CN103695439B - Gold mandarin orange FcWRKY70 gene and the application in raising drought tolerance in plants thereof - Google Patents

Abstract

The invention provides golden mandarin orange FcWRKY70 gene and improving the application in drought tolerance in plants, the WRKY transcription factor FcWRKY70 that applicant is separated from golden mandarin orange (Fortunella.crassifolia), clone obtains a multiple adverse circumstance abduction delivering, its sequence is for shown in SEQ ID NO.1, and the protein of coding is for shown in SEQ ID NO.2.This gene imports in tobacco, lemon and golden mandarin orange by applicant respectively, and the tolerance of overexpression transfer-gen plant to drought stress of acquisition significantly improves, and interferes transfer-gen plant obviously weakened to the tolerance of drought stress.The present invention is that Genes For Plant Tolerance abiotic stress Molecular design breeding provides new genetic resources, for enforcement green agriculture, water-saving agriculture provide new genetic resources, the exploitation of this genetic resources are conducive to reducing agriculture production cost, promote agricultural industry high-efficient development.

Description

Gold mandarin orange FcWRKY70 gene and the application in raising drought tolerance in plants thereof

Technical field

This patent belongs to plant genetic engineering field.Be specifically related to the WRKY transcription factor FcWRKY70 be separated from golden mandarin orange (Fortunella.crassifolia), clone obtains a multiple adverse circumstance abduction delivering, then this gene is imported to respectively in tobacco, lemon and golden mandarin orange, the tolerance of overexpression transfer-gen plant to drought stress obtained significantly improves, and interferes transfer-gen plant obviously weakened to the tolerance of drought stress.

Background technology

Plant is in process of growth, and what often can be subject to various different poor environment coerces impact, and as arid, salt, damages to plants caused by sudden drop in temperature, high temperature, and the poor and disease and pest of nutrition, has a strong impact on its grow (Gong and Liu 2013).It should be noted that the annual whole world is because arid causes crop production reduction to be about 20% (Wang et al 2003).

For adapting to various environment-stress, plant defines a series of change (Krasensky andJonak, 2012) in physiology and biological process.Such as, change metabolic process and maintain cell Peng pressure to accumulate a series of metabolite, resist adverse circumstance impact (Nishizawa et al., 2008; Verbruggen and Hermans 2008; Livingston et al., 2009; Shi et al., 2010; Fu et al., 2011a, b).In addition, the transcriptional level of lots of genes changes, by adverse circumstance abduction delivering or suppressedly to transcribe (Seki et al., 2003).Part stress response gene, by encode functional protein, promotes vegetable cell accumulation beneficial metabolites, Cell protection escapes injury (Chen and Murata 2011; Wang et al., 2011).Another portions gene exercises the function of transcriptional regulatory, the expression of modified target dna, such as transcription factor, protein kinase, Starch phosphorylase (Su et al., 2010; Mao etal., 2011; Seo et al., 2012; Cui et al., 2012)

Transcription factor plays a part indispensable (Singh et al., 2002) in transcribing in recombinant expressed process of stress response gene.Plant Genome includes a large amount of transcription factors, this wherein WRKY albumen be considered to the large gene family (Eulgem et al., 2000) of of the distinctive member composition by enormous amount of plant.Bioinformatic analysis shows, and all there is very abundant WRKY albumen in some species announcing genome sequence, wherein in Arabidopis thaliana 74, in paddy rice 109, in papaya 66, in willow 104,68 (Eulgem and Somssich 2007 in jowar; Rosset al., 2007; Pandey and Somssich 2009).Since find first WRKY albumen in sweet potato, investigators identify increasing WRKY albumen in plant.

Up to now, existing large quantifier elimination shows that WRKY albumen plays keying action in different physiological and biochemical procedure, and image signal conducts, metabolism, (Ren et al., 2008 such as lignifying; Guillaumie et al., 2010; Mao et al., 2011; Yu et al., 2013).In addition, increasing evidence shows that WRKY transcription factor take part in plant to response process (Liu et al., 2013) that is biological and abiotic stress.Such as, three members of Arabidopis thaliana II race a subtribe, AtWRYK18, AtWRKY40 and AtWRKY60, structurally with functionally form redundancy, antagonism and mutually different complexing action pattern, tackle different fungies and Micobial Disease (Xu et al., 2006; Shen et al., 2007; Pandey et al., 2010).The expression of the adjustable PHOSPHATE1 of AtWRKY6 and AtWRKY42 (PHO1), improves transgenic arabidopsis to the tolerance (Chen et al., 2009) of low-phosphorus stress.2 III race WRKY transcription factors, WRKY70 and WRKY54, in Arabidopis thaliana, collaborative negative regulation stomatal movement, regulates osmotic stress (Li et al., 2013).Also there is the allelotrope about a pair WRKY transcription factor under bacterial leaf-blight, bacterial stripe and the adverse circumstance such as arid, salt, act on diametrically opposite research report (Tao etal., 2009 in paddy rice; Tao et al., 2011).In view of WRKY transcription factor Adversity-stressed Plant process the vital role that rises, this genoid can be used as efficient gene resource, creates by transgenosis pattern the transgenic plant that resistance strengthens.So far, existing large quantity research shows, the similar gene of overexpression significantly can strengthen resistance (Fu at al., the 2011b of plant; Hao etal., 2011; Su et al., 2010).

In research before, we excavate the EST of a multiple stress response, obtain total length, find that this gene has a WRKY structural domain by the amplification of RACE technology, are potential WRKY transcription factors.Bioinformatic analysis shows that this gene is under the jurisdiction of III race WRKY transcription factor, and the most similar to the WRKY70 of Arabidopis thaliana, therefore we are by its called after FcWRKY70.It is a unknown new gene, a micromolecule polypeptide product of encoding, not containing any known conserved domain.Research finds ulcer bacteria, and external source adds SA and ABA obviously can induce the expression of FcWRKY70, and arid and Ficus caricaL also can obviously induce it to express in addition, is a very excellent genetic resources in resistance breeding.

Summary of the invention

The present invention seeks to be separated from golden mandarin orange (Fortunella.crassifolia), to clone a WRKY transcription factor by multiple adverse circumstance abduction delivering, FcWRKY70, its nucleotide sequence is as shown in SEQ ID NO:1, and the aminoacid sequence of its coding is as shown in SEQ ID NO:2.

Another object of the present invention there are provided a kind of golden mandarin orange FcWRKY70 gene and is improving the application in Genes For Plant Tolerance drought stress, imported in tobacco, lemon and golden mandarin orange by Agrobacterium-mediated genetic transformation, identify that it is in the function of dewatering and under drought stress, for plant stress-resistance Molecular design breeding provides new genetic resources, for enforcement green agriculture, water-saving agriculture provide new genetic resources, the exploitation of this genetic resources are conducive to reducing agriculture production cost and realizing environmental friendliness.

In order to achieve the above object, the present invention adopts following technical measures:

Gold mandarin orange wild-type cDNA is template, increases with following primer:

Forward primer: 5 '-CATGCCATGGATCATAATGAAAATGGAGGCCGGTC-3 ';

Reverse primer: 5 '-GGGTCACCAGTTGCAAAGGACTAGCGGTAGCAG-3 '.

Obtain a new golden mandarin orange gene FcWRKY70, its nucleotide sequence as shown in SEQ ID NO:1, FcWRKY70 total length 1196bp, wherein 83-1069bp place is the coding region of this gene; The aminoacid sequence of its coding is as shown in SEQ ID NO:2, and it comprises the open reading frame of 987bp, 328 amino acid of encoding, and iso-electric point is 5.89, and the molecular weight of prediction is 36.78kD.

Utilize agrobcterium-mediated transformation transformation of tobacco and lemon, the transfer-gen plant of acquisition, through biological function verification, shows that the FcWRKY70 gene that the present invention clones has the function improving dehydration tolerance and drought stress.In embodiments of the invention part, we describe the separation of golden mandarin orange FcWRKY70, functional verification and application.

Compared with prior art, the present invention has the following advantages:

The adverse circumstance such as arid, low temperature, salinization all can along with serious plant dehydration process, and physiology arid, affects the regional distribution of plant normal growth growth and crop.Traditional agriculture is comparatively large to the dependency of water resources, and particularly high temperature season, constrains the development of agricultural industry.Rely on the present invention effectively can improve the drought-resistance ability of plant, make it adapt to drought environment.Once obtain effective transgenic line, production effectively can reduce the consumption of pouring water, economize on resources, reduce production cost.On the other hand, the present invention can according to actual production demand for improve stock material or other, be widely used, be easy to be accepted accreditation.

Lead in plant by Agrobacterium-mediated genetic transformation by this gene, identify its function under different adverse environmental factor, for plant stress-resistance Molecular design breeding provides new genetic resources, for implementing green agriculture, water-saving agriculture provides new genetic resources.

Accompanying drawing explanation

Fig. 1 is techniqueflow chart of the present invention.

Fig. 2 is the expression schematic diagram of FcWRKY70 gene of the present invention under Different stress process.

Fig. 3 is FcWRKY70 gene Subcellular Localization fluorescence schematic diagram of the present invention.

Wherein in Fig. 3, A-B is GFP gene (contrast) imaging under light field, details in a play not acted out on stage, but told through dialogues; In Fig. 3, C-E is the imaging of FcWRKY70 gene under light field (C), details in a play not acted out on stage, but told through dialogues DAPI dyeing (D) and details in a play not acted out on stage, but told through dialogues GFP (E).

Fig. 4 is FcWRKY70 gene transcriptional activation of the present invention qualification schematic diagram.

Fig. 5 is that FcWRKY70 genophore of the present invention builds schematic diagram figure.

In Fig. 5, A is that overexpression vector builds schematic diagram; In Fig. 5, B is that interference vector builds schematic diagram.

Fig. 6 is FcWRKY70 gene transformation lemon of the present invention and regenerative process schematic diagram.

In Fig. 6, A is the stem section of Dual culture, and in Fig. 6, B is the stem section of screening and culturing; In Fig. 6, C is that resistant buds extends and expands numerous in elongation medium, and in Fig. 6, D is that resistant buds carries out root culture.Tobacco and golden mandarin orange conversion process similar with it.

Fig. 7 is that FcWRKY70 gene transgenic material PCR of the present invention identifies schematic diagram.

In Fig. 7, A is the regeneration plant PCR qualification figure (be above FcWRKY70 specific primer PCR amplification figure, lower is HPTII gene specific primer pcr amplification figure) obtained after FcWRKY70 transformation of tobacco;

In Fig. 7, B is the regeneration plant PCR qualification figure (be above FcWRKY70 specific primer PCR amplification figure, lower is HPTII gene specific primer pcr amplification figure) obtained after FcWRKY70 transforms lemon;

In Fig. 7, C is the regeneration plant PCR qualification figure (be above 35S forward primer and FcWRKY70 specific primer PCR amplification figure, lower is NPTII gene specific primer pcr amplification figure) obtained after FcWRKY70 transforms golden mandarin orange;

Gene expression analysis in Transgenic Tobacco system for the purpose of D in Fig. 7, gene expression analysis in lemon transgenic lines for the purpose of Fig. 7 E, gene expression analysis in golden mandarin orange transgenic lines for the purpose of 7F;

Fig. 8 is that FcWRKY70 transgene tobacco seedling (1#, 4#) of the present invention compares schematic diagram with wild-type drought resistance.

In Fig. 8, A is that before and after transgene tobacco in vitro seedling natural-dehydration 60 minutes, each based material phenotype compares and trypan blue dyeing difference compares;

In Fig. 8, B is that in the in vitro seedling natural-dehydration of each system 60 minutes processes, relative percentage of water loss compares;

In Fig. 8, C is that before and after pot material control water, phenotype compares;

Fig. 8 D is that after control water, each based material soil relative water content compares; Fig. 8 E samples the MDA value that each based material records after control water.

Fig. 9 is that FcWRKY70 transgenosis lemon seedling (28#, 43#) of the present invention compares schematic diagram with the dirty water separation capability of wild-type.

In Fig. 9, A is the comparison of the relative percentage of water loss in FcWRKY70 transgenic line and wild-type natural-dehydration 6 hours period;

In Fig. 9, B is that after dehydration, each based material NBT dyeing difference compares; Fig. 9 C is that after dehydration, each based material DAB dyeing difference compares.

Figure 10 is that FcWRKY70 transgenosis gold mandarin orange seedling (20#, 22#) compares schematic diagram with the dirty water separation capability of wild-type.

Embodiment

Below in conjunction with concrete enforcement, the present invention is described in detail.Implement according to following description and these, those skilled in the art can determine essential characteristic of the present invention, and when not departing from spirit and scope of the invention, can make various change and amendment to the present invention, to make its applicable various uses and condition.

Embodiment 1:

FcWRKY70 gene isolation Cloning and Expression is analyzed

In research before, we, with chip probe Cit.7937.1.S1_at sequence alignment oranges and tangerines est database HarvEST (http://harvest.ucr.edu), obtain an objective est sequence.For obtaining its full length sequence information, we have employed RACE technology.According to known frag info, design 3 ' Race and 5 ' Race primer respectively, as follows:

3’Race outer primer-GS:5’AGTGCCCACATCACCGAAAA 3’

3’Race inner primer-GS:5’TGGGGATGTGATTTCTGGAGT 3’

5’Race primer-GS:5’AAATCTTTTTCGGTGATGTGGGCA 3’

First the total serum IgE (, purchased from TAKARA company, working method to specifications for test kit) in RNAiso Plus test kit extracting gold mandarin orange blade is used.Then get 1 μ g total serum IgE sample, add 3 ' Race and react joint, with 3 ' Full RACE CoreSet Ver.2.0Kit test kit, synthesize the first chain cDNA (purchased from TAKARA company, the first chain cDNA synthesizes see test kit instruction manual test kit).First chain cDNA of gained is used for 3 ' end sequence of nest-type PRC reaction amplifying target genes.In the reaction system of Outer PCR 50 μ l, each component is as follows:

PCR reaction is at Bio-Rad Alpha ^tMunit Block Assembly For DNA systems amplification instrument completes by following program: 95 DEG C of denaturations 3 minutes; 95 DEG C of sex change, 30 seconds, 55 DEG C annealing 30 seconds, 72 DEG C extend 120 seconds (totally 20 circulations); After circulation completes, 72 DEG C extend 10 minutes.Outer pcr amplification terminates after product in-20 DEG C of preservations, for Inner pcr amplification.

In the reaction system of Inner PCR 50 μ l, each component is as follows:

PCR reaction is at Bio-Rad Alpha ^tMunit Block Assembly For DNA systems amplification instrument completes by following program: 95 DEG C of denaturations 3 minutes; 95 DEG C of sex change, 30 seconds, 55 DEG C annealing 30 seconds, 72 DEG C extend 120 seconds (totally 30 circulations); After circulation completes, 72 DEG C extend 10 minutes.A PCR band product is obtained after twice PCR reaction, after the agarose gel electrophoresis of 1.2%, reclaim test kit (purchased from Axygen company) with Axygen DNA Gel Extraction Kit gel and reclaim object band (concrete steps consult and use explanation).The DNA solution and the pMD18-T carrier (purchased from TAKARA company) that reclaim purifying carry out ligation (by specification operates), ligation cumulative volume is 10 μ l, comprise the Solution I (all in support agent box) of 5 μ l, the PCR primer of 4.5 μ l purifying and 0.5 μ l carrier T, 16 DEG C of connections are spent the night.Then with whole connection products, heat shock method transformation of E. coli DH5 α competence, and in the LB solid plate containing 100mg/L ammonia benzyl mycin screening positive clone, a picking 4-6 cloning and sequencing.

It is SMARTer that 5 ' Race reacts used kit ^tMrACE cDNA Amplification Kit (purchased from Clontech company).3 μ g total serum IgE samples are used as synthesis first chain cDNA, and concrete grammar is see test kit specification sheets.First chain cDNA of gained is used for Touchdown PCR and reacts, 5 ' cDNA end of amplifying target genes.This PCR reacts and adopts Advantage 2Polymerase Mix (purchased from Clontech company) to increase, and each component is added see specification sheets.PCR reaction is at Bio-Rad Alpha ^tMunit Block Assembly For DNA systems amplification instrument completes by following program: 94 DEG C of sex change 30 seconds, 72 DEG C extend 3 minutes (totally 5 circulations); 94 DEG C of sex change, 30 seconds, 70 DEG C annealing 30 seconds, 72 DEG C extend 3 minutes (totally 5 circulations); 94 DEG C of sex change, 30 seconds, 68 DEG C annealing 30 seconds, 72 DEG C extend 3 minutes (totally 25 circulations); After circulation completes, 72 DEG C extend 10 minutes.Subsequent experimental is with 3 ' Race, and namely the PCR primer of gained is connected to carrier T after electrophoresis reclaims, and transformation of E. coli DH5 α competence, last picking 4-6 positive colony order-checking.

Whether can respond different environmental stimuluses for analyzing FcWRKY70 gene, adopting the expression of this gene of Real-time PCR Analysis under different adverse circumstance process.Result shows, citrus processing Xcc, ABA, SA, dehydration, salt all can induce this genetic expression, and low temperature does not have obvious inducing phenomena (see Fig. 2) to its expression, show that it is a multiple induced gene in adversity, biotic stress and abiotic stress can be responded simultaneously.

Embodiment 2:

FcWRKY70 gene Subcellular Localization

Tuning on-line prediction points out that FcWRKY70 is positioned nucleus, and this research and utilization epidermal tobacco studies the Subcellular Localization of FcWRKY70 gene.Here we have selected the positioning carrier of pCAMBIA1302 vector construction FcWRKY70 gene.Utilize RT-PCR to amplify the whole ORF of FcWRKY70 gene (reading frame), and add Nco I single endonuclease digestion site respectively at its amplimer two ends.Reclaim product and pCAMBIA1302 carrier with Nco I single endonuclease digestion PCR simultaneously, carry out the recovery of digestion products afterwards respectively, gene fragment and carrier segments restructuring connect, thus obtain pCAMBIA1302-FcWRKY70-GFP recombinant plasmid.Restructuring material and control plasmid (pCAMBIA1302) proceed to Agrobacterium GV3101 subsequently respectively.

Agrobacterium is infected epidermal tobacco and carries out as follows:

1. thalline activation: pick a small amount of Agrobacterium GV3101 bacterium liquid containing recombinant plasmid and control plasmid respectively, line on Km/LB flat board, cultivates 1-2d for 28 DEG C, activation thalline.

2. from picking mono-clonal the flat board of line, be inoculated in 50ml Km/LB liquid nutrient medium, 28 DEG C, 250rpm cultivates, and between OD600=0.4-0.6, now thalli growth is in logarithmic phase, active high.

3. bacterium liquid is proceeded in 50ml centrifuge tube, the centrifugal 10min of 4000rpm, collect at the bottom of thalline to pipe.

4. remove supernatant, with 10mM MES (pH 5.6,10mM MgCl ₂, 150 μMs of AS (acetosyringone)) and damping fluid suspension thalline, make its OD600=1.0, for subsequent use.

5. in super clean bench, get the aseptic tobacco seedling (wild-type of 40-60d, Nicotiana nudicaulis) be material, the blade that gripping is of moderate size is in sterile petri dish, to remove the disposable syringe of syringe needle, resuspended good bacterium liquid is injected tobacco leaf (pros and cons, leaf back because of pore more, more easily inject bacterium liquid).

6. suck the unnecessary bacterium liquid on blade with aseptic filter paper, blade is layered on and has padded on the MS substratum of filter paper in advance to carry on the back ventricumbent mode, 28 DEG C of light culture.

After 48 hours, by NIKON ECLIPSE 90i type microscopic examination fluorescence localization situation.Result shows, all has fluorescence (Fig. 3, upper right) when control vector transforms in whole cell, and in the cell that recombinant vectors transforms, fluorescence can only detect (Fig. 3, bottom right) in core, illustrates that FcWRKY70 is the albumen of a nuclear location.

FcWRKY70 gene transcriptional activation is analyzed

Transcriptional activation activity is an essential characteristic of transcription factor, and here we have employed pGBKT7 carrier and carry out recombination to construct, and whether checking FcWRKY70 has transcriptional activation activity.The primer amplification designed with BamH I and Pst I restriction enzyme site obtains FcWRKY70 full length gene ORF, product is after BamH I and Pst I double digestion, subclone on the pGBKT7 carrier of BamH I and Pst I double digestion, obtains fusion expression vector pGBKT7-FcWRKY70 to same.By fusion expression vector and empty carrier (pGBKT7) transformed yeast strain AH109 respectively after order-checking confirmation sequence is errorless, then cultivate on different disappearance substratum.Result shows, the yeast cell that empty carrier transforms can only grow on disappearance substratum SD/-Trp, and the positive yeast cell that fusion vector transforms can grow in the disappearance substratum such as SD/-Trp/-ade and SD/-Trp/-ade/-His, (Fig. 4), illustrating that FcWRKY70 has transcriptional activation activity, is a typical transcription factor.

Embodiment 3:

Plant Transformation overexpression vector builds

According to the restriction enzyme site analysis in the multiple clone site of pCAMBIA1301 carrier and the coding region sequence of FcWRKY70 gene, select Nco I and BstE II as restriction endonuclease.First with golden mandarin orange wild-type cDNA for template amplification obtains the total length of FcWRKY70, PCR primer and pCAMBIA1301 carrier carry out double digested with Nco I and BstE II respectively and again connect into object carrier.

PCR primer double digestion system cumulative volume is 20 μ l, wherein reclaims product 16 μ l, each 1 μ l of 10 × K damping fluid (purchased from Takara) 2 μ l, Nco I and BstE II containing PCR.PCAMBIA1301 carrier double digestion system cumulative volume is 40 μ l, wherein containing pCAMBIA1301 plasmid 8 μ l, each 1 μ l of 10 × K damping fluid (purchased from Takara) 4 μ l, Nco I and BstE II, distilled water 24 μ l.After 37 DEG C of enzymes cut through night, difference purifying reclaims.Comprise 10 × T4 in ligation system and connect damping fluid 1 μ l, T4DNA ligase enzyme 1 μ l, FcWRKY70 gene and carrier pCAMBIA1301 are respectively 3 μ l and 1 μ l, and with distilled water polishing to 10 μ l, 16 DEG C of connections are spent the night.Afterwards with whole connection product conversion bacillus coli DH 5 alpha competent cells, and screen in the LB solid plate containing 100mg/L kantlex, the positive monoclonal detected through PCR send order-checking, the mono-clonal extraction recombinant plasmid that sequence is errorless, (with reference to Pehanorm Brooker, Huang Peitang translates application freeze-thaw method, " Molecular Cloning: A Laboratory guide " third edition, Science Press, 2002) recombinant plasmid to be proceeded in agrobacterium tumefaciens GV3101 and to preserve.

Plant Transformation interference vector builds

In order to obtain interfering transfer-gen plant, taking pHellsgate2 interference vector and carrying out recombination to construct.First choose the fragment design primer of about 300bp at the 5 ' end of FcWRKY70, and before special primer, add attB site universal primer, as target fragment amplimer.With golden mandarin orange wild-type cDNA for template amplification obtains target fragment, PCR primer, after reclaiming, is reacted restructuring through BP and is connected to pHellsgate2 carrier, be built into object carrier.

Universal primer sequence is as follows:

attB1：5'-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3'

attB2：5'-GGGGACCACTTTGTACAAGAAAGCTGGGT-3'

BP reacts used kit bP Clonase ^tMiI Enzyme Mix (purchased from Invitrogen company).Reaction system is 6 μ l, the PCR primer 100ng wherein after purifying and the pHellsgate2 plasmid 100ng with attP site, adds 1 μ l B/P Clonase, and add TE buffer (pH 8.0) to 6 μ l, 25 DEG C of reactions are spent the night.Get all reactant thermal shock method transformation of E. coli DH5 α competence, be applied to the LB solid plate screening positive clone containing spectinomycin (Spec) 100mg/L.Detect with 35S promoter aligning primer and reverse special primer analyze for amplimer carries out PCR to positive colony, PCR detects correct positive colony and carries out single endonuclease digestion checking respectively with XbaI and XhoI further, if consistent and about the 200bp larger than target fragment of two groups of single endonuclease digestion clip size, then in this positive colony, target fragment is exact connect ion.The positive monoclonal of exact connect ion send order-checking, the mono-clonal extraction recombinant plasmid that sequence is errorless, application freeze-thaw method is (with reference to Pehanorm Brooker, Huang Peitang translates, " Molecular Cloning: A Laboratory guide " third edition, Science Press, 2002) recombinant plasmid to be proceeded in agrobacterium tumefaciens GV3101 and to preserve.

Embodiment 4:

Tobacco genetic transformation

Agrobacterium tumefaciens mediated tobacco genetic transformation step is as follows:

1. agrobacterium tumefaciens is cultivated: get agrobacterium tumefaciens (containing overexpression vector) the bacterium liquid preserved in Ultralow Temperature Freezer, in the flat lining out activation of the LB that with the addition of 100mg/L kantlex, scraping line bacterial plaque, add in liquid MS minimum medium, 28 DEG C of 180rpm shaking culture, treat that bacterial concentration reaches OD ₆₀₀contaminate during=0.4-0.6.

2. contaminate: get aseptic wild-type tobacco material, get its blade and be cut into 0.5cm × 0.5cm size, put into cultured agrobacterium tumefaciens bacterium liquid, infect 8-10 minute, period constantly vibrates.

3. Dual culture: get the tobacco leaf after dip-dye, blots bacterium liquid unnecessary on blade with aseptic filter paper, and leaf back is elaborated downwards on the MS minimum medium containing 0.3g/L NAA and 2.25g/L 6-BA, 25 DEG C of light culture 3 days.

4. screening and culturing: through the tobacco leaf of Dual culture after 3 days, one time is washed with the aqueous solution containing 400mg/L cephamycin, then aseptic water washing 3-5 time, transfers in the MS minimum medium containing 2.5mg/L Totomycin+400mg/L cephamycin+2.25mg/L6-BA+0.3mg/L NAA and cultivates.

5. root culture: when indefinite bud grows to about 2-3cm, proceeds in the MS substratum adding 2.5mg/L Totomycin (or not adding) and 400mg/L cephamycin+0.3mg/L NAA and induces root to generate.

6. tobacco seedling proceeds to earth culture: the transformation seedlings after taking root covers with culturing bottle, takes out by root media, cleans the substratum on transformation seedlings with tap water, and plants in the Nutrition Soil of sterilizing.

Transformation of tobacco seedling used medium is in table 1.

Table 1 Transformation of tobacco seedling used medium is filled a prescription

7. positive transgenic tobacco is tentatively determined

Obtain according to the method described above turning FcWRKY70 tobacco resistant buds, extract DNA and handy PCR method qualification positive transgenic material.Concrete grammar is as follows: design primer HPTII and gene specific primer pcr amplification identify positive seedling (table 1).PCR gathers in the crops seed (T1 is for seed) after being accredited as genetically modified plantlet of transplant, 4 DEG C of vernalization treatment 3 days, get a little seed in the centrifuge tube of 1.5ml, 70% alcohol-pickled seed 30 seconds, then wash once with sterilizing distilled water, add 1ml 2.5%NaClO surface sterilization 7 minutes again, abundant vibration, 3 times are washed with sterilizing distilled water after discarding NaClO, finally use the seed of 1ml 0.1% agarose suspension sterilizing, and elaborate on interpolation 2.5mg/L Totomycin (Hyg) MS minimum medium, result shows resistance seedling energy normal growth on resistance culture base that the present invention obtains, for green, non-resistance seedling then yellow is die.Positive transgenic material is used for resistance Function Identification.

Embodiment 5:

Lemon and golden mandarin orange genetic transformation

1, lemon and golden mandarin orange seed is got, clean with clear water after soaking 20 minutes with the NaOH of 1M, then on Bechtop, soak sterilizing 15-20min with the NaClO of 2%, sterilized water washs 3 times, seed through surface sterilization aseptically peels off seed coat, be seeded on MT solid medium, light culture 3-4 week again illumination cultivation 1 week be used for transforming.

2, Agrobacterium (the overexpression vector bacterium conversion lemon containing recombinant vectors is got, interference vector bacterium transforms golden mandarin orange) bacterium liquid lines containing 100mg/L microbiotic that (overexpression vector is kantlex, interference vector is spectinomycin) solid LB media on, 28 DEG C of light culture two days; Picking list bacterium colony lines on new flat board again, 28 DEG C of light culture 2-3 days; Scrape the thalline grown with scalpel, be inoculated in not containing in antibiotic liquid MT substratum, 28 DEG C of 200rpm shaking culture are to OD ₆₀₀=0.6-0.8, adding AS (Syringylethanone) is 100 μMs (20mg/L) to final concentration, stand-by.

3, get aseptic seedling epicotyl, be mitered into 1-1.5cm long shoot section in Bechtop, the stem section cut temporarily is put in the empty triangular flask of sterilizing and (adds a small amount of water moisturizing), for transforming after Agrobacterium bacterium liquid has been cultivated.

4, be immersed in by the explant cut in the Agrobacterium bacterium liquid (the Agrobacterium bacterium containing overexpression vector transforms lemon, the Agrobacterium-mediated Transformation gold mandarin orange containing interference vector) prepared and infect 20min, constantly shake is several times therebetween.Infect rear aseptic thieving paper and blotted unnecessary bacterium liquid, explant has been elaborated on Dual culture base, 25 DEG C of light culture 3 days.

5, Dual culture is after 3 days, with aseptic washing explant 3-5 time, then blots surperficial moisture content with aseptic thieving paper, forward to add 2.5mg/L Totomycin and 400mg/L cephamycin screening culture medium on, cultivate under 25 DEG C of light culture 4 Zhou Houzai forward illumination condition to.As resistant buds >0.5cm, cut resistant buds and forward its differentiation short on substratum of sprouting to.When resistant buds >1.5cm is long, proceeded to root induction in root media, lemon and golden mandarin orange conversion process are shown in Fig. 6.

Conventional culture medium prescription:

LB solid medium: peptone 10g/L+ yeast extract 5g/L+NaCl 10g/L+ agar 15g/L

Lemon and golden mandarin orange Dual culture substratum: MT+1.0mg/L BA+20mg/L AS

Lemon screening culture medium: MT+1.0mg/L BA+400mg/L Cef+4mg/L Hyg

Lemon sprouts substratum: MT+1.0mg/L BA

Gold mandarin orange screening culture medium: MT+2.0mg/L BA, 1.0mg/L NAA+1.0mg/L KT+400mg/L Cef+25mg/L Km)

Lemon and golden mandarin orange root media: 1/2MT+0.5mg/L NAA+0.1mg/L IBA+0.5g/L gac

In conversion process, each substratum all adds agar 7.5g/L+ sucrose 35g/L, and pH is adjusted to 5.8.

Embodiment 6:

Transfer-gen plant molecule and Physiological Appraisal

1 tobacco, lemon and golden mandarin orange leaf DNA are extracted

Get appropriate blade and put into 1.5ml centrifuge tube, add liquid nitrogen, fully grind; Then cetyltriethylammonium bromide (the being called for short CTAB) DNA extraction damping fluid (formula: 100mM Tris-HCl of 1ml 65 DEG C of preheatings is added, pH 8.0,1.5M NaCl, 2mM EDTA, pH 8.0,1%PVP (polyvinylpyrrolidone), 2%CTAB, 2% mercaptoethanol), 65 DEG C of temperature baths 60-90 minute (take out centrifuge tube therebetween for every 15 minutes and put upside down mixing gently up and down); Centrifugal 10 minutes of 12000rpm; Get 600 μ l supernatants, add equal-volume chloroform, put upside down mixing 10 minutes; Centrifugal 15 minutes of 12000rpm; Get supernatant 400 μ l, add 2 times of volume precooling dehydrated alcohols, 1/10 volume 3M NaAc (sodium-acetate), mixing, place 30 minutes for-20 DEG C, centrifugal 20 minutes of 12000rpm; Abandon supernatant, with the ethanol of l ml 75%, wash precipitation 3 times, add appropriate 1 × TE solubilize.

2 positive transgenic plant PCR detect

Primer Hyg and NPT II and gene specific primer is adopted to carry out pcr amplification.Primer sequence, response procedures and system are respectively in table 1, table 2 and table 3.First Hyg primer pair tobacco regeneration plant is adopted to carry out PCR qualification, 17 transgenic lines can amplify the fragment of Hyg gene, gene specific primer is utilized to carry out PCR, wherein 14 transgenic lines can amplify the fragment (Fig. 7 A) of expection size, show that they are positive transgenic strain.Fig. 7 B is depicted as the PCR qualification of part lemon resistance seedling.Find that 10 resistance seedling materials can amplify the fragment of Hyg gene, utilize gene specific primer to carry out pcr amplification to them, wherein 7 systems can amplify the fragment (Fig. 7 B) of expection size simultaneously, show that they are all transfer-gen plants.Fig. 7 C is depicted as the PCR qualification of part gold mandarin orange resistance seedling, have the fragment that 15 resistance seedling materials can amplify NPT II gene, when utilizing 35S forward primer and gene specific reverse primer to increase, wherein 4 systems can amplify the fragment of expection size, show that they are positive transgenic strain.

Table 1, primer sequence information

Table 2, PCR response procedures

Table 3, PCR reaction system

Embodiment 7:

Semiquatitative RT-PCR assay detects the expression of FcWRKY70 gene

This research adopts semi-quantitative RT-PCR analysis transgene tobacco, the expression amount of foreign gene FcWRKY70 in lemon and golden mandarin orange plant, and transgenic line blade RNA extracts and uses Trizol test kit, and its step is as follows:

1, mortar is in-80 DEG C of freezing precoolings, gets appropriate blade material amount in mortar, and liquid nitrogen grinding is to Powdered.2, get 1.5ml centrifuge tube and add 1ml Trizol damping fluid (cracking cell also protects RNA), the powder of grinding is added in damping fluid in right amount, concuss 2min, leave standstill 10min, 4 DEG C of 12000g, centrifugal 15min.3, get supernatant, add 200 μ L chloroforms, concuss 2min, room temperature leaves standstill 10min, 4 DEG C of 12000g, centrifugal 15min.4, repeating step 3.5, get supernatant, add 500 μ L Virahols, slowly mix, leave standstill 10min, 4 DEG C of 12000g, centrifugal 10min.6, abandon supernatant, the ethanol (joining with sterilizing DEPC water) adding 1ml75% washes precipitation 3 times.Air-dry precipitation, adds 30-50 μ l DEPC water dissolution ,-80 DEG C of preservations.7, concentration and quality that 2 μ l detect RNA is got.

Get 2 μ l RNA and synthesize the first chain cDNA, used kit is First Strand cDNA Synthesis Kit (purchased from Toyobo), and concrete operations are see test kit specification sheets.Sxemiquantitative the primer be FcWRKY70 gene specific primer (F:5'-CCAAGCAAGTAAGCAGGTTCA-3 '; R:5'-TGACTCCAGAAATCACATCCC-3 '), response procedures participates in table 2 (annealing temperature 56 DEG C).The ubiquitin gene of tobacco and oranges and tangerines actin gene are respectively as reference gene, and primer is as follows:

Ubiqutin-F：5'-GGTGTTTCCAGTGGCGGACG-3’

Ubiqutin-R：5'-TCCTCCCCTCAGCTACGGGGTAT-3’

Actin-F：5'-CCAAGCAGCATGAAGATCAA-3'

Actin-R：5'-ATCTGCTGGAAGGTGCTGAG-3'

Choose tobacco 4 transgenic lines, lemon 3 transgenic lines, the expression level of golden mandarin orange 5 transgenic lines is analyzed, and result shows, in overexpression transgene tobacco and lemon institute selectable 7 be in FcWRKY70 expression level all obtain remarkable enhancing (Fig. 7 D, E).And in the transgenosis gold mandarin orange of interfering, in 5 transgenic lines, the expression of FcWRKY70 all demonstrates weakening (Fig. 7 F) in various degree.In the transgenic line that these are different, each selection 2 transgenic lines are used in follow-up transgenic resistance evaluation experimental.

Embodiment 8:

Turn the Evaluation of drought resistance of FcWRKY70 gene plant

Whether can change transfer-gen plant drought resistance, transgene tobacco for evaluating FcWRKY70, lemon and golden mandarin orange material are all subjected to Drought Stress in various degree, observe the phenotypic difference of every part of material, evaluate its drought resistance with this.

Get Transgenic Tobacco system (1# and 4#) and the in vitro seedling leaf of wild-type and be placed in natural-dehydration under room temperature condition, after 60 minutes, obviously there is wilting symptom in wild-type material blade, and similar phenotype does not appear on transgenic leaf.Be respectively that blade is used to trypan blue and dye after dehydration, wild-type leaves presents dark blue dyeing simultaneously, and transgenic leaf is slightly then light blue (Fig. 8 A), and illustrate after natural-dehydration, transgenic lines apoptosis program is far below wild-type.On the other hand, in dehydration 60 minutes processes, each system shows dehydration in various degree, after 60 minutes, the relative percentage of water loss of wild-type reaches 39.3%, transgenic lines is 30.5% and 24.6% respectively, significantly lower than wild-type, illustrates that transgenic lines water-holding power is better than wild-type (Fig. 8 B).Show that FcWRKY70 can significantly improve the anti-dehydrating ability of transgenic line accordingly.

Coerce the phenotype of lower transgenic line for investigating prolonged drought further, the tobacco material of 50 days seedling ages, after the normal pouring of 1 week, enters the control water phase.Shown in Fig. 8 C, FcWRKY70 does not affect the normal phenotype (figure is left) of transgenic line, but after control water, the phenotypic difference of wild-type and transgenic line then clearly (scheming the right side), wild-type material is wilted yellow withered, and although transgenic line growth is also subject to certain suppression, but there is not any wilting symptom, still remain good blade tension force.Random sampling testing soil water content, test result has negated the possibility (Fig. 8 D) that underground part water supply difference causes phenotypic difference.After in wild-type and transgenic line, the difference of mda (MDA) content shows to control water further, transgenic line cell is hurt degree obviously lower (Fig. 8 E).The above results shows that FcWRKY70 effectively changes the phenotype of transgene tobacco material under dehydration and drought stress, improves its resistance.

For the experimental evidence providing more horn of plenty strong illustrates the effect of FcWRKY70 under drought resisting, different oranges and tangerines materials is also used to the research carrying out strengthening or weaken transgene expression, for oranges and tangerines molecular breeding provides effective genetic resources.Get overexpression lemon material (28#, 43#) with wild-type material blade natural-dehydration under room temperature condition, each based material shows dehydration in various degree, after 6 hours, wild-type material percentage of water loss reaches 30.5%, transgenic lines bill of material reveals stronger water-holding power, and percentage of water loss is 26.7% and 22.3% (Fig. 9 A) respectively.After dehydration, get each based material and carry out NBT and DAB staining analysis respectively.Wild-type material blade half is caught mazarine, and transgenic line blade only has fragmentary blue dot staining reaction (Fig. 9 B).Same result is presented in DAB dyeing.As shown in Figure 9 C, transgenosis lemon blade distributed fragmentary light brown dyeing speck uniformly, and wild-type leaves then has large-area Vandyke brown to dye.Coloration result illustrates that natural-dehydration is after 6 hours, and have accumulated more active oxygen in wild-type material, caused seriously cell damage, transgenic line is then effectively remove unnecessary active oxygen, maintains the normal function of cell.On the other hand, design weakens the expression of FcWRKY70 in golden mandarin orange, shown in Figure 10, get that effectively to weaken be (20#, 22#) and wild-type material natural-dehydration under room temperature condition, after 6 hours, the relative percentage of water loss of wild-type is that the relative percentage of water loss of 56.2%, 20# and 22# is respectively 63.5% and 60.0%, apparently higher than wild-type, illustrate that 20# and 22# material water-holding power is compared with wild-type, obviously weakened.Research in oranges and tangerines illustrates, overexpression FcWRKY70, then lemon material anti-dehydrating ability significantly improves; Otherwise weaken the expression of FcWRKY70, the ability of golden mandarin orange material anti-dehydrating obviously dies down, and shows that FcWRKY70 effectively changes transgenosis oranges and tangerines material to the tolerance of drought stress.

SEQUENCE LISTING

 

<110> Hua Zhong Agriculture University

 

<120> gold mandarin orange FcWRKY70 gene and the application in raising drought tolerance in plants thereof

 

<130> gold mandarin orange FcWRKY70 gene and the application in raising drought tolerance in plants thereof

 

<160> 4

 

<170> PatentIn version 3.3

 

<210> 1

<211> 1196

<212> DNA

<213> gold mandarin orange

 

<400> 1

acatggggat atttaaaagc aagcatttgc cttttctctt ttgctactcc tacttggaaa 60

 

aaaaggggga aaaaaaatca taatgaaaat ggaggccggt caggccacat cttcatcttc 120

 

ttggctagag aattcatcag atcgaagaag ggccatagaa gagcttatca aaggccaaga 180

 

aatggcactg cagcttcgaa atctcattca cacatccaca aaaaaaggag aagggtcaaa 240

 

agcgatgatc atcaaccaag atctcgtggc aaatatactg agcttattta caaactctct 300

 

ttcaatattg aaaaatggtg actctgatga agcttcccaa gttcaagaac atacccaatt 360

 

gagctctcct tgctgggagg cttatttgaa gacggaggat tcaggtgaaa gcagcaagag 420

 

ctcaaccgtt aaagaccgga gaggatgcta caagagaaga aagtgtgcgg agtcatggac 480

 

agagcatagc tccactctga cagatgatgg ctttgcatgg aggaaatatg ggcaaaaagt 540

 

gattctcaat tccaaatttc caaggaatta ctttaggtgc actcataaat ttgatcaagg 600

 

ctgccaagca agtaagcagg ttcaaaggat tcaaggggaa cctccactat acagaacaac 660

 

atattatggc cgccacactt gcaaaagttt gatcaagtcc tctcaactga tgccagattc 720

 

tactactagt gatcagtgtc caatgatcag ctttggtagt gcccacatca ccgaaaaaga 780

 

ttttaaccca tttttatcat cattcccatc aataaagcag gagtcaaata aggatgatca 840

 

ggcaccttta agcgatatga ctcacaacca atcatcatca tctgatgaat atcttgtgtc 900

 

acatgacttc ccagcatttg aatcaaatga gcacatgaaa gtactttctt ctgatcatgg 960

 

ggatgtgatt tctggagtca actcatcatg cactgcttct gctcacagct tggacttggc 1020

 

ggtggatatg tctgttaact ttgatgatgt tttggagttt aatttttgat gcttagttag 1080

 

ttactgctac cgctagtcct ttgcaacttt catgtataaa cagatgagtt tgctcttaac 1140

 

tagtttctca atataatgtt agtttctgca acccctttag gtaaaaaaaa aaaaaa 1196

 

 

<210> 2

<211> 328

<212> PRT

<213> gold mandarin orange

 

<400> 2

 

Met Lys Met Glu Ala Gly Gln Ala Thr Ser Ser Ser Ser Trp Leu Glu

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Asn Ser Ser Asp Arg Arg Arg Ala Ile Glu Glu Leu Ile Lys Gly Gln

20 25 30

 

 

Glu Met Ala Leu Gln Leu Arg Asn Leu Ile His Thr Ser Thr Lys Lys

35 40 45

 

 

Gly Glu Gly Ser Lys Ala Met Ile Ile Asn Gln Asp Leu Val Ala Asn

50 55 60

 

 

Ile Leu Ser Leu Phe Thr Asn Ser Leu Ser Ile Leu Lys Asn Gly Asp

65 70 75 80

 

 

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

85 90 95

 

 

Cys Trp Glu Ala Tyr Leu Lys Thr Glu Asp Ser Gly Glu Ser Ser Lys

100 105 110

 

 

Ser Ser Thr Val Lys Asp Arg Arg Gly Cys Tyr Lys Arg Arg Lys Cys

115 120 125

 

 

Ala Glu Ser Trp Thr Glu His Ser Ser Thr Leu Thr Asp Asp Gly Phe

130 135 140

 

 

Ala Trp Arg Lys Tyr Gly Gln Lys Val Ile Leu Asn Ser Lys Phe Pro

145 150 155 160

 

 

Arg Asn Tyr Phe Arg Cys Thr His Lys Phe Asp Gln Gly Cys Gln Ala

165 170 175

 

 

Ser Lys Gln Val Gln Arg Ile Gln Gly Glu Pro Pro Leu Tyr Arg Thr

180 185 190

 

 

Thr Tyr Tyr Gly Arg His Thr Cys Lys Ser Leu Ile Lys Ser Ser Gln

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Leu Met Pro Asp Ser Thr Thr Ser Asp Gln Cys Pro Met Ile Ser Phe

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Gly Ser Ala His Ile Thr Glu Lys Asp Phe Asn Pro Phe Leu Ser Ser

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Phe Pro Ser Ile Lys Gln Glu Ser Asn Lys Asp Asp Gln Ala Pro Leu

245 250 255

 

 

Ser Asp Met Thr His Asn Gln Ser Ser Ser Ser Asp Glu Tyr Leu Val

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Ser His Asp Phe Pro Ala Phe Glu Ser Asn Glu His Met Lys Val Leu

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Ser Ser Asp His Gly Asp Val Ile Ser Gly Val Asn Ser Ser Cys Thr

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Ala Ser Ala His Ser Leu Asp Leu Ala Val Asp Met Ser Val Asn Phe

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Asp Asp Val Leu Glu Phe Asn Phe

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<212> DNA

<213> synthetic

 

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catgccatgg atcataatga aaatggaggc cggtc 35

 

 

<210> 4

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<212> DNA

<213> synthetic

 

<400> 4

gggtcaccag ttgcaaagga ctagcggtag cag 33

 

 

Claims (4)

1. the gene be separated, its sequence is for shown in SEQ ID NO.1.

2. the protein of genes encoding described in claim 1, its sequence is for shown in SEQ ID NO.2.

3. albumen described in gene described in claim 1 or claim 2 is improving the application in golden mandarin orange or lemon drought tolerance.

4. albumen described in gene described in claim 1 or claim 2 is improving the application in tobacco drought tolerance.