CN103993017B - A kind ofly control the leaf zinc finger protein gene OsRLZP of paddy rice and application thereof - Google Patents

Abstract

The present invention relates to the leaf zinc finger protein gene OsRLZP of a kind of adjusting and controlling rice and application thereof, is this gene nucleotide series as SEQ in sequence table? ID? shown in NO:1; Is its aminoacid sequence of the albumen of described genes encoding as SEQ in sequence table? ID? shown in NO:2.Is the present invention by SEQ? ID? described in NO:1, full length gene encoding sequence is inserted in eukaryotic vector, obtains the eukaryon recombinant plasmid of OsRLZP gene overexpression, by above-mentioned eukaryon recombinant plasmid rice transformation, obtains OsRLZP process LAN transfer-gen plant.Does experiment show: SEQ of the present invention? ID? NO:1 gene can control rice leaf and grow; Compared with WT lines, OsRLZP raises that arteries and veins in rotaring gene plant blade is thicker, blade narrows, and whole plant leaf is involute upright, and blade table fur reduces.Therefore, gene pairs rice leaf of the present invention is grown and is had regulating and controlling effect, and can improve in genetic breeding at plant type of rice and apply.

Description

A kind ofly control the leaf zinc finger protein gene OsRLZP of paddy rice and application thereof

Technical field

The invention belongs to plant genetic engineering field, particularly the leaf controlling gene of a kind of paddy rice and the application in crop plant type improvement genetic breeding thereof.

Background technology

Rice leaf is the deciding factor that rice yield is formed as carrying out photosynthetic major organs.Larger leaf area could form larger photosynthetic colony, but easily criticizes vertical when blade is long, wide, can reduce total light-receiving area of colony on the contrary, and the ventilation condition of impact plantation colony, be easy to pathogenic micro-organism and grow procreation.Thus, the improvement of rice leaf form is the important goal of plant type improvement always.

Rice leaf form leading indicator comprises: Leaf rolling index, inclination angle, hang down loosely degree and width of blade etc.Advances in Molecular Genetics shows, rice leaf crimpness primarily of near/far away between centers growth of rolled leaf gene regulation and control blade, the growth of bulliform cell, sclerenchymatous formation and the cuticular growth of blade etc. wherein a certain link realize; Leaf inclination mainly affects growing of pulvinus cell via leaf angle gene regulating brassinolide intracellular signaling; Blade degree of hanging down loosely may be by draping over one's shoulders the growth of phyllopodium because of arteries and veins in the control blades such as DL1; Narrow leaf gene is the growth of the synthesis of major regulatory growth hormone and polar translocation, vascular tissue and distribution then, affects blade vascular bundle number and width.But up to now, cloned leaf regulatory gene and few and between gene the research of mutual relationship also deep not enough, completely clearly can't sketch the contours the leaf molecular regulation network (Xu waits quietly, Acta Agronomica Sinica, 2013) built up and grow of paddy rice.

Due to blade appropriateness, curling colony can significantly improve upright blade, and can increasing leaf length and width, blade can be avoided to criticize again vertical, improves colony and ventilates and illumination condition, promotes that later stage dry-matter increases, and is conducive to improving economic coefficient.So, in the ideotype that rice breeding man proposes, all think that vertical flag leaf is the important component part of Ideal Rice Plant Type." heavy panicle type " Ideotype model as, Zhou Kaida thinks that " leaf inward-curl is upright " comparatively should (Sichuan Agricultural University's journal, 1995); In the ideotype of the Super High-yield Rice Hybrids of Yuan Longping, Top-three Leaves should " long, straight, narrow, recessed, thick " (hybrid rice, 1997).

Rolled Leaf Gene in Rice can be coerced as arid, insect pest etc. cause by external environment, but by the leaf rolling characteristics of Gene Handling be then can heredity, there is the breeding objective of important value.Nearly 20 rolled leaf genes are located at present, as rl1, rl2, rl3, rl4, rl5, rl6, rl7, rl9, rl10, rl11, rl12, rl (t), url1, nal3, RL-A2, SLL1, NRL1, OsAGO7, (Pan Cunhong etc., rice in China science, 2011; Zhang etc., PlantCell, 2009), wherein most gene is recessive inheritance.But the rolled leaf gene of Fine Mapping is also few, and the rolled leaf gene be cloned is just less.In the rolled leaf gene of having cloned, SHALLOT-LIKE1 (SLL1) encodes a SHAQKYF class myb transcription factor, the curling proterties of rice leaf (PlantCell such as zhang, 2009) is controlled by the programmed cell death of adjusting and controlling rice blade abaxial side sclerenchyma cell.Shi etc. (Planta, 2007) are by T-DNA labeling acts cloning rice OsAGO7 gene, and its overexpression makes leaf rolling.

Zinc finger protein (zinc-fingerprotein) is the type studying more deep in transcription factor, can name because it has in conjunction with the dactylitic texture territory-Zinc finger domain (zinc-fingerdomain) of zine ion.Zinc finger domain that is single or cluster appearance is had in zinc finger protein, rely on the changable composite of the Zinc finger domain formed in long-term evolution process, different physiological roles (the Gamsjaeger etc. such as regulatory gene is transcribed, chromatin reconstruct can be had in conjunction with DNA, RNA equimolecular, TrendsBiochemSci, 2007).Ubiquity zinc finger protein in plant, some zinc finger protein is that plant institute is peculiar, and grow regulation and control, the stress response etc. in wide participation plant each period.Paddy rice zinc finger protein gene acts on and also not illustrating in leaf development regulation and control, and this is for cloning the new leaf controlling gene resource of paddy rice, is applied to plant type improvement genetic breeding and has important value.

Summary of the invention

The object of the invention is the gene being to provide to control rice leaf and grow, the rise of described gene can affect rice leaf form.

Technical scheme of the present invention is as follows:

The gene that control paddy rice of the present invention is leaf, called after OsRLZP (OryzasativaRollingLeafZinc-fingerProtein), its nucleotide sequence is as shown in SEQ ID NO:1.

The aminoacid sequence of described gene coded protein is as shown in SEQ ID NO:2.Its encoding sequence respectively has a zinc to refer to (zinc-finger) structural domain in 68-88 amino acids, 94-117 amino acids.

The clone of this gene: according to rice mRNA sequences in GenBank (accession number: AK063705), utilizes primer-design software PrimerPremier5.0 to design PCR special primer.From rice leaf, extract total serum IgE, from paddy rice, be separated to OsRLZP gene coded sequence by reverse transcription PCR technology.

OsRLZP gene overexpression eukaryon recombinant plasmid of the present invention is inserted in carrier for expression of eukaryon pHB by full length gene encoding sequence described in SEQIDNO:1 to obtain.

By above-mentioned process LAN recombinant plasmid transformed paddy rice, obtain transfer-gen plant.Experimental result shows: SEQIDNO:1 gene of the present invention can control rice leaf and grow; Compared with WT lines, in OsRLZP rise rotaring gene plant blade, arteries and veins is thicker, blade narrows, curls inward upright, and blade face epidermal hair reduces.Therefore, gene pairs rice leaf of the present invention is grown and is had regulating and controlling effect, and can improve in genetic breeding at plant type of rice and apply.

The present invention has following beneficial effect:

1, the present invention is that the leaf regulation and control of the unifacial leaf gramineous crops such as paddy rice and genetic breeding provide a kind of new genetic resources.

2, the present invention's gene used is the own gene of paddy rice itself, so the safety performance of transgenic paddy rice is high.

3, the clone of gene of the present invention and plant transgene are ordinary method, and material requested is easy to obtain.

Accompanying drawing explanation

Fig. 1 is that the real-time quantitative PCR of OsRLZP process LAN transgenic line detects.The schematic diagram of A:OsRLZP process LAN eukaryon recombinant plasmid (pHB-OsRLZP).B: process LAN transgenic line real-time quantitative PCR detected result.Be followed successively by from left to right: the expression level of OsRLZP gene in wild-type (WT), 3 independent process LAN strains (OX-1 ~ OX-3).

Fig. 2 is OsRLZP process LAN rotaring gene plant blade scanning electronic microscope detected result.A: wild-type (WT), process LAN plant (OX-1) blade upper surface scanning electronic microscope detected result; B: wild-type, process LAN plant leaf lower surface scanning electronic microscope detected result.

Embodiment

Below in conjunction with embodiment, the invention will be further described.In following embodiment, all unreceipted specific experiment conditions, be according to normal condition well known to those skilled in the art, the molecular cloning of such as SambrookRussell: laboratory manual (NewYork:ColdSpringHarborLaboratoryPress, 1989) condition described in, or according to the condition that manufacturer advises.

Embodiment 1: the clone of rice Os RLZP gene

1, reagent

RNA extracts reagent Trizol and is purchased from Invitrogen company; ThermoScript II ReverTraAce is purchased from Toyobo company; High-fidelity DNA polymerase PrimeStar is purchased from TaKaRa company; Cloning vector pEASY ^tM-BluntSimpleCloningVector is purchased from Beijing Quanshijin Biotechnology Co., Ltd; Primer is synthesized by Shanghai Ying Jun Bioisystech Co., Ltd, and all the other reagent are import packing or domestic analytical pure product.

2, coli strain and vegetable material

Intestinal bacteria (Escherichiacoli) bacterial strain DH5 α is purchased from Beijing Quanshijin Biotechnology Co., Ltd; Paddy rice japonica rice variety Japan fine (OryzasativeL.ssp.japonicacv.Nipponbare) seed is bred by Sichuan Academy of Agricultural Sciences and is provided.

3, substratum and solution

LB substratum: Tryptones 10g/L, yeast powder 5g/L, NaCl10g/L.PH to 7.0 is adjusted, autoclaving with NaOH.

SOB substratum: Tryptones 20g/L, yeast powder 5g/L, NaCl0.58g/L, KCl0.19g/L, 100 × Mg ²⁺10mL.PH to 7.0 is adjusted, autoclaving with NaOH.

SOC substratum: SOB+20% glucose.

100 × Mg ²⁺solution: 20.33gMgCl ₂.6H ₂o and 24.65gMgSO ₄.7H ₂o constant volume is in 100mLH ₂o, autoclaving.

20% glucose solution: 20g glucose constant volume is in 100mLH ₂o, filtration sterilization.

4, method

4.1 rice leaf RNA extract

1) get fresh rice leaf, add liquid nitrogen grinding powdering, transfer 100-200mg powder is not in containing in the 1.5mLEp pipe of RNase rapidly, at once adds the mixing of 1mLTrizol extracting solution, vibration 10s, and ambient temperatare puts 5min;

2) add 0.2mL chloroform, thermal agitation 15s, ambient temperatare puts 2-3min;

3) 4 DEG C, the centrifugal 15min of 12000g, in absorption in honest and upright and thrifty 600 μ L to new EP pipe, add 0.6mL Virahol, ambient temperatare puts 10min;

4) 4 DEG C, the centrifugal 10min of 12000g, abandons supernatant, precipitates with 70% alcohol flushing twice, 4 DEG C, the centrifugal 5min of 7500g;

5) outwell ethanol, Air drying RNA precipitates 10min, is dissolved in 50 μ LRNase-freeddH ₂in O, be stored in-80 DEG C for subsequent use.

4.2RT-PCR

4.2.1RT

1) 1 μ g total serum IgE and 1 μ LpolyT is got ₁₈(10 μMs) primer mixes, and uses RNase-freeddH ₂o supplies 12.75 μ L, mixes gently;

2) 65 DEG C of insulation 5min, are transferred to immediately in ice bath, place 2min;

3) add 5 × reaction buffer 4 μ L, 10mMdNTP2 μ L, RNA inhibitor 0.25 μ L (40U/ μ L), ReverTraAce ThermoScript II 1 μ L (100U/ μ L), 42 DEG C of 1h, synthesize the first chain cDNA;

4) 95 DEG C of heating 5min, inactivation ThermoScript II, stops building-up reactions.

4.2.2PCR

The clone of rice Os RLZP gene.200 μ LEP pipes are positioned on ice, add following reagent:

Increase by following program: 98 DEG C of 2min (denaturation); 98 DEG C of 10s (sex change), 58 DEG C of 10s (renaturation), 72 DEG C of 40s (extension), described denaturation renaturation-extension 30 circulation; 72 DEG C of 5min (total elongation).Primer sequence is: RLZPF:5 '-AAGCTTCCCCGTCTGCTTCAAAACCTTC-3 '; RLZPR:5 '-GAGCTCGAAGCAGGTCTTTGCGATT-3 '.By aforesaid operations, obtain OsRLZP gene PCR amplified production.

4.3 High fidelity PCR products are connected with cloning vector pEASY-Blunt

By by described in above-mentioned 4.2 obtain OsRLZP gene PCR amplified production and cloning vector pEASY ^tM-BluntSimpleCloningVector connects (25 DEG C, 10min) by mole molecule number than 2:1, and linked system is as follows:

pEASY ^TM-BluntSimpleCloningVector(50μg/μL)1μL

PCR primer (~ 150 μ g/ μ L) 2 μ L

4.4 intestinal bacteria transform

1) from liquid nitrogen, take out intestinal bacteria (Escherichiacoli) bacterial strain DH5 α competent cell ice bath thaw;

2) product will be connected described in 4.3 and competent escherichia coli cell mixes gently, ice bath 30min;

3) 42 DEG C of thermal shocking 90s, immediately ice bath 1-2min;

4) 0.8mLSOC is added, mixing, 37 DEG C of gentle shaking culture 1h;

5) the centrifugal 1min of room temperature 13000rpm, outwells a part of supernatant liquor, stays the supernatant liquor of about 200 μ L, supernatant liquor and cell is mixed with suction nozzle, and the LB coated containing penbritin (100 μ g/mL) is dull and stereotyped, 37 DEG C of overnight incubation.

4.5 rapid cleavage method qualification recombinant clones

1) picking mono-clonal is inoculated in 500 μ L and contains in the LB nutrient solution of penbritin (100 μ g/mL), and 37 DEG C of shaking culture are to A ₆₀₀be 0.6 ~ 0.8;

2) get 200 μ L bacterium liquid in 0.5mLEP pipe, the centrifugal 1min of 13000rpm, removes supernatant, stays about 20 μ L supernatants;

3) 20 μ L2 × rapid cleavage liquid (0.2MNaOH50mL, SDS0.5g, sucrose 27.2g add distilled water to 200mL) are added, thermal agitation;

4) the centrifugal 15min of 13000rpm;

5) the direct electrophoresis of 5 μ L supernatant is got.Compared with the control, namely what electrophoresis band was delayed may be recombinant vectors.

4.6 bacterium colony PCR identify recombinant plasmid

Carry out bacterium colony PCR qualification by described in 4.5 again through the recombinant vectors of rapid cleavage method qualification, to determine that Insert Fragment is target fragment, reaction system is as follows:

Reaction conditions: 94 DEG C of 3min (denaturation); 94 DEG C of 30s (sex change), 58 DEG C of 30s (renaturation), 72 DEG C of 40s (extension), described denaturation renaturation-extension 26 circulation; 72 DEG C of 5min (total elongation).

To the recombinant vectors that bacterium colony PCR identifies, called after pEASY-OsRLZP, checks order.Sequencing result shows, obtains the OsRLZP full length gene encoding sequence being connected to pEASY-BluntSimple cloning vector.

Embodiment 2: the structure of rice Os RLZP gene overexpression recombinant plasmid

1, reagent

Plasmid extraction kit EasyPurePlasmidMiniPrepKit is purchased from Beijing Quanshijin Biotechnology Co., Ltd; Agarose gel reclaims test kit EasyPureQuickGelExtractionKit and is purchased from Beijing Quanshijin Biotechnology Co., Ltd; Restriction enzyme HindIII, SacI, T4 ligase enzyme is purchased from TaKaRa company.

Other import packing, conventional reagent are identical with embodiment 1.

2, agrobacterium strains and plant expression vector

Clontech company is purchased from for genetically modified agrobacterium tumefaciens (Agrobacteriumtumefaciens) bacterial strain EHA105; Carrier for expression of eukaryon pHB teaches laboratory structure (Mao etc., ProcNatlAcadSciUSA, 2005) by Shanghai Communications University Yang Hongquan and provides.

3, substratum

YEB substratum: yeast extract 1g/L, extractum carnis 5g/L, peptone 5g/L, sucrose 5g/L, MgSO ₄.7H ₂o0.5g/L.PH to 7.0 is adjusted, autoclaving with NaOH.

4, method

4.1 Plasmid Mini extract

Will by embodiment 1 obtain and check order, be connected to cloning vector pEASY ^tMthe OsRLZP gene recombination plasmid pEASY-OsRLZP of-BluntSimpleCloningVector carries out plasmid extraction, and leaching process carries out according to manufacturer's recommended procedure.

1) the intestinal bacteria Jie Zhong Yu Installed with recombinant plasmid pEASY-OsRLZP is had in the test tube of 5mLLB nutrient solution (containing 100 μ g/mL penbritins), cultivate 12h for 37 DEG C;

2) get 3mL bacterium liquid, the centrifugal 1min of 10000g under room temperature, exhaust supernatant.Add the colourless re-suspension liquid RB (ResuspensionBuffer) that 250 μ L contain RNaseA, vortex oscillation Eddy diffusion intestinal bacteria;

3) add blue lysate LB (LysisBuffer) of 250 μ L, gentle upset mixing 4-6 time, makes the abundant cracking of thalline, forms blue bright solution;

4) add yellow neutralizer NB (NeutralizationBuffer) of 350 μ L, mix 5-6 time gently, until form the yellow aggegation block of consolidation, room temperature leaves standstill 2min;

5) the centrifugal 5min of 15000g, careful supernatant of drawing adds in adsorption column;

6) the centrifugal 1min of 15000g, discards collection liquid;

7) add 650 μ L washingss WB (WashBuffer), the centrifugal 1min of 15000g, discards collection liquid;

8) the centrifugal 2min of 15000g, thoroughly removes residual WB;

9) adsorption column is placed in clean centrifuge tube, add 50 μ L elutriants EB (ElutionBuffer) in adsorption column central authorities, room temperature leaves standstill 1min;

10) the centrifugal 1min of 10000g, eluted dna.DNA solution is in-20 DEG C of preservations.

4.2DNA fragment enzyme cuts back to close

With restriction enzyme HindIII, SacI respectively double digestion embodiment 1 obtain recombinant plasmid pEASY-OsRLZP and plant expression vector pHB, then carry out glue recovery.DNA removal process is carried out according to manufacturer's recommended procedure.

1) cut the DNA fragmentation in sepharose, put into clean centrifuge tube;

2) add 3 times of volume sepharose lysates GSB (GelSolubilizationBuffer), in 55 DEG C of water-bath 6-10min, blob of viscose is melted completely.After blob of viscose melts completely, observe solution colour, if color is purple, add appropriate 3M sodium-acetate (pH5.2), make solution be yellow;

3) be down to room temperature (during high temperature, adsorption column is weak in conjunction with DNA ability) until gelating soln, add in adsorption column and leave standstill the centrifugal 1min of 1min, 10000g, discard collection liquid;

4) add 650 μ L washingss WB (WashBuffer), the centrifugal 1min of 10000g, discards collection liquid;

5) the centrifugal 2min of 10000g, removes residual WB;

6) uncap adsorption column standing 1min, and residual ethanol is volatilized clean, add 60 DEG C of preheating 50 μ L elutriant EB (ElutionBuffer) in adsorption column central authorities, room temperature leaves standstill 1min;

7) the centrifugal 1min of 10000g, eluted dna.By the DNA that elutes in-20 DEG C of preservations.

4.3 reclaim fragment connects conversion

To above-mentioned 4.2 reclaim pHB carrier segments, OsRLZP gene coded sequence connects.Linked system is as follows:

4h is connected in 16 DEG C.Transformation of E. coli DH5 α competent cell, operation steps is with in embodiment 1 4.4.

On picking transformation plate grow mono-clonal, as carried out bacterium colony PCR qualification as described in 4.6 in embodiment 1.Then by identify transform positive colony as carried out plasmid extraction as described in 4.1 in embodiment 2.Through the qualification of HindIII, SacI double digestion, obtain OsRLZP gene overexpression recombinant plasmid, as shown in Figure 1, called after pHB-OsRLZP.

4.4 Agrobacterium competent cell preparations

1) the mono-bacterium colony of picking Agrobacterium (Agrobacteriumtumefaciens) bacterial strain EHA105 is in 2mLYEB liquid nutrient medium (containing 50 μ g/mL Rifampins), and 28 DEG C of shaking culture are spent the night;

2) get incubated overnight liquid 500 μ L to transfer in 50mLYEB (containing 50 μ g/mL Rifampins) liquid nutrient medium, 28 DEG C of shaking culture are to OD ₆₀₀=0.5;

3) 4 DEG C of centrifugal 5min of 5000rpm collect thalline, add l0mL0.15MNaCl solution suspension thalline, ice bath 10min;

4) 4 DEG C of centrifugal 5min of 5000rpm collect thalline, with the 20mMCaCl of lmL precooling ₂solution suspension thalline, ice bath 10min;

5) competent cell prepared by is distributed into 200 μ L/ and manages, and quick-frozen lmin in liquid nitrogen, puts-80 DEG C and save backup.

4.5 Agrobacterium-mediated Transformation

1) get 200 μ L Agrobacterium competent cells, thaw on ice;

2) add the above-mentioned 4.3 pHB-OsRLZP recombinant vectorss that obtain of l μ g, flick mixing, ice bath 30min;

3) quick-frozen lmin in liquid nitrogen, 37 DEG C of water-bath 5min, then add 1mLYEB substratum, 28 DEG C of shaking culture 4h at a slow speed;

4) culture is coated on the YEB flat board containing 50 μ g/mL kantlex and 50 μ g/mL Rifampins, cultivate about 48h for 28 DEG C.

The qualification of 4.6 Agrobacterium positive colonies

The single bacterium colony of the Agrobacterium that picking as above transformation plate described in 4.5 grows, be inoculated in the YEB liquid nutrient medium containing 50 μ g/mL kantlex and 50 μ g/mL Rifampins, 28 DEG C of shaking culture are spent the night, with bacterium liquid for template carries out PCR qualification.Qualification result shows, obtains and can be used for the genetically modified positive Agrobacterium colonies with pHB-OsRLZP plasmid.

Embodiment 3: the acquisition of rice Os RLZP process LAN transfer-gen plant

1, reagent

RNA extracts reagent Trizol and is purchased from Invitrogen company; ThermoScript II ReverTraAce is purchased from Toyobo company; Real-time quantitative PCR reagent TransStart ^tMgreenqPCRSuperMix is purchased from Beijing Quanshijin Biotechnology Co., Ltd.Other reagent is import packing or domestic analytical pure product.

2, substratum

Plant tissue culture media:

(1) inducing culture: NB+2mg/L2,4-D, pH5.8 ~ 5.9; (2) Dual culture substratum: NB+2mg/L2,4-D+100 μm of ol/L Syringylethanone, pH5.2; (3) screening culture medium: NB+2mg/L2,4-D+250mg/L Pyocianil+30 ~ 50mg/L Totomycin, pH5.8 ~ 5.9; (4) division culture medium: NB+10mg/LKT+0.4mg/LNAA+250mg/L Pyocianil, pH5.8 ~ 5.9; (5) root media: 1/2MS, pH5.8 ~ 5.9.

3, method

3.1 Agrobacterium-mediated transformation paddy rice

3.1.1 the induction of embryo callus and subculture

After the fine mature seed of paddy rice wild-type Japan shells by hand, 75% ethanol disinfection 1min, vibration sterilization 25min in 25% chlorine bleach liquor, sterile purified water rinses 3 times, is inoculated on inducing culture.Induce about 7 days Callus formation under 27 DEG C of light culture conditions, excision radicle, continue to cultivate 7d, after callus is grown up, carry out succeeding transfer culture.Subculture 2 times altogether.

3.1.2 the cultivation of Agrobacterium and process

Scrape from-80 DEG C of cryogenic vials the embodiment 3 that takes a morsel obtain positive Agrobacterium colonies bacterium liquid with pHB-OsRLZP plasmid, in the YEB solid medium line containing 50mg/L kantlex and 50mg/L Rifampin, then activate at 28 DEG C of light culture 48h.The single bacterium colony got on activation flat board is transferred in the YEB liquid nutrient medium containing 50mg/L kantlex and 50mg/L Rifampin, after 28 DEG C of cultivation 48h, is suspended in 20mL and contains in the AAM substratum of 100 μMs of Syringylethanones, acutely shake 1min, leave standstill 1h.

3.1.3 Dual culture

Choose nature dispersion, color cadmium yellow, diameter be about the particulate state callus of 2-3mm in the culturing bottle of sterilizing, add the Agrobacterium bacterium liquid of above-mentioned 3.1.2 process, slightly leave standstill 30min after shake, be inoculated in Dual culture base dry callus on aseptic filter paper after, 25 DEG C of light culture 3d.

3.1.4 eccysis Agrobacterium

Callus after picking Dual culture is in wide-mouth culturing bottle, thread thalline is loseed with in aseptic water washing to water, leave standstill 1h with the sterilized water containing 250mg/L carboxylic Bian penicillin, then conversion callus is placed on aseptic filter paper and dries, move to the screening culture medium containing Totomycin.

3.1.5 the screening of callus

Transform callus to grow on sifting motion cultivation plate, every 2 pallet 1 time.Corotation plate 2 times.

3.1.6 the subculture of resistant calli and the regeneration of plant

Transform callus on sifting motion cultivation plate, grow after about 3 weeks that to be visible warty aureus kanamycin-resistant callus tissue grow from the shrivelled callus of brownization.Kanamycin-resistant callus tissue is selected on division culture medium after callus is grown up.After about 2 weeks, callus starts to turn green, then differentiates seedling.Moved to by seedling on root media, after seedling takes root and grows up to, shift out culturing bottle, the substratum cleaned on root moves to greenhouse pot culture.

The qualification of 3.2 transfer-gen plants

3.2.1 the extraction of oryza sativa genomic dna

Get wild-type respectively, OsRLZP process LAN rotaring gene plant blade extracts genomic dna.

1) liquid nitrogen grinding blade fine powder 100mg is got in 1.5mLEP pipe, often pipe adds 2 × CTAB Extraction buffer (100mMTris-HclpH8.0,20mMEDTApH8.0, the 1.4MNaCl of 500 μ L65 DEG C preheatings, 40mM beta-mercaptoethanol, 2%CTAB) mixing;

2) 65 DEG C of water bath heat preservation 60min, are cooled to room temperature, add isopyknic chloroform;

3) the centrifugal 10min of 5000g, gets supernatant liquor, adds isopyknic Virahol, and room temperature places 15min, precipitation DNA;

4) the centrifugal 10min of 12000g, abandons supernatant, dries up precipitation with after 70% alcohol flushing;

5) precipitation is dissolved in 200 μ L sterilizing ddH ₂in O, add RNaseA (10mg/mL), 37 DEG C of insulation 30min.Add isopyknic phenol/chloroform, mixing;

6) the centrifugal 10min of 12000g, gets supernatant, adds the dehydrated alcohol of 1/10 volume 3MNaAc and 2.5 times volume, and room temperature places 10min;

7) the centrifugal 10min of 12000g, abandons supernatant, dries up precipitation with after 70% alcohol flushing, is dissolved in 100 μ L sterilizing ddH ₂in O.DNA solution is in-20 DEG C of preservations.

3.2.2 positive transgenic plant is identified

Respectively with wild-type, process LAN transgenic paddy rice genomic dna for template, utilize Hygromycin resistance marker's gene (Hpt) entrained by carrier to carry out pcr amplification.Hygromycin gene special primer is:

HPTF：5'-TCGTTATGTTTATCGGCACTTTG-3'

HPTR：5'-GCGTCTGCTGCTCCATACAAG-3'

Increase by following program: 94 DEG C of 3min (denaturation); 94 DEG C of 30s (sex change), 58 DEG C of 30s (renaturation), 72 DEG C of 20s (extension), described denaturation renaturation-extension 30 circulation; 72 DEG C of 5min (total elongation).

3.2.3 the horizontal OsRLZP quantitative PCR detection of process LAN transfer-gen plant

Extract RNA after getting wild-type, process LAN rotaring gene plant blade liquid nitrogen grinding respectively, carry out reverse transcription, operation steps as described in Example 1.

Get wild-type, transfer-gen plant cDNA, with paddy rice reference gene (ACTIN, Genbank accession number X16280) for contrast, carry out the Real-time PCR Analysis of OsRLZP gene expression dose.OsRLZP gene primer is: RTRLZPF and RTRLZPR; ACTIN gene primer is: RTACTF and RTACTR.Primer sequence is as follows:

RTRLZPF：5’-CCGTCTGCTTCAAAACCTTCAAC-3’

RTRLZPR：5’-CTCCGGCCCCTTCCTGTACT-3’

RTACTF：5’-AGTGATTGCACCACCAGAAAGA-3’

RTACTR：5’-CAGGACCAGATTCATCATACTCG-3’

Real-time quantitative PCR reaction system is as follows:

Reaction conditions: 95 DEG C of 30s; 95 DEG C of 5s, 60 DEG C of 15s, 72 DEG C of 10s, 40 circulations.After amplification, 65 DEG C of 5s, each circulation increases by 0.5 DEG C, and 60 circulations, carry out solubility curve analysis.Each sample in triplicate.PCR reaction runs on Bio-RadCFX96.

3.2.4 transfer-gen plant phenotype analytical

Process LAN transgenic positive qualification strain (OX-1 ~ OX-3) and the same period are grown WT lines growing state and carries out com-parison and analysis.Get plant leaf sample and carry out tissue slice, scanning electron microscope detection.

3.2.4.1 tissue slice

Get wild-type, OsRLZP process LAN transfer-gen plant tissue sample preparation section respectively.

1) fixing: clip plant leaf tissue, with FAA stationary liquid (70% alcohol: formaldehyde: glacial acetic acid=16:1:1), is placed in 4 DEG C of fixing 24h;

2) dye: remove stationary liquid, add 50% ethanol, room temperature leaves standstill 0.5h; Be put in phenodin dye liquor the 48h that dyes; Then outwell staining fluid, embathe 2h with 30% ethanol, change 30% ethanol several therebetween to water without loose colour;

3) dewater: adopt ethanol concentration gradient to dewater stained specimens, successively through 50%, 70%, 85%, 95%, 100% ethanol dehydration, each serial dehydration 2h;

4) transparent: after dehydration, sample processes 2h with 1/2 dimethylbenzene+1/2 dehydrated alcohol (namely dimethylbenzene and dehydrated alcohol equal-volume mix liquid), then adds dimethylbenzene process 3h;

5) waxdip: material is proceeded to the small vessels that dimethylbenzene is housed, adds broken wax gradually in dimethylbenzene, adds a little broken wax again to saturated, spend the night in 37 DEG C of thermostat containers after it dissolves.Add broken wax next day to saturated, after putting 60 DEG C of thermostat container 3-5h, the wax liquid outwelled containing dimethylbenzene changes to the pure wax of fusing.Waxdip 48h, pure wax 2-3 time is changed in midway;

6) embed: waxdip sample is poured in the paper groove folded, puts into rapidly frozen water and wax stone is solidified;

7) cut into slices: finishing wax stone, with microtome, thickness 8-10 μm;

8) paster: be coated with a little bonding die agent on slide glass, puts into warm water by the wax band cut, then puts on slide glass, is placed in 37 DEG C of thermostat containers and spends the night baking sheet;

9) dewaxing and dyeing: slide glass is dewaxed 2 times in dimethylbenzene, each 0.5-1h.With neutral gum mounting after dewaxing.

3.2.4.2 prepared by blade scanning electron microscope example

1) fixing: fritter blade being cut into 3-4mm, invade rapidly in the 4% glutaraldehyde stationary liquid that phosphoric acid buffer (pH7.2) prepares, after 4 DEG C of fixing 2h, clean sample 3 times with phosphoric acid buffer (pH7.2); Then with 1% osmic acid, 4 DEG C of fixing 1h of precooling, sample is cleaned 3 times with phosphoric acid buffer (pH7.2);

2) dewater: adopt ethanol concentration gradient dehydration, successively through 30%, 50%, 70%, 80%, 90%, 95%, 100% ethanol dehydration, each concentration ethanol 15min;

3) dry: sample after dehydration is put in dry 0.5-1h in vacuum coater;

4) conductive processing: dry rear blade sample is bonded in sample table, forms metallic membrane with vacuum evaporating instrument at sample surfaces, then carry out Microscopic observation.

4, result

4.1OsRLZP process LAN transgenic rice plant is identified

4.1.1 transgenic positive rice plant is identified

Utilize Hygromycin resistance marker's gene (Hpt) sequence pair transgenosis, wild rice leaves genomic DNA carries out pcr amplification detection.Positive transgenic plant amplifies Hpt target stripe (560bp), and WT lines fails to amplify target stripe.

4.1.2 OsRLZP gene expression dose quantitative analysis in transgenic positive rice plant

Real-time PCR Analysis result shows, in 3 independent process LAN transgenic lines (OX-1 ~ OX-3) OsRLZP expression amount comparatively wild-type obviously raise, see Fig. 1.

4.2OsRLZP process LAN transgenic rice plant phenotype analytical

The field phenotype display of OsRLZP process LAN transfer-gen plant, compared with wild-type, in the blade of OsRLZP process LAN transfer-gen plant (OX-1 ~ OX-3), thicker, the blade of arteries and veins narrows, whole plant leaf involute upright (table 1).Electron microscope scanning result shows, the blade table fur quantity of OsRLZP process LAN plant reduces, and stomatal frequency comparatively wild-type reduces (Fig. 2).

Table 1 wild-type, OsRLZP process LAN transgenic line leaf morphology

Claims (2)

1. the zinc finger protein gene OsRLZP that the control paddy rice of nucleotide sequence as shown in sequence table SEQ IDNO:1 is leaf is in the developmental application of control rice leaf.

2. one kind control rice leaf grow, improve the leaf method of paddy rice, it is characterized in that: zinc finger protein gene OsRLZP complete encoding sequence according to claim 1 is inserted in carrier for expression of eukaryon and obtains eukaryon recombinant plasmid, then by described eukaryon recombinant plasmid rice transformation, transfer-gen plant is obtained.