CN101240281B - Protein encoding sequence of cucumber side branch inhibition gene cls - Google Patents

Abstract

The invention relates to a coding sequence of green cucumber collateral inhibition gene CLS in genetic engineering technology field. The coding sequence comprises coding polypeptides nucleotide sequence with cucumber collateral inhibition albumen activity, and the nucleotide sequence and 491-1666 bite nucleotide sequence in SEQ ID NO.3 have at least 701mology. The opening reading frame of coding sequence is 1175 bases from 491th to 1666th. The invention can stimulate forming of lateral bud anlage and is used for alimentary crops, floral plants, economic crop and oil crop to increase the number of branch stem thereby raise productivity.

Description

The albumen coded sequence of cucumber side branch inhibition gene cls

Technical field

What the present invention relates to is the albumen coded sequence of a kind of encoding sequence of gene engineering technology field, particularly a kind of cucumber side branch inhibition gene cls.

Background technology

Plant branching is grown consequence in phytomorph is built up.The crop branch is one of Main Agronomic Characters that influences crop yield, can reach the purpose of volume increase by the minimizing branch in the domestication process of corn, the too much or very few raising that all is unfavorable for output of tillering number in paddy rice.For cucumber, output height and side shoot number also have confidential relation.Up to the present, the cucumber side shoot is used as quantitative character research always, has located several QTLs relevant with collateral development.Obtain gene but never separate, more do not study in great detail.Obtained a plurality of genes relevant with collateral development but separated in corn, paddy rice, Arabidopis thaliana, tomato, pea, wherein the most noticeable is separation and the functional verification of tomato side shoot suppressor gene ls.The ls gene of tomato is one of gene that influences the earliest axillary meristem formation, is the important member of plant specific GRAS transcription factor family.The protein product of GRAS gene family is transcription factor, has the ability of other genetic transcription of regulation and control, and name is by three members that find the earliest, GAI, and RGA, the initial spelling of SCR forms.Typical GRAS albumen is made up of 400-700 amino acid, the VHIID structural domain that two leucine heptamer tumor-necrosis factor glycoproteinss (Leucine Heptad Repeats) surround, conservative C-latter end and length, the variable N-end of sequence, just the sequence of N-end has been given its species specificity.Cloned genes cls of the present invention also belongs to the GRAS gene family, and prediction should also be a transcription factor.

In analysis to the prior art document, " though Proc.Natl.Acad.Sci.USA NAS journal ", 1999,1,96:290-295 has delivered article " The lateral suppressor (Ls) geneof tomato encodes a new member of the VHIID protein family tomato side shoot suppressor gene Ls proteins encoded is a newcomer of VHIID family " function of tomato side shoot suppressor gene Ls has been done to elaborate; " the theoretical utilization of Theor Appl Genet genetics " is 2003, the article that 107:875-883 delivers " Comparative analysis of response to phenotypic and marker-assisted selection for multiple lateral branching incucumber (the Cucumis sativus L.) molecular marker assisted selection of many side shoots of cucumber proterties and the comparative analysis of phenotype " has carried out the QTLs location to many side shoots proterties, does not obtain gene but separate.

Summary of the invention

The objective of the invention is to overcome deficiency of the prior art, the encoding sequence of a kind of cucumber side shoot suppressor gene CLS is provided.The present invention can promote the lateral bud original hase to form, and is used for food crop, flower plant, cash crop, oil crops, increases their side shoot number, thereby increases output.

The present invention is achieved by the following technical solutions:

In one aspect of the invention, a kind of isolated cDNA molecule is provided, this molecule comprises: coding has the nucleotide sequence of the active polypeptide of cucumber side shoot arrestin, shows at least 70% homology from the nucleotides sequence of Nucleotide 491-1666 position among described nucleotide sequence and the SEQ IDNO.3.Perhaps described nucleotide sequence can be at the nucleotide sequence hybridization of 491-1666 position among moderate stringent condition (hybridization temperature is between 55 ℃-60 ℃) and the SEQ ID NO.3.Preferably, described coding is meant to have the polypeptide that nucleotides sequence is classified the aminoacid sequence of SEQ ID NO.3 as, or its conservative property variation polypeptide or its active part, or its reactive derivative.More preferably, described coding is meant the polypeptide with nucleotide sequence of 491-1666 position among the SEQ ID NO.3.

Aspect another, provide a kind of protein and peptide of isolated cucumber side shoot suppressor gene in the present invention, its coding is meant polypeptide or its conservative property variation polypeptide or its active fragments with SEQ ID No.3 aminoacid sequence.This polypeptide is that the plant lateral organ forms requisite albumen at the early expression that the plant axillary meristem forms.

The present invention also provides a kind of carrier, and it comprises above-mentioned dna molecular.A kind of nucleic acid molecule, it comprises 8-100 continuous nucleotide in the described dna molecular.

The present invention also provides a kind of usefulness above-mentioned carrier transformed host cells, and it is an eukaryotic cell.This host cell is an Arabidopis thaliana in example.

Among the present invention, " isolating ", " purifying " DNA are meant, this DNA or fragment have been arranged in the sequence of its both sides under native state separates, and refers to that also this DNA or fragment with under the native state follow the component of nucleic acid to separate, and separates with the protein of following it in cell.

Encoding sequence of the present invention comprises that coding has the nucleotide sequence of the active polypeptide of cucumber side shoot arrestin, as 491-1666 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.3.This degenerate sequence is meant, is arranged in the encoder block 491-1666 position Nucleotide of SEQ ID NO.3 sequence, and having one or more codons to be encoded, the degenerate codon of same amino acid replaces the back and the sequence that produces.Because the degeneracy of codon, thus with SEQ ID NO.3 in 491-1666 position nucleotide sequence homology be low to moderate about 70% the degenerate sequence described sequence of SEQ ID NO.3 of also encoding out.This term also comprise can with the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 491-1666 position among the SEQ ID NO.3.This term also comprise with SEQID NO.3 in from the homology of nucleotide sequence at least 70% of Nucleotide 491-1666 position, can be preferred 80%, further preferred 90%, best preferred 95% nucleotide sequence.

This term also comprises encoding to have the variant form of open reading frame sequence among the proteic SEQ ID NO.3 with cucumber side shoot suppressor gene identical function.These variant forms comprise (but being not limited to): several (are generally 1-90, preferably 1-60, further preferred 1-20, disappearance, insertion and/or the replacement of best preferred 1-10 Nucleotide, and several (are generally in 60 to hold interpolation 5 ' and/or 3 ', in can preferred 30, further in preferred 10, in best preferred 5) Nucleotide.

Encoding sequence of the present invention has polypeptide or its conservative property variation polypeptide or its active fragments of the aminoacid sequence shown in the SEQ ID NO.3, or its reactive derivative.Also comprise having and suppress variant form relevant identical function, SEQ ID NO.3 sequence with the cucumber side shoot.These variant forms comprise (but being not limited to): several (are generally 1-50, preferably 1-30, further preferred 1-20, preferred 1-10 of the 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, in can preferred 10, further in preferred 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.The present invention also comprises the active part and the reactive derivative of cucumber side shoot suppressor gene associated protein.

The variant form of cucumber side shoot suppressor gene of the present invention comprises: albumen that homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, the DNA that (hybridization temperature is higher than 65 ℃) can relevant DNA hybridization with cucumber side shoot suppressor gene under moderate (hybridization temperature 55-60 ℃) or height stringent condition are coded and polypeptide or the albumen that utilizes the antiserum(antisera) acquisition of cucumber side shoot suppressor gene.

The variation polypeptide of described cucumber side shoot suppressor gene is compared with the polypeptide with SEQ ID NO3 aminoacid sequence, has 10 at the most, can be preferred 8, and further preferred 5 amino acid are replaced by similar performance or close amino acid and are formed polypeptide.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?

Invention also comprises the analogue of side shoot suppressor gene albumen or polypeptide.The difference of these analogues and natural side shoot suppressor gene related polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.

(the not changing primary structure usually) form of modification comprises: the chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modification also comprises glycosylation, carries out glycosylation modified and polypeptide that produce in the procedure of processing as those in the synthetic and processing of polypeptide or further.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and finishes by polypeptide is exposed to.Modified forms also comprises have the phosphorylated amino acid residue sequence of (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Thereby also comprise the polypeptide that has been improved its anti-proteolysis performance or optimized solubility property by modifying.

In the present invention, can select various carrier known in the art for use, the carrier as commercially available comprises plasmid, clay etc.When production cucumber side shoot of the present invention suppresses polypeptide, the encoding sequence of cucumber side shoot suppressor gene operationally can be connected in expression regulation sequence, thereby form cucumber side shoot arrestin 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.

Also available hybridization in situ technique is analyzed the expression of cucumber side shoot arrestin CLS in the cucumber different tissues.

In addition, the present invention also provides a kind of nucleic acid molecule that can be used as probe, and this molecule has 8-100 continuous nucleotide of cucumber side shoot suppressor gene nucleotide coding sequence usually, preferably has 15-50 continuous nucleotide.This probe can be used for whether existing in the test sample the relevant nucleic acid molecule of coding cucumber side shoot suppressor gene.

The present invention also provides the method that whether exists the cucumber side shoot to suppress related nucleotide sequences in the test sample, and 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 suppresses relevant nucleotide coding sequence corresponding to the cucumber side shoot, and can be positioned at the both sides or the centre of this encoding sequence.Primer length is generally 15-50 Nucleotide.

In addition, suppress nucleotide sequence and aminoacid sequence according to cucumber side shoot of the present invention, can be on the homology basis of nucleic acid homology or marking protein, screening cucumber side shoot suppresses relevant homologous gene or homologous protein.

In order to obtain the dot matrix with the Cucumber cDNA s of cucumber side shoot inhibitory phase correlation gene, can screen the Cucumber cDNA library with dna probe, these probes are under low stringent condition, with 32P to the cucumber side shoot suppress relevant all or part of do the radioactivity mark and.The cDNA library that most is suitable for screening is the library from cucumber.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 of the gene family relevant with the inhibition of cucumber side shoot.

Cucumber side shoot of the present invention suppresses the associated nucleotide full length sequence or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.

In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.

In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.

Except producing with recombination method, the also available solid phase technique of the proteic fragment of the present invention is produced (people such as Stewart, (1969) Solid-Phase Peptide Synthesis, WHFreeman Co., San Francisco by direct peptide synthesis; Merrifield J. (1963) J.Am Chem.Soc 85:2149-2154).Can carry out by hand or automatically at external synthetic protein.For example, can (Foster City CA) synthesizes peptide automatically with the 431A type peptide synthesizer of AppliedBiosystems.Can distinguish proteic each fragment of chemosynthesis the present invention, be connected to produce the molecule of total length with chemical process then.

Utilize cucumber side shoot suppressor gene of the present invention,, can filter out with the cucumber side shoot and suppress the interactional material of relevant generation, perhaps acceptor, inhibitor or antagonist etc. by various conventional screening methods.

Plant branching is grown and have critical role in phytomorph is built up.The branch of crop be influence crop solid what and and then influence one of Main Agronomic Characters of per unit area yield.The present invention has the vegetable cell that forms the lateral meristem ability.In the early stage effect that the lateral bud original hase forms, be the developmental key gene of plant lateral meristem.Can allow the arabidopsis mutant body of no side shoot grow side shoot fully, illustrate that the conservative property of this gene in different plant species is very strong, can be used for food crop fully, see the application on leaf, the stem-watching flower and plant, the number that increases their side shoots has improved output or the ornamental value of these plants.Therefore, the present invention has very big using value.

Embodiment

Below in conjunction with specific embodiment, further set forth the present invention.These embodiment only are used to the present invention is described and are not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, for example the Sambrook equimolecular is cloned: laboratory manual (New York:Cold SpringHarbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

The clone of embodiment 1 cucumber side shoot suppressor gene

1. separate tissue (isolation)

The European greenhouse kind that cucumber s06 collects for our laboratory places 28 ℃ to germinate 24 hours cucumber, is seeded in then in the dish of cave, treats the time in general week when two cotyledons of cucumber flatten fully, prepares DNA extraction or RNA.

2.RNA separation (RNA isolation)

Get portion of tissue, grind, add the 1.5mL EP pipe that fills lysate, fully after the vibration, move in the glass homogenizer again with mortar.Move to after the homogenate in the 1.5mL EP pipe, and extracted total RNA (TRIzol Reagents, GIBCO BRL, USA).Identify total RNA quality with the denaturing formaldehyde gel electrophoresis, on spectrophotometer, measure rna content then.

3. the full-length clone of gene (Cloning of Full-length cDNA)

According to Arabidopis thaliana, tomato, the Nucleotide conserved sequence of the side shoot suppressor gene of paddy rice utilizes homologous gene clone principle, adopts RACE method (GibcoBRL test kit) to carry out the cDNA full-length clone, divides three phases to carry out:

(1)RT-PCR

PCR[BP001 (SEQ ID NO.1)+BP002 (SEQ ID NO.2)] obtain 2002BP (931bp), reclaim, be connected on the pGEMT-Easy carrier, with M13 or T7 as universal primer, adopt thing fluorescent mark (Big-Dye, Perkin-Elmer, method USA) of stopping, (Perkin-Elmer checks order on USA) at ABI 377 sequenators.Sequencing result GCG software package (Wisconsin group, USA) BLAST in and the existing database of FASTA software search (Genebank+EMBL), the homology of knowing its nucleotide sequence and proteins encoded and known cress such as Arabidopis thaliana (Arabidopsis thaliana) cucumber side shoot suppressor gene is very high, so think that tentatively it is a side shoot suppressor gene.

(2)3’-RACE

PCR[AP+BP003 (5 ' TTCATAAGACTGGTGATAGACTCTCA-3 ')] obtain 2002BP3 ' (817bp), reclaim, be connected on the T-Easy carrier, with M13 or T7 as universal primer, adopt thing fluorescent mark (Big-Dye, Perkin-Elmer, method USA) of stopping, (Perkin-Elmer checks order on USA) at ABI 377 sequenators.Sequencing result GCG software package (Wisconsin group, USA) BLAST in and the existing database of FASTA software search (Genebank+EMBL), the homology of knowing its nucleotide sequence and proteins encoded and known cress such as Arabidopis thaliana (Arabidopsis thaliana) side shoot suppressor gene is very high, so think that tentatively it is one and side shoot inhibitory phase correlation gene.

(3)5’-RACE

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

Second takes turns PCR[(AUAP+BPR005 (5 '-CACAACGGATAAGTAATTGACGCAT-3 ')) obtain 2001BP 5 ' (about 500bp) (process is with (1))

With the overlap splicing of sequencing result, the fragment that discovery procedure (1) obtains is the complete coding region of this gene.

The gene that result's proof of BLAST newly obtains from cucumber really is a gene relevant with plant branching.

By being used in combination above-mentioned 3 kinds of methods, obtained the complete encoding sequence (SEQ ID NO.3) of candidate's cucumber side shoot suppressor gene.

The sequence information and the homology analysis of embodiment 2 cucumber side shoot inhibitory phase correlation genes

The cucumber side shoot suppressor gene cDNA total length that present embodiment is new is 2175bp (SEQ ID NO.3), and wherein open reading frame is positioned at 491-1666 position Nucleotide (1175 Nucleotide).Derive the aminoacid sequence of cucumber side shoot suppressor gene according to full-length cDNA, totally 391 amino-acid residues, detailed sequence is seen SEQ ID NO.4.

Full length cDNA sequence that cucumber side shoot suppressor gene is relevant and coded protein thereof carry out Nucleotide and protein homology retrieval with blast program in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS translations+PDB+SwissProt+Superdate+PIR database, found that it and tomato side shoot suppressor gene (AF098674) only have homology (subordinate list 2) on the part section of Nucleotide, have 67% homogeny (subordinate list 3) on amino acid (AAD05242) level; Tomato side shoot suppressor gene has been proved to be the effect with tangible promotion sprouting of lateral bud, can think that the cls gene is promoting lateral bud also to have similar effect on initial.

Table 2

Query: the nucleotide sequence of cucumber side shoot suppressor gene CLS

Sbjct: the nucleotide sequence of tomato LS (AF098674)

Table 2 is that the homology of the nucleotide sequence of cucumber side shoot suppressor gene of the present invention (cls) and tomato side shoot suppressor gene (ls) compares (GAP) table.

Table 3

Query: the aminoacid sequence of cucumber side shoot suppressor gene CLS

Subjct: the aminoacid sequence of tomato LS (AAD05242)

Table 3 is that the homology of the aminoacid sequence of cucumber side shoot gene of the present invention (cls) and Arabidopis thaliana side shoot suppressor gene (ls) compares (FASTA) table.Wherein, identical amino acid marks with the amino acid monocase between two sequences, and similar amino acid marks with "+".

The Subcellular Localization of embodiment 3cls gene

GRAS family protein member's function is a transcription factor, and some the GRAS members based on having been found that have nuclear localization signal as RGA and SLR1.But with molecular biology software (pSORT, http//psort.nibb.ac.jp) analysis revealed CLS does not have the diaphragm area of striding, in order to determine whether CLS is positioned in the nuclear, has made up the fusion rotein that comprises green fluorescent protein GFP a: CLS-GFP, make up up time expression vector PA-7.Bombard with particle gun in the epidermis of onion, 24 ℃ of dark cultivations after 36 hours are observed with laser confocal microscope.The green fluorescence signal mainly detects in nuclear as a result, shows that CLS is positioned in the nuclear, and supposition is a transcription factor.

Embodiment 4 cucumber side shoot suppressor gene associated protein or polypeptide carry out the evaluation of eukaryotic cell expression and transfer-gen plant in Arabidopis thaliana

The structure that contains goal gene (cucumber side branch inhibition gene cls) expression vector

Full length cDNA sequence (SEQ ID NO.3) according to cucumber side shoot suppressor gene, design amplifies the primer that complete coding is read frame, and on the upstream and downstream primer, introduce restriction endonuclease sites (this is decided by the carrier of selecting for use) respectively, so that construction of expression vector.Amplified production with acquisition among the embodiment 1 is a template, behind pcr amplification, cDNA is cloned into intermediate carrier (as pUCM-T) with cucumber side shoot suppressor gene, further be cloned into binary expression vector (pMON530), guaranteeing to identify good expression vector under the correct prerequisite of reading frame, again it is changed in the Agrobacterium, adopt inflorescence dip method arabidopsis thaliana transformation.

1. the growth of plant: the ratio according to vermiculite 6 black earth 1 perlite 0.25 prepares matrix, install in the cave dish of diameter 8CM, with the program request of Arabidopis thaliana seed in the above, preservative film in the covering, put into 4 ℃ of refrigerating chambers, change over to after 3 days in the illumination box of the dark 8h of temperature 16h at 24 ℃ of sunshine and cultivate.When plant grows to the about 3cm of stem height, remove it and push up biochemical preface, to stimulate the growth of axillary inflorescence, note avoiding injuring axillary inflorescence.Treat that axillary inflorescence grows, its underpart spend existing pollination phenomenon to occur the time transform, before the conversion, the flower and the fruit pod that have pollinated are removed.

2. bacterium liquid preparation and infect operation

The Agrobacterium mono-clonal that picking is identified is put in 20ml and contains in the LB liquid nutrient medium of each 200ul of spectinomycin spe/ kantlex KM, and 28 ℃, it is about 0.5 that the 250rpm concussion is cultured to OD600.Transforming the day before yesterday, change the same LB liquid nutrient medium of 50ml over to and spend the night, second day, when bacterium liquid OD600 is between 1.2-1.6, take out and use.Centrifugal 15 minutes of room temperature 5000rpm, abandon supernatant, precipitation is suspended in infiltration substratum (the 1/2MS+ sucrose 5%+silwetL77 200ul/L+6BA1mg/ml) pH value 5.6 of respective volume, agrobacterium suspension is poured in the small beaker, there is the cultivation cup of Arabidopis thaliana to tip upside down on the inside with long, infect 30s, take out dark the cultivation 1 day.According to circumstances can contaminate once again.Cultivate 3-4 week, treat seed maturity after, collect seed, deposited for 2 weeks in the loft drier.

3. the screening of transformant and genetic analysis

Seed is evenly spread to solid screening culture medium (MS, 1% sucrose, 0.8% agar, PH5.7, kantlex 50mg/ml) surface.

Arabidopis thaliana is cultivated: the Arabidopis thaliana culture dish was handled two days for 4 ℃, and 22-25 ℃ of shading cultivated two days later, puts into the constant temperature culture chamber, after about two weeks, both can distinguish transformant and unconverted strain, and transformant is transplanted in the soil to collect T1 for seed.

4. the genetic analysis of transformant

Be the Arabidopis thaliana transgenosis strain that obtains isozygotying, transformant selfing three generations gets T3 and carries out the Physiology and biochemistry test for seed.

In view of the formation of Arabidopis thaliana las gene pairs lateral bud original hase has obvious facilitation, Arabidopis thaliana ls deletion mutant shows the characteristics that the rosette state does not have side shoot fully under the short day condition.Therefore complementary successful plant grew one month under the long day after short day is handled 28 days again, at first showing the rosette state has more side shoot to grow, secondly show with sxemiquantitative RT-PCR qualification result: can detect transcribing of cucumber side shoot suppressor gene in the phenotype complementary plant, but in Arabidopis thaliana ls mutant and wild-type, then detect less than.The sprouting of this proof cls gene pairs lateral bud has definite effect, and cucumber cls gene will can be used for utilizing transgenic technology to change in plant number of branch purpose research and the industrialization production.

Embodiment 5

The copy number analysis of cucumber side branch inhibition gene cls in cucumber

Adopt ordinary method from the blade of seven days seedling of cucumber, to extract DNA (with reference to " molecular cloning ", Sambrook etc., 1989), cut DNA[20 μ g (microgram)/sample with BamH and EcoRI enzyme respectively] after, go to DNA on the Hybond membrane (nylon membrane) after.Use the Amersham Pharmacia GeneImages of company ^TMContents CDP-Star ^TMLabelling module (PRN3540), we are labeled as probe with the cls gene coding region, hybridize (in 60 ℃ of hybridization 16 hours) then.Take out film, place film washing liquid I (1*SSC, 1%SDS) in, in 60 ℃ of rinsings 3 times, each 15 minutes.Change over to film washing liquid II (0.1*SSC, 1%SDS) in 60 ℃ of rinsings 3 times, each same 15 minutes.With X-ray sheet compressing tablet 60-90 minute, develop then, photographic fixing (method is with reference to Roche DIG labeled test kit specification sheets).A hybridization band only appears in result (Southernblot) on Hybond membrane, illustrate that only there is a copy in the cls gene in cucumber.

Embodiment 6

The expressed in situ of cucumber side branch inhibition gene cls in cucumber

1. the preparation of material

Get the cucumber terminal bud material FAA stationary liquid of ten days seedling ages, 4 ℃ of fixing 12-16h, through the dehydration of alcohol gradient, paraffin (Paraplast Plus, sigma) section, slice thickness 10um are advanced in embedding after the transparent processing.

2. the preparation of probe

With cucumber side shoot suppressor gene sequences Design primer YWF-TGGATCCGAACCAGAACAGCAGGAA, the Partial cDNA segment of YWR-TGTCGACGTGGCAGTAATACGAAGC amplification cucumber side branch inhibition gene cls, altogether the correct back of 454bp order-checking subclone is to the pSK carrier, extracts plasmid and uses BamHI and the SalI complete degestion masterplate as T7 and T3 polysaccharase respectively.Linearizing obtains justice and antisense probe.Probe generally is hydrolyzed into the fragment of 75-150bp size, and hydrolysis time calculates according to following formula: T (time)=(Li-Lf)/KLiLf (Li: the length of original probe; Lf: the length of terminal crossing probe; K=0.11kb/min.)

3. in situ hybridization process

(Roche, the method that USA) provides is carried out according to digoxin RNA labelling kit in the operating process of the RNA in situ hybridization of non-radioactive mark.Take pictures with Olympus BX-51 digital camera.

RNA in situ hybridization did not show before naked eyes are seen lateral bud formation just can detect the expression of CLS in axillary meristem.And opposite with the high expression level of axillary meristem, CLS does not express at apical meristem.These presentation of results CLS has played keying action in lateral meristem initial sum lateral bud forms.

Sequence that the present invention relates to and mark apportion are as follows:

(1) information of SEQ ID NO.1

(i) sequence signature:

(A) length: 23bp

(B) type: Nucleotide

(C) chain: strand

(D) topological framework: linearity

(ii). molecule type: oligonucleotide

(iii). sequence description: SEQ ID NO.1

CA(A/G)TGGCC(T/A/G)CC(A/G/T)TT(G/A)ATGCAAGC

(2) information of SEQ ID NO.2

(i) sequence signature:

(A) length: 24bp

(B) type: Nucleotide

(C) chain: strand

(D) topological framework: linearity

(ii). molecule type: oligonucleotide

(iii). sequence description: SEQ ID NO.2

(C/T)CT(T/C/A)A(A/G)(C/A)A(A/G)(G/A)AGCTT(G/A)GCTTG(C/T)GA

(3) information of SEQ ID NO.3

(i) sequence signature:

(A) length: 2175bp

(B) type: Nucleotide

(C) chain: strand

(D) topological framework: linearity

(ii). molecule type: Nucleotide

(iii). sequence description: SEQ ID NO.3

＜110〉Shanghai Communications University

＜120〉albumen coded sequence of cucumber side branch inhibition gene cls

<140>

<141>

<160>2

<170>

<210>3

<211>2175

<212>DNA

＜213〉cucumber (Cucumis sativus)

<400>3

<210>4

<211>391

<212>PRT

＜213〉CLS albumen

<400>4

Claims (3)

1. the encoding gene of a cucumber side shoot suppressor gene CLS is characterized in that, described encoding gene is the nucleotide sequence of SEQ ID NO.3.

2. according to the encoding gene of the cucumber side shoot suppressor gene CLS of claim 1, it is characterized in that, described encoding gene, its open reading frame is from 491-1666 totally 1176 bases.

3. according to the encoding gene of the cucumber side shoot suppressor gene CLS of claim 2, it is characterized in that the initiator codon of described open reading frame is ATG, terminator codon is TGA.