CN101798577B - Jatropha curcas phytoene synthetase gene sequence and application thereof in plants - Google Patents
Jatropha curcas phytoene synthetase gene sequence and application thereof in plants Download PDFInfo
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Abstract
The invention relates to the technical fields of molecular biology and genetic engineering and relates to a JcPSY protein for expressing the Jatropha curcas phytoene synthetase and a nucleotide sequence thereof. Due to the use of the Jatropha curcas PSY protein, substances interacting with the Jatropha curcas PSY protein, or receptors, inhibitors or antagonists, and the like can be screened by various conventional screening methods. The Jatropha curcas PSY protein genes can be used for improving the beta-carotene content in the plants and improving the economic values of the plants through the genetic engineering technology, and have obvious market application prospects.
Description
Technical field
The present invention relates to molecular biology, gene engineering technology field.Particularly, the present invention relates to a kind of in Cortex jatrophae, express JcPSY albumen (jatropha curcas phytoene synthetase, Jatropha curcas phytoenesynthase, JcPSY) and nucleotide sequence.
Background technology
Known, (biosynthesizing of β-Carotene) mainly betides in higher plant, algae, fungi and the bacterial body β-Hu Luobusu, can not the de novo synthesis β-Hu Luobusu in the animal body.β-Hu Luobusu is the precursor of vitamin A (Vitamin A), in human body, can be converted into vitamin A as required.It has nutrition, painted dual function, is the outstanding nourishing food additive of category-A (food additives) that the Food and Argriculture OrganizationFAO (FAO) and the foodstuff additive joint specialist council of The World Health Organization (WHO) are assert.In recent years, increasing medical research shows that β-Hu Luobusu can resist multiple cancer, particularly can reduce the lung cancer morbidity rate.β-Hu Luobusu is at the cancellation radical, enhancing human immune, and protection human health aspects such as preventing cardiovascular disease play an important role.Lyeopene is the main pigment of structure in the carrotenoid, experiment showed, that the absorption of Lyeopene helps reducing the incidence probability of prostate cancer, protect cardiovascularly, reduces ultraviolet to multiple uses such as human skin injuries.Phytoene synthetase is the biosynthetic specific enzymes of catalysis carrotenoid, and it is with synthetic first the carrotenoid-phytoene of bimolecular geranyl geranyl tetra-sodium (GGPP) catalysis.Research shows that phytoene synthase gene is the rate-limiting enzyme in the carrotenoid biosynthetic pathway, and this is proved in tamato fruit.Fray etc. import the phytoene synthetase in the tomato (PSY) gene antisense in the tomato, in the transfer-gen plant that the result obtains carotenoid content significantly reduce (Fray RG, etc.Constitutive expression of a fruit phytoene synthasegene in transgenic tomatoes causes dwarfism by redirecting metabolitesfrom the gibberellin pathway.Plant J，1995，8(5)：693-701)。Overexpression phytoene synthetase in tomato, rape, Arabidopis thaliana and yam; The level of accumulation carrotenoid significantly improves in the respective organization; β-Hu Luobusu has improved 1.9 times in tamato fruit; Average raising is 43 times in the Arabidopis thaliana seed, improves 50 times in the Semen Brassicae campestris, improves 8 times in the potato tuber.The phytoene synthetase PSY of catalysis carrotenoid pathways metabolism the first step reaction is the crucial regulatory enzyme of this pathways metabolism, and generates " bottleneck " that this step of phytoene has become the carrotenoid pathways metabolism by GGPP.Therefore; The PSY gene has become the first-selected goal gene (Zhu Changfu in the genetic engineering of plant carrotenoid at present; Deng. the application in genetically engineered of biosynthesizing of plant carrotenoid and genes involved thereof. Mol.Biol., 2004,30 (6): 609-618).Before the present invention comes forth, any cDNA sequence that discloses or reported the coding jatropha curcas phytoene synthetase of mentioning in the present patent application is not arranged as yet.The clone of relevant jatropha curcas phytoene gene and functional study receive state natural sciences fund (30771745) subsidy among the present invention.
Summary of the invention
First purpose of the present invention provides a kind of new Cortex jatrophae PSY gene, and this gene is a Cortex jatrophae PSY protein gene.
Second purpose of the present invention provides a kind of new Cortex jatrophae PSY albumen.
The 3rd purpose of the present invention provides a kind of recombinant technology that utilizes and prepares the above-mentioned new Cortex jatrophae PSY albumen and the method for nucleotide sequence.
In one aspect of the invention; A kind of isolated dna molecular is provided; This molecule comprises: coding has the nucleotide sequence of polypeptide of Cortex jatrophae PSY protein active, among described nucleotide sequence and the SEQ ID NO.1 from Nucleotide 78-1364 position the nucleotides sequence of dna molecular show at least 70% homology; Perhaps described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.1 in from the nucleotide sequence hybridization of Nucleotide 78-1364 position.Preferably, described sequence encoding has the polypeptide of the aminoacid sequence shown in the SEQ ID NO.2.More preferably, described sequence has among the SEQ ID NO.1 nucleotide sequence from Nucleotide 78-1364 position.
In another aspect of this invention, a kind of isolated Cortex jatrophae PSY protein and peptide is provided, it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQ ID NO.2 aminoacid sequence, or its reactive derivative.Preferably, this polypeptide is to have SEQ ID NO.2 polypeptide of sequence.
In another aspect of this invention, a kind of carrier is provided also, it comprises above-mentioned dna molecular.
In another aspect of this invention, also provide a kind of usefulness above-mentioned carrier transformed host cells.This host cell is yeast cell, tobacco and other vegetable cell in instance.
In another aspect of this invention, also provide a kind of generation to have the method for the polypeptide of Cortex jatrophae PSY protein active, its step is following:
(1) nucleotide sequence that coding is had a purifying of Cortex jatrophae PSY protein active polypeptide operationally is connected in expression regulation sequence; Form Cortex jatrophae PSY protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 78-1364 position among described nucleotide sequence and the SEQ ID NO.1;
(2) change the expression vector in the step (1) over to prokaryotic host cell, form the proteic reconstitution cell of Cortex jatrophae PSY;
(3) be fit to express under the condition of Cortex jatrophae PSY protein polypeptide the reconstitution cell in the culturing step (2);
(4) isolate pure basically polypeptide with Cortex jatrophae PSY protein-active.
Preferably, the nucleotide sequence that uses in the method has the sequence of 78-1364 position among the SEQ ID NO.1.
In another aspect of this invention, also provide a kind of transgenic technology of utilizing that the nucleotide sequence that coding has Cortex jatrophae PSY protein active polypeptide is transformed into plant to improve the method for β-Hu Luobusu in the plant, its step is following:
(1) nucleotide sequence of purifying that coding is had a polypeptide of Cortex jatrophae PSY protein-active operationally is connected in the expression of plants regulating and controlling sequence; Formation contains the plant expression vector of Cortex jatrophae PSY protein gene, shows at least 70% homology from the nucleotides sequence of Nucleotide 78-1364 position among described nucleotide sequence and the SEQ ID NO.1;
(2) change the expression vector in the step (1) over to Agrobacterium; The Agrobacterium that will contain expression vector is cultivated with eukaryotic host cell altogether; Under 22-28 ℃ of condition, secretly cultivate 1-2 days after, through the screening like antibiotic-screening; Acquisition contains transformant and the final regeneration of transgenic plant and the offspring thereof of Cortex jatrophae PSY protein gene, comprises plant seed and plant tissue.Contain that β-Hu Luobusu significantly improves in the transfer-gen plant of Cortex jatrophae PSY protein gene.
Preferably, the nucleotide sequence that uses in the method has the sequence of 78-1364 position among the SEQ ID No.1.The present invention also provides and PSY protein polypeptide specificity bonded antibody, and it comprises polyclonal antibody and monoclonal antibody.In the present invention; " isolating ", " purifying " or " sill is pure " 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 the active polypeptide of jatropha curcas phytoene synthetase to term " jatropha curcas phytoene synthetase (or polypeptide) encoding sequence ", like 78-1364 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.1.This degenerate sequence is meant, is arranged in the encoder block 78-1364 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 78-1364 position nucleotide sequence homology be low to moderate about 70% the degenerate sequence described sequence of SEQ ID NO.2 of also encoding out.This term also comprises can be under the moderate stringent condition, better under the height stringent condition with SEQ IDNO.1 in from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 78-1364 position.This term also comprise with SEQ ID NO.1 in from the homology of nucleotide sequence at least 70% of Nucleotide 78-1364 position, preferably at least 80%, more preferably at least 90%, at least 95% nucleotide sequence best.
This term also comprises encoding to have the variant form of open reading frame sequence among the proteic SEQ IDNO.1 with natural Cortex jatrophae PSY albumen identical function.These variant forms comprise (but being not limited to): several (are generally 1-90; 1-60 preferably, 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, " pure basically " protein or polypeptide are meant that it accounts at least 20% of the total material of sample at least, preferably at least 50%, more preferably at least 80%, and at least 90% (by dry weight or weight in wet base) best.Purity can be measured with any suitable method, as measure the purity of polypeptide with column chromatography, PAGE or HPLC method.Basically pure polypeptide is substantially free of the component of following it under the native state.
In the present invention, term " Cortex jatrophae PSY albumen or polypeptide " refers to have the SEQ ID NO.1 polypeptide of sequence of Cortex jatrophae PSY protein-active.This term also comprises the variant form that has with the SEQ ID NO.2 sequence of natural Cortex jatrophae PSY albumen identical function.These variant forms comprise (but being not limited to): several (are generally 1-50; Preferably 1-30; 1-20 more preferably, 1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20; Preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises proteic active fragments of Cortex jatrophae PSY and reactive derivative.
The variant form of Cortex jatrophae PSY protein polypeptide of the present invention comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low stringent condition can with the coded albumen of the DNA of Cortex jatrophae PSY protein D NA hybridization and the polypeptide or the albumen that utilize the serum of Cortex jatrophae PSY protein polypeptide to obtain.
In the present invention, " Cortex jatrophae PSY albumen conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID NO.1, has 10 at the most, and preferably at the most 8, more preferably 5 amino acid similar performances or close amino acid are replaced and formed polypeptide at the most.These conservative property variation polypeptide are preferably replaced according to table 1 and are produced.
Table 1
| Initial residue | Representational replacement | The preferred replacement |
| Ala(A) | Val；Leu；Ile | Val |
| Arg(R) | Lys；Gln；Asn | Lys |
| Asn(N) | Gln；His；Lys；Arg | Gln |
| Asp(D) | Glu | Glu |
| Cys(C) | Ser | Ser |
| Gln(Q) | Asn | Asn |
| Glu(E) | Asp | Asp |
| Gly(G) | Pro；Ala | Ala |
| His(H) | Asn；Gln；Lys；Arg | Arg |
| Ile(I) | Leu；Val；Met；Ala；Phe | Leu |
| Leu(L) | Ile；Val；Met；Ala；Phe | Ile |
| Lys(K) | Arg；Gln；Asn | Arg |
| Met(M) | Leu；Phe；Ile | Leu |
| Phe(F) | Leu；Val；Ile；Ala；Tyr | Leu |
| Pro(P) | Ala | Ala |
| Ser(S) | Thr | Thr |
| Thr(T) | Ser | Ser |
| Trp(W) | Tyr；Phe | Tyr |
| Tyr(Y) | Trp；Phe；Thr；Ser | Phe |
| Val(V) | Ile；Leu；Met；Phe；Ala | Leu |
The present invention also comprises the analogue of Cortex jatrophae PSY albumen or polypeptide.The difference of these analogues and natural PS Y protein 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 through various technology, as through 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-(like D-amino acid), and has non-natural analogue that exist or synthetic amino acid (like β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to above-mentioned representational polypeptide of giving an example.
Described modification (not changing primary structure usually) form comprises: the chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modification also comprises glycosylation, in the synthetic and processing of polypeptide or further, carries out glycosylation modified and polypeptide that produce in the procedure of processing like those.This modification can be carried out glycosylated enzyme (like mammiferous glycosylase or deglycosylating enzyme) and accomplishes through polypeptide is exposed to.Modified forms also comprises have the phosphorylated amino acid residue sequence of (like Tyrosine O-phosphate, Serine O-phosphate, phosphothreonine).Thereby also comprise and modified the polypeptide that has improved its proteolyze performance or optimized solubility property.
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 Cortex jatrophae PSY protein polypeptide of the present invention, can Cortex jatrophae PSY albumen coded sequence operationally be connected in expression regulation sequence, thereby form Cortex jatrophae PSY protein expression vector.
As used herein, " operationally being connected in " refer to a kind of like this situation, and promptly some part of linear DNA sequence can influence the activity of same other parts of linear DNA sequence.For example, if signal peptide DNA as precursor expression and participate in the secretion of polypeptide, signal peptide (secretion leader sequence) DNA operationally is connected in polypeptid DNA so; If transcribing of promotor control sequence, it is operationally to be connected in encoding sequence so; When if ribosome bind site is placed in the position that can make its translation, it is operationally to be connected in encoding sequence so.Generally, " operationally being connected in " means adjacent, then means in reading frame adjacent for the secretion leader sequence.
In the present invention, term " host cell " is an eukaryotic cell.Eukaryotic host cell commonly used comprises yeast cell, tobacco cell and other vegetable cell.
Whether and quantity the expression of also available Northern blotting technical Analysis Cortex jatrophae PSY protein gene product promptly analyzes the existence of the proteic rna transcription thing of Cortex jatrophae PSY in cell.
The Western engram analysis of the Northern engram analysis of Cortex jatrophae PSY RNA and Cortex jatrophae PSY specific antibody can be united use, to confirm the expression of Cortex jatrophae PSY in biological specimen.
In addition, can be used as the nucleic acid molecule of probe among the present invention, this molecule has 8-100 continuous nucleotide of Cortex jatrophae PSY albumen nucleotide coding sequence usually, preferably has 15-50 continuous nucleotide.This probe can be used for whether existing in the test sample the proteic nucleic acid molecule of coding Cortex jatrophae PSY.
The present invention relates to whether exist in the test sample method of Cortex jatrophae PSY pyrenoids nucleotide sequence, it comprises with above-mentioned probe and sample and hybridizing whether detection probes combination has taken place then.Preferably, this sample is the product behind the pcr amplification, and wherein the pcr amplification primer is corresponding to Cortex jatrophae PSY albumen nucleotide coding sequence, and can be positioned at the both sides or the centre of this encoding sequence.Primer length is generally 15-50 Nucleotide.
In addition, according to Cortex jatrophae PSY pyrenoids nucleotide sequence of the present invention and aminoacid sequence, can be on the homology basis of nucleic acid homology or marking protein, screening Cortex jatrophae PSY albumen homology gene or homologous protein.
In order to obtain the dot matrix of the Cortex jatrophae cDNAs relevant with Cortex jatrophae PSY protein gene, can screen Cortex jatrophae cDNA library with dna probe, these probes are under low stringent condition, use
32P to Cortex jatrophae PSY proteic all or part of do the radioactivity mark and.The cDNA library that most is suitable for screening is the library from Cortex jatrophae.Structure is that biology field is well-known from the method in the cDNA library of interested cell or tissue.In addition, many such cDNA libraries also can buy, for example available from Clontech, and Stratagene, Palo Alto, Cal..This screening method can be discerned the nucleotide sequence with the proteic gene family of Cortex jatrophae PSY.
Cortex jatrophae PSY pyrenoids thuja acid full length sequence of the present invention or its fragment can use the method for pcr amplification method, recombination method or synthetic to obtain usually.For the pcr amplification method; Can be disclosed according to the present invention about nucleotide sequence; Especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually need carries 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 come to obtain in large quantity relevant sequence with recombination method.This normally is cloned into carrier with it, changes cell again over to, from the host cell after the propagation, separates obtaining relevant sequence then through ordinary method.
In addition, also the method for available artificial chemosynthesis is synthesized relevant sequence.Before the application, prior art fully can be through first synthetic a plurality of polynucleotide small segments, and then connect and obtain the proteic nucleotide sequence of code book invention Cortex jatrophae PSY.Then, can be with in various existing dna moleculars (like carrier) and the cell in this nucleotide sequence introducing this area.In addition, also can will suddenly change and introduce in the protein sequence of the present invention through chemosynthesis.
Except producing with recombination method, the also available solid phase technique of the proteic fragment of the present invention is produced (people such as Stewart, (1969) Solid-Phase Peptide Synthesis, WHFreeman Co., San Francisco through 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 use 431A type peptide synthesizer (Foster City, CA) the next automatically synthetic peptide of AppliedBiosystems.Can distinguish proteic each fragment of chemosynthesis the present invention, connect to produce the molecule of total length with chemical process then.
Utilize Cortex jatrophae PSY albumen of the present invention,, can filter out with Cortex jatrophae PSY albumen interactional material takes place, perhaps acceptor, suppressor factor or short of money dose etc. through various conventional screening methods.Cortex jatrophae PSY protein gene of the present invention can be used for improving the β-Hu Luobusu in the plant through genetic engineering technique, and in order to improve the economic worth of plant, the present invention has great application prospect.
Table 2 is that the homology of the nucleotide sequence of Cortex jatrophae PSY of the present invention and sweet orange (Citrus sinensis) PSY compares (GAP) table.
Table 3 is that the homology of the aminoacid sequence of proteic aminoacid sequence of Cortex jatrophae PSY of the present invention and papaya (Carica papaya) PSY compares (FASTA) table.Wherein, identical amino acid marks with the amino acid monocase between two sequences, and similar amino acid marks with "+".
Table 2
Query: the proteic nucleotide sequence of Cortex jatrophae JcPSY
Sbjct: the proteic nucleotide sequence of sweet orange CsPSY (GenBank Accession No.DQ235260)
Table 3
Query: the proteic aminoacid sequence of Cortex jatrophae JcPSY
Sbjct: the proteic aminoacid sequence of papaya CpPSY (GenBank Accession No.ABG72805)
Description of drawings
Fig. 1 is the proteic hydrophobicity plot of Cortex jatrophae PSY.Wherein, the hydrophobicity plot amino acid whose hydrophobic value that is based on Kyte and Doolittle report is set up.
Fig. 2 is the proteic transmembrane amino acid analysis of Cortex jatrophae PSY.
Fig. 3 is the domain analyses figure of the proteic aminoacid sequence of Cortex jatrophae PSY.
Embodiment
Below in conjunction with laboratory concrete testing data and combination specific embodiment, further set forth the present invention.These embodiment only be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions in the following example; Usually according to normal condition; For example the Sambrook equimolecular is cloned: laboratory manual (New York:Cold Spring Harbor Laboratory Press; 1989) condition described in, or the condition of advising according to manufacturer.
The clone of Cortex jatrophae PSY protein gene
1. separate tissue (isolation)
Jatropha curcas seed derives from Sichuan Province's Panzhihua Region, after jatropha curcas seed is gathered, is placed on the laboratory and preserves.
2.RNA separation (RNA isolation)
The kind skin of jatropha curcas seed is peeled off, taken out kind of a benevolence, grind, behind the adding liquid nitrogen, after pulverizing, get 100mg and move in the 1.5mL EP pipe, extracted total RNA (two step cracking processs) with mortar.Identify total RNA quality with the denaturing formaldehyde gel electrophoresis, on spectrophotometer, measure rna content then.
3. the full-length clone of gene (Cloning of Full-length cDNA)
According to the amino acid conserved sequence of some plant PSY, the design degenerate primer utilizes homologous genes clone principle, SMART
TMRACE cDNA amplification method (Clonetech test kit) carries out the cDNA full-length clone, divides four-stage to carry out:
(1) clone of core sequence
PCR (JcPSYF+JcPSYR) obtains PSY-1 (422bp), reclaims, and is connected on the pMD-18T carrier; With M13F or M13R 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 is with GCG software package (Wisconsin group; USA) BLAST in and the existing DB of FASTA software search (Genebank+EMBL); The homology of PSY gene of knowing its nucleotide sequence and proteins encoded and known model plant papaya etc. is very high, so think that tentatively it is a PSY gene.
(2)3’-RACE
Result according to the amplification of core sequence designs two forward special primers (JcPSYF1 and JcPSYF2).Adopt the secondary PCR amplification method to carry out.For the first time PCR (JcPSYF1+AP) obtains the PCR product, dilute 100 times after, do template with it, carry out PCR second time (JcPSYF2+AP), obtain JcPSY-3 (850bp), reclaim connection, order-checking process same (1)).
(3)5’-RACE
Result according to the amplification of core sequence designs two reverse special primers (JcPSYR1 and JcPSYR2).Adopt the secondary PCR amplification method to carry out.For the first time PCR (JcPSYR1+UPM) obtains the PCR product, dilute 100 times after, do template with it, carry out PCR second time (JcPSYR2+NUP), obtain JcPSY-5 (678bp), reclaim, connect check order (process is (1) together).
(4) clone of encoding sequence
With the comparison of 5 ' RACE sequencing result and 3 ' RACE sequencing result and splice; Obtain the full length fragment sequence information; And design a pair of special primer and carry out JcPSY coding region (JcPSYfull-F+JcPSYfull-R) pcr amplification, obtain JcPSY coding region (1287bp) (process is with (1)).
Use above-mentioned 4 kinds of methods through combination, obtained candidate's the proteic complete encoding sequence of Cortex jatrophae PSY.Obtain on the total length basis of (comprising complete ORF at least) in splicing; Further design primer JcPSYfull-F:5-ATGACCGTCGCA TTACTATGGGTTGCA-3 (SEQ ID NO.3) is a forward primer; Oligonucleotide JcPSYfull-R:5-TTATGCCTTCCTCAA AGGAGTTGAGATTC-3 (SEQ ID NO.4) is a reverse primer; With total RNA is template, carries out RT-PCR amplification, the PCR condition be 94 ℃ 5 minutes; Carried out 35 circulations in 2 minutes with 94 ℃ 1 minute, 58 ℃ 1 minute and 72 ℃ thereupon, extended 10 minutes with 72 ℃ at last.The electrophoresis detection pcr amplification product, the acquisition expanding fragment length is 1290bp.Clone, check order with pcr amplification product by ordinary method then, obtain the sequence shown in SEQ ID NO.3 and the SEQ ID NO.4.
The sequence information and the homology analysis of Cortex jatrophae PSY protein gene
The length of the Cortex jatrophae PSY albumen full-length cDNA that the present invention is new is 1755bp, and detailed sequence is seen SEQ IDNO.1, and wherein ORF is positioned at 78-1364 position Nucleotide.Derive the proteic aminoacid sequence of Cortex jatrophae PSY according to full-length cDNA, totally 429 amino-acid residues, molecular weight 48068.52, pI are 8.96.Detailed sequence is seen SEQ ID NO.2.
Proteic full length cDNA sequence of Cortex jatrophae PSY and coded protein thereof are carried out Nucleotide and protein homology retrieval with blast program in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDStranslations+PDB+SwissProt+Superdate+PIR DB; The result find Cortex jatrophae PSY gene nucleotide sequence and other plant nucleotide sequence such as sweet orange (C.sinensis) (DQ235260); Oranges and tangerines (C.unshiu) (AF220218); Shaddock (C.maxima) (EU375852); Natsudaidai (C.paradisi) (AF152892); Calamondin (Citrofortunella mitis) (EU375851), papaya (C.papaya) etc. has certain homology.But the aminoacid sequence of Cortex jatrophae PSY and the aminoacid sequence of other plant have more extensively and higher similarity; As similarity widely being arranged with the aminoacid sequence of Cortex jatrophae PSY and the aminoacid sequence of other plant; As with papaya (C.papaya) (ABG72805), Radix Dauci Sativae (D.carota) (ABB52068), coffee (Coffea canephora) is (ABA43898); Tomato (S.lycopersicum) (ABU40771); Oranges and tangerines (C.unshiu) (AF220218), plum blossom (Prunus mume) consistence (BAF49052) is respectively 81%, 76%, 77%, 78%, 78% and 77%; Similarity is respectively 90%, 88%, 88%, 87%, 86% and 88%.
The proteic structure characteristic analysis of embodiment 3 Cortex jatrophae PSY
1. the proteic domain analyses of Cortex jatrophae PSY
(network address is: index structure territory http://www.ncbi.nlm.nih.gov/Blast.cgi) obtains the result and sees Fig. 3 at ncbi database with the proteic aminoacid sequence of Cortex jatrophae PSY.
In aminoacid sequence, exist the isopentenyl pyrophosphate synthetic enzyme (Trans-Isoprenyl DiphosphateSynthases, catalytic site Trans_IPPS) (be in the sequence table SEQ ID.1 from aminoterminal (N end) 137-404 amino acids residue.
2. Cortex jatrophae PSY protein signal peptide analysis
Plant carotenoid synthase class is encoded by cell nucleus gene; The coding translation forms the precursor (precursors) that there is transit peptide sequence in the N end; Transit peptides has the function that plant carotenoid synthase class is transported to plastid; After transport process or transhipment, then be sheared, plant carotenoid synthase class gets into plastid with maturation protein (mature proteins) form.The aminoacid sequence of JcPSY is analyzed with ChloroP software; Finding has the translocation peptide (being cTP) that is made up of 28 amino acid in the total length JcPSY aminoacid sequence; Its shearing site is between the 28th and 29 amino acid, and this also has report in plants such as Arabidopis thaliana, tomato and Sunflower Receptacle.
3. the proteic hydrophobicity analysis of Cortex jatrophae PSY
Ripe PSY to after the transit peptides shearing analyzes, and finds that it contains a plurality of water repellent regions, and especially 224-245 amino acids hydrophobicity is very strong, has significantly to stride film trend (Fig. 1).
4. the proteic transmembrane amino acid analysis of Cortex jatrophae PSY
Find that through the TMHMM prediction 223-245 amino acids of ripe PSY is a transmembrane helix structure; Show the ripe PSY of Cortex jatrophae be a N end inwardly, the C end outside stride embrane-associated protein, the 223-245 amino acids is the site (Fig. 2) of itself and film secure bond.
Embodiment 4
The activation analysis of jatropha curcas phytoene synthetase cDNA
In this embodiment, Cortex jatrophae JcPSY encoding sequence or the fragment with total length is built into commercial albumen
Among the matter fusion expression vector, to identify the activity of JcPSY.
Cortex jatrophae JcPSY construction of prokaryotic expression vector, and transformed into escherichia coli
According to the aminoacid sequence of Cortex jatrophae JcPSY, the primer of design protein-coding region, and on positive anti-primer, introduce restriction endonuclease sites (this decides according to the pBluescript II KS-vector carrier of selecting for use) respectively, so that construction of expression vector.Amplified production to obtain among the embodiment 1 is a template, behind pcr amplification, Cortex jatrophae JcPSY gene is being guaranteed to be cloned into pBluescript II KS-vector (Stratahene) under the correct prerequisite of reading frame.Identify that good expression vector utilizes CaCl
2Method changes intestinal bacteria DH10B over to, and (there is pACCAR25 Δ crtB plasmid in this cingula; Have the ctr gene cluster that derives from the synthetic carrotenoid of uredo erwinia phage (Erwiniauredovora) on the plasmid; But the ctrB that lacks in this kind is the PSY gene), Screening and Identification obtains containing the engineering bacteria DH10B-JcPSY of JcPSY expression vector.
The result observes
On the colibacillary flat board of growth; Do not change the color that the intestinal bacteria DH10B of JcPSY gene grows over to and be white on flat board; Changed color that the intestinal bacteria DH10B of JcPSY gene grows over to for yellow on flat board; Explanation has the generation of carrotenoid on the conversion flat board that has changed the JcPSY gene over to, further specify JcPSY and can replace the ctrB gene in the uredo erwinia phage (E.uredovora), promotes the generation of carrotenoid.Use derives from the PSY gene of mouse and Arabidopis thaliana and does contrast, and the color of bacterium that changes Cortex jatrophae JcPSY gene over to is darker than the color of the bacterium of the psy gene that changes mouse and Arabidopis thaliana over to, explains that Cortex jatrophae JcPSY gene has higher activity.
Embodiment 5
The functional analysis of jatropha curcas phytoene synthetase cDNA
In this step, the cDNA sequence construct of coding jatropha curcas phytoene synthetase is gone among the plant expression vector, and transformation of tobacco, to identify its function.
1. the structure of plant expression vector
According to jatropha curcas phytoene synthetase full length cDNA sequence (table 1), design amplifies the primer of entire reading frame, on positive anti-primer, introduces restriction endonuclease sites respectively.Amplified production to obtain in embodiment 1 step 3 is a template, behind pcr amplification, guarantees that the reading frame of jatropha curcas phytoene synthetase is entirely true.Use endonuclease digestion, carrier p2300 reclaims the purpose fragment respectively with also using identical endonuclease digestion.The purpose fragment of the jatropha curcas phytoene synthetase cDNA that will cut through enzyme is connected with the p2300 carrier of cutting through corresponding restriction endonuclease, and transformed into escherichia coli DH5 α cultivates 20h for 37 ℃, and the PCR that carries out recon identifies and enzyme is cut evaluation.
2. plant expression vector transforms Agrobacterium
(1) gets a competence Agrobacterium EHA105, add the about 1pg of plant expression vector that contains jatropha curcas phytoene synthetase cDNA, gently mixing;
(2) quick-frozen 2 minutes in liquid nitrogen, 37 ℃ of incubations 5 minutes;
(3) add 500 μ L YEB liquid nutrient mediums, 28 ℃ jog 2-4 hour, eliminate competence;
(4) get 50-200 μ L bacterium liquid respectively, separate application is selected on the flat board in containing suitable antibiotic YEB, is inverted for 28 ℃ and cultivates two days.
(5) identify the single bacterium colony of the Agrobacterium EHA105 that is positive; Be inoculated into and contain the 50mg/L Rifampin; In the 20ml liquid YEB substratum of that mycin of 100mg/L, to logarithmic phase, get an amount of Agrobacterium doubly with liquid MS medium dilution 20-30 in 28 ℃ of constant temperature shaking table shaking culture 30 hours.
3. genetic transformation of tobacco and regeneration
(1) get aseptic tobacco leaf, excision blade edge and Zhong Mai cultivated 2 days for 28 ℃ in Ms+1.0mg/L NAA solid medium in advance;
(2) retrieve material, put into the Agrobacterium that has goal gene that aseptic MS liquid nutrient medium diluted, soaked 15 minutes, low speed shook 15 minutes in 28 ℃ of shaking tables are arranged then;
(3) take out vanelets, inhale with aseptic filter paper and remove unnecessary bacterium liquid, 28 ℃ of dark cultivations two days in the MS solid medium;
(4) vanelets, with the aseptic washing twice that adds 100mg/L Cb, after aseptic filter paper is inhaled and removed unnecessary bacterium liquid; Change over to and contain among 100mg/L Km and the 100mg/L Cb division culture medium M1; Be cultured under 28 ℃ of light and differentiate callus, until growing bud, per therebetween 15 days subcultures once;
The bud that (5) will grow to 3-5cm changes root media 1/2MS over to and goes up root induction.
(6) after being accredited as positive transgene tobacco, continue to cultivate into seedling.
4. the extraction of transgene tobacco blade carrotenoid
(1) get each transgene tobacco blade, lyophilized powder is processed in lyophilize rapidly, puts in the vacuum drying oven and keeps in Dark Place, and measure to the greatest extent and as early as possible.
(2) accurately take by weighing lyophilized powder 1.000g in tool plug triangular flask, add extracting solution (acetone: 45mL ethanol=3: 2), shake up, cover bottle stopper, with ultrasonic extraction 20 minutes, filter in the 50mL volumetric flask also constant volume.Get 20mL filtrating and put in the separating funnel, add 10mL sherwood oil mixing, add 10mL zero(ppm) water and distribute; Getting sherwood oil puts in the rotary evaporation bottle mutually; Surplus water adds the 10mL sherwood oil reallocates, and merges 2 sherwood oils and puts on the Rotary Evaporators 40-45 ℃ of evaporate to dryness mutually; With the dissolving of 2.0mL Virahol, filter the back and go up machine mensuration.
5. high effective liquid chromatography for measuring transgene tobacco blade carrotenoid
(1) preparation of B-Serlabo standard specimen: B-Serlabo standard substance (Sigma Company products) 1.0mg, with a small amount of trichloromethane dissolving, use petroleum ether dissolution again, solution changes in the 25.0ml volumetric flask, uses the sherwood oil constant volume, and concentration is 0.04mg/ml, and refrigerator is preserved.Face the time spent, draw 1.0ml in the 10.0ml volumetric flask, add moving phase to scale, this moment, concentration was 0.004mg/ml.
(2) preparation of Lyeopene standard specimen: Pure Lycopene (Sigma Company products) 1.0mg, with a small amount of trichloromethane dissolving, use petroleum ether dissolution again, solution changes in the 25.0ml volumetric flask, uses the sherwood oil constant volume, and concentration is 0.04mg/ml, and refrigerator is preserved.Face the time spent, draw 1.0ml in the 10.0ml volumetric flask, add moving phase to scale, this moment, concentration was 0.004mg/ml.
(3) measuring method: moving phase: second eyeball: trichloromethane (92: 8).Detect wavelength: dual wavelength is measured, 470nm (O-min) and 450nm (8-13min).Flow velocity: 1.0mL/min.Column temperature: 35 ℃.Observing samples has the size variation of absorption peak at 470nm and 450nm place.
Sequence that the present invention relates to and mark apportion are following:
< 110>Fudan University
< 120>jatropha curcas phytoene synthetase gene sequence and the application in plant
<160>2
<170>PatentIn version 3.1
<210>1
<211>1755
<212>mRNA
< 213>Cortex jatrophae (Jatropha carcas L.)
<220>
<221>CDS
<222>(78)..(1364)
<223>
<400>1
GGAAATTTGC ATAGACAAGG TAAACAAAGA AAGAAAGAAA AAAATAAAAA AATAAAAATA
AAAATCTGGT TTTGAACATG ACC GTC GCA TTA CTA TGG GTT GCA ACA CCA AGT
Met Thr Val Ala Leu Leu Trp Val Ala Thr Pro Ser
1 5 10
ACA GAG GCT TCC AAT TCA TTT GGG TTT CTC CAT TCG GTT CGG GTC TTA GAT
Thr Glu Ala Ser Asn Ser Phe Gly Phe Leu His Ser Val Arg Val Leu Asp
15 20 25
TCG TCG AAA GTT GGT TCT CTA GAT CGG AAT TTG ACG TTT AAG GGA AGA GCA
Ser Ser Lys Val Gly Ser Leu Asp Arg Asn Leu Thr Phe Lys Gly Arg Ala
30 35 40 45
AAA AAG GGT AGG AGT CAA AAA TGG AAG TCA AGC TCA GTT AAT ATA GAT TTG
Lys Lys Gly Arg Ser Gln Lys Trp Lys Ser Ser Ser Val Asn Ile Asp Leu
50 55 60
AGG AAT CCT TGT ATA GGT AGT GGA AGC GAA TTC CCT GTA TTA GCA AGT ATG
Arg Asn Pro Cys Ile Gly Ser Gly Ser Glu Phe Pro Val Leu Ala Ser Met
65 70 75 80
ATT GCC AGT CCA GCT GGA GAA ATG GCT ATC TCG TCA GAG GAG AAG GTA TAC
Ile Ala Ser Pro Ala Gly Glu Met Ala Ile Ser Ser Glu Glu Lys Val Tyr
85 90 95
AAT GTG GTG ATG AAG CAG GCA GCT TTG GTT AAA AAG CAA TTA AGG TCT AAT
Asn Val Val Met Lys Gln Ala Ala Leu Val Lys Lys Gln Leu Arg Ser Asn
100 105 110
CAA GAT CTT GAT GTG AAA CCA GAT ATT GTT CTT CCA GGA AAT CTG AGT TTG
Gln Asp Leu Asp Val Lys Pro Asp Ile Val Leu Pro Gly Asn Leu Ser Leu
115 120 125 130
TTG AGT GAA GCT TAC GAT AGA TGT GGA GAA GTT TGT GCT GAG TAT GCA AAA
Leu Ser Glu Ala Tyr Asp Arg Cys Gly Glu Val Cys Ala Glu Tyr Ala Lys
135 140 145
ACA TTT TAC TTG GGA ACT TTG CTG ATG ACT TCT GAA AGA AGA AGA GCT ATC
Thr Phe Tyr Leu Gly Thr Leu Leu Met Thr Ser Glu Arg Arg Arg Ala Ile
150 155 160
TGG GCA ATA TAT GTG TGG TGT CGC AGG ACT GAT GAG CTT GTT GAT GGG CCT
Trp Ala Ile Tyr Val Trp Cys Arg Arg Thr Asp Glu Leu Val Asp Gly Pro
165 170 175 180
AAT GCT TCA CAC ATA ACG CCA ACA GCT TTA GAT AGG TGG GAG GCA AGG TTG
Asn Ala Ser His Ile Thr Pro Thr Ala Leu Asp Arg Trp Glu Ala Arg Leu
185 190 195
GAA GAC CTT TTC CGA GGT CGT CCA TTT GAT ATG TTT GAT GCT GCT TTA TCA
Glu Asp Leu Phe Arg Gly Arg Pro Phe Asp Met Phe Asp Ala Ala Leu Ser
200 205 210 215
GAC ACG GTT ACT AAA TTT CCT GTT GAC ATT CAG CCA TTC AAA GAT GTG ATT
Asp Thr Val Thr Lys Phe Pro Val Asp Ile Gln Pro Phe Lys Asp Val Ile
220 225 230
GAA GGA ATG AGG CTG GAC CTG AAG AAA ACA AGA TAT AAG AAC TTT GAT GAG
Glu Gly Met Arg Leu Asp Leu Lys Lys Thr Arg Tyr Lys Asn Phe Asp Glu
235 240 245 250
CTT TAT CTT TAC TGT TAT TAT GTT GCT GGG ACG GTT GGA TTG ATG AGT GTT
Leu Tyr Leu Tyr Cys Tyr Tyr Val Ala Gly Thr Val Gly Leu Met Ser Val
255 260 265
CCG GTC ATG GGC ATT GCA CCT GAA TCA CAG GCA CCA ACT GAG AGC GTC TAT
Pro Val Met Gly Ile Ala Pro Glu Ser Gln Ala Pro Thr Glu Ser Val Tyr
270 275 280
AAT GCT GCC TTG GCA TTA GGA ATA GCA AAT CAG CTT ACC AAC ATA CTC AGG
Asn Ala Ala Leu Ala Leu Gly Ile Ala Asn Gln Leu Thr Asn Ile Leu Arg
285 290 295 300
GAT GTG GGA GAG GAT GCA AGA AGA GGA AGG ATT TAT TTA CCA CAA GAT GAG
Asp Val Gly Glu Asp Ala Arg Arg Gly Arg Ile Tyr Leu Pro Gln Asp Glu
305 310 315
CTC GTG CAG GCT GGA CTT TCA GAT GAT GAC ATA TTT GCT GGG AAA GTG ACA
Leu Val Gln Ala Gly Leu Ser Asp Asp Asp Ile Phe Ala Gly Lys Val Thr
320 325 330 335
GAT AAA TGG AGA AAT TTT ATG AAG AGT CAA ATT AAG AGG GCA AGG ATG TTC
Asp Lys Trp Arg Asn Phe Met Lys Ser Gln Ile Lys Arg Ala Arg Met Phe
340 345 350
TTC AAT GAG GCA GAG AAA GGA GTG ACC GAG TTG AGT GCC GCA AGT AGA TGG
Phe Asn Glu Ala Glu Lys Gly Val Thr Glu Leu Ser Ala Ala Ser Arg Trp
355 360 365
CCG GTA TGG GCA TCT TTA GAA TTA TAT CAG AAA ATT CTA GAT GAG ATA GAA
Pro Val Trp Ala Ser Leu Glu Leu Tyr Gln Lys Ile Leu Asp Glu Ile Glu
370 375 380 385
GCC AAT GAT TAC AAT AAC TTC ACA AAG AGA GCA TAT GTA AGC AAA GCC AAG
Ala Asn Asp Tyr Asn Asn Phe Thr Lys Arg Ala Tyr Val Ser Lys Ala Lys
390 395 400
AAG TTA GCT TCT TTA CCA ATT GCA TAT GCA AGA TCA TTT GTT GGG CCA TCA
Lys Leu Ala Ser Leu Pro Ile Ala Tyr Ala Arg Ser Phe Val Gly Pro Ser
405 410 415 420
AGA ATC TCA ACT CCT TTG AGG AAG GCA TAAATGTCAA ATCATGAACA TCATATATAG
Arg Ile Ser Thr Pro Leu Arg Lys Ala
425
AAGAAAAGGG AAAAATACCA TAATTAATTT GTATATTAGC AACTTTTGTA ATGCAGCAAG
TGGAAGCAAG AACAGTCAAT TCAAATTTTA TTTGAAGGGA TGAACTGTAT ATATTCTGCT
AGGAACTTAA AGCTTCTTGG TTTAGAGAGT GTTTAGTGTT TTAAGAGAAG ACTCATCTAA
GAGTCTGCTT GTGCTTTTCT ACCCTCACGT AGACATGGCT GGTTAACCAA ACCGGTGGTT
CATGGCCTTT AAAAGTAGGA ACTGAGAACG GTCTTAATCA GGCTGGTTTA ATTCAATTTC
GATTCAATCG GGCTGGTTTA ATTCAATTTC GATTCTGTAA AATGAAAAAA AAAAAAAAAAA
<210>2
<211>429
<212>PRT
< 213>Cortex jatrophae (Jatropha carcas L.)
<400>2
Met Thr Val Ala Leu Leu Trp Val Ala Thr Pro Ser Thr Glu Ala Ser
1 5 10 15
Asn Ser Phe Gly Phe Leu His Ser Val Arg Val Leu Asp Ser Ser Lys
20 25 30
Val Gly Ser Leu Asp Arg Asn Leu Thr Phe Lys Gly Arg Ala Lys Lys
35 40 45
Gly Arg Ser Gln Lys Trp Lys Ser Ser Ser Val Asn Ile Asp Leu Arg
50 55 60
Asn Pro Cys Ile Gly Ser Gly Ser Glu Phe Pro Val Leu Ala Ser Met
65 70 75 80
Ile Ala Ser Pro Ala Gly Glu Met Ala Ile Ser Ser Glu Glu Lys Val
85 90 95
Tyr Asn Val Val Met Lys Gln Ala Ala Leu Val Lys Lys Gln Leu Arg
100 105 110
Ser Asn Gln Asp Leu Asp Val Lys Pro Asp Ile Val Leu Pro Gly Asn
115 120 125
Leu Ser Leu Leu Ser Glu Ala Tyr Asp Arg Cys Gly Glu Val Cys Ala
130 135 140
Glu Tyr Ala Lys Thr Phe Tyr Leu Gly Thr Leu Leu Met Thr Ser Glu
145 150 155 160
Arg Arg Arg Ala Ile Trp Ala Ile Tyr Val Trp Cys Arg Arg Thr Asp
165 170 175
Glu Leu Val Asp Gly Pro Asn Ala Ser His Ile Thr Pro Thr Ala Leu
180 185 190
Asp Arg Trp Glu Ala Arg Leu Glu Asp Leu Phe Arg Gly Arg Pro Phe
195 200 205
Asp Met Phe Asp Ala Ala Leu Ser Asp Thr Val Thr Lys Phe Pro Val
210 215 220
Asp Ile Gln Pro Phe Lys Asp Val Ile Glu Gly Met Arg Leu Asp Leu
225 230 235 240
Lys Lys Thr Arg Tyr Lys Asn Phe Asp Glu Leu Tyr Leu Tyr Cys Tyr
245 250 255
Tyr Val Ala Gly Thr Val Gly Leu Met Ser Val Pro Val Met Gly Ile
260 265 270
Ala Pro Glu Ser Gln Ala Pro Thr Glu Ser Val Tyr Asn Ala Ala Leu
275 280 285
Ala Leu Gly Ile Ala Asn Gln Leu Thr Asn Ile Leu Arg Asp Val Gly
290 295 300
Glu Asp Ala Arg Arg Gly Arg Ile Tyr Leu Pro Gln Asp Glu Leu Val
305 310 315 320
Gln Ala Gly Leu Ser Asp Asp Asp Ile Phe Ala Gly Lys Val Thr Asp
325 330 335
Lys Trp Arg Asn Phe Met Lys Ser Gln Ile Lys Arg Ala Arg Met Phe
340 345 350
Phe Asn Glu Ala Glu Lys Gly Val Thr Glu Leu Ser Ala Ala Ser Arg
355 360 365
Trp Pro Val Trp Ala Ser Leu Glu Leu Tyr Gln Lys Ile Leu Asp Glu
370 375 380
Ile Glu Ala Asn Asp Tyr Asn Asn Phe Thr Lys Arg Ala Tyr Val Ser
385 390 395 400
Lys Ala Lys Lys Leu Ala Ser Leu Pro Ile Ala Tyr Ala Arg Ser Phe
405 410 415
Val Gly Pro Ser Arg Ile Ser Thr Pro Leu Arg Lys Ala
420 425
<210>3
<211>27
<212>DNA
< 213>Cortex jatrophae (Jartropha curcas L.)
<400>3
ATGACCGTCGCATTACTATGGGTTGCA
<210>4
<211>29
<212>DNA
< 213>Cortex jatrophae (Jatropha curcas L.)
<400>4
TTATGCCTTCCTCAAAGGAGTTGAGATTC
Claims (3)
1. the proteic nucleic acid of coding jatropha curcas phytoene synthetase is characterized in that the nucleotide sequence of this nucleic acid is the nucleotide sequence shown in the SEQ ID NO.1.
2. an isolating jatropha curcas phytoene synthetase albumen is characterized in that, said proteic aminoacid sequence is the aminoacid sequence shown in the SEQ ID NO.2.
3. the proteic nucleic acid of coding jatropha curcas phytoene synthetase as claimed in claim 1 is characterized in that, said sequence is from the nucleotide sequence of Nucleotide 78-1364 position among the SEQ ID NO.1.
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| Title |
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| 何秀霞等.八氢番茄红素合成酶基因植物表达载体的构建及对人参的遗传转化.《长春理工大学学报（自然科学版）》.2007,第30卷(第4期), * |
| 侯嵘等.麻疯树八氢番茄红素合成酶基因JcPSY的克隆及分析.《中国遗传学会第八次代表大会暨学术讨论会论文摘要汇编(2004-2008)》.2008, * |
| 郑阳霞等.宁夏枸杞八氢番茄红素合成酶基因的克隆与序列分析.《林业科学》.2006,第42卷(第5期), * |
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