGACAGGCGGCGGCAGTGGGGCAGCTGCCACCAGGGGGCAAAGGGCCA

CCTGTGGTGCTCCCAGTGCAGGTCAAAGTGGTAGCATGTCAGGAAACTCATCACATCAGATGCCTCACTTGTCT

TGGCCCTGAACCAGGCCATCACCTGAGAGCCTGCTGCAGGGCCTGGCTCAGGCTTGTGTGGCAGGTAAGTGCC

GAAGTAGAAGAGGCGGAATGCTGACAAGATTGGGGCTGCAGCCATGAAGACTAGGAGATTTGCCATGGGCGC

CCCCAGCATTTGCATCACCACTGCCCACCATGCCAGCCGGGCAAACTGCCACAGGGACATGTAGCTGGACATG

AAGCTGGCGAACCAGGGGACAAGGCCGGGATTTCCCTTGTGGAAGTCAGGGTCTTTCCCCACTTCGCCAGTAT

GGTTGTGGTGCTCCCAGTGCTTGCGATGCAGCATGCTGTAGTCAAACCAGGCGTACAGTGATATGCAGATGTTG

CCAAGGAGATCATTGAGCTGCCTGTGCCTCAAAGCTATGGTGCCATGCATTGCGTCATGTGTGGTGATGAATAG

ACCAGTGTACAGGAACTCAAGTACAATGAAGACTGCAGCGATGTGCAGTAGGCTGCTGCTTCCGCCCAAAAGC

TGGGCTGTGGCTTCGGACACAGGCAACCAGTGAAGCTGGTCCATGGATGTCGGTAGCCTGATTTGAAATATTG

CGTGTAAAAACACTGCGGTCCAGGTGCCAATGATGGTCAGCGCCATCGTGATGCCCTTGGCGTCAGATGCTGG

AGGTTTGTAGGCGTGCTTTAGCGCAGGACGAGCTGCGTCTGACGACTCGGATGGCATGTGATACTGTGTCGCCC

ACGCTCTCAAGACGTCTGGGCTGGAAGCAGCTGCCTCACTGCCTTTCTGCTCGACCATTAGTGCCGATGCGACG

TGCATCTCTAGAGTCCCCCGTGTTCTCTCCAAATGAAATGAACTTCCTTATATAGAGGAAGGGTCTTGCGAAGG

ATAGTGGGATTGTGCGTCATCCCTTACGTCAGTGGAGATATCACATCAATCCACTTGCTTTGAAGACGTGGTTG

GAACGTCTTCTTTTTCCACGATGCTCCTCGTGGGTGGGGGTCCATCTTTGGGACCACTGTCGGCAGAGGCATCT

TCAACGATGGCCTTTCCTTTATCGCAATGATGGCATTTGTAGGAGCCACCTTCCTTTTCCACTATCTTCACAATA

AAGTGACAGATAGCTGGGCAATGGAATCCGAGGAGGTTTCCGGATATTACCCTTTGTTGAAAAGTCTCAATTG

CCCTTTGGTCTTCTGAGACTGTATCTTTGATATTTTTGGAGTAGACAAGTGTGTCGTGCTCCACCATGTTGACGA

AGATTTTCTTCTTGTCATTGAGTCGTAAGAGACTCTGTATGAACTGTTCGCCAGTCTTTACGGCGAGTTCTGTTA

GGTCCTCTATTTGAATCTTTGACTCCATGGCCTTTGATTCAGTGGGAACTACCTTTTTAGAGACTCCAATCTCTA

TTACTTGCCTTGGTTTGTGAAGCAAGCCTTGAATCGTCCATACTGGAATAGTACTTCTGATCTTGAGAAATATA

TCTTTCTCTGTGTTCTTGATGCAGTTAGTCCTGAATCTTTTGACTGCATCTTTAACCTTCTTGGGAAGGTATTTGA

TTTCCTGGAGATTATTGCTCGGGTAGATCGTCTTGATGAGACCTGCTGCGTAAGCCTCTCTAACCATCTGTGGG

TTAGCATTCTTTCTGAAATTGAAAAGGCTAATCTGGGGACCTGCAGGCATGCAAGCTAGCTTGGCACTGGCCGT

CGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCG

CCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATG

CTAGAGCAGCTTGAGCTTGGATCAGATTGTCGTTTCCCGCCTTCAGTTTAGCTTCATGGAGTCAAAGATTCAAA

TAGAGGACCTAACAGAACTCGCCGTAAAGACTGGCGAACAGTTCATACAGAGTCTCTTACGACTCAATGACAA

GAAGAAAATCTTCGTCAACATGGTGGAGCACGACACACTTGTCTACTCCAAAAATATCAAAGATACAGTCTCA

GAAGACCAAAGGGCAATTGAGACTTTTCAACAAAGGGTAATATCCGGAAACCTCCTCGGATTCCATTGCCCAG

CTATCTGTCACTTTATTGTGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGG

AAAGGCCATCGTTGAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTG

GAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATG

ACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGAACACGG

GGGACTCTTGACCATGGTA

(a) sequence signature:

● length: 12727bp

● type: base sequence

● chain: strand

● topological framework: ring-type

(b) molecule type: double-stranded DNA

(c) suppose: not

(d) antisense: not

(e) initial source: Haematocoocus Pluvialls

(f) specificity title: CDS

Embodiment 2

The structure of plant bivalent expression vector:

1. from Haematocoocus Pluvialls, clone β-Hu Luobusu ketonize enzyme gene (bkt) and β-Hu Luobusu '-hydroxylase gene (crtR-B).

2. the bkt that obtains and crtR-B are connected to and obtain pET-28a (+)-bkt and pET-28a (+)-crtR-B carrier on two pET-28a (+) carrier (Novagen).

3. according to a pair of primer P of carrier pET-28a (+)-bkt sequences Design ₁/ P ₂, and it is (synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd to introduce BamHI and SacI restriction enzyme site respectively at 5 ' end of upstream primer and downstream primer.), be template with plasmid pET-28a (+)-bkt, use combination of primers P ₁/ P ₂Carry out pcr amplification with La Taq archaeal dna polymerase, product cuts back to close fragment and pBI221 carrier T through BamHI and SacI enzyme ₄Dna ligase connects, and obtains the pBI221-bkt intermediate carrier, obtains pBI221-bkt carrier (as Fig. 1).Use HindIII, this carrier of EcoRI double digestion again, obtain the 35S-bkt-NOS fragment that size is about 2kb, then with plant expression vector pCAMBIA1301 (CAMBIA) equally through HindIII, EcoRI double digestion, its enzyme is cut back fragment and 35S-bkt-NOS fragment T ₄Dna ligase connects, and obtains intermediate carrier pCAMBIA1301-bkt (as Fig. 2,3), and preserves among the transformed into escherichia coli TOP10, and is stand-by.

P ₁：5′AAA GGATCCATGCACGTCGCATC?3′

P ₂：5′AAC GAGCTCTCATGCCAAGGCAG?3′

Pcr amplification reaction condition: 95 ℃ of pre-sex change 5min; 94 ℃ of sex change 1min, 59 ℃ of annealing 1min, 72 ℃ are extended 1.5min, circulate 35 times; 72 ℃ are continued to extend 10min.

4. according to carrier pET-28a (+)-a pair of primer of crtR-B sequences Design, and introduce BglII and BstEII restriction enzyme site (synthetic) respectively by Shanghai Sangon at 5 ' end of upstream primer and downstream primer, with plasmid pET-28a (+)-crtR-B is template, uses combination of primers P ₃/ P ₄Carry out pcr amplification with La Taq archaeal dna polymerase, obtain containing the crtR-B gene fragment of about 879bp of BglII and BstEII restriction enzyme site, with reclaiming behind BglII and the BstEII double digestion.The pCAMBIA1301-bkt intermediate carrier is cut through BglII and BstEII enzyme, with itself and crtR-B fragment T ₄Dna ligase connects, and transformed into escherichia coli TOP10, preserve stand-by, i.e. plant bivalent expression vector (as Fig. 4,5) shown in the base sequence among the calling sequence table SEQ ID No.1.Comprise two expressed intact boxes of bkt gene and crtR-B gene (containing CaMV 35S promoter and NOS terminator respectively) in this carrier, be two valency plant expression vectors, selection markers is Km.Further enzyme is cut and is identified demonstration, the pCAMBIA1301-bkt-crtR-B carrier produces 963bp one band (as Fig. 6) through BamHI, SacI double digestion, produce 879bp one band (as Fig. 7) through BglII, BstEII double digestion, illustrate that bkt gene and crtR-B gene successfully be cloned among the pCAMBIA1301.

P ₃：5′CCC AGATCTATGCTGTCGAAGCTGC?3′

P ₄：5′AAA GGTCACCCTACCGCTTGGACCA?3′

Pcr amplification reaction condition: 95 ℃ of pre-sex change 5min; 94 ℃ of sex change 1min, 63 ℃ of annealing 1min, 72 ℃ are extended 1.5min, circulate 35 times; 72 ℃ are continued to extend 10min.

Embodiment 3, two valency carrier transform peanut

1. transform agrobacterium tumefaciens EHA105: the plant bivalent expression vector pCAMBIA1301-bkt-crtR-B plasmid that extracts embodiment 2 gained, adopt freeze-thaw method transformed competence colibacillus Agrobacterium EHA105, coat and contain rifomycin (Rif, 50mg/L), kantlex (Km, on solid YEB substratum plate 50mg/L), 28 ℃ of dark 48h that cultivate.Picking list bacterium colony adopts and has identical antibiotic YEB liquid nutrient medium cultivation.Use combination of primers P ₁/ P ₂To the bkt gene and use combination of primers P ₃/ P ₄The crtR-B gene is carried out PCR respectively detect, two positive colonies are the Agrobacterium engineering strain EHA105/pCAMBIA1301-bkt-crtR-B that contains two valency plant expression vectors.Inoculation Agrobacterium EHA105/pCAMBIA1301-bkt-crtR-B mono-clonal contains in the YEB liquid nutrient medium of Rif (50mg/L), Km (50mg/L) at 5ml, 28 ℃, 200r/min shake overnight incubation, get 1ml and move in the 25mlYEB liquid nutrient medium and continue to be cultured to OD ₆₀₀Be about 0.65, behind the centrifugal 15min of 6000r/min, with inducing liquid nutrient medium to be suspended into OD ₆₀₀=0.65.

2. the preparation of peanut embryo leaflet and embryonic development point explant: choose the peanut seed of mature and plump, remove cotyledon, take out embryo,, use 0.1%HgCl again with 70% alcohol-pickled about 1min ₂Solution disinfection 6min, aseptic condition use aseptic water washing 3-4 time down, place 28 ℃ of incubators, and sterilized water soaks.After soaking 16-18h, the embryo leaflet of peanut embryo is downcut from its base portion, obtain the little leaf explant of embryo, make every embryo leaflet separately as far as possible, be seeded on the inducing culture.Peel off the embryo of embryo leaflet, cut plumular axis and radicle, obtain embryonic development point explant, be seeded on the inducing culture, cultivate 4d in advance.

3. two valency plant expression vectors are to the genetic transformation of peanut: inoculation Agrobacterium EHA105/pCAMBIA1301-bkt-crtR-B mono-clonal contains in the YEB liquid nutrient medium of Rif (50mg/L), Km (50mg/L) at 5ml, 28 ℃, 200r/min shake overnight incubation, get 1ml and move in the 25mlYEB liquid nutrient medium and continue to be cultured to OD ₆₀₀Be about 0.65, behind the centrifugal 15min of 6000r/min, with inducing liquid nutrient medium to be suspended into OD ₆₀₀=0.65.Pre-incubated acceptor material is put into this suspension 15min, take out and blot surface liquid, be connected on the inducing culture, 16h illumination/sky, 28 ℃ of cultivations altogether with aseptic filter paper.After cultivating 4d altogether, explant is with aseptic washing 4 times, containing cefotaxime mycin (Cef, concussion sterilization 6h in MSB liquid nutrient medium 650mg/L), blot surface-moisture, switching is kept on inducing culture and is cultivated 10d, changes screening and continuation regeneration induction bud on the inducing culture that contains Cef (500mg/L), Km (100mg/L) afterwards again over to, and every 20d succeeding transfer culture once.Behind the screening and culturing 40d, change the bud clump or the resistant buds of surviving on the bud elongation medium that contains Cef (300mg/L), Km (100mg/L) continuation cultivation, when treating that bud grows to 2～3cm left and right sides, downcut and place root media to take root.Relevant substratum sees Table 1.

4. detect: agrobacterium strains EHA105/pCAMBIA1301-bkt-crtR-B mediated transformation " flower educates 23 " peanut embryo vegetative point explant obtains the regeneration peanut plant with the screening of 100mg/L kantlex.With the positive contrast of recombinant plasmid pCAMBIA1301-bkt-crtR-B, the negative contrast of unconverted plant, respectively getting 1 μ L DNA is template, detects bkt gene and all positive plant of crtR-B gene.

Therefore bkt and two genes of crtR-B are structured on the identical carrier and are also expressed by two promoters driven respectively, by improving the expression amount of enzyme, improve the content of astaxanthin, and keep the biological activity of astaxanthin.

Table 1 is for the examination medium component

Sequence table .txt

SEQUENCE?LISTING

＜110〉Institute of Oceanology of the Chinese Academy of Sciences

＜120〉a kind of plant bivalent expression vector and construction process thereof

<130>

<160>13

<170>PatentIn?version?3.1

<210>1

<211>12727

<212>DNA

＜213〉Haematocoocus Pluvialls

<220>

<221>CDS

<222>(1)..(882)

<223>

<220>

<221>CDS

<222>(10125)..(11087)

<223>

<400>1

atg?ctg?tcg?aag?ctg?cag?tca?atc?agc?gtcaag?gcc?cgc?cgc?gtt?gaa 48

Met?Leu?Ser?Lys?Leu?Gln?Ser?Ile?Ser?Val?Lys?Ala?Arg?Arg?Val?Glu

1 5 10 15

cta?gcc?cgc?gac?atc?acg?cgg?ccc?aaa?gtc?tgc?ctg?cat?gct?cag?cgg 96

Leu?Ala?Arg?Asp?Ile?Thr?Arg?Pro?Lys?Val?Cys?Leu?His?Ala?Gln?Arg

20 25 30

tgc?tcg?tta?gtt?cgg?ctg?cga?gtg?gca?gca?cca?cag?aca?gag?gag?gcg 144

Cys?Ser?Leu?Val?Arg?Leu?Arg?Val?Ala?Ala?Pro?Gln?Thr?Glu?Glu?Ala

35 40 45

ctg?gga?acc?gtg?cag?gct?gcc?ggc?gcg?ggc?gat?gag?cac?agc?gcc?gat 192

Leu?Gly?Thr?Val?Gln?Ala?Ala?Gly?Ala?Gly?Asp?Glu?His?Ser?Ala?Asp

50 55 60

gta?gca?ctc?cag?cag?ctt?gac?cgg?gct?atc?gca?gag?cgt?cgt?gcc?cgg 240

Val?Ala?Leu?Gln?Gln?Leu?Asp?Arg?Ala?Ile?Ala?Glu?Arg?Arg?Ala?Arg

65 70 75 80

cgc?aaa?cgg?gag?cag?ctg?tca?tac?cag?get?gcc?gcc?att?gca?gca?tca 288

Arg?Lys?Arg?Glu?Gln?Leu?Ser?Tyr?Gln?Ala?Ala?Ala?Ile?Ala?Ala?Ser

85 90 95

att?ggc?gtg?tca?ggc?att?gcc?atc?ttc?gcc?acc?tac?ctg?aga?ttt?gcc 336

Ile?Gly?Val?Ser?Gly?Ile?Ala?Ile?Phe?Ala?Thr?Tyr?Leu?Arg?Phe?Ala

100 105 110

atg?cac?atg?acc?gtg?ggc?ggc?gca?gtg?cca?tgg?ggt?gaa?gtg?gct?ggc 384

Met?His?Met?Thr?Val?Gly?Gly?Ala?Val?Pro?Trp?Gly?Glu?Val?Ala?Gly

115 120 125

act?ctc?ctc?ttg?gtg?gtt?ggt?ggc?gcg?ctc?ggc?atg?gag?atg?tat?gcc 432

Thr?Leu?Leu?Leu?Val?Val?Gly?Gly?Ala?Leu?Gly?Met?Glu?Met?Tyr?Ala

130 135 140

cgc?tat?gca?cac?aaa?gcc?atc?tgg?cat?gag?tcg?cct?ctg?ggc?tgg?ctg 480

Arg?Tyr?Ala?His?Lys?Ala?Ile?Trp?His?Glu?Ser?Pro?Leu?Gly?Trp?Leu

145 150 155 160

ctg?cac?aag?agc?cac?cac?aca?cct?cgc?act?gga?ccc?ttt?gaa?gcc?aac 528

Leu?His?Lys?Ser?His?His?Thr?Pro?Arg?Thr?Gly?Pro?Phe?Glu?Ala?Asn

165 170 175

gac?ttg?ttt?gca?atc?atc?aat?gga?ctg?ccc?gcc?atg?ctc?ctg?tgt?acc 576

Asp?Leu?Phe?Ala?Ile?Ile?Asn?Gly?Leu?Pro?Ala?Met?Leu?Leu?Cys?Thr

180 185 190

ttt?ggc?ttc?tgg?ctg?ccc?aac?gtc?ctg?ggg?gcg?gcc?tgc?ttt?gga?gcg 624

Phe?Gly?Phe?Trp?Leu?Pro?Asn?Val?Leu?Gly?Ala?Ala?Cys?Phe?Gly?Ala

195 200 205

ggg?ctg?ggc?atc?acg?cta?tac?ggc?atg?gca?tat?atg?ttt?gta?cac?gat 672

Gly?Leu?Gly?Ile?Thr?Leu?Tyr?Gly?Met?Ala?Tyr?Met?Phe?Val?His?Asp

210 215 220

ggc?ctg?gtg?cac?agg?cgc?ttt?ccc?acc?ggg?ccc?atc?gct?ggc?ctg?ccc 720

Gly?Leu?Val?His?Arg?Arg?Phe?Pro?Thr?Gly?Pro?Ile?Ala?Gly?Leu?Pro

225 230 235 240

tac?atg?aag?cgc?ctg?aca?gtg?gcc?cac?cag?cta?cac?cac?agc?ggc?aag 768

Tyr?Met?Lys?Arg?Leu?Thr?Val?Ala?His?Gln?Leu?His?His?Ser?Gly?Lys

245 250 255

tac?ggt?ggc?gcg?ccc?tgg?ggt?atg?ttc?ttg?ggt?cca?cag?gag?ctg?cag 816

Tyr?Gly?Gly?Ala?Pro?Trp?Gly?Met?Phe?Leu?Gly?Pro?Gln?Glu?Leu?Gln

260 265 270

cac?att?cca?ggt?gcg?gcg?gag?gag?gtg?gag?cga?ctg?gtc?ctg?gaa?ctg 864

His?Ile?Pro?Gly?Ala?Ala?Glu?Glu?Val?Glu?Arg?Leu?Val?Leu?Glu?Leu

275 280 285

gac?tgg?tcc?aag?cgg?tag?gtgaccagct?cgaatttccc?cgatcgttca 912

Asp?Trp?Ser?Lys?Arg

290

aacatttggc?aataaagttt?cttaagattg?aatcctgttg?ccggtcttgc?gatgattatc 972

atataatttc?tgttgaatta?cgttaagcat?gtaataatta?acatgtaatg?catgacgtta 1032

tttatgagat?gggtttttat?gattagagtc?ccgcaattat?acatttaata?cgcgatagaa 1092

aacaaaatat?agcgcgcaaa?ctaggataaa?ttatcgcgcg?cggtgtcatc?tatgttacta 1152

gatcgggaat?taaactatca?gtgtttgaca?ggatatattg?gcgggtaaac?ctaagagaaa 1212

agagcgttta?ttagaataac?ggatatttaa?aagggcgtga?aaaggtttat?ccgttcgtcc 1272

atttgtatgt?gcatgccaac?cacagggttc?ccctcgggat?caaagtactt?tgatccaacc 1332

cctccgctgc?tatagtgcag?tcggcttctg?acgttcagtg?cagccgtctt?ctgaaaacga 1392

catgtcgcac?aagtcctaag?ttacgcgaca?ggctgccgcc?ctgccctttt?cctggcgttt 1452

tcttgtcgcg?tgttttagtc?gcataaagta?gaatacttgc?gactagaacc?ggagacatta 1512

cgccatgaac?aagagcgccg?ccgctggcct?gctgggctat?gcccgcgtca?gcaccgacga 1572

ccaggacttg?accaaccaac?gggccgaact?gcacgcggcc?ggctgcacca?agctgttttc 1632

cgagaagatc?accggcacca?ggcgcgaccg?cccggagctg?gccaggatgc?ttgaccacct 1692

acgccctggc?gacgttgtga?cagtgaccag?gctagaccgc?ctggcccgca?gcacccgcga 1752

cctactggac?attgccgagc?gcatccagga?ggccggcgcg?ggcctgcgta?gcctggcaga 1812

gccgtgggcc?gacaccacca?cgccggccgg?ccgcatggtg?ttgaccgtgt?tcgccggcat 1872

tgccgagttc?gagcgttccc?taatcatcga?ccgcacccgg?agcgggcgcg?aggccgccaa 1932

ggcccgaggc?gtgaagtttg?gcccccgccc?taccctcacc?ccggcacaga?tcgcgcacgc 1992

ccgcgagctg?atcgaccagg?aaggccgcac?cgtgaaagag?gcggctgcac?tgcttggcgt 2052

gcatcgctcg?accctgtacc?gcgcacttga?gcgcagcgag?gaagtgacgc?ccaccgaggc 2112

caggcggcgc?ggtgccttcc?gtgaggacgc?attgaccgag?gccgacgccc?tggcggccgc 2172

cgagaatgaa?cgccaagagg?aacaagcatg?aaaccgcacc?aggacggcca?ggacgaaccg 2232

tttttcatta?ccgaagagat?cgaggcggag?atgatcgcgg?ccgggtacgt?gttcgagccg 2292

cccgcgcacg?tctcaaccgt?gcggctgcat?gaaatcctgg?ccggtttgtc?tgatgccaag 2352

ctggcggcct?ggccggccag?cttggccgct?gaagaaaccg?agcgccgccg?tctaaaaagg 2412

tgatgtgtat?ttgagtaaaa?cagcttgcgt?catgcggtcg?ctgcgtatat?gatgcgatga 2472

gtaaataaac?aaatacgcaa?ggggaacgca?tgaaggttat?cgctgtactt?aaccagaaag 2532

gcgggtcagg?caagacgacc?atcgcaaccc?atctagcccg?cgccctgcaa?ctcgccgggg 2592

ccgatgttct?gttagtcgat?tccgatcccc?agggcagtgc?ccgcgattgg?gcggccgtgc 2652

gggaagatca?accgctaacc?gttgtcggca?tcgaccgccc?gacgattgac?cgcgacgtga 2712

aggccatcgg?ccggcgcgac?ttcgtagtga?tcgacggagc?gccccaggcg?gcggacttgg 2772

ctgtgtccgc?gatcaaggca?gccgacttcg?tgctgattcc?ggtgcagcca?agcccttacg 2832

acatatgggc?caccgccgac?ctggtggagc?tggttaagca?gcgcattgag?gtcacggatg 2892

gaaggctaca?agcggccttt?gtcgtgtcgc?gggcgatcaa?aggcacgcgc?atcggcggtg 2952

aggttgccga?ggcgctggcc?gggtacgagc?tgcccattct?tgagtcccgt?atcacgcagc 3012

gcgtgagcta?cccaggcact?gccgccgccg?gcacaaccgt?tcttgaatca?gaacccgagg 3072

gcgacgctgc?ccgcgaggtc?caggcgctgg?ccgctgaaat?taaatcaaaa?ctcatttgag 3132

ttaatgaggt?aaagagaaaa?tgagcaaaag?cacaaacacg?ctaagtgccg?gccgtccgag 3192

cgcacgcagc?agcaaggctg?caacgttggc?cagcctggca?gacacgccag?ccatgaagcg 3252

ggtcaacttt?cagttgccgg?cggaggatca?caccaagctg?aagatgtacg?cggtacgcca 3312

aggcaagacc?attaccgagc?tgctatctga?atacatcgcg?cagctaccag?agtaaatgag 3372

caaatgaata?aatgagtaga?tgaattttag?cggctaaagg?aggcggcatg?gaaaatcaag 3432

aacaaccagg?caccgacgcc?gtggaatgcc?ccatgtgtgg?aggaacgggc?ggttggccag 3492

gcgtaagcgg?ctgggttgtc?tgccggccct?gcaatggcac?tggaaccccc?aagcccgagg 3552

aatcggcgtg?acggtcgcaa?accatccggc?ccggtacaaa?tcggcgcggc?gctgggtgat 3612

gacctggtgg?agaagttgaa?ggccgcgcag?gccgcccagc?ggcaacgcat?cgaggcagaa 3672

gcacgccccg?gtgaatcgtg?gcaagcggcc?gctgatcgaa?tccgcaaaga?atcccggcaa 3732

ccgccggcag?ccggtgcgcc?gtcgattagg?aagccgccca?agggcgacga?gcaaccagat 3792

tttttcgttc?cgatgctcta?tgacgtgggc?acccgcgata?gtcgcagcat?catggacgtg 3852

gccgttttcc?gtctgtcgaa?gcgtgaccga?cgagctggcg?aggtgatccg?ctacgagctt 3912

ccagacgggc?acgtagaggt?ttccgcaggg?ccggccggca?tggccagtgt?gtgggattac 3972

gacctggtac?tgatggcggt?ttcccatcta?accgaatcca?tgaaccgata?ccgggaaggg 4032

aagggagaca?agcccggccg?cgtgttccgt?ccacacgttg?cggacgtact?caagttctgc 4092

cggcgagccg?atggcggaaa?gcagaaagac?gacctggtag?aaacctgcat?tcggttaaac 4152

accacgcacg?ttgccatgca?gcgtacgaag?aaggccaaga?acggccgcct?ggtgacggta 4212

tccgagggtg?aagccttgat?tagccgctac?aagatcgtaa?agagcgaaac?cgggcggccg 4272

gagtacatcg?agatcgagct?agctgattgg?atgtaccgcg?agatcacaga?aggcaagaac 4332

ccggacgtgc?tgacggttca?ccccgattac?tttttgatcg?atcccggcat?cggccgtttt 4392

ctctaccgcc?tggcacgccg?cgccgcaggc?aaggcagaag?ccagatggtt?gttcaagacg 4452

atctacgaac?gcagtggcag?cgccggagag?ttcaagaagt?tctgtttcac?cgtgcgcaag 4512

ctgatcgggt?caaatgacct?gccggagtac?gatttgaagg?aggaggcggg?gcaggctggc 4572

ccgatcctag?tcatgcgcta?ccgcaacctg?atcgagggcg?aagcatccgc?cggttcctaa 4632

tgtacggagc?agatgctagg?gcaaattgcc?ctagcagggg?aaaaaggtcg?aaaaggtctc 4692

tttcctgtgg?atagcacgta?cattgggaac?ccaaagccgt?acattgggaa?ccggaacccg 4752

tacattggga?acccaaagcc?gtacattggg?aaccggtcac?acatgtaagt?gactgatata 4812

aaagagaaaa?aaggcgattt?ttccgcctaa?aactctttaa?aacttattaa?aactcttaaa 4872

acccgcctgg?cctgtgcata?actgtctggc?cagcgcacag?ccgaagagct?gcaaaaagcg 4932

cctacccttc?ggtcgctgcg?ctccctacgc?cccgccgctt?cgcgtcggcc?tatcgcggcc 4992

gctggccgct?caaaaatggc?tggcctacgg?ccaggcaatc?taccagggcg?cggacaagcc 5052

gcgccgtcgc?cactcgaccg?ccggcgccca?catcaaggca?ccctgcctcg?cgcgtttcgg 5112

tgatgacggt?gaaaacctct?gacacatgca?gctcccggag?acggtcacag?cttgtctgta 5172

agcggatgcc?gggagcagac?aagcccgtca?gggcgcgtca?gcgggtgttg?gcgggtgtcg 5232

gggcgcagcc?atgacccagt?cacgtagcga?tagcggagtg?tatactggct?taactatgcg 5292

gcatcagagc?agattgtact?gagagtgcac?catatgcggt?gtgaaatacc?gcacagatgc 5352

gtaaggagaa?aataccgcat?caggcgctct?tccgcttcct?cgctcactga?ctcgctgcgc 5412

tcggtcgttc?ggctgcggcg?agcggtatca?gctcactcaa?aggcggtaat?acggttatcc 5472

acagaatcag?gggataacgc?aggaaagaac?atgtgagcaa?aaggccagca?aaaggccagg 5532

aaccgtaaaa?aggccgcgtt?gctggcgttt?ttccataggc?tccgcccccc?tgacgagcat 5592

cacaaaaatc?gacgctcaag?tcagaggtgg?cgaaacccga?caggactata?aagataccag 5652

gcgtttcccc?ctggaagctc?cctcgtgcgc?tctcctgttc?cgaccctgcc?gcttaccgga 5712

tacctgtccg?cctttctccc?ttcgggaagc?gtggcgcttt?ctcatagctc?acgctgtagg 5772

tatctcagtt?cggtgtaggt?cgttcgctcc?aagctgggct?gtgtgcacga?accccccgtt 5832

cagcccgacc?gctgcgcctt?atccggtaac?tatcgtcttg?agtccaaccc?ggtaagacac 5892

gacttatcgc?cactggcagc?agccactggt?aacaggatta?gcagagcgag?gtatgtaggc 5952

ggtgctacag?agttcttgaa?gtggtggcct?aactacggct?acactagaag?gacagtattt 6012

ggtatctgcg?ctctgctgaa?gccagttacc?ttcggaaaaa?gagttggtag?ctcttgatcc 6072

ggcaaacaaa?ccaccgctgg?tagcggtggt?ttttttgttt?gcaagcagca?gattacgcgc 6132

agaaaaaaag?gatctcaaga?agatcctttg?atcttttcta?cggggtctga?cgctcagtgg 6192

aacgaaaact?cacgttaagg?gattttggtc?atgcattcta?ggtactaaaa?caattcatcc 6252

agtaaaatat?aatattttat?tttctcccaa?tcaggcttga?tccccagtaa?gtcaaaaaat 6312

agctcgacat?actgttcttc?cccgatatcc?tccctgatcg?accggacgca?gaaggcaatg 6372

tcataccact?tgtccgccct?gccgcttctc?ccaagatcaa?taaagccact?tactttgcca 6432

tctttcacaa?agatgttgct?gtctcccagg?tcgccgtggg?aaaagacaag?ttcctcttcg 6492

ggcttttccg?tctttaaaaa?atcatacagc?tcgcgcggat?ctttaaatgg?agtgtcttct 6552

tcccagtttt?cgcaatccac?atcggccaga?tcgttattca?gtaagtaatc?caattcggct 6612

aagcggctgt?ctaagctatt?cgtataggga?caatccgata?tgtcgatgga?gtgaaagagc 6672

ctgatgcact?ccgcatacag?ctcgataatc?ttttcagggc?tttgttcatc?ttcatactct 6732

tccgagcaaa?ggacgccatc?ggcctcactc?atgagcagat?tgctccagcc?atcatgccgt 6792

tcaaagtgca?ggacctttgg?aacaggcagc?tttccttcca?gccatagcat?catgtccttt 6852

tcccgttcca?catcataggt?ggtcccttta?taccggctgt?ccgtcatttt?taaatatagg 6912

ttttcatttt?ctcccaccag?cttatatacc?ttagcaggag?acattccttc?cgtatctttt 6972

acgcagcggt?atttttcgat?cagttttttc?aattccggtg?atattctcat?tttagccatt 7032

tattatttcc?ttcctctttt?ctacagtatt?taaagatacc?ccaagaagct?aattataaca 7092

agacgaactc?caattcactg?ttccttgcat?tctaaaacct?taaataccag?aaaacagctt 7152

tttcaaagtt?gttttcaaag?ttggcgtata?acatagtatc?gacggagccg?attttgaaac 7212

cgcggtgatc?acaggcagca?acgctctgtc?atcgttacaa?tcaacatgct?accctccgcg 7272

agatcatccg?tgtttcaaac?ccggcagctt?agttgccgtt?cttccgaata?gcatcggtaa 7332

catgagcaaa?gtctgccgcc?ttacaacggc?tctcccgctg?acgccgtccc?ggactgatgg 7392

gctgcctgta?tcgagtggtg?attttgtgcc?gagctgccgg?tcggggagct?gttggctggc 7452

tggtggcagg?atatattgtg?gtgtaaacaa?attgacgctt?agacaactta?ataacacatt 7512

gcggacgttt?ttaatgtact?gaattaacgc?cgaattaatt?cgggggatct?ggattttagt 7572

actggatttt?ggttttagga?attagaaatt?ttattgatag?aagtatttta?caaatacaaa 7632

tacatactaa?gggtttctta?tatgctcaac?acatgagcga?aaccctatag?gaaccctaat 7692

tcccttatct?gggaactact?cacacattat?tatggagaaa?ctcgagcttg?tcgatcgaca 7752

gatccggtcg?gcatctactc?tatttctttg?ccctcggacg?agtgctgggg?cgtcggtttc 7812

cactatcggc?gagtacttct?acacagccat?cggtccagac?ggccgcgctt?ctgcgggcga 7872

tttgtgtacg?cccgacagtc?ccggctccgg?atcggacgat?tgcgtcgcat?cgaccctgcg 7932

cccaagctgc?atcatcgaaa?ttgccgtcaa?ccaagctctg?atagagttgg?tcaagaccaa 7992

tgcggagcat?atacgcccgg?agtcgtggcg?atcctgcaag?ctccggatgc?ctccgctcga 8052

agtagcgcgt?ctgctgctcc?atacaagcca?accacggcct?ccagaagaag?atgttggcga 8112

cctcgtattg?ggaatccccg?aacatcgcct?cgctccagtc?aatgaccgct?gttatgcggc 8172

cattgtccgt?caggacattg?ttggagccga?aatccgcgtg?cacgaggtgc?cggacttcgg 8232

ggcagtcctc?ggcccaaagc?atcagctcat?cgagagcctg?cgcgacggac?gcactgacgg 8292

tgtcgtccat?cacagtttgc?cagtgataca?catggggatc?agcaatcgcg?catatgaaat 8352

cacgccatgt?agtgtattga?ccgattcctt?gcggtccgaa?tgggccgaac?ccgctcgtct 8412

ggctaagatc?ggccgcagcg?atcgcatcca?tagcctccgc?gaccggttgt?agaacagcgg 8472

gcagttcggt?ttcaggcagg?tcttgcaacg?tgacaccctg?tgcacggcgg?gagatgcaat 8532

aggtcaggct?ctcgctaaac?tccccaatgt?caagcacttc?cggaatcggg?agcgcggccg 8592

atgcaaagtg?ccgataaaca?taacgatctt?tgtagaaacc?atcggcgcag?ctatttaccc 8652

gcaggacata?tccacgccct?cctacatcga?agctgaaagc?acgagattct?tcgccctccg 8712

agagctgcat?caggtcggag?acgctgtcga?acttttcgat?cagaaacttc?tcgacagacg 8772

tcgcggtgag?ttcaggcttt?ttcatatctc?attgcccccc?gggatctgcg?aaagctcgag 8832

agagatagat?ttgtagagag?agactggtga?tttcagcgtg?tcctctccaa?atgaaatgaa 8892

cttccttata?tagaggaagg?tcttgcgaag?gatagtggga?ttgtgcgtca?tcccttacgt 8952

cagtggagat?atcacatcaa?tccacttgct?ttgaagacgt?ggttggaacg?tcttcttttt 9012

ccacgatgct?cctcgtgggt?gggggtccat?ctttgggacc?actgtcggca?gaggcatctt 9072

gaacgatagc?ctttccttta?tcgcaatgat?ggcatttgta?ggtgccacct?tccttttcta 9132

ctgtcctttt?gatgaagtga?cagatagctg?ggcaatggaa?tccgaggagg?tttcccgata 9192

ttaccctttg?ttgaaaagtc?tcaatagccc?tttggtcttc?tgagactgta?tctttgatat 9252

tcttggagta?gacgagagtg?tcgtgctcca?ccatgttatc?acatcaatcc?acttgctttg 9512

aagacgtggt?tggaacgtct?tctttttcca?cgatgctcct?cgtgggtggg?ggtccatctt 9572

tgggaccact?gtcggcagag?gcatcttgaa?cgatagcctt?tcctttatcg?caatgatggc 9432

atttgtaggt?gccaccttcc?ttttctactg?tccttttgat?gaagtgacag?atagctgggc 9492

aatggaatcc?gaggaggttt?cccgatatta?ccctttgttg?aaaagtctca?atagcccttt 9552

ggtcttctga?gactgtatct?ttgatattct?tggagtagac?gagagtgtcg?tgctccacca 9612

tgttggcaag?ctgctctagc?caatacgcaa?accgcctctc?cccgcgcgtt?ggccgattca 9672

ttaatgcagc?tggcacgaca?ggtttcccga?ctggaaagcg?ggcagtgagc?gcaacgcaat 9732

taatgtgagt?tagctcactc?attaggcacc?ccaggcttta?cactttatgc?ttccggctcg 9792

tatgttgtgt?ggaattgtga?gcggataaca?atttcacaca?ggaaacagct?atgaccatga 9852

ttacgcccga?tctagtaaca?tagatgacac?cgcgcgcgat?aatttatcct?agtttgcgcg 9912

ctatattttg?ttttctatcg?cgtattaaat?gtataattgc?gggactctaa?tcataaaaac 9972

ccatctcata?aataacgtca?tgcattacat?gttaattatt?acatgcttaa?cgtaattcaa 10032

cagaaattat?atgataatca?tcgcaagacc?ggcaacagga?ttcaatctta?agaaacttta 10092

ttgccaaatg?tttgaacgat?cggggaaatt?cg?tca?tgc?caa?ggc?agg?cac?cag 10145

Ser?Cys?Gln?Gly?Arg?His?Gln

295 300

gcc?acg?ccc?gga?cag?gcg?gcg?gca?gtg?ggg?cag?ctg?cca?cca?ggg?ggc 10193

Ala?Thr?Pro?Gly?Gln?Ala?Ala?Ala?Val?Gly?Gln?Leu?Pro?Pro?Gly?Gly

305 310 315

aaa?ggg?cca?cct?gtg?gtg?ctc?cca?gtg?cag?gtc?aaa?gtg?gta?gca?tgt 10241

Lys?Gly?Pro?Pro?Val?Val?Leu?Pro?Val?Gln?Val?Lys?Val?Val?Ala?Cys

320 325 330

cag?gaa?act?cat?cac?atc?aga?tgc?ctc?act?tgt?ctt?ggc?cct?gaa?cca 10289

Gln?Glu?Thr?His?His?Ile?Arg?Cys?Leu?Thr?Cys?Leu?Gly?Pro?Glu?Pro

335 340 345

ggc?cat?cac?ctg?aga?gcc?tgc?tgc?agg?gcc?tgg?ctc?agg?ctt?gtg?tgg 10337

Gly?His?His?Leu?Arg?Ala?Cys?Cys?Arg?Ala?Trp?Leu?Arg?Leu?Val?Trp

350 355 360

cag?gta?agt?gcc?gaa?gta?gaa?gag?gcg?gaa?tgc?tga?caa?gat?tgg?ggc 10385

Gln?Val?Ser?Ala?Glu?Val?Glu?Glu?Ala?Glu?Cys Gln?Asp?Trp?Gly

365 370 375

tgc?agc?cat?gaa?gac?tag?gag?att?tgc?cat?ggg?cgc?ccc?cag?cat?ttg 10433

Cys?Ser?His?Glu?Asp Glu?Ile?Cys?His?Gly?Arg?Pro?Gln?His?Leu

380 385 390

cat?cac?cac?tgc?cca?cca?tgc?cag?ccg?ggc?aaa?ctg?cca?cag?gga?cat 10481

His?His?His?Cys?Pro?Pro?Cys?Gln?Pro?Gly?Lys?Leu?Pro?Gln?Gly?His

395 400 405 410

gta?gct?gga?cat?gaa?gct?ggc?gaa?cca?ggg?gac?aag?gcc?ggg?att?tcc 10529

ValAla?Gly?His?Glu?Ala?Gly?Glu?Pro?Gly?Asp?Lys?Ala?Gly?Ile?Ser

415 420 425

ctt?gtg?gaa?gtc?agg?gtc?ttt?ccc?cac?ttc?gcc?agt?atg?gtt?gtg?gtg 10577

Leu?Val?Glu?Val?Arg?Val?Phe?Pro?His?Phe?Ala?Ser?Met?Val?Val?Val

430 435 440

ctc?cca?gtg?ctt?gcg?atg?cag?cat?gct?gta?gtc?aaa?cca?ggc?gta?cag 10625

Leu?Pro?Val?Leu?Ala?Met?Gln?His?Ala?Val?ValLys?Pro?Gly?Val?Gln

445 450 455

tga?tat?gca?gat?gtt?gcc?aag?gag?atc?att?gag?ctg?cct?gtg?cct?caa 10673

Tyr?Ala?Asp?Val?Ala?Lys?Glu?Ile?Ile?Glu?Leu?Pro?Val?Pro?Gln

460 465 470

agc?tat?ggt?gcc?atg?cat?tgc?gtc?atg?tgt?ggt?gat?gaa?tag?acc?agt 10721

Ser?Tyr?Gly?Ala?Met?His?Cys?Val?Met?Cys?Gly?Asp?Glu Thr?Ser

475 480 485

gta?cag?gaa?ctc?aag?tac?aat?gaa?gac?tgc?agc?gat?gtg?cag?tag?gct 10769

Val?Gln?Glu?Leu?Lys?Tyr?Asn?Glu?Asp?Cys?Ser?Asp?Val?Gln Ala

490 495 500

gct?gct?tcc?gcc?caa?aag?ctg?ggc?tgt?ggc?ttc?gga?cac?agg?caa?cca 10817

Ala?Ala?Ser?Ala?Gln?Lys?Leu?Gly?Cys?Gly?Phe?Gly?His?Arg?Gln?Pro

505 510 515

gtg?aag?ctg?gtc?cat?gga?tgt?cgg?tag?cct?gat?ttg?aaa?tat?tgc?gtg 10865

Val?Lys?Leu?Val?His?Gly?Cys?Arg Pro?Asp?Leu?Lys?Tyr?Cys?Val

520 525 530

taa?aaa?cac?tgc?ggt?cca?ggt?gcc?aat?gat?ggt?cag?cgc?cat?cgt?gat 10913

Lys?His?Cys?Gly?Pro?Gly?Ala?Asn?Asp?Gly?Gln?Arg?His?Arg?Asp

535 540 545

gcc?ctt?ggc?gtc?aga?tgc?tgg?agg?ttt?gta?ggc?gtg?ctt?tag?cgc?agg 10961

Ala?Leu?Gly?Val?Arg?Cys?Trp?Arg?Phe?Val?Gly?Val?Leu Arg?Arg

550 555 560

acg?agc?tgc?gtc?tga?cga?ctc?gga?tgg?cat?gtg?ata?ctg?tgt?cgc?cca 11009

Thr?Ser?Cys?Val Arg?Leu?Gly?Trp?His?Val?Ile?Leu?Cys?Arg?Pro

565 570 575

cgc?tct?caa?gac?gtc?tgg?gct?gga?agc?agc?tgc?ctc?act?gcc?ttt?ctg 11057

Arg?Ser?Gln?Asp?Val?Trp?Ala?Gly?Ser?Ser?Cys?Leu?Thr?Ala?Phe?Leu

580 585 590 595

ctc?gac?cat?tag?tgc?cga?tgc?gac?gtg?cat?ctctagagtc?ccccgtgttc 11107

Leu?Asp?His Cys?Arg?Cys?Asp?Val?His

600

tctccaaatg?aaatgaactt?ccttatatag?aggaagggtc?ttgcgaagga?tagtgggatt 11167

gtgcgtcatc?ccttacgtca?gtggagatat?cacatcaatc?cacttgcttt?gaagacgtgg 11227

ttggaacgtc?ttctttttcc?acgatgctcc?tcgtgggtgg?gggtccatct?ttgggaccac 11287

tgtcggcaga?ggcatcttca?acgatggcct?ttcctttatc?gcaatgatgg?catttgtagg 11347

agccaccttc?cttttccact?atcttcacaa?taaagtgaca?gatagctggg?caatggaatc 11407

cgaggaggtt?tccggatatt?accctttgtt?gaaaagtctc?aattgccctt?tggtcttctg 11467

agactgtatc?tttgatattt?ttggagtaga?caagtgtgtc?gtgctccacc?atgttgacga 11527

agattttctt?cttgtcattg?agtcgtaaga?gactctgtat?gaactgttcg?ccagtcttta 11587

cggcgagttc?tgttaggtcc?tctatttgaa?tctttgactc?catggccttt?gattcagtgg 11647

gaactacctt?tttagagact?ccaatctcta?ttacttgcct?tggtttgtga?agcaagcctt 11707

gaatcgtcca?tactggaata?gtacttctga?tcttgagaaa?tatatctttc?tctgtgttct 11767

tgatgcagtt?agtcctgaat?cttttgactg?catctttaac?cttcttggga?aggtatttga 11827

tttcctggag?attattgctc?gggtagatcg?tcttgatgag?acctgctgcg?taagcctctc 11887

taaccatctg?tgggttagca?ttctttctga?aattgaaaag?gctaatctgg?ggacctgcag 11947

gcatgcaagc?tagcttggca?ctggccgtcg?ttttacaacg?tcgtgactgg?gaaaaccctg 12007

gcgttaccca?acttaatcgc?cttgcagcac?atcccccttt?cgccagctgg?cgtaatagcg 12067

aagaggcccg?caccgatcgc?ccttcccaac?agttgcgcag?cctgaatggc?gaatgctaga 12127

gcagcttgag?cttggatcag?attgtcgttt?cccgccttca?gtttagcttc?atggagtcaa 12187

agattcaaat?agaggaccta?acagaactcg?ccgtaaagac?tggcgaacag?ttcatacaga 12247

gtctcttacg?actcaatgac?aagaagaaaa?tcttcgtcaa?catggtggag?cacgacacac 12307

ttgtctactc?caaaaatatc?aaagatacag?tctcagaaga?ccaaagggca?attgagactt?12367

ttcaacaaag?ggtaatatcc?ggaaacctcc?tcggattcca?ttgcccagct?atctgtcact?12427

ttattgtgaa?gatagtggaa?aaggaaggtg?gctcctacaa?atgccatcat?tgcgataaag?12487

gaaaggccat?cgttgaagat?gcctctgccg?acagtggtcc?caaagatgga?cccccaccca?12547

cgaggagcat?cgtggaaaaa?gaagacgttc?caaccacgtc?ttcaaagcaa?gtggattgat?12607

gtgatatctc?cactgacgta?agggatgacg?cacaatccca?ctatccttcg?caagaccctt?12667

cctctatata?aggaagttca?tttcatttgg?agagaacacg?ggggactctt?gaccatggta?12727

<210>2

<211>293

<212>PRT

＜213〉Haematocoocus Pluvialls

<400>2

Met?Leu?Ser?Lys?Leu?Gln?Ser?Ile?Ser?Val?Lys?Ala?Arg?Arg?Val?Glu

1 5 10 15

Leu?Ala?Arg?Asp?Ile?Thr?Arg?Pro?Lys?Val?Cys?Leu?His?Ala?Gln?Arg

20 25 30

Cys?Ser?Leu?Val?Arg?Leu?Arg?Val?Ala?Ala?Pro?Gln?Thr?Glu?Glu?Ala

35 40 45

Leu?Gly?Thr?Val?Gln?Ala?Ala?Gly?Ala?Gly?Asp?Glu?His?Ser?Ala?Asp

50 55 60

Val?Ala?Leu?Gln?Gln?Leu?Asp?Arg?Ala?Ile?Ala?Glu?Arg?Arg?Ala?Arg

65 70 75 80

Arg?Lys?Arg?Glu?Gln?Leu?Ser?Tyr?Gln?Ala?Ala?Ala?Ile?Ala?Ala?Ser

85 90 95

Ile?Gly?Val?Ser?Gly?Ile?Ala?Ile?Phe?Ala?Thr?Tyr?Leu?Arg?Phe?Ala

100 105 110

Met?His?Met?Thr?Val?Gly?Gly?Ala?Val?Pro?Trp?Gly?Glu?Val?Ala?Gly

115 120 125

Thr?Leu?Leu?Leu?Val?Val?Gly?Gly?Ala?Leu?Gly?Met?Glu?Met?Tyr?Ala

130 135 140

Arg?Tyr?Ala?His?Lys?Ala?Ile?Trp?His?Glu?Ser?Pro?Leu?Gly?Trp?Leu

145 150 155 160

Leu?His?Lys?Ser?His?His?Thr?Pro?Arg?Thr?Gly?Pro?Phe?Glu?Ala?Asn

165 170 175

Asp?Leu?Phe?Ala?Ile?Ile?Asn?Gly?Leu?Pro?Ala?Met?Leu?Leu?Cys?Thr

180 185 190

Phe?Gly?Phe?Trp?Leu?Pro?Asn?Val?Leu?Gly?Ala?Ala?Cys?Phe?Gly?Ala

195 200 205

Gly?Leu?Gly?Ile?Thr?Leu?Tyr?Gly?Met?Ala?Tyr?Met?Phe?Val?His?Asp

210 215 220

Gly?Leu?Val?His?Arg?Arg?Phe?Pro?Thr?Gly?Pro?Ile?Ala?Gly?Leu?Pro

225 230 235 240

Tyr?Met?Lys?Arg?Leu?Thr?Val?Ala?His?Gln?Leu?His?His?Ser?Gly?Lys

245 250 255

Tyr?Gly?Gly?Ala?Pro?Trp?Gly?Met?Phe?Leu?Gly?Pro?Gln?Glu?Leu?Gln

260 265 270

His?Ile?Pro?Gly?Ala?Ala?Glu?Glu?Val?Glu?Arg?Leu?Val?Leu?Glu?Leu

275 280 285

Asp?Trp?Ser?Lys?Arg

290

<210>3

<211>82

<212>PRT

＜213〉Haematocoocus Pluvialls

<400>3

Ser?Cys?Gln?Gly?Arg?His?Gln?Ala?Thr?Pro?Gly?Gln?Ala?Ala?Ala?Val

1 5 10 15

Gly?Gln?Leu?Pro?Pro?Gly?Gly?Lys?Gly?Pro?Pro?Val?Val?Leu?Pro?Val

20 25 30

Gln?Val?Lys?Val?Val?Ala?Cys?Gln?Glu?Thr?His?His?Ile?Arg?Cys?Leu

35 40 45

Thr?Cys?Leu?Gly?Pro?Glu?Pro?Gly?His?His?Leu?Arg?Ala?Cys?Cys?Arg

50 55 60

Ala?Trp?Leu?Arg?Leu?Val?Trp?Gln?Val?Ser?Ala?Glu?Val?Glu?Glu?Ala

65 70 75 80

Glu?Cys

<210>4

<211>9

<212>PRT

＜213〉Haematocoocus Pluvialls

<400>4

Gln?Asp?Trp?Gly?Cys?Ser?His?Glu?Asp

1 5

<210>5

<211>74

<212>PRT

＜213〉Haematocoocus Pluvialls

<400>5

Glu?Ile?Cys?His?Gly?Arg?Pro?Gln?His?Leu?His?His?His?Cys?Pro?Pro

1 5 10 15

Cys?Gln?Pro?Gly?Lys?Leu?Pro?Gln?Gly?His?Val?Ala?Gly?His?Glu?Ala

20 25 30

Gly?Glu?Pro?Gly?Asp?Lys?Ala?Gly?Ile?Ser?Leu?Val?Glu?Val?Arg?Val

35 40 45

Phe?Pro?His?Phe?Ala?Ser?Met?Val?Val?Val?Leu?Pro?Val?Leu?Ala?Met

50 55 60

Gln?His?Ala?Val?Val?Lys?Pro?Gly?Val?Gln

65 70

<210>6

<211>28

<212>PRT

＜213〉Haematocoocus Pluvialls

<400>6

Tyr?Ala?Asp?Val?Ala?Lys?Glu?Ile?Ile?Glu?Leu?Pro?Val?Pro?Gln?Ser

1 5 10 15

Tyr?Gly?Ala?Met?His?Cys?Val?Met?Cys?Gly?Asp?Glu

20 25

<210>7

<211>16

<212>PRT

＜213〉Haematocoocus Pluvialls

<400>7

Thr?Ser?Val?Gln?Glu?Leu?Lys?Tyr?Asn?Glu?Asp?Cys?Ser?Asp?Val?Gln

1 5 10 15

<210>8

<211>25

<212>PRT

＜213〉Haematocoocus Pluvialls

<400>8

Ala?Ala?Ala?Ser?Ala?Gln?Lys?Leu?Gly?Cys?Gly?Phe?Gly?His?Arg?Gln

1 5 10 15

Pro?Val?Lys?Leu?Val?His?Gly?Cys?Arg

20 25

<210>9

<211>7

<212>PRT

＜213〉Haematocoocus Pluvialls

<400>9

Pro?Asp?Leu?Lys?Tyr?Cys?Val

1 5

<210>10

<211>28

<212>PRT

＜213〉Haematocoocus Pluvialls

<400>10

Lys?His?Cys?Gly?Pro?Gly?Ala?Asn?Asp?Gly?Gln?Arg?His?Arg?Asp?Ala

1 5 10 15

Leu?Gly?Val?Arg?Cys?Trp?Arg?Phe?Val?Gly?Val?Leu

20 25

<210>11

<211>6

<212>PRT

＜213〉Haematocoocus Pluvialls

<400>11

Arg?Arg?Thr?Ser?Cys?Val

1 5

<210>12

<211>30

<212>PRT

＜213〉Haematocoocus Pluvialls

<400>12

Arg?Leu?Gly?Trp?His?Val?Ile?Leu?Cys?Arg?Pro?Arg?Ser?Gln?Asp?Val

1 5 10 15

Trp?Ala?Gly?Ser?Ser?Cys?Leu?Thr?Ala?Phe?Leu?Leu?Asp?His

20 25 30

<210>13

<211>6

<212>PRT

＜213〉Haematocoocus Pluvialls

<400>13

Cys?Arg?Cys?Asp?Val?His

1 5

Claims (5)

1. plant bivalent expression vector, it is characterized in that: shown in the base sequence among the plant bivalent expression vector sequence table SEQ ID No.1, it is inserted on the pCAMBIA1301 expression vector jointly for β-Hu Luobusu ketonize enzyme gene (bkt) and β-Hu Luobusu '-hydroxylase gene (crtR-B), the plant bivalent expression vector that obtains.

2. by the described plant bivalent expression vector of claim 1, it is characterized in that: described plant bivalent expression vector total length is 12727bp, and bkt and crtR-B expression of gene are by the CaMV35S promotor startup of upstream separately.

3. the construction process by the described plant bivalent expression vector of claim 1 is characterized in that: at first, clone β-Hu Luobusu ketonize enzyme gene (bkt) and β-Hu Luobusu '-hydroxylase gene (crtR-B) from Haematocoocus Pluvialls; Secondly, the bkt that obtains and crtR-B are connected to obtain pET-28a (+)-bkt and pET-28a (+)-crtR-B carrier on two pET-28a (+) carrier; Once more, be template with pET-28a (+)-bkt, P ₁And P ₂Be the primer PCR amplification, product cuts back to close fragment and pBI221 carrier T through BamHI and SacI enzyme ₄Dna ligase connects, and obtains the pBI221-bkt intermediate carrier, uses HindIII, this intermediate carrier of EcoRI double digestion and pCAMBIA1301 carrier afterwards again, and uses T ₄The dna ligase connection obtains the pCAMBIA1301-bkt intermediate carrier; At last, be template with pET-28a (+)-crtR-B, P ₃And P ₄Be the primer PCR amplification, product and pCAMBIA1301-bkt intermediate carrier are cut through BglII and BstEII enzyme, and the fragment of recovery is by T ₄On the pCAMBIA1301-bkt carrier after dna ligase is connected to crtR-B gene forward enzyme and cuts, i.e. plant bivalent expression vector shown in the base sequence among the calling sequence table SEQ ID No.1.

4. by the construction process of the described plant bivalent expression vector of claim 3, it is characterized in that: described P ₁, P ₂, P ₃, P ₄Primer is respectively: P ₁: 5 ' AAA GGATCCATGCACGTCGCATC 3 ', P ₂: 5 ' AAC GAGCTCTCATGCCAAGGCAG 3 ', P ₃: 5 ' CCC AGATCTATGCTGTCGAAGCTGC 3 ', P ₄: 5 ' AAA GGTCACCCTACCGCTTGGACCA 3 '.

5. press the construction process of the described plant bivalent expression vector of claim 3, it is characterized in that: described bkt upstream region of gene has 35S promoter, the downstream has the NOS termination signal, is built into the 35S-bkt-NOS fragment, and this fragment oppositely is inserted on the pCAMBIA1301 carrier; The crtR-B upstream region of gene has 35S promoter, and the downstream has the NOS termination signal, constitutes the 35S-crtR-B-NOS fragment, and this fragment forward is inserted on the pCAMBIA1301 carrier.

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