CN107022551B - A kind of regulation arabidopsis seedling stage trophosome is big, early blossoming and the increased corn gene of grain weightZmGRAS37And its application - Google Patents

Abstract

Big, early blossoming that the present invention provides a kind of regulation arabidopsis seedling stage trophosomes and the increased corn gene of grain weightZmGRAS37And its application.The present invention separates from corn and is cloned into geneZmGRAS37, willZmGRAS37CDNA sequence be connected to 35S promoter starting expression vector pPZP211 on, arabidopsis thaliana transformation is infected using Agrobacterium infestation method, the experimental results showed that model plant arabidopsis can be made to occur trophosome in seedling stage is big for the gene, and there are early blossoming and grain the characters such as to increase again.Phylogenetic analysis is the results show that direct homologous gene in model plant arabidopsis without the gene, but the homology of the gene and gramineae plant sorghum, corn is higher, illustrates that the gene is GRAS family gene specific to gramineae plant；In view of conservative of the gene in gramineae plant and the phenotype presented in transgenic arabidopsis, it was demonstrated that the gene has potential application.

Description

A kind of regulation arabidopsis seedling stage trophosome is big, early blossoming and the increased corn gene of grain weightZmGRAS37And its application

Technical field

The invention belongs to field of plant genetic project technology, and in particular to a kind of regulation arabidopsis seedling stage trophosome is big, early The increased corn gene of colored and grain weightZmGRAS37And its application.

Background technique

The agricultural production of China's the Yellow River and Huai He River sea region is mainly the crop rotation of wheat and corn.Since the production of corn is intended to wheat After receipts machine live streaming and it is mature after machine harvest, there is an urgent need to cultivate breeding time short corn variety in breeding work.Crop battalion Feeding body can increase greatly the photosynthetic efficiency of crop significantly, 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 it obtains more More trophosome is big, early blossoming and the increased genetically modified crops of grain weight, can promote the gramineae plants such as corn, rice significantly Molecular design breeding work.

GRAS albumen is distinctive protein family in plant, its name derives from three of first discovery in plant kingdom GRAS protein memberGAI [GA (GIBBERELLICACID)-INSENSITIVE], RGA (REPRESSOR OF GAI) andSCR (SCARECROW) (Pysh et al., 1999).GRAS gene family is referred to as " green revolution " of gene, to the family The research of race has had more than 10 years history, has now been found that GRAS protein family is related to plant transcription level modulation and control Multi-signal transduction pathway (the Bolle of plant growth and developmentet al. 2004).Currently, in 294 kinds of terrestrial plants It was found that GRAS gene has 1035 kinds of sequence (Sunet al. 2012).The analysis carried out to the GRAS albumen of different plant species is taken off Show that they have a similar subfamily branch, including ten subfamilies, the title of these subfamilies are 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, needs subsequent research.

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

In addition to the presence of specific motif proves that GRAS family member can be used as transcription regulatory factor (Pyshet al. 2000, Bolle 2004) outside, the nuclear location of most of GRAS albumen alternatively bright this point.In addition to PAT1 subfamily it Outside, other GRAS transcription factor families member is most of all enters nuclear signal (NLS) (Bolle 2004) containing one, therefore GRAS Albumen can one of the condition as transcription regulatory factor be exactly whether it has nuclear localization signal.

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

(1) GRAS transcription factor protein is related to the growth of root.GRAS gene family member's AtSCR gene of first discovery Mutant, since the afunction of the gene leads to the growth failure of root, the function and root cortical cell of SCR gene are not The radiation growth of symmetrical fissions and root is related.In addition, another GRAS transcription factor family member short root(SHR) base Because being also proved to related to the radiation growth of root.

(2) GRAS transcription factor family is related to the growth of axillary meristem.Schumacher et al. was in 1999 The afunction mutant for the tomato Ls gene mentioned in work shows plant without branch, the development of axillary bud separate living tissue early stage It is blocked, and in the structure of Ls gene includes the VHIID motif of GRAS transcription factor family, sufficiently prove GRAS transcription factor man Race is related with the growth of axillary meristem.The gene in arabidopsis and 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 family and axillary point The growth of raw tissue has substantial connection.

(3) GRAS transcription factor family is related to the holding of the characteristic of shoot apical meristem.HAM subfamily is the Asia GRAS man One of family member, Stuurman et al. have found in the mutant of the petunia hairy meristem (ham) of discovery in 2002 It no longer keeps the indiscriminate characteristic of shoot apical meristem, and the growth stopping of shoot apical meristem and axillary meristem is led The stopping for causing plant organ development, in addition, HAM mutant forms leaf more smaller than wild type.Illustrate that HAM subfamily is dividing It plays an important role in some approach of raw tissue growth.

(4) GRAS family member is related to the conductive process of phytochrome signal.PAT1 subfamily is GRAS family weight Member is wanted, the mutant of arabidopsis PAT1 makes the signal transduction process exception of its phytochrome A, chalcone synthase and chlorophyll The expression of synthetic proteins is severely impacted, and the lower axle for also resulting in mutant extends, but is not affected under other light, Therefore PAT1 gene is single-minded related to the signal transduction of phytochrome A.

(5) GRAS family member is related to gibberellin signal transduction.Gibberellin usually promote the elongation of stem, the formation of flower with And the germination process of seed.GAI expression product in GRAS gene family plays 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 (20%-27%) of protein member is many higher.This also turns out that this part albumen participates in the process of gibberellin signal transduction.

(6) GRAS protein family can be used as transcription factor and work.In addition to PAT1 subfamily is the egg of cytoplasm positioning Ultrawhite, most of GRAS albumen is all the albumen of nuclear location.Find there are some cores in GRAS protein structure in a recent study Positioning signal, this is also that GRAS albumen works as transcription factor and provides more ample evidence.

GRAS family gene plays extremely important and unique effect in the growth and development process of plant.Therefore, for The research of GRAS family protein has a very important significance.And the research of GRAS family member in corn is not goed deep into, it is beautiful GRAS family member in rice lacks at present in terms of growth and development can disclose the document retrieved.

Summary of the invention

The case where for the above-mentioned prior art, the object of the present invention is to provide a kind of regulation arabidopsis seedling stage trophosomes Greatly, early blossoming and the increased corn gene of grain weightZmGRAS37And its application, the present invention are separated and are cloned into from cornGRAS37 Gene, and be named asZmGRAS37, willZmGRAS37Full-length cDNA be connected to 35S promoter starting 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 the increased phenotype of grain weight.

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

Big, early blossoming that the present invention provides a kind of regulation arabidopsis seedling stage trophosomes and the increased corn gene of grain weightZmGRAS37,Its nucleotide sequence is as shown in SEQ ID No:1, and CDS sequence is as shown in SEQ ID NO:2, the albumen of coding The amino acid sequence of matter is as shown in sequence SEQ ID NO:3.

Described in amplificationZmGRAS37The primer sequence of gene are as follows:

Upstream primer 5 '-GGTACCATGGCCGCCGAGCCGGCGGATGTC-3 '；

Downstream primer 5 '-AAGCTTTCATTTTGCAGCCCACGCAGACAT-3 '.

The present invention also provides the genesZmGRAS37Application in the regulation big phenotype of plant seedling stage trophosome.

It is further:ZmGRAS37Transgenic positive Arabidopsis plant blade diameter, blade compared with wild type in seedling stage Cell become larger, fresh weight, dry weight increase.

The present invention also provides the genesZmGRAS37Application in regulation plant early blossoming phenotype.

It is further:ZmGRAS37Transgenic positive Arabidopsis plant flowering time compared with wild type proposes first three days.

The present invention also provides the genesZmGRAS37Increase the application in phenotype again in regulation plant grain.

It is further:ZmGRAS37Transgenic positive Arabidopsis plant seed diameter compared with wild type increases and mass of 1000 kernel Increase.

Compared with prior art, advantages of the present invention and have the technical effect that the present invention willZmGRAS37Full length gene cDNA It is connected on the expression vector of over-express vector starting, infects arabidopsis thaliana transformation 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 is the results show that mould Direct homologous gene in formula plant Arabidopsis thaliana without the gene, but the gene and gramineae plant sorghum, corn homology compared with Height illustrates that the gene is GRAS family gene specific to gramineae plant；Literature search is not found about the gene function The research of aspect.In view of conservative of the gene in gramineae plant and the phenotype presented in transgenic arabidopsis, recognize It is had potential application for the gene.

Detailed description of the invention

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

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

Fig. 3 A band is the target gene being cloned intoZmGRAS37Schematic diagram, size 2001bp；Fig. 3 B band is mesh Gene connect the obtained positive colony of pEASY-T1 cloning vector；Fig. 3 C is that sequencing correctly hasGRAS37Gram of gene Grand vector plasmid digestion result schematic diagram；Fig. 3 D is that purpose gene is connected to the obtained positive colony of pPZP211 expression vector；Figure 3 E be withGRAS37The expression vector plasmid enzyme restriction result schematic diagram of gene.

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

Fig. 5 A is transgenic line35S::ZmGRAS37It (is from left to right respectively planted with wild type WT in different time Grow 14,18,21 days afterwards) phenotypic map planted in same alms bowl.

Fig. 5 B is transgenic line35S::ZmGRAS37With wild type WT different time (after plantation grow 14,18,21, 28 days) phenotypic map of single-strain planting in different flowerpot.

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

Fig. 6 B is transgenic line35S::ZmGRAS37With wild type WT growth 18 days (35S::ZmGRAS37Expose flower Bud) when diameter data statistics, and be found to have significant difference.

Fig. 6 C is respectively transgenic line35S::ZmGRAS37With wild type WT growth 18 days (35S::ZmGRAS37Dew Bud out) Shi Xianchong and dry weight (80 degrees Celsius are dried 24 hours) data statistics, and be found to have significant difference.

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

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

Fig. 7 C is the data statistics of bed board cell number under the visual field A.

Fig. 7 D is the data statistics of blade area under the visual field B.

Fig. 7 E figure is the data statistics of total bed board cell in the two blade.

Fig. 8 A is transgenic line35S::ZmGRAS37With table of the wild type WT when growing 21 days (WT exposes bud) Type figure.

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

Fig. 8 D is transgenic line 35S::ZmGRAS37With five in wild type WT and the expression quantity for related gene of blooming QRT testing result.

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

Fig. 9 B is the statistics of the two mass of 1000 kernel.

Specific embodiment

Technical solution of the present invention is further explained in following embodiment, according to above description and these implementations Example, those skilled in the art can determine essential characteristic of the invention, and without departing from spirit and scope of the invention the case where Under, various changes and modifications can be made to the present invention, so that it is applicable in 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 plant cell in, introduction method be all it is well known in the art, these methods include but are not limited to: mediated by agriculture bacillus Conversion method, particle bombardment, electrization, Ovary injection etc..The antibiotic of the positive seedling of the screening that the present invention selects is mould to block that Element；In addition to this, of the present invention is state of the art.

The present invention obtains total serum IgE from corn first, and reverse transcription obtains the first chain of cDNA, utilizes it as mould later Plate is usedZmGRAS37Corresponding specific primer carries out the amplification of Phusion high fidelity enzyme to the cDNA of acquisition.

The extraction and purifying of corn RNA can directly adopt prior art, and documented technique in the present invention can also be used； The synthesis of the first chain of cDNA is also identical situation, can also use existing technique or kit, but both above-mentioned Preferably using concrete technology documented by the present invention.

Embodiment 1: the acquisition of transgenic line

(1) cornGRAS37Sequence analysis, clone and the vector construction of gene

The present invention finds gene using Maize genome database website MaizeGDBGRAS37, using known to NCBI analysis This gene possesses a conservative C-terminal GRAS structural domain as all GRAS protein family members；GRAS37Genome Sequence is 2258 bp(sequences as shown in SEQ ID NO.1), and CDS sequence is 2001 bp(sequences such as SEQ ID NO.2 It is shown), it willGRAS37Amino acid sequence (sequence is as shown in SEQ ID NO.3) and corn, arabidopsis, rice and sorghum in All GRAS protein family members construct chadogram, as a result as shown in Figure 1.Fig. 1 phylogenetic analysis is the results show that nothing in arabidopsis The direct homologous gene of the gene, but the gene and the homology of gramineae plant sorghum are higher, therefore illustrate that the gene is standing grain A gene specific to graminaceous plant.

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

(1) extraction of RNA: being the total serum IgE that century TRIzon extracts corn using health.

1. fresh plant tissue material is taken to be fully ground in liquid nitrogen, 1ml TRIzon, whirlpool is added in every 30-50mg tissue Oscillation mixes, and is placed at room temperature for 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 tube.

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

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

5. isometric isopropanol is added, it is mixed by inversion, is placed at room temperature for 10 minutes.

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

7. 75% ethyl alcohol (matching while using) that 1mL RNase-Free water is prepared is added, washing precipitating.(washable twice).

8.4 DEG C, 12000g is centrifuged 5 minutes, is abandoned supernatant (as far as possible all blotting supernatant), drying at room temperature 5-10 minutes, is added Enter water dissolution precipitating of the 30-100 μ L without RNase, -80 DEG C of refrigerators can be placed in after precipitating dissolution and saved for a long time.

(2) synthesis of the first chain of reverse transcription cDNA: RNA concentration is measured after the RNA of extraction is dissolved, is then used TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix reverse transcription reagent box carries out Reverse transcription.

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

(3)GRAS37The clone of gene:

GRAS37Gene primer sequence:

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

Downstream primer 5 '-AAGCTTTCATTTTGCAGCCCACGCAGACAT-3'；(SEQ ID NO.5)；

It is wherein restriction enzyme site at underscore, the restriction enzyme site of upstream primer is Kpn1, and the restriction enzyme site of downstream primer is HindⅢ。

It is expanded using Phusion high fidelity enzyme, reaction system are as follows: 10 × reaction buffer, 2.5 μ L, deoxyribose core Acid (dNTP) 2 μ L, 1 μ L of upstream primer, 1 μ L, Phusion high fidelity enzyme of downstream primer, 0.5 0.75 μ L, cDNA template of μ L, DMSO 1 μ L, water polishing to 25 μ L.

PCR reaction condition are as follows: 95 DEG C initial denaturation 5 minutes；

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

Extend 5 minutes after 72 DEG C；

16 DEG C of heat preservations.

After reaction, agarose gel electrophoresis is carried out, after detecting purpose band, as shown in Figure 3A, glue is cut and carries out Glue recycling, glue recovery method according to the fast-type Ago-Gel DNA QIAquick Gel Extraction Kit (Cat#DP1722) of BioTeke company into Row.

The product end of high fidelity enzyme amplification is flat end, just can be carried out T-A clone, reactant after needing to add polyA Be as follows: 10 × reaction buffer, 1.5 μ L, 1.2 μ L, Easy Taq enzyme of DNA (dNTP), 0.15 μ L, glue recycling produce 15 μ L of object polishing.Later again 72 DEG C react 30 minutes.

(4) above-mentioned 4 μ L tailing product is taken to be attached with pEASY-T1 cloning vector, operating procedure is according to Quan Shi King Company Product pEASY-T1 specification carry out, then connection product using heat shock method convert e.colistraindh5α, containing card It is grown overnight on the LB plate of that mycin.Picking white single bacterium colony is crossed on LB plate, carries out bacterium colony PCR, reaction system As above, positive bacteria is chosen to fall in LB liquid medium overnight, as shown in Figure 3B.

(5) extraction of Plasmid DNA: being that the small extracts kit of century high purity plasmid (CW0500A) extracts plasmid using health DNA。

(6) sequencing: this work is carried out in Beijing Liuhe Huada Genomics Technology Co., Ltd.

(7) it the building of expression vector: is correctly had with Kpn1 and III digestion of Hind sequencingGRAS37The plasmid of gene is such as Shown in Fig. 3 C and the pPZP211 empty carrier with 35S, after one hour of 37 DEG C of digestions, agarose gel electrophoresis, excision are carried out Correct band carries out glue recycling, the glue recovery product T4 DNA ligase of Thermo company is connected, connection product conversion DH5 α bacterial strain is grown overnight on the LB plate containing SPE.Picking white single bacterium colony is crossed on LB plate, carries out bacterium colony PCR chooses positive bacteria and falls in LB liquid medium overnight, as shown in Figure 3D.The extraction of Plasmid DNA: being that century is high using health The small extracts kit of Pure Plasmid extracts Plasmid DNA, and digestion is identified, as shown in FIGURE 3 E.Building obtains35S::GRAS37Cross table Up to carrier.

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

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

(1) arabidopsis floral infects

Infect formula of liquid (5 ml system)；

0.25 g Silwet(of sucrose is toxic, is protected from light) 1.5 ul ddH2O are settled to 5 ml；

Infect step:

1. selecting PCR and the Agrobacterium single colonie that is positive being identified in digestion, add 20ml or so LB (spectinomycin adds rifampin) In culture medium, it is incubated overnight.

2. arabidopsis flowering time is generally in 11-13 point, young plant to be infected requires wet condition；It therefore should be in morning 9- Young plant to be infected is watered with water by 10 points or so.

3. under room temperature, 5000rpm is centrifuged 5 minutes, collect thallus 2-3 times, thallus is resuspended with 8-10 ml infected liquid.

4. around noon, by opened flower immerse infected liquid in 6-7 second, be spaced 5-7 days after can Secondary Infection to mention High infect efficiency.

5. dark treatment, humidity package 24 hours.

(2) screening of transgenic positive seedling

1. the seed that single plant receives the arabidopsis after infecting is T0 generation.

2.T0 is cultivated on culture medium containing kanamycin for seed, selects green seed in matrix, what single plant received Seed is T1 generation.

3.T1 is cultivated on culture medium containing kanamycin for seed, and select green seedling: Huang Miao is about in the strain of 3:1 For green seed in matrix, the seed that single plant receives is T2 generation.

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

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

Embodiment 2: the phenotype that transgenic line has trophosome big in seedling stage

(1): being overexpressed ZmGRAS37The culture of transgenic arabidopsis

Now match 70% ethanol disinfection 5 minutes of arabidopsis seed, then with hypochlorite disinfectant 10 minutes of 2.6%, It is finally rinsed 5-7 times with the ddH2O containing Tween-20, the aseptic seed of disinfection is laid on 1/2MS solid medium.4 It DEG C is protected from light processing 3 days, 22 DEG C of long-day incubator is placed in and grows 7 days, then Arabidopsis thaliana Seedlings are transferred to containing nutrient solution Matrix in continue to cultivate.Long-day conditions are dark for 16h illumination/8h, and 22 DEG C.

Experimental result is as shown in Figure 5 and Figure 6, and Fig. 5 A, B, which can illustrate transgenic positive plant in seedling stage, has trophosome big Phenotype.The phenotype that Fig. 6 A, B, C can illustrate transgenic positive plant in seedling stage and have trophosome big.

(2): being overexpressed ZmGRAS37Transgenic line bed board cellular morphology, size observation

(1) it decolourizes, is transparent

1. take respectively fresh WT,ZmGRAS37Blade immerse 14% glacial acetic acid, 84% dehydrated alcohol mixed solution in Overnight, it decolourizes.

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

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

(2) film-making, observation

Material standing time is longer, 3-4 days or so, material is pulled out before observation in the dehydrated alcohol for be placed on 70% into Row rinsing, 10% glycerol carry out tabletting, are observed with Differential interference contrast microscope (DIC).

Experimental result is as shown in fig. 7, conclusion as can be drawn from Figure 7, and it is big to be overexpressed strain bed board cell ratio WT, but number There is no significant differences for mesh, to cause the blade ratio WT for being overexpressed strain big.

Embodiment 3: transgenic line has the phenotype of early blossoming

(1): being overexpressed ZmGRAS37The detection of transgenic line floral genes expression quantity

According to the requirement of qRT-PCR design of primers, Beacon Designer 7 and Primer Premier are used The software designs gene specific primers such as 5.0.SYBR Green Design option is selected, file, list entries, operation are established BLAST

After search sequence and Template structure search tool, Primer is run Search tool selects optimal primer sequence (first guarantee that primer specificity considers further that and avoid formwork structure influence).Primer is upper The synthesis of Hai Shenggong bioengineering Services Co., Ltd, is purified using PAGE.

Using dedicated 96 orifice plate of qRT-PCR (Axygen, the U.S.) and high transparency sealed membrane (Axygen, the U.S.), Quantitative fluorescent PCR instrument Icycler real-time PCR system(Bio-Rad, the U.S.) qRT-PCR analysis is carried out, 3 repetitions of each sample.Reverse transcription product is template, and reaction system is referring to SYBR Green Realtime PCR Master Mix(QPK-201) specification, reaction condition is as follows:

(1) 95.0 DEG C of 60s；(2) 95.0 DEG C of 10s；(3) 58.0 ± 5.0 DEG C of 10s；(4) 72.0 DEG C of 15s；(5) Plate Read；(6) 65 DEG C of for 20s of Incubate at；(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 be used, and verifies primer according to melt curve analysis and expands The specificity of increasing, simple spike thinks specific amplified, if any bimodal, appropriate adjustment annealing temperature and primer dosage, if still not Energy specific amplified, redesigns primer.It is successively diluted, is diluted 4 times altogether, with 5 by 10 times of concentration using hybrid template The sample of concentration constructs relative standard's curve, and the amplification efficiency and aim sequence for verifying all primers are in this concentration range Whether there is the relationship of linear amplification.

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

Experimental results are shown in figure 8, and the number of lotus throne leaf when the two starts to expose bud is shown in Fig. 8 B, finds lotus throne There is no apparent difference for number of sheets mesh；Fig. 8 C figure is shown the two and starts the time for exposing bud, it can be seen that transgenosis sun The property plant blossom time 3 days more early than wild type.Fig. 8 D display inhibits the expression quantity of floral genes FLC significantly to lower, Accelerate bloom The expression quantity of FT significantly raises in gene, and the gene expression amount variation of excess-three Accelerate bloom is unobvious.In conclusion turning base Because of strain35S::ZmGRAS37With the phenotype of early blossoming compared with wild type WT.

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

(1): being overexpressed ZmGRAS37Transgenic line seed morphology, grain count again

Transgenic line 35S: is carried out under entity anatomical lens:ZmGRAS37With the observation of wild type WT seed morphology, such as scheme Shown in 9A；The electronic balance of a ten thousandth precision carries out mass of 1000 kernel statistics to the two, as shown in Figure 9 B.Fig. 9 the experimental results showed that It is big to be overexpressed strain seed ratio WT.

The above embodiments are merely illustrative of the technical solutions of the present invention, rather than is limited；Although referring to aforementioned reality Applying example, invention is explained in detail, for those of ordinary skill in the art, still can be to aforementioned implementation Technical solution documented by example is modified or equivalent replacement of some of the technical features；And these are modified or replace It changes, the spirit and scope for claimed technical solution of the invention that it does not separate the essence of the corresponding technical solution.

SEQUENCE LISTING

<110>Shandong Agricultural University

<120>a kind of regulation arabidopsis seedling stage trophosome is big, early blossoming and the increased corn gene ZmGRAS37 of grain weight and its answers

With

<130>

<160> 5

<170> PatentIn version 3.3

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<211> 2258

<212> DNA

<213>corn

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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 (5)

1. the application of the maize gene ZmGRAS37 that regulates Arabidopsis thaliana seedling stage vegetative body large, early flowering and grain weight increase in the regulation plant seedling stage vegetative body large phenotype, it is characterized in that: ZmGRAS37 transgenic positive Arabidopsis plant is in Compared with the wild type, the leaf diameter and leaf cells became larger, and the fresh weight and dry weight increased at the seedling stage. 2. Application of a maize gene ZmGRAS37 that regulates vegetative size, early flowering and grain weight increase in Arabidopsis seedling stage in regulating plant early flowering phenotype. 3 . The application of the gene ZmGRAS37 according to claim 2 in regulating the early flowering phenotype of plants, characterized in that: ZmGRAS37 transgenic positive Arabidopsis thaliana plants have an earlier flowering time than wild type by three days. 4. Application of a maize gene ZmGRAS37 , which regulates vegetative size, early flowering and grain weight increase in Arabidopsis seedling stage, in regulating the phenotype of plant grain weight increase. 5 . The application of the gene ZmGRAS37 according to claim 4 in regulating the increased grain weight phenotype of plants, wherein: ZmGRAS37 transgenic positive Arabidopsis plants have increased seed diameter and increased 1000-grain weight compared with wild type. 6 .