CN102604948B - Separation and application of salicylic acid-induced citrus sinensis osbeck promoter GSTU19P - Google Patents

Abstract

The invention belongs to the technical field of plant genetic engineering. The invention specifically relates to separation and identification of a promoter. A salicylic acid-induced promoter GSTU19P can be separated from GSTU19 genes which are separated from citrus sinensis osbeck, the nucleotide sequence of the promoter is as shown in SEQ ID NO: 1 (sequence identity number: 1), the length of the nucleotide sequence is 249bp, and the promoter has obvious salicylic acid induced activity. The induced promoter GSTU19P can be used for plant stress resistance or secondary metabolism and other physiological processes.

Description

Separation and the application of the Cara Cara promotor GSTU19P of an Induced by Salicylic Acid

Technical field

The invention belongs to the plant gene engineering technology field.Be specifically related to Whitfield's ointment (SA) inducible promoter in the Cara Cara, the clone of GSTU19 gene promoter and space expression.This promotor be separated into agriculture production and biological study provides a controlled expression regulation element.Described inducible promoter plant opposing coerce with physiological process such as secondary metabolism in bringing into play important effect.

Background technology

Agricultural is the basic industry of a country, and under the ever-increasing situation of world population, it is particularly important that supply of food becomes.The traditional agriculture volume increase has so not only increased the cost of agricultural-food mainly by fertilising and applying pesticides weeding deinsectization, returns environment and brings very big burden.The improvement of crops quality now more and more depends on the Protocols in Molecular Biology of continuous development.Many genetic resourceses have all become the important weapon of transgenic technology improvement crop varieties, but these genes are its expression of promoters driven of using composing type in transfer-gen plant more, this phraseology is owing to run counter to the natural physiological activity rule of plant, not only do not reach genetically modified intended purposes, even may cause very big injury to the physiological activity of transgenic plant self.As the overexpression (Li of disease-resistant gene Pto in tomato, et al.2002) overexpression (Blanco of disease-resistant related gene NPR1 gene and in the Arabidopis thaliana, et al.2005) all the growths of transgenic plant is caused side effect, phenomenons such as cape horn fever spot and plant be short and small occurred.Based on transgenosis to the influence of plant-growth and the consideration of security, people wish in the genetically modified while enough safety control to be arranged, so that utilize induction type and do not drive expression of exogenous gene in edible position expression promoter and become optimal selection.

Gene is successfully transcribed, and needs series of factors such as promotor, RNA polymerase, transcription factor to participate in together.TATA in the eukaryote and CAAT box, they have determined initiation site and the efficient of transcribing, and are responsible for and the RNA polymerase combination, start the basic transcription process.And other driving gene is called as cis-acting elements at the element of specific time and space expression.The structure that drives specific expressed cis element can't provide a unified structure owing to the difference of function has nothing in common with each other, and the structure of these elements may have the characteristics of himself, and they have played conclusive effect to gene specific expressed.The existence of these specificity elements has brought certain degree of difficulty for the separation of specificity promoter, but these functional zone have determined the specificity of promotor just.

Thiadiazolidine isomerase (GSTs) is a class soluble proteins that extensively exists in the organism, and they are by a huge gene family coding.Gene sequence characteristic according to them can be divided into seven big class: Phi, Tau, Theta, Zeta, Lambda, DHAR (L-dehydroascorbic acid) and TCHQD (tetrachloro-p-phenylene's diphenol dehalogenase) to GSTs.Wherein Tau class GSTs (GSTU) is plant specific, and opposing coerce with physiological processs such as secondary metabolism in bringing into play important effect.Forefathers discover that GST is induced by multiple factor, and Whitfield's ointment is exactly one of them.(salicylic acid SA) is the lower a kind of endogenous phenols micromolecular compound (Raskin 1992) of plant materials intensive amount to Whitfield's ointment, and the content in the monocotyledons body is than the height (Raskin, et al.1990) of dicotyledons.SA has the important physical effect in plant, comprise influence stomatal movement, seed germination, fruit yield, ionic absorption, heat production, bloom, the biosynthesizing of sexual differentiation and ethene suppressing etc., thereby SA and its esters are considered to the plant hormone that a class has significant application value.SA can induce various plants that virus, fungi and Micobial Disease are produced resistance (Fan Zhijin, et al.2004).SA brings out systemic acquired resistance (systemic acquired resistance, SAR) one of signaling molecule (Yalpani andRaskin 1993), relate to also anaphylaxis (the hypersensitive reaction of involved in plant, HR) and SAR reaction, in the SAR of plant signal transduction, play keying action.SA directly or indirectly regulates and control Expression of Related Genes in the plant materials growth course as plant hormone and signaling molecule.

From above result of study as can be known the GSTs gene opposing coerce with physiological process such as secondary metabolism bringing into play important effect, SA can induce that the GSTs gene is early stage to be started and express, and has isolated the cis-acting elements of the several SA of being subjected to indirect adjustments and controlss at present.Whitfield's ointment is the small molecule material in the plant materials simultaneously, is considered to a kind of new plant hormone.Consider that from the biological safety angle this type of promotor is used to having very promising prospects of land for growing field crops use.The present invention is material with the Cara Cara, with Whitfield's ointment blade is handled, and the GSTU19 gene of separating clone up-regulated expression adopts the method for chromosome walking to separate its promotor.Scan by the promoter element analysis software, determine the relevant controlling element of abduction delivering in this promoter sequence, and then make up 3 deletion sequence expression vectors of this promotor, adopt agriculture bacillus mediated vacuum infiltration method, protoplastis transient expression to carry out functional verification, draw the GSTU19 gene promoter and contain the Induced by Salicylic Acid related elements at-700bp to-300bp sequence, its promotor is the inducible promoter that SA induces.

Summary of the invention

The object of the invention is isolated the inducible promoter that is subjected to Whitfield's ointment (SA) induction regulating controlling exactly from Cara Cara.Thereby further the resistance for the breed improvement of crop, raising plant provides the genetic resources of usefulness.Behind bigcatkin willow acid treatment Cara Cara blade, GSTU19 gene up-regulated expression is remarkable, the promotor (GSTU19P) of separating this gene then, by the carrier construction arabidopsis thaliana transformation, the Arabidopis thaliana protoplastis can both be induced its expression, so this promotor might obtain important use the land for growing field crops farm crop.

The present invention is achieved through the following technical solutions:

The applicant separates the promotor that obtains the GSTU19 gene of up-regulated after the Induced by Salicylic Acid by gene clone technology from navel orange, its nucleotide sequence is shown in sequence table SEQ NO:1, and sequence length is 2072bp.The applicant is with this promotor called after GSTU19P.

Promotor GSTU19P of the present invention is expressed by Induced by Salicylic Acid.Cara Cara GSTU19 gene is subjected to salicylic abduction delivering, therefore use the method for chromosome walking to clone the promotor GSTU19P of GSTU19 gene, find to exist the multiple element relevant with Induced by Salicylic Acid in the GSTU19P promotor by bioinformatic analysis, as by the W-box of WRKY transcription factor institute combination, the ACGT element that can be identified by the bZIP proteinoid and ARR1 binding member etc.Transient expression and the Induced by Salicylic Acid of this promotor in the Arabidopis thaliana protoplastis handled experimental result and also shown, no matter the GSTU19P promotor is to drive GFP or the expression of GUS is influenced by Induced by Salicylic Acid all.Show that GSTU19P of the present invention is the salicylic acid inducible promotor.

The key step that obtains promotor shown in the present is as follows:

(1) gradient of Whitfield's ointment (SA) concentration for the treatment of is set, selects suitable SA concentration to handle the Cara Cara blade.

(2) by quantitative fluorescent PCR screening up-regulated gene.

(3) utilize the chromosome walking technology to clone the nucleotide sequence of GSTU19P promotor.

(2) utilize bioinformatics method to analyze this promoter structure.

(4) make up GSTU19P expression vector and arabidopsis thaliana transformation.

(5) transgenic arabidopsis is carried out resistance screening and transplanting.

(6) positive plant is carried out the active detection of GUS.

(7) protoplastis to Arabidopis thaliana carries out the transient expression conversion.

The present invention clone's GSTU19P promotor may further be the product genetic improvement of plant, and the resistance that improves plant provides the genetic resources of usefulness.Promotor application of the present invention also comprises by salicylic response regulation and control Cara Cara is resisted coerces or secondary metabolism.

More detailed technological invention details is provided by following examples, but is not the protection domain that limits this invention:

Description of drawings

Sequence table SEQ ID NO:1 is the nucleotide sequence of the GSTU19P promotor of separating clone of the present invention.

Fig. 1: technological line figure of the present invention.

Fig. 2: the different concns Whitfield's ointment is handled the form (handling back 7 days situation) of Cara Cara blade.Among the figure: CK, be contrast, namely pure water is handled; 1 is 10mmol/L SA processing; 2 are 5mmol/L SA processing.

Fig. 3: real-time quantitative PCR detects SA and handles back GSTU19 expression conditions.Among the figure: blue expression water treatment group sample; Purple is represented 5mmol/LSA treatment group sample; The time point that numeral is sampled after representing respectively to handle again.

Fig. 4: adopt PLACE software (public use software) to analyze the GSTU19P structure.

Fig. 5: make up deletion fragment checking GSTU19P sequence deletion fragment synoptic diagram.

Fig. 6: the structure synoptic diagram of expression vector pCAMBIA1391+GSTU19P of the present invention and pCAMBIA1301+GSTU19P carrier.

Fig. 7: the structure synoptic diagram of expression vector pCAMBIA1302+GSTU19P carrier of the present invention.

Fig. 8: the promoter deletion carrier connects GFP arabidopsis thaliana transformation protoplastis and detects promotor response SA activity.

Fig. 9: the promoter deletion carrier connects GUS arabidopsis thaliana transformation protoplastis and detects promotor response SA activity.

Embodiment

Selection and the separation of embodiment 1 Induced by Salicylic Acid candidate gene

Present embodiment use the Whitfield's ointment of 0mmol/L, 1mmol/L, 5mmol/L, 4 concentration of 10mmol/L be processed into the term Cara Cara (kind for " early red ", from Hua Zhong Agriculture University oranges and tangerines plantation resource garden, this kind has been awarded Chinese Plants kind power, kind power number be CN20060194.6, authorizes day: young sprout blade on May 1st, 2008).Per two strains are a repetition.Treatment time is at 8 o'clock in the morning.Observe every day, and week back observation blade situation has been determined 5mmol/L SA for handling the concentration (see figure 2) of Cara Cara tree body.

Be keyword search citrus est database (HarvEST:Citrus ver.0.51) with GSTU, obtain 5 close sequences, these form a unigene by DNAstar (common software), utilize this sequence to design primers with Primer Premier 5.0, expand with the RT-PCR method total length.5mmol/L SA handles Cara Cara blade extracting RNA and reverse transcription, and the first chain cDNA of gained is used for amplification GSTU19 full length gene.The RNA extracting uses the Trizol test kit (available from Invitrogen company, operate according to the process specifications that this test kit provides), utilize the total RNA sample of 3 μ g of extracting through the DNaseI of 1U (Amplification Grade, available from Invitrogen company) room temperature treatment is after 15 minutes, add 1 μ l EDTA (25mM), in 65 ℃ of incubations 10 minutes.The first chain cDNA's is synthetic with MBI reverse transcription test kit (article No.: K1621 is available from Fermentas company, according to the operation of test kit specification sheets).Amplification gene GSTU19 primer is to being: Forward, 5 '-TTCTGTCACAATGGCGGACG-3 '; Reverse primer: 5 '-GGAATAAGCAGGCAGCACGA-3 '.Comprise 100ng cDNA in the reaction system of 25 μ l, 1 * damping fluid, 2.5mM MgCl2,0.25mM dNTP, (aforementioned damping fluid and Taq polysaccharase Lithuania) add the above-mentioned primer of 0.5 μ M available from Fermentas company to 0.5U Taq polysaccharase.PCR is reflected on ABI 9700 (Applied Biosystem) the amplification instrument and finishes by following program: 94 ℃, and 5 minutes, 94 ℃ of sex change 30 seconds, 60 ℃ of annealing 50 seconds, 72 ℃ were extended 35 circulations 90 seconds; 72 ℃ were extended 15 minutes after circulation was finished.Produce a single PCR band product, behind 1% agarose gel electrophoresis, use

Dna gel reclaims test kit (available from Omega company, the U.S.) and reclaims special band, and extraction step is consulted and used explanation.The dna solution and the pMD18-T carrier (company limited is TaKaRa company available from precious biotechnology Dalian) that reclaim purifying carry out ligation, the by specification operation, the mol ratio of inserting GSTU19 gene and pMD18-T carrier in the ligation system is that 3: 1 ligation cumulative volumes are 10 μ l, 2 * damping fluid (available from precious biotechnology Dalian company limited) comprising 5 μ l, 4.5 the PCR product of μ l purifying, 0.5 μ l T carrier.16 ℃ of connections of spending the night.Get 10 μ l and connect product, adopt thermal shock method (Sambrook, et al.2002) transformed into escherichia coli DH5 α, screening positive clone in the LB solid plate that contains 50mg/L ammonia benzyl mycin, 5 cloning and sequencings of picking (genome company finishes by the Shanghai associating), sequencing result shows that it is 1060bp (seeing shown in the SEQ ID NO:2) that the present invention clones the GSTU19 full length gene.For whether analyzing the GSTU19 gene to salicylic processing response, adopt real-time quantitative PCR to analyze the expression of this gene under the Whitfield's ointment different treatment.The result shows that the GSTU19 expression amount is fast rise in 4 hours but, and peaks in the time of 8 hours, is (Fig. 3) more than 9 times of original state, shows that it is a Whitfield's ointment answer candidate gene.

Clone and the bioinformatic analysis of embodiment 2GSTU19 promotor

Adopt the chromosome walking method to obtain promotor (the GenomeWalkerTM Universal Kit of GSTU19 gene, Clontech, USA), GSTU19cDNA sequence according to oranges and tangerines in the HarvEST-citrus database, be two antisense primer: GSP1 of stencil design and GSP2 with it, two sense primer AP1 and AP2 (seeing Table 2) are a pair of primer with GSP1 and AP1, according to the reaction conditions of table 1, carry out first round PCR reaction.The PCR system is: 2.5 μ l, 10 * LA Taq Buffer, and 0.5 μ l 10mM dNTP, 0.5 μ l, 10 μ M GSP1 and AP1 primer, 1U LA Taq enzyme, 0.5 μ l DNA adds deionized water and mends to 25 μ l.For increasing the specificity of reaction, the PCR product of the employing first round is template after diluting 50 times, is that primer is right with GSP2 and AP2, adopts nest-type PRC to carry out second amplification of taking turns.The PCR reaction system is: 2.5 μ l, 10 * LA Taq Buffer, and 0.5 μ l 10mM dNTP, 0.5 μ l, 10 μ M GSP2 and AP2 primer, 1U LA Taq enzyme, 0.5 μ l DNA adds deionized water and mends to 25 μ l.

Table 1PCR reaction conditions

After PCR finishes, utilize 1% agarose gel electrophoresis detection PCR product, utilize

Dna gel reclaims test kit (available from Omega company, the U.S.) and reclaims special band, and extraction step is consulted and used explanation.The dna solution and the pMD18-T carrier (company limited is TaKaRa company available from precious biotechnology Dalian) that reclaim purifying carry out ligation, the by specification operation, the mol ratio that reclaims fragment and pMD18-T carrier in the ligation system is that 3: 1 ligation cumulative volumes are 10 μ l, 2 * damping fluid (available from precious biotechnology Dalian company limited) comprising 5 μ l, 4.5 the PCR product of μ l purifying, 0.5 μ l T carrier.16 ℃ of connections of spending the night.Get 10 μ l and connect product, adopt thermal shock method (Sambrook, et al.2002) transformed into escherichia coli DH5 α, screening positive clone in the LB solid plate that contains 50mg/L ammonia benzyl mycin, 5 cloning and sequencings of picking (genome company finishes by the Shanghai associating).It is 2072bp (seeing shown in the SEQ ID NO:1) that the present invention clones the GSTU19P full length sequence.Use the online software (Higo of PLACE (http://www.dna.affrc.go.jp/PLACE/signalscan.html), et al.1998) sequence of the 2072bp of analysis GSTU19P, find to have many TATA-box and CAAT-box element near 3 ' end, and in-200bp, three GATA-box (see figure 4)s are arranged.In-300bp, there is class inducibility element a---TGAC.TGAC is the core parts (see figure 4) of W-box, and W-box is the binding site of WRKY transcription factor institute specific recognition, is present in the promotor of many stress-inducing genes and pathogenesis-related proteins (PR) (Xie, et al.2005).Also having ocs element (TGACG), is to be present in many GST promotors and relevant with the Induced by Salicylic Acid element (Chen andSingh 1999) of inducing.Distal promoter beyond-1000bp also exists many epigamic promoter element (see figure 4)s, such as ARR1-bindingelement, GTAC (GTAC is the core sequence of CuRE (copper response element)) (Quinn, et al.2002), TTATCC (a kind of sugared response element (SRE)) (Tatematsu, et al.2005), also have the recognition site (see figure 4) of many MYB proteinoids.

Embodiment 3 utilizes the GSTU19P promotor that Arabidopis thaliana is carried out genetic transformation

1) vector construction: in order to verify the space expression of GSTU19P, multiple clone site and GSTU19P sequence according to pCAMBIA1391 carrier (buying from Australian CAMBIA laboratory), going out 3 different carriers (Fig. 5 and Fig. 6) of clip size of amplification design according to the principle of general design primer with Primer Premier 5.0 software designs is respectively 329bp (being numbered Del 1), 702bp (being numbered Del 2), 1856bp (being numbered Full-length).The primer of Del1 carrier construction is F1 and R1, and the primer of Del2 carrier construction is F2 and R1, and the primer of Full-length carrier construction is F3 and R1 (seeing Table 2).DNA with Cara Cara (kind is " early red ") is that template is carried out pcr amplification, and the extracting method of DNA is according to Cheng Yun river reported method (Cheng, et al.2003).The annealing temperature of pcr amplification is 60 ℃.The PCR reaction conditions is: 95 ℃, and 5min; 95 ℃, 30s; 56 ℃, 30s; 72 ℃, 90s; 72 ℃, 10min.35 circulations.The double digestion system: the reaction cumulative volume is 20 μ l, wherein contains the purified product 10 μ l of PCR, 10 * G damping fluid (available from MBI company), 2 μ l, each 1 μ l of EcoR I and Sal1, distilled water 6 μ l.Cutting the back purifying that spends the night at 37 ℃ of enzymes reclaims.The double digestion system of pCAMBIA1391 carrier: the reaction cumulative volume is 20 μ l, wherein contain through plasmid and extract the pCAMBIA1391 carrier DNA 8 μ l that obtain, 10 * G damping fluid (available from MBI company), 2 μ l, each 1 μ l of EcoR I and Sal1 adds distilled water 8 μ l.Cutting the back purifying that spends the night in 37 ℃ of enzymes reclaims.The different fragments of insertion GSTU19P and the mol ratio of carrier pCAMBIA1391 are 3: 1 in the ligation system, the reaction cumulative volume is 10 μ l, wherein contain 10 * Buffer, 1 μ l, T4DNA ligase enzyme 1 μ l, the double digestion of GSTU19P reclaims product 4 μ l, the double digestion of pCAMBIA1391 carrier reclaims product 2 μ l, distilled water 2 μ l.16 ℃ of reactions 14-16 hour.Connect product transformed into escherichia coli bacterial strain DH5 α, screening positive clone in containing the LB solid plate of 50mg/L kantlex, the extracting plasmid carries out that enzyme is cut and PCR identifies, not sudden change is determined in order-checking, acquisition contains the recombinant clone that inserts the purpose fragment, with its called after pCAMBIA1391-Full-length, pCAMBIA1391-Del1, the pCAMBIA1391-Del2 recombinant vectors, (Sambrook etal.2002) imports to recombinant vectors pCAMBIA1391 series among the Agrobacterium EHA105 to use freeze-thaw method.

2) conversion of Arabidopis thaliana: adopt inflorescence infestation method (Zhang, et al.2006) arabidopsis thaliana transformation: treat that the Arabidopis thaliana plant grows to when more inflorescence appears in 8～15cm and can infect.(Agrobacterium pCAMBIA1391-Del2) (EH105) is inoculated in the 3mL liquid LB substratum for pCAMBIA1391-Full-length, pCAMBIA1391-Del1, and 28 ℃, 250r/min shakes bacterium 24h will to have the purpose fragment.Inoculation 1mL incubated overnight liquid contains in the 100mL liquid LB substratum to 250mL, and 28 ℃, 250r/min shakes bacterium, until logarithmic phase OD600=0.8～1.6.Cultivate bacterium liquid branch and be filled in the 50mL centrifuge tube 4000r/min, the centrifugal collection thalline of 15min.Add the resuspended thalline of 5% sucrose, suction is beaten thalline is dispersed into individual cells repeatedly gently, adds 0.05%Sillwet L-77 before transforming.The Arabidopis thaliana inflorescence is dipped in 30sec～2min in the bacterium liquid that contains Agrobacterium, and the Arabidopis thaliana after infecting changes normal growth over to after secretly cultivating 24h.Treat can gather behind the Arabidopis thaliana seed maturity, the seed of results is placed in the 2mL centrifuge tube in batches, 4 ℃ of preservations.Heating for dissolving solid 1/2MS substratum at first treats that the substratum temperature is down to below 60 ℃, adds 50mg/mill Pyocianil and 50mg/mL hygromycin B, and making final concentration is 75mg/L Pyocianil and 75mg/L hygromycin B.Be layered on behind the mixing on the plate, blow more than half an hour at Bechtop.With 70% alcohol-pickled Arabidopis thaliana seed 1min, use 2% chlorine bleach liquor's disinfection seed, 4～5min more earlier, sterilized water washs 3～4 times, uses the 0.1% agar suspension culture Arabidopis thaliana seed of the bacterium of going out, inhales repeatedly to beat with pipettor and evenly broadcasts on resistant panel.Seal with the Parafilm film, at 25 ℃ temperature condition, relative humidity between 75% to 80%, the illumination of 16 hours every days.When true leaf grows to 4～5, wash residual substratum on the root off, to be transplanted in the training flower bud matrix (the Pei Lei company in Zhejiang), epiphragma 2d preserves moisture, and can carry out subsequent experimental according to the experiment needs subsequently.Plant division results seed after the plant maturation.Del1 and Del2 obtain T1 that 10 strains and 15 strains identify through PCR respectively for the transgenic positive seedling, with identical screening method screening T2 for the resistance seedling, with T2 for positive seedling analysis promoter expression pattern.

3) interpretation of result: by positive seedling is carried out the GUS tissue staining.It is stronger in the expression of the bottom of first pair of blade and stem under the driving of the different fragments length of GSTU19P promotor that the result presents gus gene, and each segment can both be to the evoked response of SA.Wherein the climax leaves blade tip of Del 2 positive seedlings is expressed also very strong.Disappearance promotor Del 1 expresses very strong before and after handling, and illustrate-function of promoter fragment with regard to having had very strong startup to transcribe of 300bp.For Del 2, though more intense expression is also arranged, the stem that can see Del 2 does not have the gus gene expression to compare with Del 1 and Full-length, and this phenomenon may be because exist the element of control tissue specific expression between the 700bp-300bp of GSTU19P promoter sequence

Embodiment 4 utilizes the GSTU19P promotor that the Arabidopis thaliana mesophyll protoplast is transformed transient expression

Make up promoter vector: (being numbered Full-length), 702bp (being numbered Del 2), 329bp (being numbered Del 1) that to design 3 different fragments of clip size be respectively 1856bp are loaded into above-mentioned fragment respectively on carrier pCAMBIA1301 and the pCAMBIA1302 (available from Australian CAMBIA laboratory); With its called after pCAMBIA1301-Full-length, pCAMBIA1301-Del1, pCAMBIA1301-Del2, pCAMBIA1302-Full-length, pCAMBIA1302-Del1, pCAMBIA1302-Del2 (seeing Fig. 6 and Fig. 7), pCAMBIA1301 serial carrier (Full-length in the present embodiment, Del1 and Del2) and the method for transformation of the technical program of the used primer of pCAMBIA1302 serial carrier (Full-length, Del1 and Del2), amplification condition, vector construction and Agrobacterium described with embodiment 3.Adopt PEG-calcium mediated method (Yoo, et al.2007) arabidopsis thaliana transformation mesophyll protoplast (Fig. 8).Concrete grammar is: choose the Arabidopis thaliana plant that grows to big robust growth of 3-4 week, get its 5th to the 7th true leaf, downcut the wide tissue of 0.5～1mm with blade at the blade middle part, the tissue that will downcut is put into enzymolysis solution fast, submergence fully.With vacuum drier (going up the grand experimental installation DZF-6050 of company limited of Nereid, the by specification operation) vacuum infiltration 30min.Under the room temperature more than the enzyme digestion reaction 3h.Quality at the protoplastis of microscopically Detection and Extraction.Then with isopyknic W5 Solution dilution enzymolysis solution, filter the not leaf tissue of enzymolysis with the blood nylon membrane filtering net of 75 μ m.Centrifugal 2min removes supernatant under the 100g room temperature.With blood counting chamber counting protoplastis number.With W5 Solution resuspension, make that cell concn is 2 * 10 ⁵Individual/ml, place 30min on ice.Remove supernatant, use MMG Solution resuspension again, make that cell concn is 2 * 10 ⁵Individual/ml, obtain the mesophyll protoplast of Arabidopis thaliana, under room temperature, place.10 μ L plasmid DNA (10-20ug) are joined the 2ml centrifuge tube, add 100 μ L Arabidopis thaliana mesophyll protoplasts (about 2 * 10 again ⁴Individual), mixing gently.Add 110 μ l, 30% polyvinyl alcohol (PEG) solution again, gently mixing.Room temperature is placed 5～15min.With 440 μ l W5 Solution dilution, put upside down the mixing termination reaction.The centrifugal 2min of 100g under the room temperature.Remove supernatant, the Arabidopis thaliana mesophyll protoplast is added contain in 6 well culture plates of 1mL WI Solution, incubated at room temperature 3-8h, the conversion back adds the 0.1mmol/L Whitfield's ointment, cultivates 16h, the protoplastis after centrifugal 2min collects conversion under the 100g.The medicine configuration that present embodiment is all and working method are according to reported method such as above-mentioned Yoo (Yoo, et al.2007).

The Arabidopis thaliana mesophyll protoplast that pCAMBIA1302 series is transformed is used for the GFP Fluirescence observation, and its result as shown in Figure 8.As shown in Figure 8, the protoplastis that utilizes the pCAMBIA1302 empty carrier to transform, fluorescence intensity is the strongest, can start the GFP expression of gene though the GSTU19 promotor is described, and intensity does not have the active strong of CaMV35S promotor.The contrast Whitfield's ointment handles and untreated two picture group sheets can find that the fluorescence intensity that contrasts among the Del 1 is greater than treatment group; Whitfield's ointment is handled back GFP and is then expressed byer force among 2 groups of the Del, illustrate-promoter fragment of 300bp possessed the function that startup is transcribed; And-700～-may exist Induced by Salicylic Acid element (Fig. 8) between the 300bp.

Meanwhile, present embodiment is also done parallel GUS active level analysis with the Arabidopis thaliana mesophyll protoplast that pCAMBIA1301 series transforms, and the method for its conversion is with the method for transformation of pCAMBIA1302.The fluorescent quantitative measurement of GUS activity is with reference to the method (Jefferson of Jefferson, et al.1987), its result as shown in Figure 9, as shown in Figure 9, there be not the acid-treated control group of bigcatkin willow, the GUS expression intensity of pCAMBIA1301 empty carrier is better than other three recombinant vectorss, explanation is without any under the situation about handling, though the GSTU19P promotor can be expressed by promotor gene, intensity does not have active strong (the pCAMBIA1301 empty carrier starts GUS by the CaMV35S promotor expresses) of CaMV35S promotor.

The nucleotide sequence of the relevant primer of table 2 the present invention design

Compare group and treatment group again being added with under the prerequisite of promotor that the CaMV35S promotor is constitutive expression, no matter whether use Whitfield's ointment, do not influence promoter activity (see among Fig. 9 1301 groups).In the disappearance group of Del1, Whitfield's ointment is handled back GUS and is expressed slightly rising; Whitfield's ointment is handled back GUS and is expressed grow in Del 2 disappearance groups, is not add 5 times of Whitfield's ointment treatment group; And in the Full-length group, the GUS expression amount before and after it is handled all is the same, shows that the promoter fragment of GSTU19P promotor-300bp of the present invention has possessed the function that startup is transcribed; And-700～-may there be Induced by Salicylic Acid element (Fig. 9) between the 300bp.

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Claims (3)

1. one by the promotor of the Cara Cara GSTU19 gene of Induced by Salicylic Acid, and its nucleotide sequence is shown in sequence table SEQ ID NO:1.

2. the application of the described promotor of claim 1 in the Arabidopis thaliana genetic improvement.

3. the described application of claim 2, comprising by to salicylic response regulation and control Arabidopis thaliana opposing coerce or secondary metabolism in application.

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