CN106086024A - SlIP L gene photo effect promoter - Google Patents

Abstract

The invention provides a kind of isolated in Fructus Lycopersici esculenti, can be with the photo effect promoter of TOMV coat protein interaction albumen slIP L.The nucleotide sequence of SEQ ID NO.1 in this promoter tool sequence table, the function of this promoter is can to drive gene expression under the induction of different photoperiods, can play a significant role in the function aspects analyzing light controlling gene.The photo effect promoter of the slIP L that the present invention obtains can be carried out guiding gene by the regulation and control of different photoperiods and produce expression in various degree in plant, thus analyze and the function of light regulation and control related gene, significant to research Plant Light regulation and control related gene function aspects.

Description

SlIP-L gene photo effect promoter

Technical field

The present invention relates to the gene promoter of a kind of gene engineering technology field, the light efficiency of a kind of slIP-L Answer promoter.

Background technology

Fructus Lycopersici esculenti (Lycopersicon esculentum Mill.) is that annual production in the world is the highest, cultivates widest vegetables One of dish crop, is also the important object in the disciplinary study such as hereditism, thremmatology and biological engineering and test material, and it is being lost Biography is studied the most deep in theory, and its result of study is usually used for reference for other crops.It is wide in variety, purposes is wide, nutrition Abundant, extensively plant in various places, China north and south.

But in recent years, it is continuously increased the variation with planting form along with cultivated area, the tomato disease of China also day Benefit is serious, causes heavy losses to tomato production.The disease species of Fructus Lycopersici esculenti is various, occurs more serious disease just to have in the world Kind more than 40, the tomato virus disease that wherein TOMV (Tomato mosaic virus, ToMV) causes is to cause Fructus Lycopersici esculenti raw Produce one of important disease of the underproduction.This virus is one of member of Tobamovirus (Tobamovirus).Its host range Very wide, many plants such as Solanaceae, Cruciferae, grass family, Chenopodiaceae, pulse family can be infected, Fructus Lycopersici esculenti is its main host plants.

IP-L (Interaction Protein L) be filter out from Tobacco cDNA library with ToMV CP interaction Host's albumen, its sequence reaches with exciton response protein (AB040409) homology of a unknown function in common cigarette 100%.Zhang C Z etc. are (biological at " Biochemical and Biophysical Research Communications " Chemistry and biophysics communication) 2008 years volume 374 253-257 page delivered entitled " Characterization of a specific interaction between IP-L,a tobacco protein localized in the Thylakoid membranes, and Tomato mosaic virus coat protein " (analyze IP-L location and Function) paper.Article points out that IP-L is positioned at Thylakoid membrane, may participate in photosynthesis.We are from tomato dna Group cloned the homologous genes slIP-L (KP791801) of IP-L, this gene and Nicotiana tabacum L. IP-L nucleotide and amino acid whose with The essential amino acids site that source property is respectively in 99.8% and 99.4%, and Nicotiana tabacum L. IP-L with ToMV CP interaction does not all occur Sudden change.Show that slIP-L also interacts with ToMV CP.Fructus Lycopersici esculenti antiviral gene be mainly obtained by collecting in a large number Fructus Lycopersici esculenti Germ plasm resource carries out antiviral evaluation, then, determines that Cloning of blast resistance gene identifies antiviral gene, finally by dividing by biotechnology Sub-marker-assisted breeding obtains antiviral New Tomato Variety.The anti-ToMV gene having been found that in Fructus Lycopersici esculenti is mainly dominant disease-resistant Gene Tm-1, Tm-2 and Tm-22 (or Tm-2a), by the molecular marker label of these disease-resistant genes, have been obtained for preferably Anti-ToMV kind.But along with the continuous variation of cause of disease, these resistant genes obviously can not meet the demand of Anti-virus Disease Breeding, finds New disease-resistant related gene has become the key of Fructus Lycopersici esculenti Anti-virus Disease Breeding.Limited germ plasm resource is made by disease resistance evaluation Excavate the bottleneck of more multi-resistance ToMV gene.At present, by host's gene of screening with virus interaction, and carry out Function Identification and resist Characteristic of disease evaluation is the new way obtaining antiviral gene.Since slIP-L is and ToMV CP interaction, thereby increases and it is possible to participate in light cooperation With, the regulation of light.

At present, research gene overexpression and knock out the 35S that the promoter used by gene is all cauliflower mosaic virus and open Mover, although it can successfully start up the expression of gene in Fructus Lycopersici esculenti, but the regulation and control of not light, in the function of research light controlling gene Aspect is restricted.Fructus Lycopersici esculenti is currently also found that the most polygenic promoter, but majority is organizing specific expression promoter, or By hormone and thermoinducible promoter, not yet there is the report of light regulation and control promoter.Therefore the promoter of slIP-L is cloned to grinding Study carefully Plant Light regulation and control related gene function aspects significant.

Summary of the invention

It is an object of the invention to overcome the deficiency of the Fructus Lycopersici esculenti promoter being currently known, it is provided that can be by the different photoperiods Regulation and control carry out guiding gene in plant, produce expression in various degree, thus analyze and light regulation and control related gene function SlIP-L gene promoter.

The technical scheme is that a kind of slIP-L gene promoter, the sequence of this promoter is SEQ ID:1.This opens Mover derives from Fructus Lycopersici esculenti, is regulated and controled by the photoperiod.

The preparation method of this slIP-L gene promoter:

Step one, cultivates tomato seedling to the 4-6 leaf phase；

Step 2, uses CTAB method to extract Fructus Lycopersici esculenti STb gene；

Step 3, TAIL-PCR method cloning promoter genome sequence；

Step 4, analyzes the cis acting element in promoter, determines the type of slIP-L gene promoter；

Step 5, the vector construction of slIP-L gene promoter；

Step 6, the plant expression vector photoinduction of Agrobacterium tumefaciens mediated slIP-L gene promoter gus gene fusion Transient expression gus on Ben Shi cigarette；

This preparation method also includes: step 7, the GUS determination of activity in Ben Shi cigarette of the slIP-L gene promoter and dyeing Detection.

This promoter is being analyzed and the application in light regulation and control related gene function.

The Agrobacterium tumefaciems EHA105 that the present invention relates to is at " Huang Yali, Jiang Xiliang, Yunlong, field, Guo Ping, Zhu Changxiong；Root The research of the trichoderma harzianum genetic transformation that cancer is agriculture bacillus mediated, Chinese biological engineering magazine, 2008,28 (3): 38-43 " document Disclosed in.Agrobacterium tumefaciems EHA105 can obtain by disclosing commercially available commercial channel, as can be public from Australia CAMBIA Department buys, and strain number is Gambar1.

Beneficial effects of the present invention:

The present invention clones the slIP-L gene promoter obtained, and can start and high efficient expression external source base with the induction of light Cause, can apply to analyze and in light regulation and control related gene function.

Accompanying drawing explanation

Fig. 1 nested PCR amplification IP-L gene promoter sequence, M:Marker V；1: nest-type PRC second takes turns product；2: nest Formula PCR first round product.

Fig. 2 PCR detects promoter sequence, M:Marker V；1: do not add template PCR system；2: containing template PCR system.

Fig. 3 promoter region cis acting element.

The GUS that under the conditions of Fig. 4 photoinduction, slIP-L gene promoter starts expresses.

The sequence chart of the promoter of Fig. 5 present invention.

Detailed description of the invention

1. potted plant sowing tomato seeds in greenhouse, cultivated to the 4-6 leaf phase；

2. the extraction (improvement CTAB method) of tobacco gene group DNA: take 1g fresh tomato blade and grind rapidly in liquid nitrogen Become powder, with the spoon of pre-cooling, powder is transferred quickly in the 50mL centrifuge tube of pre-cooling, be subsequently adding the CTAB of 10mL preheating Buffer, is simultaneously introduced mercaptoethanol extremely final concentration of 1% (v/v), jog incubation 10-15min in 65 DEG C of water-baths；Add The chloroform of volume: isoamyl alcohol (24:1, v/v), reverse mixing under room temperature；15-20 DEG C, 6000rpm is centrifuged 25-30min, with cutting Supernatant is transferred in new centrifuge tube by the Tip head of tip；Add the isoamyl alcohol of at least 2/3 volume, be simultaneously introduced NaAc to end Concentration is 0.1M, rotates mixing ,-20 DEG C of precipitation 1-2h gently；Go out bulk DNA with Tip choicest and be transferred to 1.5mL centrifuge tube In, 6000rpm is centrifuged 10min, removes residual liquid；With 70% ethanol (900 μ L) and the 0.1MKAc/HAc buffer of pre-cooling (100 μ L) washs 2 times, and 6000rpm is centrifuged 10min and removes residual liquid；By soaked in absolute ethyl alcohol DNA5min of pre-cooling, 12000rpm is centrifuged 5-10min, removes supernatant, and 37 DEG C are dried；Dissolve the DNA being dried with ddH2O, add RNase to final concentration 20 μ g/mL, 37 DEG C are incubated overnight；DNA is transferred in 15mL centrifuge tube, adds 2mL TE, 1mL 10M NH4Ac and 8mL pre- Cold dehydrated alcohol, mixes gently；Go out bulk DNA with Tip choicest and be transferred in 1.5mL centrifuge tube, by the anhydrous second of pre-cooling Alcohol soaks, and 12000rpm is centrifuged 5min, removes surplus liquid, 37 DEG C of dry 5-10min as far as possible, uses ddH₂O dissolves dry again Dry DNA；Measuring DNA concentration with ultraviolet spectrophotometer, the agarose gel electrophoresis with 1% detects DNA mass.

Primer used by table 1 the present embodiment

3., with the Strategies For The Cloning listed by table 2, first prepared tomato dna group DNA is carried out single endonuclease digestion with several groups of enzymes, Then the fragment purification selecting 2000-3000bp reclaims.Reclaim post-fragment to connect to through the corresponding carrier T limiting cleavage, with Connecting product is that template carries out nest-type PRC.As it is shown in figure 1, only using in Sac I enzyme action the product that connects amplification to about 1500bp fragment, does template by PCR primer after dilution and carries out second and take turns nest-type PRC, and result has been got back the most special product (Fig. 1).Enter in pMD 18-T carrier to check order by this fragment purification recovery rear clone.Obtain after sequencing result is spliced The fragment of slIP-L upstream 955bp.For verifying the reliability of this result further, devise primer according to sequence after splicing SlIP-LP-F, and with tomato dna group DNA as template, slIP-LP-F and slIP-L-R carry out PCR for primer.As in figure 2 it is shown, In containing the system that tomato dna group DNA is template, Successful amplification has arrived the specific band of about 1400bp, and this fragment is surveyed Sequence shows the full length sequence containing slIP-L.It is not added with in the comparison of template then having no any band, shows the 955bp expanded Fragment is IP-L upstream region of gene promoter sequence really.

Table 2 IP-L promoter Strategies For The Cloning

4.IP-L promoter bioinformatic analysis

Utilize molecular biology software and online software (http://bioinformatics.psb.ugent.be/ Webtools/plantcare/htmL/) the slIP-L promoter sequence obtained is carried out preliminary bioinformatic analysis.Only With promoter sequence analysis, multiple cis acting element is contained in application PlantCARE instrument on-line prediction slIP-L promoter region, Including multiple core promoter element CAAT-box and TATA-box.Additionally this promoter is different types of possibly together with 8 Light response element, such as: the Box 4 in normal chain, L-box；The ATCT-motif of minus strand, G-box, GA-motif, GT1-motif, MRE, chs-Unit 1m1 etc. (table 3).With slIP-L transcriptional start site for+1, its cis acting unit in promoter normal chain Part position is as shown in Figure 3.

The promoter region cis acting element of PlantCARE on-line prediction applied by table 3

The vector construction of 5.slIP-L gene promoter: with the primer introducing restriction enzyme site listed by table 1 with aforementioned acquisition SlIP-L gene promoter be that template carries out PCR, amplified production, after Pst I and BamH I enzyme action, is connected to through same enzyme On double p BI121 carriers cut, replace 35S promoter.Successfully it is built into the plant of slIP-L gene promoter gus gene fusion Expression vector.

6. will proceed to Agrobacterium tumefaciems containing the plant binary expression vector of slIP-L gene promoter and gus gene fusion, Performing PCR of going forward side by side is verified.Result shows, the most successfully imports in Agrobacterium tumefaciens strain containing plant binary expression vector.

7. by the Agrobacterium tumefaciems 5 μ L containing slIP-L gene promoter and the plant binary expression vector of gus gene fusion Bacterium solution adds 5mL and contains in kanamycin and streptomycin YEP culture medium, 225rpm/min, 30 DEG C, cultivates 12-16h；

From 5mL bacterium solution, take 1mL add the acetosyringone 5 μ L that concentration is 20 μMs, join 25mL and contain kanamycin With 225rpm/min in streptomycin YEP culture medium, cultivate 12-16h for 30 DEG C；

Taking 2mL from 25mL Agrobacterium bacterium solution to join in 50mL centrifuge tube, room temperature 5000rpm/min is centrifuged 15min, abandons Supernatant；

By resuspended with newly configured buffer for precipitation and regulate OD₆₀₀Value is to 0.6, and room temperature stands 3h；

Take and cultivate the Ben Shi cigarette to 5-6 leaf phase robust growth, spile at vacuum side of blade syringe needle, with removing syringe needle 2mL needle tubing is drawn agrobacterium suspension and is expelled in blade, and each injection point injects 30 μ L OD₆₀₀It it is the bacteria suspension of 0.6.Illumination Under the conditions of induce GUS express.

8. with GUS dye solution by X-gluc dyeing liquor be 50 × concentrated solution dilute 50 times, be made into GUS dyeing liquor.Will The Ben Shi Tobacco Leaves of abduction delivering GUS is immersed in GUS dyeing liquor, is incubated 1 hour in 25-37 DEG C.Proceed in 70% ethanol Decolour 2-3 time, white to negative control material.Naked eyes or basis of microscopic observation GUS expression.As shown in Figure 4, in photo-induction It can be seen that blue gus protein in Ben Shi cigarette under the conditions of leading, and under dark condition, GUS can not successful expression, have no blue.

Above one embodiment of the present of invention is described in detail, but described content has been only the preferable enforcement of the present invention Example, it is impossible to be considered the practical range for limiting the present invention.All impartial changes made according to the present patent application scope and improvement Deng, within all should still belonging to the patent covering scope of the present invention.

Claims (5)

1. a slIP-L gene promoter, it is characterised in that the sequence of this promoter is SEQ ID:1.

SlIP-L gene promoter the most according to claim 1, it is characterised in that this promoter derives from Fructus Lycopersici esculenti, light The regulation and control in cycle.

3. the preparation method of the slIP-L gene promoter described in claim 1: it is characterized in that, comprise the following steps:

(1) tomato seedling is cultivated to the 4-6 leaf phase；(2) CTAB method is used to extract Fructus Lycopersici esculenti STb gene；(3) TAIL-PCR method cloning promoter Genome sequence；(4) analyze the cis acting element in promoter, determine the type of slIP-L gene promoter；(5)slIP-L The vector construction of gene promoter；(6) plant of Agrobacterium tumefaciens mediated slIP-L gene promoter gus gene fusion is expressed and carries Body photoinduction is transient expression gus on Ben Shi cigarette.

4. according to the preparation method belonging to claim 3, it is characterised in that: also include, step (7), slIP-L gene promoter GUS determination of activity in Ben Shi cigarette and dyeing detection.

5. the slIP-L gene promoter described in claim 1 or 3 is being analyzed and the application in light regulation and control related gene function.