CN1844397A

CN1844397A - Phophomannose isomerase gene and its coded protein and use

Info

Publication number: CN1844397A
Application number: CN 200610079044
Authority: CN
Inventors: 陈蕾; 徐建勇
Original assignee: BEIFANGJIESHI BIOLOGICAL SCIO-TECH Co Ltd BEIJING
Current assignee: Shanghai Longyu Industrial Co. Ltd.
Priority date: 2006-04-30
Filing date: 2006-04-30
Publication date: 2006-10-11
Anticipated expiration: 2026-04-30
Also published as: CN100374569C

Abstract

The invention discloses a gene and its protein of phosphomannose isomerase, and its application. The purpose is that providing a gene and its protein of phoshomannose isomerase, and applying the gene and protein in selecting trans-genic plant. The gene has one of nucleotide sequences as follow: 1) the SEQ ID NO: 1 DNA sequence in sequence table; 2) coding the DNA sequence of SEQ ID NO: 2 in the sequence table; 3) the nucleotide sequence that can hybrid with restrict DNA sequence of SEQ ID NO: 1 in the sequence table at high strict condition. Mannose has no harm for human and animal, degradation-liable in environment and selected marker system has the advantages of product yield safety, the selecting regent price is cheap, the selecting procedure is simplicity and the selected marker system don't impact the metabolism balance of transformation plant, the trans-genic plant grow vigorous, significance effect selection. The selected marker system has potential and extensive application outlook in trans-genic plant study and its market process; it may be the leading selected marker system in plant transformation.

Description

A kind of Phophomannose isomerase gene and proteins encoded thereof and application

Technical field

The present invention relates to a kind of selectable marker gene and proteins encoded thereof and application, particularly relate to a kind of Phophomannose isomerase gene and proteins encoded thereof and its application in the screening transgenic plant.

Background technology

In the plant genetic engineering operation, the screening of transfer-gen plant is vital link.At present, generally utilize the gene that certain toxicant (as microbiotic, weedicide etc.) is had a resistance transfer-gen plant to be screened, be about to toxicant and join in the substratum, can kill unconverted vegetable cell as selective marker.Although can screen transfer-gen plant effectively in this way, also exist great defective simultaneously.As everyone knows, in the gene transformation operation, have only the cell of few part to be transformed, obtain exogenous dna fragment, most cells still is in non-conversion conditions, need be removed by selectivity.Though traditional screening method can be killed non-transformed cell, these dead cells may discharge toxicant, and transformant is caused damage; A large amount of dead cells also can form the barrier between transformant and the culture, hinders the dietetic alimentation and the normal growth thereof of transformant, causes transformation efficiency to reduce (Joersbo, M.﹠amp; Okkels, F.T.1996.Plant Cell Rep 16:219-221); Screening system based on microbiotic and weedicide also will; False positive plant (Girgi, M.etal.2002.Mol Breed 10:243-252 appear in the escaping phenomena that also occurs non-transformed cell through regular meeting; Zhang, P.﹠amp; Puonti-Kaerlas, J.2000.PlantCell Rep 19:1041-1048); In addition, use microbiotic, weedicide, cause the transfer-gen plant phenotype unusual sometimes, physical environment is polluted, even also can injure human beings'health.The use of anti-herbicide gene may cause the appearance of antiweed weeds, and increases the use of weedicide in environment, causes ecological crisis.Particularly after the transgenic plant cells cracking, the antibiotic resistant gene of encoding might be obtained by bacterium, causes bacterium to develop immunity to drugs, thereby publilc health is constituted a threat to.

Summary of the invention

The purpose of this invention is to provide a kind of Phophomannose isomerase gene.

Phophomannose isomerase gene provided by the present invention is named as KL manA, derives from a carat dimension saccharomyces lactis (Kluyveromyces lactis), is one of following nucleotide sequence:

1) SEQ ID № in the sequence table: 1 dna sequence dna;

2) SEQ ID № in the code sequence tabulation: 2 dna sequence dna;

3) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 1 dna sequence dna hybridization that limits.

The rigorous condition of described height be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, wash film under 60 ℃ of conditions.

SEQ ID № in the sequence table: 1 by 1358 based compositions, and its encoding sequence is from 5 ' end 11-1297 bit base, SEQ ID № in the code sequence tabulation: 2 amino acid residue sequence.

Phophomannose isomerase gene encoded protein of the present invention (KL manA) has one of following amino acid residue sequences:

1) the SEQ ID № in the sequence table: 2;

2) with SEQ ID № in the sequence table: 2 amino acid residue sequence is through replacement, disappearance or the interpolation of one to ten amino-acid residue and have the protein of phosphomannose transformation function.

SEQ ID № in the sequence table: 2 are made up of 428 amino-acid residues.

Contain expression carrier of the present invention, transgenic cell line and host bacterium and all belong to protection scope of the present invention.

Arbitrary segmental primer is to also within protection scope of the present invention among the amplification KL manA.

Another object of the present invention provides a kind of carrier that is used to screen transgenic plant.

The carrier that is used to screen transgenic plant provided by the present invention is the plant binary expression vector that contains described Phophomannose isomerase gene (selectable marker gene).

The described carrier that is used for screening transgenic plant also contains the promotor and the terminator of mannose isomerase gene.The selection of described promotor and terminator is diversified, described promotor can be cauliflower mosaic virus (CaMV) 35S promoter, Actin promotor or the Ubiquitin promotor etc. in the composition type expression promoter, and described terminator can be Tnos terminator or T35S terminator etc.

The carrier that sets out that is used for making up described screening vector can be any one can be at the plant binary expression vector of plant expression alien gene, as pCAMBIA1300, pBI121, pCAMBIA1301 or Bin19 etc.

Be the carrier that sets out with pCAMBIA1300, the screening vector of structure is pC1300 (KLmanA).

The 3rd purpose of the present invention provides the screening method of a kind of transgenic plant.

The screening method of transgenic plant provided by the present invention, be target gene and promotor thereof and terminator to be connected into have in the above-mentioned screening vector that contains Phophomannose isomerase gene, obtain the transfer-gen plant screening vector of target gene, again this carrier is transformed plant, through the seminose screening, obtain transfer-gen plant.

In above-mentioned screening method, the promotor of described target gene and terminator can be selected according to actual needs.

The described transfer-gen plant screening vector that comprises target gene can be transformed plant tissue, to place through the tissue of conversion again and filter out the resistance tissue on the screening culture medium that contains seminose, then the resistance tissue is placed on the regeneration culture medium that contains seminose and break up again, subsequently the transgenic seedling that differentiates root and bud is cultivated, obtained the transfer-gen plant of target gene.

When described plant tissue is plant callus, the transfer-gen plant screening vector of described target gene is transformed plant callus, to place on the callus screening culture medium that contains seminose through the callus of conversion again and filter out resistant calli, then resistant calli is placed on the regeneration culture medium that contains seminose and break up again, subsequently the transgenic seedling that differentiates root and bud is cultivated, obtained the transfer-gen plant of target gene; The described callus screening culture medium that contains seminose is to add 2 on the basis of MS minimum medium, 4-dichlorphenoxyacetic acid 0.5-8mg/L, 6-benzyl aminopurine 0-0.5mg/L, kinetin 0-0.5mg/L, caseinhydrolysate 0.5-2g/L, sucrose 0-15g/L, seminose 10-30g/L and microbiotic, described microbiotic is Pyocianil or Reflin, and wherein, the concentration of Pyocianil is 250mg/L; The regeneration culture medium that contains seminose is to have added 6-BA 0.1-0.5mg/L on the basis of MS minimum medium, kinetin 0.1-0.5mg/L, sucrose 0-15g/L, seminose 5-20g/L and microbiotic, described microbiotic is Pyocianil or Reflin, wherein, the concentration of Pyocianil is 250mg/L.

In addition, also the transfer-gen plant screening vector of described target gene can be transformed the plant propagation organ, and the results seed, the seed after will sprouting again places the enterprising row filter of the screening culture medium that contains seminose, obtains the transfer-gen plant of target gene; The described screening culture medium that contains seminose is to have added seminose 1.5-2.0g/L on the basis of MS minimum medium.

Seminose in the above-mentioned substratum is the combination of pure seminose or seminose and other carbohydrate, and described seminose can be the D-seminose.

The described transfer-gen plant screening vector that carries target gene can transform plant callus or plant propagation organ by using methods such as agrobacterium-mediated transformation, particle bombardment, electric shocking method, pollen tube introductory technique or liposome fusion method; Described Agrobacterium can be any one agrobacterium tumefaciens or Agrobacterium rhizogenes.

The screening method of above-mentioned transgenic plant all is suitable for all plants, both has been applicable to monocotyledonss such as corn, paddy rice, wheat, also is applicable to dicotyledonss such as Arabidopis thaliana, tobacco, cotton.

The invention provides a kind of Phophomannose isomerase gene and proteins encoded thereof.Can mannose isomerase gene of the present invention (KL manA) as the selection markers gene, utilize plant expression vector with this gene-transformed plant callus or plant propagation organ, through screening on the substratum that contains seminose (screening reagent) and differentiation, obtain transfer-gen plant.The principle of this screening method is: in the substratum of seminose as main carbon source, the seminose that cell absorbed is converted into the 6-phosphomannose by hexokinase, transgenic cell is expressed phosphomannose isomerase can be converted into fructose-1, 6-diphosphate with the 6-phosphomannose, for the cell growth provides carbon source, not genetically modified cell has then accumulated a large amount of 6-phosphomannoses, its cell growth is suppressed, thereby reach the screening purpose.Compare with traditional genetic transformation system of selection, when using the seminose selecting and labelling system, non-transformed cell is in starvation, and is not to be killed, so select the less necrotic tissue that occurs in the culturing process, more helps growth and the regeneration of genetically modified organism.Seminose does not have harm to people and animals, easily degraded in environment, and this selecting and labelling system has product safety (enzyme of coding has been widely used in foodstuffs industry), selective agent is cheap, select procedure is simple, and do not influence the metabolic balance that transforms plant, the transfer-gen plant growth is vigorous, advantages such as screening effect is remarkable have potential, application prospects in transgenic plant research and market-oriented process, be expected to develop into leading selecting and labelling system in the Plant Transformation.

The present invention will be further described below in conjunction with specific embodiment.

Description of drawings

Fig. 1 is the physical map of plasmid pC1300 (KLmanA)

Fig. 2 is the physical map of plasmid pC1300 (KLmanA) ∷ gus

Embodiment

Method therefor is ordinary method if no special instructions among the following embodiment, can reference: Sambrook et al., and 2001, Molecular cloning:A Laboratory Manual, it is synthetic that the primer sequence is given birth to worker company by Shanghai.Carat dimension saccharomyces lactis (Kluyveromyces lactis) bacterial classification is bought from Microbe Inst., Chinese Academy of Sciences.General biochemical reagents are purchased in Beijing chemical reagents corporation, T ₄Dna ligase, restriction enzyme etc. are purchased the company in TaKaRa.

The clone of embodiment 1, Phophomannose isomerase gene KL manA

Predict its Phophomannose isomerase gene sequence and design primer that according to carat dimension saccharomyces lactis genome sequence add restriction enzyme Xho I recognition site at the primer two ends, primer sequence is as follows:

P1 (upstream primer): 5 '- CTCGAGTACCATGCCACAGCTTTTCAG (band underscore base is a restriction enzyme Xho I recognition site);

P2 (downstream primer): 5 '- CTCGAGCTTGTCGATCGACAGATCATACTGTTTCTTTGATTGTG GTTC (band underscore base is a restriction enzyme Xho I recognition site)

Genomic dna with carat dimension saccharomyces lactis is a template, under the guiding of primer P1 and P2, and pcr amplification.50 μ l PCR reaction systems are: 5 μ l, 10 * PCR reaction buffer, and each 0.5 μ l of 10 μ M primer P1 and P2,100ng DNA, 1 μ l 10mM dNTPs, 2.5U pfu archaeal dna polymerase (Shen, Shanghai energy betting office) adds sterilized water to 50 μ l.The PCR reaction conditions is: 94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 1 minute, totally 30 circulations.After reaction finishes, the PCR product is carried out 0.8% agarose gel electrophoresis to be detected, reclaim the also dna fragmentation of the about 1.36kbp of purifying, connect EcoR V site into cloning vector pBluescript II KSM (Genebank Accession No.X52329), again with recombinant vectors transformed into escherichia coli DH5 α competent cell, screening positive clone, the upgrading grain, with positive colony plasmid called after pBS ∷ KL manA, it is checked order, sequencing result shows that deriving from carat ties up the nucleotide sequence that the mannose isomerase gene (called after KL manA) of saccharomyces lactis has sequence 1 in the sequence table, sequence 1 in the sequence table is by 1358 based compositions, its encoding sequence is from 5 ' end 11-1297 bit base, the amino acid residue sequence of sequence 2 in the code sequence tabulation, sequence 2 in the sequence table is made up of 428 amino-acid residues, with this albumen called after KL manA.

Embodiment 2, structure are used to screen the carrier of transgenic plant

With restriction enzyme Xho I plasmid pBS ∷ KL manA being carried out enzyme cuts; recovery and purifying contain the dna fragmentation of the 1.36kb of KL manA; be inserted in the Agrobacterium binary vector pCAMIBA1300 (Genbank Accession No.AF234296) that same enzyme enzyme is cut; with recombinant vectors transformed into escherichia coli DH5 α competent cell; screening positive clone; the upgrading grain; the positive colony plasmid called after pC1300 (KLmanA) that will contain KL manA; its physical map as shown in Figure 1; in this carrier; KL manA is subjected to the regulation and control of gene expression in plants element CaMV 35S enhancement type promotor and CaMV 35S polyA tailing signal; can be in vegetable cell effective expression; in addition, this plasmid also contains multiple clone site and intestinal bacteria lacZ expression cassette thereof, is convenient to carry out the clone of foreign gene.

The screening of embodiment 3, gusA transgenic tall fescue grass

One, makes up the transfer-gen plant screening vector that contains gusA (target gene)

With restriction enzyme EcoRI and Hind III plasmid vector pBI121 (Genbank AccessionNo.AF485783) being carried out enzyme cuts, reclaim the also dna fragmentation that contains gusA expressed intact external member of purifying 3kb, and be inserted on the special-purpose screening vector pC1300 (KLmanA) that the embodiment 2 of same enzyme double digestion makes up, with recombinant vectors transformed into escherichia coli DH5 α competent cell, screening positive clone, the upgrading grain, the positive colony plasmid that will contain gusA expressed intact external member, called after pC1300 (KLmanA) ∷ gus, its physical map as shown in Figure 2.

Two, the screening of gusA transgenic tall fescue grass

Plasmid pC1300 (KLmanA) ∷ gus is transformed Agrobacterium LBA4404 with freeze-thaw method, obtain containing the Agrobacterium of this plasmid, called after LBA4404-(KLmanA) ∷ gus uses the Agrobacterium infestation method that pC1300 (KLmanA) ∷ gus is transformed the turfgrass Festuca Arundinacea again, and concrete grammar is as follows:

1) cultivation of Agrobacterium

Agrobacterium LBA4404-(KLmanA) ∷ gus is seeded to YEB substratum (beef extract 5g/L, yeast extract paste 1g/L, peptone 5g/L, sucrose 5g/L, the MgSO that 5mL contains kantlex 50mg/L and rifomycin 50mg/L ₄7H ₂O 0.5g/L, agar 15g/L, pH 5.8) in, shaking culture was transferred 100 μ l to the fresh YEB substratum that contains kantlex 50mg/L and rifomycin 50mg/L after 24 hours under 28 ℃, 180rpm, and 28 ℃ of concussions were cultivated about 12 hours, treated bacterium liquid OD ₆₀₀Value is about at 0.8 o'clock, and 5000rpm collected thalline in centrifugal 10 minutes, and thalline is re-suspended in the MS liquid nutrient medium that contains the 0.1mM Syringylethanone.

2) the Festuca Arundinacea callus induces

Choose tall grass (the Festuca arundinacea Schreb.) seed of mature and plump, embryo is peeled, after the crosscut into two, it is inserted callus inducing medium (on the basis of MS minimum medium, add 2, the 4-dichlorphenoxyacetic acid (2,4-D) 5mg/L, caseinhydrolysate 0.5g/L, pH 5.8), every fortnight changes ware once with callus.And before infecting, callus is transferred to the callus state and adjusts on the substratum and (on the basis of MS minimum medium, add 2,4-D 0.5-2mg/L, cytokinin-like substance 6-benzyl aminopurine (6-BA) 0.1-0.5mg/L, kinetin (KT) concentration 0.1-0.5mg/L, caseinhydrolysate concentration is at 0.5g/L, and pH 5.8).

3) callus infects and screens

Place Agrobacterium LBA4404-(KLmanA) the ∷ gus bacterium liquid of step 1) to soak 10 minutes the callus of adjusting state, remove bacterium liquid, switching is gone on the callus inducing medium, 25 ℃ cultivate 3 days altogether after, with the callus screening culture medium of adding seminose callus is screened and (on the basis of MS minimum medium, to add 2,4-dichlorphenoxyacetic acid (2,4-D) 2mg/L, 6-benzyl aminopurine 0.1mg/L, caseinhydrolysate 0.5g/L, sucrose 5g/L, seminose 15g/L, Pyocianil 250mg/L), 2-3 is after week, and is as seen fresh, the resistant calli of ivory buff grows out.

4) regeneration

The dark cultivation after one month, place the regeneration culture medium that adds seminose (on the basis of MS minimum medium, to add cytokinin-like substance 6-BA 0.5mg/L resistant calli, KT 0.3mg/L, sucrose 5g/L, seminose 10g/L, Pyocianil 250mg/L) carry out differentiation culture on, the visible differentiation of transgenic calli is sprouted and root, and the non-transgenic contrast does not have normal bud and root to produce.With the callus that differentiates root and bud routinely culture condition proceed to cultivate, obtain transgenosis regeneration tall grass.

5) detection of transfer-gen plant

Again the transgenosis regeneration tall grass that obtains through screening is carried out glucuronidase activity dyeing experiment, with the non-transgenic plant is contrast, concrete grammar is: clip tall grass blade is soaked in (1%X-Gluc in the X-Gluc reaction solution, 2.5mM the Tripotassium iron hexacyanide, 2.5mM yellow prussiate of potash, 0.05%Triton-X 100, and pH 7.0), put 37 ℃ and be incubated 12-24 hour down, decolour with dehydrated alcohol again.The gusA of result in the transgenic tall fescue grass that screening obtains can correctly express, and presents blueness, and the screening positive rate reaches 30%.

The screening of embodiment 4, gusA transgenic arabidopsis

Now with Agrobacterium LBA4404-(KLmanA) the ∷ gus arabidopsis thaliana transformation of petal dip method with embodiment 3 structures, concrete grammar is as follows:

1) cultivation of Agrobacterium

Agrobacterium LBA4404-(KLmanA) ∷ gus is inoculated in 5mL to be contained in the LB liquid nutrient medium of kantlex 50mg/L and rifomycin 50mg/L, shaking culture is about 48 hours under 28 ℃, 180rpm, then bacterium liquid is poured in 1 liter of LB liquid nutrient medium, continued to be cultured to OD ₆₀₀Be 1.2-1.8 (needing 48 hours approximately), after cultivating end, the 5000rpm room temperature was collected thalline in centrifugal 10 minutes, somatic cells was resuspended in 1 liter of conversion fluid (1/2MS macroelement, 1/2MS trace element again, 1/2 * molysite, 1 * B5 is organic, 5% sucrose, 1mg/L 6-BA, 50 μ lSilwetL-77 transfer pH to 5.8 with NaOH) in.

2) conversion of Arabidopis thaliana and cultivation

When treating the seed germination of Arabidopis thaliana (Arabidopsis thaliana Columbia) and being cultured to the high 3-10cm of stem, go its terminal inflorescence, to stimulate the growth of axillary inflorescence, after continued growth 7-9 days, keep petal, be partially submerged in more than Arabidopis thaliana base the lifes portion step 1) acquisition the Agrobacterium suspension in, put into vacuum pump, be evacuated to 0.5Bar, kept 5 minutes.Take out transformed plant then, it is disposed across in the plastic tub, build with plastic film, keeping humidity, lucifuge 22 ℃ of following constant temperature culture 1 day, is erect then and is cultivated, treat the pod flavescence after, carefully collect seed.

3) screening of transgenic positive plant

MS be will be tiled in through the seed of surface sterilization and substratum (1 * MS macroelement, 1 * B selected ₅Trace element, 1 * molysite, 1 * B ₅Organic element, 100mg inositol, agar 7.8g, add the 1.8g/L seminose again after regulating pH value to 5.8 with KOH) in, density is 100-200 grain seed/ware, 4 ℃ of vernalization 3 days, move into culturing room again at constant temperature (22 ℃), (light intensity is 30-40 μ mol.m in illumination in 24 hours ^-2.s ^-1) cultivate down, one week back screening true leaf health (being deep green), root is stretched to the plant in the substratum, go in the MS substratum that contains the 1.8g/L seminose, cultivate and move into soil after 10 days, transfer-gen plant has the growth of tangible root generation and seedling, and the non-transgenic contrast does not have normal root to produce and the seedling growth.

4) detection of transfer-gen plant

Again the transgenosis regeneration Arabidopis thaliana that obtains through screening is carried out glucuronidase activity dyeing experiment, with the non-transgenic plant is contrast, and detection method is identical with embodiment 3, and the gusA of result in the transgenic arabidopsis that screening obtains can correctly express, present blueness, screening rate reaches 80%.

Sequence table

<160>2

<210>1

<211>1358

<212>DNA

＜213〉carat dimension saccharomyces lactis (Kluyveromyces lactis)

<400>1

ctcgagtacc?atgccacagc?ttttcaggtt?agatgctggc?ttccagcagt?acgattgggg 60

taagatcggt?tcatcctctg?cagttgctca?gtttgctgct?cattcagacc?catcagtaaa 120

aattgatgaa?cagaaaccat?atgccgaact?atggatgggt?actcatcata?aggttccatc 180

ttataatcat?gatactaaag?aatcgttgcg?tgatttgatt?gaagctgatc?cagttggaat 240

gttgggtcaa?ggcaacgtcg?acaagtttgg?ttctatgaag?gaattaccat?tcttgtttaa 300

agttttatcc?attaaaaaag?tgttatctat?ccaagcccat?ccagataagg?ctttagctaa 360

agtcttacat?ttcaacgatc?ctgccaatta?tcctgatgat?aaccacaaac?cggaaatggc 420

gttggcagtc?accgattttg?aaggtttctg?tggattcaag?ccattggaag?agattgcaga 480

tgagttgcaa?cgtatcccag?aattgagaaa?tatcgttggg?gatgaagttt?ccgaaacttt 540

tatcaacaat?attaacccag?aggcagtgaa?ggattctgct?gatgatgcca?agaacaagaa 600

actcttgcaa?caagtattca?gtaaagttat?gaacgcttct?gataaagtgg?tggtagaaaa 660

cgcacgtgcg?ttgatcaaga?gagctcatga?atctcctgct?gatttcaaca?aggacacatt 720

gccacaactc?ttaatagact?tgaatgaaca?gttcccagat?gatgttggtt?tgttctgtgg 780

tgggttactt?ctaaaccatt?gtaacttgaa?agcaggtgaa?gcaatcttct?tgagagcgaa 840

ggacccacac?gcgtacattt?ctggtgacat?catcgaatgt?atggctgctt?ctgataacgt 900

cgttagagca?ggtttcacac?caaagttcaa?ggacgttaaa?aacttggtcg?aaatgttgac 960

atacacttac?gattccgtcg?aaaaacaaaa?gatgtctcca?gaaaactttg?aaagatcttc 1020

tggccaaggt?aaatctgttc?tatttaatcc?accaattgag?gagtttgcag?tattgtacac 1080

caccttccag?gatggtgtcg?gtacgagaca?ctttgagggt?ttacacggtc?cttccattgt 1140

gatcaccacc?aagggtaacg?gtttcatcaa?gacaggtgat?ttgaaattga?aagcggaacc 1200

gggttttgtc?ttcttcatcg?cacctggaac?tgaagtcgac?ttcattgcag?acgacaccga 1260

ctttaccaca?tacagagcat?ttgtcgaacc?aaattaaata?aatgttttga?gaaccacaat 1320

caaagaaaca?gtatgatctg?tcgatcgaca?agctcgag 1358

<210>2

<211>428

<212>PRT

＜213〉carat dimension saccharomyces lactis (Kluyveromyces lactis)

<400>2

Met?Pro?Gln?Leu?Phe?Arg?Leu?Asp?Ala?Gly?Phe?Gln?Gln?Tyr?Asp?Trp

1 5 10 15

Gly?Lys?Ile?Gly?Ser?Ser?Ser?Ala?Val?Ala?Gln?Phe?Ala?Ala?His?Ser

20 25 30

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

35 40 45

Met?Gly?Thr?His?His?Lys?Val?Pro?Ser?Tyr?Asn?His?Asp?Thr?Lys?Glu

50 55 60

Ser?Leu?Arg?Asp?Leu?Ile?Glu?Ala?Asp?Pro?Val?Gly?Met?Leu?Gly?Gln

65 70 75 80

Gly?Asn?Val?Asp?Lys?Phe?Gly?Ser?Met?Lys?Glu?Leu?Pro?Phe?Leu?Phe

85 90 95

Lys?Val?Leu?Ser?Ile?Lys?Lys?Val?Leu?Ser?Ile?Gln?Ala?His?Pro?Asp

100 105 110

Lys?Ala?Leu?Ala?Lys?Val?Leu?His?Phe?Asn?Asp?Pro?Ala?Asn?Tyr?Pro

115 120 125

Asp?Asp?Asn?His?Lys?Pro?Glu?Met?Ala?Leu?Ala?Val?Thr?Asp?Phe?Glu

130 135 140

Gly?Phe?Cys?Gly?Phe?Lys?Pro?Leu?Glu?Glu?Ile?Ala?Asp?Glu?Leu?Gln

145 150 155 160

Arg?Ile?Pro?Glu?Leu?Arg?Asn?Ile?Val?Gly?Asp?Glu?Val?Ser?Glu?Thr

165 170 175

Phe?Ile?Asn?Asn?Ile?Asn?Pro?Glu?Ala?Val?Lys?Asp?Ser?Ala?Asp?Asp

180 185 190

Ala?Lys?Asn?Lys?Lys?Leu?Leu?Gln?Gln?Val?Phe?Ser?Lys?Val?Met?Asn

195 200 205

Ala?Ser?Asp?Lys?Val?Val?Val?Glu?Asn?Ala?Arg?Ala?Leu?Ile?Lys?Arg

210 215 220

Ala?His?Glu?Ser?Pro?Ala?Asp?Phe?Asn?Lys?Asp?Thr?Leu?Pro?Gln?Leu

225 230 235 240

Leu?Ile?Asp?Leu?Asn?Glu?Gln?Phe?Pro?Asp?Asp?Val?Gly?Leu?Phe?Cys

245 250 255

Gly?Gly?Leu?Leu?Leu?Asn?His?Cys?Asn?Leu?Lys?Ala?Gly?Glu?Ala?Ile

260 265 270

Phe?Leu?Arg?Ala?Lys?Asp?Pro?His?Ala?Tyr?Ile?Ser?Gly?Asp?Ile?Ile

275 280 285

Glu?Cys?Met?Ala?Ala?Ser?Asp?Asn?Val?Val?Arg?Ala?Gly?Phe?Thr?Pro

290 295 300

Lys?Phe?Lys?Asp?Val?Lys?Asn?Leu?Val?Glu?Met?Leu?Thr?Tyr?Thr?Tyr

305 310 315 320

Asp?Ser?Val?Glu?Lys?Gln?Lys?Met?Ser?Pro?Glu?Asn?Phe?Glu?Arg?Ser

325 330 335

Ser?Gly?Gln?Gly?Lys?Ser?Val?Leu?Phe?Asn?Pro?Pro?Ile?Glu?Glu?Phe

340 345 350

Ala?Val?Leu?Tyr?Thr?Thr?Phe?Gln?Asp?Gly?Val?Gly?Thr?Arg?His?Phe

355 360 365

Glu?Gly?Leu?His?Gly?Pro?Ser?Ile?Val?Ile?Thr?Thr?Lys?Gly?Asn?Gly

370 375 380

Phe?Ile?Lys?Thr?Gly?Asp?Leu?Lys?Leu?Lys?Ala?Glu?Pro?Gly?Phe?Val

385 390 395 400

Phe?Phe?Ile?Ala?Pro?Gly?Thr?Glu?Val?Asp?Phe?Ile?Ala?Asp?Asp?Thr

405 410 415

Asp?Phe?Thr?Thr?Tyr?Arg?Ala?Phe?Val?Glu?Pro?Asn

420 425

Claims

1, Phophomannose isomerase gene is one of following nucleotide sequence:

1) SEQ ID № in the sequence table: 1 dna sequence dna;

2) SEQ ID № in the code sequence tabulation: 2 dna sequence dna;

2, the described Phophomannose isomerase gene encoded protein of claim 1 has one of following amino acid residue sequences:

1) the SEQ ID № in the sequence table: 2;

3, being used to screen the carrier of transgenic plant, is the plant binary expression vector that contains the described Phophomannose isomerase gene of claim 1.

4, carrier according to claim 3 is characterized in that: the promotor and the terminator that contain the mannose isomerase gene in the described carrier; Described promotor is cauliflower mosaic virus CaMV 35S promoter, Actin promotor or Ubiquitin promotor; Described terminator is Tnos terminator or T35S terminator.

5, according to claim 3 or 4 described carriers, it is characterized in that: the carrier that sets out that is used to make up described carrier is pCAMBIA1300, pBI121, pCAMBIA1301 or Bin19; With pCAMBIA1300 is that the set out plant binary expression vector of vector construction is pC1300 (KLmanA).

6, the described gene of claim 1 is as the application of transgenic plant selection markers.

7, application according to claim 6, it is characterized in that: the step of described screening is, target gene and promotor thereof and terminator are connected in the described carrier of claim 3, obtain the transfer-gen plant screening vector of target gene, again this carrier is transformed plant, through the seminose screening, obtain transgenic plant.

8, application according to claim 7, it is characterized in that: the transfer-gen plant screening vector of described target gene is transformed plant callus, to place on the callus screening culture medium that contains seminose through the callus of conversion again and filter out resistant calli, then resistant calli is placed on the regeneration culture medium that contains seminose and break up again, subsequently the transgenic seedling that differentiates root and bud is cultivated, obtained the transfer-gen plant of target gene; The described callus screening culture medium that contains seminose is to add 2 on the basis of MS minimum medium, 4-dichlorphenoxyacetic acid 0.5-8mg/L, 6-benzyl aminopurine 0-0.5mg/L, kinetin 0-0.5mg/L, caseinhydrolysate 0.5-2g/L, sucrose 0-15g/L, seminose 10-30g/L and microbiotic, described microbiotic is Pyocianil or Reflin, and wherein, the concentration of Pyocianil is 250mg/L; The regeneration culture medium that contains seminose is to have added 6-BA 0.1-0.5mg/L on the basis of MS minimum medium, kinetin 0.1-0.5mg/L, sucrose 0-15g/L, seminose 5-20g/L and microbiotic, described microbiotic is Pyocianil or Reflin, wherein, the concentration of Pyocianil is 250mg/L.

9, application according to claim 7, it is characterized in that: the transfer-gen plant screening vector of described target gene is transformed the plant propagation organ, and the results seed, the seed after will sprouting again places the enterprising row filter of the screening culture medium that contains seminose, obtains the transfer-gen plant of target gene; The described screening culture medium that contains seminose is to have added seminose 1.5-2.0g/L on the basis of MS minimum medium.

10, according to claim 6 or 7 or 8 or 9 described application, it is characterized in that: described plant comprises monocotyledons and dicotyledons.