CN101886078A - Inducible promoter containing S box as well as construction method and application to genetic engineering - Google Patents

Abstract

The invention provides an inducible promoter containing an S box as well as a construction method and the application to genetic engineering. The inducible promoter at least contains a nucleotide sequence shown as SEQ ID No.1. The construction method comprises the following step of: fusing a stress-response-stressed S box identified in dicotyledon parsley with a CaMV35S promoter core sequence to construct an inducible promoter. The inducible promoter can well response the invasion of rice blast fungus, the feeding of rice cnaphalocrocis medinalis and the mechanical damage in transgenic rice, has obvious response activity to the treatment of small-molecule compounds with disease resistance after separation identification and is weak to express or does not express the response activity of abiotic stress treatment, such as drought, low temperature, high temperature, and the like. The inducible promoter has very important application values in the disease-resistant and pest-resistant genetic engineering of monocotyledon rice, and transgenic plant strains also have very important application values in the high throughput screening of small-molecule compounds for inducing plants to resist diseases.

Description

Contain inducible promoter and the structure and the application on genetically engineered of S box

Technical field

The present invention relates to a kind of inducible promoter and structure and the application on genetically engineered, related more specifically to a kind of application of inducible promoter in the disease-resistant worm genetically engineered of paddy rice monocotyledon rice of the S of containing box, belong to the plant gene engineering technology field.

Background technology

Pathogenic micro-organisms such as fungi, bacterium, virus infect the decline that often causes various crop yields and quality.Cultivating and promote disease-resistant variety is to prevent and treat crop pest, the high and stable yields of assurance crop approach the most safely and effectively.Utilize plant self disease-resistant gene, cultivated many disease-resistant varieties by conventional breeding and molecular mark method.Yet resistant gene utilizes between kind has certain limitation.Transgenic method can overcome above-mentioned limitation, has opened up a new way for the crop disease-resistant breeding, also is one of important channel of plant germplasm innovation.At present, employed promotor great majority are constitutive promoter in transgenic technology, if use plant tissue specificity promoter or pathogeny evoked plant promoter not only can obtain the purpose product, make it, nor can have side effects.

Plant gene promoter is the dna sequence dna that is positioned at structure gene 5 ' end upstream, contains cis-acting elements, is determining specificity, direction and efficient that downstream gene is transcribed, is the center of transcriptional control.There are various feature member in the higher plant promoter region, comprises transcription initiation site, TATA box, initiation factor and different cis-acting elements etc.Wherein, the TATA frame is to depend on rna plymerase ii to transcribe necessaryly, is determining transcription initiation, and is regulating and control the behavior of upstream activator; The function of initiation factor is similar to the TATA box, can influence the direction of transcribing and the location of starting point, but generally can not change the TATA activity that comprises 2 core sequence factors in the promotor.The many proterties of plant and the structure and the control methods thereof of promotor are closely related.Therefore, the research promotor not only can also can promote the plant new resources that have important utility value from the excavation of genetic molecule basis for genetically engineered provides important controlling element.

Transcriptional profile according to plant promoter can be divided into it constitutive promoter, organizing specific type promotor and inducible promoter.

The genetic expression of constitutive promoter regulation and control is not expressed in plant with inducing of certain material by the space-time restriction.Constitutive promoter cauliflower mosaic virus (CaMV) 35S promoter commonly used is to use maximum promotors in the present plant genetic engineering in the dicotyledons, and it orders about transgenosis and produces constitutive expression.It is an allos virus type promotor, has higher expression activity in transgenic plant.Constitutive promoter can be applicable to estimate the biological function of a gene, transgene expression quantity and activity thereof, but the subject matter in the practical application of improvement genetically modified crops is: the gene that (1) constitutive promoter drives all has in various degree in each tissue of plant expresses, it drives foreign gene and all can express in various tissues and all etap of plant, produce a large amount of heterologous proteins or meta-bolites at plant interior accumulation, broken the original metabolic balance of plant, increased the metabolism burden of plant, some product even poisonous to growth and development of plant, caused the huge waste on the matter and energy, also can change the form of plant simultaneously, influence plant and grow normally, even cause death; (2) the plant all cells may all enter the defence pattern, even still carry out disease resistance response when no cause of disease infects; (3) transgene product of constitutive promoter regulating and expressing can accumulate in the edible plant tissue of humans and animals, but biological safety requires that transgene product can not be arranged.

If replace constitutive promoter with tissue, organ Idiotype and pathogeny evoked type promotor, make foreign gene can be regularly, fixed point, in plant, express quantitatively, can overcome the problem that constitutive promoter brings.Therefore, the research of organizing specific type promotor and pathogeny evoked type promotor is the important content and the current focus of research both at home and abroad of plant genetic engineering with using.The best method of utilizing promotor may be to utilize an endogenous resistance gene promoter, so both can reduce the metabolism burden of plant, can avoid the false activation of plant defense reaction again.

Be only to express and only start disease-resistant related gene and express than constitutive expression and the better expression pattern of organizing specific type expression at infection site at the when and where of needs.Pathogeny evoked type promotor can reach above-mentioned comparatively ideal results, prevents that " fleeing from necrocytosis " phenomenon from occurring, and eliminated the side effect to plant-growth and development impact.The pathogeny evoked type promotor of a kind of ideal should be rapidly by wide spectrum wild-type cause of disease infect activate, and broad spectrum resistance is arranged.Yet because plant has been taked different defense mechanisms to various pathogenic bacteria, many important plant disease-resistant molecular mechanisms are still indeterminate, so present isolating most of pathogeny evoked type promotors seldom can satisfy above-mentioned requirements, still have the phenomenon of slight " fleeing from necrocytosis ".Therefore, press for separation or the pathogeny evoked type promotor of artificial constructed ideal.The development of plant disease-resistant Mechanism Study, transcription group and promotor clone technology is for artificial constructed new pathogeny evoked type promotor is laid a good foundation.

The major cause that current spendable pathogeny evoked type promotor lacks is that the various natural pathogeny evoked type promotors of separation are less.Compare with natural pathogeny evoked type promotor, artificial constructed pathogeny evoked type promotor not only has more broad spectrum, and can select flexibly according to various objectives, as eliminating the promoter expression element, can change the intensity of promotor and the quality and quantity of pathogeny evoked expressing gene.Discover that it is conservative relatively that promoter element is defendd signal in different plant varieties.Therefore, can artificial constructed cause of disease promotor by collecting various plant promoter elements.

Promoter component has many different types, comprises pathogeny evoked type functional element, organizing specific type functional element, cell-specific type functional element and manually sets up the required minimal promoter section of promotor.Wherein pathogeny evoked type functional element is most important aspect disease-resistant.In the known plant transcription factor family many members that play an important role are arranged in disease resistance response, comprising WRKYs, ERFs, bZIPs, Mybs, Dofs, bHLHs, Whirly, SR and DBP1 etc.These transcription factors and PR gene specific bonded site are cis-acting elements, are very useful for setting up the promotor functional element.

In plant defense Expression of Related Genes process, cis-acting elements is by bringing into play important regulating and controlling effect with transcription factor mutual: environmental stimulus is through after a series of signal transduction, transmitting, activate the mutual work of defence associated transcription factor and special cis-acting elements, thereby realize relevant defense function expression of gene.At present, the resistance by transgenic technology raising plant has become research strategy important in the plant genetic engineering.Above-mentioned important regulating and controlling effect in view of cis-acting elements, use with the transgenosis of the resistant gene of early stage constitutive expression and to compare, use the resistant gene of inducible promoter mediation to have many-sided advantage undoubtedly, identify that therefore resistance relevant (particularly bacterial pathogen is relevant) inducible promoter has become the focus of present molecular biology of plants research.So far, had been found that several be subjected to Different Kinds of Pathogens inductive GCCbox (AGC-CGCC) and be subjected to the pathogeny evoked Wbox of wound, part ([T] TGAC[C/T].Often also contain GCCbox at the defensin gene promoter region, it is similar with the Sbox (AGCCACC) of regulation and control fungal induction expression to the JERE element (AGACCGCC) of regulation and control jasmonic, exciton abduction delivering.To be people such as Kirsch in 2000 found a new pathogenic fungi inductive cis-acting elements that is subjected to from Parsley EL17 gene promoter with S box (core sequence AGCCACC), be a class GCC (AGCCGCC) element, with jasmonic response element JERE(AGACCGCC), arid response element DRE/CRT(TACCGACAT) all have a high similarity.These several cis-acting elements can both specificity be incorporated into the AP2/EREBP transcription factor.

At present, use in the genetically engineered plants of constitutive promoter, the continuous expression that changes disease-resistant related gene over to may cause uncontrollable defensive raction, for example " flees from necrocytosis ".Utilizing transgenic method to improve another subject matter that crop yield faces is that the background that promotor causes is expressed, have switch and cause of disease infect the site can be rapidly momently the promotor of inducing anti-disease genetic expression be more satisfactory promotor.In addition, the separation of endogenesis promoter can reduce background with utilization and express, but the controlling element of some promotor is not only regulated and control pathogeny evoked expression of gene but also regulate and control background and express usually, thereby the separation of the pathogeny evoked Idiotype promotor of ideal is also relatively more difficult so far.Yet along with the deep development to promotor functional element and the research of disease-resistant regulatory mechanism, successfully the possibility of separation or artificial constructed better promotor can be increasing.

Studies show that, with the S box identified in the dicotyledonss such as potato, Arabidopis thaliana, HERBA CORIANDRI, Vinca and CaMV35S core promoter (46～+ 8bp) merge constructed inducible promoter can be in protoplastiss such as tobacco, Arabidopis thaliana moment express or in transfer-gen plant by pathogenic bacteria or bring out son and handle rear drive GUSGenetic expression.This result shows the vital role of S box element in abduction delivering on the one hand, illustrates also that on the other hand the signal transduction path of the conjugated protein and upstream of this cis-acting elements has conservative property in above-mentioned multiple dicotyledons.But, still lack the direct evidence that in paddy rice and monocotyledons element such as S box is replied pathogenic bacteria or exciton so far.Further in paddy rice, carry out the S box and reply the evaluation and the applied research of pathogenic bacteria, ET, JA signal, have crucial meaning: (1) is expected to find out the cis-acting elements of replying disease, worm, with these elements is bait, can further separate its corresponding transcription factor gene; (2) owing to leaving its host's promotor, cis-acting elements still can keep the function that it replys pathogenic bacteria or incitant, can utilize these elements and suitable promotor core sequence (as the core sequence of gene promoters such as CaMV35S, Act1), make up response pathogen infection or JA(insect's food-taking) inducible promoter.

Summary of the invention

The invention provides a kind of inducible promoter and structure and the application on genetically engineered of the S of containing box, will make greater efforts to promote engineered development of paddy disease-resistant and application.

The inducible promoter that contains the S box of the present invention, it contains the nucleotide sequence shown in the SEQIDNo.1 at least.

Construction process is as follows:

The present invention with the S box of replying environment stress (its sequence is CAGCCACCAAAGAGGACCCAGAA) identified in the dicotyledons parsley and CaMV35S promotor core sequence (46～+ 8bp) merge, be built into inducible promoter.Described CaMV35S promotor core sequence is TCGACCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGAC.

That the inducible promoter background that contains the S box of the present invention is expressed is low, speed of answer fast, longer duration, expression intensity are moderate.Above-mentioned inducible promoter is combined with disease-resistant genes such as (worms), be built into the conversion carrier rice transformation, can make the external source resistant gene abduction delivering of the transgenic paddy rice of acquisition, both brought into play the effect of resistant gene in disease-resistant (worm), can avoid again since the waste of the material that causes of resistant gene overexpression, energy and resistant gene expression product to the disadvantageous effect of transgenic plant cells itself.

Remarkable advantage of the present invention:

The inducible promoter of the S of containing box of the present invention can reply preferably in transgenic paddy rice that rice blast fungus infects, Cnaphalocrocis medinali(rice leaf roller) is got food and physical abuse, the processing of the micromolecular compound with resistant effect of isolation identification (as ethrel, phenylpropyl alcohol thiadiazoles, methyl jasmonate) also had significantly replys activity, and to abiotic stresses such as arid, low temperature, high temperature handle to reply specific activity more weak or do not express.This inducible promoter is disease-resistant at monocotyledon rice, have important application in the worm genetically engineered, and its transgenic plant strain ties up to also has important use to be worth in the disease-resistant micromolecular compound high flux screening of inducing plant.

Description of drawings

Fig. 1 is the plasmid map of moment expression vector pBT10-GUS;

Fig. 2 is the pDNOR-207 entry vector;

Fig. 3 is a pMDC-163 purpose carrier;

Fig. 4 is the structure iron of moment expression vector pBT10-GUS;

Fig. 5 is the purpose fragment PCR detection of transgenic paddy rice T0 for plant; 1-13 is a transfer-gen plant amplified production band, and 14 is the wild-type amplification band; 15 is the plasmid amplification band;

Fig. 6 infects T1 for the painted result of GUS after plant 5-7 days for Pyricularia oryzae; Wherein 1 expression is after 5 days, and 2 expressions are after 6 days, and 3 expressions are after 7 days, and CK is contrast;

Fig. 7 for Cnaphalocrocis medinali(rice leaf roller) get food T1 for the plant leaf different time sections after the result of GUS histochemical stain; Wherein behind the 1 expression 3h, behind the 2 expression 5h, behind the 3 expression 24h.

Embodiment

1 vector construction process and commentaries on classics

1.1 materials and methods

Material

1.1.1.1 bacterial strain and carrier: intestinal bacteria ( Escherichiacoli) DH5 а and the laboratory preservation of life science institute of DB3.1(University Of Agriculture and Forestry In Fujian); Agrobacterium tumefaciens ( Agrobacteriumtumefaciems) EHA105 bacterial strain (preserve in life science institute of University Of Agriculture and Forestry In Fujian laboratory); PBT10-GUS moment expression vector (see figure 1) is by (professor Weisshaar is so kind as to give, Sprenger-HausselsandWeisshaar, 2000); PDNOR-207 entry vector (see figure 2) and pMDC-163 purpose carrier (see figure 3) are all available from Invitrogen company.

1.1.1.2 biochemical reagents: DNA reclaims test kit fast and plasmid extraction kit is given birth to worker's biotechnology company limited available from Shanghai, XbaI, SacI, SpeI, PfuTaqEnzyme, RTaqEnzyme, T4 ligase enzyme and dNTP etc. are available from TaKaRa company, and Gateway vector construction test kit is available from Invitrogen company, and gentamicin, kantlex, Rifampin, carboxylic Bian penicillin, Totomycin, X-gluc etc. are Sigma company product.Other chemical reagent is homemade chemical pure.

1.1.1.3 primer:Primer sequence is given birth to worker company by Shanghai and synthesized, and is as shown in table 1.

Table 1 primer title and nucleotide sequence

1.1.1.4 sequence is synthetic: according to the S box sequence among the Ohme-TakagiandShinshi, (base of wherein ruling is what add up, and the double-stranded both sides of positive anti-chain annealing synthetic are had respectively XbaIWith SpeITwo restriction enzyme sites) entrust Shanghai Bo Ya biotech company synthetic, as shown in table 2 respectively.

The title and the nucleotide sequence of the synthetic chain of table 2

1.1.1.5 explant: japonica rice variety Japan fine ( Oryzasativassp.japonica) mature embryo institute evoked callus for transforming used explant.

1.1.1.6 biological and abiotic stress is handled: (bacterial strain is Pyricularia oryzae Guy11) conidium, rice leaf roller larvae.

Method:

1.1.2.1 make up the inducible promoter moment expression vector that contains two times of boxes: synthetic among the 1.1.1.4 is had XbaIWith SpeIOn the corresponding restriction enzyme site of the S box double chain oligonucleotide fragment insertion pBT10-GUS of restriction enzyme site (as shown in Figure 4), obtain to contain the carrier pBT10-X-GUS(X-S box of single copy S box, down together), pBT10-X-GUS uses respectively with carrier XbaI/ SacIWith SpeI/ SacIThe combination enzyme is cut (37 ° of enzymes are cut 3-5h), reclaims the fragment contain the S box T4 ligase enzyme connects by using, transformed into escherichia coli DH5 а and checking, copies placed in-line moment expression vector pBT10-XX-GUS thereby obtain to have 2 S boxes.

1.1.2.2 make up the inducible promoter plant expression vector that contains two times of boxes: the CaMV35S core promoter adjacent with its downstream according to the last duple S box of the moment expression vector pBT10-XX-GUS that builds among the above-mentioned 1.1.2.1 (46～+ 8bp) fragment designs the inboard primer of Gateway, and utilization Gateway vector construction technology is (with reference to the Gateway of Invitrogen company ^TMTechnology) makes up plant expression vector, with the pDNOR-207 carrier is entry vector, thereby pMDC-163 is the purpose carrier obtains can be used for the inducible expression carrier pMDC-XX-163 that plant genetic transforms, expression vector conversion agrobacterium strains EHA105 is standby, and (the pMDC-XX-163 vector plasmid of getting about 1ug joins in the EHA105 competent cell, behind the mixing, ice bath 30min; Liquid nitrogen flash freezer 3min; Take out back 37 ℃ of water-bath 5min; Ice bath 2min; Add the not YEP liquid nutrient medium of added with antibiotic of 400ul, 28 ℃, 180rpm shakes training 4h; Be coated in the YEP solid medium that contains 50mg/L kantlex and Rifampin after the taking-up, cultivate for 28 ℃ and form single bacterium colony, picking mono-clonal bacterium colony carries out the PCR checking).

1.1.2.3 plant expression vector transforms Agrobacterium and to the genetic transformation of paddy rice:Contain the Agrobacterium EHA105 bacterium liquid of goal gene pMDC-XX-163 with the inoculating needle picking, lining concentration is the AB solid medium of 50mg/L kantlex and Rifampin, places 28 ℃ of dark culturing 3-4d.Mono-clonal of picking places 28 ℃ of shaking tables in the AAM liquid nutrient medium from above-mentioned AB substratum, is 0.1 until the OD value.The Japanese fine seed of getting shelling is with 70% ethanol disinfection 1-2min, aseptic water washing 5 times, then with 2.5% chlorine bleach liquor (every 50ml2.5% chlorine bleach liquor contains a Tween-20) sterilization 15min, with aseptic water washing 5 times, be placed on the filter paper of sterilizing and dry, receive the N6D+2.4D inducing culture again, place 32 ℃ of illumination box illumination cultivation 5-7d to induce the generation callus.Taking-up is through the seed of inducing culture, pulls out that to immerse above-mentioned OD behind the bud be (containing AS10-20mg/L) 10min in 0.1 the AAM bacterium liquid, during rock gently, the bacterium liquid on callus surface is blotted with filter paper in the back.Callus is transferred on the aseptic filter paper that contains the AAM liquid nutrient medium on the 2N6-AS substratum, and 25 ℃ of dark are cultivated 3d altogether.Callus after cultivating is altogether cleaned 5-6 all over not occurring until there being muddiness in sterilized water, then with the sterile water wash 30min that contains the 500mg/L Pyocianil, blot with filter paper, callus is placed (Totomycin that contains 400mg/L Pyocianil, 2mg/L2.4-D and 50mg/L) 32 ℃ of 2 weeks of illumination screening and culturing on the N6D screening culture medium.To transfer to through the callus of screening and culturing and (contain 0.02mg/L α-NAA, 2mg/LKT, 50mg/L Totomycin and 400mg/L Pyocianil) on the RE-III substratum 32 ℃ of illumination differentiation culture 2-3 weeks.Getting seedling that differentiation comes out transfers on the HF root media 30 ℃ of root inductions and cultivated for 1 week.

1.1.2.4 the extraction of transgenic paddy rice DNA and Molecular Detection: the extraction reference literature of oryza sativa genomic dna (sambrookandrussel, 2002) method.With the inboard special primer sequence of Gateway it is carried out PCR and detect (as shown in Figure 5), among the figure with the positive contrast of expression vector pBT10-XX-GUS, with the negative contrast of non-transgenic paddy rice, as can be seen from the figure 1～13 the PCR band the same with positive control occur, and all positive transgenic plant are described.

1.1.2.5 transfer-gen plant is biological and abiotic stress is handled: get T1 for the aqueous solution (concentration 50mg/L) the resistance screening every day 12h illumination of full seed through containing Totomycin, 25 ℃, cultivate and get the green little seed that can normally sprout behind the 7-8d in the plastics small flower that fills soil, as base manure, urea topdresses twice after emerging with urea and fertilizer.The 3-4 leaf adopts spray inoculation method inoculum density during the phase be that (bacterial strain is for the Pyricularia oryzae of 105/mL Guy11) move to hot-house culture a couple of days of 25 ℃ and 80% humidity behind the conidium.Rice leaf roller larvae is removed rice leaf roller larvae after getting the young leaflet tablet different time sections of food T1 for the transfer-gen plant 5-6 leaf phase from blade.

1.1.2.6 histochemical stain: biology is coerced the method that detects according to (Jefferson, 1987) with the abiotic stress response result and is disposed the GUS staining fluid, stores in-20 ℃ of lucifuges.Handle back clip 5-6cm blade through above-mentioned biology and abiotic stress and place 12h in 37 ℃ and use 70% ethanol decolorization again, during change the ethanol several.Its photo is got in the back bat fully of waiting to decolour.

2S box replying to biological environment stresses such as Cnaphalocrocis medinali(rice leaf roller) and rice blast

T1 grows into the 3-4 leaf for the S-GUS transfer-gen plant, and (bacterial strain is by rice blast during the phase Guy11) conidium infects the back and cultivate the expression detected gus reporter gene in 5-7 days respectively and find, along with the increase disease of inoculation back incubation time becomes seriously gradually, and the expression amount of GUS is also along with increase, especially the serious zone of disease.Remove near do not detect the active (see figure 6) of tangible GUS with other positions of outer leafs, scissors clip position the T1 generation plant of the same age of infecting without Pyricularia oryzae.

Get food T1 for the young leaflet tablet of transgenic paddy rice 5-6 leaf phase plant with rice leaf roller larvae, gently Cnaphalocrocis medinali(rice leaf roller) is removed from blade behind pick up counting when beginning to get food 3h, 5h, the 24h.Clip blade 5-6cm immerses and detect the variation that above-mentioned different time sections is got food back gus gene expression amount in the GUS liquid respectively behind 37 ℃ of 12h, get the food place Cnaphalocrocis medinali(rice leaf roller) and can detect tangible GUS activity, increase the corresponding (see figure 7) that increases of GUS expression along with getting food time lengthening affected area.

3 conclusions

The 35S promoter that will contain the S cis-acting elements connects goes up gus reporter gene importing paddy rice, under biological environment stresses such as Cnaphalocrocis medinali(rice leaf roller) and rice blast, observe that GUS has obtained certain expression in the transgenic paddy rice, also exist in this explanation paddy rice can discern the S element element of S core sequence (or contain), and with it in conjunction with the AP2/EREBP class transcription factor of impelling reporter gene GUS to express.

＜110〉University Of Agriculture and Forestry In Fujian

＜120〉contain inducible promoter and the structure and the application on genetically engineered of S box

<160>1

<210>1

<211>77

<212>DNA

＜213〉artificial sequence

<220>

<400>1

cagccaccaaagaggacccagaatcgaccgcaagacccttcctctatataaggaagttca60

tttcatttggagaggac77

 

 

 

Claims (5)

1. inducible promoter that contains the S box, it contains the nucleotide sequence shown in the SEQIDNo.1 at least.

2. the construction process of an inducible promoter as claimed in claim 1 is characterized in that: S box of replying environment stress and the CaMV35S promotor core sequence identified in the dicotyledons parsley are merged, be built into inducible promoter.

3. the construction process of inducible promoter according to claim 2, it is characterized in that: the nucleotides sequence of described S box is classified CAGCCACCAAAGAGGACCCAGAA as.

4. the construction process of inducible promoter according to claim 2, it is characterized in that: described CaMV35S promotor core sequence is TCGACCGCAAGACCCTTCCTCTATATAAGG

AAGTTCATTTCATTTGGAGAGGAC。

5. the application in an inducible promoter that contains the described inducible promoter of claim 1 or make up by the described method of claim 2, the worm genetically engineered disease-resistant at monocotyledon rice.

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