CN1262654C - Corn height related gene and coding protein and uses - Google Patents

Abstract

The present invention discloses a sequence with maize plant height genes, a coding protein and an application of the gene, which aims to provide a gene relevant to the height of maize plants and a coding protein thereof, and a method for cultivating short-stalked maize by utilizing the gene. The gene relevant to the height of maize plants is one of the following nucleotide sequences: (1) the DNA sequence of SEQID No. 1 in the sequence table, (2) the polyribonucleotide of the protein sequence SEQID No. 2 in the coding sequence table and (3) the DNA sequence for encoding the identical functional proteins and having more than 95% homology with the DNA sequence limited by SEQID No. 1 in the sequence table. The gene relevant to the height of maize plants exerts important action in genetic improvement of maize.

Description

With corn plant height genes involved and proteins encoded and application

Technical field

The present invention relates in the genetically engineered field application with corn plant height genes involved and proteins encoded thereof, particularly cultivating the application of downgrading in the corn with corn plant height genes involved ZmDWF1 and proteins encoded thereof and this gene for one.

Background technology

In the genetic improvement of corn, plant height is crucial proterties, and it and corn plant type, planting density are in close relations, and the desirable plant height of quick breeding corn is the target that breeding man has painstakingly been pursued since long-term.What use in the domestic and international corn breeding for a long time, all is the nanism shape of polygene decision.In order to reach the ideal plant height, often to select, thereby seriously influence the breeding efficiency of corn through too much generation.Though also found the short living mutant of many single-genes in corn, up to the present, people carried out more careful genetic research to 28 short living genes at least, wherein 26 be positioned (Lin etc., 1995).But most short living genes or since with other bad gene close linkage, perhaps because one because of multiple-effect, every individuality that has these genes often shows the plant nanism, is not easy to breeding utilization.Therefore people think that always the single-gene nanism does not have utility value on corn breeding.

Though the nanism shape to plant has carried out a large amount of research, for a long time, people also understand seldom the mechanism of plant control plant height.In recent years, people have cloned several dwarfing genes (dwf1/dim/cbb1, dwf2/bri1/cbb2, dwf3/cbb3/cpd, dwf4, det2 etc.) with modern molecular biology techniques such as T-DNA labeling acts in Arabidopis thaliana, and have studied it and cause short molecule mechanism.Discover that the sudden change of these genes has influenced the biosynthesizing or the signal transduction of brassinolide.

Brassinolide is a kind of plant steroid hormone, and it has physiological effect widely in plant materials, and its effect comprises: cell elongation, cell fission, vascular bundle differentiation, pollen tube growth, fertilization, aging and environment-stress reaction etc.Except that Arabidopis thaliana, people have cloned two pea gene-1s of control brassinosteroid synthetic kb (homologous gene of dwf1) and 1K (homologous gene of det2) and two paddy gene OsDIM (homologous gene of dwf1) and brd1 gene.Though a large amount of short living mutant is also arranged in corn, does not up to the present see clone as yet with brassinolide biosynthesizing or signal conduction genes involved.

At present, only in a few crops such as pea, cotton, cloned the homologous gene of DWF1/DIM gene.L.Tao and N.Kameya etc. have cloned from paddy rice and have obtained this homologous gene, but have only submitted aminoacid sequence in GeneBank to.In addition, recently people such as Isabell Greevel clone the homologous gene Seladin-1 that obtains the DWF1/DIM gene from the mankind, and the product of this genes encoding can be resisted neuron degeneration and the oxidative pressure that the Alzheimer dementia causes and coerces.

Summary of the invention

The purpose of this invention is to provide a kind of and corn plant height genes involved and proteins encoded thereof.

A kind of and corn plant height genes involved provided by the present invention, name is called ZmDWF1, derives from Zea corn (Zea mays L.), is one of following nucleotide sequences:

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

2) SEQ ID № in the code sequence tabulation: the open reading frame of 2 protein sequences.

CDNA sequence in the sequence 1 is by 2005 based compositions, and the open reading frame of this gene is from 5 ' end the 127th to the 1815th bit base.

Described and corn plant height genes involved, be that reverse transcription cDNA with total RNA of corn is a template, amplification by 5 ' RACE and 3 ' RACE and whole gene cDNA obtains, and the used primer of described 5 ' RACE is the universal amplification primer AAP (Abridged Anchor Primer:5 '-GGC CAC GCG TCG ACT AGT ACG GGI IGG GIIGGG IIG-3 ') that simplifies, the SEQ ID № in the sequence table: 3 and sequence table in sequence 4; The used primer of described 3 ' RACE is the SEQ ID № in AP (Adapter primer:5 '-GGC CAC GCG TCG ACT AGT ACT TTT TTT TTT TTTTTT T-3 '), the sequence table: 5 and sequence table in sequence 6; The primer of the amplification of described total length corn plant height genes involved cDNA is the SEQ ID № in the sequence table: 7 and sequence table in sequence 8.

A kind of and corn plant height related gene coded protein, name is called ZmDWF1, derive from Zea corn (Zea maysL.), be to have SEQ ID № in the sequence table: the protein of 2 amino acid residue sequences, or with SEQ ID №: 2 amino acid residue sequence is through replacement, disappearance or the interpolation of one or several amino-acid residue and have the № with SEQ ID: 2 amino acid residue sequence is identical active by SEQ ID №: 2 deutero-protein.

The protein that the amino acid residue sequence of sequence 2 is made up of 562 amino-acid residues in the sequence table.

Contain expression carrier of the present invention and clone, as recombinant vectors pT-MD, p3301F all belongs to protection scope of the present invention with the intestinal bacteria (E.coli) (DH5 α) that contain this gene.

Arbitrary segmental primer is to also within protection scope of the present invention among the amplification ZmDWF1.

Second purpose of the present invention provides a kind of method of downgrading corn of cultivating.

The method of corn is downgraded in cultivation provided by the present invention, be in the reticent corn with the plant height genes involved, suppress this expression of gene, the corn that obtains downgrading.

ZmDWF1 gene in the reticent corn can pass through accomplished in many ways, as double-stranded RNA perturbation technique (Double-stranded RNA interference, RNAi) method of silencer, the method of sense-rna silencer, the method of plant viral vector mediated gene silencing, T-DNA inserts sudden change, the method of silencer of the present invention is not limited to above-mentioned several method, as long as can all can by reticent ZmDWF1, be preferably the method for RNAi silencer, as obtaining recombinant vectors on the conversion carrier, as p3301RNAi with being cloned into the form of double-stranded RNA i with corn plant height genes involved, by Agrobacterium osmose process maize transformation, the corn that obtains downgrading.

The inventor is a target sequence with the dwf1 gene of Arabidopis thaliana, obtains the EST fragment (471 Nucleotide) of a corn, and it and Arabidopis thaliana dwf1/dim gene have 77% homology.With RACE technology (Rapidamplification cDNA ends, the terminal rapid amplifying technology of cDNA) obtaining 3 ' of this cDNA of corn holds and 5 ' end fragment, and the clone has obtained corn ZmDFF1 full length gene cDNA, because Argine Monohydrochloride sequence of deriving with brassinolide biosynthesis related genes DWF1 of ZmDWF1 and Arabidopis thaliana has very high homology (86%), therefore infers that ZmDWF1 of the present invention is also relevant with brassinolide biosynthesizing in the corn.The present invention simultaneously also initial analysis its expression characterization at corn different tissues different development stage, and the phenotype after further being suppressed with the expression of RNAi gene studies corn ZmDWF1, the construct that utilizes this gene of success makes the plant height of corn obtain dwarfing, this transcription product that shows corn ZmDWF1 double-stranded RNA i construct among the present invention disturbs endogenous ZmDWF1 in the corn probably, it is synthetic to influence the Semen Maydis oil rape lactone, thereby causes the dwarfing of its plant height.Of the present invention and corn plant height genes involved will play a significant role in the genetic improvement of corn.

Description of drawings

Figure 1A is the pcr amplification product electrophoretogram of 5 ' RACE of ZmDWF1

Figure 1B is the pcr amplification product electrophoretogram of 3 ' RACE of ZmDWF1

Fig. 1 C is the pcr amplification product electrophoretogram of the full-length cDNA of ZmDWF1

Fig. 2 is that the Southern blot of ZmDWF1 gene in the corn gene group analyzes collection of illustrative plates

Fig. 3 A is for detecting the Northernblot collection of illustrative plates of ZmDWF1 gene expression in each tissue of corn different development stage

Fig. 3 B is the histogram of ZmDWF1 gene expression amount in each tissue of corn different development stage

The collection of illustrative plates synoptic diagram of Fig. 4 plant expression vector p3301RNAi

Fig. 5 observes for the phenotype that transgenic corn plant is downgraded plant type

Fig. 6 A is the transgenic corn plant T0 bar gene PCR amplified fragments electrophoretogram in generation

Fig. 6 B is transgenic corn plant T ₁The bar gene PCR amplified fragments electrophoretogram in generation

Fig. 7 A is the goal gene T of transformed plant ₁The Southern blot analytical results figure in generation

Fig. 7 B is the goal gene T of transformed plant ₂The Southern blot analytical results figure in generation

Fig. 8 A is transgenic corn plant T ₁The albumen coomassie brilliant blue staining electrophoretogram in generation early stage (1 month)

Fig. 8 B is the proteic Western blot collection of illustrative plates with Fig. 8 A moderate

Fig. 8 C is transgenic corn plant T ₁Albumen coomassie brilliant blue staining electrophoretogram for late period (2 first quarter moons)

Fig. 8 D is the proteic Western blot collection of illustrative plates with Fig. 8 C moderate

Embodiment

Material

1. bacterial strain, plant virus and plasmid

Intestinal bacteria (E.coli) (DH5 α) and Agrobacterium LBA4404 are available from vast company.The p3301 carrier is available from Australian CAMBIA.

2. toolenzyme and biochemical reagents

Various restriction enzymes, T-Easy support agent box and random primer test kit are available from Promega company; The dNTP mixture is available from Takara company; T ₄Dna ligase is available from BioLabs; T ₄Archaeal dna polymerase available from Takara company, naphthylacetic acid, penbritin (Amp), kantlex (Km), Streptomycin sulphate (Sm) and Reflin (Cef) available from glad through company of section; Weedicide Bastar is available from Japanese Mingzhi company; Isotropic substance α- ³²P dCTP is available from the inferior brightness in Beijing biotech firm; Nylon membrane is available from Amersham company.

3. instrument HITACHI spectrophotometer, incubator, vacuum pump, group are trained chamber, incubator, pcr amplification instrument (GeneAmp PCR System 9700), 511 type enzyme micro-plate readers, pcr amplification instrument (GeneAmp PCR System9700);

4. substratum

Related content sees that application number is " 02158758.2 ", and denomination of invention is the Chinese patent application of " a kind of method of corn being carried out gene transformation ", and wherein screening culture medium has only been used weedicide Bastar wherein.

5.PCR primer

M1：5’-AAG CAG CAC GAC GAG AA -3’

M2：5’-ATG CCA TAG AAA AGG TCA GA -3’

M3：5’-GAC CCA TGT TGA CAA GGG GCT CG -3’(SEQ ID №：3)

M4：5’-GGG GCT CGA CCT TGG CAA CC -3’(SEQ ID №：4)

M5：5’-GCA GAT GGC CGG GTC GTC AGA G -3’(SEQ ID №：5)

M6：5’-GGG TCG TCA GAG CCA CCA AGG ACA A-3’(SEQ ID №：6)

M7：5’-CAT CTA GAT CAG AGC CGT AGC AAC CAC CA -3’(SEQ ID №：7)

M8：5’-GTT GAT CAA CAA CGC TGG ACA TCA ACG ACA A-3’(SEQ ID №：8)

M9：5’-CTC ACG TGG AAA GAA ACC GCT GCT GCT AA-3’

M10：5’-GTA GAT CTC ATC AAC GAC AAT GAC TGA CCG-3’

M11：5’-CGC TCT CCC TTA TGC GAC TCC-3’

M12：5’-CTG GAC ATC AAC GAC AAT GAC TGA C-3’

M13：5’-GGC GGT CTG CAC CAT CGT CA-3’

M14：5’-GTA CCG GCA GGC TGA AGT CCA-3’

M15：5’-TTG GAT CCC CGA GGG ACG GTG ACC-3’

M16：5’-TTT CTC GAG AAT GGG CTA AGC AAA ACG-3’

It is synthetic to give birth to the worker by Shanghai.

Embodiment 1, with the clone of corn plant height genes involved ZmDWF1

The acquisition of A.EST sequence and confirmation

Dwarf1/dim full length gene with Arabidopis thaliana is compared in the other_ests storehouse of GeneBank, has found the EST of a corn chip cDNA 471bp, with the similarity of Arabidopis thaliana be 77%.This fragment probably is exactly dwarf1/dim homogenic part in corn.So design respectively at these EST two ends primer (M1, M2), extract total RNA reverse transcription of corn radicle and plumule according to a conventional method after, carry out pcr amplification with the RT-PCR method, reaction mixture is as follows:

H ₂O 10.15μl

10 * PCR damping fluid, 2.0 μ l

The cDNA 2.0 μ l of reverse transcription

M1(2μM) 2.0μl

M2(2μM) 2.0μl

dNTPmix(250μM) 1.6μl

Taq enzyme (4U/ μ l) 0.25 μ l

Amount to 20.0 μ l

Reaction conditions: Preserve.

With reference to T-Easy support agent box specification sheets with the PCR product cloning to the T-Easy carrier.Through order-checking, with the est sequence similarity of corn chip cDNA 471bp in the storehouse be 99%, thereby confirmed that this EST is dwarf1/dim homogenic part in corn.

B.5 ' amplification of RACE

At this est sequence 5 ' end design nested primers M3, M4, for guaranteeing the specificity of primer, the selective annealing temperature is 66 ℃, so also make they and 5 ' RACE test kit (GIBCOBRL NO.18374-058, available from the white biotech firm in Yuanping City, Beijing) annealing temperature of the primer AUAP that provides is approaching substantially, and they carry out pcr amplification with the AUAP primer that partners respectively.Be used for second after first round product dilutes 500 times and take turns template, first round reaction mixture is as follows: H ₂O 10.15 μ l

10 * PCR damping fluid, 2.0 μ l

The cDNA 2.0 μ l of reverse transcription

AUAP(10μM) 2.0μl

M4(10μM) 2.0μl

dNTPmix(250μM) 1.6μl

Taq enzyme (4U/ μ l) 0.25 μ l

Amount to 20.0 μ l

Reaction conditions:

Preserve.

Second to take turns reaction mixture as follows:

H ₂O 33.5μl

10×PCR buffer 5.0μl

10mM dNTP mix 1.0μl

25mM MgCl ₂ 3.0μl

M3(10μM) 2.0μl

AUAP Primer(10μM) 2.0μl

Dilute 100 times first round PCR product 5.0 μ l

Taq enzyme (5U/ μ l) 0.5 μ l

Total 50μl

The PCR response procedures is consistent with the first round, and annealing temperature is 68 ℃.

Amplified production shown in Figure 1A, shows the fragment that obtains an about 520bp and an about 480bp through 0.8% agarose gel electrophoresis result, and wherein the fragment of 520bp is consistent with the sequence size of supposition.Among Figure 1A, M1 is GeneRuler ^TM1kb bp DNA ladder (MBI-SM0313); 1 is 5 ' RACE product.Reclaim this fragment, be cloned among the pGEM@T-Easy Vector (available from Promega company) and order-checking according to ordinary method, with the dwarf1/dim similarity be 82%, can determine that basically institute's calling sequence is exactly an aim sequence.

C.3 ' amplification of RACE

For obtaining the 3 ' sequence of holding of ZmDWF1,3 ' end design primer (M5 at est sequence, M6), equally for the specificity that improves primer and with 3 ' RACE test kit (GIBCOBRL NO.18373-019, available from the former white biotech firm in Beijing) in the AP primer annealing temperature unanimity that provides, adopt 68 ℃ of higher annealing temperatures, and carry out pcr amplification with the AP primer that partners respectively.Be used for second after first round product dilutes 1000 times and take turns template, first round reaction mixture is as follows:

H ₂O 10.15μl

10 * PCR buffered soln, 2.0 μ l

The cDNA 2.0 μ l of reverse transcription

AP(10μM) 2.0μl

M6(10μM) 2.0μl

dNTPmix(250μM) 1.6μl

Taq enzyme (4U/ μ l) 0.25 μ l

Amount to 20.0 μ l

Reaction conditions: Preserve.

Second to take turns reaction mixture as follows:

H ₂O 10.15μl

10 * PCR buffered soln, 2.0 μ l

Dilute 1000 times first round PCR product 2.0 μ l

AP(10μM) 2.0μl

M5(10μM) 2.0μl

dNTPmix(250μM) 1.6μl

Taq enzyme (4U/ μ l) 0.25 μ l

Amount to 20.0 μ l

Pcr amplification program and first round basically identical are just reduced to 66 ℃ with annealing temperature.

Amplified production is through 0.8% agarose gel electrophoresis, and the result shows the fragment that obtains an about 1.3kb and the fragment of an about 750bp shown in Figure 1B, and wherein the fragment of 1.3kb is consistent with the sequence size of supposition.Among Figure 1B, M2 is GeneRuler ^TM100bp ladder plus (MBI-SM0241); 2 is 3 ' RACE product.Reclaim this fragment, be cloned into pGEM according to ordinary method ^@Among the T-Easy Vector (available from Promega company) and the order-checking, with the dwarf1/dim similarity be 72%, can determine that basically institute's calling sequence is exactly an aim sequence.

D. the amplification of total length MzDWF1 cDNA

Although the full sequence of MzDWF1 is clear substantially, yet they are three sections different sequences after all.In order to obtain the total length of MzDWF1 cDNA, design respectively at the two ends of known array primer (M7, M8), extract total RNA reverse transcription of corn radicle and plumule according to a conventional method after, carry out pcr amplification with the RT-PCR method, reaction mixture is as follows:

H ₂O 10.15μl

10 * PCR damping fluid, 2.0 μ l

The cDNA 2.0 μ l of reverse transcription

M7(2μM) 2.0μl

M8(2μM) 2.0μl

dNTPmix(250μM) 1.6μl

Taq enzyme (4U/ μ l) 0.25 μ l

Amount to 20.0 μ l

Reaction conditions:

Preserve.

Amplified production shown in Fig. 1 C, shows the cDNA clone who has obtained comprising the ZmDWF1 of whole coding region by RT-PCR, the about 1850bp of total length through 0.8% agarose gel electrophoresis result.Among Fig. 1 C, M2 is GeneRuler ^TM100bp ladder plus (MBI-SM0241); 3 is ZmDWF1 full-length cDNA product.Reclaim this fragment, be cloned into pGEM according to ordinary method ^@Among the T-Easy Vector (available from Promega company), with this carrier called after pT-MD.Match the order-checking of hundred victory biotech firms through Beijing and show that the nucleotide sequence of ZmDWF1 is shown in the sequence in the sequence table 1, its coding has the protein of the aminoacid sequence shown in the sequence 2 in the sequence table.

Embodiment 2, Southern blot analyze the copy number of ZmDWF1 in the corn gene group

Extract corn gene group DNA according to the CTAB method, get 15 μ g corn gene group DNAs respectively, through BamHI, HindIII, XbaI, DraI, EcoRI and the EcoRV enzyme that spends the night is cut, and is transferred to the Southern blotting membrane after enzyme is cut the product electrophoresis, is that probe is hybridized with the ZmDWF1 total length.The result shows at EcoRV (R) that as shown in Figure 2 HindIII (S) enzyme is cut has one to hybridize band clearly in the product, and these two kinds of enzymes all do not have the point of contact in the ZmDWF1 gene.In addition, EcoRI (R) (S) and BamHI (R) enzyme (S) cut and contain two in the product and hybridize band clearly or be less than two bands, and these two kinds of enzymes all have only a point of contact in the ZmDWF1 gene.Although in gene, do not have or a restriction enzyme site only arranged (EcoRV HindIII), has 2-3 band even more in the enzyme slitting band at remaining enzyme.The possible cause that this situation occurs is the point of contact that enzyme is arranged in the intron of this gene, and gene has been divided into several sections.Therefore, infer a copy that in the corn gene group, has only the ZmDWF1 gene.Simultaneously, this results of hybridization also shows: the ZmDWF1 gene has polymorphism in corn disease-resistant variety (p138) and susceptible variety (5003).Among Fig. 2, R is corn disease-resistant variety P138, and S is a corn susceptible variety 5003.

Embodiment 3, ZmDWF1 are at the expression of corn different development stage different tissues

Extracting total RNA in each tissue of corn different development stage according to a conventional method, accurately quantitatively, change the Northern blotting membrane with 5 μ g mRNA behind the enrichment mRNA, is that probe is hybridized with the ZmDWF1 total length then.Wherein plumule (ES) and radicle (ER) are gathered behind seed germination 48hr; Spire (YL), young stem (YS), young root (YR) are gathered after one month in plantation, and young root is crown; Mature leaf (ML), ripe stem (MS), matured root (MR), ripe tassel (PT), female fringe (PE), filigree (PS) were gathered in the pollination phase; The tip of a root 1,2,3,4 (T1, T2, T3, T4) is gathered from main radicle behind seed germination 48hr, is 0-0.5cm, 0.5-1.0cm, 1.0-2.0cm, 2.0-3.0cm successively; The tender tassel of children (YT) is not gathered before tassel exposes blade as yet.Results of hybridization is shown in Fig. 3 A and Fig. 3 B, and Fig. 3 A shows that the ZmDWF1 gene grows the hybrid belt that a treaty 2.0kb is all arranged in each tissue of different times basically at corn, and the ZmDWF1 cDNA's that this obtains by splicing with embodiment 1 is big or small consistent.Although showing the ZmDWF1 gene, Fig. 3 B all expresses in each period, but expression amount obviously has the specificity of developmental stage and tissue: except plumule, compare with other organ, ZmDWF1 expresses stronger in root, and be about as much as the young root tip of a root (0-0.5cm) (comprising root cap and tip of a root meristematic tissue) and position, maturation zone express the strongest, near the expression of facing regional ZmDWF1 mutually of the tip of a root but very a little less than.And the expression of ZmDWF1 is all more weak in reproductive organ.Because one of ZmDWF1 and Arabidopis thaliana have very high homology with the brassinolide biosynthesis related genes, these discoveries may point out that brassinolide mainly synthesizes the especially young tender and vigorous root tissue of growing in the corn in root.

Embodiment 4, the high corn of downgrading of trainer

1, the structure of corn conversion carrier

According to the restriction enzyme site analysis, cut processing pT-MD with NcoI and NotI enzyme, the big fragment that obtains is inserted between the NcoI and NotI of plasmid pET30a (+) (purchase) from Novagen, form a new plasmid pET70D, can be used for prokaryotic expression.Be template then with pET70D, (M9, M10) fragment amplification that will contain total length goal gene (ZmDWF1) comes out with primer.With this fragment and and plant conversion carrier p3301 (buy from Australian CAMBIA company) simultaneously with PmlI with BglII is two cuts and recovery respectively its product T ₄The dna ligase connection (16 ℃) of spending the night.With connecting product, filter out positive colony containing on the LB substratum of 50mg/L kantlex by heat shock method transformed into escherichia coli (E.coli) (DH5 α).The alkaline lysis method is extracted the plasmid DNA of positive colony routinely, and with M9, M10 is that primer carries out the PCR evaluation.The target gene fragment of the 2.0kb size that negative contrast do not have has all appearred in the result in the amplified reaction that with the positive colony is template.Plasmid called after p3301F after the connection.

With pET70D is template, and (M11, M12) entire segment (about 2.4kb) that will contain about the 500bp that contains multiple clone site after total length goal gene (ZmDWF1) and the pET70D promotor increases with primer.Amplified fragments reclaim the back with Klenow (the big fragment of dna polymerase i) 37 ℃ handle 30 minutes after, 75 ℃ of deactivations 10 minutes are cut p3301F with the PmlI enzyme simultaneously and are reclaimed respectively, with the product T of recovery ₄The dna ligase connection (16 ℃) of spending the night.By heat shock method transformed into escherichia coli (E.coli) (DH5 α), on the LB substratum that contains kantlex (50mg/L), filter out positive colony with the connection product that obtains.Extract the plasmid DNA of positive colony according to a conventional method, utilize four kinds of different restriction endonucleases singly to cut (NcoI, EcoRI, PstI and KpnI) respectively and carry out enzyme and cut evaluation, the result institute section of section size is all correct.Plasmid called after p3301RNAi (its collection of illustrative plates such as Fig. 4) after the connection

The reaction system of above-mentioned pcr amplification and reaction conditions are all identical with D among the embodiment 1.Enzyme is cut and ligation is all carried out with reference to " molecular cloning experiment guide " (Sa nurse Brooker is compiled, cold spring harbor laboratory, calendar year 2001, the third edition) and the specification sheets of agents useful for same.

2, the cultivation of transgenic corns

Extract the plasmid DNA of p3301RNAi according to a conventional method in a large number, get 1ug and transform Agrobacterium LBA4404 competent cell, 28 ℃, YEB substratum (containing Km 50mg/L, Sm 125mg/L) are gone up and were cultivated two days.Select six clone M9, the M10 primer, the PCR that does bacterium liquid identifies, all amplifies the target stripe that size is about 2000bp.

The Agrobacterium that will contain expression vector p3301RNAi is inoculated in the YEB substratum and (contains Km 50mg/L, Sm 125mg/L), carry out shaking culture with 250rpm, being cultured to OD600 at 28 ℃ is 0.6-0.8, bacterium liquid is placed centrifuge tube, centrifugal 5min and collect thalline under 5000rpm, the thalline of collecting is washed with the D-inf liquid nutrient medium that adds 100 μ M Syringylethanones, to remove remaining YEB substratum, at last Agrobacterium is suspended among the D-inf that adds 100 μ M Syringylethanones, the OD value is about 0.3-0.4, and is standby.(or cultivated 2-3 days with the AB solid medium, collect thalline, the thalline of collecting is washed with the D-inf liquid nutrient medium that adds 100 μ M AS, at last Agrobacterium is suspended among the D-inf that adds 100 μ M AS, the about 0.3-0.4 of OD value, placement can be used for transforming half an hour.)

Maize immature embryos and callus were put in the D-inf solution after one hour, wash once with D-inf, immerse in the D-inf Agrobacterium bacterium liquid that adds 100 μ M again, with vibrator vibration 30 seconds, and place 5 fens kinds, and take out and blot with sterilization filter paper, be put on the D-AS substratum, cultivate 3 days altogether 25 ℃ of dark, and establish contrast.

Rataria and the callus cultivated altogether after 3 days are moved in the sterilized water of the Reflin that contains 250mg/L, wash 1-3 time, immersion 30 minutes in the sterilized water of the Reflin that contains 250mg/L for the last time.Take out vegetable material, blot, put to the callus of induce substratum DN6 of the Reflin that contains 250mg/L and carried out following processing after the last week with aseptic filter paper:

1) low pressure is selected transformation receptor moved into and is added with on the screening culture medium LSD1.5 of corresponding microbiotic (Reflin 250mg/L), earlier contain Bastar 5mg/L low select to depress cultivated for 2 weeks.

2) high pressure is selected to select every the wheel for 3 weeks through 2-3 wheel Bastar 10mg/L high pressure again.Each subculture notes eliminating the callus that is brown and water stainization, and broken with the tweezers folder the normal callus of growth, separately selects to cultivate.

3) inducing then of embryoid forwards the resistant calli of choosing on the inducing embryoid body substratum D1.5DM to, and embryoid can appear in 3 weeks.

4) regeneration is transferred to the embryoid that forms again on the division culture medium RM and breaks up, and culture condition is 28 ℃, illumination every day 3000Lx light intensity illumination 16 hours, and it is born again to have seedling soon.

5) during seedling hardening regenerated plantlet length to 3 slice leaf, seedling can be divided and be transplanted in the Cans, and at indoor cultivation.

6) after regrowth is transplanted for the first time and treated that seedling grows young leaves and root, seedling is taken out from Cans, tap water washes down substratum, transplants in the small flower that is mixed with nutrition soil and vermiculite (1: 3), and initial 3-5 days, seedling should hide plastics film, to keep humidity

7) transplant when maize seedling grows 2-3 sheet young leaves again the second time of regrowth, it is moved into big Tanaka's normal growth, to the preceding bagging of blooming, artificial pollination is normally cultivated to the seed results, and the result is shown in Fig. 5 and table 1, show with the not genetically modified adjoining tree of cultivating under the same conditions and compare T ₁Generation and T ₂Short respectively for the average plant height of corn in contrast 54.94cm, 54.93cm (Fig. 5).Among Fig. 5, A, B represent transfer-gen plant (T ₁Generation) shows dwarfing (50 days) in various degree; C represents normal plant (50 days).

Table 1.T ₁Generation and T ₂Average plant height for corn

Sample	Average plant height (cm)	Sample number	Minimum value	Maximum value
					CK T 1 T 2	214.2±4.1 159.06±3.4 159.07±4.0	18 110 76	190 70 61	248 200 245

The Molecular Detection of embodiment 5, transgenic corns

1, PCR certification mark gene bar

Because goal gene itself exists in corn, so carry out the integration situation of the detection of bar gene with definite purpose construct.Concrete grammar is as follows:

DNA trace extracts: be equipped with in the Eppendoff pipe that the ice-cold CTAB of 400ul extracts damping fluid (not containing beta-mercaptoethanol) with the broken about 1-2cm of Circular glass rod milling one ²Fresh T ₀And T ₁For transgenic corn plant or transgenic corn plant young leaflet tablet not.The CTAB that adds 65 ℃ of preheatings of 500ul extracts damping fluid (containing beta-mercaptoethanol) mixing, and 65 ℃ of insulation 90min put upside down mixing frequently.Add 450ul chloroform/primary isoamyl alcohol after waiting to be chilled to room temperature, put upside down mixing to solution and be the milkiness shape---but not vibration.Centrifugal 2min layering.Liquid phase is transferred in the clean Eppendoff pipe, adds 3ul RNase A, be incubated 30min under the room temperature.Add the 600ul Virahol, put upside down mixing.Centrifugal 10min deposit D NA removes supernatant in the Eppendorf centrifuge.Use 800ul 76% ethanol successively, 0.2mol/L sodium acetate and 100ul70% washing with alcohol precipitation.Centrifugation 5min inhales then and removes residual ethanol.The vacuum pumping pump dry DNA is dissolved in DNA in the 50ul TE damping fluid 4 ℃ of preservations at last.

The reaction system of pcr amplification (the pcr amplification primer still is M13 and M14) all with embodiment 1 in identical, and

Reaction conditions is: 94 ℃, and 5min; Preserve.

Get PCR product 10ul and carry out electrophoresis detection.With plasmid p3301RNAi is over against photograph, and transfer-gen plant is not negative contrast, and the result shows at T shown in Fig. 6 A and Fig. 6 B ₁, T ₂For all there being size to be the goal gene band of 500bp in most of sample, negative contrast does not then have.Among Fig. 6 A and Fig. 6 B ,+plasmid p3301RNAi represented over against photograph; M is 1kb Ladder; The negative contrast of-expression, not genetically modified corn; 1-7 among Fig. 6 A and Fig. 6 B is respectively T ₁, T ₂For transgenic corn plant.

2, the integration of the RNAi construct of Southern hybridization testing goal gene (ZmDWF1)

Detect the transgenic corn plant of marker gene bar or total DNA of transgenic corn plant not in a large amount of extraction steps 1 of SDS method, in 1.5ml Eppendorf pipe, carry out enzymolysis:

Add following component:

Genomic dna 15-30ug

10×Buffer 10ul

HindIII restriction endonuclease 10ul

DdH ₂The O make up water is to 100ul

37 ℃ of enzymolysis 12 hours, whether enzymolysis is complete to get the 1ul electrophoresis detection, as not exclusively, then adds enzyme and Buffer; After enzyme cuts, electrophoresis, commentaries on classics nylon membrane.Preserve or be directly used in hybridization for 4 ℃.

Label probe:

Full-length cDNA with ZmDWF1 is a template, and with M15, M16 is a primer, reaction system all with embodiment 1 in identical, reaction conditions is 94 ℃, 5min;

Preserve.

Carry out PCR, obtain the special fragment 30ng of goal gene of about 960bp, mend to 32ul sex change 5min in the boiling water, ice bath 5min with sterilized water.Press the Prime-a-Gene Labelling System Kit explanation of Promega company, add successively:

5 * mark damping fluid 10ul

DA.T.G (every kind of 0.5mM) 2ul

BSA(10mg/ml) 2ul

Klenow fragment (5units/ul) 1ul

α- ³²P dCTP(10uCi/ul) 3ul

Cumulative volume 50ul

37 ℃ are incubated three hours, sex change 10min in the boiling water, and ice bath 10min, standby.

Prehybridization and hybridization:

The Hybond membrane that 5 * SSC prewets is put into hybrid pipe, and the one side that has a DNA adds 10ml and is preheating to 65 ℃ Church damping fluid and (contains 1%BSA, 1mM EDTA, 0.25M Na inwards ₂HPO ₄-NaH ₂PO ₄(pH 7.2), 7%SDS) assorted in advance 5hr adds the good probe of sex change, more than 65 ℃ of hybridization 16h.After hybridization finishes, use 2 * SSC+0.5%SDS successively, 1 * SSC+0.5%SDS, 0.5 * SSC+0.5%SDS, 0.1 * SSC+0.1%SDS washes film at 65 ℃, each 15min, after washing, filter paper blots the moisture on film surface, preservative film is wrapped, put into magazine ,-70 ℃ of radioautograph, the time of autography decides according to signal is strong and weak.This Southern results of hybridization is shown in Fig. 7 A and Fig. 7 B, show and occurred different band in the negative contrast in the transgenic corn plant that detects marker gene bar in the step 1, new band occurred, the RNAi construct of illustration purpose gene has been incorporated in the corn gene group.Among Fig. 7 A ,+expression is over against photograph, and the target gene PCR of 960bp reclaims fragment; The negative contrast of-expression, not transgenic corn plant; 1-8 is the transgenic corn plant that detects marker gene bar in the step 1.Among Fig. 7 B ,+expression is over against photograph, and the target gene PCR of 960bp reclaims fragment; The negative contrast of-expression, not transgenic corn plant; 1-7 is a transfer-gen plant.

3, the expression of Western blot method testing goal albumen (ZmDWF1 encoded protein)

With phosphate buffered saline buffer [200mM NaCl, 50mM NaH ₂PO ₄(pH7.0), 10mM MgCl ₂, 10% glycerine, 5mM beta-mercaptoethanol, 0.5mM PMSF] and add that 2%SDS extracts transgenic corn plant (comprising the transgenic corn plant that detects marker gene bar in the step 1) T ₁Generation, T ₂Generation and transgenic corn plant total protein not.Sample 40 μ g total proteins [protein quantification is with reference to Bradford method (Bradford, 1976)] carry out the SDS-PAGE electrophoresis with the Bio-Rad device on every point sample hole, change film and colour developing according to the Bio-Rad process specifications.Wherein, transgenic corn plant T ₁In generation, carried out 8% SDS-PAGE electrophoresis, transgenic corn plant T ₂In generation, carried out 12% SDS-PAGE electrophoresis.The result shows that the expression of the target protein in the dwarfed plant has been subjected to inhibition even has detected less than target protein the about 65KD of target protein size shown in Fig. 8 A, Fig. 8 B, Fig. 8 C and Fig. 8 D.Among Fig. 8 A and Fig. 8 B, 1 for downgrading serious transfer-gen plant, and 2 is the transfer-gen plant of medium dwarfing, and 3,4 is normal plant of the same age (height); Among Fig. 8 C and Fig. 8 D, PreM is prestaining marker; 1 is normal plant (height); 2 for downgrading serious transfer-gen plant (corresponding respectively with 1 among Fig. 8 A and Fig. 8 B); 3 is the transfer-gen plant (corresponding respectively with 2 among Fig. 8 A and Fig. 8 B) of medium dwarfing.

Sequence table

<160>8

<210>1

<211>2005

<212>DNA

＜213〉Zea corn (Zea mays L.)

<400>1

ccgcaggcag catctgcatc tctcgcccac ctccgctccg cctactgctg ctggtggtag 60

ggaggcggag aaggaggccc ttgcgcccgc ccgccggccg gatcagagcc gtagcaacca 120

ccacccatgg cggacgtgca cgaacctttg gtgcgccgta agaggaagaa ggttttggtg 180

gactacttgg tgaagttccg atggatcctc gtgatcttcg tggtccttcc tatttcaact 240

ctgatctact tcaacatctt cctgggcgac atgtggtccg ccatgaagtc ggagaagaag 300

cgccagaagc agcacgacga gaacgtgcag aaggtcgtga agcggctcaa gcagaggaac 360

ccgaagaagg acggtcttgt ttgcacggcc aggaagccct ggatcgctgt tggcatgcgc 420

aacgtggact acaagcgtgc gaggcatttc gaggtcgacc tttcttcctt caggaacatc 480

cttgagatcg acaaagagag gatggttgcc aaggtcgagc cccttgtcaa catgggtcag 540

ataaccagag ctacctgccc aatgaacctt gcccttgcgg tcgtcgccga gctcgacgac 600

ctcactgttg gtgggctgat caacggttac ggcatcgagg ggagctctca cctctatggc 660

cttttctccg acacggttgt cgcgatggag gttgttctcg cagatggccg ggtcgtcaga 720

gccaccaagg acaacgagta ctctgacctt ttctatggaa ttccctggtc ccagggaaca 780

ctggggttcc ttgtctctgc agagatcaag ctgatcccca tcaaggagta catgaagctc 840

acctacactc cagtcaaggg gggtctaaag gagatcgcgc aggcctacgc ggattctttc 900

gctccgaggg acggtgaccc ggcaaaggtc cctgactttg ttgaagggat ggtgtacaca 960

gagagcgagg gtgtcatgat gacgggcgtg tacgcttcga aagaagaggc gaagaagaag 1020

ggcaacaaga tcaactgcgt ggggtggtgg tttaagccct ggttctacca gcacgctcag 1080

acggcgctga ataggggcga gtttgtggag tacatcccga cgagggagta ctaccaccgg 1140

cacacccggt gcctgtactg ggaggggaag ctgatcctgc ccttcggcga ccagttctgg 1200

ttcaggttcc tgctgggctg gctgatgcca ccgaaggtgt ccctgctgaa ggcgacccag 1260

ggcgaggcta tcaggaacta ctaccacgac aaccatgtga tccaggacat gctggtgccg 1320

ctgtacaagg ttggggatgc gctggagttc gtgcaccgcg agatggaggt gtatcctctg 1380

tggctgtgcc ctcaccggct gtacaagctg ccggtgaaga cgatggtgta cccggagcct 1440

gggttcgagc accagcacag gcagggcgac gcgagctacg cacagatgtt cacggacgtg 1500

ggcgtgtact acgcccccgg ggcggtgctg aggggggagg agttcaacgg cgcggaggct 1560

gtgcacaggc tggagcagtg gctgatcgag aaccacagct accagccgca gtacgcggtg 1620

tcggagctga acgagaagga ctcctggcgc atgttcgacg cgtcgcacta cgagcactgc 1680

cgccaaaagt acggggcggt gggcacgttc atgagcgtgt actacaagtc caagaagggg 1740

cgcaagacgg agaaggaggt gcaggaggcg gaggcggcca tactggagcc ggcctacgcg 1800

gacgaggagg cctaaagctc gtggtcgttt tgcttagccc attttaatta gaacttgatg 1860

gatgtagtgt gtgtctgtct gaagtcattt taattagaac tcttaaagct cgtggtcggt 1920

cggtcagtca gtcagtcatt gtcgttgatg tccagcgttg tgtttttttt atattctcta 1980

atggaatctc tcagattgat tcggg 2005

<210>2

<211>562

<212>PRT

＜213〉Zea corn (Zea mays L.)

<400>2

Met Ala Asp Val His Glu Pro Leu Val Arg Arg Lys Arg Lys Lys Val

1 5 10 15

Leu Val Asp Tyr Leu Val Lys Phe Arg Trp Ile Leu Val Ile Phe Val

20 25 30

Val Leu Pro Ile Ser Thr Leu Ile Tyr Phe Asn Ile Phe Leu Gly Asp

35 40 45

Met Trp Ser Ala Met Lys Ser Glu Lys Lys Arg Gln Lys Gln His Asp

50 55 60

Glu Asn Val Gln Lys Val Val Lys Arg Leu Lys Gln Arg Asn Pro Lys

65 70 75 80

Lys Asp Gly Leu Val Cys Thr Ala Arg Lys Pro Trp Ile Ala Val Gly

85 90 95

Met Arg Asn Val Asp Tyr Lys Arg Ala Arg His Phe Glu Val Asp Leu

100 105 110

Ser Ser Phe Arg Asn Ile Leu Glu Ile Asp Lys Glu Arg Met Val Ala

115 120 125

Lys Val Glu Pro Leu Val Asn Met Gly Gln Ile Thr Arg Ala Thr Cys

130 135 140

Pro Met Asn Leu Ala Leu Ala Val Val Ala Glu Leu Asp Asp Leu Thr

145 150 155 160

Val Gly Gly Leu Ile Asn Gly Tyr Gly Ile Glu Gly Ser Ser His Leu

165 170 175

Tyr Gly Leu Phe Ser Asp Thr Val Val Ala Met Glu Val Val Leu Ala

180 185 190

Asp Gly Arg Val Val Arg Ala Thr Lys Asp Asn Glu Tyr Ser Asp Leu

195 200 205

Phe Tyr Gly Ile Pro Trp Ser Gln Gly Thr Leu Gly Phe Leu Val Ser

210 215 220

Ala Glu Ile Lys Leu Ile Pro Ile Lys Glu Tyr Met Lys Leu Thr Tyr

225 230 235 240

Thr Pro Val Lys Gly Gly Leu Lys Glu Ile Ala Gln Ala Tyr Ala Asp

245 250 255

Ser Phe Ala Pro Arg Asp Gly Asp Pro Ala Lys Val Pro Asp Phe Val

260 265 270

Glu Gly Met Val Tyr Thr Glu Ser Glu Gly Val Met Met Thr Gly Val

275 280 285

Tyr Ala Ser Lys Glu Glu Ala Lys Lys Lys Gly Asn Lys Ile Asn Cys

290 295 300

Val Gly Trp Trp Phe Lys Pro Trp Phe Tyr Gln His Ala Gln Thr Ala

305 310 315 320

Leu Asn Arg Gly Glu Phe Val Glu Tyr Ile Pro Thr Arg Glu Tyr Tyr

325 330 335

His Arg His Thr Arg Cys Leu Tyr Trp Glu Gly Lys Leu Ile Leu Pro

340 345 350

Phe Gly Asp Gln Phe Trp Phe Arg Phe Leu Leu Gly Trp Leu Met Pro

355 360 365

Pro Lys Val Ser Leu Leu Lys Ala Thr Gln Gly Glu Ala Ile Arg Asn

370 375 380

Tyr Tyr His Asp Asn His Val Ile Gln Asp Met Leu Val Pro Leu Tyr

385 390 395 400

Lys Val Gly Asp Ala Leu Glu Phe Val His Arg Glu Met Glu Val Tyr

405 410 415

Pro Leu Trp Leu Cys Pro His Arg Leu Tyr Lys Leu Pro Val Lys Thr

420 425 430

Met Val Tyr Pro Glu Pro Gly Phe Glu His Gln His Arg Gln Gly Asp

435 440 445

Ala Ser Tyr Ala Gln Met Phe Thr Asp Val Gly Val Tyr Tyr Ala Pro

450 455 460

Gly Ala Val Leu Arg Gly Glu Glu Phe Asn Gly Ala Glu Ala Val His

465 470 475 480

Arg Leu Glu Gln Trp Leu Ile Glu Asn His Ser Tyr Gln Pro Gln Tyr

485 490 495

Ala Val Ser Glu Leu Asn Glu Lys Asp Ser Trp Arg Met Phe Asp Ala

500 505 510

Ser His Tyr Glu His Cys Arg Gln Lys Tyr Gly Ala Val Gly Thr Phe

515 520 525

Met Ser Val Tyr Tyr Lys Ser Lys Lys Gly Arg Lys Thr Glu Lys Glu

530 535 540

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

545 550 555 560

Glu Ala

562

<210>3

<211>23

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>3

gacccatgtt gacaaggggc tcg 23

<210>4

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>4

ggggctcgac cttggcaacc 20

<210>5

<211>22

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>5

gcagatggcc gggtcgtcag ag 22

<210>6

<211>25

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>6

gggtcgtcag agccaccaag gacaa 25

<210>7

<211>29

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>7

catctagatc agagccgtag caaccacca 29

<210>8

<211>31

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>8

gttgatcaac aacgctggac atcaacgaca a 31

Claims (8)

1, a kind of and corn plant height related gene coded protein, be to have SEQ ID № in the sequence table: the protein of 2 amino acid residue sequences, or with SEQ ID №: 2 amino acid residue sequence is through replacement, disappearance or the interpolation of one or several amino-acid residue and have the № with SEQ ID: 2 amino acid residue sequence is identical active by SEQID №: 2 deutero-protein.

2, protein according to claim 1 is characterized in that: described and corn plant height related gene coded protein is the SEQ ID № in the sequence table: 2.

3, a kind of and corn plant height genes involved is one of following nucleotide sequences:

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

2) SEQ ID № in the code sequence tabulation: the polynucleotide of 2 protein sequences.

4, gene according to claim 3 is characterized in that: described and corn plant height genes involved is the SEQ ID № in the sequence table: 1.

5, contain the described expression carrier of claim 3.

6, the transgenic cell line that contains the described gene of claim 3.

7, a kind of method of downgrading corn of cultivating is the described and corn plant height genes involved of claim 3 in the reticent corn, suppresses this expression of gene, the corn that obtains downgrading.

8, the application of the described gene of claim 3 in the genetic improvement of corn.

Images