CN101818170B - Plant expression vector for vascular peculiar promoter to control antimicrobial protein gene and method for cultivating greensickness-resistant cotton - Google Patents
Plant expression vector for vascular peculiar promoter to control antimicrobial protein gene and method for cultivating greensickness-resistant cotton Download PDFInfo
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Abstract
The invention provides a plant expression vector for a vascular peculiar promoter to control two types of antimicrobial protein gene and a method for cultivating greensickness-resistant cotton. In the method, motherwort antimicrobial protein genes LjAMP2 and LjAMP2 controlled by a vascular tissue peculiar promoter AAP2 are integrated into a cotton genome to realize the high-amount expression of the antimicrobial protein gene in the vascular tissue of the transgenic cotton root and properly express in the vascular tissues of other parts of the plant so as to obviously improve the resistance to greensickness by cotton. After performing homozygosis to defoliating and nondefoliating greensickness bacteria inoculated by a T2 generation plant greenhouse disease pool, the transgenic cotton obtained with the method compares the defoliating and nondefoliating greensickness bacteria with Non-GMO, and disease index can be respectively lowered by 40% and 80%.
Description
Technical field
The invention belongs to plant gene engineering technology field.Be specifically related to dimension pipe specific promoter the antibiotic protein gene plant expression vector of controlling and the transgene cotton preparation method who contains above-mentioned expression vector; In addition, the invention still further relates to a kind of genetic engineering technique that utilizes and improve the method for cotton to resistance to verticillium wilt.
Technical background
Cotton is most important natural fiber crop in the world.In China, cotton is always the valuable cargo involving the interests of the state and the people, Cotton in China industry relates to the income of approximately 200,000,000 people in the countryside, is related to the foreign exchange earning of 1,900 ten thousand textile workers employment and more than 1,000 hundred million dollars, and textile industry and even the whole national economic development are all had to very important effect.But from nineteen thirty-five cotton verticillium wilt import into after China, the harm that Cotton in China is produced increases the weight of year by year, grave illness time loss gined cotton surpasses 400,000 tons, direct economic loss reaches more than 60 hundred million yuan (Chen Jieyin etc., 2005).The verticillium serious cotton field of causing harm, output can the underproduction more than 70%, simultaneously the serious fibrous quality that reduces also.This global great disease of cotton verticillium wilt, seriously hampers the development that Cotton in China is produced.Cotton verticillium wilt (cotton Verticillium wilt) belongs to soil-borne vascular bundle disease, be characterized in distributing wide, harm is heavy, host range is wide, route of transmission is many, the survival time is of a specified duration, is one of destructive disease of tool in Cotton Production.In production, never find desirable Prevention Technique and control method, be therefore referred to as " cancer " (Chen Jieyin etc., 2005) of cotton.Vascular bundle due to the pathogenic bacteria harm cotton plant of cotton verticillium wilt, still lacks effectively preventing medicament so far.Selection and popularization disease-resistant variety, is that China Ye Shi world respectively produces cotton national defence to control verticillium most economical effectively, and is unique effective approach (Gu Benkang etc., 1996).
China breeding expert is through the effort of decades, utilize conventional breeding means, obtained some and verticillium has been there are to the local variety of certain resistance against diseases, but because verticillium wilt pathogen variation is fast, microspecies are many, have obvious Virulence, for the resistance of wide spectrum cotton variety of this specific character of verticillium wilt pathogen, substantially do not have, therefore just existing at a regional representation is disease-resistant cotton seed, under another different physiology and geographical conditions, may there is the problem that disease resistance is lost.On the other hand, owing to lacking the anti-source of high resisting verticillium in upland cotton cultivar, directly caused Cotton in China resisting verticillium breeding process slow, particularly the breeding of anti-defoliation verticillium does not have progress substantially.In addition, different geographical seed exchanges and causes pathogenic bacteria more and more in kind in the localities, has formed compound population.All there is differentiation and the raising of virulence in the verticillium dahliae of various places, the appearance of strong virulence fungus strain is the one of the main reasons that causes cotton verticillium wilt to increase the weight of year by year.For this reason, the problem of resisting verticillium resource shortage and to find new disease resistant and breeding method extremely urgent in solve producing, be badly in need of obtaining there is wide spectrum and durable resistance anti-source to alleviate the massive losses that in Cotton Production, verticillium causes.
In breeding method, the breeding of Cotton in China anti-blight, adopt disease garden systematic breeding and sexual hybridization to obtain good result, but it is undesirable to be applied to resisting verticillium breeding effect.Therefore, adopt the multiple breeding methods such as biotechnology, accelerating resisting verticillium breeding process becomes inevitable.Utilize modern biotechnology separation, clone and transform resistant gene, can improve single traits at all, its advantage is that directional property is strong, the sibship restriction of anti-source is enriched, is not subject in genotype source, breeding cycle is short, nonexistence shape negative correlation is chain in theory, and a time cloning can be used for repeatedly transforming.Adopting the disease resistance of genetically engineered improvement plant is an important channel (Rommens and Kishore, 2000) of control of plant disease.
At present, the genetically engineered research of verticillium wilt resistance of cotton by same wins initial success.The report of succeed at mainly concentrates on chitinase gene and β-1,3 glucanase genes, has utilized multiple authentication method screening transgenic offspring, has obtained transgene cotton strain (happy brocade China etc., 2002 of anti-or resistance to verticillium; Wu Jia and etc., 2004; Cheng Hongmei etc., 2005).But, obtain the cotton resource to verticillium tool high resistance and stable durable resistance, also need further to find new disease-resistant gene.
Conventionally in the selection of disease-resistant gene, there are two kinds of strategies.The one, utilize R (Resistance) gene of plant, though R gene has been cloned nearly 40 kinds, but R gene shows as the specialization resistance of disease-resistant variety to pathogenic bacteria physiological strain, so its disease resistance has very strong specificity, and easily the variation because of pathogenic bacterium colony physiological strain is overcome (Xiaoyan Tang etc., 1996; Yuelin Zhang etc., 2003; Caldo R.A. etc., 2004; John M.McDowell, 2003).The 2nd, utilize the gene with wide spectrum and durable resistance.The disease-resistant transgenic plant obtaining in this way, possesses advantage (Cornelissen etc., 1993 such as the anti-multiple diseases of energy, the difficult forfeiture of disease resistance; Osusky M. etc., 2000), so this strategy is paid attention to widely.Yet can filter out this gene with wide spectrum and durable resistance, also just become the key of these tactful success or failure.
Antibacterial protein or polypeptide (AMP, antimicrobial protein or peptide) be that a class is by biogenic albumen or the polypeptide with anti-microbial activity, be distributed widely in animal, plant and microorganism, there is has a broad antifungal spectrum, the feature such as anti-microbial activity is high and resistance is lasting.In addition, the antibacterial mechanisms of antibacterial peptide is special, mainly with form duct on film, causes the mode that entocyte leaks to destroy pathogenic bacteria, causes pathogenic bacteria to be difficult to produce resistance (Yeaman etc., 2003).The antibiotic protein gene of different sources is widely used in genetic engineering of plant for disease resistance, and has obtained the multiple transgenic plant that disease resistance improves, and the application of antibiotic protein gene in plant genetic engineering more and more come into one's own.In this strategy, most widely used is to utilize the disease resistance that improves plant from the antibacterial protein of plant self.More and more research shows, the antibacterial protein of expressing from plant in transgenic plant can obviously improve the disease resistance of plant.As, Gao etc. (2000) overexpression in transgenic Rhizoma Solani tuber osi can obviously improve the resistance of potato to verticillium from the antibacterial peptide gene of clover.Ja Choon Koo etc. (2002) proceed to tobacco by the antibacterial peptide gene from morning glory seed, realize its overexpression in tobacco, can significantly improve the resistance of transgene tobacco to balck shank.Utilizing antibiotic protein gene to improve cotton verticillium wilt resistance has report few in number, comprises, Fiona Murray etc. (1999) proceed to the glucose oxidase gene from Talaromyces flavus in cotton, the T isozygotying
3for plant, resistance to verticillium wilt is improved.Y.Q.Wang etc. (2004) utilize pollen tube passage method that the antifungus protein gene GAFP from rhizoma Gastrodiae is imported to three color cotton cultivars, and the breeding selection through 2 years has obtained two strains that resistance to verticillium wilt is improved.Research shows that the Motherwort Herb antibiotic protein gene utilizing in the present invention proceeds to tobacco and tomato, can obviously improve transfer-gen plant to black shank, the resistance of the multiple diseases such as early blight of tomato (XingyongYang etc., 2007,2008; Li Xianbi etc., 2007).
In plant genetic engineering, controlling gene is expressed in the selection of promotor, usually first selects the CaMV35S promotor of constitutive expression, realize foreign gene overexpression with the function of research gene, but this promotor easily causes exogenous gene expression too high.Part antibacterial peptide gene is crossed high expression level and is easily caused that vine growth and development is abnormal.As, CEMA gene overexpression, easily causes transgene cotton leaf malformation and sterile (Guo Yulong etc., 2003).Fiona Murray etc. (1999) are by the overexpression in cotton of the glucose oxidase gene from Talaromyces flavus, and T isozygotys
3for plant, cotton verticillium wilt resistance is strengthened, but too high plant root and the growing of seed of expression amount is affected, root shortens, lateral root forms and reduces, and seed amount reduces, and seed germination rate reduces.In plant genetic engineering, control the expression of foreign gene except selecting CaMV35S promotor, usually use inducible promoter, organizing specific expression promotor.Disease-resistant gene engineering can be according to the feature of different diseases, and the needed target antibacterial protein of specifically expressing, to resist the intrusion of pathogenic bacteria or the growth of inhibition pathogenic bacteria.As, Wu Jia and etc. (2004) utilize the brightly yellowish pinta virus promoter of ring CoYMV regulation and control chitinase gene, realize chitinase gene specifically expressing in the vascular tissue of transgene cotton, reach and improve the resistance of cotton to verticillium.(2007) the abduction delivering MsrA2 genes in tobacco such as Dmytro P.Yevtushenko, can effectively improve the resistance of transgene tobacco.Rajesh Narhari Patkar (2006) etc., utilize inducible promoter PAL regulation and control ns-LTP-like gene to express in paddy rice, can effectively improve the resistance of transgenic paddy rice to multiple diseases such as rice blast.
Verticillium dahliae mainly starts to infect from the root of plant, then along with the vascular tissue at plant root, stem and each position of blade is expanded gradually to plant top, in the suitable situation of condition, can be developed to plant top, causes whole plant morbidity even dead.If the present invention study find can expression inhibiting verticillium wilt pathogen growth in cotton vascular tissue antibacterial protein, verticillium wilt pathogen is controlled within the specific limits, or is stoped its further growth in plant, just can reach the object of resisting verticillium.
Summary of the invention
An object of the present invention is to provide AAP2 promotor and control the plant expression vector of two kinds of antibiotic protein gene LjAMP1 and LjAMP2.Utilize gene engineering method, AAP2 promotor and LjAMP1 or LjAMP2 antibiotic protein gene are built in suitable expression vector simultaneously, obtained the plant expression vector of AAP2 promoter to control antimicrobial protein gene of the present invention.The plant expression vector obtaining has the constitutional features shown in Fig. 7 P5-AAP2-LjAMP1 and Fig. 8 P5-AAP2-LjAMP2.These two carriers are proceeded to respectively in plant, can obtain the transgenic plant of vascular-specific expression LjAMP1 and LjAMP2 gene.
Another object of the present invention is to provide the transformant that simultaneously contains dimension pipe specific promoter AAP2 and antibiotic protein gene LjAMP1 or LjAMP2.Utilize conventional gene engineering method, above-mentioned plant expression vector is transformed to suitable host and can obtain transformant of the present invention.In a concrete embodiment of the present invention, plant expression vector is proceeded to and in agrobacterium tumefaciens lba4404, obtained transformant of the present invention by electric shocking method.In addition, utilizing agrobacterium tumefaciens-mediated transformation to transform the transgenic cell line that contains above-mentioned recombinant plant expression vector obtaining is also the protection domain of this invention.
Another object of the present invention is to provide a kind of method of cultivating resisting verticillium transgene cotton.The method of cultivation resisting verticillium transgene cotton provided by the present invention, be to utilize agrobacterium tumefaciens-mediated transformation that recombinant plant expression vector P5-AAP2-LjAMP1 and P5-AAP2-LjAMP2 are imported in cotton cells, after tissue culture and transgenic plant Molecular, regeneration obtains the transgene cotton that resistance to verticillium wilt is improved.
The object of the present invention is achieved like this: a kind of plant expression vector of tieing up pipe specific promoter control antibiotic protein gene, it is characterized in that, for containing dimension pipe specific promoter AAP2, control the plant expression vector of antibiotic protein gene LjAMP1, extract total DNA from Arabidopis thaliana after, use pcr amplification, be connected with pMD18 carrier again, transform bacillus coli DH 5 alpha competent cell, screening positive clone obtains the promotor of Arabidopis thaliana AAP2 gene, the long 2131bp of this promotor, with it, replace the constitutive promoter CaMV35S on plant expression vector p5-LjAMP1, build a new expression of plants body, called after p5-AAP2-LjAMP1.
A kind of plant expression vector of tieing up pipe specific promoter control antibiotic protein gene, it is characterized in that, for containing dimension pipe specific promoter AAP2, control the plant expression vector of antibiotic protein gene LjAMP2, with the promotor from Arabidopis thaliana AAP2 gene, replace the constitutive promoter CaMV35S on plant expression vector p5-LjAMP2, build a new expression of plants body, called after p5-AAP2-LjAMP2.
Further, described plant expression vector, utilizes electric shocking method to transform agrobacterium tumefaciens, the recombinant bacterium that contains p5-AAP2-LjAMP2 of acquisition.
Further, described plant expression vector, utilizes agrobacterium tumefaciens-mediated transformation to carry out genetic transformation, the transgenic cell line that contains plant expression vector described in p5-AAP2-LjAMP2 of acquisition.
Further, the application of described plant expression vector in preparation transgenosis verticillium wilt-resistant cotton.
Further, the transgene cotton preparation method who contains plant expression vector described in p5-AAP2-LjAMP2, comprises the following steps:
(1) dimension pipe specific promoter AAP2 sequence, antibiotic protein gene LjAMP1 or LjAMP2 sequence are operationally inserted respectively in expression vector, built plant expression vector;
(2) the described plant expression vector of step (1) is proceeded to host agrobacterium tumefaciens, obtain transformant;
(3) by transformant, plant expression vector is integrated in cotton, obtains transgene cotton.
Further, a kind of method of cultivating verticillium wilt-resistant cotton, it is characterized in that, above-mentioned p5-AAP2-LjAMP2 plant expression vector is proceeded to respectively to cotton gene group, realize antibiotic protein gene specifically expressing in transgene cotton vascular tissue, predominant expression in root, improves the resistance against diseases of cotton to verticillium especially.
The transgenic cotton floral material of cultivating is less than 15 to non-defoliation verticillium disease index, compared with wild-type contrast, reduces more than 80%.
Beneficial effect of the present invention is, utilizes genetic engineering technique first to build the plant expression vector of AAP2 promotor control LjAMP1 and LjAMP2 antibiotic protein gene, then these plant expression vectors is proceeded to cotton gene group.Regeneration plant after the checking of strict molecular biology, the T of 4-6 sheet true leaf
0for seedling, in the defoliation verticillium wilt pathogen of the indoor inoculation high density of artificial climate, through Resistance Identification and screening, obtained the T that resistance improves
0for strain.T
1generation and T
2in generation,, respectively at phytotron and greenhouse disease pool inoculation defoliation and non-defoliation verticillium wilt pathogen, is further identified the disease resistance of transgene cotton.T
2in generation,, disease-resistant qualification result showed, transgenic line contrasted and compared with wild-type seedling stage, and the disease index of inoculation defoliation verticillium can reduce by 40% left and right, and the disease index of inoculating non-defoliation can reduce more than 80%.The disease index demonstration of plant strain growth later stage stem interior tissue, the disease index of bottom stem interior tissue compared with the control, can reduce more than 60%, and middle part and top can reduce more than 85%.The browning phenomenon of transgenic line plants stems interior tissue mainly concentrates on the middle part vascular tissue of bottom stem section, and around xylem does not partly have macroscopic illness, and all serious brownization in inner each position of wild-type adjoining tree stem.Illustrating that LjAMP1 or LjAMP2 express in vascular tissue can effectively suppress verticillium dahliae and grow in plant inside, and then improves the resistance of cotton to verticillium.Plant strain growth is observed and the result of fibrous quality detection shows, LjAMP1 and LjAMP2 specifically expressing in transgene cotton vascular tissue neither affects growing of plant and fertility, does not also affect cotton fiber quality simultaneously.Therefore, the transgene cotton that the method for utilizing the present invention to cultivate verticillium wilt-resistant cotton obtains not only can improve the resistance against diseases to verticillium, and the material obtaining can be applied to Cotton Production.
The present invention utilizes the dimension pipe specific promoter AAP2 from Arabidopis thaliana to control antibiotic protein gene LjAMP1 and LjAMP2, reach the object of antibiotic protein gene specifically expressing in transgenic cotton plant vascular tissue, effectively control verticillium dahliae further infecting and expanding in plant, can effectively control the development of disease, can alleviate again and even eliminate the harm that antibiotic protein gene overexpression causes vine growth and development simultaneously.
The inventive method is simple and easy to do, and effect is remarkable, and the verticillium wilt-resistant cotton material of acquisition can provide new resource for verticillium wilt resistance of cotton by same breeding, serves Cotton Production, and produces huge economic benefit.The inventive method is not only applicable to cultivate the cotton of resisting verticillium, is equally applicable to the cultivation that other has the disease-resistant material of vascular tissue Characteristics of Damage yet.
Accompanying drawing explanation
Fig. 1 is pMD18-AAP2 collection of illustrative plates.
Fig. 2 is pBI121-AAP2 plant expression vector construction schema.
Fig. 3 is the PCR part amplification of gus gene in AAP2::GUS transgene tobacco;
M: molecular weight marker Marker 15; P: the positive control that the plasmid of take is template; H
2o: the water of take replaces the negative control that DNA is template; 1-12: different transgene tobacco strain numberings.
Fig. 4 is AAP2::GUS transgene tobacco GUS histochemical stain result.
Fig. 5 is the pcr amplification result of AAP2 promotor in transgene cotton;
M15:DNA molecular weight marker Marker15; 1: the positive control that the plasmid of take is template; 2: the negative control that replaces DNA with water; 3-10: different transgene cotton strains.
Fig. 6 is AAP2::GUS transgene cotton GUS histochemical stain result;
The seed rip cutting of A:20d; The embryo of B:30d; C: young stem crosscut; D: blade; E: root.
Fig. 7 is P5-AAP2-LjAMP1 plant expression vector construction schema.
Fig. 8 is P5-AAP2-LjAMP2 plant expression vector construction schema.
Fig. 9 is the schema that pBI121 plant expression vector transform P5 carrier as.
Figure 10 is AAP2::LjAMP1 and AAP2::LjAMP2 transgene cotton GUS histochemical stain result.
Figure 11 is the pcr analysis of LjAMP1 gene in AAP2::LjAMP1 transgenic cotton plant;
Bp: base pair; M:Marker 2000; 1: the contrast of wild-type plant; 2: water negative control; 3: plasmid DNA positive control; 4-13:GUS sun transfer-gen plant.
Figure 12 is the pcr analysis of LjAMP2 in AAP2::LjAMP2 transgenic cotton plant;
Bp: base pair; M:Marker 2000; 1: the contrast of wild-type plant; 2: plasmid DNA positive control; The positive transfer-gen plant of 3-12:GUS; 13: water negative control.
Figure 13 is the quantitative PCR detection result of LjAMP1 gene in AAP2::LjAMP1 transgene cotton.
Figure 14 is the quantitative PCR detection result of LjAMP2 gene in AAP2::LjAMP2 transgene cotton.
Figure 15 is the indoor inoculation defoliation of artificial climate verticillium wilt pathogen 15d, AAP2::LjAMP1 transgene cotton T
0the sickness rate in generation and disease index;
WT: the wild-type plant of regeneration; AAP2-GUS: unloaded carrier transgenic cotton plant; AAP2-LjAMP1-2, AAP2-LjAMP1-3, AAP2-LjAMP1-4, AAP2-LjAMP1-5, AAP2-LjAMP1-6, AAP2-LjAMP1-7 and AAP2-LjAMP1-9:AAP2::LjAMP1 transgene cotton T
0for strain.
Figure 16 is the indoor inoculation defoliation of artificial climate verticillium wilt pathogen 15d, AAP2::LjAMP2 transgene cotton T
0the sickness rate in generation and disease index;
WT: the wild-type plant of regeneration; AAP2-GUS: unloaded carrier transgenic cotton plant; AAP2-LjAMP2-1, AAP2-LjAMP2-2, AAP2-LjAMP2-3 and AAP2-LjAMP2-6:AAP2::LjAMP2 transgene cotton T
0for strain.
Figure 17 is phytotron inoculation defoliation verticillium wilt pathogen 15d, the phenotype of plant.
Figure 18 is phytotron inoculation defoliation verticillium wilt pathogen 15d, the inner illness of plant.
Figure 19 is sickness rate and the disease index that greenhouse disease pool is inoculated non-defoliation verticillium wilt pathogen 25d plant.
Figure 20 is the phenotype that greenhouse disease pool is inoculated non-defoliation verticillium wilt pathogen 25d plant.
Figure 21 is sickness rate and the disease index of greenhouse disease pool inoculation defoliation verticillium wilt pathogen 25d plant.
Figure 22 is the phenotype of greenhouse disease pool inoculation defoliation verticillium wilt pathogen 25d plant.
Figure 23 is the plant strain growth later stage stem interior tissue disease index of inoculation defoliation verticillium wilt pathogen.
Figure 24 is the plant strain growth later stage stem interior tissue disease index of the non-defoliation verticillium wilt pathogen of inoculation.
Figure 25 is the illness of plant strain growth later stage stem interior tissue.
Embodiment
Below in conjunction with specific embodiments and the drawings, the present invention is described in further detail, but below explanation does not limit the present invention, any to distortion of the present invention and change, only otherwise depart from spirit of the present invention, all should belong to the defined scope of claims of the present invention.
Medicine and reagent in the invention process example is not specifically described is domestic conventional chemical reagent, material method be not specifically described all with reference to < < molecular cloning experiment guide > > (Sambrook and Russell, 2001).
The extraction of embodiment 1 DNA of plants and RNA
1, the extraction of plant genome DNA
Choose Arabidopis thaliana, tobacco or cotton (tomato, paddy rice etc. all can) fresh tissues of plants 0.5g, in liquid nitrogen, pulverize rapidly, CTAB extracting solution (the 100mmol/L Tris-HCl (pH8.0) that adds 65 ℃ of preheatings of 3mL, 20mmol/LEDTA (pH8.0), 1.5mol/L NaCl, 2%CTAB (W/V), 4%PVP40 (W/V) and 2% mercaptoethanol (V/V), PVP and mercaptoethanol add before using), quick oscillation mixes.65 ℃ of water-bath 30min, then add 1mL 5mol/LKAc, after ice bath 20min, use isopyknic chloroform: primary isoamyl alcohol (24: 1) extracting 1 time (10,000r/min, 4 ℃ of centrifugal 5min), get supernatant liquor, add-20 ℃ of pre-cold isopropanols of 2/3 times of volume, mix, standing about 30min, with glass rod, choose flocks, 75% ethanol is rinsing several repeatedly, then uses dehydrated alcohol rinsing 1 time, air-dry, be resuspended in 500 μ L TE.Add 1 μ L RNaseA (10mg/mL), process 1h for 37 ℃.Use again phenol (pH8.0): chloroform: primary isoamyl alcohol (25: 24: 1) and chloroform: each extracting of primary isoamyl alcohol (24: 1) 1 time (10,000r/min, 4 ℃ of centrifugal 5min), get supernatant liquor, ethanol precipitation.Precipitate after 75% ethanol rinsing air-dryly, be dissolved in 200 μ L TE ,-20 ℃ save backup.
2, the extraction of RNA
Choose about 3g fresh cotton flowering plant material, in liquid nitrogen, wear into rapidly fine powder, pack 50mL centrifuge tube into, add 15mL RNA extracting solution (2%CTAB (W/V), 2%PVP (W/V), 100mmol/L Tris-HCl (pH8.0), 0.5g/L Spermidine, 2.0mol/L NaCl, 2% mercaptoethanol (V/V adds before use)), put upside down and mix, 65 ℃ of water-bath 3min, during mix 2～3 times.Chloroform: primary isoamyl alcohol (24: 1) extracting 2 times (10,000r/min, room temperature 5min), are got supernatant liquor, add 1/4 volume 10mol/L LiCl solution, place after 6h for 4 ℃, and 12,000r/min, 4 ℃ of centrifugal 20min, abandon supernatant liquor.Precipitation is dissolved with the SSTE solution of 500 μ L.Then phenol (pH4.5): chloroform: primary isoamyl alcohol (25: 24: 1) and chloroform: each extracting of primary isoamyl alcohol (24: 1) 1 time (10,000r/min, room temperature, 5min), adds the dehydrated alcohol of 1/10 volume 3mol/L NaAc solution and 2.5 times of volumes, more than-70 ℃ of refrigerator precipitation 30min, 12,000r/min, 4 ℃ of centrifugal 10min, abandon supernatant liquor, precipitate air-dry.Add the DEPC of 200 μ L to process water dissolution, be stored in-80 ℃ standby.
The clone of embodiment 2 dimension pipe specific promoter AAP2
1, the PCR of vascular-specific expression promotor AAP2 clone
According to the feature of Arabidopis thaliana Amino acid permease gene2 (AAP2) gene, take arabidopsis thaliana genomic dna as template, sequence 1 and sequence 2 are the promoter sequence of primer amplification AAP2 gene, and the reaction system that builds 25 μ L is: 10 * ExPCR buffer is (without Mg
2+) 2.5 μ L; 2.5mmol/L dNTPs 2 μ L; 25mmol/L MgCl2 (magnesium chloride) 2 μ L; Primer 1 (5 μ mol/L) 2 μ L; Primer 2 (5 μ mol/L) 2 μ L; Ex Taq archaeal dna polymerase 1U; The about 60ng of genomic dna.
PCR reaction conditions: 94 ℃ of 5min; 94 ℃ of 1min; 50 ℃ of 1min; 72 ℃ of 2min 30sec (every circulation primary temperature reduces by 1 ℃); 94 ℃ of 1min; 40 ℃ of 1min; 72 ℃ of 2min 30sec (25 circulations); 72 ℃ of 10min.
2, the recovery of amplified production
PCR product electrophoresis on sepharose is quantitative, reclaims test kit (Roche) reclaim amplified fragments with DNA fragmentation, and all operations is all undertaken by test kit specification sheets.
3, connect and transform
The AAP2 that pcr amplification is obtained reclaims fragment and is cloned into pMD18-T (TaKaRa) carrier (Fig. 1) by ligase enzyme test kit specification sheets.Then will connect product and transform escherichia coli DH5a competent cell, screening positive clone according to a conventional method, identifies the fragment of plasmid and insertion.
4, sequential analysis
The AAP2 promoter fragment of acquisition is carried out to sequencing, and result shows, PCR gained fragment total length 2148bp, and the long 2131bp of AAP2 promoter fragment, sequencing result is shown in sequence 11.
Embodiment 3 dimension pipe specific promoter AAP2 plant expression vector constructions
1, dimension pipe specific promoter AAP2 plant expression vector construction
For analyzing the expression characterization of AAP2 dimension pipe specific promoter, AAP2 promoter sequence is replaced to the CaMV35S promoter sequence on pBI121 carrier, obtain plant expression vector pBI121-AAP2.Vector construction schema is shown in Fig. 2.
2, integrate the agrobacterium tumefaciens host's of pBI121-AAP2 expression vector conversion
Adopt freeze-thaw method that pBI121-AAP2 plant expression vector is imported in agrobacterium tumefaciens.Specific procedure is: the plasmid DNA of 0.5～1 μ g is joined in 100 μ L competent cells, mix rear ice bath 30min.After liquid nitrogen flash freezer 5min, 37 ℃ of temperature are bathed 5min, immediately ice bath 2min.Add 1000 μ L liquid YEB, 200r/min, 28 ℃ cultivate after 3～5h, centrifugal collection thalline, and with 100 μ L liquid YEB, (5g/L sucrose+1g/L is yeast extract+10g/L tryptone+0.5g/L MgSO for bacterium for bacterium
47H
2o, pH7.0) resuspended thalline evenly coat the YEB flat board containing 125mg/L Sm (Streptomycin sulphate) and 50mg/L Km (kantlex).
The expression characteristic of embodiment 4 AAP2 promotors in tobacco
1, tobacco genetic transformation substratum
Minimum medium: MSB0 (MS is inorganic+and B5 is organic+30g/L sucrose, pH5.8).Solid medium adds agar (Murashige and Skoog, 1962 of 6g/L; Gamborg etc., 1968);
Be total to culture medium MSB1:MSB0+2.0mg/L 6-BA (6-benzyl aminopurine)+0.1mg/L NAA (α-naphthaleneacetic acid)+6g/L agar;
Screening culture medium MSB2:MSB1+500mg/L Cef+50mg/L Km+6g/L agar first;
Subculture medium MSB3:MSB1+200mg/L Cef+50mg/L Km+6g/L agar;
Root media MSB4:MSB0+0.1mg/L NAA+200mg/L Cef+50mg/L Km+6g/L agar.
2, the genetic transformation of tobacco
(1) transform the preparation with During Agrobacterium liquid
Utilize method of scoring to obtain the single bacterium colony of Agrobacterium of integration plant expression vector pBI121-AAP2, then picking list colony inoculation enters to add 50mg/L Km and 125mg/L Sm 10mL liquid YEB, 28 ℃, 200rpm overnight incubation, then in 5% ratio, bacterium liquid is inoculated into 20mL containing antibiotic liquid YEB, 28 ℃, 200rpm are cultured to OD600 and are about 0.5.Get the centrifugal 5min of 5mL bacterium liquid 6000rpm and collect thalline, then use the resuspended thalline of 5mL MSB0 liquid nutrient medium, resuspended bacterium liquid to be the During Agrobacterium liquid of contaminating explant.
(2) tobacco genetic transformation
Method for transformation with reference to (1985) such as Horsch is also improved.Concrete operations are:
Mercuric chloride (the HgCl of tobacco seed 0.1%
2) sterilization 5-8min, aseptic water rinses 5-6 time, and 25 ℃, photoperiod of 16h illumination/8h dark are sprouted approximately one month in solid MSB0.The aseptic seedling young leaflet tablet of robust growth, is cut into the leaf dish of about 0.5cm * 0.5cm.During Agrobacterium immersion is dyed leaf dish 5min.The bacterium liquid that inclines, aseptic thieving paper sucks the unnecessary bacterium liquid of leaf panel surface, then inoculates into the common cultivation MSB1 substratum that is covered with one deck filter paper 25 ℃ of dark 2d that cultivate.After having cultivated altogether, leaf dish is inoculated and in screening culture medium MSB2, carried out differentiation culture, 25 ℃, photoperiod of 16h illumination/8h dark are cultivated 2 weeks, then leaf dish subculture are entered to the generation of MSB3 substratum callus induction, and every 2 weeks subcultures once.Produce after Km resistance young shoot, young shoot is cut and inoculates the root media into MSB4, obtain Km resistance regeneration plant, and breed in greenhouse.
3, the PCR of transgene tobacco checking
Take sequence 3 and sequence 4 is primer, and each strain tobacco gene group DNA is template amplification gus gene, to verify whether gus gene has been integrated into transgenic tobacco plant.The genomic dna that 25 μ L amplification reaction systems comprise about 50ng tobacco, 2.5 μ L 10 * PCR buffer, 2 μ L 2.5mmol/L dNTPs, 1.5 μ L 25mmol/L MgCl
2(magnesium chloride), each 1 μ L of the upstream and downstream primer of 5 μ mol/L, 1U Taq archaeal dna polymerase.Amplification program is: 94 ℃, and 5min; 94 ℃, 30sec, 56 ℃, 30sec, 72 ℃, 2min30sec, 30 circulations; 72 ℃ are extended 10min.Amplified production 1% agarose gel electrophoresis detects, and part amplification is shown in Fig. 3.In 20 Km resistance strains that regeneration obtains, there are 15 strain amplifications to obtain the specific band of 1810bp left and right, illustrate that AAP2::GUS has been incorporated into the genome of tobacco.
4, the expression characteristic of gus gene in transgene tobacco
Method with reference to Jefferson (1978), tender of pBI121-AAP2 transgenic tobacco plant children, stem, leaf, floral organ and the seed of the PCR test positive of take reaction are material, cut a little tissue and put into GUS staining fluid (500mg/L X-Gluc, 0.1mol/L K
3fe (CN)
6, 0.1mol/L K
4fe (CN)
6, 1%Triton X-100 (v/v), 0.01mol/L Na
2eDTA, 0.1mol/L phosphoric acid buffer (pH7.0)) carry out GUS histochemical stain.Result shows (Fig. 4), and the gus gene of AAP2 regulation and control mainly concentrates on interior expression of vascular tissue of root, stem and the blade vein etc. of transgene tobacco.In addition, the epidermis of young tender seed also has the expression of gus gene.
The expression characteristic of embodiment 5 AAP2 promotors in cotton
1, the conventional substratum of Agrobacterium tumefaciens mediated Cotton Transformation
Minimum medium: MSB (MS inorganic salt+B5 is organic) (T.Murashige, 1962; O.L.Gamborg, 1968);
Seed germination substratum: 1/2MSB+20g/L sucrose+6g/L agar, tap water preparation, natural pH;
Be total to culture medium: MSB+0.5mg/L IAA (indolylacetic acid)+0.1mg/L KT (6-Furfurylaminopurine)+30g/L glucose+100 μ mol/L Syringylethanone+2.0g/L Gelrite (Sigma), pH5.4;
The de-bacterium culture medium of screening: MSB+0.5mg/L IAA+0.1mg/L KT+75mg/L Km+500mg/L cef (cephamycin)+30g/L glucose+2.0g/L Gelrite, pH5.8;
Calli induction media: MSB+0.5mg/L IAA+0.1mg/L KT+75mg/L Km+200mg/L cef+30g/L glucose+2.0g/L Gelrite, pH5.8;
Embryo callus subculture inducing culture: MSB+0.1mg/L KT+30g/L glucose+2.0g/L Gelrite, pH5.8;
Fluid suspension culture base: MSB+1.91g/L saltpetre+0.1mg/L KT+30g/L glucose, pH5.8;
Body embryo maturation medium: MSB+15g/L sucrose+15g/L glucose+0.1mg/L KT+2.5g/L Gelrite, pH6.0;
Seedling substratum: SH+0.4g/L activated carbon+20g/L sucrose, pH6.0.(Schenk?&?Hildebrandt，1972)
2, Cotton Transformation concrete operation method
(1) transform the acquisition of explant and the preparation of During Agrobacterium liquid
Upland cotton seed shells, and seed benevolence 0.1% mercuric chloride sterilizing 10min, after aseptic water rinsing 5-6 time, is inoculated in seed germination substratum, 28 ℃ of dark 5-7d that cultivate.Aseptic hypocotyl is cut into the long segment of 3-5mm, as transforming explant.
Preparation method by embodiment 4 tobacco genetic transformation During Agrobacterium liquid obtains Cotton Transformation During Agrobacterium liquid.
(2) induction of hypocotylar genetic transformation and embryo callus subculture
Explant dyes 20min by During Agrobacterium immersion, bacterium liquid inclines, with aseptic filter paper, suck the unnecessary bacterium liquid in explant surface again, hypocotyl segment after dip-dye is inoculated in common culture medium, 26 ℃ of dark 2d that cultivate, hypocotyl is seeded to the de-bacterium culture medium of screening, the calli induction media of the additional kantlex (Km) of the follow-up substitution of 20d and cephamycin (cef) carries out the induction of callus, interval 20d subculture once, the follow-up substitution embryo callus subculture of 60d inducing culture, after obtaining embryo callus subculture, carry out fluid suspension culture, to obtain the consistent embryo callus subculture of raised growth.
(3) induction of body embryo and seedling are cultivated
The embryo callus subculture of fluid suspension culture, 30 order stainless steel sift net filtrations, the embryo callus subculture under sieve is inoculated into body embryo maturation medium dispersedly, and about 15d produces a large amount of body embryos, and its subculture is entered to SH substratum, promotes the further seedling of body embryo.The regrowth of 3-4 leaf is transplanted into greenhouse and breeds.
3, the Molecular Detection of transgene cotton
Take regeneration plant young leaflet tablet as the total DNA of material extraction plant, and take wild-type plant as contrast.Take DNA as template, and sequence 1 and sequence 2 are primer, amplification AAP2 promoter sequence.In 13 Km resistant cotton strain seedling, there are 8 strain amplifications to obtain the specific band (Fig. 5) of 2000bp left and right, show that AAP2 promotor has been incorporated into cotton gene group.
4, the expression characteristic of AAP2 promotor in transgene cotton
In plant expression vector, utilize AAP2 promotor to control gus gene, therefore, can utilize GUS histochemical staining method to study rapidly the expression characteristic of AAP2 promotor in transfer-gen plant.With reference to the method for Jefferson (1978), tender of transgene cotton children, stem, leaf, floral organ and the rataria of PCR test positive of take carries out GUS histochemical stain as material.Result shows (Fig. 6), and the gus gene that AAP2 promotor is controlled mainly concentrates on interior expression of middle part vascular tissue of Radix Gossypii.In addition, the expression of gus gene on the inner seed coat of rataria seed, detected, and the expression of gus gene in mesophyll cell, plumule and cotyledon, do not detected.
The structure of embodiment 6 AAP2::LjAMP1 and AAP2::LjAMP2 plant expression vector
1, the clone of LjAMP1 and LjAMP2 gene
The LjAMP1 of purifying and LjAMP2 antibacterial protein carry out n terminal amino acid sequencing, on the basis of order-checking, utilize 3 ' RACE (Schaeler et al., 1995) and YADE (Xiao Yuehua etc., 2002) technology is cloned its full-length gene, see sequence 12 and sequence 13, and respectively LjAMP1 and LjAMP2 are cloned into pUC-18 carrier.Sequential analysis shows, it is the open reading frame of 348bp that LjAMP1 contains long, and LjAMP2 contains that to grow be the open reading frame of 288bp.All operations is all undertaken by test kit specification sheets.
2, the structure of AAP2::LjAMP1 and AAP2::LjAMP2 plant expression vector
In order to build AAP2::LjAMP1 and AAP2::LjAMP2 plant expression vector, first utilize BamH I and SacI double digestion pBI121, pUC-LjAMP1 and pUC-LjAMP2 carrier respectively, with LjAMP1 and LjAMP2 gene order, substitute respectively the gus gene sequence on pBI121 carrier, recycling EcoR I and HindIII be double digestion pBI121-LjAMP1, pBI121-LjAMP2 and P5 expression vector respectively, and the Expression element of 35S being controlled to LjAMP1 and LjAMP2 gene is cloned into respectively P5 plant expression vector.And then the 35S promoter of target gene Expression element is replaced into AAP2 promotor, obtain AAP2::LjAMP1 and AAP2::LjAMP2 plant expression vector, and difference called after P5-AAP2-LjAMP1 and P5-AAP2-LjAM2.Build schema and see Fig. 7 and Fig. 8.All restriction enzymes all, purchased from Roche company, have operated according to working instructions.
P5 plant expression vector is to reelect conventional pBI121 carrier to obtain, and the schema of transformation is shown in Fig. 9.
The acquisition of embodiment 7 AAP2::LjAMP1 and AAP2::LjAMP2 transgene cotton
P5-AAP2-LjAMP1 and P5-AAP2-LjAMP2 plant expression vector are after electric shock transformation method proceeds to agrobacterium tumefaciens lba4404, according to the Cotton Transformation of embodiment 5 and renovation process, AAP2::LjAMP1 and AAP2:::LjAMP2 are proceeded to cotton gene group.Go through the induction of Km kanamycin-resistant callus tissue, embryo callus subculture and body embryo, body embryo seedling, then obtains AAP2::LjAMP1 and AAP2::LjAMP2 transgenic cotton plant.
The Molecular Detection of embodiment 8 AAP2::LjAMP1 and AAP2::LjAMP2 transgene cotton
1, the GUS histochemical stain of transfer-gen plant
AAP2::LjAMP1 and AAP2::LjAMP2 plant expression vector contain the gus gene that 35S promoter is controlled, therefore, first transfer-gen plant can utilize GUS histochemical staining method to carry out Rapid identification, after tender root, stem and the vanes GUS dye liquor of transfer-gen plant children dyes, occur that the blueness shown in Figure 10 is transgenic positive plant, otherwise be non-transgenic plant.
2, antibiotic protein gene LjAMP1 and the LjAMP2 pcr analysis in transgene cotton
Take Young Cotton leaflet tablet as material, extract total DNA of transfer-gen plant and wild-type plant, then take total DNA as template, sequence 5 and 6 is primer amplification LjAMP1 gene fragment, and sequence 7 and 8 is primer amplification LjAMP2 gene fragment.Pcr amplification adopts 25 μ l reaction systems to carry out.
PCR reacts the total system of 25 μ l, comprises 1 * PCR buffer, the MgCl of 1.5mmol/L
2, the dNTPs of 0.2mmol/L, upstream and downstream primer is 0.2 μ mol/L, 25ng DNA, 1U Taq archaeal dna polymerase.
PCR reaction parameter: 94 ℃ of pre-amplification 5min, then 94 ℃, 30s; 55 ℃, 30s; 72 ℃, 60s; 30 circulations, finally again 72 ℃ extend 10min.Amplified production 1.0% agarose gel electrophoresis detects.The part amplification of LjAMP1 and LjAMP2 is shown in respectively Figure 11 and 12.Result shows, all transfer-gen plants through GUS test positive can increase and obtain the special band of target of LjAMP1 or LjAMP2.
3, antibiotic protein gene LjAMP1 and the LjAMP2 quantitative RT-PCR analysis in transgene cotton
The young tender plant root of two true leaves of take, stem and blades are material, extract respectively the RNA of transgenosis and wild-type plant root, stem and blade, a chain cDNA of synthetic each sample RNA of by specification, then take cDNA as template increase respectively LjAMP1 or LjAMP2 specific fragment.The upstream and downstream primer of LjAMP1 gene is respectively sequence 5 and sequence 6, and the upstream and downstream primer of LjAMP2 gene is respectively sequence 7 and sequence 8.The cotton histone HIS3 gene of take is interior mark.The primer of HIS3 is sequence 9 and sequence 10 (Zhu YQ etc., 2003).
25 μ L reaction systems comprise 1 μ L cDNA mono-chain product, 0.2 μ mol/L special primer and 1 * iQ
tMsYBR GreenSupermix.Linear amplification program: 94 ℃ of 5min; 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 1min, 40 circulations of increasing.
Quantitative RT-PCR result shows (Figure 13 and 14), and LjAMP1 and LjAMP2 gene can effectively carry out transcriptional expression in transgenic cotton plant, and a large amount is expressed in root, in stem, expresses in right amount.Wild-type plant root, stem and Ye Nei do not detect the expression of LjAMP1 and LjAMP2 gene.
The disease-resistant authentication method of embodiment 9 transgene cottons to verticillium
1, the preparation of pathogenic bacteria for disease-resistant evaluation inoculation
Defoliation and non-defoliation verticillium wilt pathogen that a little solid PDA of picking preserves are inoculated the substratum into liquid PD, 180rpm, 25 ℃ of shaking culture 7d, in the ratio of 10% (bacterium liquid/PD substratum), inoculate the substratum into liquid PD again, 180rpm, 25 ℃ of shaking culture 10d, filter and remove mycelia and the impurity in bacteria-removing liquid with four layers of sterile gauze, and deionized water is adjusted defoliation verticillium wilt pathogen spore concentration and reached 10
8individual/ml, non-defoliation verticillium wilt pathogen spore concentration reaches 10
9individual/ml is as inoculation bacterium liquid.
2, the indoor disease-resistant evaluation pathogenic bacterium inoculation method of artificial climate
T
0for transgene cotton 4-6 sheet true leaf, all the other are 3-4 sheet true leaf from generation to generation, and growth phase adopts and hinders root filling bacterium liquid method inoculation pathogenic bacterium consistent seedling plant.While transplanting engagement alms bowl, bacterium liquid 100mL is slowly watered in every strain, allows the moistening soil group that has cotton plants of bacterium liquid as far as possible, and inoculation is watered permeable two days later, to keep the humidity of soil in basin alms bowl.After inoculation, in 20 ℃ of (night)-25 ℃ (daytime), humidity more than 80%, is grown under the dark photoperiod condition of cultivating of 14h illumination/10h, and inoculation 15d is by (0 grade: cotton plants appearance is without illness of 5 grades of sick grade standard; 1 grade: cotton plant blade is aobvious illness below 1/3; 2 grades: cotton plant blade 1/3-2/3 shows illness; 3 grades: cotton plant blade more than 2/3 aobvious illness; 4 grades: illness all appears in cotton plant blade, blade and flower abscission are serious or plant polished rod is even dead) add up the sick level of plant, and calculate sickness rate and the disease index of plant.T
0it is contrast that wild-type and unloaded carrier transgene cotton seedling are all take in each inoculation of generation.All the other each take from generation to generation wild-type and not the separated GUS feminine gender non-transgenic plant of homozygous lines be contrast.Defoliation verticillium wilt pathogen inoculum density is 10
8individual spore/ml, non-defoliation verticillium wilt pathogen inoculum density is 10
9individual spore/ml.
Sickness rate=(there is illness plant quantity/inoculation plant sum) * 100%;
Disease index=[∑ (sick progression * strain number)/(total strain number of 4*)] * 100
3, disease-resistant evaluation pathogenic bacterium inoculation method in greenhouse
T
2while entering greenhouse for homozygous lines 3-4 sheet true leaf seedling replanting, employing is hindered root filling bacterium liquid method and is inoculated respectively defoliation and non-defoliation verticillium wilt pathogen.By the density plantation of 2500 plants/acre, every strain pouring verticillium wilt pathogen liquid 200ml during transplanting allows the drenched nutrition of bacterium liquid group as far as possible, then in seedling, holds up moistening soil around, transplant water two days later permeable, to guarantee seedling normal growth in greenhouse.After inoculation 25d, add up sickness rate and the disease index of plant.And in the plant strain growth later stage, cane is divided into upper, middle and lower three parts, then cut open bar by (0 grade: stem inside is without variable color of 5 grades of sick grade standard; 1 grade: the dimmed brown of cane partly accounts for below 25% of section; 2 grades: variable color partly accounts for 50%; 3 grades: variable color partly accounts for more than 70%; 4 grades: the whole dimmed brown of cane xylem) the sick level of statistics cane different sites interior tissue, and calculate disease index.It is contrast that the inoculation of take is located away from the not negative plant of GUS and the wild-type plant of Transgenic wheat line, and the contrast of wild-type plant is not inoculated in setting.In greenhouse, inoculation test is arranged by random district group, and in triplicate, each repeats 30 strain seedling to each material.Defoliation verticillium wilt pathogen inoculum density is 10
8individual spore/ml, non-defoliation verticillium wilt pathogen inoculum density is 10
9individual spore/ml.
The indoor transgene cotton T of embodiment 10 artificial climate
0resistance for seedling to verticillium
Artificial climate is indoor, AAP2::LjAMP1 transgenic line plant inoculation different from AAP2::LjAMP2 defoliation verticillium wilt pathogen 15d, the sickness rate of part strain and disease index are compared with the control, all significantly reduce, wherein the sickness rate of AAP2-LjAMP1-5 and AAP2-LjAMP2-1 strain and disease index are all that the sickness rate of 0, AAP2-LjAMP1-4 and AAP2-LjAMP2-2 and disease index are all lower than 50% and 50 (Figure 15 and 16).Inoculation 15d, wild-type adjoining tree seriously falls ill, most leaf abscissions, and disease-resistant plant leaf does not have illness, leaf look bud green, growth normal (Figure 17).The sickness rate of transgenic cotton plant, disease index and illness show, AAP2::LjAMP1 and AAP2::LjAMP2 transgene cotton T
0in generation, is to the good resistance of defoliation verticillium tool.
Stem section and the petiole of the disease-resistant plant of transgenosis of take is material, respectively it carried out to crosscut and rip cutting, and stem section and the petiole of wild-type adjoining tree of the inoculation same period of take is contrast, the illness of stereoscopic Microscopic observation stem interior tissue.Result shows (Figure 18), and disease-resistant plants stems and petiole inside do not have obvious browning phenomenon, illustrates that this part plants stems inside is not subject to infecting of verticillium wilt pathogen or infects slightly, does not occur obvious illness.And adjoining tree stem and the serious browning of petiole interior tissue, and produced chocolate patch.
Embodiment 11 transgene cotton T
2resistance for seedling greenhouse disease pool to verticillium
T
1for seedling, respectively at phytotron, proceed after disease-resistant evaluation, the transgene cotton strain that disease resistance improves, in warm indoor propagation, is screened the T that isozygotys
2for strain plant, and this part strain plant is further carried out to disease-resistant evaluation.
One, the resistance of CpTI-Bt Transgenic Cotton at Seedling in greenhouse disease pool
1, the resistance of transgene cotton to non-defoliation verticillium
T isozygotys
2for 3-4 sheet true leaf seedling greenhouse disease pool, inoculate non-defoliation verticillium wilt pathogen 25d, the wild-type plant of inoculation all falls ill, the sickness rate of transgenic line compared with the control, reduces more than 60% significant difference (0.01 < P < 0.05); The disease index of transgenic line contrasts with wild-type to compare and can reduce more than 80.0%, and extremely significantly (P < 0.01) (Figure 19) for difference.Illustrate, LjAMP1 and LjAMP2 gene specifically expressing in transgenic cotton plant vascular tissue can effectively improve the resistance of cotton to non-defoliation verticillium.The phenotype of inoculation 25d plant is shown in Figure 20, and illness has all appearred in wild-type plant leaf, and transgenic cotton plant blade illness is not obvious or illness is slight.
2, the resistance of transgene cotton to defoliation verticillium
T isozygotys
2for 3-4 sheet true leaf seedling, inoculate type verticillium wilt pathogen 25d, wild-type inoculation adjoining tree all falls ill, and disease index reaches 93.7.The disease index of transfer-gen plant contrasts with wild-type to compare and can reduce by 40% left and right (Figure 21).Illustrate that LjAMP1 and LjAMP2 gene specifically expressing in transgenic cotton plant vascular tissue can improve the resistance of cotton to defoliation verticillium.The phenotype of inoculation 25d plant is shown in Figure 22, there is obscission because of the infection blade of verticillium wilt pathogen in wild-type plant, the wild-type plant of most inoculations is because of serious morbidity death, and transgenic cotton plant blade illness is not obvious or slightly morbidity, and plant strain growth is normal.
Two, transgene cotton T
2resistance for the stem interior tissue performance of plant strain growth later stage
1, the resistance of transgene cotton to non-defoliation verticillium
Greenhouse disease pool is inoculated after the wild-type and transgenic line plant results fiber and seed of non-defoliation verticillium wilt pathogen, and plant cane is divided into upper, middle and lower three parts, and then vertical profile stem section, adds up the disease index of stem interior tissue, the results are shown in Figure 24.The disease index on AAP2-LjAMP1-4 strain plant bottom, middle part and top be respectively 37.5,12.5 and 12.5, AAP2-LjAMP2-2 strain plant be respectively 50.0,37.5 and 25.0, all significantly lower than 100.0 of wild-type plant.Result shows, two transgene cotton strains of AAP2-LjAMP1-4 and AAP2-LjAMP2-2 have good disease resistance to non-defoliation verticillium.
2, the resistance of transgene cotton to defoliation verticillium
After wild-type plant inoculation defoliation verticillium wilt pathogen, plant seriously falls ill, gradually dead after leaf abscission, and this part plant is cutd open the disease index of stem upper, middle and lower interior tissue after bar and all reaches 100.0.And after transgenic line inoculation defoliation verticillium wilt pathogen, the slight morbidity in early stage of most plant, plant strain growth process Leaf illness does not increase the weight of, and to later stage plant strain growth there is illness in normal or lower blade.After results fiber and seed, plant cane is divided into upper, middle and lower three parts, the disease index of stem interior tissue is shown in Figure 23.The disease index on AAP2-LjAMP1-4 strain plant bottom, middle part and top be respectively 62.5,45.0 and 20.0, AAP2-LjAMP2-2 strain plant be respectively 50.0,30.0 and 22.5, all significantly lower than 100.0 of wild-type contrast.Result shows, two strains of AAP2-LjAMP1-4 and AAP2-LjAMP2-2 have good disease resistance to defoliation verticillium.
3, the illness of plant strain growth later stage stem inside
The inner whole brownization variable colors of wild-type contrast cotton plants stem of greenhouse disease pool inoculation defoliation and non-defoliation verticillium wilt pathogen.The brownization variable color of transfer-gen plant stem inside mainly concentrates on plant lower central vascular tissue, and around xylem and middle and upper part do not have obvious brown spot (Figure 25).The illness explanation of plants stems inside, LjAMP1 and LjMP2 specifically expressing in cotton vascular tissue can obviously suppress defoliation and the growth of non-defoliation verticillium wilt pathogen in plant, and verticillium is shown to good resistance.
Above-mentioned example shows, the present invention utilizes dimension pipe specific promoter AAP2 to control Motherwort Herb antibiotic protein gene LjAMP1 and LjAMP2, realizing antibiotic protein gene a large amount in transgene cotton root expresses, in stem, express in right amount, can effectively improve the resistance of transgene cotton to defoliation and non-defoliation verticillium.The inventive method is simple and easy to do, and effect is remarkable, has good market outlook.
Sequence table
Sequence 1
AAP2 promotor clone primer 1:5 '-TCTAGAGTTAAACCGAACTATTTTTGATTGCT-3 ' (30bp)
Sequence 2
AAP2 promotor clone primer 2: 5 '-ACTAGTATGAGAGAAAGAGAAAGAGAGAACA-3 ' (31bp)
Sequence 3
Gus gene PCR checking primer 1:5 '-GTCAGTCCCTTATGTTACGTCCTGTAGA-3 ' (28bp)
Sequence 4
Gus gene PCR verifies primer 2: 5 '-GCCAGGAGAGTTGTTGATTCATTG-3 ' (24bp)
Sequence 5
LjAMP1 gene PCR detects primer 1:5 '-TGCACAATGCTGATCGCG-3 ' (18bp)
Sequence 6
LjAMP1 gene PCR detects primer 2: 5 '-TGGCAAGCGTTTTCAGGC-3 ' (18bp)
Sequence 7
LjAMP2 gene PCR detects primer 1:5 '-CCAGTTCCGCTCCTGCCAAAG-3 ' (21bp)
Sequence 8
LjAMP2 gene PCR detects primer 2: 5 '-CGATACACCTCCTCGCTCT-3 ' (19bp)
Sequence 9
Cotton HIS3 gene amplification primer 1:5 '-GAAGCCTCATCGATACCGTC-3 ' (20bp)
Sequence 10
Cotton HIS3 gene amplification primer 2:5 '-CTACCACTACCATCATGGC-3 ' (19bp)
Sequence 11
AAP2 promotor full length sequence (2148bp, containing the restriction enzyme site adding)
1?TCTAGACTCA?AAAGCTCCAG?TCTTAGATAA?GGTGAATACA?TGGAAGGAAC?GGTCAAAGAC?GTGTAAGCAG
71?CCCGGAAAGC?AGGTGAAGGG?TGTCGCGCAC?CCCTTTTAAA?TATCGACTTC?ACCCTTGAAA?GCTCGGAGTA
141?CACCACAGAT?GCATCACTAA?AGATTTTGTC?GGCACGCTGA?AGTAAGCTAT?CTCGTGCCTC?CTCATAAGCT
211?GAAGTCTCAC?AGTCACTCTC?ATCATCGGTG?CTTGAGTCTC?CTTCTACTAT?CAGGGAATAT?CCATCAGCCT
281?GCTTTCTCTA?TTCTAGAATA?GCTGAAGCAT?GTCTTTCAGT?AATTACCTTC?ATCTGATCTT?CAATTGCCGT
351?GATTAAGTAA?CCGTTTTTCT?GCGAAAGAAA?GAAGATTGAC?AACAGCCGTT?TGATGGAAAT?GAAAGCACCA
421?GATACACAGA?CAAATGCAAC?ACATATTCCC?TTGGAAAAGG?ACAAAAGTAA?AAGAGCAGGC?AGAAGAGGAG
491?TACCTGCATA?AAGTTGCGGA?AAACAGATTT?GAAGTCACTG?ATTTCCTGCA?AAGAGGCATA?CTTGTAAACA
561?GGAGCAGATG?GACAACTTTC?AGCAGCTGCG?ATGCTCATAG?TCAAATTCTC?ATCAGGTTCC?TGGATCATCA
631?GAAACCAAAT?TAGATAGAAC?AATAAATAGA?AAGAAGGCAA?GTTCAAAGAG?CAATACCTTA?GCAAGCTCCT
701?CTGGTCTCCC?GGCATACATC?TGCGGTTGCT?CCTGTGGCAT?ACGCGAATTC?ACTCTTTCAT?CATAAGCATT
771?GCATATTCTA?TCAAGCGCAG?CCATCTTATC?ATCAGCTGTT?TGCGGCATCG?GACAGACGAA?GACATAAGAC
851?AAACCATCCA?TACCAATGCT?GCGCACTCGC?TCACCGTTGA?ATACTATCTC?CATGGGCCTC?TCATCCTGAC
921?TATGTTCCTT?CAAACGCTGC?TGCATCTGCT?CTTGCGTATA?AAATTCGTCC?CCCCTTGATA?AGTCGAGCAG
991?CCAAGAGGAT?TTGGCATGGT?CTCTGCCCTT?CGCATCGTCG?TCGTTCTTCG?TGGGAATGGC?CGGGAGCTTT
1061?CTACGTTTCC?ACGGGTAAAG?ATCAGAAGAG?GAAGGTTTCG?CCGGGTAAAG?ATCAGAAGAG?GAAGGTTTCG
1131?CCGCGGCGGT?TGCATCTTCA?CCGTCGATTT?CATCGTTACA?GCGACGCCGG?TAATTCCTAG?GTTGCTTAGT
1201?TCCCATTCTC?TCTCTAAAAT?TAGGGCTCGA?AATGAATTGT?TGAACAAGAT?AGAGATCTTT?TTCTGATCCC
1271?CGTCGAACAT?TTATTCAAGG?CCAAAAAAAG?CACACGGGAA?TTTAGAGTAC?CAATACATAT?CAAAACCTAA
1341?TGGGCTTTGA?ATGGTTGCAT?GTGTGTGTTT?ATTTCTGATA?TGCAAAGCGA?TCGATAGTCT?TTTCCATACA
1411?AGTGTAAACT?GTAAACAACG?TAATTAAGCA?TAACAATACA?ACTCTTTCTT?CTCTTTTTTT?TTGTAAACAC
1481?AAAATAAAAT?TACATCAATT?CATGCTTTTC?CTAGTTCATC?TGACATTTTC?CAAAATTCAT?GTTCCATTGA
1551?GTCCCTAATA?CTTGTTCATA?TTCATATTAG?GGTACATGAA?TAAAAGTTAT?CATTCTTGAA?ACTACTAAAT
1621?TTTCATAGTT?TATTTTTCTT?CTTTTCGTTT?CACTTTCGAA?CAAAACACTA?CGCGTGGCAT?TTGCAATGAA
1691?TTCCACATTA?TATGGAATAA?CACCATGATG?AACATTCTAC?ATATATAATT?ATTATGTTTA?AGCACTTAGA
1761?CAGCATAAAT?TCTTTCTAAT?TATATAAATC?TAACCTTGTT?ACATTGTACA?TCTATAAATT?ACTTGAAGAA
1831?ATAACGAGTT?CTATTTCTTT?TTAAAAATTA?AAAATACTAT?ACCATATCTC?AGTGATTAAG?TTGAACCAAA
1901?AGGTACGGAG?GAGAAACAAG?CATTTGATTC?TTCCTTATTT?TATTTTATTC?ATCTCTCACT?AATGATGGTG
1971?GAGAAAAAAA?GAAAATACCT?AACAAACAAA?TATATATTGT?CATACAAAAA?TATTTCTATA?TTTTTAGTTA
2051?ATTAGTTTAT?ATTCCTCACT?TTTCAGGGCT?TATATAAGAA?AGTGAGCAAA?CACAAATCAA?AATGCAGCAG
2201?CAAATACTAT?CATCACCCAT?CTCCTTAGGA?TTCCTAGTCA?TAACTAGT
Sequence 12
LjAMP1 full length gene sequence (348bp)
1?ATGGCTGCCT?TGATCAAGTT?GATGTGCACA?ATGCTGATCG?CGGCGGCGGT?GGTTGCTCCG?TGGCTGAGGC
71?GGCGATAGGG?TGCAACACGG?TGGCTTCCAA?GATGGCCCCA?TGTCTACCGT?ACGTCACCGG?AAAAGGGCCG
141?CTCGGCGGGT?GCTGCGGTGG?CGTAAAGGGT?CTCATCGAC?GCCGCACGGA?CCACGCCGGA?TAGGCAGGCG
211?GTTTGCAACT?GCCTGAAAAC?GCTTGCCAAG?TCGTACTCCG?GCATCAACCT?CGGCAACGCC?GCCGGACTCC
281?CCGGAAAATG?TGGTGTCAGC?ATTCCTTACC?AGATCAGCCC?TAATACTGAT?TGCTCAAAGG?TGCACTGA
Sequence 13
LjAMP2 full length sequence (288bp)
1?ATGCTGCAGG?GTCGCCAGTT?CCGCTCCTGC?CAAAGCTACC?TTAGGCAGCG?TGGGAATGTT?CTAGAAATGG
71?CCACCGGAAA?CCCTCAGTCG?CAGACGGTTG?AAGAATGCTG?CGAGAGTCTG?AAGGATATTG?AGCGGAAACA
141?GCAGCAATGC?GGGTGTGAAG?CCATCAAGCA?CGCGATGAGG?CAGATGCAGG?GGGGGCAGAG?CGAGGAGGTG
211?TATCGAAAGG?CTAGGATGCT?GCCACGTACT?TGCGGATTAA?GGTCACAGCA?ATGCCAGTTT?AATGTTATCT
281?TTGTGTAG
Claims (8)
1. tie up pipe specific promoter and control a plant expression vector for antibiotic protein gene, it is characterized in that, for containing dimension pipe specific promoter AAP2, control antibiotic protein gene
ljAMP1plant expression vector, described carrier p5-AAP2-LjAMP1, its construction step is as follows: with the promotor from Arabidopis thaliana AAP2 gene, replace the constitutive promoter CaMV35S on plant expression vector p5-LjAMP2, build a new expression of plants body, called after p5-AAP2-LjAMP2;
Wherein, promotor AAP2 is obtained by sequence 1 and sequence 2 amplifications, and described promotor AAP2 is as shown in sequence 11;
ljAMP1for the nucleotide sequence shown in gene order 12;
P5 plant expression vector is to reelect conventional pBI121 carrier to obtain; Near pBI121 right margin, there is a selectable marker gene
nPTII, and Nos promotor and terminator; Near left margin under the control of CaMV35S promotor
gUSreporter gene; Transformation process is divided into following steps:
(1) first utilize
ecor I enzyme carries out single endonuclease digestion, and pBI121 carrier is cut;
(2) otch is scabbled, recycling DNA ligase connects, original to remove on pBI121 carrier
ecorI restriction enzyme site, the carrier called after pBI121-R after this step;
(3) utilize
hind III is carried out single endonuclease digestion, will remove
ecothe pBI121-R carrier of R I restriction enzyme site cuts again;
(4) in incision, adding two ends is
hind III-
ecothe joint of R I restriction enzyme site is introduced simultaneously
sali and
kpni restriction enzyme site, then utilizes DNA ligase to connect, and finally obtains p5 plant expression vector.
2. tie up pipe specific promoter and control a plant expression vector for antibiotic protein gene, it is characterized in that, for containing dimension pipe specific promoter AAP2, control antibiotic protein gene
ljAMP2plant expression vector, described carrier is p5-AAP2-LjAMP1, its construction step is as follows: with the promotor from Arabidopis thaliana AAP2 gene, replace the constitutive promoter CaMV35S on plant expression vector p5-LjAMP2, build a new expression of plants body, called after p5-AAP2-LjAMP2;
Wherein, promotor AAP2 is obtained by sequence 1 and sequence 2 amplifications, and described promotor AAP2 is as shown in sequence 11;
ljAMP2for the nucleotide sequence shown in gene order 13;
P5 plant expression vector is to reelect conventional pBI121 carrier to obtain; Near pBI121 right margin, there is a selectable marker gene
nPTII, and Nos promotor and terminator; Near left margin under the control of CaMV35S promotor
gUSreporter gene; Transformation process is divided into following steps:
(1) first utilize
ecor I enzyme carries out single endonuclease digestion, and pBI121 carrier is cut;
(2) otch is scabbled, recycling DNA ligase connects, original to remove on pBI121 carrier
ecorI restriction enzyme site, the carrier called after pBI121-R after this step;
(3) utilize
hind III is carried out single endonuclease digestion, will remove
ecothe pBI121-R carrier of R I restriction enzyme site cuts again;
(4) in incision, adding two ends is
hind III-
ecothe joint of R I restriction enzyme site is introduced simultaneously
sali and
kpni restriction enzyme site, then utilizes DNA ligase to connect, and obtains p5 plant expression vector.
3. plant expression vector according to claim 1 and 2, is characterized in that, described plant expression vector can be used in electric shocking method and transforms agrobacterium tumefaciens, obtains the recombinant bacterium that contains claim 1 or 2 plant expression vectors.
4. plant expression vector according to claim 1 and 2, is characterized in that, described plant expression vector can be used in agrobacterium tumefaciens-mediated transformation and carries out genetic transformation, obtains the transgenic cell line contain plant expression vector described in claim 1 or 2.
5. the application of the plant expression vector described in claim 1 or 2 in preparation transgenosis verticillium wilt-resistant cotton.
6. a preparation method who contains the transgene cotton of plant expression vector, comprises the following steps:
(1) will tie up pipe specific promoter AAP2 sequence, antibiotic protein gene
ljAMP1or
ljAMP2sequence is operationally inserted respectively in expression vector, builds plant expression vector;
(2) the described plant expression vector of step (1) is proceeded to host agrobacterium tumefaciens, obtain transformant;
(3) by transformant, plant expression vector is integrated in cotton, obtains transgene cotton;
Wherein,
ljAMP1plant expression vector be the nucleotide sequence shown in gene order 12;
ljAMP2plant expression vector be the nucleotide sequence shown in gene order 13.
7. a method of cultivating verticillium wilt-resistant cotton, it is characterized in that, plant expression vector described in claim 1 and 2 is proceeded to respectively to cotton gene group, realize antibiotic protein gene specifically expressing in transgene cotton vascular tissue, predominant expression in root, improves the resistance against diseases of cotton to verticillium especially.
8. cultivate according to claim 7 the method for verticillium wilt-resistant cotton, it is characterized in that, the transgenic cotton floral material of cultivation is less than 15 to non-defoliation verticillium disease index, compared with wild-type contrast, reduces more than 80%.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001020008A2 (en) * | 1999-09-16 | 2001-03-22 | Universiteit Leiden | Vascular-specific promoters |
| WO2006085965A2 (en) * | 2004-06-30 | 2006-08-17 | Pioneer Hi-Bred International, Inc. | Methods of protecting plants from pathogenic fungi |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001020008A2 (en) * | 1999-09-16 | 2001-03-22 | Universiteit Leiden | Vascular-specific promoters |
| WO2006085965A2 (en) * | 2004-06-30 | 2006-08-17 | Pioneer Hi-Bred International, Inc. | Methods of protecting plants from pathogenic fungi |
Non-Patent Citations (4)
| Title |
|---|
| Brigitte Hirner et al..Developmental control of H+/amino acid perease gene experssion during seed development of Arabidopsis.《The Plant Journal》.1998,第14卷(第5期),535-544. * |
| Kanniah Rajasekaran et al..Disease resistance conferred by the expression of a gene encoding a synthetic peptide in transgenic cotton plants.《Plant Biotechnology Journal》.2005,545-554. * |
| 吴家和等.转几丁质酶和葡聚糖酶基因棉花的获得及其对黄萎病的抗性.《遗传学报》.2004,第31卷(第2期),183-188. * |
| 李先碧等.超量表达益母草种子抗菌蛋白提高蕃茄的抗病性.《植物保护学报》.2007,第34卷(第4期),353-358. * |
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