CN102353635B - Method for determining individual eichhornia crassipes plant dry weight by phosphoenolpyruvate carboxylase (PEPC) activity determination, and use thereof - Google Patents
Method for determining individual eichhornia crassipes plant dry weight by phosphoenolpyruvate carboxylase (PEPC) activity determination, and use thereof Download PDFInfo
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Abstract
The invention relates to a method for determining individual eichhornia crassipes plant dry weight by phosphoenolpyruvate carboxylase (PEPC) activity determination, and a use thereof. The method is characterized in that individual eichhornia crassipes plant dry weight is determined by determination of PEPC activity of functional leaves of an eichhornia crassipes plant in an exuberant growth period; the exuberant growth period of eichhornia crassipes is a period from July to September every year; and the functional leaves of eichhornia crassipes comprise all leaves but an eichhornia crassipes heart leaf and the oldest leaf. The method can determine individual eichhornia crassipes plant dry weight by determination of PEPC activity of functional leaves of an eichhornia crassipes plant in an exuberant growth period, so that an identification index of harvest of large-scale artificial planting of eichhornia crassipes can be determined.
Description
Technical field
The present invention relates to a kind of total dry weight new detecting method and application thereof to Eichhornia crassipes in the vigorous period, especially introduce PEPC enzyme assay technology and judge the Eichhornia crassipes total dry weight, be used for managed the growth period of artificial breeding Eichhornia crassipes.
Background technology
Eichhornia crassipes (Eichhornia crassipes) belongs to the vascular bundle hydrophyte, is commonly called as water hyacinth, has flourishing root system, vigorous growth and breeding and superpower receptivity.It is for feed the earliest that Eichhornia crassipes is introduced China.But, the states such as Eichhornia crassipes original producton location Brazil, Argentina and Peru generally are distributed in the world's most areas between south latitude 32.3 degree and north latitude 32 degree, and are surprising 25～35 ℃ of lower speeds of growth, usually just can increase to 600,000 from 10 in 8 months, be one of the fastest plant of the growth of generally acknowledging.Calculate and find, as long as conditions permit, the individual plant Eichhornia crassipes can produce 1.4 hundred million plant division in 1 year, can be paved with 140hm
2The water surface, fresh weight can reach 28000t, as seen its repopulate and diffusivity are extremely strong, for the ecological safety of its utilization has brought very high risk.At present, 17 provinces and cities of south China all exist serious Eichhornia crassipes to spread unchecked problem, and Eichhornia crassipes also becomes one of the world's ten large evil grass.Also be used in recent years the culture matrix of edible fungi and biogas fermentation etc., [Hu Changwei particularly is widely used in the eutrophied water treatment engineering, Sun Zhandong, Li Jianlong, Deng. the application [J] of Eichhornia crassipes in heavily contaminated river course, city is repaired. the environmental engineering journal, 2007,1 (12): P.51-56; Qi Yumei, Gao Weisheng. Eichhornia crassipes purifies water and aftertreatment technology is inquired into [J]. the environmental science progress, 1999,7 (2): P.136-140], make it become a kind of important resource [Zhou Wenbing that utilizes of modern Efficient Eco-agriculture, Tan Liangfeng, Liu Dahui, etc. Eichhornia crassipes and research of utilization progress [J] thereof, Hua Zhong Agriculture University's journal, 2005,24 (4): P.423-428; Contain Jing, keep root, Zhu Puping, etc. the farmland utilization research [J] of high eutrophication plant Eichhornia crassipes. Chinese Ecological Agriculture journal, 2010,18 (1): P.46-49], also provide new outlet for people effectively control these species.Along with the increase in the artificial breeding of Eichhornia crassipes place, cultivation work person finds that the Eichhornia crassipes form of different location breeding is variant, such as lobe numbers, plant height and root length etc.Although the speed of growth of Eichhornia crassipes is very fast, the direction of growth then has difference, and when plant is less, and when the water surface of open spaciousness was arranged, it was the shape horizontal growth of crawling, and waits to be full of after the whole water surface, then changes the plant longitudinal growth into.When the Eichhornia crassipes growth is subject to the growing environment restriction, the plant old plant that then can come off preferentially guarantees the nutrition supply of newborn plant, if untimely results and the old and feeble plant of leaving comes off in water, then may cause twice pollution of water body, therefore, judging exactly the optimum growh state of Eichhornia crassipes, gather timely, then is that Eichhornia crassipes is in the problem of the efficient most critical that utilizes of eutrophic water.How to judge Eichhornia crassipes at the upgrowth situation of different regions, determine suitable results period, also become the key issue that Eichhornia crassipes resource efficient technique of rainwater utilization is promoted in China vast eutrophic water area.
The phoenix eyes Nelumbo has 8 Plants in the world, but the result of study according to cytology and molecular genetics, what be distributed at present China only has a kind of [Wang Guangxi from state, South America Brazil, Wang Hui is diligent. the STUDY ON THE KARYOTYPE of Eichhornia crassipes [J]. and Wuhan University Journal (natural science edition), 1989,4:P.131-132; REN MX, ZHANG Q G﹠amp; ZHANG D Y.Random amplified polymorphic DNAmarkers reveal low genetic variation and a single dominant genotype inEichhornia crassipes populations throughout China[J] .Weed Research, 2005,45:P.236-244], this shows that the Eichhornia crassipes platymiscium is relevant with the ecological condition of China in the distribution of China.Wang Guirong and spring recent studies on some environmental baseline on Eichhornia crassipes biological productivity impact [Li Xuebao, what light source, Wu Zhenbin, Deng. Eichhornia crassipes, the research [J] of the some photosynthesis parameters of water peanut and enzyme. the hydrobiont journal, 1995,19 (4): P.333-336], and find Eichhornia crassipes to the adaptability of low temperature than the poor [Li Xuebao of water peanut (Alternanthera philoxeroides), what light source, Wu Zhenbin, Deng. Eichhornia crassipes, the research [J] of the some photosynthesis parameters of water peanut and enzyme. hydrobiont journal, 1995,19 (4): P.333-336].Zhu Hui etc. show from the invasion of Eichhornia crassipes and the angle of photosynthetic capacity, it is machine-processed with stronger Photosynthetic Response that the Eichhornia crassipes of In Guangdong Province has higher light-use power, this is that it has the physiological foundation [Zhu Hui that power is invaded in more trunk offering than large Piao, Ma Ruijun, the comparison of 2 kinds of aquatic instruction plant Photosynthetic Characteristics [J], Journal of Northwest Sci Tech University of Agriculture and Forestry (natural science edition), 2010,38 (5): P.193-198].There are some researches show that the photosynthetic capacity of In Nanjing Leaves of Eichhornia Crassipes in August is significantly higher than paddy rice, and with corn near [king is full for Li Xia, Ren Chenggang, etc.Strong and the temperature-responsive [J] of Jiangsu Province Leaves of Eichhornia Crassipes Photosynthetic, Jiangsu agricultural journal, 2010,26 (5): P.943-947], these researchs all are Eichhornia crassipes to the performance when the utilization of resources of flash of light preceding an earthquake temperature, and Eichhornia crassipes has high light efficiency characteristic under high light intensity and hot conditions.Wang Qiang etc. studies show that, the luminous energy that two line system is higher and CO
2Utilization ratio, stronger anti-light suppress ability and higher C
4Path enzyme phosphoric acid enol pyruvic acid carboxylase (Phosphoenolpyruvate carboxylase, PEPC) expression is the major reason [Wang Qiang of its Super-high-yielding, Lu Congming, Zhang Qide, etc. the photosynthesis of Super High-yield Rice Hybrids two line system, light suppress and C
4Path enzyme characteristic [J] is rushed state's science [C collects], 2002,32 (6): P.482-487].PEPC is at C
4Play concentrated CO in the plant
2To improve the effect of photosynthetic efficiency, especially at C
4The photosynthetic gene of type pepc imports C
3The plant paddy rice, and behind the high expressed, the PEPC enzyme just becomes the screening index of an important high light efficiency of paddy rice, and enjoy plant physiologist and crop breeding scholar extensive concern, become hot issue [Ku MSB, the Sakae A of crop breeding scholar research, Mika N, et al.High-level expression of maize phosphoenolpyruate carboxylase in transgenicrice plants[J] .Nature Biotechnology, 1999,17:P.76～78; Li Xia etc., the PEPC enzymatic activity is as the research of paddy rice breeding of high photosynthetic efficiency screening index, Jiangsu agricultural journal, 2008,24 (5): P.559-564].
We studies show that, Eichhornia crassipes is the aquatic emergent aquactic plant that is fit to the high light efficiency of high light intensity and hot conditions.Eichhornia crassipes artificial cultivation worker detects its upgrowth situation normally with reference to the assay method of hydrophyte dry matter accumulation, the fresh weight of results of regular determination plant, by oven for drying to constant weight, measure the dry weight of plant, determine at last its suitable gathering period, the method among a small circle research in the laboratory is fit to.But the continuous expansion along with the scope of the large-scale cultivation of Eichhornia crassipes; and because the high-biomass of Eichhornia crassipes; moisture is very high; eugonic especially period, the leaf volume of plant was very large; dry weight only accounts for 1/10th of fresh weight usually; therefore; because unique biology of Eichhornia crassipes forms; certainly will need to increase instrument and equipment and the drying times of long period such as large volume baking oven that oven dry is weighed and mated; add altering a great deal owing to the growth of Eichhornia crassipes form; one of objective judgement also needs to increase different sampling spots than the increase soil fertility accumulation of biomass in district of macrospecies; therefore the quantity of test sample also increases greatly, has greatly increased the workload that Eichhornia crassipes cultivation work person quantitatively detects the Eichhornia crassipes upgrowth situation.
For a long time, people's expectation can have a kind of more convenient succinct detection method to substitute the fresh weight of the results of regular determination plant of very complicated, to constant weight, measures the method for total dry weight by oven for drying, is used for the management of Eichhornia crassipes, determines best harvest time.
Also there is recently the people that modern Remote Sensing Technical is used for detection to Eichhornia crassipes scale breeding; but the spectrum that remote sensing technology is normally launched by plant leaf; judge the situation of growth such as green blade; but this method is also only relatively favourable to the crawl judgement of upgrowth situation of shape of the level of Eichhornia crassipes; longitudinal growth after the water surface is full of then can't be judged to Eichhornia crassipes; and this method also needs large-scale instrument and equipment; and by the related data of satellite etc.; not only cost is high; and need special personnel operation, should use certain difficulty.
The technology that growth detects in view of the Eichhornia crassipes of present use and the deficiency of means have limited Eichhornia crassipes to a certain extent at the vast eutrophic water recycling of China.
Summary of the invention
The object of the invention is to: the length consuming time and the large deficiency that waits of workload that exist for the technology that at present Eichhornia crassipes is detected and means, give, the inconvenience that provides the Eichhornia crassipes growing season management to bring provides a kind of method and application in the Eichhornia crassipes growing season management thereof of judging the Eichhornia crassipes total dry weight by the PEPC enzyme assay.
We studies show that, the Rice Growing later stage is the critical period that determines rice yield, bloom and rear photosynthetic 70% all be used to form output, wherein the photosynthesis of sword-like leave accounts for more than 50% of whole leaf photosynthesis, with the ground of rice plant and the physiological performance of under ground portion inseparable relation is arranged.Research is further found out, PEPC enzymatic activity and the Net Photosynthetic Rate of rice leaf have good correlativity, especially between critical period-boot stage that output forms early stage (blooming between rear 5-10 days) 10:00-12:00 in the morning, external source pepc gene paddy rice high level expression is consistent with the high value apparition performance of Net Photosynthetic Rate, and relatively stable, determine that sword-like leave PEPC activity can be used as the reliable molecular breeding index of the high photosynthetic rate of paddy rice.We find in the research of the photosynthesis characteristics of research different regions Eichhornia crassipes, physiological foundation at the high-biomass of the Eichhornia crassipes of different ecological area growth is that it has high photosynthesis characteristics characteristic, and the performance of the PEPC enzymatic activity of the accumulation of total dry weight and blade has concord, determined that accordingly utilization of the present invention is under same Ecological Area nature light and temperature condition, emphasis is at the growth period of Eichhornia crassipes key, respectively the functional leaf PEPC enzymatic activity of its different growing stage is carried out periodic monitoring, the technology of simultaneously maximum net photosynthetic rate of functional leaf and total dry weight etc. being carried out replicate determination provides theoretical foundation, by under same ecologic environment, mensuration and the data analysis of different times physiology and morphology index, the relation of research PEPC enzymatic activity and high photosynthetic rate and dry matter accumulation, to determine that the PEPC enzymatic activity is as the condition determination of the high growth indexes of Eichhornia crassipes, this upgrowth situation for high-biomass such as screening and detection Eichhornia crassipes is significant, and will triage techniques system that set up the high light efficiency germplasm of the efficient Eichhornia crassipes high-biomass of high flux all be played an important role.
The object of the present invention is achieved like this: a kind of method by PEPC enzyme assay judgement Eichhornia crassipes total dry weight, it is characterized in that, and judge its total dry weight by the PEPC enzymatic activity of measuring Eichhornia crassipes functional leaf in the breeding phase; Described Eichhornia crassipes functional leaf material refers to: remove the lobus cardiacus of Eichhornia crassipes and other blades behind the old and feeble blade of last a slice.
In the present invention: the PEPC enzyme assay to the functional leaf of Eichhornia crassipes in the breeding phase comprises:
A) the functional leaf material that takes by weighing 0.2g Eichhornia crassipes to be measured is put into the mortar of precooling, adds the thick enzyme Extraction buffer of pH7.8: 50mM/L Tris-HCl, 5mM/L dithiothreitol (DTT), the 1mM/L magnesium chloride, 2% polyvinylpyrrolidone grinds, through 4 layers of filtered through gauze, the lapping liquid that filters is collected centrifuge tube, at 4 ℃, under the 12000rpm, centrifugal 20min, get supernatant, be crude enzyme liquid;
B) measuring chlorophyll content: get 40 μ l crude enzyme liquids and be added in the 960 μ l absolute ethyl alcohols, under 649nm, 663nm and 665nm, measure absorbance value respectively,
Chlorophyll content Chl=Chlorophyll-a Content+content of chlorophyll b;
Chlorophyll a (mg/L)=12.7 * A
665-2.59 * A
649
Chlorophyll b (mg/L)=22.9 * A
649-4.67 * A
663
In the formula: A
665Refer to the light absorption value that the OD value is measured at 665nm, A
649Refer to the light absorption value that the OD value is measured under 649nm, A
663Refer to the light absorption value that the OD value is measured under 663nm;
C) the NADH absorbance changes: add the 50mmol Hepes-KHO of pH8.0 in the reaction system, and the 10mmol sodium bicarbonate, 5mmol magnesium chloride, 0.2mmol NADH-Na
2, 50 μ l crude enzyme liquids, the 1.5u malic dehydrogenase to 1ml, adds 5mmolPEP with the distilled water constant volume behind the mixing, the decline numerical value of the optical density of 1 340nm of every 15s record in ultraviolet spectrophotometer record 90s,
D) activity of PEPC enzyme (μ mol/mg.chl.h)=Δ OD
340* 10
3/ [6.3 * Chl * (the reaction system amount/crude enzyme liquid scale of construction) * reaction time)].
In the present invention: the functional leaf material of Eichhornia crassipes to be measured comes from sampling spot, and sampling spot is distributed in the resample area in the tested breeding zone, and resample area is not less than 6m
2
In the present invention: the PEPC enzymatic activity of total dry weight and functional leaf is proportionate, and the coefficient of determination between them is r
2=0.889**, related coefficient is r=0.943**.
A kind of application of said method is characterized in that: by the total dry weight that the mensuration to the PEPC enzymatic activity of the functional leaf of vigorous period Eichhornia crassipes obtains, determine the identification of indicator of Eichhornia crassipes large-scale artificial breeding phase results.
The invention has the advantages that, when judging the total dry weight of vigorous period Eichhornia crassipes, utilize ripe PEPC enzymatic activity detection technique, with respect to traditional Eichhornia crassipes total dry weight detection method, not only sampling is few, and weak point consuming time, has greatly reduced the workload that detects.The Eichhornia crassipes that the active detection technique of PEPC enzyme is used for large-scale artificial breeding animated period manages, and can greatly increase work efficiency.
Description of drawings
Fig. 1 is the variation of the Pmax of Eichhornia crassipes functional leaf in growth period of In Nanjing breeding in 2009;
Fig. 2 is the variation of the PEPC enzymatic activity of Eichhornia crassipes functional leaf in growth period of In Nanjing breeding in 2009;
Fig. 3 is the variation of Eichhornia crassipes total dry weight in growth period of In Nanjing breeding in 2009;
Fig. 4 is the correlativity of the activity of 2009 Eichhornia crassipes material total dry weights and PEPC enzyme;
Fig. 5 is the correlativity of 2009 Eichhornia crassipes material total dry weights and maximum net photosynthetic rate Pmax;
Fig. 6 is the activity of 2009 Eichhornia crassipes material PEPC enzymes and the correlativity of maximum net photosynthetic rate Pmax;
Fig. 7 is the variation of Eichhornia crassipes total dry weight, blade PEPC enzymatic activity and maximum net photosynthetic rate in growth period of In Nanjing July in 2010～breeding in September
Embodiment:
The breeding of Eichhornia crassipes material
Eichhornia crassipes (Eichhornia crassipes) belongs to Pontederiaceae phoenix eyes Nelumbo, is a kind of the floating life in the hydrophyte of the water surface.Under field conditions (factors), most of shaky, mainly by division propagation, axil has axillalry bud, can extract stolon out, and its top grows stem, and leaf and root system become new strain.(test site water body total nitrogen is 3.82mgL in this test Nanjing
-1, total phosphorus is 0.09mgL
-1, water temperature is 26-30 ℃) the artificial breeding sample plot of Eichhornia crassipes carry out.Eichhornia crassipes is every square metre of 2kg at the initial stocking rate on May 1st, 2009.Adopt mao bamboon fence plot experiment, the residential quarter area is 6m
2Grow after 2 months, July, choosing single, the number of blade is 9 ± 3, and the green healthy and strong plant that white fibrous root is 10 carries out the mensuration of plant forms and photosynthetic physiology ecological index in different growing stages.
The selection of Leaves of Eichhornia Crassipes minute
Got respectively Eichhornia crassipes functional leaf (removing lobus cardiacus and last old and feeble blade) in the different growing stages of Eichhornia crassipes every 30 days, and 10:00-12:00 gets Leaves of Eichhornia Crassipes the morning, every measuring point is got 10 samples, and each sample determination 3 times carries out respectively the mensuration of indices.
Embodiment 3
Leaves of Eichhornia Crassipes maximum net photosynthetic rate (P
Max) variation measured
The portable photosynthetic analyzer of LI-6400 that adopts U.S. LI-COR company to produce is open system, uses red blue-light source to measure, and light quantum flux density (PPFD) is 1600 μ mol/m
2.s, flow velocity is made as 500 μ mol/s, outdoor May～November the natural conditions condition determination be fine morning the natural temperature on average of 10:00～12:00 be (23～35 ℃) ℃, relative humidity is 67%～79%, light intensity is 1800 ± 50 μ mol/m
2.s, CO
2Concentration is 390.0 ± 10.5 μ mol/mol, has measured respectively the P of Different Leaf-position Leaf Blades
MaxAs can be seen from Figure 1, the Eichhornia crassipes P in November from May of beginning to put in a suitable place to breed to last results
MaxVariation present unimodal variation tendency, wherein be P July～September
MaxMaximum three months.
Embodiment 4
The year of Leaves of Eichhornia Crassipes PEPC enzyme assay changes
Method according to the people such as Gonzalez (1984).Key step is as follows, gets the functional leaf material that takes by weighing the Eichhornia crassipes to be measured in 0.2g different growth month is put into the mortar of precooling, adds thick enzyme Extraction buffer: 50mM/L Tris-HCl (pH7.8), the 5mM/L dithiothreitol (DTT), lmM/L magnesium chloride, 2% polyvinylpyrrolidone, grind, through 4 layers of filtered through gauze, the lapping liquid of filtration is collected centrifuge tube, at 4 ℃, under the 12000rpm, centrifugal 20min gets supernatant, is crude enzyme liquid.
Measuring chlorophyll content: get 40 μ l crude enzyme liquids and be added in the 960 μ l absolute ethyl alcohols, under 649nm, 663nm and 665nm, measure absorbance value respectively,
Chlorophyll content (Chl)=Chlorophyll-a Content+content of chlorophyll b
Chlorophyll a (mg/L)=12.7 * A
665-2.59 * A
649
Chlorophyll b (mg/L)=22.9 * A
649-4.67 * A
663
In the formula: A
665Refer to the light absorption value that the OD value is measured at 665nm; A
649Refer to the light absorption value that the OD value is measured under 649nm; A
663Refer to the light absorption value that the OD value is measured under 663nm.
The NADH absorbance changes: add 50mmol Hepes-KHO (pH8.0), 10mmol sodium bicarbonate, 5mmol magnesium chloride, 0.2mmol NADH-Na in the reaction system
2(nicotinamide adenine dinucleotide sodium salt), 50 μ l crude enzyme liquids, 1.5u malic dehydrogenase, with the distilled water constant volume to lml, add 5mmolPEP (phosphoenolpyruvic acid) behind the mixing, the decline of optical density under the 340nm in ultraviolet spectrophotometer record 90s (1 numerical value of every 15s record)
The activity of PEPC enzyme (μ mol/mg.chl.h)=Δ OD
340* 10
3/ [6.3 * Chl * (the reaction system amount/crude enzyme liquid scale of construction) * reaction time)].
As can be seen from Figure 2, from May of beginning to put in a suitable place to breed to last results November Eichhornia crassipes functional leaf PEPC enzymatic activity variation also present unimodal variation tendency, wherein be three months of enzymatic activity maximum August-October.
The year of Eichhornia crassipes unit area dry weight biomass estimation changes
With reference to Li Weiguo and Wang Jianbo
[14]Method [Li Weiguo, Wang Jianbo, illumination and nitrogen are on the impact of the growth of adventitious plant Eichhornia crassipes and physiological property. Wuhan University Journal (Edition), 2007,53 (4): 457-462], the different number of days that plant is cultivated was gathered in the crops afterwards every 30 days, took out 10 plant individuals from each place; After the washing, use the filter paper suck dry moisture, oven dry 24h is the electronic balance weighing dry weight of 0.0001g with sensibility reciprocal to constant weight in 85 ℃ of lower baking ovens, averages.
As can be seen from Figure 3, from May of beginning to put in a suitable place to breed to last results November the Eichhornia crassipes total dry weight variation also present unimodal variation tendency, wherein be three months of enzymatic activity maximum September-November.
Eichhornia crassipes growth measurement result's in 2009 analysis
In conjunction with above-mentioned result of study, further total dry weight in the maximum net photosynthetic rate of the PEPC enzymatic activity of different month Eichhornia crassipes functional leaf, functional leaf and the different breeding sampling spot unit area is carried out correlation analysis, from seeing Fig. 4,5,6 find out, total dry weight is proportionate with PEPC enzymatic activity and the maximum net photosynthetic rate of functional leaf respectively, and the coefficient of determination is respectively r
2=0.8894**, r
2=0.036, related coefficient is r=0.943**, r=0.190, and PEPC enzymatic activity and maximum net photosynthetic rate also are proportionate, and the coefficient of determination is respectively r
2=0.1677, related coefficient is r=0.410, wherein put the period total dry weight in a suitable place to breed and be utmost point marked positive correlation with the PEPC enzymatic activity of functional leaf respectively, further analyze, the correlativity of the photosynthetic parameters in the vigorous July of breeding-September, total dry weight is utmost point marked positive correlation with PEPC enzymatic activity and the maximum net photosynthetic rate of functional leaf respectively, and the coefficient of determination is respectively r
2=0.935**, r
2=0.786**, related coefficient is r=0.967**, r=0.887**, PEPC enzymatic activity and maximum net photosynthetic rate also are utmost point marked positive correlation, and the coefficient of determination is respectively r
2=0.1677, related coefficient is r=0.0.744**, and as seen, the PEPC that measures functional leaf in Eichhornia crassipes vigorous period July-September is active, and the mensuration of alternative different times total dry weight is as the reliable physical signs of Eichhornia crassipes Biomass Accumulation.
Judge the application of the method for Eichhornia crassipes total dry weight by the PEPC enzyme assay
On the basis of acquisition test figure in 2009, carried out the test of practical application at the artificial breeding sample plot of the Eichhornia crassipes of Nanjing in 2010.On June 1st, 2010, the initial stocking rate of Eichhornia crassipes was every square metre of 2kg, adopted mao bamboon fence plot experiment, and the residential quarter area is 6m
2Respectively at July, August and September, choosing single, the number of blade is 9 ± 3, the green healthy and strong plant that white fibrous root is 10, carry out the mensuration of plant total dry weight and maximum net photosynthetic rate, and choose 2 leaves of blade, measure the PEPC enzymatic activity of blade, as seen from Figure 7, when September, the PEPC enzymatic activity of Leaves of Eichhornia Crassipes is the highest, and the total dry weight of its plant is also maximum, and maximum Net Photosynthetic Rate is also the highest, and, be TN 5.17mgL at the initial concentration of water body nutrient
-1, TP 0.36mgL
-1, K 10.67mgL
-1Under the condition, the Eichhornia crassipes plant is at breeding 60d, contains N, P, the K amount reaches respectively 4.5%, 0.7%, 8.0%, and 60d water hyacinth N, P, K absorb total amount and reach respectively 57.57,8.95 and 111.14gm
-2And judge its dry weight according to the variation of the PEPC enzymatic activity of Leaves of Eichhornia Crassipes, determine the time of the best results of Eichhornia crassipes, make N, the P of water body in the pilot region, the clearance rate of K be respectively 75%, 71% and 86%, as seen, even in the most vigorous period in the Eichhornia crassipes growth, also water body is had good removing effect.
Claims (4)
1. the method by PEPC enzyme assay judgement Eichhornia crassipes total dry weight is characterized in that, judges its total dry weight by the PEPC enzymatic activity of measuring functional leaf in the Eichhornia crassipes vigorous period; Described Eichhornia crassipes vigorous period referred to during 7 annual～September; Described Eichhornia crassipes functional leaf material refers to: remove the lobus cardiacus of Eichhornia crassipes and other blades behind the old and feeble blade of last a slice:
PEPC enzyme assay to the functional leaf of Eichhornia crassipes in the vigorous period comprises:
A) the functional leaf material that takes by weighing 0.2g Eichhornia crassipes to be measured is put into the mortar of precooling, adds the thick enzyme Extraction buffer of pH7.8: 50mM/L Tris-HCl, 5mM/L dithiothreitol (DTT), the 1mM/L magnesium chloride, 2% polyvinylpyrrolidone grinds, through 4 layers of filtered through gauze, the lapping liquid that filters is collected centrifuge tube, at 4 ℃, under the 12000rpm, centrifugal 20min, get supernatant, be crude enzyme liquid;
B) measuring chlorophyll content: crude enzyme liquid is got 40 μ l and is added in the 960 μ l absolute ethyl alcohols, measures absorbance value respectively under 649nm, 663nm and 665nm,
Chlorophyll content Chl=Chlorophyll-a Content+content of chlorophyll b;
Chlorophyll a=12.7 * A
665-2.59 * A
649
Chlorophyll b=22.9 * A
649-4.67 * A
663
In the formula: A
665Refer to the light absorption value that the OD value is measured at 665nm, A
649Refer to the light absorption value that the OD value is measured under 649nm, A
663Refer to the light absorption value that the OD value is measured under 663nm;
C) the NADH absorbance changes: add the 50mmol Hepes-KHO of pH8.0 in the reaction system, and the 10mmol sodium bicarbonate, 5mmol magnesium chloride, 0.2mmol NADH-Na
2, 50 μ l crude enzyme liquids, 1.5 μ malic dehydrogenases to 1ml, add 5mmolPEP with the distilled water constant volume behind the mixing, the decline numerical value of the optical density of 1 340nm of every 15s record in ultraviolet spectrophotometer record 90s,
D) activity of PEPC enzyme=Δ OD
340* 10
3/ [6.3 * Chl * (the reaction system amount/crude enzyme liquid scale of construction) * reaction time].
2. the method for judging the Eichhornia crassipes total dry weight by the PEPC enzyme assay according to claim 1, it is characterized in that: the functional leaf material of Eichhornia crassipes to be measured comes from sampling spot, sampling spot is distributed in the resample area in the tested breeding zone, and resample area is not less than 6m
2
3. according to claim 2ly judge the method for Eichhornia crassipes total dry weight by the PEPC enzyme assay, it is characterized in that: the PEPC enzymatic activity of total dry weight and functional leaf is proportionate.
4. as claimed in claim 3 application of method; it is characterized in that: in Eichhornia crassipes vigorous growth; judge total dry weight by the mensuration to the PEPC enzymatic activity of the functional leaf of Eichhornia crassipes, determine again the identification of indicator of Eichhornia crassipes large-scale artificial breeding phase results.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1704476A (en) * | 2004-05-31 | 2005-12-07 | 福建农林大学 | Nucleic acid molecules encoding sugarcane phosphoenolpyruvate carboxylase and use thereof |
| JP4243014B2 (en) * | 2000-12-01 | 2009-03-25 | 株式会社荏原製作所 | Plant growth measuring device |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4243014B2 (en) * | 2000-12-01 | 2009-03-25 | 株式会社荏原製作所 | Plant growth measuring device |
| CN1704476A (en) * | 2004-05-31 | 2005-12-07 | 福建农林大学 | Nucleic acid molecules encoding sugarcane phosphoenolpyruvate carboxylase and use thereof |
Non-Patent Citations (4)
| Title |
|---|
| PEPC酶活性作为水稻高光效育种筛选指标的研究;李霞等,1;《江苏农业学报》;20081031;第24卷(第05期);559-564 * |
| 不同负载量对苹果光合速率及干物质生产的影响;冉辛拓等,1;《华北农学报》(第18期);131-132 * |
| 江苏地区凤眼莲叶片光合作用对光照度和温度的响应;李霞等,1;《江苏农业学报》;20101031;第26卷(第05期);943-947 * |
| 苹果苦痘病发病机理与防治措施;王召元等,1;《河北果树》;20080915;第39卷(第05期);902-909 * |
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