CN108048470A

CN108048470A - Structure and the application of a kind of pineapple AcWRKY28 genes and its overexpression vector

Info

Publication number: CN108048470A
Application number: CN201810111886.5A
Authority: CN
Inventors: 秦源; 蔡汉阳; 张曼; 何晴; 柴梦楠; 赵丽华
Original assignee: Fujian Agriculture and Forestry University
Current assignee: Fujian Agriculture and Forestry University
Priority date: 2018-02-05
Filing date: 2018-02-05
Publication date: 2018-05-18

Abstract

The present invention provides structure and the applications of a kind of pineapple AcWRKY28 genes and its overexpression vector, the gene nucleotide series are as shown in SEQ ID No.1, pineapple AcWRKY28 gene cDNAs with CaMV35S promoter core sequences are merged, are built into the overexpression vector of AcWRKY28.By the overexpression vector Transformed E HA105 Agrobacteriums, the inflorescence that arabidopsis is infected using inflorescence method obtains transgenic arabidopsis, can improve the resistance that infects of the transgenic arabidopsis to sclerotinite.

Description

Structure and the application of a kind of pineapple AcWRKY28 genes and its overexpression vector

Technical field

The present invention relates to the structure of a kind of pineapple AcWRKY28 genes and its overexpression vector and its in genetic engineering Using having related more specifically to application of the pineapple gene in the anti-sclerotinite genetic engineering of arabidopsis, belonged to plant gene Field of engineering technology.

Background technology

Plant growth, development and be to environment response its specific genetic system and residing environmental condition interaction under It is realized by complicated physiological and biochemical procedure, is always subjected to more strict regulation and control, and finally makes plant in specific environment The lower chance for obtaining maximum existence and procreation.Plant is when the various adverse circumstances of response, particularly biotic stress, and internal one The gene of range of defensive reaction can all be activated, such as calcium messenger system, plant endogenous hormones, phosphokinase system, transcription factor Wait the signal transduction of compositions.These defense reactions be plant growth, development and adapt to environment conditioning important step, plant cell The various information of sensing external environment and the integration of signal can be carried out, transferred, most at last signal pass into nucleus adjust it is related The expression of gene makes plant generate the biochemical reactions for being conducive to that it is survived and biography is spread out.

Pineapple（Ananas comosus）It is one of typical important fruit tree in many tropical and subtropical countries, alive boundary treaty has 61 states There is cultivation in family and area.Pineapple has stronger drought-resistant and disease resistance in production, and the generation of pest and disease damage is relatively Lack.Therefore, it can be separated from pineapple and clone disease-resistant relevant gene applied on other plant or crop, improved and plant The disease resistance of object has critically important utility value.Plant, which participates in disease-resistant gene, to be had very much, for example, Receptor-like protein ki-nase base Cause, film positioning associated receptor albumen, calcium singaling GAP-associated protein GAP and transcription factor.But people answer pineapple disease-resistant gene With or it is seldom.

In view of pilot process of the WRKY gene in plant disease-resistant signal, plays a part of signal amplification, it can be by the external world Signal amplification step by step simultaneously hands on, and separation clone's WRKY gene and functional analysis are that understand pineapple in depth disease-resistant from pineapple Mechanism and the premise that other plant or crop genetic improvement are carried out by technique for gene engineering.

Since WRKY gene is found in plant, there are more and more researchs to show that WRKY gene is being planted The signal pathway of many degeneration-resistant aspects is participated in object, and critically important effect is played in these signal pathways.There are many forefathers Report is all in arabidopsis, rice and potato isotype plant, and the report on pineapple is not yet.We are separated This WRKY gene overexpresses the resistance that can significantly enhance to arabidopsis to sclerotinite in arabidopsis.

The content of the invention

It is an object of the invention to provide a kind of pineapple AcWRKY28 genes in the anti-sclerotinite genetic engineering of arabidopsis Using by the development and application of the anti-sclerotinite genetic engineering of arabidopsis of making greater efforts to promote.

To achieve the above object, the present invention adopts the following technical scheme that：

The pineapple AcWRKY28 genes of the present invention, the gene is at least containing the nucleotide sequence as shown in SEQ ID No.1.It is described The albumen of pineapple AcWRKY28 gene codes, amino acid sequence is as shown in SEQ ID No.2.

Construction method containing the genophore is as follows：

Pineapple AcWRKY28 gene cDNAs with CaMV35S promoter core sequences are merged, are built into the overexpression of AcWRKY28 Carrier.The CaMV35S promoter core sequences are CGACCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGAC.The pineapple AcWRKY28 genes CDNA is that the gene obtained is separated from the cDNA library of pineapple leaf.

Application of the gene in the anti-sclerotinite genetic engineering of arabidopsis.By the overexpression vector Transformed E HA105 Agrobacterium, the inflorescence that arabidopsis is infected using inflorescence method obtain transgenic arabidopsis, can improve transgenic arabidopsis to sclerotinite The resistance infected.

The advantage of the invention is that：

Overexpressions of the pineapple AcWRKY28 of the present invention in arabidopsis significantly improves transgenosis plan compared with wildtype Arabidopsis thaliana The anti-sclerotinite ability of southern mustard, the mutant containing this gene have highly important application in Genes For Plant Tolerance sclerotinite genetic engineering Value.

Description of the drawings

Fig. 1 is the conserved functional domains in pineapple AcWRKY28.

Fig. 2 is the phylogenetic tree of pineapple AcWRKY28 and other WRKY gene family members.

Fig. 3-1 is entry vector pENTER.

Fig. 3-2 overexpression vectors pWGB502.

Fig. 4 is pineapple AcWRKY28 transfer-gen plants and comparison of the WT lines to sclerotinite resistance.

Specific embodiment

Embodiment 1

1 vector construction process

1.1 material

Pineapple kind is the MD2 gold pineapples that this laboratory preserves, and is preserved by straits joint study institute of University Of Agriculture and Forestry In Fujian laboratory； Arabidopsis col-0 kinds are preserved by straits joint study institute of University Of Agriculture and Forestry In Fujian laboratory；Pineapple leaf cDNA is Fujian agricultural Straits joint study institute of university laboratory structure（Illustrate to operate according in Invitrogen companies kit）, kit is purchased from Invitrogen companies of the U.S..PCR correlations dNTP,TaqEnzyme, PCR buffer are purchased from Dalian TaKaRa Products, reverse transcription And Real-Time PCR kits are purchased from Dalian TaKaRa companies.Gentamicin, kanamycins, rifampin, carboxylic Bian penicillin Deng for Sigma Products.Other chemical reagent are that domestic chemistry is pure.

1.2 method

1.2.1 the separation of pineapple AcWRKY28 candidate genes

The cDNA sequence being sequenced first according to pineapple designs primer, primer sequence P1：5’- CACCATGTCACAAGAGAGAGGAGAG-3’；P2： 5’-TCATGGTTGATTTTGGTGAATG-3’.Then PCR skills are used Art carries out the clone of sequence, and sequencing result shows the length of the cDNA positive colonies as 1011 bp, is 336 containing 1 length The opening code-reading frame of a amino acid is (shown in SEQ ID NO.2）.

Blastp search results show to contain in the putative amino acid sequence of the cDNA positive colonies all conservative The homology for the WRKY albumen identified in WRKYGQK functional domains, with other plants is up to more than 41%（Fig. 1）, by the cDNA Positive colony is temporarily named as AcWRKY28.As can be seen that our separated AcWRKY28 and oil palm on homologous chadogram The height of the tetraploid rice of EgWRKY28 and bajiao banana MaWRKY28（Fig. 2）.

1.2.2 pineapple AcWRKY28 genes overexpression vector is built

The present invention is built dicotyledon and is turned base using pENTER connections and gateway technologies (Walhout et al., 2000) Because with overexpression vector.

First according to pENTER vector constructions Technology design and synthesize special primer AcWRKY28-F: 5’- CACCATGTCACAAGAGAGAGGAGAG-3’； AcWRKY28-R: 5’-TCATGGTTGATTTTGGTGAATG-3’.Then root According to PCR amplification program, PCR amplification program program is as follows：

95 DEG C of 1 cycle of pre-degeneration 2min

It is denatured 94 DEG C of 15 s

Anneal 55 DEG C of 30s

Extend 1 min, 72 DEG C of 35 cycles

Extend 72 DEG C of 10 min

Agarose gel electrophoresis test strip quality and glue recovery purifying PCR product.

Then target gene is cloned by entry vector pENTER by pENTER reaction systems（See Fig. 3-1）On formed into Door clone, pENTER-AcWRKY28, after sequencing testing goal gene order is errorless, then by LR reaction systems by target gene Clone is transferred to overexpression vector pGWB502（Such as Fig. 3-2）, target gene is so also just cloned in the CaMV35S of Ti-plasmids Promoter downstream forms the overexpression vector containing target gene, it can be achieved that its overexpression in transfer-gen plant, finally joins According to molecular cloning guide by the further Transformed E HA105 Agrobacteriums of overexpression vector containing target gene, to be ready for use on tobacco something lost Pass conversion.PENTER coupled reaction systems：

PCR products 100 ng （1.0μl）

Vector (pENTER) 30-50 ng（0.25μl）

Solution I 0.25 μl

Total 1. 5μl

25 DEG C of warm bath are stayed overnight, and add in Proteinase K after reaction, and reaction product is converted DH5 α competence by 37 DEG C of digestion 10min Cell.

LR reaction systems：

Entry clone 50-100 ng（0.5μl）

Destination vector 100 ng（0.5μl）

LR Enzyme mix 0. 25 μl

Total 1.25 μl

25 DEG C of warm bath are stayed overnight, and add in Proteinase K, 37 DEG C of digestion 10min, by reaction product conversion DH5 α impressions after reaction State cell.

The acquisition of arabidopsis transfer-gen plant and Analysis of Resistance

2.1 arabidopsis genetic transformations and T2 are for the harvest of transgenic seed

(1) picking single bacterium colony is inoculated in 5ml LB fluid nutrient mediums (Rif containing 50mg/L and 100mg/L Spec), 28 DEG C 200rmp/min shaken cultivations are stayed overnight；(2) take 2ml bacterium solutions be transferred to 100ml LB fluid nutrient mediums (containing 50mg/LRif and 100mg/L Spec), 28 DEG C of 200rmp/min shaken cultivations to OD600 values are 1.0-1.5 or so；(3) 6000rmp/min, 10min is centrifuged, abandons supernatant；(4) 5% sucrose solutions of 1ml suspend, 6000rmp/min, centrifuge 2min, abandon supernatant；(5) repeat Step 4；(6) add in 5% sucrose solution and thalline is resuspended, until OD600 values are 0.4-0.7 or so；(7) 0.05% volume is pressed before impregnating Than adding in 77 surfactants of silwet；(8) watering before impregnating, and the pod tied is cut off, petal is soaked in above-mentioned 1min in solution；(9) plant is inverted, dark culturing for 24 hours, is taken out, normal growth culture.Containing hygromycin（100 mg/L） MS culture mediums on screen resistance seedling, and antagonism seedling genomic DNA carry out PCR detections, with determine hygromycin selection Reliability.A series of transgenic line is obtained, wherein obtaining 27 plants of AcWRKY28 transfer-gen plants.All AcWRKY28 Strain is selfed harvest T1 for transgenic seed with bagging.The positive plant in T1 generations is sowed and harvests T2 for transgenic seed, Using T2 transgenic arabidopsis phenotypic analysis and Function Identification are carried out for transgenic seed.

Pineapple AcWRKY28 participates in regulation and control phytopathogen defense reaction process

The present invention applies sclerotinite（Now it is stored in straits joint study institute of University Of Agriculture and Forestry In Fujian laboratory）Base is turned to AcWRKY28 Cause and wild type control carry out sclerotinite inoculation experiments, find AcWRKY28 transgenic arabidopsis compared with wild type pair after 2 days According to the apparent resistance shown to pathogen infection, it is anti-to generate stronger allergy in AcWRKY28 transgenic arabidopsis inoculation position Answer necrotic plaque（Fig. 4）.It is anti-to infecting for sclerotinite to illustrate that AcWRKY28 genes overexpression is remarkably improved transgenic arabidopsis Property.

As a result

Present invention discover that be remarkably improved transgenic arabidopsis anti-to infecting for sclerotinite for pineapple AcWRKY28 genes overexpression Property, which has highly important application value in Genes For Plant Tolerance sclerotinite genetic engineering.

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 should all belong to the covering scope of the present invention.

SEQUENCE LISTING

<110>University Of Agriculture and Forestry In Fujian

<120>Structure and the application of a kind of pineapple AcWRKY28 genes and its overexpression vector

<130> 7

<160> 7

<170> PatentIn version 3.3

<210> 1

<211> 1011

<212> DNA

<213>The nucleotide sequence of pineapple AcWRKY28

<400> 1

atgtcacaag agagaggaga gctctaccac caccaacatg gtcacctctt tctcaatgag 60

atgatcaata gtggtggcga cctctcgtgc ttcttctcgc accacgaggc gatgtcggtg 120

gccgacgcgg cgccgttcgc ggggttcgcc gactacgact acggcgctgc gctgtcgcgg 180

gccgccttcg gcgacgtgcg gtgcgacgag ctcgggctct tcgacgcccc gcccgcgaag 240

cccgaattgc tcatcgacgt cgacgctgcg attgactacg gcgctgttgt cggtcgacgt 300

cacggtttgg atgtttcgat aggggggggt ggtggtggtg gcgacggggg gaccggcccg 360

gtgacgccga attcgtcggt gtcgtcgtcg tcgtcggagg cgggcggaga cgaggagtcc 420

gggggacgga gcaagaagga tcggataaag gaggaagaag aggaaggtgg ggaggagaag 480

caactacaag ctagaggaag tgatgatcag ggaggtgaaa agtctaagaa actagtgaaa 540

gcaaaaaaca agggggagaa gaggcaaagg gaacctcgct tcgctttcat gacgaaaagc 600

gaggtcgacc atctggaaga cggatatcgg tggaggaagt atggccagaa ggcggtcaag 660

aatagccctt atccaaggag ctactatcgg tgcacgacgc agaagtgctc ggtgaagaag 720

agggtggagc ggtcgcacga ggacccgagc atcgtgatca cgacgtacga gggccggcac 780

acgcaccaga gccccgcgaa cctccgaggg agctcgcact tcctggcgcc gtcgccgagc 840

tttcgggccc aggagtttct taaccctagc agccaccacc acatgagcac aaaccctacc 900

atgtacctgc caattagcct accccctcct ctccagcagc tccaagtact ccctgactat 960

ggtctccttc aggacatcat cccctccttc attcaccaaa atcaaccatg a 1011

<210> 2

<211> 336

<212> PRT

<213>The amino acid sequence of pineapple AcWRKY28

<400> 2

Met Ser Gln Glu Arg Gly Glu Leu Tyr His His Gln His Gly His Leu

1 5 10 15

Phe Leu Asn Glu Met Ile Asn Ser Gly Gly Asp Leu Ser Cys Phe Phe

20 25 30

Ser His His Glu Ala Met Ser Val Ala Asp Ala Ala Pro Phe Ala Gly

35 40 45

Phe Ala Asp Tyr Asp Tyr Gly Ala Ala Leu Ser Arg Ala Ala Phe Gly

50 55 60

Asp Val Arg Cys Asp Glu Leu Gly Leu Phe Asp Ala Pro Pro Ala Lys

65 70 75 80

Pro Glu Leu Leu Ile Asp Val Asp Ala Ala Ile Asp Tyr Gly Ala Val

85 90 95

Val Gly Arg Arg His Gly Leu Asp Val Ser Ile Gly Gly Gly Gly Gly

100 105 110

Gly Gly Asp Gly Gly Thr Gly Pro Val Thr Pro Asn Ser Ser Val Ser

115 120 125

Ser Ser Ser Ser Glu Ala Gly Gly Asp Glu Glu Ser Gly Gly Arg Ser

130 135 140

Lys Lys Asp Arg Ile Lys Glu Glu Glu Glu Glu Gly Gly Glu Glu Lys

145 150 155 160

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

165 170 175

Lys Leu Val Lys Ala Lys Asn Lys Gly Glu Lys Arg Gln Arg Glu Pro

180 185 190

Arg Phe Ala Phe Met Thr Lys Ser Glu Val Asp His Leu Glu Asp Gly

195 200 205

Tyr Arg Trp Arg Lys Tyr Gly Gln Lys Ala Val Lys Asn Ser Pro Tyr

210 215 220

Pro Arg Ser Tyr Tyr Arg Cys Thr Thr Gln Lys Cys Ser Val Lys Lys

225 230 235 240

Arg Val Glu Arg Ser His Glu Asp Pro Ser Ile Val Ile Thr Thr Tyr

245 250 255

Glu Gly Arg His Thr His Gln Ser Pro Ala Asn Leu Arg Gly Ser Ser

260 265 270

His Phe Leu Ala Pro Ser Pro Ser Phe Arg Ala Gln Glu Phe Leu Asn

275 280 285

Pro Ser Ser His His His Met Ser Thr Asn Pro Thr Met Tyr Leu Pro

290 295 300

Ile Ser Leu Pro Pro Pro Leu Gln Gln Leu Gln Val Leu Pro Asp Tyr

305 310 315 320

Gly Leu Leu Gln Asp Ile Ile Pro Ser Phe Ile His Gln Asn Gln Pro

325 330 335

<210> 3

<211> 53

<212> DNA

<213>CaMV35S promoters

<400> 3

cgaccgcaag acccttcctc tatataagga agttcatttc atttggagag gac 53

<210> 4

<211> 25

<212> DNA

<213>Artificial sequence

<400> 4

caccatgtca caagagagag gagag 25

<210> 5

<211> 22

<212> DNA

<213>Artificial sequence

<400> 5

tcatggttga ttttggtgaa tg 22

<210> 6

<211> 25

<212> DNA

<213>Artificial sequence

<400> 6

caccatgtca caagagagag gagag 25

<210> 7

<211> 22

<212> DNA

<213>Artificial sequence

<400> 7

tcatggttga ttttggtgaa tg 22

Claims

A 1. pineapple AcWRKY28 gene, it is characterised in that：The gene nucleotide series are as shown in SEQ ID No.1.
2. the albumen of pineapple AcWRKY28 gene codes as described in claim 1, amino acid sequence such as SEQ ID No.2 institutes Show.
3. a kind of construction method of the overexpression vector of the gene as described in requiring right 1, it is characterised in that：By pineapple AcWRKY28 CDNA and CaMV35S the promoter core sequence of gene merge, and are built into the overexpression vector of pineapple AcWRKY28 genes.
4. a kind of application if any gene described in claim 1 in the anti-sclerotinite genetic engineering of arabidopsis.