CN106146635A - Semen Maydis ZmSTP1 albumen and encoding gene thereof and application - Google Patents

Abstract

The present invention relates to biology field, specifically provide the application in promoting root system of plant monosaccharide to absorb of a kind of Semen Maydis ZmSTP1 albumen (as shown in SEQ ID No.1) and encoding gene (as shown in SEQ ID No.2) thereof.The present invention obtains an albumen with sugared absorption function first from important cereal crops Semen Maydis, and this gene is expressed at the tip of a root of Semen Maydis, has the function absorbing and transporting multiple monosaccharide；This gene is proceeded to model plant arabidopsis and can improve the transfer-gen plant absorbability to specific sugar, significantly improve earth culture biomass and seed production, there is important application prospect；Simultaneously from the point of view of nitrogen efficiency is improved by carbohydrate, it is expected to improve plant nitrogen efficiency especially, cultivates the efficient crops of nitrogen.

Description

Semen Maydis ZmSTP1 albumen and encoding gene thereof and application

Technical field

The present invention relates to biology field, specifically, relate to Semen Maydis ZmSTP1 albumen And the application that encoding gene is in promoting root system of plant monosaccharide to absorb.

Background technology

Carbohydrate is also known as saccharide compound, is that nature existence is most, distribution is the widest The organic compound that one class is important.Saccharide is photosynthetic product, is again respiratory Substrate, provides carbon skeleton and energy, the therefore supply of sugar for biochemical reactions in plant C N metabolism, dry-matter accumulation, crop yield are formed most important.Sugar one in plant Part is directly inhaled from photosynthesis and the degraded of starch, a part by root system from soil Receive.The saccharide that wherein root system directly the absorbs accumulation meaning weight to plant total carbohydrates Greatly.The neutral sugars such as glucose, fructose, sucrose due to character such as molecular weight is big, hydrophilic, Their transdermal delivery need film transport protein participation (Ludewig and Fl ü gge, 2013 Frontiers in Plant Science 4,231).A series of sugar it is found that at present on cell membrane Transdermal delivery albumen, their biochemical function and characterization of molecules are not quite similar.In arabidopsis Speculate exist more than 50 kind of monosaccharide transport protein and 20 kind of two HUCEP-8 (Lalonde et al., 2004Annual Review of Plant Biology 55,341 372).Wherein there are 14 kinds of lists HUCEP-8 belongs to STP (Sugar Transporter Subfamily) protein family (Johnson And Thomas, 2007Molecular Biology and Evolution 24, 2412–2423).The STP albumen of existing functional study is mainly expressed at plant storehouse organ, performance Go out high affine transportation characterization (Km 10-100mm) (Buttner 2007FEBS Letters 581,2,318 2324), indicate them to transport in storehouse, accumulate the main of photosynthate Feature.AtSTP1 can heavily absorb monosaccharide (Sherson et al., the 2000That root system is discharged Plant Journal 24,849 857), the accumulation of Atstp1 mutant monosaccharide reduces, to gala Sugar, mannose insensitive (Sherson et al., 2000The Plant Journal 24, 849–857)；AtSTP13 mainly expresses at cortex, endodermis, is responsible for heavily absorbing the abiotic side of body Compel the epidermis cell that causes impaired and release monosaccharide material (Nour-Eldin et al., 2006 Plant Methods 2,17 25；Yamada et al., 2011The Journal of Biological Chemistry 286 (50), 43,577 43586).Overexpression AtSTP13 gene Arabidopsis plant, the absorption of glucose is increased, sugar content significantly improves, finally biological Amount significantly improves (Schofield et al., 2009Plant, Cell and Environment 32,271 285).Therefore, absorption and the accumulation of sugar are served the heaviest by HUCEP-8 The effect wanted.Saccharide also regulates and controls circadian clock, affects phytohormone synthesis, system of defense And developmental process (Bolouri Moghaddam and Van den Ende, 2013Journal of Experimental Botany 64 (6), 1,439 1449).

Nitrogen is the required mineral element of the plant with carbon close association, global nitrogen fertilizer amount in 2014 It is about 1.1 hundred million tons, estimates that reaching 2.25 hundred million tons could meet grain peace to the year two thousand fifty demand Complete demand (Frink 1999P Natl Acad Sci USA., 96 (4): 1175-1180；Tilman Et al., 2011P Natl Acad Sci USA., 108 (50): 20260-20264).A large amount of throwings Enter nitrogenous fertilizer cause the series of environmental problems such as soil acidification, body eutrophication (Guo et al., 2010Science, 327 (5968): 1008-1010)；On the other hand, nitrogen shortage remains Main restricting factor (Diels et al., the 2001Agronomy of developing country's agricultural production Journal, 93:1191-1199；Vitousek 2009).Nitrogen deficiency can significantly reduce chlorophyll Synthesis and dry-matter accumulation, affect the growth of genitals, ultimately result in Severe Reduction (Marschner 1995Mineral Nutrition of Higher Plants, 2nd edn).Carbon nitrogen Metabolism is most important metabolic process in plant, and carbon metablism is in close relations with nitrogen assimilation, and (Song builds The people, 1998 Plant Physiology Communications).Spatially see, carbon metablism and NO₂ ^-Assimilation occurs In chloroplast, nitrogen metabolism needs carbon source and the energy that carbon metablism provides, it is also desirable to carbon metablism synthesizes Keto acid carry out synthesizing amino acid as skeleton.The activity of nitrate reductase (NR) is also by carbon water Impact (Cheng et al., the 1986Metabolism 35,10 14 of compound；Cheng et Al., 1992PAcad Sci 89,1861-1864；Vincentz et al.1993).In Fructus Lycopersici esculenti Research shows the absorption by increasing sucrose and utilization so that the expression of nitrate reductase increases Add, and then accelerate nitrogen metabolism process, add amino acid whose synthesis rate (Morcuende et Al.1998Planta 206,394 409；Halford et al.2004Journal of Experimental Botany 55,35-42).It addition, root system nitrogen absorbs also by solubility in root Carbohydrate supply impact (Tolley et al., 1988Journal of Experimental Botany 1988,39:613-622；Tolley et al., 1991Botanical Gazette 152: 23-33)。

As important cereal crops, Semen Maydis plays an important role in world food safety, And huge relative to other cereal crops yield potential (Chen et al., 2014Nature). But so far, the HUCEP-8 in Semen Maydis rarely has report, this research is cloned from Semen Maydis To HUCEP-8 STP1 gene, by its complementation to yeast shows to glucose (Glc), The response that fructose (Frc), mannose (Man) are strong, has part to ring to galactose (Gal) Should, show that ZmSTP1 can absorb multiple monosaccharide after proceeding in yeast system, but to difference list The absorbability of sugar is different.Overexpression shows to arabidopsis, processes multiple sugar and has sound Should.Showing as nourishing and growing and reproductive growth is affected, including Biomass, grain yield shows Writing and increase, sugar content increases, sucrose transporter AtSUC2, chlorophyll a and chlorophyll b Synthesis associated protein AtCAB1 expression the most substantially increases.It can be primverose efficient accumulation base Because engineering provides important candidate gene, simultaneously for improving plant, particularly cereal crops Nitrogen uptake efficiency also improves effect, has important practical value and directly Economic benefit.

Summary of the invention

In order to solve problems of the prior art, it is an object of the invention to provide a kind of Semen Maydis ZmSTP1 albumen and encoding gene application in promoting root system of plant monosaccharide to absorb thereof.

In order to realize the object of the invention, present invention firstly provides a kind of Semen Maydis ZmSTP1 albumen, Derive from the Semen Maydis (zea mays L.) of Zea, the aminoacid sequence of described albumen such as SEQ Shown in ID No.1.

Present invention also offers the encoding gene ZmSTP1 of described albumen, its nucleotide sequence is such as Shown in SEQ ID No.2.

Present invention also offers described ZmSTP1 gene in promoting root system of plant monosaccharide to absorb Application.

Specifically, described application is by described ZmSTP1 gene transferred plant, promotes plant Root system monosaccharide efficient absorption accumulates, and then improves phytomass and/or grain yield.

Further, described ZmSTP1 gene proceeds to plant cell by recombinant expression carrier. Available existing plant expression vector construction contains the recombinant expression carrier of ZmSTP1 gene.

When using ZmSTP1 gene constructed recombinant plant expression vector, at its transcription initiation nucleoside Can be plus any enhancement mode promoter or constitutive promoter before acid, such as cauliflower mosaic Poison CAMV35S promoter, the ubiquitin promoter (Ubiquitin) of Semen Maydis, they can individually make With or be used in combination with other plant promoter；Additionally, use the gene constructed plant of the present invention During expression vector, it be also possible to use enhancer, including translational enhancer or transcriptional enhancer, these Enhancer region can be ATG initiation codon or neighboring region start codon etc., but must Need to be identical with the reading frame of coded sequence, to ensure the correct translation of whole sequence.Described translation The source of control signal and start codon is widely, can be natural, it is also possible to be to close Become.Translation initiation region can come from transcription initiation region or structural gene.

As preferably, described recombinant expression carrier is to interleave in the multiple clone site of pPT-HYG Enter the recombiant plasmid pSuper1300+-ZmSTP that described ZmSTP1 gene obtains.

Present invention also offers the recombinant expression carrier containing described ZmSTP1 gene, transgenic Cell line and recombinant bacterium.

Present invention also offers a kind of method promoting root system of plant monosaccharide efficient absorption to accumulate, Described method is for proceed to aforementioned ZmSTP1 gene in plant cell by recombinant expression carrier.

As preferably, described recombinant expression carrier is to interleave in the multiple clone site of pPT-HYG Enter the recombiant plasmid that described gene obtains.

The beneficial effects of the present invention is:

The present invention is cloned into Semen Maydis ZmSTP1 gene, by studying this gene in Semen Maydis Qualitatively and quantitatively expression characterization, finds that this gene is mainly expressed at the corn seedling tip of a root.And then use Transgenic technology by ZmSTP1 channel genes model plant arabidopsis (Columbia), result Show that multiple monosaccharide is processed and has response by it, shows as nourishing and growing and reproductive growth is by shadow Ringing, including Biomass, grain yield dramatically increases, and sugar content increases；Sucrose transporter SUC2, chlorophyll a and chlorophyll b synthesis related gene CAB1 expression substantially increase. The HUCEP-8 gene that the present invention clones from Semen Maydis is the sugared efficient absorption of staple crops And accumulate the genetic resources providing more effectively fruit, at the nutrient efficient of genetic engineering improvement plant Performance study will play a significant role.The angle simultaneously from carbohydrate, nitrogen efficiency improved Consider, be expected to improve plant nitrogen efficiency especially, cultivate the efficient crops of nitrogen.

Accompanying drawing explanation

Fig. 1 is the phylogenetic analysis of ZmSTP1 gene of the present invention；

Fig. 2 is the ZmSTP1 gene of the present invention quantitative expression analysis in different tissues；

Fig. 3 a is the tissue expression positioning analysis of ZmSTP1 gene of the present invention；

Fig. 3 b is the Subcellular Localization of ZmSTP1 gene of the present invention；

Fig. 4 is that the allos checking EBY.VW4000 yeast mutants that has complementary functions has complementary functions table Type analysis；

Fig. 5 is the plant phenotype analysis (plate is trained 18 days) of arabidopsis overexpression ZmSTP1 gene；

Fig. 6 is plant lotus throne leaf phenotype analytical (the plate training of arabidopsis overexpression ZmSTP1 gene 18 days)；

Fig. 7 is that the plant of arabidopsis overexpression ZmSTP1 gene is to variable concentrations inorganic nitrogen supply Response analysis (plate is trained 18 days)；

Fig. 8 is the plant lotus throne leaf phenotype statistical analysis of arabidopsis overexpression ZmSTP1 gene (earth culture 35 days)；

Fig. 9 is the biomass of arabidopsis overexpression ZmSTP1 gene, grain yield statistics Analyze (earth culture 55 days)；

Figure 10 is that plate is trained and the arabidopsis sugar content of overexpression ZmSTP1 gene under the conditions of earth culture Measure；

Figure 11 is the quantitative expression analysis of AtSuc2 and AtCAB1.

Detailed description of the invention

Following example are used for illustrating the present invention, but are not limited to the scope of the present invention.

In following embodiment, method therefor is if no special instructions, is conventional method.

The preparation of material

1, bacterial strain and instrument plasmid

Material used in the embodiment of the present invention includes: competent escherichia coli cell DH5 α (Code No.CB101, Tian Gen biochemical technology company limited), Agrobacterium tumefaciems GV3101 (purchased from Tian Gen biochemical technology company limited) and EBY.VW4000 yeast mutants；TA gram Grand carrier:19-T Vector (Code No.D102, Takara company), pSuper1300+ -Kanamycin, pDR195 (Zi Huan institute teacher Yuan Lihang give)；pUC-GFP(Takara Bio).

2, toolenzyme and biochemical reagents

Various restricted enzyme are purchased from NEB company；Various Taq enzyme and Trizol RNA are little Amount extracts test kit purchased from Takara company；DNTP mixture is purchased from the raw work in Shanghai；T4DNA Ligase is purchased from Promega company；Plain agar sugar gel DNA reclaims test kit (Code No.DP209, Tian Gen biochemical technology company limited)；The little extraction reagent kit of plasmid is purchased from (Code No.DP103, Tian Gen biochemical technology company limited) ampicillin (Amp), kanamycin (Kan), rifampicin (Rif) is purchased from glad through company of section.

3, pcr amplification primer thing

ZmSTP1-pDR-L:5 '-CTCGAGATGGCCGGCGGTGGCATCGTG-3 '

ZmSTP1-pDR-R:5 '-GGATCCTCACGCGTCGGCCCCCTTGG-3 '

ZmSTP1-RT-L:5 '-GTCTTCATCGCCTTCTTCCTG-3 '

ZmSTP1-RT-R:5 '-TTGGTGTCGCTGCCGTTT-3 '

ZmUbiquitin-L:5 '-GTTGAAGCTGCTGCTGTATCTGG-3 '

ZmUbiquitin-R:5 '-GCGGTCGCACGATAGTTTTG-3 '

ZmSTP1-Situ-F:5 '-GATTTAGGTGACACTATAGAATGCTCCA

AAAACGGCAGCGACA-3′

ZmSTP1-Situ-R:5 '-TGTAATACGACTCACTATAGGGGTACTAT

TGCTTGGTGGTG-3′

ZmSTP1-GFP-F:5 '-TCTAGAATGGCCGGCGGTGGCATCGTG-3 '

ZmSTP1-GFP-R:5 '-GGATCCCGCGTCGGCCCCCTTGGTG-3 '

AtAct2-L:5 '-TGATGCACTTGTGTGTGACAA-3 '

AtAct2-R:5 '-GGGACTAAAACGCAAAACGA-3 '

AtSUC2-L:5 '-GGATCGCTTGGTTCCCTTTC-3 '

AtSUC2-R:5 '-GGAGTCAGAGCTGGTGCTTTGG-3 '

AtCAB1-L:5 '-CCCATTTCTTGGCTTACAACAAC-3 '

AtCAB1-R:5 '-TCGGGGTCAGCTGAAAGTCCG-3 '.

Embodiment 1, the clone of primverose transporter gene ZmSTP1 gene

The monosaccharide transport protein aminoacid sequences such as arabidopsis, Oryza sativa L., Semen Tritici aestivi obtain data base NCBI, Maizesequence and Uniprot, use ClustalW1.8 to compare (Thompson et al.1994Nucleic Acids Research 22,4673-4680).Evolve Tree analysis shows nearest with AtSTP1 sibship, therefore by this unnamed gene is ZmSTP1 (Fig. 1).

1, Total RNAs extraction

Take 200mg fresh B73 Zea mays root, grind in liquid nitrogen；Add 1ml test kit to carry The Trizol extracting solution of confession, room temperature is shaken 5 minutes；Add 200 μ l chloroform, concussion 30 seconds, 4 DEG C, 12000 leave the heart 15 minutes；Take supernatant, add 0.5ml isopropanol, room Gentle and quiet put 1 hour, 4 DEG C, 12000 leave the heart 15 minutes；Take precipitation, add 1ml 70% Ethanol, shakes 1 minute, 4 DEG C, 10000 leave the heart 10 minutes；Supernatant is abandoned in suction, and precipitation is put Fume hood dries up, adds 50 μ l DEPC water dissolution precipitations.1% sepharose electrophoresis detection RNA Quality, simultaneously by spectrophotometric determination RNA concentration.

2, mRNA purification

Take 500 μ g total serum IgE and join in a new centrifuge tube without RNase, add 1ml The combination liquid that test kit provides；It is placed in 65 DEG C 10 minutes, forwards to the most at once put 1 point on ice Clock；Liquid is transferred in the centrifuge tube containing oligo (dT) resin that test kit provides, room temperature jog 20 minutes, room temperature 4000 left the heart 10 minutes, and careful suction abandons supernatant, is repeated 2 times；Then Add 0.3ml and combine the resuspended resin of liquid, transfer in the spin-column pipe that test kit provides, Adding 500 μ l and combine liquid washing, 4000 turns of room temperatures are centrifuged 10 seconds, survey the OD of eluate₂₆₀, If greater than 0.05, the most again add 500 μ l and combine liquid washing, until eluate OD₂₆₀Little In 0.05；Add 200 μ l eluents, soft hanged resin, spin-column pipe is forwarded to On one new centrifuge tube, room temperature 4000 leaves the heart and collects mRNA in 10 seconds.It is eventually adding 10 μ l 2mg/ml glucosides, 30 μ l 2M sodium acetates, 600 μ l dehydrated alcohol, place 30 minutes for-80 DEG C, 4 DEG C, 14000 leave the heart 20 minutes, abandon supernatant, 70% ethanol is washed once, with 20 μ l TE Dissolve.Take out 0.5 μ l and measure OD₂₆₀And calculate concentration.

3, cDNA the first chain synthesis

Taking 5 μ g mRNA, the reverse transcription carried with test kit carries out reverse transcription.Specific as follows: It is 100ng/ μ l that the Biotion-attB2-oligo (dT) that taking 1 μ l test kit provides is added to 10 μ L concentration The mRNA solution that obtains of upper step purification in, place 5 minutes, forward rapidly 3 points on ice to for 70 DEG C Clock, is subsequently adding following component:

Reference reagent box operation instructions set following reaction condition: 25 DEG C 10 minutes, 42 DEG C 60 Minute, 70 DEG C 10 minutes, ice bath 2 minutes.

4, ZmSTP1 clone

With specific clone primer (ZmSTP1-OE-F, ZmSTP1-OE-R) and above cDNA STP1 is cloned, specific operation process for template:

Addition following component in 200 μ l centrifuge tubes:

The laggard performing PCR of centrifugal mixing, PCR response procedures is as follows:

Normal Agarose Gel detection is reclaimed, and is centrifuged column type test kit operating procedure according to sky root.

Add A tail, addition following component in 200 μ l centrifuge tubes:

Purification DNA product 7 μ l

After shaking up, hatch 30min for 72 DEG C.Use19-T TA Cloning Kit, will The am-plified fragments reclaimed is cloned on T Vector, construction recombination plasmid.

16 DEG C overnight connect, and thermal shock converts to 50 μ L competent cells.37 DEG C, 150rpm Shaking table activation vibration 60min；Draw 100 μ l bacterium solution and be coated on LB+Amp solid medium On flat board, it is put in overnight incubation (12-16 hour) in 37 DEG C of constant incubators.Picking Dan Ke Grand, carry out checking order-checking.With the correct plasmid that checks order as template, add not according to subsequent experimental Same restriction enzyme site, repeats 4 operations, is connected to by the ZmSTP1 containing restriction enzyme site19-T On carrier, check order, protect bacterium after order-checking is correct standby.

Embodiment 2, the Real-Time pcr analysis Semen Maydis each tissue expression of ZmSPT1 gene are special Property.

Material to be tested is planted in village experiment centre in China Agricultural University, and to weaving silk, one week after is gathered in the crops not With the corn sample of tissue, it is placed on rapidly in liquid nitrogen, takes back laboratory and be positioned over-80 degree ice Case is standby.

Quantitative result shows, ZmSTP1 expression in root the highest (Fig. 2).

Attached Real-Time PCR operating procedure:

1, the total serum IgE (method is with embodiment 1) in different disposal sample root is extracted；

2,50 μ g total serum IgE are taken, with DNase I (TaKaRa company, catalog number (Cat.No.): D2215) Removing genomic DNA, method is as follows:

Reaction system (50 μ l):

37 DEG C are reacted 30 minutes；

Add 150 μ l DEPC water, add 200 μ l phenol/chloroform/isoamyl alcohol (25:24:1), Fully mixing；

4 DEG C, 12000rpm is centrifuged 10 minutes, takes upper strata and moves in new centrifuge tube；

Add 200 μ l chloroforms/isoamyl alcohol (24:1), fully mix；

4 DEG C of 12000rpm are centrifuged 10 minutes, take upper strata and move in new centrifuge tube；

Add the 3M NaAc (pH=5.2) of 20 μ l, add 500 μ l pre-cooling dehydrated alcohol ,-20 DEG C Place 60 minutes；

4 DEG C, 12000rpm is centrifuged 15 minutes, reclaims precipitation, and 70% pre-cooled ethanol washes precipitation 2 times；Each 4 DEG C, 7500rpm is centrifuged 5 minutes；

Drying up, DEPC water weight is molten.

3, conventional method reverse transcription synthesis cDNA the first chain (method is with embodiment 1).

4, Real-time PCR detects gene abundance, and TOYOBO company selected by reagent SYBR Green Realtime PCR Master Mix (catalog number (Cat.No.) 91620F3), quantitative PCR INSTRUMENT MODEL Bio-Rad iCycler iQ5system (BIO-RAD company), inversion product is dilute Release 10 times as Real-time pcr template

Reaction system:

PCR response procedures: 50 DEG C 2 minutes, 95 DEG C 10 minutes, 45 circulations (95 DEG C 15 Second, 61 DEG C 30 seconds, 72 DEG C 1 minute)；

Melt curve analysis step: 95 DEG C 15 seconds, with circulation in 10 seconds, each circulation increased The speed of 0.5 DEG C is warmed up to 95 DEG C from 60 DEG C, carries out 70 circulations；

With ZmUbi as internal reference, relative quantification algorithm is used to calculate ZmSTP1 at different tissues In relative expression quantity.

ZmSPT1 is expressed and determines by embodiment 3, utilization in situ hybridization, green fluorescent protein technology Position is analyzed.

1, the preparation of vegetable material

Plant culturing Hoagland culture fluid, tri-leaf period, corn seedling took tip of a root sample.Clip Tip of a root 0.5-1cm puts in FAA fixative that (composition every 100ml fixative contains: 50% ethanol 90ml, glacial acetic acid 5ml, formaldehyde 5ml)；

Then carrying out vegetable material dehydration, transparent, waxdip, method is as follows:

Discarding FAA fixative, DEPC washes twice；

50% ethanol, 50% ethanol+10% tert-butyl alcohol, 50% ethanol+20% tert-butyl alcohol, 50% Ethanol+35% tert-butyl alcohol, 50% ethanol+50% tert-butyl alcohol, 25% ethanol+75% tert-butyl alcohol, 25% Ethanol+75% tert-butyl alcohol+0.1% Eosin Y), 100% tert-butyl alcohol respectively processes 2 hours；

Proceed to 2/3 tert-butyl alcohol+1/3 paraffin oil is placed 4 hours；

Pour out 1/3 tert-butyl alcohol paraffin oil mixed liquor, supplement the paraffin of same volume 60 DEG C thawing in upper Layer, forms the wax lid of solidification, places 12 hours for 60 DEG C, (being repeated 3 times)；

Pour out whole liquid, add pure thawing paraffin, 60 DEG C, 8 hours, (being repeated 2 times)；

Then embed on 65 DEG C of boiling hot plates.

2, the synthesis of probe and purification

Rna probe synthesis is carried out, with reference to Takara company while preparing vegetable material The using method of T7-RNA polymerase (catalog number (Cat.No.): P2075), concrete operations are as follows:

The synthesis of Hua Da genome company is containing T7 promoter and the forward primer of ZmSTP1:

5′-GATTTAGGTGACACTATAGAATGCTCCAAAAACGGCAGC GACA-3′

Reverse primer:

5-TGTAATACGACTCACTATAGGGGTACTATTGCTTGGTGGT G-3′；

From plasmid, the DNA profiling containing T7 promoter is amplified with KOD enzyme, external turn Recording into rna probe, reaction system is as follows:

Totally 20 μ l, mixing, react 2 hours at 37 DEG C；

Add 4 μ l DNase I (TaKaRa company, catalog number (Cat.No.) D2215) 37 DEG C reaction 15 Minute remove genomic DNA；

Reaction is placed on ice after terminating, and adds 0.8 μ l 0.5M EDTA (pH=8.0) and terminates reaction；

Add 2 μ l 5M LiCl, the dehydrated alcohol of 75 l-20 DEG C of pre-coolings of μ, place after mixing In-20 DEG C 2 hours；

13000rpm, 4 DEG C are centrifuged 15 minutes；

Supernatant discarded, adds the washing with alcohol precipitation of 50 μ l 70% pre-coolings, 13000rpm 4 DEG C Centrifugal 5 minutes；

Supernatant discarded, dries up precipitation；

Adding 24 μ l DEPC-H2O to dissolve ,-80 DEG C save backup.

3, film-making:

Embedded cured piece, cuts out 8-10 μm slice, thin piece with Fructus mail micromali space QP-4 type microtome. The wax band (containing plant sample) intercepting correct position is bonded at the slide glass (Sigma of poly-D-lysine Company, catalog number (Cat.No.) P0425-72EA) on, wax band is put into 45 DEG C of roasting sheet platforms in DEPC water Upper exhibition sheet, sucks unnecessary water after exhibition sheet is abundant, and in 40 DEG C of baking ovens, roasting sheet 24-48 hour, makes Section is fully dried.

Section dewaxing: slice, thin piece wash in dimethylbenzene 3 times each 5 minutes, dehydrated alcohol 2 times each 2 Minute, 95% ethanol, 85% ethanol, 70% ethanol, 50% ethanol, 30% ethanol the most each 1 Minute, DEPC washing 2 times, each 1 minute.

E.C. 3.4.21.64 processes: add E.C. 3.4.21.64 reaction buffer (100 in a color jar MM Tris-HCl pH 7.5,50mM EDTA) and E.C. 3.4.21.64 to final concentration 1 μ g/ml, Putting into the slice, thin piece that pretreatment is good, 37 DEG C are incubated 20 minutes；

Microscope slide is washed 2 times with DEPC, each 1 minute；

Acetylation processes: be placed in color jar by microscope slide, adds 40ml dissolved with 100 μ l The 0.1mol/L triethanolamine solution (pH=8.0) of acetic anhydride, room temperature is placed 10 minutes；Fall Falling solution, 2 × SSC solution washes twice, each 7 minutes；

At room temperature, successively with different dilution ethanol waters (30%, 50%, 70%, 85% and 95%) washing slice, thin piece, every grade 1 minute, make tissue slice be dehydrated.Then with new Dehydrated alcohol is washed 2 times, each 2 minutes.Room temperature is dried.

4, hybridization

Hybridization solution forms: every slice, thin piece, with 200 μ l hybridization solutions, comprises: 100 μ l deionization first Amide, 20 μ l 10 × hybridization buffer (100mM Tris pH 7.5,10mM EDTA, 3 M NaCl), 24 μ l 50% dextran sulfates, 20 μ l 10 × Blocking Solution, 250 μ g salmon sperm dna, 5 μ l probes, 36.5 μ l 50 × Denhardt ' s solution.Lucifuge hybridization 16-30 Hour.

Rinse: slice, thin piece immerses in 2 × SSC and makes cover plate come off, and then room temperature is placed 30 minutes； 2 × the SSC renewed, 65 DEG C 1 hour；0.1 × SSC, 65 DEG C 1 hour.

Close: drying the microscope slide back side with absorbent paper, be placed in wet box, every adds 2ml 1% Confining liquid (every 400 milliliters contain Boehringer Block reagent 2g, 0.1M Tris-HCl, PH=7.5；With 0.15M NaCl), room temperature is placed 1 hour.

Balance: remove confining liquid, every add 1ml develop a film liquid (100mM Tris-HCl pH=7.5, 150mM NaCl, 0.3%Triton X-100,1%BSA) balance 15 minutes.

Antibody adsorbs: removes balance liquid, adds 400 μ l antibody-solutions (every 400 μ l antibody Solution contains: 399 μ l develop a film liquid, the Anti-DIG-AP that 1.32 μ l test kits provide), room temperature is miscellaneous Hand over 2 hours or 4 DEG C overnight.

Develop a film: slide is washed 3 times in liquid of developing a film, each 10 minutes.

Colour developing forward horizontal stand: in colorbuffer soak 5 minutes (1ml colorbuffer formula: 100 μ l 1M Tris-HCl pH9.5,20 μ l 5M NaCl, 860 μ l ddH2O, 0.1g are poly-own Enol).

Colour developing: add 20 μ l NBT/BCIP, every dropping 200-500 μ l in colorbuffer Nitrite ion, develop the color at room temperature dark in wet box 0.5-4h, and when microscopy, positive signal is pale red Or rufous, and background without obvious color time get final product stopped reaction, with water rinse 3 times, every time 5 minutes.After neutral gum mounting, the positive signal of rufous becomes blue or bluish violet.

As shown in Figure 3 a, for probe results of hybridization, the upside of two figures is tip of a root rip cutting figure to result, Downside is tip of a root sectional view.Result shows that ZmSTP1 has expression at whole Corn Root Tip Cells, this It is consistent for mediate root absorbing sugared biological function with it from environment.

Utilize PUC-GFP carrier, 35S promoter, build the restructuring matter merging ZmSTP1 The expression vector of grain, injects tobacco leaf, transient expression ZmSTP1, positions it, Result shows that it has expression (Fig. 3 b) on cell membrane and nucleus.

Embodiment 4, ZmSTP1 yeast allos have complementary functions checking

XhoI and BamHI restriction enzyme site is contained at the two ends of pDR195 carrier.Take 1-1.5ml Bacterium solution, with reference to the explanation of sky root plasmid little extraction reagent kit, extracts plasmid, selects XhoI and BamHI By ZmSTP1 gene fromScaling off on 19-T, electrophoresis reclaims, and is connected into pDR195 Between XhoI and BamHI restriction enzyme site on carrier, through order-checking choose that forward is connected into gram Grand.

EBY.VW4000 yeast mutants, in default of all of hexose and galactose sugar transport Albumen, cause can not surviving in the environment that monosaccharide is sole carbon source (Wieczorke et al., 1999), but can grow in the environment that maltose is carbon source, use this characteristic we Detect the function of ZmSTP1.Build yeast pDR195 recombiant plasmid through above operation to import To EBY.VW4000 yeast mutants, under carbon source certain environment, analyze yeast activity.

Yeast expression carrier proceeds to EBY.VW4000 yeast mutants

By in yeast-inoculated to be transformed to 5ml liquid YPD medium, in 30 DEG C, 200rpm Shaken overnight is cultivated.Measure concentration to OD₆₀₀=0.5 is inoculated in 50mlYPD culture medium, In 30 DEG C, 200rpm shaken cultivation to OD₆₀₀=2,3000 × g are centrifuged 5min and collect cell, Discard culture supernatants, cell suspension in 25ml sterilized water, then centrifugal collecting cell, Abandon water, cell suspension in 1ml sterilized water, be transferred in aseptic 1.5ml centrifuge tube, Centrifugal, remove supernatant, add 1ml sterilized water suspension cell,

According to inversion quantity (about 200 μ l) subpackage cell suspension, recentrifuge 1-2min precipitates Cell, with pipettor careful sucking-off supernatant, then, adds the conversion mixed liquor of premix:

After acutely vibration mixes completely to cell, it is placed in 42 DEG C of water-baths thermal shock at least 40min (Suga and Hatakeyama, 2005), high speed centrifugation 30sec, remove to convert and mix Close liquid, add 0.2-1.0ml sterilized water, extract suspension precipitation with pipettor the most gently.

The pDR-EBY.VW4000 proceeding to empty carrier is negative control, wild type PDR-23344c is positive control.Cultivate at the enterprising row filter of solid agar medium after conversion, Culture medium is 6.7g/L YNB (yeast nitrogen base), and wherein uracil is by ammonium sulfate (0.5 G/L) maltose (20g/L) substitutes.Growth medium adds on the basis of screening culture medium 2% glucose, fructose, galactose or mannose are as unique carbon source, ammonium sulfate (0.5g/L) As nitrogen source.Take OD₆₀₀The yeast liquid of=1.0, is diluted to 10^-1, 10^-2, 10^-3, 10^-4 Four gradients, inhale before 10 μ l drop in culture medium with liquid-transfering gun, are inverted for 30 DEG C and cultivate 3 days Observe phenotype.Each process, with different monoclonals in triplicate.Distinctness is formed with mutant Contrast, import ZmSTP1 yeast at glucose (Glc), fructose (Frc) and mannose (Man) growth is substantially, and under the conditions of galactose (Gal) is supplied, upgrowth situation also obtains one Determine degree and improve (Fig. 4), show that ZmSTP1 can absorb after proceeding in yeast system multiple Monosaccharide, but different to the absorbability of different monosaccharide.

Attached yeast plasmid DNA extraction method:

Picking growth bacterial plaque accesses in the 0.5ml YNB fluid medium containing 1mM Arg, 30 DEG C, 230rpm shaken cultivation is overnight；4,000rpm are centrifuged 5 minutes collection thalline；

Outwell supernatant, with the fresh resuspended thalline of fluid medium (cumulative volume about 50 μ l), The most often pipe adds the lysozyme soln of 10 μ l concentration 10mg/ml, fully vibration make solution with Thalline mixes completely；

By test tube at 30 DEG C, 230rpm shaken cultivation 60 minutes；

Often pipe adds 10 μ l 20%SDS, and acutely vibration makes fully to mix for 1 minute；

Sample being put into-20 DEG C 2 hours, take out and melt, acutely vibration makes fully to crack；

With TE buffer (pH=7.0), every pipe volume added to 200 μ l；

Add 200 μ l phenol: imitative: isoamyl alcohol (25:24:1), acutely vibration 5 minutes；14,000rpm Centrifugal 10 minutes, supernatant is transferred in new centrifuge tube；

Add 8 μ l 10M NH₄Ac and 500 μ l dehydrated alcohol；

Putting in-80 DEG C of refrigerators 1 hour, 14,000rpm are centrifuged 10 minutes；

Supernatant discarded, dries up precipitation, with 20 μ l H₂O dissolution precipitation；

Taking 0.5 μ l plasmid to forward in E.coli competent cell (DH5 α bacterial strain), 37 DEG C are shaken bacterium, Extracting plasmid, enzyme action identifies the order-checking of Hou Song company.

Embodiment 5, structure, conversion and the phenotype thereof of ZmSTP1 arabidopsis overexpression vector are divided Analysis.

Wildtype Arabidopsis thaliana col is converted with recombinant expression carrier pSuper1300+-ZmSTP1. Concrete grammar: take be accredited as the positive Agrobacterium bacterium solution 0.5ml be inoculated in 500ml YEB In fluid medium, in 28 DEG C of shaken cultivation to OD₆₀₀To 0.5.5000rpm 4 DEG C is centrifuged Within 15 minutes, collect thalline.With infiltration buffer (1 × MS a great number of elements, 5% sugarcane of 200ml Sugar) resuspended thalline, add silwet L-77 (GE company, article No.: S5505) to final concentration 0.2%.1 × a great number of elements contains 1.65g/L NH₄NO₃, 1.9g/L KNO₃, 0.44g CaCl₂ 2H₂O, 0.37g/L MgSO₄7H₂O and 0.17g/L KH₂PO₄.Just open after bolting The flower of the arabidopsis of flower is dipped in re-suspension liquid and infects 30 seconds.Plant, lucifuge is wrapped up with freshness protection package Place at 16 DEG C 24 hours, then upright normal growth, until results T₀For seed.

1, the screening of transgenic positive plant

Due to proceed to the Arabidopsis plant of pSuper1300+-ZmSTP1 carrier have hygromycin resist Property, so with normal growth, and can not proceed on the MS solid medium containing hygromycin The wild type seeds of gene can not normal growth death.Converting transfer-gen plant in the present age is T₀ In generation, by this T₀The seed produced for plant selfing and the plant grown up to by it are T₁Generation.Mixed Close and collect T₁For seed, it is seeded on the MS solid medium containing 50 μ g/ml hygromycin, Plantlet of transplant continued growth in basin of screening energy normal growth, individual plant sowing.T₂For seed again Individual plant results T after 1 hygromycin resistance screening₃For seed.The most again through once resisting Property screening, what all individualities can grow is to turn the homozygote of pSuper1300+-ZmSTP1 to plant Strain, stays standby.

2, the Molecular Detection of transgenic arabidopsis

PCR detects: extract T respectively₃In generation, turns pSuper1300+-ZmSTP1 gene arabidopsis The total serum IgE of homozygous plants, under oligo (dT) guides, reverse transcription becomes the first chain cDNA, with ZmSTP1-RT-L and ZmSTP1-RT-R is that primer carries out RT-PCR detection in transgenic plan The expression of ZmSTP1 gene in the mustard of south.

3, transfer-gen plant monosaccharide absorbability is analyzed

To T₃Vernalization, surface sterilizing is carried out, at ATS sugar-free solid agar medium for seed Carry out send out Seedling cultivate (Schofield et al., 2009Plant, Cell and Environment 32, 271-285).Process after 4 days, choose 3 strains of the consistent ZmSTP1-OE of growing way and Col-0 seedling moves to square (13 × 13cm²) in screening culture medium.Screening culture medium is for it Before send out Seedling culture medium, add the sugar of variable concentrations as carbon source simultaneously, be respectively as follows: glucose (Glc), fructose (Frc), sucrose (Suc), ribose (Rib), galactose (Gal), inositol (MI), xylose (Xyl), mannose (Man).Sugar-free ATS culture medium is negative control, adds Add the ATS of trimethyl glucose (glucalogue, it is impossible to by hexokinase phosphorylation) Culture medium is positive control.Glc (2,5,10mM), Frc (5,10mM), Suc (5mM) or Rib (5,55mM) supply under, lotus throne number of sheets showed increased；High concentration Glc, Under Frc, Gal, Suc, Xyl, Rib, Man, MI, lotus throne number of sheets mesh reduces (number According to row the most entirely).Lotus throne leaf diameter increases under low concentration Glc, Frc, Rib carbon source, Reduce under high concentration Glc, Frc, Gal, Suc, Xyl, Rib, Man or MI, Final Biomass the most dramatically different (Fig. 5, Fig. 6).Add 9 at 55mM different carbon source simultaneously Or 1mM NO₃ ^-Under nitrate nitrogen, growth is substantially suppressed (Fig. 7), illustrates that overexpression is planted Strain is more sensitive to nitrogen.

Earth culture test in, transgenic arabidopsis and wild type be planted in short-day under the conditions of (8/16 Day/night, 84 μm ol m^-2s^-1, 22 DEG C), until 35 days.After 35 days, short-day changes For long-day (16/8h day/night, 56 μm ol m^-2s^-1, 22 DEG C) until 55 days are ripe. To the first inflorescence height, plant fresh weight, dry weight, every seed weight is added up.Solubility Sugar chloroform/methanol method extracts (Antonio et al., 2008Rapid Communications in Mass Spectrometry 22,1399-1407), with liquid chromatograph spectrophotometry (HP 1100, Agilent Technologies, Palo Alto, CA, USA), according to Focks etc. The method of people test kit (Sigma-Aldrich, St.Louis, USA) measures content of starch. Under the conditions of short-day, ZmSTP1 overexpression plant vegetative growth is promoted, shows as blade Increase, petiole length increases, number increases, and lotus throne leaf diameter dramatically increases (Fig. 8).Long day Under the conditions of according to, overexpression plant shows as inflorescence number to be increased, and dry weight increases, and leaf color is more Deeply (Fig. 9).AtSUC2 (sucrose transporter 2) up-regulated expression (figure in overexpression plant 10), show that ZmSTP1 affects the transhipment of other saccharides；ZmSTP1 is also possible to and light and way Being correlated with in footpath, chlorophyll a and chlorophyll B synthesis associated protein AtCAB1 are in overexpression plant Raise (Figure 11).To sum up showing, ZmSTP1 may be by affecting the transhipment of other saccharides Yield is finally increased with regulating and controlling photosynthetic pathway.

Attached: arabidopsis is cultivated and transgenic seedlings screening

Take appropriate arabidopsis seed in 1.5mL centrifuge tube, add deionized water.Make water as far as possible Do not had all seeds, be placed in 4 DEG C of refrigerator vernalization 2 days.Seed is carried out disinfection by super-clean bench, Add 75% ethanol sterilizing 1 minute, clean one time with aquesterilisa, add 2% sodium hypochlorite Sterilizing 2 minutes, rinses 5-7 time with aquesterilisa.With 10 μ L liquid-transfering guns, seed is uniformly put In 1/2MS (or 1/2MS+50mg/ml hygromycin) culture medium, seed-bearing training will be put Foster base is vertically arranged in culturing room's (illumination: 100 μ E m^-2s^-1, the photoperiod: 16h daytime/8h is black Night, temperature 22/20 DEG C, humidity 100%) middle cultivation.By preferable for growing way seedling replanting to dress Have in the flowerpot of moistening Nutrition Soil (m Vermiculitum/m Nutrition Soil=1:1).The 4 every basins of strain, often dish 12 Basin, flowerpot surface covers one layer of preservative film, to prevent water point excessive vaporization from affecting growth of seedling. Within every 5 days, water 400ml water, throw off preservative film after 10 days, within every 3 days, water 400ml water.

Take T₀For arabidopsis seed vernalization, sterilization, seed uniform application is screened at 1/2MS (containing 50 μm ol/L hygromycin) in culture medium.Wrap up one layer of black plastic film, be positioned over reality Test room, dark culturing 5-7 days, if seedling stem is extracted out the highest, base may be turned for what there is resistance Because of seedling, each transgenic plant selects 12 strain seedling, numbering, moves to the training of 1/2MS culture medium Support 3-5 days, after it grows 4 spires, move to that Nutrition Soil is placed in culturing room cultivate, complete Its whole period of duration.Repeat above operation until obtaining the T isozygotied₃For seed.

Although, the most with a general description of the specific embodiments the present invention has been made in detail Most description, but on the basis of the present invention, it can be made some modifications or improvements, this is to this It is apparent from for skilled person.Therefore, on the basis without departing from spirit of the present invention Upper these modifications or improvements, belong to the scope of protection of present invention.

Claims (9)

1. Semen Maydis ZmSTP1 albumen, it is characterised in that the aminoacid sequence of described albumen is such as Shown in SEQ ID No.1.

2. the encoding gene of albumen described in claim 1, it is characterised in that described gene Nucleotide sequence is as shown in SEQ ID No.2.

3. the application in promoting root system of plant monosaccharide to absorb of the gene described in claim 2.

Application the most according to claim 3, it is characterised in that described in claim 2 In gene transferred plant, promote the accumulation of root system of plant monosaccharide efficient absorption, and then improve plant Biomass and/or grain yield.

Application the most according to claim 4, it is characterised in that described gene is by weight Group expression vector proceeds to plant cell.

Application the most according to claim 5, it is characterised in that described recombinant expressed load Body is to insert the recombiant plasmid that described gene obtains between the multiple clone site of pPT-HYG.

7. contain the recombinant expression carrier of gene described in claim 2, transgenic cell line and Recombinant bacterium.

8. the method promoting that root system of plant monosaccharide efficient absorption accumulates, it is characterised in that Gene described in claim 2 is proceeded in plant cell by recombinant expression carrier.

Method the most according to claim 8, it is characterised in that described recombinant expressed load Body is to insert the recombiant plasmid that described gene obtains between the multiple clone site of pPT-HYG.