CN102776233A - Method for culturing witloof high-quality new species by using plant genetic engineering technology - Google Patents
Method for culturing witloof high-quality new species by using plant genetic engineering technology Download PDFInfo
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Abstract
The invention discloses a method for culturing a witloof high-quality new species by using a plant genetic engineering technology. The method comprises the following steps of: A1, obtaining a witloof sterile seedling; A2, pre-culturing leaf blade explants; A3, preparing a transforming bacterium liquid; A4, dip-dyeing and transforming an exogenous gene; A5, screening and regenerating a resistance bud; and A6, identifying and screening the witloof high-quality new species. The method has the advantages of short culturing period, high efficiency, low cost, little influence on important economical characters of witloof, and is beneficial to acceleration of the progress of culturing the witloof variety in China. The method realizes green pollution-free production of high-quality feeds through increasing content of sulfur containing amino acid of the witloof and lowering the cost of the feed, provides an enough green protein raw material source for development of animal husbandry and life of people, and is wide in application prospect.
Description
Technical field
The present invention relates to the gene engineering technique field, in particular a kind of method of utilizing plant gene engineering technology to cultivate witloof high-quality (containing methionine(Met) and halfcystine) new germ plasm.
Background technology
Sulfur-containing amino acid content in forage plant is less, and particularly halfcystine and methionine(Met) often become the first restricted indispensable amino acid.The forage grass kind of cultivating high sulfur-containing amino acid is one of research focus of current forage grass quality-improving.Witloof (Cichorium intybus L.) is a composite family Cichorium per nnial herb, and flexibility is strong, and happiness warm and moist weather when temperature reaches 5 ℃, can be grown by normal growth.Cold tolerance is strong, the low temperature of seedling ability-8 ℃.Soil is required not strict, in the wasteland, the hillside fields all can grow, all parts of the country all are fit to plant.Period of use, is long, owing to turn green spring early, winter dormancy is late; Period of use, reach 8 months (4～November), and autumn in summer arid season is when most of herbages are withered and yellow; It is verdure that witloof still can keep; Can solve aquaculture spring and autumn and green fodder contradiction in short supply, once sowing is not being received under the situation of planting and can utilized for many years continuously.Witloof is widely used in feed, vegetables, pharmacy and sugaring raw material; Be at present than the cash crop of tool exploitation future and the aquaculture green fodder of first-selection; In recent years, witloof a large amount of cultivations in a lot of areas, oneself becomes a kind of extremely welcome high-grade health vegetables and novel good forage grass.But witloof sulfur-containing amino acid particularly methionine(Met) and cysteine content is very low, has limited it and has utilized more widely.Witloof genetic background is complicated; It is very big to cultivate protein-high and high sulfur-containing amino acid kind consumption wealth consumption power not only consuming time and difficulty with the conventional breeding method; Therefore; Utilize some high sulfur-containing amino acid protein genes, create witloof high-quality new germ plasm, and the sulfur-containing amino acid level has crucial meaning in the raising witloof protein through the genetic engineering means.Utilizing transgenic technology improvement witloof quality particularly to improve sulfur amino acid content does not also report at present.
Recombinant protein gene zeolin constitutes phaseollin gene phaseiolin through the N end that connection peptides is connected to corn seed alcohol soluble protein gene γ-Zein; This gene is rich in methionine(Met) and halfcystine; For having improved the stability and its expression of enhancing of zeolin gene in the transgenic plant, added the resident protein signal of endoplasmic reticulum (KDEL) in the recombination front.Zeolin recombinant protein gene coding region has 1578 Nucleotide, 526 aminoacid sequences of encoding.Its protein molecular weight is 58.0kDa.
Summary of the invention
Technical problem to be solved by this invention is that the deficiency that is directed against prior art provides a kind of method of utilizing plant gene engineering technology to cultivate witloof high-quality new germ plasm.
Technical scheme of the present invention is following:
A kind of method of utilizing plant gene engineering technology to cultivate witloof high-quality new germ plasm may further comprise the steps: the acquisition of A1, the aseptic seedling of witloof; A2, leaf explant are cultivated in advance;
The preparation of A3, transformed bacteria liquid: the single bacterium colony of Agrobacterium LBA4404 that contains the foreign gene plasmid with aseptic toothpick or inoculating needle picking; Being inoculated into 50 mL contains in the liquid YMB substratum of Vetstrep (Str, 50 mg/L) and kantlex (Kan, 50 mg/L); 28 ℃ of shaking culture 16～24h are to logarithmic phase; Culture in centrifugal 10 min of 4000 ~ 5000 r/min, is removed supernatant, collect thalline; With the resuspended OD600=0.4Abs that is precipitated to of 1/2MS aseptic liquid nutrient medium, be used to infect conversion; Or directly be used to infect with 1/2MS aseptic liquid nutrient medium dilution bacterium liquid.Said YMB substratum: 0.5g/L K
2HPO
4+ 0.2g/L MgSO
47H
2O+0.5g/L NaCl+10g/LMannitol+0.4g/LYeast extract, pH7.5;
The dip-dye of A4, foreign gene transforms: under the aseptic condition, choose the good preparatory cultivation explant of growth conditions and put into the bacterium liquid for preparing, leaf explant is fully contacted with bacterium liquid, and be put in concussion dip-dye 10min on the concussion incubator; Contaminate the back and taken out explant; Inhale the unnecessary bacterium liquid of defoliation sheet surface attachment with sterilization filter paper; Be inoculated into be lined with one deck aseptic filter paper and be added with Syringylethanone (AS, on common substratum 100umol/L), in dark, cultivating 2-3d altogether has visible microcolony to naked eyes; After having cultivated altogether, explant is inoculated on the substratum identical with preparatory cultivation recovers to cultivate 1-2d; Said culture medium altogether is: MS minimum medium+6-BA 2.0mg/L+IBA 0.2mg/L+AS 100 μ mol/L+30g/L sucrose+8g/L agar, pH5.4～5.8;
Screening of A5, resistant buds and regeneration: after recovering to cultivate, will transform explant change over to contain the selective agent Totomycin (Hyg, 25mg/L) and fungistat cephamycin (Cef; 500 mg/L) screening and culturing is up to growing resistant buds in the screening culture medium, and is long during to 1cm when resistant buds, changes over to contain selective agent Totomycin (Hyg; 15mg/L) with fungistat cephamycin (Cef; 250 mg/L) expand numerous longly to 2-3cm in the expansion breeding culture medium, change over to again and contain the selective agent Totomycin (Hyg is 10mg/L) with fungistat cephamycin (Cef to bud; 250 mg/L) carry out root culture in the root media, up to growing up to complete plantlet;
Said screening culture medium is: MS minimum medium+2.0mg/L6-BA+0.2mg/LIBA+0.01mg/L TDZ+25mg/L Hyg+500 mg/L Cef+30g/L sucrose+8g/L agar; PH5.8～6.0;
Expand breeding culture medium: MS minimum medium+2.0mg/L 6-BA+0.2mg/LIBA+0.01mg/L TDZ+15mg/L Hyg+500 mg/L Cef+30g/L sucrose+8g/L agar; PH5.8～6.0;
Root media is: 1/2MS minimum medium+0.1mg/LNAA+10mg/L Hyg+250 mg/L Cef+ sucrose 15g/L+8g/L agar, pH5.8-6.0;
The evaluation and the screening of A6, witloof high-quality germplasm materials.
Described method in the steps A 1, is selected full chicory seed, carries out the ethanol surface of 75 % successively and soaks 30s; Mercuric chloride soaked 10 minutes, during do not stop to shake, outwell mercuric chloride, sterilized water washes repeatedly; Soak seed 12h with sterilized water at last, outwell soak solution, blot surface-moisture with aseptic filter paper; On being inoculated in the triangular flask that autoclaved Ms substratum is housed, being put in the greenhouse and sprouting, obtain aseptic seedling; Said aseptic seedling substratum is: MS minimum medium+sucrose 30g/L+ agar 8g/L, pH 5.8～6.0.
Described method in the steps A 2, is chosen witloof aseptic seedling young leaflet tablet, is cut into 0.5 * 0.5cm fritter, it is inoculated in cultivates 2-3d in the inducing culture in advance, cultivates blade edge and begins to expand; Said inducing culture is: MS basic medium+6-BA 2.0mg/L+IBA 0.2mg/L+30g/L sucrose+8g/L agar, pH is 5.8-6.0.
Have that cultivation period is short, efficient is high, cost is low, to advantages such as the Main Agronomic Characters influence of witloof own are little, this technology will speed up the process of China's witloof breed of variety.This technology reduces feed cost through improving the witloof sulfur amino acid content, realizes the green non-pollution production of high-quality feed, for animal husbandry development and people's life provide competent green protein raw material to originate, has a extensive future.
Through method of the present invention; Determination data result (shown in the table 1) is illustrated in 12 strains that detected to be changeed in the zeolin gene witloof; The sulfur-containing amino acid total content comparison that 2 strain materials are arranged has been according to having improved 7.87% and 13.7% respectively, chooses the transgenic witloof plant that this 2 strain methionine(Met) and Gelucystine total content be significantly higher than contrast and is witloof high-quality new germ plasm.
Description of drawings
Fig. 1 is the screening and the regeneration of resistant buds;
Fig. 2 is the root culture of resistant buds;
Fig. 3 is for changeing zeolin gene witloof refining seedling
Fig. 4 is for transforming the witloof DNA extraction;
Fig. 5 expands numerous plant RNA for the transgenic witloof and extracts;
Fig. 6 is for changeing the PCR electrophoresis detection of zeolin gene witloof; Annotate: M:DL 2000 DNA marker; P:pCB-zeolin (plasmid of positive control); CK: negative control (non-transgenic witloof); 1～5 transgenic witloof, this figure is identical with the numerous P of expansion;
Fig. 7 expands numerous plant RT-PCR detection for changeing Zeolin gene witloof, annotates: M:DL 2000 DNA marker; P: plasmid pCB-GFP-zeolin (positive control); CK: negative control (non-transgenic witloof); 1～4 expands numerous plant for the transgenic witloof;
Fig. 8 expands numerous plant Southern blotting detection for changeing zeolin gene witloof, and annotate: CK is ddH
2O, 1 and 2 is transformed plant;
Fig. 9 is for changeing field performance of zeolin gene witloof and screening.
Embodiment
Below in conjunction with specific embodiment, the present invention is elaborated.
1, the acquisition of the aseptic seedling of witloof.Select full chicory seed, carry out the ethanol surface of 75 % successively and soak 30s, mercuric chloride soaked 10 minutes; Do not stop during this time to shake, outwell mercuric chloride, sterilized water washes repeatedly; Soak seed 12h with sterilized water at last, outwell soak solution, blot surface-moisture with aseptic filter paper; On being inoculated in the triangular flask that autoclaved Ms substratum is housed, being put in the greenhouse and sprouting, obtain aseptic seedling.
Said aseptic seedling substratum is: MS minimum medium+sucrose 30g/L+ agar 8g/L, pH 5.8～6.0.
2, leaf explant is cultivated in advance.Choose witloof aseptic seedling young leaflet tablet, be cut into 0.5 * 0.5cm fritter, it is inoculated in cultivates 2-3d in the inducing culture in advance, cultivate blade edge and begin to expand.Said inducing culture is: MS basic medium+6-BA 2.0mg/L+IBA 0.2mg/L+30g/L sucrose+8g/L agar, and pH is 5.8-6.0;
3, the preparation of transformed bacteria liquid.Single bacterium colony (the reference: Zhang Yu of Agrobacterium LBA4404 that contains the foreign gene plasmid with aseptic toothpick or inoculating needle picking; White history and; The structure of .pCB-zeolin-GFP expression vectors such as Li Cong and transient expression [J]. Chinese agronomy circular, 2012,28 (03): 233-239.), be inoculated into 50 mL and contain Vetstrep (Str, 50 mg/L) and kantlex (Kan; 50 mg/L) in the liquid YMB substratum, 28 ℃ of shaking culture 16～24h are to logarithmic phase, with culture in centrifugal 10 min of 4000 ~ 5000 r/min; Remove supernatant; Collect thalline,, be used to infect conversion with the resuspended OD600=0.4Abs that is precipitated to of 1/2MS aseptic liquid nutrient medium; Or directly be used to infect with 1/2MS aseptic liquid nutrient medium dilution bacterium liquid.Said YMB substratum: 0.5g/L K
2HPO
4+ 0.2g/L MgSO
47H
2O+0.5g/L NaCl+10g/LMannitol+0.4g/LYeast extract, pH7.5.
4, the dip-dye of foreign gene transforms.Under the aseptic condition, choose the good preparatory cultivation explant of growth conditions and put into the bacterium liquid for preparing, leaf explant is fully contacted with bacterium liquid, and be put in concussion dip-dye 10min on the concussion incubator; Contaminate the back and taken out explant; With sterilization filter paper inhale defoliation sheet surface attachment unnecessary bacterium liquid (note blade not in air exposure duration oversize; Can not inhale too driedly); Be inoculated into be lined with one deck aseptic filter paper and be added with Syringylethanone (AS, on common substratum 100umol/L), in dark, cultivating 2-3d altogether has visible microcolony to naked eyes; After having cultivated altogether, explant is inoculated on the substratum identical with preparatory cultivation recovers to cultivate 1-2d.Said culture medium altogether is: MS minimum medium+6-BA 2.0mg/L+IBA 0.2mg/L+AS 100 μ mol/L+30g/L sucrose+8g/L agar, pH5.4～5.8.
5, resistant buds screening and regeneration.After recovering to cultivate; To transform explant change over to contain the selective agent Totomycin (Hyg, 25mg/L) with the screening culture medium of fungistat cephamycin (Cef, 500 mg/L) in screening and culturing up to growing resistant buds; As shown in Figure 1; As can be seen from the figure, after 2 weeks, transformant has grown the resistant buds that has foreign gene to the explant of conversion through the 25mg/LHyg resistance screening; Statistics draws Hyg resistant buds incidence average out to 13.52% after 4 weeks, has improved 3.58 times than 2.95% under the conventional conversion condition.Long during when resistant buds to 1cm, change over to and contain the selective agent Totomycin (Hyg is 15mg/L) with fungistat cephamycin (Cef; 250 mg/L) expand numerous long to 2-3cm in the expansion breeding culture medium to bud; Change over to again and contain the selective agent Totomycin (Hyg 10mg/L) with in the root media of fungistat cephamycin (Cef, 250 mg/L) carries out root culture; Up to growing up to complete plantlet; As shown in Figure 2, resistant buds is taken root in the root media that contains suitable cephalo and Totomycin and is grown fine, and statistics draws rooting rate and reaches 92.56 %.
Said screening culture medium is: MS minimum medium+2.0mg/L6-BA+0.2mg/LIBA+0.01mg/L TDZ+25mg/L Hyg+500 mg/L Cef+30g/L sucrose+8g/L agar; PH5.8～6.0;
Expand breeding culture medium: MS minimum medium+2.0mg/L 6-BA+0.2mg/LIBA+0.01mg/L TDZ+15mg/L Hyg+500 mg/L Cef+30g/L sucrose+8g/L agar; PH5.8～6.0;
Root media is: 1/2MS minimum medium+0.1mg/LNAA+10mg/L Hyg+250 mg/L Cef+ sucrose 15g/L+8g/L agar, pH5.8-6.0.
6, the evaluation of witloof high-quality germplasm materials and screening.Transgenic resistance plantlet reaches the contrast of transgenic regenerated plant and after refining seedling, transplants to the greenhouse flowerpot; As shown in Figure 3; Behind the transgenic seedling refining seedling that as can beappreciated from fig. 3 draws through screening plant become to live good, draw the refining seedling through statistical study after survival rate of plant up to more than 95%.To becoming plant alive to extract DNA, as shown in Figure 4, carry out PCR and detect; The PCR detected result is as shown in Figure 6; Show that tentatively the zeolin gene has been imported in most of witloof genome, statistical results show is through the positive plant of hygromycin selection, and the positive rate that PCR detects is 46.15%; Witloof material to the PCR test positive carries out Southern hybridization detection, and detected result (shown in Figure 7) shows that the zeolin gene advances in the witloof genome with the form random integration of single copy; Adopt the TRIzol method; The all positive transgenic line of above detection is extracted RNA (shown in Figure 5) carry out the RT-PCR detection; Detected result (shown in Figure 8) shows that the witloof material that changes the zeolin gene all amplifies the expection band of the 890bp identical with the plasmid size; Explain that positive transgenic witloof material is transcribed into mRNA with the external source goal gene, has obtained expression in nucleic acid level.The positive transfer-gen plant of stably express is transplanted to the field mark of listing; Cradle to peduncle-growing period for rapeseed at lotus throne; Oxydrolysis is pulverized in oven dry; Measure the content (shown in Figure 9) of methionine(Met) and Gelucystine; Determination data result (shown in the table 1) is illustrated in 12 strains that detected to be changeed in the zeolin gene witloof, and the sulfur-containing amino acid total content comparison that 2 strain materials are arranged has been according to having improved 7.87% and 13.7% respectively, chooses the transgenic witloof plant that this 2 strain methionine(Met) and Gelucystine total content be significantly higher than contrast and is witloof high-quality new germ plasm.
Table 12 strain transgenic line sulfur amino acid contents are measured the result
Annotate: because material halfcystine in treating processes transforms for Gelucystine, so only survey the total content of Gelucystine.
Should be understood that, concerning those of ordinary skills, can improve or conversion, and all these improvement and conversion all should belong to the protection domain of accompanying claims of the present invention according to above-mentioned explanation.
Claims (3)
1. a method of utilizing plant gene engineering technology to cultivate witloof high-quality new germ plasm is characterized in that, may further comprise the steps: the acquisition of A1, the aseptic seedling of witloof; A2, leaf explant are cultivated in advance;
The preparation of A3, transformed bacteria liquid: the single bacterium colony of Agrobacterium LBA4404 that contains the foreign gene plasmid with aseptic toothpick or inoculating needle picking; Being inoculated into 50 mL contains in the liquid YMB substratum of Vetstrep (Str, 50 mg/L) and kantlex (Kan, 50 mg/L); 28 ℃ of shaking culture 16～24h are to logarithmic phase; Culture in centrifugal 10 min of 4000 ~ 5000 r/min, is removed supernatant, collect thalline; With the resuspended OD600=0.4Abs that is precipitated to of 1/2MS aseptic liquid nutrient medium, be used to infect conversion; Or directly be used to infect with 1/2MS aseptic liquid nutrient medium dilution bacterium liquid; Said YMB substratum: 0.5g/L K
2HPO
4+ 0.2g/L MgSO
47H
2O+0.5g/L NaCl+10g/LMannitol+0.4g/LYeast extract, pH7.5;
The dip-dye of A4, foreign gene transforms: under the aseptic condition, choose the good preparatory cultivation explant of growth conditions and put into the bacterium liquid for preparing, leaf explant is fully contacted with bacterium liquid, and be put in concussion dip-dye 10min on the concussion incubator; Contaminate the back and taken out explant; Inhale the unnecessary bacterium liquid of defoliation sheet surface attachment with sterilization filter paper; Be inoculated into be lined with one deck aseptic filter paper and be added with Syringylethanone (AS, on common substratum 100umol/L), in dark, cultivating 2-3d altogether has visible microcolony to naked eyes; After having cultivated altogether, explant is inoculated on the substratum identical with preparatory cultivation recovers to cultivate 1-2d; Said culture medium altogether is: MS minimum medium+6-BA 2.0mg/L+IBA 0.2mg/L+AS 100 μ mol/L+30g/L sucrose+8g/L agar, pH5.4～5.8;
Screening of A5, resistant buds and regeneration: after recovering to cultivate, will transform explant change over to contain the selective agent Totomycin (Hyg, 25mg/L) and fungistat cephamycin (Cef; 500 mg/L) screening and culturing is up to growing resistant buds in the screening culture medium, and is long during to 1cm when resistant buds, changes over to contain selective agent Totomycin (Hyg; 15mg/L) with fungistat cephamycin (Cef; 250 mg/L) expand numerous longly to 2-3cm in the expansion breeding culture medium, change over to again and contain the selective agent Totomycin (Hyg is 10mg/L) with fungistat cephamycin (Cef to bud; 250 mg/L) carry out root culture in the root media, up to growing up to complete plantlet;
Said screening culture medium is: MS minimum medium+2.0mg/L6-BA+0.2mg/LIBA+0.01mg/L TDZ+25mg/L Hyg+500 mg/L Cef+30g/L sucrose+8g/L agar; PH5.8～6.0;
Expand breeding culture medium: MS minimum medium+2.0mg/L 6-BA+0.2mg/LIBA+0.01mg/L TDZ+15mg/L Hyg+500 mg/L Cef+30g/L sucrose+8g/L agar; PH5.8～6.0;
Root media is: 1/2MS minimum medium+0.1mg/LNAA+10mg/L Hyg+250 mg/L Cef+ sucrose 15g/L+8g/L agar, pH5.8-6.0;
The evaluation and the screening of A6, witloof high-quality germplasm materials.
2. method according to claim 1 is characterized in that, in the steps A 1, selects full chicory seed; Carry out the ethanol surface of 75 % successively and soak 30s, mercuric chloride soaked 10 minutes, during do not stop to shake, outwell mercuric chloride; Sterilized water washes repeatedly, soaks seed 12h with sterilized water at last, outwells soak solution, blots surface-moisture with aseptic filter paper; On being inoculated in the triangular flask that autoclaved Ms substratum is housed, being put in the greenhouse and sprouting, obtain aseptic seedling; Said aseptic seedling substratum is: MS minimum medium+sucrose 30g/L+ agar 8g/L, pH 5.8～6.0.
3. method according to claim 1 is characterized in that, in the steps A 2, chooses witloof aseptic seedling young leaflet tablet, is cut into 0.5 * 0.5cm fritter, it is inoculated in cultivates 2-3d in the inducing culture in advance, cultivates blade edge and begins to expand; Said inducing culture is: MS basic medium+6-BA 2.0mg/L+IBA 0.2mg/L+30g/L sucrose+8g/L agar, pH is 5.8-6.0.
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| CN103555757A (en) * | 2012-12-25 | 2014-02-05 | 西北大学 | Cichorium intybus chloroplast transformation system establishment method |
| CN105200003A (en) * | 2015-10-26 | 2015-12-30 | 中国热带农业科学院热带生物技术研究所 | In-vitro regeneration and genetic transformation method of emperor bananas and in-vitro regeneration culture medium of emperor bananas |
| CN115386591A (en) * | 2022-09-05 | 2022-11-25 | 中国科学院华南植物园 | Molecular breeding method of monochantia sonchifolia |
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2012
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103555757A (en) * | 2012-12-25 | 2014-02-05 | 西北大学 | Cichorium intybus chloroplast transformation system establishment method |
| CN103555757B (en) * | 2012-12-25 | 2015-07-08 | 西北大学 | Cichorium intybus chloroplast transformation system establishment method |
| CN105200003A (en) * | 2015-10-26 | 2015-12-30 | 中国热带农业科学院热带生物技术研究所 | In-vitro regeneration and genetic transformation method of emperor bananas and in-vitro regeneration culture medium of emperor bananas |
| CN105200003B (en) * | 2015-10-26 | 2022-05-13 | 中国热带农业科学院热带生物技术研究所 | Method for in-vitro regeneration and genetic transformation of emperor bananas and in-vitro regeneration culture medium thereof |
| CN115386591A (en) * | 2022-09-05 | 2022-11-25 | 中国科学院华南植物园 | Molecular breeding method of monochantia sonchifolia |
| CN115386591B (en) * | 2022-09-05 | 2024-05-28 | 中国科学院华南植物园 | Molecular breeding method of single herba Cichorii |
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Application publication date: 20121114 |