CN109468333A

CN109468333A - Citrus laccase family gene CsiLAC4 and its application

Info

Publication number: CN109468333A
Application number: CN201811457508.9A
Authority: CN
Inventors: 黄镜浩; 林雄杰; 胡菡青; 王贤达; 范国成
Original assignee: Pomology Research Institute Fujian Academy of Agricultural Sciences
Current assignee: Pomology Research Institute Fujian Academy of Agricultural Sciences
Priority date: 2018-11-30
Filing date: 2018-11-30
Publication date: 2019-03-15

Abstract

The invention discloses a kind of citrus laccase family genesCsiLAC4And its application, belong to field of plant genetic project technology.Citrus laccase family gene of the present inventionCsiLAC4Preparation method obtains total serum IgE the following steps are included: extracting from Citrus leaf；(2) RNA reverse transcription obtained by step (1) is obtained into cDNA；(3) using gained cDNA as template, first round PCR amplification is carried out with forward primer F1 and reverse primer R1；(4) using first round volume increase object as template, forward primer F1 and reverse primer R2 nested PCR amplification obtain the citrus laccase family geneCsiLAC4.Citrus laccase family geneCsiLAC4In terms of improving the plant poison of resistance to boron ability, new genetic resources are provided for plant resistant abiotic stress molecular breeding.

Description

Citrus laccase family gene CsiLAC4 and its application

Technical field

The invention belongs to field of plant genetic project technology, and in particular to citrus laccase family geneCsiLAC4And its it answers With.

Background technique

Boron (boron, B) is plant growth, development and one of carries out the indispensable microelement of zoogamy.But boron is suitable The concentration range for closing plant growth is very narrow, thus easily occurs lacking or intoxicating phenomenon.Boron, which is poisoned, usually to be occurred in boron-rich soil In.It is raw in the serious agricultural in arid and semi-arid lands or boracic emission of industrial pollutants largely irrigated using desalinated seawater In state system, boron murder by poisoning is the major reason for leading to the multiple kinds of crops underproduction and quality decline.Even if in boron deficiency ecotope, no Appropriate boron fertilizer application also often causes the borism phenomenon of plant.It has recently been demonstrated that in China's southeastern coast citrus main product Area, the improper method of application of boron fertilizer is the key factor for causing citrus boron to be poisoned.

Citrus is the economic fruit of world rankings first.China's citriculture area and annual output rank the world the at present One.It is a kind of physiological disturbance to happen suddenly in citriculture production in recent years that citrus boron, which is poisoned, equal in America, Europe and Asia It has been reported.2015, there is 800,000 mu nearly to the gentle whole county in Fujian of scholar of citrus orchard to carry out random sampling, discovery is super It is exceeded to cross tree body boron content in 74% orchard, boron, which is poisoned, becomes urgent problem in Orange Producing.National citrus producing region boron It poisons that a situation arises in recent years is more universal.Cultivating high-quality, degeneration-resistant citrus new varieties is the most effective of solution citrus boron murder by poisoning Means.However, conventional breeding methods are unable to satisfy modern Citrus Industry hair since citrus genetic background is complicated, the generation cycle is long Open up the demand to excellent new varieties.

Studies have shown that plant inter-species or the boron planted between interior different lines poison tolerance significant difference.But how is plant The mechanism for resisting boron murder by poisoning is still not clear.It is " based on transhipment egg that presently most the resistance to boron of the generally accepted plant of people, which poisons mechanism, White boron outlet mechanism ".In gramineae plant, barley (Hordeum vulgare) by turning in tip of a root up-regulated expression outlet Albumen (BOR family) is transported to reduce to the absorption of boron or lower expression outlet transhipment receptor (NIP family) and reduce in root system boron to ground The transhipment on top, or through up-regulated expression outlet transporter gene BOR2 in blade, the accumulation of internal boron is reduced, thus acquisition pair The tolerance that boron is poisoned.Similar mechanism is then also confirmed in arabidopsis.But " outlet mechanism " is without method interpretation such as mandarin orange " total boron content having the same but shows completely different boron and poisons tolerance in Different Individual present in many species such as tangerine Property " this phenomenon.Ours recent studies have shown that, the laccase family gene regulated and controled in citrus by miR397CsiLAC4It may ginseng It is specific expressed in xylem with the secondary metabolism of lignin, it is likely to reduced transhipment of the boron from xylem to bast, from And reduce murder by poisoning of the excessive boron to phloem tissue.Therefore, it studiesCsiLAC4Biological function and its boron stress under act on machine It manages and mechanism is poisoned to the resistance to boron of parsing xylophyta, and cultivate and all have highly important meaning for resistance to boron murder by poisoning new varieties Justice.Currently, there is not yet relevant research is reported.

Summary of the invention

The purpose of the present invention is to provide citrus laccase family genesCsiLAC4And its application.

In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:

The present invention provides citrus laccase family genesCsiLAC4, citrus laccase family geneCsiLAC4Nucleotide sequence such as Shown in SEQ ID No. 1.In the present invention, the citrus laccase family geneCsiLAC4Preferably derive from ' mandarin orange ' (Citrus sinensis), the citrus laccase family geneCsiLAC4Open reading frame comprising 1668 bp.

Preferably, the citrus is ' mandarin orange ' or ' sour shaddock '.

Citrus laccase family gene in the present inventionCsiLAC4Preparation method the following steps are included: (1) from Citrus leaf It extracts and obtains total serum IgE；(2) RNA reverse transcription obtained by step (1) is obtained into cDNA；(3) using gained cDNA as template, drawn with forward direction Object F1 and reverse primer R1 carries out first round PCR amplification；(4) using first round volume increase object as template, forward primer F1 and reversely draw Object R2 nested PCR amplification obtains the citrus laccase family geneCsiLAC4。

The Citrus leaf is that ' mandarin orange ' blade is grown directly from seeds using ' mandarin orange ' that final concentration of 400 μM of boron poisons processing 15 weeks The extraction of blade at 1/3 plant height of seedling, the citrus RNA is mentioned using the plant tissue RNA of molecular biology field routine Take method.Reverse transcription uses the conventional method of molecular biology field, preferably uses SMART RACE cDNA Amplification Kit(Clontech) reverse transcription reagent box progress, explanation of the specific method step referring to the kit Book.

The sequence of the forward primer F1 is as shown in SEQ ID No .3, and specially 5 '- CGGATCCATGGACTCCTGGGTT

CGGCTTCT-3'；The sequence of the reverse primer R1 is as shown in SEQ ID No. 4, and specially 5 '- GCTACATCCTTCT

TCTCCAGCAAAC-3'；The sequence of the reverse primer R2 is as shown in SEQ ID No. 5, specially 5 '-CGAGCTCT TAACACTTTGGAAGATCACTTGGAGGT-3’。

The nested PCR amplification system is preferably 25 μ L systems, and the amplification system includes 1 μ L " 100 × dilution Template cDNA " or " 500 × first round PCR product ", 2 × Trans Taq^RDNA polymerase High Fidelity (HiFi), 1.0 μM of forward primers and 1.0 μM of reverse primer.2× Trans Taq^RDNA polymerase High Fidelity (HiFi) is preferably purchased from Beijing Quan Shi King Company.The program of heretofore described amplified reaction is preferably: 95 DEG C 3 min of initial denaturation；95 DEG C of 30 s of denaturation, 62 DEG C of 30 s of annealing, 72 DEG C of 90 s of extension, 35 recycle, 72 after the completion of circulation DEG C extend 3 min.

Amplification obtains the citrus laccase family geneCsiLAC4Afterwards, the product preferably obtained amplification carries out agar Sequence verification obtains the citrus laccase family gene after sugared gel electrophoresis, recyclingCsiLAC4Nucleotide sequence.

To citrus laccase family geneCsiLAC4Nucleotide sequence analyzed, obtain citrus laccase family geneCsiLAC4The protein of coding, the amino acid sequence of the protein is as shown in SEQ ID No. 2.It is describedCsiLAC4Coding Protein include 555 amino acid, isoelectric point 7.15, theoretical molecular weight be 60.39 KDa.Protein of the present invention Subcellular localization in cell wall, belong to extracellular protein；The lignin time intercrescence under plant boron poison stress can effectively be enhanced At ability, to improve the resistance to boron toxicity of plant.

The present invention provides the citrus laccase family genesCsiLAC4Application.

In the present invention, the plant preferably includes arabidopsis.When plant of the present invention is arabidopsis, including such as Lower step: 1) citrus laccase family gene is obtainedCsiLAC4；2) by citrus laccase family geneCsiLAC4With plasmid vector Connection obtains overexpression carrier；3) overexpression carrier obtained by step 2 is transferred in Agrobacterium tumefaciems and obtains recombination crown gall Agrobacterium；4) recombination Agrobacterium tumefaciems obtained by step 3) is infected into arabidopsis floral, obtains and is overexpressed citrus laccase family geneCsiLAC4Arabidopsis.

Above-mentioned steps 1) obtain citrus laccase family geneCsiLAC4Preferably: using ' mandarin orange ' leaf cDNA as mould Plate carries out nested PCR amplification and obtains citrus laccase family geneCsiLAC4；The nested PCR amplification obtains mandarin orange in the present invention Tangerine laccase family geneCsiLAC4Specific method and step referring to above-mentioned citrus laccase family geneCsiLAC4Acquisition side Method.

Above-mentioned steps 2) by citrus laccase family geneCsiLAC4It is connect with carrier and obtains overexpression carrier；By step 1) by the citrus laccase family geneCsiLAC4It is connect with carrier and obtains overexpression carrier, in the present invention the carrier Preferably pCHF3 carrier, the construction method of the overexpression carrier is the following steps are included: by citrus laccase family geneCsiLAC4It carries out double digestion respectively with pCHF3 carrier and obtains endonuclease bamhi and digestion carrier；Then by the endonuclease bamhi and institute It states the connection of digestion carrier and obtains overexpression carrier.

Construct the combination of restriction enzyme used in double digestion during overexpression carrier be preferably Bam H I and Sac I.In the present invention, the citrus laccase family geneCsiLAC4The preferably temperature of digestion is 37 DEG C, double enzymes The time cut is preferably 2 h；Described in constructing during overexpression carrierCsiLAC4The system total volume of digestion is preferably 50 μ L includes insertionCsiLAC41.5 μ g, 10 × CutSmart buffer of pEASY-T plasmid, 5 μ L, Bam H I and Sac I Each 1 μ L.In the present invention, the citrus laccase family geneCsiLAC4After double digestion, agarose gel recovery purifying digestion is produced Object obtains citrus laccase family geneCsiLAC4Endonuclease bamhi.In the present invention, the preferable temperature of the pCHF3 double digestion is 37 DEG C, the preferred time of the pCHF3 double digestion is 2 h；The carrier double enzyme digestion reaction volume is preferably 50 μ L, includes 1.5 μ g, 10 × CutSmart buffer of pCHF3 carrier, 5 μ each 1 μ L or Bam H I of L, Bam H I and Asc I and Each 1 μ L of Sac I, distilled water supply system to 50 μ L.For the present invention after the pCHF3 double digestion, glue absorption method purifies digestion Product obtains digestion pCHF3 carrier.

The present invention is obtaining the citrus laccase family geneCsiLAC4By enzyme after endonuclease bamhi and pCHF3 digestion carrier Slice section is connect with digestion carrier obtains overexpression carrier.In present invention connection reaction, the citrus laccase family geneCsiLAC4The molar ratio of endonuclease bamhi and the digestion pCHF3 carrier is 3:1.The totality of the connection reaction in the present invention Product is preferably 10 μ L, including 10 × connection Buffer 1 μ L, T₄1 μ L of DNA ligase, double digestion citrus laccase family base CauseCsiLAC42 ul of segment is recycled, double digestion pCHF3 recycles 6 μ L of carrier.In the present invention, the preferred temperature of the connection reaction Degree is 4 DEG C；The time of the connection reaction is preferably 16 h.The present invention collects the connection in the connection after reaction Product obtains overexpression carrier.

The overexpression carrier is preferably converted coli strain after obtaining overexpression carrier by the present invention Whether DH5 α, recombinant expression carrier described in the bacterium colony PCR sequence verification method validation using this field routine construct success.

The overexpression carrier is transferred to Agrobacterium tumefaciems after obtaining pCHF3 overexpression carrier by the present invention Recombination Agrobacterium tumefaciems is obtained in EHA105；In the present invention, the method for the overexpression carrier being transferred to Agrobacterium tumefaciems Preferably freeze-thaw method, the method and step of specific freeze-thaw method is referring to (Pehanorm Brooker, Huang Peitang are translated, " molecule gram in the present invention Grand experiment guide " third edition, Science Press, 2002).

The recombination Agrobacterium tumefaciems is infected arabidopsis after obtaining the recombination Agrobacterium tumefaciems by the present invention, is obtained It is overexpressed citrus laccase family geneCsiLAC4Arabidopsis.In the present invention, the recombination Agrobacterium tumefaciems infects arabidopsis Method preferentially be inflorescence dip method, the specific steps reference of specific inflorescence dip method in the present invention: (Zhang X, Henr iques R, Lin S, Niu Q, Chua N. Agrobacterium-mediated transformation ofArabidopsis thaliana using the floral dip method. Nature Protocols, 2006, 1 (2): 1-6. doi:10.1038/nprot.2006.97)

The present invention has preferably also carried out the PCR verifying of transgenic positive plant after obtaining the transgenic arabidopsis strain. The PCR verifying is preferably with the specific primer PCR augmentation detection of Kanamycin resistant gene in the present invention.It is obtaining Arabidopsis strain in, the segment of expected size can be amplified, then be shown to be positive transgenic strain.

Beneficial effects of the present invention: citrus laccase family gene provided by the inventionCsiLAC4, through transgenosis functional verification Have the function of improving the plant poison of resistance to boron ability, is overexpressed citrus laccase family geneCsiLAC4It can be effectively reduced and turn base Absorption and transhipment because of plant root to boron, to improve the tolerance of plant pair boron murder by poisoning.Citrus paint provided by the invention Enzyme family geneCsiLAC4New genetic resources are provided for plant resistant abiotic stress molecular breeding, which opens Hair is using with advantageously reducing boron industrial pollution area, the salt-soda soil that a large amount of application desalinated seawaters are irrigated and arid and semi-arid The agriculture production cost in area and realization are environmental-friendly；Citrus laccase family geneCsiLAC4It can be applied to resistance to boron murder by poisoning and turn base Because of the cultivation of plant variety, citrus laccase family geneCsiLAC4It is overexpressed genetically modified plants, the lignin under boron stress conditions Content it is higher than wild type, total boron content and free state boron concentration are lower in plant body, and grow, development condition it is more preferable, table Bright citrus laccase family geneCsiLAC4The genetically modified plants poison of resistance to boron capability improving.

Detailed description of the invention

Fig. 1 boron poisons processing Citrus leaf total serum IgE；C. sinensis: ' mandarin orange '； C. grandis: ' sour shaddock '.

The long-term boron of Fig. 2 poisons processing to ' mandarin orange ' and ' in sour shaddock ' bladeCsiLAC4The influence of expression；Control: 10 μM of boric acid processing；B-toxicity:400 μM of boric acid processing；C. sinensis: ' mandarin orange '； C. grandis: ' acid Shaddock '.

Fig. 3 boron poisons the influence to ' mandarin orange ' and ' sour shaddock ' cell wall Contents of Main Components；C. sinensis: ' mandarin orange '；C. grandis: ' sour shaddock '；(a): cell wall crude extract；(b): pectin；(c): hemicellulose 1；(d)；Hemicellulose 2；(e): Cellulose；(f): lignin；Different letters show difference up to the level of signifiance (P< 0.05, independent sample t- Test)

The subcellular localization of Fig. 4 CsiLAC4 albumen；Light: the cellular morphology under light microscopic；GFP: green florescent signal； DAPI: nuclear signal；Merged: it is overlapped image.

Fig. 5CsiLAC4The PCR verification result of transgenic positive plant；dH₂O: negative control；pCHF3-CsiLAC4- GFP: positive control.

The processing pair of Fig. 6 difference boron concentrationCsiLAC4The influence of transgenic arabidopsis phenotype；A: different boron concentration (B Concentration plant above ground portion fresh weight (Fresh weight), root long (Root length), epicotyl length) are handled (Hypocotyl length) statistical result；Wild type: wildtype Arabidopsis thaliana；OXCsiLAC4:CsiLAC4The quasi- south of transgenosis Mustard；B: the influence of different boron concentration processing epicotyl length；C: the influence of different boron concentration processing root longs.

The processing pair of Fig. 7 difference boron concentrationCsiLAC4Transgenic arabidopsis overground part boron content (A) and epicotyl vascular bundle knot The influence of structure (B)；Light background figure is the epicotyl cross section shapes under light microscopic in B, and dark-background figure is under fluorescence microscope Epicotyl vascular bundle in lignin autofluorescence (clear zone)；Total B: total boron content；Free B: free state boron content； Wild type: wildtype Arabidopsis thaliana；OXCsilAC4:CsiLAC4Transgenic arabidopsis.

Specific embodiment

Below with reference to embodiment to citrus laccase family gene provided by the inventionCsiLAC4And its application progress is detailed Explanation.

1 citrus laccase family gene of embodimentCsiLAC4The clone of full-length cDNA

According to citrus laccase family geneCsiLAC4Gene order use 5 .0 design primer of Primer premier, in conjunction with RT-PCR and nested PCR method amplify its overall length from mandarin orange.Detailed step is as follows:

Material is plantation in ' mandarin orange ' seedling of Inst. of Fruit Trees, Fujian Prov. Academy of Agricultural Sciences's citrus Greenhouse, seedling age 11 Week.After imposing 400 μM of boric acid Hoagland's nutrient solutions of addition every 1 day and carrying out boron murder by poisoning processing in 15 weeks by a definite date, fetch bit The the 20th -25 Cheng Ye (1/3 plant height position) more than base portion is carried out with liquid nitrogen quick-frozen immediately.Using TRIzol method Total serum IgE is extracted, the specific method is as follows:

Preparation before experiment: use 10%(v/v) all kinds of liquid transfer gun heads, centrifuge tube needed for DEPC water treatment experiment, mortar and Pestle.Required experimental article is intended to 12 h or more of advanced processing, and autoclave sterilization is dried for standby.

1. taking ' mandarin orange ' blade, liquid nitrogen is poured into mortar, is rapidly fully ground into sample powdered, takes 0.1 g group It knits powder to be transferred quickly in the 1.5 mL centrifuge tubes equipped with 1 mL TRIzol extracting solution, rear lysis at room temperature 5-10 fullys shake min。

2. 200 μ L chloroforms are added, 15 s are acutely shaken, are placed at room temperature for 2-3 minutes, 4 DEG C, 12,000 × g centrifugation 10 min。

3. shifting in upper strata aqueous phase to another 1.5 clean mL centrifuge tubes, 0.5 mL isopropanol is added, is stored at room temperature 10 Min, 4 DEG C, 12,000 × g is centrifuged 10 min.

4. incline supernatant, 1 mL 75%(v/v is added) ethyl alcohol, shakes 5-10 s, 4 DEG C, 7,500 × g is centrifuged 5 min.

5. it is 4. primary to repeat step.

6. incline supernatant, centrifuge tube is inverted in superclean bench or ventilating kitchen and is air-dried RNA precipitate 5-10 min.

7. 50 ul DEPC processing water is added, it is placed at room temperature for 5-10 min dissolution, 1 uL is taken to be examined with Nano-Drop instrument Survey concentration is 1350 ng/uL.(Fig. 1) is stored in -80 DEG C of ultralow temperature ices after agarose gel electrophoresis detection purity and integrality It is spare in case.

8. taking 1 μ g total serum IgE sample, 1 U DNase I, 37 DEG C of processing 30min are added and are placed on ice.SMART™ RACE cDNA Amplification Kit(Clontech) reverse transcription synthesis the first chain of full-length cDNA.Resulting first chain CDNA is used for citrus laccase family geneCsiLAC4The nested PCR amplification of gene.

First round PCR system:

PCR program:

Second wheel (nido) PCR system:

PCR program:

Amplified production generates single band after the agarose gel electrophoresis of 0.8wt%, cuts glue and with QIAquick Gel Extraction Kit by " use Illustrate " the special purpose band of step recycling.The segment of recovery purifying is attached with pEASY-T1 carrier, gene in linked system It is that 3:1 is connect with the molar ratio of carrier.Reacting total volume is 5 μ L, wherein 1 μ L, PCR purified product of pEASY-T1 carrier, 4 μ L.4 DEG C of connections overnight.Thermal shock method is transformed into E. coli competent DH5 α, carries out PCR verifying with objective gene sequence primer Simultaneously (completion of You Shenggong bio-engineering corporation) is sequenced in (nest-type PRC program when expanding with said gene is consistent).

The long-term boron of embodiment 2 is poisoned under stressCsiLAC4The qRT-PCR of gene analyze and its with cell wall secondary metabolism Relationship

In order to analyze, boron poisons sensitive ' sour shaddock ' and boron poisons citrus laccase family gene in insensitive ' mandarin orange 'CsiLAC4Gene The response modes that stress is poisoned to long-term boron, use Real-time round pcr pairCsiLAC4The expression pattern of gene is divided Analysis.

Use 5 .0 software design of Primer premierCsiLAC4Gene and reference geneβ-actinQRT- PCR special primer,CsiLAC4QRT-PCR special primer forward and reverse primer sequence be respectively SEQ ID No. 6 and SEQ Shown in ID No. 7；β-actinQRT-PCR special primer forward and reverse primer sequence be respectively 8 He of SEQ ID No. Shown in SEQ ID No. 9.

It is extracted respectively using TRIzol method and was imposed 10 μM of boric acid (Control) of addition or 400 μM of boric acid every 1 day (B-toxicity) boron that Hoagland's nutrient solution carries out 15 weeks by a definite date boron Stress treatments poisons sensitive ' sour shaddock ' and boron poison The total serum IgE of insensitive ' mandarin orange ' blade of evil, the synthesis of the first chain of cDNA is referring to Maxima First Strand cDNA Synthesis Kit(Thermo Scientific, USA) reverse transcription reagent box operation manual carry out.10 μ L's It is added in qRT-PCR reaction system: 5 μ L 2 × Mix, 0 .1 μ L cDNA, just, to each 0.25 μ L of primer, 4.4 μ L water； Withβ-actinFor reference gene.The program of quantitative PCR is as follows:

In ' mandarin orange ' blade, long-term boron murder by poisoning processing causesCsiLAC4The significant up-regulation of expression；And in ' boron in sour shaddock ' blade Murder by poisoning causesCsiLAC4Expression is significant to lower (Fig. 2).' in sour shaddock ' bladeCsiLAC4Significant lower causes cell wall dry matter Specific gravity decline, mainly causes pectin, hemicellulose 1(HC1) and hemicellulose 2(HC2) component content decline, but do not reduce Klason content of lignin.Although being had no significant effect on the contrary, boron is poisoned to ' mandarin orange ' blade cell wall dry matter percentage, It isCsiLAC4Up-regulated expression cause cell wall Contents of Main Components occur significant changes, be mainly shown as HC1 content reduce and Klason content of lignin increases (Fig. 3).

3 citrus laccase family gene of embodimentCsiLAC4The subcellular localization of gene

According toCsiLAC4The nucleotide sequence and pCHF3-GFP carrier figure of gene,CsiLAC4Add respectively before and after gene order Enter Bam H I and Sac I restriction enzyme site.The sequence of restriction enzyme site is as follows:

Primer sequence with restriction enzyme site is as follows:

3 forward primer of SEQ ID No.: CGGATCCATGGACTCCTGGGTTCGGCTTCT, wherein underscore part is Bam H I restriction enzyme site；

10 reverse primer of SEQ ID No.: CGGCGCGCCAACACTTTGGAAGATCACTTGGAGGT, wherein underscore part For Asc I restriction enzyme site.

Correctly contained with sequencing resultCsiLAC4The pEASY-T of gene extracts plasmid as template, with addition restriction enzyme site Primer amplification, PCR program used be example 1 in nest-type PRC program.

CsiLAC4Sequence 3 ' eliminates two bases of AA, and purpose removes terminator codon TAA, gene is allowed to merge with GFP. PCR product is through 0.8%(wt) agarose gel electrophoresis, plastic recovery kit recycling.The PCR fragment of purifying send sequencing company, surveys Itself and pCHF3-GFP vector plasmid are subjected to double digestion with Bam H I and Asc I respectively after sequence confirmation.Digestion system is 50 μ L, includingCsiLAC435 μ L or pCHF3-GFP carrier of PCR purified product, 1.5 μ g, 10 × CutSmart buffer, 5 μ L, Each 1 μ L of Bam H I and Asc I, distilled water complement to total volume 50 μ L, 37 DEG C of 2 h of digestion.Glue recycles the production after digestion ObjectCsiLAC4Gene and pCHF3-GFP carrier.The two is through T₄DNA ligase connection, linked system are 10 μ L, wherein 10 × Connect Buffer 1 μ L, T₄1 μ L of DNA ligase, double digestionCsiLAC42 ul of segment is recycled, double digestion pCHF3 recycling carries 6 μ L of body, 4 DEG C overnight.E. coli competent DH5 α is converted, confirmation sequence is further sequenced in picking positive colony.It will obtain Recombinant vector be named as pCHF3-CsiLAC4-GFP。

Freeze-thaw method (Pehanorm Brooker, Huang Peitang are translated, " Molecular Cloning:A Laboratory guide " third edition, Science Press, and 2002 Year) by recombinant vector pCHF3-CsiLAC4-GFP and pCHF3-GFP empty plasmid is directed respectively into Agrobacterium tumefaciems EHA105 and obtains Take recombination Agrobacterium tumefaciems.Positive restructuring EHA105 clone is with containing final concentration of 20 mg/L rifampin and final concentration of 50 mg/L The LB liquid medium of kanamycins is at 28 DEG C, 220 rpm constant-temperature table shake culture, 48 h.Culture solution is through 10,000 rpm Be centrifuged 1 min collect thallus, mass percent concentration be 5% sucrose solution (contain percent by volume be 0.02% Silwet L-77, Final concentration of 100 mM acetosyringone) resuspension (OD₆₀₀=0.5).Re-suspension liquid and onion endepidermis co-culture 48-72 h.It is logical The position of GFP fluorescence is crossed to determine the subcellular localization situation of CsiLAC4 albumen.As a result as shown in figure 4, wherein CsiLAC4- The green fluorescence that GFP is generated is distributed on the outer wall of onion epidermis cell；The green fluorescence of empty carrier is mainly distributed on nucleus In interior and cytoplasm, and do not find green fluorescence in cell wall.ShowCsiLAC4The assignment of genes gene mapping is on cell wall, being one Extracellular protein.

4 citrus laccase family gene of embodimentCsiLAC4Improving the application in the arabidopsis poison of resistance to boron ability

(1) plant conversion carrier constructs

TIANprep Mini Plasmid Kit II(TIANGEN, Beijing) the small extraction reagent kit extraction sequencing confirmation of plasmid is just True containsCsiLAC4The pEASY-T plasmid and pCHF3-GFP vector plasmid of gene order, respectively with Bam H I and Sac I into Row double digestion.Digestion system is 50 μ L, respectively includes containingCsiLAC4The pEASY-T plasmid or pCHF3-GFP of gene order 1.5 μ g, 10 × CutSmart buffer of carrier, 5 μ L, Bam H I and Asc each 1 μ L of I, distilled water complement to total volume 50 μ L, 37 DEG C of 2 h of digestion.Glue recycles the product after digestionCsiLAC4Genetic fragment and pCHF3-GFP carrier, the two is passed through T₄DNA ligase connection, linked system are 10 μ L, include 10 × connection Buffer 1 μ L, T₄1 μ L of DNA ligase, double enzymes It cutsCsiLAC4Segment and the 3:1 calculating in molar ratio of double digestion pCHF3 recycling carrier are recycled, 4 DEG C overnight.Convert Escherichia coli sense By state DH5 α, positive colony further confirms that sequence through sequencing company.Obtained overexpression carrier is named as pCHF3- CsiLAC4。

Overexpression carrier pCHF3-CsiLAC4 is imported into the freeze thawing in the method and example 3 of Agrobacterium tumefaciems EHA105 Method is identical.It is stored in -80 DEG C of refrigerators after LB Liquid Culture using glycerol through the pCHF3-CsiLAC4 bacterial strain of PCR confirmation.

(2) the arabidopsis genetic transformation of mediated by agriculture bacillus and transgenic plant screening

1. the culture of wildtype Arabidopsis thaliana: wildtype Arabidopsis thaliana seed stores 3-4 d in 4 DEG C of refrigerators with breaking dormancy.Then Seed is seeded in the cm plastic tub of 8 cm × 8.Basin mouth is wrapped with preservative film.Culture in 20 DEG C of growth cabinets, light/ The dark period is the h of 16 h/8.Preservative film is needled after seed sprouting to grow by bud.

2. Agrobacterium is cultivated: the Agrobacterium tumefaciems bacterium solution saved in ultra low temperature freezer (- 80 DEG C) is taken, it is dense eventually being added to Degree is crosses on the LB plating medium of 50 mg/L kanamycins, scraping scribing line bacterial plaque, is added liquid LB base after 28 DEG C of cultures In basal culture medium, 28 DEG C, 250 rpm shaken cultivations reach OD to bacterial concentration₆₀₀When=0.8,10,000 rpm are centrifuged 1 min Thallus is collected, mass percent concentration is that (the Silwet L-77 containing concentration of volume percent 0.02%, end are dense for 5% sucrose solution Degree is the acetosyringone of 100 mM) it is resuspended floating to OD₆₀₀=0.5.Freshly prepared bacteria suspension is for disseminating.

3. arabidopsis floral is disseminated: when 4/5 arabidopsis floral in basin it is long to 10 cm long when, make inflorescence full flowerpot back-off Portion is immersed in freshly prepared bacteria suspension, is taken out after 10 s of gentle agitation bacteria suspension, is shrugged off extra bacterium solution, with plastic film or guarantor Fresh film seals inflorescence together with flowerpot, and room temperature is horizontal positioned with moisturizing 16-24h.Remove covering film and by flowerpot again Moving into culture in 20 DEG C of growth cabinets, (light dark cycles continue culture 1 month for the h of 16 h/8), stop watering, wait kind T1 is collected for seed after folder is mature.

4. the screening of transgenic arabidopsis: the T1 of harvest for arabidopsis seed after 4 DEG C of vernalization treatment breaking dormancies, 70% (v/v) 1 min of alcohol treatment, 0.1%(v/v) for HgCl(containing 0.05% (v/v) Tween-20), 10 min of disinfection, sterile water is thorough Rinsing 5 times, 0.05%(wt) it agar suspension seed and is seeded in containing final concentration of 50 mg/L kanamycins and final concentration of 100 On the MS solid selection medium plate of mg/L cephalosporin.Plate is placed in 20 DEG C of continuous illumination incubators and cultivates 7-10 T1 is continued to cultivate and carries out PCR verifying by d for the small transplantation of seedlings plant of positive transgenic arabidopsis in the Nutrition Soil of sterilizing.

5. the PCR of transgenic arabidopsis is verified: take T1 for positive transgenic Arabidopsis leaf, after liquid feeding nitrogen is fully ground, Take 0.1 g powder Plant DNA Mini Kit(Omega) DNA is extracted according to " operation instruction " step.Use Primer 5 .0 software design Kanamycin resistance label gene forward direction of premier, reverse primer, sequence is respectively such as SEQ ID No. Shown in 11 and SEQ ID No. 12.

PCR system:

PCR program:

(3)CsiLAC4The resistance to boron of transgenic arabidopsis poisons Function detection

It collectsCsiLAC43 T1 of transgenic arabidopsis are seeded in respectively after vernalization containing final concentration for the selfed seed (T2) of strain To be screened on the MS solid selection medium plate of 50 mg/L kanamycins and final concentration of 100 mg/L cephalosporin, The homozygous selfed seed (T3) for harvesting T2 strain is stored in 4 DEG C of refrigerators and is used as resistance to boron murder by poisoning Function detection.

In order to identifyCsiLAC4The whether resistance to boron of transgenic arabidopsis poisons stress, by arabidopsis control series (wild type) andCsiLAC4Transgenic arabidopsis T3 system carries out boron and poisons Stress treatment.When detection, wild type (Wild type) and T3 are for seed Through 70%(v/v) alcohol treatment 1 min, 0.1%(w/v) HgCl(contains 0.05%(v/v) Tween-20) and 10 min of disinfection, sterile water Rinsing 5 times, is seeded in the 1/2 MS solid medium tablets added with various concentration boric acid.Plate is placed in 20 DEG C of continuous light After cultivating 14 d in incubator, its phenotype is observed, and measures overground part fresh weight, root long, epicotyl length respectively.Wild type is quasi- Southern mustard root long significantly shortens with the increase of boric acid concentration in culture medium, andCsiLAC4Transgenic arabidopsis is lower than 2 in final concentration The shortening trend of the lower root long of mM boric acid processing is unobvious, and it is raw only significant root occur in the case where final concentration is higher than 2 mM boric acid treatment conditions It is long to inhibit.Wild type andCsiLAC4Transgenic arabidopsis overground part fresh weight is with the increase of boric acid concentration in culture medium in decline Trend, but the processing of each boric acid concentrationCsiLAC4Transgenic arabidopsis overground part fresh weight is all remarkably higher than wild type.Wild type Arabidopsis epicotyl length increases with boric acid concentration is presented growth trend, but unobvious；CsiLAC4Transgenic arabidopsis is dense eventually Degree is lower than the extremely significant growth of epicotyl length under 2 mM boric acid treatment conditions, subsequent (Fig. 6) on a declining curve.Our result table Bright, T3 is obviously showed than wild type for transgenic plant and is poisoned more resistant to boron.

The tolerance that plant poisons boron with intracorporal boron content --- the concentration of especially free state boron is related.Pass through survey Determine wildtype Arabidopsis thaliana withCsiLAC4The boron concentration of transgenic line arabidopsis aerial part, it has been found that be lower than in final concentration Under the boric acid treatment conditions of 2 mM, total boron of wild type and transgenic plant and the content of free state boron are with the boron in culture medium Concentration increases and significantly rises, but under same boric acid level, and the total boron and free state boron concentration of transgenic plant are significant respectively Lower than the total boron and free state boron concentration (Fig. 7 A) of wild type.Show to be overexpressedCsiLAC4Gene can effectively enhance and turn base Because the resistance to boron of plant poisons ability.

CsiLAC4It is under the jurisdiction of laccase gene family, participates in the secondary metabolism of lignin.It is handled with various concentration boric acid upper Plumular axis is material, carries out the anatomical research of vascular tissue, the results showed that, 10 μM of boric acid processing pairCsiLAC4Transgenic line Vascular tissue secondary metabolism has no significant effect, but with the increase of boric acid concentration in culture medium, apparent wood occurs for bundle sheath Quality secondary deposit；When boric acid concentration reaches 2 mM, the not redeposited lignin of bundle sheath, but the vessel element in xylem Quantity dramatically increases (Fig. 7 B).ShowCsiLAC4The secondary synthesis regulation of lignin that gene participates in needs the participation of high concentration boron.

The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with Modification, is all covered by the present invention.

SEQUENCE LISTING

<110>Inst. of Fruit Trees, Fujian Prov. Academy of Agricultural Sciences

<120>citrus laccase family gene CsiLAC4 and its application

<130> 12

<160> 12

<170> PatentIn version 3.3

<210> 1

<211> 1668

<212> DNA

<213>artificial sequence

<400> 1

atggactcct gggttcggct tcttcttctc gtagcatgcc tttttccggc tctcgtcgag 60

tgccgggttc gccattacaa gttcaatgtg gtgatgaaga atagcactaa attgtgttca 120

tccaagccaa ttgtcacagt taatggcaag ttcccgggac ccactctcta tgcgagggaa 180

catgacacag tgctggtcaa agtagtcaac catgtcaaat ataatgtcac tatccactgg 240

catggtgtcc ggcaactgag aaccggctgg gccgatggac cagcatacat aacacaatgt 300

ccaattcagt cagggcatag ctatgtgtac aatttcacaa tcacaggcca acggggcaca 360

cttctttggc atgcacacat tctctggctc cgggccaccg tacacggcgc tatcgtcatc 420

ttgccgaagc gcggcgttcc ttaccctttc cccaaacccc acgaggaagt cgtcgtcgtc 480

ctcgccgagt ggtggaaatc cgacactgaa gctgtcatca accaagcttt gcggtccgga 540

ttggccccta atgtctctga ttcccacacc attaacggac aaccagggcc catctccagt 600

tgctcgtcac agggaggctt tacattgcct gtggacagcg gcaaaaccta catgctacga 660

ataatcaatg ctgcactcaa tgaagagcta tttttcaaaa tagcaggaca caagctaact 720

gttgttgaag ttgatgccac ttatgttaaa ccattcaaaa cagacactat tgtaatagct 780

ccaggccaaa ccaccaatgt cctgctctct gctgacaaaa catccggaaa atacctcgtt 840

gccgcctcgc cgttcatgga cgctccgatt gcagtagaca acgcgaccgc caccgccacg 900

ttacactact ccggcacact cgccagctcc gccacaactc tcaccagcac tcctccgaaa 960

aacggcaccg ccattgccaa caaatttatt gactctctcc gtagcttgaa ctccaagaaa 1020

tacccagcta aagtcccaca aacagttgat cacaatctct tgttcacagt tggacttgga 1080

gttaacccct gcccttcttg caaagccggt aacggcagcc gtgttgtggc ttcaatcaac 1140

aacgtcacat ttgttatgcc aacgattgcg ttgcttcagg ctcatttttt caacatcagt 1200

ggagttttca ctaccgattt tcctggcaac ccaccgcata cttataactt taccggcact 1260

ccaaaaaatc tgcagacttc caatggaaca aaagcttaca ggctagctta caactctaca 1320

gttcagctaa ttttacaaga tactggaatc atagcccctg agaaccatcc agtccattta 1380

cacggattca atttctttgc cgtcggtaag ggacttggta acttcaatcc caagaaagat 1440

cctaagaagt ttaatcttgt tgatcctgtt gaaaggaaca caattggagt cccatctggg 1500

ggatgggttg ctatcagatt cagcgcagat aatccaggag tatggtttat gcattgccat 1560

ctggaagtac acacaacatg gggattgaag atggcatttt tggttgacaa tggcaaaggc 1620

cctaatgagt cacttttgcc acctccaagt gatcttccaa agtgttaa 1668

<210> 2

<211> 555

<212> PRT

<213>artificial sequence

<400> 2

Met Asp Ser Trp Val Arg Leu Leu Leu Leu Val Ala Val Leu Phe Pro

1 5 10 15

Ala Leu Val Glu Val Arg Val Arg His Tyr Lys Phe Asn Val Val Met

20 25 30

Lys Asn Ser Thr Lys Leu Val Ser Ser Lys Pro Ile Val Thr Val Asn

35 40 45

Gly Lys Phe Pro Gly Pro Thr Leu Tyr Ala Arg Glu His Asp Thr Val

50 55 60

Leu Val Lys Val Val Asn His Val Lys Tyr Asn Val Thr Ile His Trp

65 70 75 80

His Gly Val Arg Gln Leu Arg Thr Gly Trp Ala Asp Gly Pro Ala Tyr

85 90 95

Ile Thr Gln Val Pro Ile Gln Ser Gly His Ser Tyr Val Tyr Asn Phe

100 105 110

Thr Ile Thr Gly Gln Arg Gly Thr Leu Leu Trp His Ala His Ile Leu

115 120 125

Trp Leu Arg Ala Thr Val His Gly Ala Ile Val Ile Leu Pro Lys Arg

130 135 140

Gly Val Pro Tyr Pro Phe Pro Lys Pro His Glu Glu Val Val Val Val

145 150 155 160

Leu Ala Glu Trp Trp Lys Ser Asp Thr Glu Ala Val Ile Asn Gln Ala

165 170 175

Leu Arg Ser Gly Leu Ala Pro Asn Val Ser Asp Ser His Thr Ile Asn

180 185 190

Gly Gln Pro Gly Pro Ile Ser Ser Val Ser Ser Gln Gly Gly Phe Thr

195 200 205

Leu Pro Val Asp Ser Gly Lys Thr Tyr Met Leu Arg Ile Ile Asn Ala

210 215 220

Ala Leu Asn Glu Glu Leu Phe Phe Lys Ile Ala Gly His Lys Leu Thr

225 230 235 240

Val Val Glu Val Asp Ala Thr Tyr Val Lys Pro Phe Lys Thr Asp Thr

245 250 255

Ile Val Ile Ala Pro Gly Gln Thr Thr Asn Val Leu Leu Ser Ala Asp

260 265 270

Lys Thr Ser Gly Lys Tyr Leu Val Ala Ala Ser Pro Phe Met Asp Ala

275 280 285

Pro Ile Ala Val Asp Asn Ala Thr Ala Thr Ala Thr Leu His Tyr Ser

290 295 300

Gly Thr Leu Ala Ser Ser Ala Thr Thr Leu Thr Ser Thr Pro Pro Lys

305 310 315 320

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

325 330 335

Asn Ser Lys Lys Tyr Pro Ala Lys Val Pro Gln Thr Val Asp His Asn

340 345 350

Leu Leu Phe Thr Val Gly Leu Gly Val Asn Pro Val Pro Ser Val Lys

355 360 365

Ala Gly Asn Gly Ser Arg Val Val Ala Ser Ile Asn Asn Val Thr Phe

370 375 380

Val Met Pro Thr Ile Ala Leu Leu Gln Ala His Phe Phe Asn Ile Ser

385 390 395 400

Gly Val Phe Thr Thr Asp Phe Pro Gly Asn Pro Pro His Thr Tyr Asn

405 410 415

Phe Thr Gly Thr Pro Lys Asn Leu Gln Thr Ser Asn Gly Thr Lys Ala

420 425 430

Tyr Arg Leu Ala Tyr Asn Ser Thr Val Gln Leu Ile Leu Gln Asp Thr

435 440 445

Gly Ile Ile Ala Pro Glu Asn His Pro Val His Leu His Gly Phe Asn

450 455 460

Phe Phe Ala Val Gly Lys Gly Leu Gly Asn Phe Asn Pro Lys Lys Asp

465 470 475 480

Pro Lys Lys Phe Asn Leu Val Asp Pro Val Glu Arg Asn Thr Ile Gly

485 490 495

Val Pro Ser Gly Gly Trp Val Ala Ile Arg Phe Ser Ala Asp Asn Pro

500 505 510

Gly Val Trp Phe Met His Val His Leu Glu Val His Thr Thr Trp Gly

515 520 525

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

530 535 540

Leu Leu Pro Pro Pro Ser Asp Leu Pro Lys Val

545 550 555

<210> 3

<211> 30

<212> DNA

<213>artificial sequence

<400> 3

cggatccatg gactcctggg ttcggcttct 30

<210> 4

<211> 25

<212> DNA

<213>artificial sequence

<400> 4

gctacatcct tcttctccag caaac 25

<210> 5

<211> 35

<212> DNA

<213>artificial sequence

<400> 5

cgagctctta acactttgga agatcacttg gaggt 35

<210> 6

<211> 22

<212> DNA

<213>artificial sequence

<400> 6

cgtcacaggg aggctttaca tt 22

<210> 7

<211> 22

<212> DNA

<213>artificial sequence

<400> 7

tggtttaaca taagtggcat ca 22

<210> 8

<211> 23

<212> DNA

<213>artificial sequence

<400> 8

agaactatga actgcctgat ggc 23

<210> 9

<211> 22

<212> DNA

<213>artificial sequence

<400> 9

gcttggagca agtgctgtga tt 22

<210> 10

<211> 35

<212> DNA

<213>artificial sequence

<400> 10

cggcgcgcca acactttgga agatcacttg gaggt 35

<210> 11

<211> 23

<212> DNA

<213>artificial sequence

<400> 11

acgttgtcac tgaagcggga agg 23

<210> 12

<211> 24

<212> DNA

<213>artificial sequence

<400> 12

ggcgataccg taaagcacga ggaa 24

Claims

1. citrus laccase family geneCsiLAC4, it is characterised in that: the citrus laccase family geneCsiLAC4Nucleotide Sequence is as shown in SEQ ID No. 1.

2. citrus laccase family gene according to claim 1CsiLAC4, which is characterized in that citrus laccase family GeneCsiLAC4The protein of coding, amino acid sequence is as shown in SEQ ID No.2.

3. citrus laccase family gene as described in claim 1CsiLAC4Application in terms of improving the plant poison of resistance to boron ability.

4. application according to claim 3, which is characterized in that the plant includes arabidopsis or citrus；The citrus is ' mandarin orange ' or ' sour shaddock '.

5. application according to claim 4, which is characterized in that when the plant is arabidopsis, comprising the following steps:

1) citrus laccase family gene described in claim 1 is obtainedCsiLAC4；

2) by gained citrus laccase family geneCsiLAC4It is connect with plasmid vector and obtains overexpression carrier；

3) recombination Agrobacterium tumefaciems will be obtained in overexpression vector introduction Agrobacterium tumefaciems obtained by step 2；4) by step 3) gained recombination Agrobacterium tumefaciems infects arabidopsis, obtains and is overexpressed citrus laccase family geneCsiLAC4Arabidopsis.

6. application according to claim 5, which is characterized in that step 1) are as follows: using Citrus leaf cDNA as template, carry out nest Formula PCR amplification obtains citrus laccase family geneCsiLAC4；Nested PCR amplification the primer is to including forward primer F1 and instead To primer R1 and R2；The sequence of the forward primer F1 is as shown in SEQ ID No .3；The sequence such as SEQ of the reverse primer R1 Shown in ID No .4；The sequence of the reverse primer R2 is as shown in SEQ ID No .5.