CN1596657A - Method of reducing collection of cadmium in plant and its application - Google Patents
Method of reducing collection of cadmium in plant and its application Download PDFInfo
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- CN1596657A CN1596657A CN 03157195 CN03157195A CN1596657A CN 1596657 A CN1596657 A CN 1596657A CN 03157195 CN03157195 CN 03157195 CN 03157195 A CN03157195 A CN 03157195A CN 1596657 A CN1596657 A CN 1596657A
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Abstract
A method for decreasing the collection of Cd in plant features that three-valence La ions are applied to the growth medium of plant. It can be used to produce low-Cd agriculture products.
Description
Technical field
The present invention relates to a kind of method and application thereof that heavy metal accumulates that reduce in plant, particularly a kind ofly reduce method and the application thereof that cadmium accumulates in plant.
Background technology
Since the industrial revolution, the pollution of global toxic heavy metal aggravates rapidly, and wherein heavy metal cadmium is one of serious environmental pollutant, has every year 15000 tons of cadmiums to be produced, and is used for battery, alloy and dyestuff.The chronic environment that is exposed to cadmium pollution can cause the infringement of kidney and bone, also has the danger of cause cancer.Researching and proposing Trace Cadmium for one that in August, 2003, Nature-Medicine delivered has the estrogenic effect of simulation, even quite low dosage also will cause large-scale influence to health, may bring out the cancer of the uterus and breast cancer (Johnson MD, Kenney N, Stoica A, Hilakivi-Clarke L, Singh B, Chepko G, Clarke R, Sholler PF, LirioAA, Foss C, Reiter R, Trock B, Paik S ﹠amp; Martin MB.Cadmium mimics the in vivoeffects of estrogen in the uterus and mammary gland.Nature Medicine, 2003.9:1081-1084).According to estimates, only in the U.S., remove toxic heavy metal with traditional method and pollute and to spend at least 2,000 hundred million dollars.The phytoremediation technology of developed recently can utilize plant that the enrichment of heavy metal is removed and lower the pollution of heavy metal to environment, this technology is easy to operate, expense is cheap, and good governance role (Prasad is arranged for the pollution of the unable low concentration on a large scale that tackles of chemical method, M.N.V and Freitas, H.M.O.Feasible biotechnological and bioremediation strategies for serpentine soilsand mine spoils.EJB Electronic J.Biotechnology, 1999.2:36-50).But heavy metal super-enriched plant known today is very few, and often biomass is little, bioaccumulation efficiency is low and its habit of growth is not understood, and these have all limited phytoremediation technology application in practice.In the planting process of heavy metal accumulation plant, how improve it to bioaccumulation efficiency of heavy metal report not also by easy and inexpensive method.
At present, the quantity that municipal refuse, sewage, mud etc. are used for agricultural production is more and more many, more in particular for the city suburbs agricultural production.Relaxed the contradiction fertile, that water is in short supply on the one hand, for contribution has been made in the increasing both production and income of agricultural production; Bring heavy metal pollution in various degree then on the other hand soil and crops, influence the yield and quality of crops directly or indirectly, and enter the human body harm people's health by food chain.Especially near the vegetables heavy metal pollution suburb nearby, city and the industrial and mining enterprises is more and more serious.Vegetable soil is with the historical prolongation that plants vegetables, the increase of amount of cure and the pollution of urban industry, and the content of heavy metal has the trend that obviously increases, and wherein the content of beary metal of part vegetables has surpassed food hygienic standard.
Vegetables are indispensable food in the numerous people daily life, and the content of beary metal of vegetables is directly connected to the healthy of people.Soil pollution to vegetables influences bigger cadmium pollution, mercury pollution, pollution of chromium and arsenic contamination arranged, wherein particularly serious with cadmium pollution.Martin etc. are up-to-date discovers that cadmium can produce estrogen effect (Johnson MD, KenneyN in all safe doses (per kilogram of body weight 7 micrograms weekly) scope in world health organisation recommendations, Stoica A, Hilakivi-Clarke L, Singh B, Chepko G, Clarke R, Sholler PF, LirioAA, Foss C, Reiter R, Trock B, Paik S ﹠amp; Martin MB.Cadmium mimics the in vivoeffects of estrogen in the uterus and mammary gland.Nature Medic ine.2003.9:1081-1084).
Rare-earth elements of lanthanum can improve plant to the adaptive capacity of poor environment or increase resistance to adverse circumstance, as low temperature, high temperature, arid and salt marsh etc.Other there are some researches show, rare earth element can alleviate heavy metal lead to the injury effect of plant (Peng An, lanthanum coerce down the influence of wheat seedling activities of antioxidant enzymes to lead for Pang Xin, Wang Donghong. Environmental Chemistry, 2002,21 (4): 318-323).In the face of the reality that present heavy metal pollution of soil still can not be removed fully in the quite a long time, the task of top priority is to study the technology that how to reduce heavy metal accumulation in the vegetables.Vegetables are comparatively complicated to the mechanism that heavy metal absorbs, accumulates and detoxifies, someone adds the toxicity (Pang Xin that lanthanum can be alleviated heavy metal on plants by plant physiology research supposition, Wang Donghong, Peng An, lanthanum is coerced down the influence of wheat seedling activities of antioxidant enzymes to lead. Environmental Chemistry, 2002,21 (4): 318-323; Zhang Jiarong, Gu Yuehua, Zhao Guiwen, the rare earth seed soaking is to the biological effect of rape seed sprouting and seedling early growth. rare earth, 1999,30 (3): 55-57; Zhou Qing, Huang Xiaohua, yellow steel industry etc., La uses and the environmental organism journal the influence of Pb injury soybean seedling, and 1999,5 (1): 22--25).Nearest studies show that plant complexing element and phytochelatin synthase gene play an important role in the tolerance mechanism of vegetables to heavy metal.But about the relation of lanthanum and this expression of gene and the accumulation of plant heavy metal does not still have report both at home and abroad.
The innovation and creation content
The purpose of this invention is to provide a kind of method and application thereof that cadmium accumulates that reduce in plant.
The method that reduction cadmium provided by the present invention accumulates in plant is to apply the trivalent lanthanum ion in containing the plant growth medium of cadmium.
Described plant can be crops, vegetables, and ornamental plants, trees etc. are preferably vegetables, are the leaf vegetables of leaf vegetables, particularly Cruciferae especially, most preferably are romaine lettuce.The trivalent lanthanum ion can be from La (NO
3)
3, La (Ac)
3, LaCl
3Or, be preferably La (NO based on the RE microbial fertilizer of above-mentioned composition
3)
3The concentration of trivalent lanthanum ion is 0.02-0.06mg/L or 0.02-0.06mg/kg somatomedin, is preferably 0.04mg/L or 0.04mg/kg plant growth medium.Plant growth medium can be soil, sand, any materials that are fit to plant growing such as Sickle stone.
Method of the present invention has reduced the accumulation of plant to cadmium effectively.The romaine lettuce seedling applies the 0.04mg/L lanthanum nitrate under cadmium is coerced after, the trivalent lanthanum ion can suppress the accumulation of romaine lettuce to the harmful heavy metal cadmium, the particularly accumulation in the vegetables cauline leaf.The existing lanthanum that studies show that all be safe and harmless to environment and animal, and the cadmium concentration that applies among the present invention is far below this critical value when using critical value 30mg/kg mold.Method of the present invention can be widely used in produces low cadmium crops.
Description of drawings
Figure 1A is the sprouting photo of romaine lettuce seed under the different disposal condition
Figure 1B is the long and long statistic histogram of stem of fresh weight, root of different disposal romaine lettuce bud
Fig. 2 coerces down the histogram that influences of 20 days romaine lettuce fresh weight of growth, root length and stem length to cadmium for lanthanum
Fig. 3 A is the content histogram of cadmium in the romaine lettuce root after different disposal
Fig. 3 B is the content histogram of cadmium in the romaine lettuce cauline leaf after different disposal
Fig. 4 is LsPCS1 fragment nucleotide sequence and encoding amino acid sequence
Fig. 5 A romaine lettuce cauline leaf RT-PCR electrophoretogram
Fig. 5 B romaine lettuce root RT-PCR electrophoretogram
Fig. 6 A be in the Chinese leaf under lanthanum is coerced cadmium phytochelatin synthase gene expression influence histogram
Fig. 6 B be in the romaine lettuce root under lanthanum is coerced cadmium phytochelatin synthase gene expression influence histogram
Embodiment
Material
1, vegetable material and plasmid
Used vegetable material is U.S.'s balling romaine lettuce (Lactuca sativa); Main agents is import or homemade analytical reagent.Cloning vector pGEM-TEasy is available from Promega company.
2, main biochemical reagents and enzyme
Restriction endonuclease, dna molecular amount object of reference are available from Huamei Bio-Engrg Co.; DNA reclaims kit and purchases the biotech company to Shanghai Shen You; Taq enzyme, dNTPs and RT-PCR kit are available from Takara company; X-Gal, IPTG are available from Promega company; SuperScript II reverse transcriptase is available from Invitrogenn company.
The influence of the romaine lettuce seed germination under embodiment 1, lanthanum are coerced cadmium
With the romaine lettuce seed with 70% alcohol immersion 30 seconds, 0.1%HgCl
2Aqueous solution soaking 10 minutes is used aseptic water washing 5 times then, and in the plate that is lined with filter paper, sprout the 28 ℃ of dark places of solution (processing and code see Table 1) that drip different disposal with the seed dibbling.After 5 days, the seedling of gathering, flushing back suck dry moisture claim its fresh weight, measure the length of its root and stem.
Table 1, various processing and concentration of treatment
The code process concentration of treatment
The CK contrast---
The La lanthanum is handled 0.04mg/L La (NO
3)
3
The Cd cadmium is handled 0.05mM CdCl
2
Cd+La cadmium lanthanum is handled 0.05mM CdCl
2+ 0.04mg/L La (NO
3)
3
The romaine lettuce seed is in 0.05mM CdCl
2Coerce down and sprout after 5 days, compare with the seed under the normal condition, the young shoot fresh weight reduces, root is long and stem is long reduces, and wherein obvious variation is the inhibition to root growth.The result shows to add 0.04mg/L La (NO simultaneously shown in Figure 1A and Figure 1B
3)
3, the root of romaine lettuce obviously than only long with the root of cadmium processing, illustrates that the trivalent lanthanum ion has antagonism to the toxicity of cadmium to a certain extent; On rectangular of fresh weight, stem, lanthanum ion has also slowed down the toxicity of cadmium to a certain extent.
The influence of the romaine lettuce plant strain growth under embodiment 2, lanthanum are coerced cadmium
Romaine lettuce (Lactuca sativa) seed is handled back germination and emergence in the quartz sand of cleaning according to the method for disinfection in the step 1, get the consistent seedling of growth after 10 days, clean up, move in polypots, every alms bowl is contained the sand that 4/5 volume is cleaned, nutriment 1/2MS (CaCl
2Free) medium joins in the sand in the mode of solution, cultivates photoperiod 16/8h, 25 ℃ of cultivation temperature in the greenhouse.Plant strain growth begins after 20 days to handle.6 processing are established in test, and the concentration of cadmium ion and lanthanum ion saw Table 2 during each was handled.Each is handled respectively at 1h, 2h, and 6h, 12h, 24h, 48h and 96h divide 7 sub-samplings, gather root and cauline leaf respectively, and each sample is equipped with four parts.A copy of it is used to measure cadmium content.
Table 2, various processing and concentration of treatment
The code process concentration of treatment
The CK contrast---
The La lanthanum is handled 0.04mg/L La (NO
3)
3
The Cd cadmium is handled 0.5mM CdCl
2
Cd+La cadmium lanthanum is handled 0.5mM CdCl
2+ 0.04mg/L La (NO
3)
3
Coerce down at cadmium, the most tangible poisoning symptom of romaine lettuce plant is a blade wilting chlorosis, poor growth even stagnation.But add the toxicity that lanthanum can be alleviated cadmium to a great extent simultaneously, plant does not see tangible wilting.The result shows at cadmium and coerces down that the plant of cadmium processing is compared fresh weight with adjoining tree, root is long and stem is long all significantly descends as shown in Figure 2, and the plant fresh weight that the cadmium lanthanum is handled, root length and stem length have only reduction very by a small margin.Illustrate that adding the trivalent lanthanum ion can improve the tolerance of romaine lettuce to heavy metal cadmium.
Embodiment 3, apply lanthanum is coerced down the accumulation of cadmium in the romaine lettuce to cadmium effect
With after being used to measure the romaine lettuce root of cadmium content and cauline leaf among the embodiment 2 and cleaning, complete 65 ℃ of oven dry down under 105 ℃ with deionized water.After sample is pulverized, take by weighing in right amount, add 3ml nitric acid and in microwave dissolver, clear up 15min, be settled to after clearing up in the 25ml volumetric flask, with sampling Graphite Furnace Atomic Absorption spectrophotometric determination cadmium content (cadmium wavelength: 228.8nm).
The result shows that cadmium content is almost nil in the romaine lettuce plant that contrasts and only add lanthanum shown in Fig. 3 A and Fig. 3 B.Add cadmium in nutrient solution, cadmium content just significantly increases in the romaine lettuce plant, and in the root cadmium content obviously than high in the cauline leaf; And along with the prolongation of time, the content of the cadmium in romaine lettuce root and the cauline leaf increases.But the content of cadmium is starkly lower than the Cd processing in the romaine lettuce that Cd+La handles.Wherein when cadmium was coerced 12h, cadmium content was respectively 205.8 μ g/gDW and 165.8 μ g/g DW in root and the cauline leaf; Add the trivalent lanthanum ion when cadmium is coerced, cadmium content is 185.1 μ g/g DW and 73.8 μ g/g DW in romaine lettuce root and the cauline leaf, is reduced to 89.9% and 44.5% respectively.When cadmium was coerced 96h, cadmium content was respectively 709.7 μ g/g DW and 416.8 μ g/g DW in romaine lettuce root and the cauline leaf; Add the trivalent lanthanum ion when cadmium is coerced, cadmium content is 556.6 μ g/g DW and 255.6 μ g/g DW in romaine lettuce root and the cauline leaf, is reduced to 78.4% and 53.7% respectively.Illustrate that applying the trivalent lanthanum ion has reduced its absorption and accumulation to cadmium in to the heavy metal cadmium tolerance having improved romaine lettuce.
The molecule mechanism of embodiment 4, lanthanum effect
1, the clone of LsPCS1 fragment and order-checking
(1) preparation of the total DNA of romaine lettuce
Extract buffer solution: 5ml Tris (1M, pH 8.0), 5ml EDTA (0.5M, pH 8.0), 6.25mlNaCl (4M), 3.3ml 20%SDS, 1.180 β-Mecapto ethanol are settled to 50ml.
Get 0.2g romaine lettuce complete stool and in liquid nitrogen, be ground into powdery, add 800 μ l and extract buffer solution (65 ℃) concuss mixing, 65 ℃ of water-baths 20 minutes, per 5 minutes mixings are once.Add 250 μ l, 5 M KAc (precooling), put upside down mixing rapidly and placed 5 minutes on ice.4 ℃, 12000rpm, centrifugal 5min, supernatant change new pipe over to, and 4 ℃, 12000rpm, centrifugal 5min thoroughly goes precipitation.Supernatant changes the centrifuge tube that contains 600 μ l isopropyl alcohols over to, puts upside down mixing at least 10 times (RNA precipitation).4 ℃, behind the centrifugal 15min of 12000rpm, remove supernatant.Add 500 μ l deionized water dissolvings precipitation, use isopyknic phenol: chloroform: isoamyl alcohol (25: 24: 1) extracting, 4 ℃, the centrifugal 5min of 12000rpm, supernatant precipitates with 2 times of volume ethanol.75% ethanol, washing precipitation and the centrifugal tube wall that add 4 ℃ of precoolings of 1ml.4 ℃, the centrifugal 5min of 12000rpm abandons supernatant, blots with blotting paper.Add an amount of Milli-Q water, gentle vibration is carried out ultra-violet analysis and is measured to dissolving fully.
(2) clone of LsPCS1 fragment
According to the cDNA sequence comparative result of the phytochelatin synthase gene of the arabidopsis of having cloned, soybean, rape etc., at the conserved regions design degenerate primer, upstream primer and downstream primer are respectively:
5’-TGGAAGGG(AGCT)CC(CT)TGGAG(AGCT)TGG-3’，
5’-TGCCGTCGCAG(A/G)TGCA(G/A)(T/A)ACCT-3’
With total DNA of romaine lettuce as template, carry out pcr amplification, in the reaction system of 20 μ L, add the about 10ng of template DNA successively, 10 * PCR buffer solution, 2 μ l, 2.5mmol/L dNTP2 μ l, each 1 μ l of 20mmol/L upstream and downstream primer, Taq archaeal dna polymerase 1U, the enterprising performing PCR amplification of PTC200 type amplification instrument of producing in MJ Research company.Reaction condition is 95 ℃ of pre-sex change 5min, the 94 ℃ of sex change 30sec that at every turn circulate, and 60 ℃ of annealing 30min, 72 ℃ are extended 1min, totally 30 circulations, last 72 ℃ are extended 10min again.Total DNA cloning product electrophoresis detection on 1% Ago-Gel, and with the GDS 8000 gel images analytical systems record of taking pictures.
(3) recovery of specific amplified fragment and cloning and sequencing
The specific amplified fragment is through the low melting-point agarose gel electrophoresis, and recovery, purifying are cloned on the pGEM-TEasy carrier according to a conventional method, and Transformed E .coli DH5 α, after the blue hickie screening, carry out EcoR I enzyme and cut evaluation, PCR identifies that bacterial strain is delivered to the order-checking of Shanghai Shen You biotech company.The result as shown in Figure 4, show and from the romaine lettuce genomic DNA, amplify a segment, sequencing result shows, size is 421bp, 115 amino acid of encoding, carry out the autoploidy comparative result and show in GenBank, the plain synthase gene autoploidy of this fragment and other plant complexing reaches 85%, wherein comprises the intron of a 76bp.With this fragment called after LsPCS1, and in GenBank registration (Accession No.AF506283, the protein sequence of nucleotide sequence and supposition is seen Fig. 4).
The influence of the romaine lettuce phytochelatin synthase gene expression under 2, lanthanum is coerced cadmium
(1) extracting of romaine lettuce seedling leaf, the total RNA of root
Adopt total RNA of TRIZOL total RNA extraction agent box (GIBCOBRL company product) extracting romaine lettuce seedling leaf, root.CK, La, Cd, Cd+La (CdCl are set
20.5mM, La (NO
3)
30.04mg/L) 4 processing, respectively at the sample of gathering behind 1h, 2h, 6h, 12h, 24h, the 48h, extract RNA in cauline leaf and the root and carry out sxemiquantitative RT-PCR and detect.Take by weighing and organize 0.1mg to put into stone roller alms bowl, in liquid nitrogen, be ground to Powderedly, go to the homogenate pipe and add RNA extraction buffer 1ml with the liquid nitrogen precooling.The homogenate pipe is inserted on ice static ice bath 20 minutes.Add equal-volume phenol chloroformic solution, firmly shook ice bath 20 minutes 1 minute.4 ℃, 12000rpm, centrifugal 20min.From every pipe, draw supernatant to another centrifuge tube.Add 1/10 volume 3mol/l NaAc (pH4.5) respectively, add equal-volume acidic phenol chloroformic solution, firmly shook centrifuge tube 1 minute, ice bath 20 minutes.4 ℃, 12000rpm, centrifugal 20min.From every pipe, draw supernatant to another centrifuge tube, add the isopropyl alcohol of equal-volume-20 ℃ precooling, the mixing postprecipitation.4C behind the centrifugal 20min of 12000rpm, removes supernatant.75% ethanol, washing precipitation and the centrifugal tube wall that add 4 ℃ of precoolings of 1ml.4 ℃, the centrifugal 5min of 12000rpm abandons supernatant, blots with blotting paper.Add 25 μ l Milli-Q water, gentle vibration is carried out ultra-violet analysis and is measured to dissolving fully.RNA is placed-20 ℃ of preservations standby.
(2) RT-PCR detects the expression of LsPCS1
1. different disposal cDNA's is synthetic
By the synthetic reverse transcription of following composition
2×mRNA?Selective?PCR?Buffer?25μl，
MgCl
2 10μl，
DNTP/ homologue mixture 5 μ l,
AMV reverse transcriptase 1 μ l,
Downstream special primer 1 μ l,
ddH
2O 6μl。
Carry out reverse transcription reaction by following condition: 30 ℃ of 10min, 45 ℃ of 30min, 5 ℃ of 5min.
2. the clone of romaine lettuce actin genetic fragment
Design pair of degenerate primers according to dicotyledon actin gene conservative districts such as arabidopsiss: the upstream, 5 '-TTTGCTGGAGATGATGCTCC-3 ', the downstream, 5 '-GTGGTACGACCACTGGCATA-3 ', cDNA is a template with the romaine lettuce contrast, carries out pcr amplification, obtain the band of size about a 400bp, through recovery, connect, check order, comparing, prove that the actin gene with dicotyledon has high autoploidy, be the actin genetic fragment of romaine lettuce.The actin expression of gene is more stable, do not stimulate or tissue is different changes because of external environment, in RT-PCR as confidential reference items.
3. PCR reaction:
According to the sequence of LsPCS, design a pair of special primer:
The upstream, 5 '-GCTTGACTGTTGCGAGCCTTTG-3 '
Downstream 5 '-ATCAATGCCATGTCCCTTCCAGCA-3 '
CDNA with different disposal is a template, is undertaken by following program: 95 ℃ of 30sec, 60 ℃ of 30sec, 72 ℃ 50 seconds, 23 circulations.PCR all is provided with corresponding positive and negative contrast, promptly with total DNA be template as PCR over against photograph, not add template as negative contrast.The result shows that with the first synthetic chain cDNA as template, special primer amplifies a band about 300bp shown in Fig. 5 A and Fig. 5 B.
The PCR product of getting equivalent carries out electrophoresis, through Alphaimager scanning and utilize BiolD that the band of each swimming lane is carried out integration.Measure corresponding actin gene expression amount with method, and both are compared, the expression of LsPCS1 in romaine lettuce cauline leaf and the root is analyzed.The result shows that the transcriptional level of LsPCS1 in adjoining tree cauline leaf and the root is all lower shown in Fig. 6 A and Fig. 6 B; Coerce down at heavy metal cadmium, the transcriptional level of LsPCS1 obviously raises, and the speed of LsPCS1 mRNA accumulation in the root and amount are all than high in the cauline leaf.The similar trend of cadmium accumulation in this and the plant.Add the trivalent lanthanum ion when cadmium is coerced after, the expression of LsPCS1 is higher; Particularly in handling 24 hours root, the expression of LsPCS1 is 6.4 times of contrast, 1.7 times of coercing of cadmium; But the accumulation of corresponding cadmium drop to that cadmium coerces 57%.This shows that the trivalent lanthanum ion may be the murder by poisoning that reduces heavy metal on plants in heavy metal detoxification by the phytochelatin synthase expression of gene that promotes to play an important role, and its result shows as that plant is improved the resistance of heavy metal and accumulation reduces.
Claims (10)
1, a kind ofly reducing the method that cadmium accumulates in plant, is to apply the trivalent lanthanum ion in containing the plant growth medium of cadmium.
2, method according to claim 1 is characterized in that: described plant is crops, vegetables, ornamental plants, trees.
3, method according to claim 2 is characterized in that: described plant is vegetables.
4, method according to claim 3 is characterized in that: described plant is a romaine lettuce.
5, according to claim 1,2,3 or 4 described methods, it is characterized in that: described trivalent lanthanum ion is from La (NO
3)
3, La (Ac)
3Or LaCl
3Or based on the RE microbial fertilizer of above-mentioned composition.
6, method according to claim 5 is characterized in that: described trivalent lanthanum ion is La (NO
3)
3
7, according to claim 1,2,3 or 4 described methods, it is characterized in that: the concentration of described trivalent lanthanum ion is 0.02-0.06mg/L or 0.02-0.06mg/kg somatomedin.
8, method according to claim 7 is characterized in that: the concentration of described trivalent lanthanum ion is 0.04mg/L or 0.04mg/kg plant growth medium.
9, method according to claim 1 is characterized in that: described plant growth medium is a soil, sand or Sickle stone.
10, the application of the described method of claim 1 in producing low cadmium plant.
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| CN109287659A (en) * | 2018-11-12 | 2019-02-01 | 武汉大学 | Lanthanum is as the agent of crop cut cadmium and cadmium transporter gene expression inhibiting agent and the application of increasing agent |
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| CN109940031A (en) * | 2019-03-20 | 2019-06-28 | 浙江大学 | Witchweed lactone is alleviating Cd stress to the application in plant generation cadmium poisoning |
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