CN104450771B - A kind of breeding method of the transgenic plant of low heavy metal accumulation - Google Patents

Abstract

The present invention relates to the cultivation of transgenic plant, specifically provides a kind of breeding method of the transgenic plant of low heavy metal accumulation, transport outward channel protein by heavy metal in heterogenous expression yeast to cultivate the safe crops of low accumulation heavy metal.The breeding method drives yeast Ycf1p genes heterogenous expression in the cell membrane of crop root system especially by epiblem cell membrane specificity promoter, enable plant that the heavy metal for entering root system of plant is discharged to plant through the metal extroversion transport protein on cell membrane external, obtain the crops of low heavy metal accumulation, so as to reach on the soil of heavy metal pollution, by planting the crop varieties of low absorption, low accumulation heavy metal, production meets the purpose of the agricultural product of safety criterion.Wherein, as shown in SEQ ID No.1, the nucleotide sequence of Prp1 promoteres is as shown in SEQ ID No.2 for the nucleotide sequence of the yeast Ycf1p genes.

Description

A kind of breeding method of the transgenic plant of low heavy metal accumulation

Technical field

The present invention relates to the cultivation of transgenic plant, specifically, is related to a kind of transgenic plant of low heavy metal accumulation Breeding method.

Background technology

In China, with the development of industrial and agricultural production, heavy metal pollution of soil problem is increasingly projected.Soil is to plant The storage vault of thing growth needs nutrient substance, thus the number of heavy metals in soil content determines agriculture to a great extent The contaminated degree of crop.The improvement in heavy metal pollution farmland is current one of difficult point and hot research field in the world.Science Family using fixed, filter, multiple Physicochemical methods such as ion exchange and insoluble fixation, it is intended to the soil of purification of heavy metal pollution Earth, but the application of these technology needs equipment and the technology of high price.Moreover as cultivated soil, pollute slight and contaminated area Practical application is difficult where big.In recent years, for the research work that heavy metal pollution farmland is administered is concentrated mainly on for planting The super tired foliage filter screening that thing is repaired, these plant major parts are wild, and the speed of growth is slow, and Biomass is low, and repair time is long, There is uncertain and plantation difficulty in Field information.Hyperaccumulative plant in heavy-metal contaminated soil is put in actual production Clean-up effect can not show a candle to the effect obtained under laboratory condition.In view of the actual national conditions of China, by large area-agriculture of slight pollution Field stops farming, carries out prolonged phytoremediation or other cost intensive engineering reparations are clearly unpractical. 2006S.Herbette et al. proposes the concept of the kind pollution-safe cultivars (PSCs) of pollution safety.I.e. Contaminated soil is grown in, accumulating level of the specific pollutants in edible part is sufficiently low, meets quality standard, is available for safety Edible kind.Reducing increasingly and the further serious present situation of soil heavy metal cadmium in the face of farmland resource, screening and cultivation Low absorption, the crop gene type of low accumulation heavy metal in soil, cultivate low amounts accumulation heavy metal in particular with genetic engineering Crop varieties become the main direction of studying for ensureing crop production safety.In recent years, pollute safe kind and attract research The bigger concern of person and breeding companies and interest.

Most of heavy metals are the nonessential trace element of plant, have very strong toxicity to plant.Exceed a certain amount meeting Suppress plant cell growth, inhibited oxidation phosphorylation, initiated oxidation to coerce, affect photosynthesis, damage kernel and affect plasma membrane The vigor of ATP enzyme.Some plants resist heavy metal poison by induced synthesis chelating peptide, metallothionein, plant stress protein etc. Evil, the approach such as the plant also having then is fixed by cell wall, vacuole separation, glandular secretion is resisting murder by poisoning.The metabolism of plant is produced Thing is stored after forming complex with heavy metal in vacuole in the cell, suppresses toxic insults.

Gene is to determine most important factor of the plant to heavy metal-polluted soil absorption and accumulation characteristic, low-accumulation plants heavy metal Rejection mechanism have been generally acknowledged that including two aspects, one be reduce root heavy metal absorption；Two is that heavy metal is logical in root Compartmentation preservation is crossed, is shifted to overground part so as to limit.But Plant Tolerance, transhipment and cadmium are complicated processes, relate to And transport protein family, chelating protein family, the participation of many aspects such as antioxidant system.At present, can apply in plant Genetic improvement is engineered, and its main metal transport proteins of heavy metal Rejection mechanism and key regulator appear in the newspapers not yet Road.

Content of the invention

In order to solve problems of the prior art, it is an object of the invention to provide a kind of low heavy metal accumulation turns base Breeding method because of plant.

In order to realize the object of the invention, present invention firstly provides a kind of cultivation side of the transgenic plant of low heavy metal accumulation Method, comprises the steps：

(1) structure of recombinant expression carrier：

By in yeast Ycf1p gene clonings to Agrobacterium plant binary expression vector, in the presence of restricted enzyme Carrier T of the connection containing Prp1 promoteres, obtains the expression vector for expressing Ycf1p genes under Prp1 promoters drivens；

(2) structure of transfer-gen plant：

Gained expression vector is proceeded in plant callus, is converted with the culture fluid containing derivant and Agrobacterium, Material after conversion through co-culturing-screen-break up-take root-exercise of transgenic seedling and transplanting, screening transgenic is positive to plant Strain；

Wherein, the nucleotide sequence of the yeast Ycf1p genes is as shown in SEQ ID No.1, the nucleoside of Prp1 promoteres Acid sequence is as shown in SEQ ID No.2.

Further, described step (1) is specially：

Ycf1p is cloned on intermediate carrier pGEM-5Z with restriction endonuclease SacI and SalI and obtains pYcf1p-5Z, will After pYcf1p-5Z is with SalI and SpeI enzyme action, Ycf1p genetic fragments is reclaimed, then is connected to through SalI and SpeI through T4 ligases On plant expression vector pCAMBIA1301 after enzyme action, then by BamHI enzyme action expression vector pCAMBIA1301 and contain Prp1 The carrier T of promoter, obtains marking with hygromycin in the case where epiblem cell membrane specific promoter Prp1 drives as screening after connection The expression vector pCAMBIA1301-pRP1-Ycf1p of note.

Present invention also offers a kind of carrier for cultivating the transgenic plant of low heavy metal accumulation, the carrier contains Yeast Ycf1p genes and epiblem cell membrane specific promoter Prp1；

Wherein, as shown in SEQ ID No.1, epiblem cell membrane is special for the nucleotide sequence of the yeast Ycf1p genes The nucleotide sequence of 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 yeast Ycf1p genes answering in the transgenic plant for cultivating low heavy metal accumulation With specially：

By in yeast Ycf1p gene clonings to Agrobacterium plant binary expression vector, in the presence of restricted enzyme Carrier T of the connection containing Prp1 promoteres, obtains the expression vector for expressing Ycf1p genes under Prp1 promoters drivens；

Gained expression vector is proceeded in plant callus, is converted with the culture fluid containing derivant and Agrobacterium, Material after conversion through co-culturing-screen-break up-take root-exercise of transgenic seedling and transplanting, screening transgenic is positive to plant Strain, gained transgenic positive plant will can be conveyed into by Active transport and passively planting by expressing yeast Ycf1p genes The heavy metal of thing root system is discharged to plant in vitro through the metal extroversion transport protein on cell membrane, so as to reduce plant body weight The purpose of tenor.

Breeding method of the present invention is can apply in any crops that can carry out transgenic, in the tool of the present invention In body embodiment, implement technical scheme by taking Oryza sativa L. as an example.

In order to preferably improve the productivity of contaminated soil, preferably described plant is Oryza sativa L..

The beneficial effects of the present invention is：

The present invention from the productivity for improving contaminated soil and ensures that the angle of crop production safety is started with, by epiblem Cell membrane specificity promoter drives yeast Ycf1p genes heterogenous expression in the cell membrane of crop root system, render transgenic to plant Thing obtains the toleration and repulsive force to specific heavy metal.The inventive method is cultivation low heavy metal accumulation, high patience, increment Big crop varieties provide important application and theoretical foundation, also the productivity for raising contaminated soil and guarantee agricultural product security Production provides effective approach.

Description of the drawings

Fig. 1 is pCAMBIA1301-pRP1-Ycf1p expression vectors collection of illustrative plates of the present invention.

Fig. 2 is the strain Ycf1p gene southern results of hybridization of transgenic paddy rice difference described in the embodiment of the present invention 1.

Fig. 3 is that transgenic paddy rice difference described in the embodiment of the present invention 1 strain Ycf1p gene RT-PCR and northern are miscellaneous Knot fruit.

Contents of the Fig. 4 for heavy metal cadmium in rice grain in different transgenic lines in the embodiment of the present invention 1.

Specific embodiment

Following examples are used for the present invention to be described, but are not limited to the scope of the present invention.

The structure of 1 recombinant vector of embodiment

Ycf1p is cloned on intermediate carrier 5Z with restriction endonuclease SacI and SalI and obtains pYcf1p-5Z, by p Ycf1p-5Z After with SalI and SpeI enzyme action, the Ycf1p genetic fragments of recovery, T4 ligases are connected to through the plant after SalI and SpeI enzyme action On expression vector pCAMBIA1301, by BamHI enzyme action expression vector and the carrier T containing Prp1 promoteres, obtain after link The expression vector with hygromycin as selection markers arrived in the case where Prp1 epiblem cell membrane specific promoter drives PCAMBIA1301-pRP1-Ycf1p (see Fig. 1), with Quick post large quantity extracting plasmids, tests for Oryza sativa L. Agrobacterium-mediated Transformation.

The structure of 2 transfer-gen plant of embodiment

1st, the preparation of transgenic plant receptor

The acquisition of mature embryo callus：Mature seed is removed clever shell, aseptically, is soaked with 70% ethanol 3-5min, aseptic water washing 3 times soak 20min, aseptic water washing 3～5 in mass fraction is proceeded to for 0.1% mercuric chloride solution Secondary, with 20% sodium hypochlorite immersion 20min, constantly rock therebetween, can be placed in shaking table, aseptic water washing 3～5 times is placed in Wipe dry on aseptic filter paper, moves on calli induction media, and 27 DEG C of light cultures removed bud after about three days, when scultellum is swollen after one week Greatly, remove the endosperm fraction of the grain of rice when endosperm softens, it is seen that yellow wound healing grows, and the embryo callus for peeling, is placed in and lures Lead in culture medium, once, after subculture 2-3 time, growth selection is rapid, quality is crisp, the wound healing that color is vivid for 15 days or so subcultures Organize for Agrobacterium-mediated Transformation.

2nd, Agrobacterium-mediated transformation Mature Embryos of Rice wound healing

(1) 200 μ l competent cells are taken, the plasmid DNA for adding 1 μ g to build, quick-freezing 1min in liquid nitrogen, 37 DEG C of water-baths 5min, is subsequently adding 1ml YEB culture medium, 28 DEG C of shaken cultivation 4h at a slow speed；30s is centrifuged with 10,000rpm, supernatant is abandoned, is added 0.1ml YEB culture medium suspension cell again, coats (100 μ g/ml Kan, 125 μ g/ml Sm) on YEB flat boards, 28 DEG C of trainings Support about 48h.

(2) single bacterium colony grown on picking flat board, is inoculated in YEB liquid mediums (100 μ g/ml Kan, 125 μ g/ml Sm, in), 28 DEG C of shaken cultivation are overnight；Extract plasmid DNA in a small amount, performing PCR amplification identification is entered as template with plasmid DNA.

(3) wound healing for having induced is proceeded in fresh culture medium, illumination cultivation 3 days.The Agrobacterium for shaking is taken, collects bacterium, Suspended and diluted certain multiple with the YEB culture medium containing 100umol acetosyringones.

(4) suspension 20-30 minutes in the Agrobacterium suspension medium for having diluted wound healing immersion.Wound healing is taken out, filter paper is used Unnecessary bacterium solution is blotted, is put in co-cultivation culture medium, light culture 3 days or so, until there is bacterium colony to grow.

(5) use the Mannitol sterile liquid oscillation cleaning of 0.1M twice, cleaned twice with the cephamycin liquid of 500mg/L, extremely Supernatant is clarified completely.

(6) wound healing is taken out, sucks unnecessary liquid, be placed on recovery media (500mg/L cef), light culture recovers three My god.

(7) wound healing is proceeded to and sieve on Antibiotics of Low Concentration (30mg/L hygromycin) and the screening culture medium of 500mg/L cef Select one month or so.

(8) wound healing is proceeded to and sieve on high concentration antibiotic (40mg/L hygromycin) and the screening culture medium of 500mg/L cef Select one month or so.

(9) wound healing is proceeded to and sieve on high concentration antibiotic (50mg/L hygromycin) and the screening culture medium of 500mg/L cef Select one month or so.

(10) division culture medium in MS minimal medium+3mg/L 6-BA+1mg/L NAA is shown in that light breaks up.

(11) screen on the root media of 1/2MS culture medium+50mg/L hygromycin and take root.

(12) grow to the 3-4 leaf phases and during more flourishing root system, by seedling on 1/2MS Transgenic Rice Seedlings growth mediums Move in small flower, outer housing plastic bag heat and moisture preserving；After moving to greenhouse white silk Seedling 5-7d, remove plastic bag；Land for growing field crops is moved into after one week, Until blossoming and bearing fruit.

3rd, the screening of transgenic positive plant

Especially by PCR to transfer-gen plant in Prp1 promoteres, on Ycf1p genes and pCAMBIA1301 carriers The positive rate of HYG screening-genes is detected that preliminary screening obtains positive plant S7, S9, S25, S30 and S37.By RT- Integration and expression of the method for PCRSouthern, Northern to the target gene in the transfer-gen plant of preliminary screening Detected, obtained the positive strain of stable expression.Southern results (see Fig. 2) show different strain genes of interest Ycf1p Insertion situation, the expression intensity of the different strain Ycf1p genes of RT-PCR with Northern results (see Fig. 3 and Fig. 4) display, its Middle S7, S9 and S37 this four strains, Ycf1p genes have higher expression, and the expression of S25 strains is taken second place, and S30 strains Ycf1p genes in Oryza sativa L. are not expressed substantially.

4th, the content of in plant cadmium element is detected

(1) the long-term monitoring point in somewhere is selected to carry out field experiment, the continuous 3 years monitor values of the Cadmium in Soil of the monitoring point are put down Average is 1.86mg/kg, faces more than the Cadmium in Soil of national standard of soil environment quality middle peasant production of forestry and plant normal growth Dividing value 1.0mg/kg.The Ycf1p trans-genetic hybrid rice seed that turns of the different strains processed through sterilization is directly seeded in soil.Wait to plant After son germination one week, according to size and the growing way situation thinning of seedling, each process is repeated 3 times and carries out random district's groups arrangement, Further checking test is carried out in land for growing field crops to the low accumulation cadmium characteristic of the transgenic positive rice strain for screening.

(2) heavy metal cadmium constituent content mensure, according to GB/T 17141-1997, vegetable cadmium content is according to GB/ for Soil K+adsorption T 5009.15-2003, with thermoelectricity M6 atomic absorption spectrophotometers to rice grain and soil in cadmium content detect. The present embodiment is 1.93mg/kg for examination Cadmium in Soil content, and same plot compares strain positive strain different with Ycf1p genes are turned It is that cadmium content is respectively 0.0680,0.0996,0.0695,0.0895,0.0998 in S7, S9, S25, S30 and S37 rice grain Arrive 0.705mg/kg.

(3) evaluate whether Oryza sativa L. has higher cadmium patience and relatively low accumulation from the angle of agricultural product security, that is, exist Under higher cadmium pollution can normal growth and Biomass without being remarkably decreased, and cadmium content is relatively low in the plant body.Especially by The edible part of Oryza sativa L. is less than relevant standard《Pollutants in food limitation GB2762-2012》Cadmium limit standard in middle regulation rice It is 0.2mg/kg evaluating.Rice strain S7, S9, S25 and the S37 obtained by this method, compares with control, is exceeding country's soil In the Cd-polluted farmland of earth grade III Standard, heavy metal preferably tolerance and good low characteristic of accumulation is shown, can conduct 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 the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.

Claims (5)

1. a kind of breeding method of the transgenic paddy rice of heavy metal low cadmium-accumulation, it is characterised in that comprise the steps：

(1) structure of recombinant expression carrier：

By in yeast Ycf1p gene clonings to Agrobacterium plant binary expression vector, connect in the presence of restricted enzyme Carrier T containing Prp1 promoteres, obtains the expression vector for expressing Ycf1p genes under Prp1 promoters drivens；

(2) structure of transfer-gen plant：

Gained expression vector is proceeded in plant callus, is converted with the culture fluid containing derivant and Agrobacterium, converted Rear material through co-culturing-screen-break up-take root-exercise of transgenic seedling and transplanting, screening transgenic positive plant；

Wherein, the nucleotide sequence of the yeast Ycf1p genes is as shown in SEQ ID No.1, the nucleotides sequence of Prp1 promoteres Row are as shown in SEQ ID No.2.

2. breeding method according to claim 1, it is characterised in that described step (1) is specially：

Ycf1p is cloned on intermediate carrier pGEM-5Z with restriction endonuclease SacI and SalI and obtains pYcf1p-5Z, by pYcf1p-5Z After with SalI and SpeI enzyme action, Ycf1p genetic fragments are reclaimed, then is connected to through the plant after SalI and SpeI enzyme action through T4 ligases On thing expression vector pCAMBIA1301, then carried with the T containing Prp1 promoteres by BamHI enzyme action expression vector pCAMBIA1301 Body, obtains the expression vector pCAMBIA1301- with hygromycin as selection markers under Prp1 promoters drivens after connection pRP1-Ycf1p.

3. a kind of carrier for cultivating the transgenic paddy rice of heavy metal low cadmium-accumulation, it is characterised in that the carrier contains ferment Female Ycf1p genes and epiblem cell membrane specific promoter Prp1；

The nucleotide sequence of the yeast Ycf1p genes as shown in SEQ ID No.1, epiblem cell membrane specific promoter Prp1 Nucleotide sequence as shown in SEQ ID No.2.

4. the engineering bacteria containing carrier described in claim 3.

5. yeast Ycf1p genes cultivate heavy metal low cadmium-accumulation transgenic paddy rice in application, it is characterised in that include as Lower step：

(1) structure of recombinant expression carrier：

By in yeast Ycf1p gene clonings to Agrobacterium plant binary expression vector, connect in the presence of restricted enzyme Carrier T containing Prp1 promoteres, obtains the expression vector for expressing Ycf1p genes under Prp1 promoters drivens；

(2) structure of transfer-gen plant：

Gained expression vector is proceeded in plant callus, is converted with the culture fluid containing derivant and Agrobacterium, converted Rear material through co-culturing-screen-break up-take root-exercise of transgenic seedling and transplanting, screening transgenic positive plant；

, as shown in SEQ ID No.1, the nucleotide sequence of Prp1 promoteres is such as the nucleotide sequence of the yeast Ycf1p genes Shown in SEQ ID No.2.