CN107022551A - One kind regulates and controls big arabidopsis seedling stage trophosome, early blossoming and the increased corn gene ZmGRAS37 of grain weight and its application - Google Patents

Abstract

Regulate and control big arabidopsis seedling stage trophosome, early blossoming and the increased corn gene of grain weight the invention provides one kindZmGRAS37And its application.The present invention separates and is cloned into gene from cornZmGRAS37, willZmGRAS37CDNA sequence be connected to 35S promoter startup expression vector pPZP211 on, arabidopsis thaliana transformation is infected using Agrobacterium infestation method, test result indicates that model plant arabidopsis can be made to occur trophosome in seedling stage is big for the gene, and the character such as there is early blossoming and grain to increase again.Phylogenetic analysis result shows, the direct homologous gene without the gene in model plant arabidopsis, but the homology of the gene and grass sorghum, corn is higher, and it is GRAS family genes specific to grass to illustrate the gene；In view of conservative of the gene in grass and the phenotype presented in transgenic arabidopsis, it was demonstrated that the gene has potential application value.

Description

One kind regulates and controls big arabidopsis seedling stage trophosome, early blossoming and the increased corn gene of grain weightZmGRAS37And its application

Technical field

The invention belongs to field of plant genetic, and in particular to one kind regulation and control arabidopsis seedling stage trophosome is big, early Flower and the increased corn gene of grain weightZmGRAS37And its application.

Background technology

The agricultural production of China's the Yellow River and Huai He River sea region is mainly the crop rotation of wheat and corn.Because the production of corn is intended to wheat Machine is harvested after machine is live and ripe after receipts, in the urgent need to cultivating breeding time short corn variety in breeding work.Crop is sought Foster body can significantly increase greatly the photosynthetic efficiency of crop, improve the yield of the crop for the purpose of obtaining trophosome；Appropriateness Early blossoming can shorten the breeding time of crop, improve the adaptability of kind, thus be important crop economical character.Therefore obtain more Many trophosomes are big, early blossoming and grain weigh increased genetically modified crops, can be obviously promoted the grasses such as corn, paddy rice Molecular design breeding work.

GRAS albumen is distinctive protein family in plant, and its name derives from three found first in plant kingdom GRAS protein membersGAI [GA (GIBBERELLICACID)-INSENSITIVE], RGA (REPRESSOR OF GAI) andSCR (SCARECROW) (Pysh et al., 1999).GRAS gene families are referred to as " green revolution " of gene, to the family The research of race has had more than the history of 10 years, has now been found that GRAS protein families are related to plant transcription level modulation and control Multi-signal transduction pathway (the Bolle of growth and development of plantset al. 2004).At present, in 294 kinds of terrestrial plants It was found that, GRAS genes have 1035 kinds of sequence (Sunet al. 2012).The analysis that the GRAS albumen of different plant species is carried out is taken off Show that they have a similar subfamily branch, including ten subfamilies, the title of these subfamilies is with family inside A member or a general motif come what is named, be respectively：AtLAS (Arabidopsis LATERAL SUPPRESSOR), AtSCL (Arabidopsis SCR-like), HAM (HAIRY MERISTEM), AtSCR (Arabidopsis SCR), DLT (DWARF AND LOW TILLERING), AtSCL3, DELLA, AtPAT1 [Arabidopsis pat (phytochrome A signal transduction) 1-1], AtSHR [Arabidopsis SHR (SHORTROOT)] and LISCL (Lilium longiflorum SCR-like) (Bolle et al. 2004, Tian et al.2004, Sun et al. 2011, Lim et al. 2005, Sanchez et al. 2007).At present Still some GRAS albumen has not found suitable subfamily, it is necessary to follow-up research.

There is very big difference, the place collection of main difference on nucleotide sequence and length in each member of GRAS protein families In in N-terminal, comprising by proline, glutamic acid, tyrosine, serine, glycine, aspartic acid and alanine etc. constitute it is same Property paradigmatic structure domain, but their C-terminal has very high homology, and C-terminal conserved sequence mainly has：Leucine Heptad Repeat（LHR）, VHIID, LeucineHeptad Repeat（LHR）, PFYRE and SAW motifs (Hrichet al. 2009)。

Except the presence of specific motif proves that GRAS family members can be used as transcription regulatory factor（Pyshet al. 2000, Bolle 2004）Outside, the nuclear location of most of GRAS albumen can also illustrate this point.Except PAT1 subfamilies it Outside, other GRAS transcription factor families member largely enters nuclear signal containing one（NLS）（Bolle 2004）, therefore GRAS Can albumen be exactly whether it has nuclear localization signal as one of condition of transcription regulatory factor.

GRAS transcription factor family albumen is related to the various aspects of plant growth, and regulation is played in multiple growth courses and is made With, including the signal transduction of hormone, phytomorph development multiple processes such as build up.Mainly include following aspect：

（1）GRAS transcription factor proteins are related to the growth of root.GRAS gene family member's AtSCR genes of first discovery it is prominent Variant, because the afunction of the gene causes the growth failure of root, the functions of SCR genes is asymmetric with root cortical cell The radiation growth of division and root is related.In addition, another GRAS transcription factor family member short root（SHR）Gene It is proved to related to the radiation growth of root.

（2）GRAS transcription factor families are related to the growth of axillary meristem.Schumacher et al. was in 1999 The afunction mutant for the tomato Ls genes mentioned in work shows plant without branch, the development of axillary bud separate living tissue early stage It is blocked, and the VHIID motifs of GRAS transcription factor families is included in the structure of Ls genes, fully proves GRAS transcription factors man Race is relevant with the growth of axillary meristem.The gene in arabidopsis and paddy rice in homologous gene AtLAS/SCL1 and The mutant of OsMOCI afunction, which equally shows axillary bud, to be sprouted.This also illustrates GRAS transcription factor families and axillary point The growth of raw tissue has substantial connection.

（3）GRAS transcription factor families are related to the characteristic holding of shoot apical meristem.HAM subfamilies are the sub- families of GRAS One of race member, the petunia hairy meristem that Stuurman et al. had found in 2002（ham）Mutant in find It no longer keeps the indiscriminate characteristic of shoot apical meristem, and the growth of shoot apical meristem and axillary meristem stops leading The stopping of plant organ development is caused, in addition, HAM mutant forms the leaf smaller than wild type.Illustrate that HAM subfamilies are dividing Played an important role in some approach of raw tissue growth.

（4）GRAS family members are related to the conductive process of phytochrome signal.PAT1 subfamilies are GRAS families weights Member is wanted, arabidopsis PAT1 mutant makes its phytochrome A signal transduction process exception, chalcone synthase and chlorophyll The expression of synthetic proteins is severely impacted, and also results in the lower axle extension of mutant, but is not affected under other light, Therefore PAT1 genes are single-minded related to phytochrome A signal transduction.

（5）GRAS family members are related to gibberellin signal transduction.Gibberellin usually promote the extending of stem, flower formation with And the germination process of seed.GAI expression products in GRAS gene families play negative regulation during gibberellin signal transduction Effect, participate in gibberellin signal transduction process GRAS albumen between homology（55%-76%）Than with other GRAS The homology of protein member（20%-27%）It is many higher.This also demonstrates that this Partial Protein participates in the process of gibberellin signal transduction.

（6）GRAS protein families can work as transcription factor.Except PAT1 subfamilies are the eggs of cytoplasm positioning Ultrawhite, most of GRAS albumen is all the albumen of nuclear location.Find there are some cores in GRAS protein structures in nearest research Positioning signal, this also works there is provided more ample evidence for GRAS albumen as transcription factor.

GRAS family genes play extremely important and unique effect in the growth and development process of plant.Therefore, for The research tool of GRAS family proteins is of great significance.It is beautiful and the research to GRAS family members in corn is not goed deep into GRAS family members in rice lack at present at aspect of growing can disclose the document retrieved.

The content of the invention

For the situation of above-mentioned prior art, it is an object of the invention to provide a kind of regulation and control arabidopsis seedling stage trophosome Greatly, early blossoming and the increased corn gene of grain weightZmGRAS37And its application, it is of the invention to separate and be cloned into from cornGRAS37 Gene, and be named asZmGRAS37, willZmGRAS37Full-length cDNA be connected to 35S promoter startup expression vector On pPZP211, arabidopsis thaliana transformation is infected using Agrobacterium, it is found that the gene can make model plant arabidopsis seedling stage nutrition occur Body is big, early blossoming and grain weigh increased phenotype.

For achieving the above object, the present invention is achieved using following technical scheme：

Regulate and control big arabidopsis seedling stage trophosome, early blossoming and the increased corn gene of grain weight the invention provides one kindZmGRAS37, Its nucleotide sequence such as SEQ ID No:Shown in 1, CDS sequences such as SEQ ID NO:Shown in 2, the amino acid of its protein encoded Sequence such as sequence SEQ ID NO:Shown in 3.

Amplification is describedZmGRAS37The primer sequence of gene is：

- the GGTACCATGGCCGCCGAGCCGGCGGATGTC-3 ' of sense primer 5 '；

- the AAGCTTTCATTTTGCAGCCCACGCAGACAT-3 ' of anti-sense primer 5 '.

Present invention also offers described geneZmGRAS37Application in the regulation and control big phenotype of plant seedling stage trophosome.

Further：ZmGRAS37Transgenic positive Arabidopsis plant blade diameter, blade compared with wild type in seedling stage Cell become big, fresh weight, dry weight increase.

Present invention also offers described geneZmGRAS37Application in regulation and control plant early blossoming phenotype.

Further：ZmGRAS37Transgenic positive Arabidopsis plant flowering time compared with wild type shifts to an earlier date three days.

Present invention also offers described geneZmGRAS37Application on regulation and control plant grain again increase phenotype.

Further：ZmGRAS37Transgenic positive Arabidopsis plant seed diameter increase and mass of 1000 kernel compared with wild type Increase.

Compared with prior art, advantages of the present invention and have the technical effect that：The present invention willZmGRAS37Full length gene cDNA On the expression vector for being connected to over-express vector startup, arabidopsis thaliana transformation is infected using Agrobacterium, it is found that the gene overexpression can So that model plant arabidopsis seedling stage big trophosome, early blossoming and grain occurs and increases isophenous again, phylogenetic analysis result is shown, mould Direct homologous gene without the gene in formula plant Arabidopsis thaliana, but the gene and grass sorghum, corn homology compared with Height, it is GRAS family genes specific to grass to illustrate the gene；Literature search is not found on the gene function The research of aspect.In view of conservative of the gene in grass and the phenotype presented in transgenic arabidopsis, recognize There is potential application value for the gene.

Brief description of the drawings

Fig. 1 is the chadogram constructed by GRAS protein family members in corn, arabidopsis, paddy rice and sorghum.

Fig. 2 is the schematic diagram and restriction enzyme site of the expression vector built.Wherein, expression vector is pPZP211, promoter For 35S, target geneZmGRAS37The restriction enzyme site at two ends is respectively Kpn1 and Hind III.

Fig. 3 A bands are the target gene being cloned intoZmGRAS37Schematic diagram, size is 2001bp；Fig. 3 B bands are Positive colony obtained by target gene connection pEASY-T1 cloning vectors；Fig. 3 C correctly carry for sequencingGRAS37Gene Cloning vector plasmids digestion result schematic diagram；Fig. 3 D are positive gram that purpose gene is connected to obtained by pPZP211 expression vectors It is grand；Fig. 3 E be withGRAS37The expression vector plasmid enzyme restriction result schematic diagram of gene.

Fig. 4 is the identification of the positive seedling of transgenic line；No. 1 swimming lane is wild type WT, and remaining swimming lane is transgenic line.

Fig. 5 A are transgenic line35S::ZmGRAS37With wild type WT in different time（It is respectively from left to right to plant Afterwards growth 14,18,21 days）The phenotypic map planted in same alms bowl.

Fig. 5 B are transgenic line35S::ZmGRAS37With wild type WT in different time（After plantation growth 14,18,21, 28 days）The phenotypic map of single-strain planting in different flowerpots.

Fig. 6 A are transgenic line35S::ZmGRAS37With wild type WT in growth 18 days（35S::ZmGRAS37Expose flower Bud）When phenotypic map.

Fig. 6 B are transgenic line35S::ZmGRAS37With wild type WT in growth 18 days（35S::ZmGRAS37Expose flower Bud）When diameter data statistics, and find have significant difference.

Fig. 6 C are respectively transgenic line35S::ZmGRAS37With wild type WT in growth 18 days（35S::ZmGRAS37Dew Go out bud）Shi Xianchong and dry weight（80 degrees Celsius are dried 24 hours）Data statistics, and find have significant difference.

Fig. 7 A are transgenic line 35S under Differential interference contrast microscope::ZmGRAS37With wild type WT bed board cells Phenotypic map.

Fig. 7 B are transgenic line 35S under entity anatomical lens::ZmGRAS37With the phenotypic map of wild type WT leaf blade sizes.

Fig. 7 C are the data statistics of bed board cell number under the A visuals field.

Fig. 7 D are the data statistics of B visuals field lower blade area.

Fig. 7 E figures are the data statistics of total bed board cell in both blades.

Fig. 8 A are transgenic line35S::ZmGRAS37With wild type WT in growth 21 days（WT exposes bud）When table Type figure.

Fig. 8 B, C are transgenic line35S::ZmGRAS37With the statistics of wild type WT early blossoming data.

Fig. 8 D are transgenic line 35S::ZmGRAS37With five in the wild type WT expression quantity with related gene of blooming QRT testing results.

Fig. 9 A are transgenic line 35S under entity anatomical lens::ZmGRAS37With the size of wild type WT seeds.

The statistics of Fig. 9 B mass of 1000 kernel for both.

Embodiment

Technical scheme is further explained in following examples, description and these implementations more than Example, those skilled in the art can determine the essential characteristic of the present invention, and in the situation without departing from spirit and scope of the invention Under, various changes and modifications can be made to the present invention, so that it is applicable various uses and condition；The present invention carries above-mentioned expression Body is imported into model plant arabidopsis cell, and introduction method is Agrobacterium-medialed transformation method.Wherein by above-mentioned expression vector Import in plant cell, introduction method is all well known in the art, and these methods are included but are not limited to：It is agriculture bacillus mediated Conversion method, particle bombardment, electrization, Ovary injection etc..The antibiotic for the positive seedling of screening that the present invention is selected is mould to block that Element；In addition, of the present invention is state of the art.

The present invention obtains total serum IgE from corn first, and reverse transcription obtains the chains of cDNA first, mould is utilized it as afterwards Plate, is usedZmGRAS37Corresponding specific primer carries out Phusion high-fidelity enzymatic amplifications to the cDNA of acquisition.

Corn RNA extraction and purifying can directly use existing process, also can be using technique described in the present invention； The synthesis of the chains of cDNA first is also identical situation, it would however also be possible to employ existing technique or kit, but both above-mentioned It is preferred to use the concrete technology described in the present invention.

Embodiment 1：The acquisition of transgenic line

（One）CornGRAS37Sequence analysis, clone and the vector construction of gene

The present invention finds gene using Maize genome database website MaizeGDBGRAS37, this base is understood using NCBI analyses Because as all GRAS protein family members, possessing a conservative C-terminal GRAS domain；GRAS37Genome sequence Total length is 2258 bp（Sequence is as shown in SEQ ID NO.1）, and CDS sequences are 2001 bp（Sequence such as SEQ ID NO.2 institutes Show）, willGRAS37Amino acid sequence（Sequence is as shown in SEQ ID NO.3）With institute in corn, arabidopsis, paddy rice and sorghum There is GRAS protein families member to build chadogram, as a result as shown in Figure 1.Fig. 1 phylogenetic analysis results show that nothing should in arabidopsis The direct homologous gene of gene, but the gene and the homology of grass sorghum are higher, therefore illustrate that the gene is grass A gene specific to section plant.

According to the corn foundGRAS37The CDS primers of gene, are cloned, cloning process is as follows：

（1）RNA extraction：It is the total serum IgE for extracting corn in century TRIzon using health.

1. taking fresh plant tissue material to be fully ground in liquid nitrogen, per 30-50mg, tissue adds 1ml TRIzon, whirlpool Vibration is mixed, and room temperature is placed 5 minutes, is kept completely separate protein nucleic acid compound.

2.4 DEG C, 12000g is centrifuged 10 minutes, takes supernatant into new RNase-Free centrifuge tubes.

3. adding 400 μ L chloroforms into above-mentioned centrifuge tube, acutely concussion 15 seconds, room temperature is placed 2-3 minutes.(chloroform can be used Extracting is twice).

4.4 DEG C, 12000g is centrifuged 15 minutes, and the superiors' colourless aqueous phase is transferred to a new RNase-Free by liquid layered In centrifuge tube.

5. adding isometric isopropanol, overturn and mix, room temperature is placed 10 minutes.

6.4 DEG C, 12000g is centrifuged 10 minutes, abandons supernatant.

7. add 75% ethanol that 1mL RNase-Free water is prepared（Matching while using）, washing precipitation.（It is washable twice）.

8.4 DEG C, 12000g is centrifuged 5 minutes, abandons supernatant（Supernatant is all blotted as far as possible）, drying at room temperature 5-10 minutes, plus Enter water dissolving precipitations of the 30-100 μ L without RNase, can be placed in -80 DEG C of refrigerators after precipitation dissolving and preserve for a long time.

（2）The synthesis of the chains of reverse transcription cDNA first：RNA concentration will be determined after the RNA dissolvings of extraction, then used TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix reverse transcription reagent box is carried out Reverse transcription.

5 μ g total serum IgEs are taken, 2 × reaction buffer 10 μ L, primer oligo dT is added（0.5μg/μL）1 μ L, the μ of reverse transcriptase 1 L, removes the μ L of genome enzyme 1, and moisturizing to 20 μ L is incubated 30 minutes, 85 DEG C of enzymes are inactivated 5 minutes at 42 DEG C.

（3）GRAS37The clone of gene：

GRAS37Gene primer sequence：

Sense primer 5 '-GGTACCATGGCCGCCGAGCCGGCGGATGTC-3’；（SEQ ID NO.4）；

Anti-sense primer 5 '-AAGCTTTCATTTTGCAGCCCACGCAGACAT-3’；（SEQ ID NO.5）；

It is restriction enzyme site wherein at underscore, the restriction enzyme site of sense primer is Kpn1, and the restriction enzyme site of anti-sense primer is Hind Ⅲ。

Expanded using Phusion high-fidelity enzymes, reaction system is：The μ L of 10 × reaction buffer 2.5, deoxyribose core Acid（dNTP）2 μ L, the μ L of sense primer 1,1 μ L, Phusion high-fidelities enzyme of anti-sense primer 0.5,0.75 μ L, cDNA templates of μ L, DMSO 1 μ L, water polishing to 25 μ L.

PCR reaction conditions are：95 DEG C of pre-degenerations 5 minutes；

95 DEG C are denatured 15 seconds, and 58 DEG C are annealed 15 seconds, and 72 DEG C extend 2 minutes, totally 35 circulations；

Extend 5 minutes after 72 DEG C；

16 DEG C of insulations.

After reaction terminates, enter row agarose gel electrophoresis, detect after purpose band, as shown in Figure 3A, cut glue and carry out Glue reclaim, glue reclaim method is according to the fast-type Ago-Gel DNA QIAquick Gel Extraction Kits of BioTeke companies（Cat#DP1722）Enter OK.

The product end of high-fidelity enzymatic amplification is flat end, it is necessary to which T-A clones, reactant could be carried out by adding after polyA System is as follows：The μ L of 10 × reaction buffer 1.5, DNA（dNTP）The μ L of 1.2 μ L, Easy Taq enzyme 0.15, glue reclaim production The μ L of thing polishing 15.Afterwards again 72 DEG C react 30 minutes.

（4）Above-mentioned 4 μ L tailings products are taken to be attached with pEASY-T1 cloning vectors, operating procedure is according to Quan Shi King Companies Product pEASY-T1 specifications carry out, then connection product using heat shock method convert e.colistraindh5α, containing card Grow and stay overnight on the LB flat boards of that mycin.Picking white single bacterium colony carries out bacterium colony PCR, reaction system in the flat lining outs of LB As above, choose positive bacterium colony to stay overnight in LB fluid nutrient mediums, as shown in Figure 3 B.

（5）The extraction of DNA：The use of health is the century small extracts kit of high-purity plasmid（CW0500A）Extract plasmid DNA。

（6）Sequencing：Originally it is operated in Beijing Liuhe Huada Genomics Technology Co., Ltd's progress.

（7）The structure of expression vector：Correctly carried with Kpn1 and the digestions of Hind III sequencingGRAS37The plasmid of gene is such as Shown in Fig. 3 C and the pPZP211 empty carriers with 35S, after one hour of 37 DEG C of digestions, enter row agarose gel electrophoresis, cut off Correct band carries out glue reclaim, and glue reclaim product is connected with the T4 DNA ligases of Thermo companies, connection product conversion DH5 α bacterial strains, grow on the LB flat boards containing SPE and stay overnight.Picking white single bacterium colony carries out bacterium colony in the flat lining outs of LB PCR, chooses positive bacterium colony and is stayed overnight in LB fluid nutrient mediums, as shown in Figure 3 D.The extraction of DNA：The use of health is that century is high The small extracts kit of Pure Plasmid extracts DNA, and digestion is identified, as shown in FIGURE 3 E.Structure is obtained35S::GRAS37Cross table Up to carrier.

（8）2.5 μ L plasmids are taken to convert Agrobacterium EHA105, arabidopsis is infected in preparation.

（Two）：Arabidopsis floral infects, is overexpressed ZmGRAS37The screening of transgenic arabidopsis positive seedling

（1）Arabidopsis floral infects

Infect formula of liquid（5 ml systems）；

The g Silwet of sucrose 0.25（It is poisonous, lucifuge）1.5 ul ddH2O are settled to 5 ml；

Infect step：

1. select PCR and Agrobacterium single bacterium colony, plus 20ml or so LB (spectinomycin adds rifampin) cultures that are positive are identified in digestion In base, incubated overnight.

2. arabidopsis flowering time is general in 11-13 points, young plant to be infected requires wet condition；Therefore should be in morning 9- Young plant to be infected is poured water by 10 points or so.

3. under room temperature condition, 5000rpm is centrifuged 5 minutes, collects thalline 2-3 times, infecting liquid with 8-10 ml is resuspended thalline.

4. around noon, the flower immersion opened is infected in liquid 6-7 seconds, interval after 5-7 days can Secondary Infection to carry High infect efficiency.

5. dark treatment, humidity parcel 24 hours.

（2）The screening of transgenic positive seedling

1. the seed that individual plant receives the arabidopsis after infecting is T0 generations.

2.T0 is cultivated for seed on the culture medium containing kanamycins, selects green seed in matrix, what individual plant was received Seed is T1 generations.

3.T1 is cultivated for seed on the culture medium containing kanamycins, selects green seedling：Huang Miao is about 3：In 1 strain Green seed is in matrix, and the seed that individual plant is received is T2 generations.

4.T2 is cultivated for seed on the culture medium containing kanamycins, selects the strain kind of entirely green seedling in matrix In, the seed received is the seed of the transgenic line of homozygosis, extracts the genome of transgenic line, and base is turned with PCR detections Because of strain, as a result as shown in Figure 4.

The seed of the transgenic line of homozygosis is selected in all experiments below.

Embodiment 2：Transgenic line, which ties up to seedling stage, has the big phenotype of trophosome

（One）：It is overexpressed ZmGRAS37The culture of transgenic arabidopsis

Arabidopsis seed 70% ethanol disinfection now matched somebody with somebody 5 minutes, then with 2.6% hypochlorite disinfectant 10 minutes, finally Rinsed 5-7 times with the ddH2O containing Tween-20, the aseptic seed of sterilization is laid on 1/2MS solid mediums.4 DEG C are kept away Light is handled 3 days, and the long-day incubator for being positioned over 22 DEG C is grown 7 days, and Arabidopsis thaliana Seedlings then are transferred into the base containing nutrient solution Continue to cultivate in matter.Long-day conditions are 16h illumination/8h dark, 22 DEG C.

As shown in Figure 5 and Figure 6, transgenic positive plant can be explained in Fig. 5 A, B has trophosome big to experimental result in seedling stage Phenotype.Transgenic positive plant, which can be explained, in Fig. 6 A, B, C has the big phenotype of trophosome in seedling stage.

（Two）：It is overexpressed ZmGRAS37Transgenic line bed board cellular morphology, size observation

（1）Decolourize, it is transparent

1. take respectively fresh WT,ZmGRAS37Blade immerse 14% glacial acetic acid, in the mixed solution of 84% absolute ethyl alcohol overnight, Decolourized.

2. sample is respectively cleaned twice in 70%, 99.5% absolute ethyl alcohol respectively.

3. cleaned blade is put into following solution carry out it is transparent：Chloraldurate 200g, glycerine 20g, distilled water 50g。

(2) film-making, observation

Material standing time is longer, 3-4 days or so, pulls material out before observation and is floated in the absolute ethyl alcohol for be placed on 70% Wash, 10% glycerine carries out tabletting, uses Differential interference contrast microscope（DIC）Observed.

Experimental result is as shown in fig. 7, conclusion as can be drawn from Figure 7, and it is bigger than WT to be overexpressed strain bed board cell, but number Mesh does not have significant difference, so as to cause the blade for being overexpressed strain bigger than WT.

Embodiment 3：Transgenic line has the phenotype of early blossoming

（One）：It is overexpressed ZmGRAS37Transgenic line floral genes expression quantity is detected

According to the requirement of qRT-PCR design of primers, Beacon Designer 7 and Primer Premier 5.0 etc. are used Software for Design gene specific primer.SYBR Green Design options are selected, file is set up, list entries runs BLAST

After search sequence and Template structure search instruments, Primer search works are run Tool, selects optimal primer sequence（First ensure that primer specificity is considered further that and avoid formwork structure influence）.Primer is in Shanghai life work life Thing Engineering Service Co., Ltd synthesizes, and is purified using PAGE.

Using special 96 orifice plates of qRT-PCR（Axygen, the U.S.）With high transmission rate sealed membrane（Axygen, the U.S.）, Quantitative fluorescent PCR instrument Icycler real-time PCR system（Bio-Rad, the U.S.）QRT-PCR analyses are carried out, 3 repetitions of each sample.Reverse transcription product is template, and reaction system is with reference to SYBR Green Realtime PCR Master Mix（QPK-201）Specification, reaction condition is as follows：

（1）95.0℃ 60s；（2）95.0℃ 10s；（3）58.0±5.0℃ 10s；（4）72.0℃ 15s；（5）Plate Read；（6）Incubate at 65℃ for 20s；（7）65 DEG C of Melting curve from 95 DEG C of to, read 0.5 DEG C of every, hold 1s；（8）End；Wherein（2）（3）（4）50-60 cycles.

Multiple samples are mixed, first time amplification is carried out, whether detection primer can use, verify that primer expands according to melt curve analysis The specificity of increasing, simple spike is to think specific amplified, if any bimodal, appropriate adjustment annealing temperature and primer consumption, if still not Can specific amplified, redesign primer.Diluted, diluted 4 times altogether, with 5 successively by 10 times of concentration using hybrid template The sample of concentration builds relative standard's curve, verifies the amplification efficiency of all primers, and aim sequence is in this concentration range Whether there is the relation of linear amplification.

Using tubulin as internal reference, adjusting each template concentrations makes the difference of internal reference Ct values be less than 2.Each gene magnification is equal There is internal reference to expand simultaneously, Ct values are read under implied terms, each sample makees three repetitions, and data analysis is using double standard curves Method, calculates average expression amount and relative deviation, is mapped with Excel.Utilize 2 simultaneously^-ΔΔCtSubstantially calculate relative with internal reference Expression quantity, determines the gene expression abundance of gene.Relative expression quantity calculates fold change=2^–△△CT, △ △ CT=(C herein_T-gen– C_T-tubulin)_Processing–(C_T-gene–C_T-tubulin)_Control。

Experimental result is as shown in figure 8, the number of lotus throne leaf when the two starts to expose bud, discovery lotus throne is shown in Fig. 8 B Number of sheets mesh does not have obvious difference；Fig. 8 C figures are shown both and start to expose the time of bud, it can be seen that transgenosis sun 3 days more early than wild type of the plant blossom time of property.The expression quantity that Fig. 8 D displays suppress floral genes FLC is significantly lowered, Accelerate bloom FT expression quantity is significantly raised in gene, and the gene expression amount change of its excess-three Accelerate bloom is not obvious.In summary, base is turned Because of strain35S::ZmGRAS37There is the phenotype of early blossoming compared with wild type WT.

Embodiment 4：Transgenic line has the increased phenotype of grain weight

（One）：It is overexpressed ZmGRAS37Transgenic line seed morphology, grain are counted again

Transgenic line 35S is carried out under entity anatomical lens::ZmGRAS37With the observation of wild type WT seed morphologies, such as Fig. 9 A institutes Show；The electronic balance of a ten thousandth precision carries out mass of 1000 kernel statistics to both, as shown in Figure 9 B.Fig. 9 is test result indicates that cross table It is bigger than WT up to strain seed.

The above embodiments are merely illustrative of the technical solutions of the present invention, rather than is limited；Although with reference to foregoing reality Apply example the present invention is described in detail, for the person of ordinary skill of the art, can still implement to foregoing Technical scheme described in example is modified, or carries out equivalent substitution to which part technical characteristic；And these are changed or replaced Change, the essence of appropriate technical solution is departed from the spirit and scope of claimed technical solution of the invention.

SEQUENCE LISTING

<110>Shandong Agricultural University

<120>One kind regulates and controls big arabidopsis seedling stage trophosome, early blossoming and the increased corn gene ZmGRAS37 of grain weight and its should

With

<130>

<160> 5

<170> PatentIn version 3.3

<210> 1

<211> 2258

<212> DNA

<213>Corn

<400> 1

cgaccatggc cgccgagccg gcggatgtcg gcgactccga gcccgagccc ttctccccgt 60

cggacttcct caaccttggt cccccaacgc ctcgcctcga cagccccgcc ctgcagctgc 120

acgacgacga tgacgacctc gtgctcctct tcatctcgcg catgctcatg gaagaggaga 180

cggacggctt cttcgagcag taccacccgg accaccccgc gctgctccag gcccagcagc 240

ccttcgccga catcctcgct ggcgccgccg ccgccgacgg cagccgaggg acctctgtag 300

ctggctcccc tgcattcgcc gccaacgcta tctggcccta cgatccagtc cagctctccc 360

agctgcttct ttctaggacc tacccggccg acacggcggc ataccccagc gccgccgccg 420

actcgatggc gtcctctgcc gggtccggtg ctggcacggt aaacatggac atgctcaacc 480

tggcgttcct caagggcatg gaggaggcca gcaagttctt gccggccacc agcaacaacc 540

atgaccaccg gcgccaagcg acggaaacat gcgacaagcg ggtggacgaa tcgtcgccgt 600

tgctccaaca gagtgcaagc aaaggccgca agaaccacag gcacgctcct tattggggtg 660

aggatgagga ggccgagaca ggcaggagga gctgcaaggt gatggcggtc gaaacagagg 720

aaagcgagat ggtggaggag ttcgtccaga gcggatacga ctcgctgcac gagcagatga 780

tggccatgac cctcagcacg gccggcagca ggaaagggaa ggggaaaggg aaagggagcg 840

ccaacgaggc cgtggacctg cgcaccttgc tgatccactg cgcgcaggcc gtggccgccg 900

gcaaccgccc cagcgccgcg gacctgctgg gcaagatcag ggcgcgctcc tcgccgaggg 960

gagacgccac gcagaggcta gcgcactgct tcgccggggg gctggaggcg cggctcgcgg 1020

gcactgggac ccagcagctc acggcggcga cgaagcgcgc ctccgccgtg gagatcctca 1080

gggcctacca gctctacctg gcggcctgca gcttcacagc gatggcgtac aagttctcca 1140

acctggccat ctgcaaggcc gtcggcggcg gcgggaggaa gaaggtgcac atcgtggact 1200

acggcgacca ctactacggg ttccagtggc cgtcgttgct gggctactgg ggtagcctgg 1260

aggctgggcc gcccgaggtg aggatcaccg ccatcgactt ccccgagccc gggttccgcc 1320

cggatgctcg gctccaggcg accgggcggc ggctcacatg cttcgctcgc cggcatggcg 1380

tccccttgag gttccacggc atcgaggcca ggtgggacgc ggtttcggtg gacgagctga 1440

gcatcgagcg cgacgaggtg ctcgtcgtga acggcctgtt cagtctcggt aggatgcagg 1500

agcaggagca ggacgacgtg gatagggata gccggccgag ccctagggac actgtcctcg 1560

gtaacgtccg gaagatgcgg cccgacgtgt tcgtcctgtg cgtggagaac agctcgtacg 1620

gcgcgccctt gttcgtgacg cggttccggg aggcgctctt ctactactcg gcgctgttcg 1680

acatgatgga cgcggtggcg gcgcgggacg acgacgaccg cgtgctggtg gagcagcacc 1740

tgtttgggca gcgcgccttg aacgccatcg cgtgcgaggg ctcggacagg gtggagcggc 1800

cggagacgta ccggcagtgg caggtgcgga acgaacgggc agggctgagg cagctcccgt 1860

tggatcctga tgcggttcgg gccatcagaa ggaaggtgaa ggacaagtac cacagggact 1920

tgttcatcga tgaggatcag cagtggctcc tgcaagggtg gaagggacga gtactctacg 1980

ccatgtctgc gtgggctgca aaatgatacg tacactagtt taacaaagtt caaagaaaac 2040

accattaata tctatggctc atgttcaaaa aacgtttgat tccgagaatt gtaatctttc 2100

ttcttctttt ttatgaaggg acgtagaaag agtatataat taattttatg gattacttgt 2160

ttgactgtcc taacaagtct ggtcacgtga aggacttccc tatctgttgt aacatcactc 2220

ggagtttcct tgggtctgga aaaaggaaac tacttagc 2258

<210> 2

<211> 2001

<212> DNA

<213>Corn

<400> 2

atggccgccg agccggcgga tgtcggcgac tccgagcccg agcccttctc cccgtcggac 60

ttcctcaacc ttggtccccc aacgcctcgc ctcgacagcc ccgccctgca gctgcacgac 120

gacgatgacg acctcgtgct cctcttcatc tcgcgcatgc tcatggaaga ggagacggac 180

ggcttcttcg agcagtacca cccggaccac cccgcgctgc tccaggccca gcagcccttc 240

gccgacatcc tcgctggcgc cgccgccgcc gacggcagcc gagggacctc tgtagctggc 300

tcccctgcat tcgccgccaa cgctatctgg ccctacgatc cagtccagct ctcccagctg 360

cttctttcta ggacctaccc ggccgacacg gcggcatacc ccagcgccgc cgccgactcg 420

atggcgtcct ctgccgggtc cggtgctggc acggtaaaca tggacatgct caacctggcg 480

ttcctcaagg gcatggagga ggccagcaag ttcttgccgg ccaccagcaa caaccatgac 540

caccggcgcc aagcgacgga aacatgcgac aagcgggtgg acgaatcgtc gccgttgctc 600

caacagagtg caagcaaagg ccgcaagaac cacaggcacg ctccttattg gggtgaggat 660

gaggaggccg agacaggcag gaggagctgc aaggtgatgg cggtcgaaac agaggaaagc 720

gagatggtgg aggagttcgt ccagagcgga tacgactcgc tgcacgagca gatgatggcc 780

atgaccctca gcacggccgg cagcaggaaa gggaagggga aagggaaagg gagcgccaac 840

gaggccgtgg acctgcgcac cttgctgatc cactgcgcgc aggccgtggc cgccggcaac 900

cgccccagcg ccgcggacct gctgggcaag atcagggcgc gctcctcgcc gaggggagac 960

gccacgcaga ggctagcgca ctgcttcgcc ggggggctgg aggcgcggct cgcgggcact 1020

gggacccagc agctcacggc ggcgacgaag cgcgcctccg ccgtggagat cctcagggcc 1080

taccagctct acctggcggc ctgcagcttc acagcgatgg cgtacaagtt ctccaacctg 1140

gccatctgca aggccgtcgg cggcggcggg aggaagaagg tgcacatcgt ggactacggc 1200

gaccactact acgggttcca gtggccgtcg ttgctgggct actggggtag cctggaggct 1260

gggccgcccg aggtgaggat caccgccatc gacttccccg agcccgggtt ccgcccggat 1320

gctcggctcc aggcgaccgg gcggcggctc acatgcttcg ctcgccggca tggcgtcccc 1380

ttgaggttcc acggcatcga ggccaggtgg gacgcggttt cggtggacga gctgagcatc 1440

gagcgcgacg aggtgctcgt cgtgaacggc ctgttcagtc tcggtaggat gcaggagcag 1500

gagcaggacg acgtggatag ggatagccgg ccgagcccta gggacactgt cctcggtaac 1560

gtccggaaga tgcggcccga cgtgttcgtc ctgtgcgtgg agaacagctc gtacggcgcg 1620

cccttgttcg tgacgcggtt ccgggaggcg ctcttctact actcggcgct gttcgacatg 1680

atggacgcgg tggcggcgcg ggacgacgac gaccgcgtgc tggtggagca gcacctgttt 1740

gggcagcgcg ccttgaacgc catcgcgtgc gagggctcgg acagggtgga gcggccggag 1800

acgtaccggc agtggcaggt gcggaacgaa cgggcagggc tgaggcagct cccgttggat 1860

cctgatgcgg ttcgggccat cagaaggaag gtgaaggaca agtaccacag ggacttgttc 1920

atcgatgagg atcagcagtg gctcctgcaa gggtggaagg gacgagtact ctacgccatg 1980

tctgcgtggg ctgcaaaatg a 2001

<210> 3

<211> 666

<212> PRT

<213>Corn

<400> 3

Met Ala Ala Glu Pro Ala Asp Val Gly Asp Ser Glu Pro Glu Pro Phe

1 5 10 15

Ser Pro Ser Asp Phe Leu Asn Leu Gly Pro Pro Thr Pro Arg Leu Asp

20 25 30

Ser Pro Ala Leu Gln Leu His Asp Asp Asp Asp Asp Leu Val Leu Leu

35 40 45

Phe Ile Ser Arg Met Leu Met Glu Glu Glu Thr Asp Gly Phe Phe Glu

50 55 60

Gln Tyr His Pro Asp His Pro Ala Leu Leu Gln Ala Gln Gln Pro Phe

65 70 75 80

Ala Asp Ile Leu Ala Gly Ala Ala Ala Ala Asp Gly Ser Arg Gly Thr

85 90 95

Ser Val Ala Gly Ser Pro Ala Phe Ala Ala Asn Ala Ile Trp Pro Tyr

100 105 110

Asp Pro Val Gln Leu Ser Gln Leu Leu Leu Ser Arg Thr Tyr Pro Ala

115 120 125

Asp Thr Ala Ala Tyr Pro Ser Ala Ala Ala Asp Ser Met Ala Ser Ser

130 135 140

Ala Gly Ser Gly Ala Gly Thr Val Asn Met Asp Met Leu Asn Leu Ala

145 150 155 160

Phe Leu Lys Gly Met Glu Glu Ala Ser Lys Phe Leu Pro Ala Thr Ser

165 170 175

Asn Asn His Asp His Arg Arg Gln Ala Thr Glu Thr Cys Asp Lys Arg

180 185 190

Val Asp Glu Ser Ser Pro Leu Leu Gln Gln Ser Ala Ser Lys Gly Arg

195 200 205

Lys Asn His Arg His Ala Pro Tyr Trp Gly Glu Asp Glu Glu Ala Glu

210 215 220

Thr Gly Arg Arg Ser Cys Lys Val Met Ala Val Glu Thr Glu Glu Ser

225 230 235 240

Glu Met Val Glu Glu Phe Val Gln Ser Gly Tyr Asp Ser Leu His Glu

245 250 255

Gln Met Met Ala Met Thr Leu Ser Thr Ala Gly Ser Arg Lys Gly Lys

260 265 270

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

275 280 285

Leu Ile His Cys Ala Gln Ala Val Ala Ala Gly Asn Arg Pro Ser Ala

290 295 300

Ala Asp Leu Leu Gly Lys Ile Arg Ala Arg Ser Ser Pro Arg Gly Asp

305 310 315 320

Ala Thr Gln Arg Leu Ala His Cys Phe Ala Gly Gly Leu Glu Ala Arg

325 330 335

Leu Ala Gly Thr Gly Thr Gln Gln Leu Thr Ala Ala Thr Lys Arg Ala

340 345 350

Ser Ala Val Glu Ile Leu Arg Ala Tyr Gln Leu Tyr Leu Ala Ala Cys

355 360 365

Ser Phe Thr Ala Met Ala Tyr Lys Phe Ser Asn Leu Ala Ile Cys Lys

370 375 380

Ala Val Gly Gly Gly Gly Arg Lys Lys Val His Ile Val Asp Tyr Gly

385 390 395 400

Asp His Tyr Tyr Gly Phe Gln Trp Pro Ser Leu Leu Gly Tyr Trp Gly

405 410 415

Ser Leu Glu Ala Gly Pro Pro Glu Val Arg Ile Thr Ala Ile Asp Phe

420 425 430

Pro Glu Pro Gly Phe Arg Pro Asp Ala Arg Leu Gln Ala Thr Gly Arg

435 440 445

Arg Leu Thr Cys Phe Ala Arg Arg His Gly Val Pro Leu Arg Phe His

450 455 460

Gly Ile Glu Ala Arg Trp Asp Ala Val Ser Val Asp Glu Leu Ser Ile

465 470 475 480

Glu Arg Asp Glu Val Leu Val Val Asn Gly Leu Phe Ser Leu Gly Arg

485 490 495

Met Gln Glu Gln Glu Gln Asp Asp Val Asp Arg Asp Ser Arg Pro Ser

500 505 510

Pro Arg Asp Thr Val Leu Gly Asn Val Arg Lys Met Arg Pro Asp Val

515 520 525

Phe Val Leu Cys Val Glu Asn Ser Ser Tyr Gly Ala Pro Leu Phe Val

530 535 540

Thr Arg Phe Arg Glu Ala Leu Phe Tyr Tyr Ser Ala Leu Phe Asp Met

545 550 555 560

Met Asp Ala Val Ala Ala Arg Asp Asp Asp Asp Arg Val Leu Val Glu

565 570 575

Gln His Leu Phe Gly Gln Arg Ala Leu Asn Ala Ile Ala Cys Glu Gly

580 585 590

Ser Asp Arg Val Glu Arg Pro Glu Thr Tyr Arg Gln Trp Gln Val Arg

595 600 605

Asn Glu Arg Ala Gly Leu Arg Gln Leu Pro Leu Asp Pro Asp Ala Val

610 615 620

Arg Ala Ile Arg Arg Lys Val Lys Asp Lys Tyr His Arg Asp Leu Phe

625 630 635 640

Ile Asp Glu Asp Gln Gln Trp Leu Leu Gln Gly Trp Lys Gly Arg Val

645 650 655

Leu Tyr Ala Met Ser Ala Trp Ala Ala Lys

660 665

<210> 4

<211> 30

<212> DNA

<213>Artificial sequence

<400> 4

ggtaccatgg ccgccgagcc ggcggatgtc 30

<210> 5

<211> 29

<212> DNA

<213>Artificial sequence

<400> 5

aagctttcat tttgcagccc acgcagaca 29

Claims (8)

1. one kind regulates and controls big arabidopsis seedling stage trophosome, early blossoming and the increased corn gene of grain weightZmGRAS37,It is characterized in that： Its nucleotide sequence such as SEQ ID No:Shown in 1, CDS sequences such as SEQ ID NO:Shown in 2, the amino acid of its protein encoded Sequence such as sequence SEQ ID NO:Shown in 3.

2. corn gene according to claim 1ZmGRAS37,It is characterized in that：Amplification is describedZmGRAS37Gene draws Thing sequence is：

- the GGTACCATGGCCGCCGAGCCGGCGGATGTC-3 ' of sense primer 5 '；

- the AAGCTTTCATTTTGCAGCCCACGCAGACAT-3 ' of anti-sense primer 5 '.

3. the gene described in claim 1ZmGRAS37Application in the regulation and control big phenotype of plant seedling stage trophosome.

4. gene according to claim 3ZmGRAS37Application in the regulation and control big phenotype of plant seedling stage trophosome, it is special Levy and be：ZmGRAS37Transgenic positive Arabidopsis plant seedling stage compared with wild type blade diameter, blade cell become Greatly, fresh weight, dry weight increase.

5. the gene described in claim 1ZmGRAS37Application in regulation and control plant early blossoming phenotype.

6. gene according to claim 5ZmGRAS37Application in regulation and control plant early blossoming phenotype, it is characterised in that：ZmGRAS37Transgenic positive Arabidopsis plant flowering time compared with wild type shifts to an earlier date three days.

7. the gene described in claim 1ZmGRAS37Application on regulation and control plant grain again increase phenotype.

8. gene according to claim 7ZmGRAS37Application on regulation and control plant grain again increase phenotype, its feature exists In：ZmGRAS37Transgenic positive Arabidopsis plant seed diameter increase and mass of 1000 kernel increase compared with wild type.