CN102398990A - Use of transgenic terrestrial plants in eutrophic water restoration - Google Patents

Use of transgenic terrestrial plants in eutrophic water restoration Download PDF

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Publication number
CN102398990A
CN102398990A CN2010102775641A CN201010277564A CN102398990A CN 102398990 A CN102398990 A CN 102398990A CN 2010102775641 A CN2010102775641 A CN 2010102775641A CN 201010277564 A CN201010277564 A CN 201010277564A CN 102398990 A CN102398990 A CN 102398990A
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water
transgenic
phosphorus
terrestrial plant
plant
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CN2010102775641A
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许雷
邱枫
兰军
徐汉卿
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许雷
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Abstract

The invention belongs to the field of biological phosphorus accumulating technology, and relates to a method for removing soluble phosphorus from eutrophic water by using transgenic terrestrial plants. According to the present invention, the transgenic terrestrial plants comprise tobacco in Solanaceae and strawberry in Rosaceae, and the imported exogenous gene is polyphosphate kinase gene (ppk); after the transgenic plants are induced by water roots, a foam plate sealed with a nylon net is adopted to fix the transgenic plants, which float on the water surface; in the high concentration soluble phosphorus (50 mg/ml)-containing water, the maximum phosphorus removal rates of the transgenic tobacco and the transgenic strawberry are respectively 57.4% and 41.38%; in the low concentration soluble phosphorus (100 mug/ml)-containing water, the maximum phosphorus removal rates of the transgenic tobacco and the transgenic strawberry are respectively 98.8% and 94.3%. The method of the present invention is applicable for efficient biological removal of the phosphorus, biological treatment of the phosphorus-rich wastewater, biological restoration of the eutrophic water, and biological purification of the eutrophic water.

Description

The purposes of transgenic terrestrial plant in the eutrophication water body is repaired
Technical field
The invention belongs to biology and gather the phosphorus technical field; Relate to a kind of method of utilizing the transgenic terrestrial plant to carry out the removal of eutrophication water body titanium pigment; The high-performance bio that is used for phosphorus is removed, and the biological sewage of rich phosphorus is handled the biological prosthetic of eutrophication water body and biopurification.
Background technology
China is the very poor country of a water resources, and the Freshwater resources total amount is about 28000 billion cubic meters, and the occupancy volume per person is about 2300 cubic metres, is merely 1/4 of world average level, arranges the 110th in the world, is one of the most in great shortage country of global water resources per capita.And the pollution of serious day by day water makes the water situation more severe.The whole pollution situation of the Yellow River, the Changjiang river, Huaihe River and Haihe River four large watersheds has become normality.It is five types and bad five types that 26% water quality is arranged in the 7 big water systems, and 7 is five types and bad five types in the 9 big lakes.
The principal mode of China's water pollution at present is: the water body eutrophication that excessive phosphorus content causes, and the outbursts such as blue-green algae that cause thus; Because body eutrophication causes blue algae bloom to cause the dead phenomenon of function of water body to be diffused into inland lake in all parts of the country from the Dian Chi, Yunnan, the one and another beautiful water body of paralysing was made the matter worse with regard to rare water resources originally.
Phosphorus is excessive to be the eutrophication water body, especially the inducement of inland lake eutrophication.Phosphorus is the nutrition source of algae and waterplant, and its content is determining the Yield of algae usually, and existing research shows, cause in all kinds of nutrition goods and materials of body eutrophication, and be limiting factor usually with phosphorus.Traditional biological phosphate-eliminating technology is to have adopted anaerobism (A/O) and anaerobic/anoxic/aerobic (A/A/O) active sludge; In recent years then begin to adopt with the activated sludge be matrix improvement enhanced biological phosphorus removal (enhanced biological phosphorus removal; EBPR), the cardinal principle of its dephosphorization is that polyP bacteria strain in the activated sludge is polymerized to soluble phosphate insoluble and is collected in endobacillary poly phosphorus.But activated sludge and EBPR mainly are applicable to sewage work and carry out the rough handling of sewage; Be difficult to the original position reparation of the eutrophication natural water body that phosphorus causes; Its reason is following: (1) activated sludge such as direct input natural water body; The phosphorus of its enrichment still in mud, is easy to be hydrolyzed to phosphoric acid salt again by poly phosphorus, gets into water body once more; (2) EBPR Technology Need aeration and anaerobism circulation, this can't realize at natural water body; (3) to gather phosphorus limited in one's ability for polyP bacteria itself; The phosphorus decomposing enzyme that phosphorus enzyme and catalysis poly phosphorus is hydrolyzed to soluble phosphate that gathers that catalysis phosphoric acid salt is polymerized to poly phosphorus in all polyP bacterias of finding has at present been formed an operon; Promotor in this operon is phosphorus regulation and control promotors; This promotor induces required phosphorus concentration to be higher than the phosphorus concentration in the national V class water quality standard far away, and the phosphorus concentration that therefore brings out the natural water body eutrophication is difficult to start and gathers phosphorus enzyme expression of gene; Gather being composed in series of phosphorus enzyme and phosphorus decomposing enzyme equally and make that gathering phosphorus expression of enzymes phosphorus decomposing simultaneously enzyme expresses synchronously, this causes gathering phosphorus and phosphorus decomposing running balance, is difficult in the enrichment of carrying out phosphorus in the natural water body.Therefore will be correlated with importing such as gathering the phosphorus gene through transgenic technology can be in the water body suspension culture but not the plant of hydrocoles bait is the important channel of effectively carrying out the polluted-water reparation; The research of carrying out this respect will be for effectively repairing polluted-water; Enlarge water resources capable of using, effectively carry out environmental management work, technical support is provided, have huge social and economic implications.
Summary of the invention
The objective of the invention is to practical problems and demand in the production practice; A kind of novel water body dephosphorization technology is provided; This technology be utilize to integrate the floating cultivation of water body of the transgenic Lu Sheng Solanaceae tobacco plant that gathers phosphorus enzyme gene and Rosaceae strawberry plant and plantation around the water body remove the water body titanium pigment with prevent or reduce titanium pigment from land leaching entering water body, the minimizing of realization water body titanium pigment content; Another object of the present invention is: provide and use the biology in situ renovation method that technology of the present invention is carried out the eutrophication water body of P pollution.
Technical scheme of the present invention is summarized as follows:
The present invention provides a kind of floating cultivation of the water surface that utilizes the terrestrial plant that changes the ppk gene to carry out the technology of biomagnification eutrophication water body titanium pigment.
1, the clone of ppk gene: (" Chinese microorganism strain is preserved management committee common micro-organisms center ", culture presevation number are the polyP bacteria of having by oneself with the applicant: CGMCCNo.1805) clone complete ppk gene for aimed strain.
2, through the Agrobacterium plasmid vector of subclone technique construction insertion ppk gene.
3, realize the conversion of ppk gene pairs tobacco, strawberry through leaf dish infestation method; Accomplish the regeneration of transgene tobacco, strawberry through plant tissue culture technique.
The plant expression vector that (1) will contain target gene changes among the Agrobacterium LBA4404.
(2) picking list bacterium colony activation in the YEB liquid nutrient medium is spent the night.
(3) getting 1ml bacterium liquid is inoculated in the 50mlYEB liquid nutrient medium and is cultured to the OD value for 0.8-1.0;
(4) 5000rpm is centrifugal, and 6 minutes, collecting precipitation;
(5) deposition is resuspended in the 80mlYEB liquid nutrient medium, the OD value of Agrobacterium is between 0.5-0.6 at this moment.
(6) will blade infected putting into sterilized water cleans;
(7) putting into the Agrobacterium for preparing infected 6-8 minute;
(8) filter paper blots and puts into MS+2.0mg/L 6-BA+0.2mg/L IAA, and dark is cultivated 2 days (tobacco) altogether; Filter paper blots puts into MS+3.0mg/L 6-BA+0.1mg/L 2.4-D, and dark is cultivated 3 days (strawberry) altogether;
(9) use the liquid MS of the Pyocianil that contains 500mg/L to clean 2-3 time in the blade of dark culturing.
(10) filter paper blots and puts into MS+2.0mg/L 6-BA+0.2mg/L IAA+500mg/L Carb+100mg/LKm (tobacco); Filter paper blots puts into MS+3.0mg/L 6-BA+0.1mg/L 2.4-D+500mg/LCarb+10mg/LKm (strawberry);
(11) the tobacco seedling that differentiates is put into MS+500mg/L Carb+100mg/LKm;
The strawberry seedling that differentiates is put into MS+0.5mg/L 6-BA+0.2mg/L IBA+500mg/L Carb+10mg/LKm.
4, detect through antibiotic-screening, PCR and Southern hybridization, obtain to change ppk gene masculine seedling
5, water root is induced: except that transgene tobacco, strawberry go to move on the water planting bed behind the terrestrial root, and aerating oxygen, progressively reduce oxygen feeding amount behind the formation adventive root, up to not oxygenation state.
6, the phosphorus ability of the gathering difference between more different transgenic plant strains system, screening gathers the highest transgenic seedling of phosphorus ability, realizes the volume production of transgenic seedling through fast breeding technique.
7, the volume production transfer-gen plant is colonizated on the floating plate, and floating plate below cover nylon wire is protected root, carries out water root and induces, and after water root grows, moves into the aquarium of culturing fish and carries out the reparation of eutrophication water body.
Efficiently gather the transgene tobacco of phosphorus, the soil that strawberry is colonizated in rich titanium pigment, carry out the soil original position and gather phosphorus.
The biology of transfer-gen plant gathers the phosphorus purposes
The purposes of this plant is that biology gathers phosphorus, is applicable to the removal phosphorus of trade effluent, life and agricultural area source sewage; The biological prosthetic and the biopurification of removal in common water body and the aquaculture water and relevant eutrophication water.
Embodiment
Embodiment 1: the water planting transgene tobacco is removed titanium pigment (high phosphorus) in the water body
The water body of phosphorous 50mg/L, water body volume 0.63m 3(1.5 * 0.6 * 0.7); Surface-area 0.9m 2Raise fingerling brocade carp (50g/ tail); Raise quantity: 15 tails; Feeding one day 2 times, feeding amount 1.5g/ time; 90 strains of water planting transgene tobacco seedling; Sampling in 3 days is 1 time at interval; Measuring the supernatant phosphorus content with molybdenum-antimony anti-spectrophotometric method, gather the phosphorus effect according to what transgene tobacco was calculated in the decline of supernatant phosphorus content, is contrast with the sample of suspension culture non-transgenic tobacco (quantity, growth conditions consistent).
The dephosphorization result of transgene tobacco seedling is as shown in table 1.
Table 1 transgene tobacco seedling is to the removal of water systems'phosphorus
Embodiment 2: the water planting transgene tobacco is removed titanium pigment (low-phosphorous) in the water body
The water body of phosphorous 100ug/L, water body volume 0.63m 3(1.5 * 0.6 * 0.7); Surface-area 0.9m 2Raise fingerling brocade carp (50g/ tail); Raise quantity: 15 tails; Feeding one day 2 times, feeding amount 1.5g/ time; 90 strains of water planting transgene tobacco seedling; Sampling in 3 days is 1 time at interval; Measuring the supernatant phosphorus content with molybdenum-antimony anti-spectrophotometric method, gather the phosphorus effect according to what transgene tobacco was calculated in the decline of supernatant phosphorus content, is contrast with the sample of suspension culture non-transgenic tobacco (quantity, growth conditions consistent).
The dephosphorization result of transgene tobacco seedling is as shown in table 2.
Table 2 transgene tobacco seedling is to the removal of water systems'phosphorus
Embodiment 3: water planting transgenic strawberry is removed titanium pigment (high phosphorus) in the water body
The water body of phosphorous 50mg/L, water body volume 0.63m 3(1.5 * 0.6 * 0.7); Surface-area 0.9m 2Raise fingerling brocade carp (50g/ tail); Raise quantity: 15 tails; Feeding one day 2 times, feeding amount 1.5g/ time; 90 strains of water planting transgenic strawberry seedling; Sampling in 3 days is 1 time at interval; Measuring the supernatant phosphorus content with molybdenum-antimony anti-spectrophotometric method, gather the phosphorus effect according to what the transgenic strawberry was calculated in the decline of supernatant phosphorus content, is contrast with the sample of suspension culture non-transgenic tobacco (quantity, growth conditions consistent).
The dephosphorization result of transgene tobacco seedling is as shown in table 3.
Table 3 transgenic strawberry seedling is to the removal of water systems'phosphorus
Embodiment 4: water planting transgenic strawberry is removed titanium pigment (low-phosphorous) in the water body
The water body of phosphorous 100ug/L, water body volume 0.63m 3(1.5 * 0.6 * 0.7); Surface-area 0.9m 2Raise fingerling brocade carp (50g/ tail); Raise quantity: 15 tails; Feeding one day 2 times, feeding amount 1.5g/ time; 90 strains of water planting transgenic strawberry seedling; Sampling in 3 days is 1 time at interval; Measuring the supernatant phosphorus content with molybdenum-antimony anti-spectrophotometric method, gather the phosphorus effect according to what the transgenic strawberry was calculated in the decline of supernatant phosphorus content, is contrast with the sample of suspension culture non-transgenic tobacco (quantity, growth conditions consistent).
The dephosphorization result of transgene tobacco seedling is as shown in table 2.
Table 4 transgene tobacco certain kind of berries seedling is to the removal of water systems'phosphorus

Claims (4)

1. one kind is utilized the transgenic terrestrial plant to carry out the method that eutrophication water body titanium pigment is removed, and its characteristic is following:
1): the chromosomal integration external source of this transgenic terrestrial plant is gathered phosphorus gene-gather phosphorus enzyme gene (ppk).
2): induce through aquatic, this transgenic terrestrial plant can grow in water body
3): terrestrial plant grows in water body through being fixed on the floatation of foam plate at first
4): a space is sealed out by nylon wire in the floating plate below; Space moisture and nutritive element can freely exchange with net is outer in the net; The water root of the interior space of net tolerable terrestrial plant fully stretches and contacts water body, but can prevent to touch roots of plants in the hydrocoles entering net
5): the transgenic terrestrial plant of the floating cultivation of water body can effectively be removed titanium pigment in the water body
6): these transgenic plant can be induced without water root and directly plant around water body, and enrichment flows to the phosphoric of water body by land, form the protection isolation strip of water body.
7): the transgenic terrestrial plant is: Solanaceae tobacco, Rosaceae strawberry
2. purposes according to claim 1, the foreign gene that this transgenic terrestrial plant imports is for gathering phosphorus enzyme gene (ppk).
3. purposes according to claim 1, this transgenic terrestrial plant can directly grow in water body through the floating cultivation of cystose, and can be used for high-performance bio and gather phosphorus, and the biological sewage of rich phosphorus is handled the biological prosthetic of eutrophication water body and biopurification.
4. purposes according to claim 1, this transgenic terrestrial plant can directly be planted zones such as beach around the water body, dykes and dams, hold back the titanium pigment element and flow to water body from land, be used for natural water body maintenance, prevent and repair the eutrophication water body.
CN2010102775641A 2010-09-10 2010-09-10 Use of transgenic terrestrial plants in eutrophic water restoration Pending CN102398990A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105145464A (en) * 2015-07-18 2015-12-16 常州大学 Method for removing antibiotics in aquaculture water
CN107746850A (en) * 2017-10-26 2018-03-02 海南师范大学 A kind of Polyphosphate kinase gene and the application in sewage dephosphorization
CN109609542A (en) * 2018-12-28 2019-04-12 博域环保技术研究院(南京)有限公司 The genetic engineering application of Polyphosphate kinase gene ppk1 in rice

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105145464A (en) * 2015-07-18 2015-12-16 常州大学 Method for removing antibiotics in aquaculture water
CN107746850A (en) * 2017-10-26 2018-03-02 海南师范大学 A kind of Polyphosphate kinase gene and the application in sewage dephosphorization
CN107746850B (en) * 2017-10-26 2018-07-24 海南师范大学 A kind of Polyphosphate kinase gene and the application in sewage dephosphorization
CN109609542A (en) * 2018-12-28 2019-04-12 博域环保技术研究院(南京)有限公司 The genetic engineering application of Polyphosphate kinase gene ppk1 in rice

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Application publication date: 20120404