CN104087596B - Fructus Lycii ERF transcription and encoding gene thereof and degeneration-resistant application - Google Patents

Abstract

The invention discloses a kind of Fructus Lycii ERF transcription and encoding gene thereof and degeneration-resistant application, the LcERF gene of coding Fructus Lycii ERF transcription, is the nucleotide sequence shown in SEQ ID NO.1.The present invention isolates the global cDNA of coding ERF transcription gene from Fructus Lycii, it is connected on plant expression vector, utilize Agrobacterium infestation method transformation of tobacco, obtain transfer-gen plant, transfer-gen plant has been carried out resistance analysis, result shows that ERF transcription can respond adverse circumstance signal, is effectively improved the Salt And Alkali Tolerance ability of plant.

Description

Fructus Lycii ERF transcription and encoding gene thereof and degeneration-resistant application

Technical field

The invention belongs to genetic engineering field, be specifically related to a kind of encode the LcERF gene of Fructus Lycii ERF transcription, this base Because of coding protein, include the degeneration-resistant application of the recombinant vector containing this gene and gene.

Background technology

The adverse circumstance of plant include abiotic stress (such as arid, saline and alkaline and low temperature etc.) and biotic stress (as fungus, antibacterial, virus and Disease, insect pest and the Weed infestation that nematicide etc. cause).The most arid, saline and alkaline and low temperature is to affect terrestrial plant growth and limit work The main abiotic stress factor of produce amount.Different from mobile animal, plant the most directly lives in complicated and changeable oneself So in environment.These adverse circumstances all can make plant be subject to physiological damage in various degree, even results in Plant death time serious.In order to Adapting to these stressful environmental, plant develops in very long evolutionary process and the resistance mechanism to these abiotic stress, such as: Pore closedown, cell membrane and the change etc. of cytoplasmic components.Plant is under the effect of biotic and abiotic stress, it is possible to pass through signal Pathway, makes the expression of some genes be regulated and controled by adverse circumstance, thus makes response to coercing.

It is generally believed that plant to the resistance of arid and the adverse circumstance such as high salt by controlled by multiple genes, therefore, utilize technique for gene engineering to lead Although entering individual feature gene can increase certain single resistance of plant, but can not the most comprehensively improve the resistance of plant. The regulation switch that transcription factor is expressed as functional gene, can regulate accurately different genes, believes in plant adverse circumstance Number transmittance process plays the effect of key, so by the effect strengthening certain transcription factor, can promote multiple and degeneration-resistant have The functional gene closed is expressed, and this is the very effective approach making plant stress-resistance character obtain comprehensive improvement.

AP2/ERF albuminoid is a class transcription factor specific to higher plant, ERF (ethylene-responsive element Binding factor) class transcription factor is a subfamily of AP2/ERF transcription factor family, each member containing one by greatly The very conservative DNA binding domain of about 58-59 aminoacid composition.From structural analysis of protein, ERF class transcription factor contains 4 functional domains, i.e. DNA binding domain (DNA-binding domain), transcriptional regulatory domain (transcription regulation Domain) (include activating and suppression territory), oligomerisation site (oligomerization) and nuclear localization signal (nuclear Localization signal, NLS).Wherein, the ERF/AP2DNA binding structural domain of high conservative contains by 58-59 amino Acid residue composition comprise 3 β-pleated sheets and 1 α spiral, by with genes of interest upstream promoter region in rich in GC's Cis acting element (such as DRE element, GCC-box etc.) interacts, and participates in regulation and control plant cell cycle, growth promoter, thin Born of the same parents' propagation, secondary metabolism, biology and the multi-signal approach such as abiotic stress response and Plant hormone signal conduction, coercing letter Number transmittance process plays an important role.ERF transcription can regulate and control multiple and plant resistant arid, high salt, low temperature and cause of disease The expression of the functional gene that bacterium etc. are relevant, strengthens the resistance of plant on the whole, improves its stability, changes for genetic engineering The resistance of reverse of good plant has huge potential using value, and will have huge application prospect.

Research shows, the expression of major part ERF gene is all inverse by arid, saline and alkaline, low temperature, freeze injury, disease and mechanical wounding etc. The induction that border is coerced.Fructus Lycii (Lycium chinense Mill) is the important plant of Solanaceae of one being grown in arid, semiarid zone, It is distributed widely in northwest China and other warm tropic countries, such as Korea S, Japan and some European countries.Except it is heavy Outside the medicinal and nutritive value wanted.Fructus Lycii also has huge adaptive capacity to environment, is a kind of important Salt tolerant plants kind. Therefore in Fructus Lycii, clone new ERF class transcription factor gene, study its basic biological characteristics and function, can be whole planting Thing adversity gene regulated and control network and stress response reaction mechanism are provided fundamental basis, and provide certain thing for Crop Improvement resistance Matter basis.

Summary of the invention

It is an object of the invention to provide a kind of LcERF gene encoding Fructus Lycii ERF transcription.

Second object of the present invention is to provide the protein of this gene code a kind of.

Third object of the present invention is to provide a kind of recombinant vector containing this gene.

Fourth object of the present invention is to provide a kind of host cell containing this recombinant vector.

Final object of the present invention is to provide the purposes of this gene.

Technical scheme is summarized as follows:

A kind of LcERF gene encoding Fructus Lycii ERF transcription, it is the nucleotide sequence shown in SEQ ID NO.1.

The protein of said gene coding, it is the aminoacid sequence shown in SEQ ID No.2.

A kind of recombinant vector pCAMBIA2300-LcERF containing said gene, it contains the nucleoside shown in SEQ ID NO.1 Acid sequence.

A kind of host cell containing recombinant vector pCAMBIA2300-LcERF.

Said gene is preparing the degeneration-resistant application of transgene tobacco.

Advantages of the present invention:

The present invention isolates the global cDNA of coding ERF transcription gene from Fructus Lycii, is connected on plant expression vector, Utilize Agrobacterium infestation method transformation of tobacco, it is thus achieved that transfer-gen plant, transfer-gen plant has been carried out resistance analysis, result table Bright ERF transcription can respond adverse circumstance signal, is effectively improved the Salt And Alkali Tolerance ability of plant.

Accompanying drawing explanation

Fig. 1. with P1, P2 primer PCR electrophoretogram to Fructus Lycii cDNA.

Fig. 2 .LcERF gene connects the PCR the result of pMD18-T carrier.

Fig. 3 .pMD18-T-LcERF carrier schematic diagram.

Fig. 4 .pCAMBIA2300-LcERF carrier schematic diagram.

Fig. 5. transgene tobacco Genomic PCR.

Fig. 6 under the growth conditions of 200mmol/L NaCl, transgene tobacco Seedling and wild type control Seedling figure.

Fig. 7 under the growth conditions of 300mmol/L NaCl, transgene tobacco Seedling and wild type control Seedling figure.

Detailed description of the invention

Below by specific embodiment, the present invention is further illustrated.

Specific experiment method involved in following embodiment if no special instructions, is conventional method or illustrates according to manufacturer The condition of book suggestion is implemented.

Embodiment 1

The clone of ERF transcription LcERF gene in Fructus Lycii

Utilize Trizol reagent, from the fresh Fructus Lycii blade of 100mg, extract totalRNA, according to the Unigene of transcript profile Sequential design forward primer, in Fructus Lycii, the forward primer of ERF transcription LcERF gene is: LcERFf: 5-' ATGTATCAACCCATTTCTACTGAGTTAGC-3 ' (SEQ ID NO.3), utilizes 3 ' FULLRACE Core The amplification of Set Ver.2.0 (TaKaRa, Japan) test kit obtains complete gene order.Concrete steps: 1. with totalRNA as mould Plate, utilizes 3 ' RACE Adaptor primers to carry out reverse transcription reaction, synthesizes 1st Strand cDNA, and reaction system is as follows:

Reaction condition: 42 DEG C, 60min；70 DEG C, 15min.

Downstream primer 3 ' the RACE out primer:5 ' provided with test kit further according to the forward primer (SEQ ID NO.3) of gene -TACCGTCGTTCCACTAGTGATTT-3 ' (SEQ ID NO.4), with 1 st Strand cDNA as template, is carried out PCR reacts, and reaction system is as follows:

Reaction condition is as follows: 94 DEG C, 4min；94 DEG C, 30Sec；56 DEG C, 30Sec；72 DEG C, 1min；72 DEG C, 10min； 30 circulations.Obtain encoding the cDNA fragment of the LcERF gene (SEQ ID NO.1) of Fructus Lycii ERF transcription, see Fig. 1.The protein (SEQ ID No.2) of the LcERF gene code of Fructus Lycii ERF transcription

Embodiment 2

Building process (the pMD of this process use TaKaRa of cloning vehicle pMD18-T-LcERF^TM18-T Vector Cloning Ki test kit)

Being connected with pMD18-T carrier by LcERF gene shown in sequence table SEQ ID NO.1, reaction system is as follows:

The cDNA fragment 4 μ L of LcERF gene

PMD18-T carrier 1 μ L

Solution I 5μL

Reaction condition: 16 DEG C, 30min.Connect product Transformed E-Coli.DH5 α competence, be coated on containing 40 μ l 25mg/ml X-Gal, 16 μ l50mg/ml IPTG, 100mg/L Amp LB Agar Plating on cultivate, form single bacterium colony. Select white colony, use bacterium colony PCR method to confirm the length scale of Insert Fragment in PMD18-T carrier, such as Fig. 2, and in advance Phase is consistent, and this carrier is sent to the order-checking of Hua Da genome company, and we have obtained this gene base sequence of 678bp, at NCBI Carry out blast, high with Solanaceae homology, it is shown to be this gene and clones successfully, Fig. 3 is shown in by pMD18-T-LcERF carrier schematic diagram.

Embodiment 3

The structure of binary plant expression vector pCAMBIA2300-LcERF

First with pMD18-T-LcERF plasmid as template, P3 (SEQ ID No.5) P4 (SEQ ID No.6) be respectively on Downstream primer, expands LcERF gene, and its reaction condition is: 94 DEG C, 4min；94 DEG C, 30Sec；56 DEG C, 30Sec； 72 DEG C, 1min；72 DEG C, 10min, 30 circulations, P3 introduces BamHI restriction enzyme site (GGATC), in P4 Introducing SalI restriction enzyme site (GTCGAC), then PCR primer and pCAMBIA2300 empty carrier plasmid use BamHI respectively With SalI double digestion, being connected by the digestion products of the two, linked system is as follows:

Connect product Transformed E-Coli.DH5 α, coat on the LB flat board containing 100mg/L concentration kana resistance.37 DEG C of cultivations, After 12h, picking list bacterium colony carries out bacterium colony PCR checking, by bacterium positive for bacterium colony PCR checking, shakes bacterium and extracts plasmid, and enzyme action is identified Obtaining purpose band, finally send the order-checking of Hua Da gene sequencing company, result is showing that carrier pCAMBIA2300-LcERF is just building Really, Fig. 4 is shown in by pCAMBIA2300-LcERF carrier schematic diagram.

Embodiment 4

Structure for the Agrobacterium engineering strain C58:pCAMBIA2300-LcERF of plant transgene.

Prepared by Agrobacterium competent cell

1. by mono-for Agrobacterium C58 colony inoculation in 5mLYEP fluid medium, 28 DEG C, 180r/min shaken cultivation

2. above-mentioned bacterium solution is proceeded in 100mLYEP fluid medium, 28 DEG C, 180r/min, shaken cultivation to (OD₆₀₀Value is about It is 0.5)

3., after ice bath 30min, 4 DEG C, 4000r/min is centrifuged 10min, collects thalline, is resuspended in the H of 20mL pre-cooling₂In O

4.4 DEG C, 4000r/min is centrifuged 10min, collects thalline, is resuspended in 10% glycerol of pre-cooling, often pipe 200 μ L quick-freezing, Be stored in-80 DEG C standby.

Plant expression vector electroporated

1. the C58 competent cell that-80 DEG C are taken out is placed on ice so that it is slowly melt；

2. add 2 μ L plasmids, mixing, ice bath 5min；

3. it is transferred to shock by electricity in cup；

4. electroporated instrument parameter is set: 1500V, 0.2s, electroporated；

5. room temperature stands and adds 500 μ LYEB fluid mediums after 2min, 28 DEG C, 180r/min shaken cultivation 3h；

6. take 50 μ L bacterium solution and coat on the YEB flat board containing 100mg/L kanamycin and 100mg/L rifampicin resistance, fall Horizontalization plate, 28 DEG C of cultivations, 48h, until seeing single bacterium colony clearly.

7. picking list bacterium colony, bacterium colony PCR verifies, obtains the Agrobacterium engineering strain C58 for plant transgene: pCAMBIA2300-LcERF。

Embodiment 5

Agriculture bacillus mediated Nicotiana tabacum L. genetic transformation

1. the aseptic culture of tobacco seedling: select full, healthy tobacco seed, soaks 1min with volumetric concentration 75% ethanol water, Peace tiformin (effective chlorine 2.5%) the aqueous solution sterilizing 8min of 25%, rinsed with sterile water three times, seed is placed on MS solid culture In base (pH=5.8), 25 DEG C of light are cultivated, the 16h/8h photoperiod.

The culture medium that this experiment is used is as follows:

2. the Agrobacterium-mediated Transformation of Nicotiana tabacum L.

(1) preparation of bacterium solution is infected

A. picking positive Agrobacterium list bacterium colony, is inoculated in the 5ml YEP fluid medium containing 100mg/L kanamycin, in 28 DEG C, the shaker overnight of 200r/min cultivates.

B. next day, take 3ml bacterium solution, be inoculated in the 50mlYEP fluid medium containing 100mg/L kanamycin, at bacterium solution In vigorous period (OD₆₀₀=0.6), into time, bacterium solution is poured 50ml centrifuge tube, at 3500r/min, centrifugal 10min at 4 DEG C, Abandon supernatant, collect thalline.Resuspended with the MS fluid medium (pH=5.8) of equivalent, make OD₆₀₀=0.9.

(2) outer implant is contaminated

A. tobacco leaf is removed master pulse and limb edge, then blade is cut into 0.5 × 0.5cm size, immerse the agriculture bar prepared Bacterium bacterium solution, soaks 15min, therebetween shake 3 times, makes blade be fully contacted bacterium solution, takes out blade, with aseptic filter paper exhaustion Unnecessary bacterium solution, blade face down, blade back be inoculated in upward in common training culture medium, about 25 DEG C light culture 2 days.

B. being transferred in screening culture medium by blade, within 20 days, change primary screening culture medium, induction of resistance bud produces, and works as resistant buds From callus, grow to about 1m, from wound healing, cut resistant buds, be inoculated in resistance seedling rooting culture medium.

3. resistance transplantation of seedlings

The resistance Seedling that in resistance seedling rooting culture medium, root growth is good, vitality is vigorous is taken out, rinses culture medium with tap water (reduce root system damage), is planted in frog stone that mass ratio is 3:1 and the compost that fertile soil mixes, cover film as far as possible At 25 DEG C, relative humidity 75%15 days, then open thin film, periodically water, apply fertilizer, be allowed to normal growth in greenhouse.

Embodiment 6

Transgene tobacco Molecular Detection and resistance detection

The PCR detection of transgene tobacco genomic DNA: 1.CTAB method extracts the Nicotiana tabacum L. STb gene that embodiment 6 obtains；2. with Genomic DNA is that template carries out PCR detection, and primer is P3, P4, reaction condition: 94 DEG C, 4min；94 DEG C, 30Sec； 56 DEG C, 30Sec；72 DEG C, 1min；72 DEG C, 10min；30 circulations.

Take above-mentioned PCR primer 5 μ l and carry out electrophoresis detection, Fig. 5, illustrate that LcERF gene the most successfully proceeds to Nicotiana tabacum L..

At greenhouse (25 DEG C) normal growth, 4 months big transgene tobacco Seedlings and wild type control Seedling, water respectively and contain The water of the NaCl of 200mmol/L and 300mmol/L concentration, irrigates, and continues under conditions of keeping the humidity of 75% in greenhouse After growing one month, monitoring finds, under the growth conditions of 200mmol/LNaCl, wild type control Seedling plant strain growth is the slowest (Fig. 6), Biomass is relatively fewer, and transgene tobacco can normal growth, Biomass is big, it is possible to blossom and bear fruit, 300mmol/L Under conditions of the NaCl of concentration, wild-type tobacco is little to normal growth.Although the growth of transgene tobacco is also by certain journey The suppression of degree, but obvious unlike wild-type tobacco, substantially can normal growth (Fig. 7).

Claims (5)

1. the LcERF gene encoding Fructus Lycii ERF transcription, it is characterised in that it is the nucleotide shown in SEQ ID NO.1 Sequence.

2. the protein of gene code described in claim 1, it is characterised in that it is the aminoacid sequence shown in SEQ ID No.2.

3. the recombinant vector pCAMBIA2300-LcERF containing gene described in claim 1, it is characterised in that it is containing having the right Require the nucleotide sequence shown in SEQ ID NO.1 described in 1.

4. the host cell containing claim 3 recombinant vector pCAMBIA2300-LcERF.

5. the gene described in claim 1 is improving the application of transgene tobacco Saline alkali tolerance.