CN104450771A - Cultivation method of heavy-metal and low-accumulation transgenic plants - Google Patents
Cultivation method of heavy-metal and low-accumulation transgenic plants Download PDFInfo
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Abstract
The invention relates to a cultivation method of transgenic plants and particularly provides a cultivation method of heavy-metal and low-accumulation transgenic plants. According to the cultivation method, heavy-metal and low-accumulation safe crops are cultured by virtue of heavy metal outward-transport passage proteins in heterologous expression yeast; particularly, the heterologous expression of yeast Ycflp genes in cell membranes of roots of the crops are driven by specific promoters of cell membranes of roots and epidermis, so that heavy metals entering the roots of the plants can be exported out of plant bodies by virtue of metal outward-transport proteins on the cell membranes by the plants, the heavy-metal and low-accumulation crops are obtained, and the purpose of producing agricultural products accordant with safety standards on heavy-metal-polluted soil is achieved by planting low-absorption, low-accumulation and heavy-metal crops; the nucleotide sequence of the yeast Ycflp genes is represented by SEQ ID No.1, and the nucleotide sequence of Prp1 promoters is represented by SEQ ID No.2.
Description
Technical field
The present invention relates to the cultivation of transgenic plant, specifically, relate to the method for cultivation of a kind of transgenic plant of low heavy metal accumulation.
Background technology
In China, along with the development of industrial and agricultural production, heavy metal pollution of soil problem is more and more outstanding.Soil is the storage vault that plant-growth needs nutritive substance, and thus the number of heavy metals in soil content determines the contaminated degree of farm crop to a great extent.The improvement in heavy metal contamination farmland is current one of difficult point and hot research field in the world.Scientists utilize fixing, filter, ion-exchange and the insoluble multiple Physicochemical method such as fixing, attempt the soil of purification of heavy metal pollution, but the application of these technology needs equipment and the technology of high price.Moreover as cultivated soil, pollute slight and that contaminated area is large place and be difficult to practical application.In recent years, the research work for the improvement of heavy metal contamination farmland mainly concentrated on the super tired foliage filter screening for phytoremediation, and these plant major parts are wild, the speed of growth is slow, biomass is low, and repair time is long, and Field information exists uncertain and plantation difficulty.The effect obtained under the decontamination effect improving putting into the hyperaccumulative plant in heavy-metal contaminated soil in actual production can not show a candle to laboratory condition.In view of the actual national conditions of China, by big area-farmland of slight pollution stops farming, carries out long phytoremediation or other cost intensive engineering reparation is obviously unpractical.The people such as 2006S.Herbette propose the concept of the kind pollution-safe cultivars (PSCs) polluting safety.Namely grow at contaminated soil, specific pollutants is enough low at the accumulating level of edible part, meets quality standard, can for the kind of safe edible.In the face of increasingly reducing and the further serious present situation of soil heavy metal cadmium of farmland resource, screen and cultivate the crop gene type of low absorption, low accumulation heavy metal in soil, the crop varieties particularly utilizing genetically engineered to cultivate low amounts accumulation heavy metal has become the main direction of studying ensureing crop production safety.In recent years, pollute safe kind and attract the larger concern of investigator and breeding companies and interest.
Most of heavy metal is the nonessential trace element of plant, has very strong toxicity to plant.Exceed certain amount and can suppress plant cell growth, inhibited oxidation phosphorylation, initiated oxidation is coerced, and affects photosynthesis, damage kernel and the vigor affecting plasma membrane ATPase.Some plants resist heavy metallic poison by induced synthesis chelating peptide, metallothionein(MT), plant stress protein etc., and the approach such as the plant also had then is fixed by cell walls, vacuole separation, glandular secretion resist murder by poisoning.Be stored at after the meta-bolites of plant and heavy metal form complex body in intracellular vacuole, suppress toxic insults.
Gene determines that plant is to the most important factor of heavy metal-polluted soil absorption and accumulation characteristic, and the Rejection mechanism of low-accumulation plants heavy metal it has been generally acknowledged that and comprises two aspects, and one is the absorption reducing root heavy metal; Two is that heavy metal is preserved by compartmentation at root, thus restriction is shifted to overground part.But Plant Tolerance, transhipment and cadmium are complicated processes, relate to the participation of the many aspects such as translocator family, chelating protein family, antioxidant system.At present, can be applied to genetic improvement engineered in plant, its main metal transport proteins of heavy metal Rejection mechanism and key regulator have not yet to see report.
Summary of the invention
In order to solve problems of the prior art, the object of this invention is to provide the method for cultivation of a kind of transgenic plant of low heavy metal accumulation.
In order to realize the object of the invention, first the present invention provides the method for cultivation of a kind of transgenic plant of low heavy metal accumulation, comprises the steps:
(1) structure of recombinant expression vector:
By yeast Ycf1p gene clone on Agrobacterium plant binary expression vector, under the effect of restriction enzyme, connect the carrier T containing Prp1 promotor, obtain the expression vector of expressing Ycf1p gene under Prp1 promoters driven;
(2) structure of transfer-gen plant:
Gained expression vector is proceeded in plant callus, transforms with the nutrient solution containing inductor and Agrobacterium, the material after conversion through the exercise of Dual culture-screen-break up-take root-transgenic seedling and transplanting, screening transgenic positive plant;
Wherein, the nucleotide sequence of described yeast Ycf1p gene is as shown in SEQ ID No.1, and the nucleotide sequence of Prp1 promotor is as shown in SEQ ID No.2.
Further, described step (1) is specially:
With restriction endonuclease SacI and SalI Ycf1p is cloned on intermediate carrier pGEM-5Z and obtains pYcf1p-5Z, after pYcf1p-5Z SalI and SpeI enzyme are cut, reclaim Ycf1p gene fragment, be connected on the plant expression vector pCAMBIA1301 after SalI and SpeI enzyme is cut through T4 ligase enzyme again, cut expression vector pCAMBIA1301 by BamHI enzyme again and containing the carrier T of Prp1 promotor, obtain after connection taking Totomycin as the expression vector pCAMBIA1301-pRP1-Ycf1p of selection markers under epiblem cytolemma specific promoter Prp1 drives.
Present invention also offers the carrier of a kind of transgenic plant for cultivating low heavy metal accumulation, described carrier contains yeast Ycf1p gene and epiblem cytolemma specific promoter Prp1;
Wherein, the nucleotide sequence of described yeast Ycf1p gene is as shown in SEQ ID No.1, and the nucleotide sequence of epiblem cytolemma specific promoter Prp1 is as shown in SEQ ID No.2.
Present invention also offers a kind of engineering bacteria containing aforementioned bearer.
The present invention still further provides the application of yeast Ycf1p gene in the transgenic plant cultivating low heavy metal accumulation, is specially:
By yeast Ycf1p gene clone on Agrobacterium plant binary expression vector, under the effect of restriction enzyme, connect the carrier T containing Prp1 promotor, obtain the expression vector of expressing Ycf1p gene under Prp1 promoters driven;
Gained expression vector is proceeded in plant callus, transform with the nutrient solution containing inductor and Agrobacterium, material after conversion is through the exercise of Dual culture-screen-break up-take root-transgenic seedling and transplanting, screening transgenic positive plant, gained transgenic positive plant is by expressing yeast Ycf1p gene, the export-oriented translocator of the metal of heavy metal on cytolemma being entered root system of plant by active transport and passive transport can be discharged to outside plant materials, thus reach the object reducing plant materials heavy metal content.
Method of cultivation of the present invention can be applied to any can carrying out in genetically modified farm crop, in the specific embodiment of the present invention, implements technical scheme of the present invention for paddy rice.
In order to improve the productivity of contaminated soil better, preferred described plant is paddy rice.
Beneficial effect of the present invention is:
The present invention is from improving the productivity of contaminated soil and ensureing that the angle of crop production safety is started with, drive yeast Ycf1p gene heterogenous expression in the cytolemma of farm crop root system by epiblem cytolemma specificity promoter, the acquisition of render transgenic plant is to the tolerance of specific heavy metal and repulsive force.The inventive method be cultivate low heavy metal accumulation, high patience, crop varieties that increment is large provide important application and theoretical foundation, also for improving the productivity of contaminated soil and ensureing that crop production safety provides effective approach.
Accompanying drawing explanation
Fig. 1 is pCAMBIA1301-pRP1-Ycf1p expression vector collection of illustrative plates of the present invention.
Fig. 2 is the different strain Ycf1p gene of transgenic paddy rice described in the embodiment of the present invention 1 southern results of hybridization.
Fig. 3 is the different strain Ycf1p gene RT-PCR of transgenic paddy rice described in the embodiment of the present invention 1 and northern results of hybridization.
Fig. 4 is the content of heavy metal cadmium in rice grain in different transgenic line in the embodiment of the present invention 1.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.
The structure of embodiment 1 recombinant vectors
With restriction endonuclease SacI and SalI Ycf1p is cloned on intermediate carrier 5Z and obtains pYcf1p-5Z, after p Ycf1p-5Z SalI and SpeI enzyme are cut, the Ycf1p gene fragment reclaimed, T4 ligase enzyme is connected on the plant expression vector pCAMBIA1301 after SalI and SpeI enzyme is cut, expression vector and the carrier T containing Prp1 promotor is cut by BamHI enzyme, obtain after link obtaining take Totomycin as the expression vector pCAMBIA1301-pRP1-Ycf1p (see Fig. 1) of selection markers under Prp1 epiblem cytolemma specific promoter drives, with Quick post large quantity extracting plasmid, test for paddy rice Agrobacterium-mediated Transformation.
The structure of embodiment 2 transfer-gen plant
1, the preparation of transgenic plant acceptor
The acquisition of mature embryo callus: mature seed is removed clever shell, aseptically, 3-5min is soaked with in 70% alcohol, aseptic water washing 3 times, be soak 20min in 0.1% mercuric chloride solution proceeding to massfraction, aseptic water washing 3 ~ 5 times, clorox with 20% soaks 20min, constantly rock therebetween, can be placed in shaking table, aseptic water washing 3 ~ 5 times, be placed in wipe dry on aseptic filter paper, move on calli induction media, 27 DEG C of light culture removed bud after about three days, when scultellum expands after one week, the endosperm fraction of the grain of rice is removed during endosperm deliquescing, visible yellow color callus grows, the embryo callus peeled, be placed on inducing culture, about 15 days subcultures once, after subculture 2-3 time, growth selection is rapid, quality is crisp, the callus that color is vivid is used for Agrobacterium-mediated Transformation.
2, Agrobacterium-mediated transformation Mature Embryos of Rice callus
(1) get 200 μ l competent cells, add the plasmid DNA that 1 μ g builds, quick-frozen 1min in liquid nitrogen, 37 DEG C of water-bath 5min, then add 1ml YEB substratum, 28 DEG C of shaking culture 4h at a slow speed; With the centrifugal 30s of 10,000rpm, abandon supernatant, add 0.1ml YEB substratum Eddy diffusion cell, coat (100 μ g/ml Kan, 125 μ g/ml Sm) on YEB flat board, cultivate about 48h for 28 DEG C.
(2) single bacterium colony picking flat board grown, be inoculated in YEB liquid medium (100 μ g/ml Kan, 125 μ g/ml Sm), 28 DEG C of shaking culture are spent the night; Extracting plasmid DNA in a small amount, is that template carries out pcr amplification qualification with plasmid DNA.
(3) callus of having induced is proceeded in fresh substratum, illumination cultivation 3 days.Get the Agrobacterium shaken, collection bacterium, suspends with the YEB substratum containing 100umol Syringylethanone and dilutes certain multiple.
(4) callus is immersed in the Agrobacterium suspension medium diluted the 20-30 minute that suspends.Take out callus, with filter paper, unnecessary bacterium liquid is blotted, be put on Dual culture substratum, light culture about 3 days, until there is bacterium colony to grow.
(5) with the N.F,USP MANNITOL sterile liquid oscillation cleaning twice of 0.1M, with the cephamycin liquid cleaning twice of 500mg/L, clarify completely to supernatant.
(6) take out callus, suck unnecessary liquid, be placed on (500mg/Lcef) on recovery media, light culture recovers three days.
(7) screening about month in screening culture medium callus being proceeded to Antibiotics of Low Concentration (30mg/L Totomycin) and 500mg/L cef.
(8) screening about month in screening culture medium callus being proceeded to high density microbiotic (40mg/L Totomycin) and 500mg/L cef.
(9) screening about month in screening culture medium callus being proceeded to high density microbiotic (50mg/L Totomycin) and 500mg/L cef.
(10) see that light breaks up at the division culture medium of MS minimum medium+3mg/L 6-BA+1mg/L NAA.
(11) screen on the root media of 1/2MS substratum+50mg/L Totomycin and take root.
(12) on 1/2MS Transgenic Rice Seedlings growth medium, grow to the 3-4 leaf phase and root system is more flourishing time, seedling is moved in small flower, outer cover plastics bag heat and moisture preserving; After moving to greenhouse white silk seedling 5-7d, remove plastics bag; Land for growing field crops is moved into, until blossom and bear fruit after one week.
3, the screening of transgenic positive plant
Detect the positive rate of the HYG screening-gene on the Prp1 promotor in transfer-gen plant, Ycf1p gene and pCAMBIA1301 carrier especially by PCR, preliminary screening obtains positive plant S7, S9, S25, S30 and S37.Detected by the incorperation and expression situation of method to the target gene in the transfer-gen plant of preliminary screening of RT-PCRSouthern, Northern, obtain the positive strain of stably express.Southern result (see Fig. 2) shows the insertion situation of different strain goal gene Ycf1p, RT-PCR and Northern result (see Fig. 3 and Fig. 4) shows the expression intensity of different strain Ycf1p gene, wherein these four strains of S7, S9 and S37, Ycf1p gene all has higher expression level, S25 strain is expressed and is taken second place, and the Ycf1p gene in S30 strain paddy rice is not expressed substantially.
4, the content of cadmium element in plant is detected
(1) the long-term monitoring point in somewhere is selected to carry out field experiment, the Cadmium in Soil continuous 3 years monitor value mean value of this monitoring point is 1.86mg/kg, exceedes the Cadmium in Soil threshold value 1.0mg/kg of national standard of soil environment quality middle peasant production of forestry and plant normal growth.The Ycf1p trans-genetic hybrid rice seed that turns of the different strains through the bacterium process that disappears directly is seeded in soil.Until seed germination after one week, according to size and the thinning of growing way situation of seedling, each process repeats 3 times and carries out the group arrangement of random district, carries out further proof test to the low accumulation cadmium characteristic of the transgenic positive rice strain screened in land for growing field crops.
(2) heavy metal cadmium constituent content measures, and Soil K+adsorption is according to GB/T 17141-1997, and vegetables cadmium content, according to GB/T 5009.15-2003, detects the cadmium content in rice grain and soil with thermoelectricity M6 atomic absorption spectrophotometer.The present embodiment is 1.93mg/kg for examination Cadmium in Soil content, and in same plot contrast strain positive strain S7 different from turning Ycf1p gene, S9, S25, S30 and S37 rice grain, cadmium content is respectively 0.0680,0.0996,0.0695,0.0895,0.0998 to 0.705mg/kg.
(3) evaluate paddy rice from the angle of agricultural product security and whether there is higher cadmium patience and lower accumulation volume, namely under higher cadmium pollution can normal growth and biomass without remarkable decline, and cadmium content is lower in plant materials.That 0.2mg/kg evaluates especially by the edible part of paddy rice lower than cadmium limit standard in regulation rice in related standards " pollutants in food limitation GB2762-2012 ".Rice strain S7, S9, S25 and S37 of being obtained by present method, compare with contrast, in the Cd-polluted farmland exceeding national soil grade III Standard, show heavy metal and tolerate preferably and good low characteristic of accumulation, can as the alternative kind of Cd-polluted farmland safety in production.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (6)
1. a method of cultivation for the transgenic plant of low heavy metal accumulation, is characterized in that, comprises the steps:
(1) structure of recombinant expression vector:
By yeast Ycf1p gene clone on Agrobacterium plant binary expression vector, under the effect of restriction enzyme, connect the carrier T containing Prp1 promotor, obtain the expression vector of expressing Ycf1p gene under Prp1 promoters driven;
(2) structure of transfer-gen plant:
Gained expression vector is proceeded in plant callus, transforms with the nutrient solution containing inductor and Agrobacterium, the material after conversion through the exercise of Dual culture-screen-break up-take root-transgenic seedling and transplanting, screening transgenic positive plant;
Wherein, the nucleotide sequence of described yeast Ycf1p gene is as shown in SEQ ID No.1, and the nucleotide sequence of Prp1 promotor is as shown in SEQ ID No.2.
2. method of cultivation according to claim 1, is characterized in that, described step (1) is specially:
With restriction endonuclease SacI and SalI Ycf1p is cloned on intermediate carrier pGEM-5Z and obtains pYcf1p-5Z, after pYcf1p-5Z SalI and SpeI enzyme are cut, reclaim Ycf1p gene fragment, be connected on the plant expression vector pCAMBIA1301 after SalI and SpeI enzyme is cut through T4 ligase enzyme again, cut expression vector pCAMBIA1301 by BamHI enzyme again and containing the carrier T of Prp1 promotor, obtain after connection taking Totomycin as the expression vector pCAMBIA1301-pRP1-Ycf1p of selection markers under Prp1 promoters driven.
3. for cultivating a carrier for the transgenic plant of low heavy metal accumulation, it is characterized in that, described carrier contains yeast Ycf1p gene and epiblem cytolemma specific promoter Prp1;
The nucleotide sequence of described yeast Ycf1p gene is as shown in SEQ ID No.1, and the nucleotide sequence of epiblem cytolemma specific promoter Prp1 is as shown in SEQ ID No.2.
4. the engineering bacteria containing carrier described in claim 3.
5. the application of yeast Ycf1p gene in the transgenic plant cultivating low heavy metal accumulation, is characterized in that, comprise the steps:
(1) structure of recombinant expression vector:
By yeast Ycf1p gene clone on Agrobacterium plant binary expression vector, under the effect of restriction enzyme, connect the carrier T containing Prp1 promotor, obtain the expression vector of expressing Ycf1p gene under Prp1 promoters driven;
(2) structure of transfer-gen plant:
Gained expression vector is proceeded in plant callus, transforms with the nutrient solution containing inductor and Agrobacterium, the material after conversion through the exercise of Dual culture-screen-break up-take root-transgenic seedling and transplanting, screening transgenic positive plant;
The nucleotide sequence of described yeast Ycf1p gene is as shown in SEQ ID No.1, and the nucleotide sequence of Prp1 promotor is as shown in SEQ ID No.2.
6. application according to claim 5, is characterized in that, described plant is paddy rice.
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| CN108192914A (en) * | 2018-01-30 | 2018-06-22 | 西安泽睿环境科技有限公司 | Turn the initiative and application of the heavy metal super-enriched transgenic engineering rape of S.plumbizincicola SpNramp5 genes |
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| CN108192914A (en) * | 2018-01-30 | 2018-06-22 | 西安泽睿环境科技有限公司 | Turn the initiative and application of the heavy metal super-enriched transgenic engineering rape of S.plumbizincicola SpNramp5 genes |
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Effective date of registration: 20220803 Address after: 100097 No. 9 middle garden, Shuguang garden, Beijing, Haidian District Patentee after: BEIJING ACADEMY OF AGRICULTURE AND FORESTRY SCIENCES Address before: Room 511, Comprehensive Building, Beijing Academy of Agriculture and Forestry, No. 9 Shuguang Garden Middle Road, Haidian District, Beijing 100097 Patentee before: BEIJING RESEARCH CENTER FOR AGRICULTURAL STANDARDS AND TESTING |