CN112553367A - Method for identifying heat resistance of alfalfa by using ALA6 - Google Patents
Method for identifying heat resistance of alfalfa by using ALA6 Download PDFInfo
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Abstract
The invention discloses a method for identifying the heat resistance of alfalfa by using ALA6, which researches 12P in different parts (roots, stems and leaves) of the alfalfa of 'Deckino' (Medicago sativa L. 'Deqin') under different stress high temperature (30 ℃, 35 ℃, 40 ℃) and stress time (0d, 2d and 4d)4Influence of type ATPase (Aminophopholipid ATPase, ALA 1-ALA 12) on the relative expression level. The results show that: stress for 0-4 days at 35 ℃, and alfalfa P4The ATP-type enzyme gene is mostly shown to continuously rise with the extension of the stress time. After the root and the stem are stressed for 4 days at 35 ℃ and the leaf is stressed for 4 days at 30 ℃, the relative expression level of ALA6 is the highest, namely 408.50, 51.50 and 17.66 respectively. ALA6 was most heat-resistant related by membership function and cluster analysisHigh; ALA2 has high heat resistance correlation; ALA1, ALA4 and ALA5 have a low heat resistance correlation; ALA3 and ALA 7-ALA 12 have the lowest correlation to heat resistance. The results of the comprehensive experiments suggest that the ALA6 gene is related to the heat resistance of alfalfa.
Description
Technical Field
The invention relates to the field of alfalfa heat resistance research, and particularly relates to a method for identifying alfalfa heat resistance by using ALA 6.
Background
The P-type ATP enzyme widely exists in prokaryotes and eukaryotes, is a membrane protein with a conserved structure and mainly mediates ATPVarious small cations are relied upon for transport across membranes. It contains five subclasses (P1-P5), of which the fourth subclass (P4) is mainly responsible for the transport of phospholipids, present only in eukaryotes. The phospholipid bilayer of the cell membrane is composed mainly of Phosphatidylcholine (PC), Phosphatidylserine (PS), Phosphatidylethanolamine (PE), and Phosphatidylinositol (PI). P4The ATP enzyme participates in the regulation of a membrane transport system mainly by regulating the phospholipid balance on a membrane, and responds to the temperature change in an organism. P is found in Saccharomyces cerevisiae (Saccharomyces cerevisiae)4The ATP-type enzyme Drs2p is involved in the transport of fluorescently labeled analogs (NBD-aminophospholipids), regulating the transmembrane activity of PE and PS; sec14p regulates PI and PC transport; dnf1 and Dnf2 regulate the process of phospholipid endocytosis at low temperatures; ALA2 compensates for the sensitivity of deletion mutants to low temperatures, primarily transporting PS. P4The ATP-type enzymes have 12 members in Arabidopsis thaliana (Arabidopsis thaliana) and are named ALA 1-ALA 12 (Aminopholipid ATPase). The functional deletion of ALA3 in Arabidopsis can influence the sensitivity of Arabidopsis to temperature, and mainly transports PE and PC; ALA6 overexpression plants have stronger heat resistance than wild type plants and deletion mutants; in the ALA6/7 double mutant, the pollen tube is easy to be damaged at high temperature in the day/low temperature at night; ALA10 can increase the content of monogalactosyl diglyceride, and regulate low temperature tolerance, mainly PC. The cold tolerance of cotton (Gossypium hirsutum) is affected by the down-regulation of the gene GbPATP which has high homology with Arabidopsis ALA 1.
Alfalfa (Medicago sativa L.) is a kind of high-quality bean grass of cool season type with wide cultivation, high nutritive value and good palatability, and is also a good grass seed for improving nitrogen circulation of ecosystem, recovering vegetation and improving water and soil, but the high temperature limits the large-area application of alfalfa in tropical zone and subtropical zone[19-23]. Research shows that high temperature stress reduces the membrane stability of alfalfa, but influences the stable P of the cell membrane4The ATP ase gene is rarely reported. The previous researches show that the alfalfa of the 'Deckino' is distributed in pieces in the dry and hot river valley of the Yunnan Jinsha river, adapts to local soil and climatic conditions, and has certain heat resistance. Alfalfa is used in the present studyMelissitus as a material, exploratory analysis of P4The expression characteristics of the ATP enzyme gene at different stress high temperature, time and different parts provide reference for screening the alfalfa heat-resistant related genes.
Disclosure of Invention
In order to solve the problems of the prior art, the invention aims to provide a method for identifying the heat resistance of alfalfa by using ALA6,
in order to achieve the purpose, the technical scheme of the invention is as follows:
a method for identifying the heat resistance of alfalfa by using ALA6 comprises the following steps:
step one, material selection treatment
Selecting alfalfa; sowing seeds in a flowerpot, placing the flowerpot in an illumination incubator with a substrate of sterilized peat soil, and pouring Hoagland nutrient solution;
step two, high temperature stress method
Subjecting alfalfa of certain seedling age to different air high temperature and time stresses, and measuring 12P of root, stem and leaf4Type ATPase Gene: relative expression levels of ALA 1-ALA 12;
step three, measuring the relative conductivity
Referring to the method of leaf good red and slightly improving, 0.20g of roots, stems and leaves are respectively weighed and put into 3 test tubes, 15mL of distilled water is added into the test tubes, the test tubes are placed for 30min at room temperature, a conductivity instrument is used for measuring the conductivity S1, then the test tubes are placed in a boiling water bath for 20min, after cooling, the conductivity instrument is used for measuring the conductivity S2 and the conductivity S0 of the distilled water, and the relative conductivity is calculated;
relative conductivity (%) - (S1-S0)/(S2-S0). times.100%
Step four, measuring the content of MDA
Respectively weighing 0.20g of roots, stems and leaves, putting the roots, the stems and the leaves into a mortar, adding a small amount of quartz sand and 2mL of distilled water, grinding the mixture until the mixture is homogenized, transferring the mixture into a 20mL graduated test tube, washing the mortar twice by using 3mL of distilled water, and pouring the extract; then another graduated test tube is taken, and 5mL of distilled water is added as a blank control; respectively adding 5mL of 0.50% thiobarbituric acid solution into the extracting solution and the blank control, heating for 15min on a boiling water bath, taking out the test tube, cooling, and recording the volume of the liquid; transferring the liquid into a centrifuge tube, centrifuging at 3000rpm for 10min by a centrifuge, taking the supernatant into a 1cm cuvette, measuring the light absorption values at the wavelengths of 600nm, 532nm and 450nm, and calculating the content of MDA;
step six, extraction and reverse transcription of total RNA of roots, stems and leaves
According to
The method of the Super total RNA extraction kit comprises the steps of respectively extracting the total RNA of roots, stems and leaves after treatment at different stress temperatures and time, running 1% agarose gel electrophoresis (0.27g agarose and 27ml 1 XTAE), and observing the RNA quality; the two RNA bands are obvious and do not disperse, which indicates that the RNA is not obviously degraded, and the next step of total RNA reverse transcription can be carried out
Synthesizing cDNA by reverse transcription of an RT Master Mix Kit;
step seven, P4Fluorescent primer design and relative expression quantity determination of type ATPase gene
Using alfalfa Actin (Actin) as internal reference, according to the registered P in NBCI4The sequence of the type ATPase gene is referenced[8]Primers were designed using Primer design tool online software (https:// www.ncbi.nlm.nih.gov/tools/Primer-blast/index. cgilink _ LOC ═ BlastHome) (table 1), and synthesized by TSINGKE bio-corporation; by using
Method for measuring P in alfalfa roots, stems and leaves by using qPCR Master Mix (2X) kit4Relative expression level of type ATPase gene; the used instrument is Quantstudio 5.0, and the reaction system is
qPCR Master Mix,2x 10uL,10uM Primer F 0.4uL,10uM Primer R 0.4uL,CXR 100x★0.2uL,Template DNA2 uL,ddH2O7 uL. The reaction program is Hold Stage at 50 ℃ for 2min and 95 ℃ for 10 min; PCR Stage 95℃5s，60℃15s，72℃34s；Melt Curve Stage 95℃15s，60℃1min，95℃15s。P4The relative expression level of the type ATPase gene was calculated by 2-ΔΔCtA method;
TABLE 1 Gene primers selected for testing
Step eight, data processing and analysis
Performing data analysis by using Excel, SPSS 17.0 and DPS 2006 software; and calculating membership function values.
Further, in the step one, specifically: the test material was alfalfa of 'decheng'; sowing seeds in a flowerpot (the substrate is sterilized peat soil), placing in an illumination incubator at 23 deg.C/20 deg.C (white/night), with a photoperiod of 16h/8h (white/night), relative humidity of 65%, and light intensity of 400 μmol · m-2·s-1Pouring 200mL of Hoagland nutrient solution every 2 days;
further, in the second step, 130d seedling age is selected, specifically, the stress temperature is 23 ℃/20 ℃ (Control), 30 ℃/25 ℃, 35 ℃/25 ℃, 40 ℃/25 ℃, the stress time is 2d and 4d respectively, the photoperiod is 16h/8h (white/night), the relative humidity is 65%, and the light intensity is 400 mu mol.m-2·s-1Pouring 200mL of Hoagland nutrient solution every 2 days; further, in the fourth step, the specific calculation method of the MDA concentration of the extract and the MDA content of the fresh tissue is as follows:
the extract had MDA concentration (μmol · L-1) ═ a 532-a 600)/(0.155 × L) -0.56 × a450
Fresh tissue MDA content (nmol. g-1) ═ MDA concentration of extract solution × V (mL)/W (g)
Wherein A600, A532 and A450 respectively represent absorbance values of a solution to be detected at wavelengths of 600nm, 532nm and 450nm, L is a cuvette thickness (cm), V is a total volume (mL) of an extracting solution, and W is a sample weight (g);
further, in the eighth step, the method for calculating the membership function value specifically includes: membership function value (r (Xi)) ═ Xi-Xmin)/(Xmax-Xmin) Wherein X represents a measurement of a sample, XminIs the minimum value of the measured value of the sample, XmaxIs the maximum value of the measurement value of the sample; degree of membership ═ Σ (R (X)1)+R(X2)+…+R(Xi) N, where n is the number of samples.
Compared with the prior art, the invention has the beneficial effects that:
the invention discloses a method for identifying the heat resistance of alfalfa by using ALA6, which researches 12P in different parts (roots, stems and leaves) of the alfalfa of 'Deckino' (Medicago sativa L. 'Deqin') under different stress high temperature (30 ℃, 35 ℃, 40 ℃) and stress time (0d, 2d and 4d)4Influence of type ATPase (Aminophopholipid ATPase, ALA 1-ALA 12) on the relative expression level. The results show that: stress for 0-4 days at 35 ℃, and alfalfa P4The ATP-type enzyme gene is mostly shown to continuously rise with the extension of the stress time. After the root and the stem are stressed for 4 days at 35 ℃ and the leaf is stressed for 4 days at 30 ℃, the relative expression level of ALA6 is the highest, namely 408.50, 51.50 and 17.66 respectively. ALA6 has the highest heat-resistant correlation through membership functions and cluster analysis; ALA2 has high heat resistance correlation; ALA1, ALA4 and ALA5 have a low heat resistance correlation; ALA3 and ALA 7-ALA 12 have the lowest correlation to heat resistance. The results of the comprehensive experiments suggest that the ALA6 gene is related to the heat resistance of alfalfa.
Drawings
FIG. 1 is a graph showing the effect of different stress temperatures and times on the relative conductivity of alfalfa at different locations; note: at different stress temperatures and times, different lower case letters indicated significant differences (P < 0.05), the same below.
FIG. 2 is a graph showing the effect of different stress temperatures and times on the MDA content of alfalfa at different positions;
FIG. 3 is a diagram of total RNA agarose gel electrophoresis;
FIG. 4 is a cluster diagram showing the heat-resistance correlation of the alfalfa P4 type ATPase gene
Detailed Description
The technical scheme of the invention is further described in detail by combining the drawings and the detailed implementation mode:
as shown in figure 1 of the drawings, in which,
test example:
1 materials and methods
1.1 test materials
The test material was alfalfa. Sowing seeds in a flowerpot (the substrate is sterilized peat soil), placing in an illumination incubator at 23 deg.C/20 deg.C (white/night), with a photoperiod of 16h/8h (white/night), relative humidity of 65%, and light intensity of 400 μmol · m-2·s-1200mL of Hoagland nutrient solution is poured every 2 days.
1.2 high temperature stress methods
After the alfalfa with the age of 130d is stressed by different air high temperature and time, 12P of roots, stems and leaves are respectively measured4Relative expression levels of type ATPase genes (ALA 1-ALA 12). Stress temperature of 23 deg.C/20 deg.C (Control), 30 deg.C/25 deg.C, 35 deg.C/25 deg.C, 40 deg.C/25 deg.C, stress time of 2d and 4d, light cycle of 16h/8h (white/night), relative humidity of 65%, and light intensity of 400 μmol/m-2·s-1200mL of Hoagland nutrient solution is poured every 2 days.
1.3 relative conductivity measurement
Referring to the method of leaf good red and slightly improving, 0.20g of roots, stems and leaves are respectively weighed and put into 3 test tubes, 15mL of distilled water is added into the test tubes, the test tubes are placed for 30min at room temperature, a conductivity instrument is used for measuring the conductivity S1, then the test tubes are placed in a boiling water bath for 20min, after cooling, the conductivity instrument measures the conductivity S2 and the conductivity S0 of the distilled water, and the relative conductivity is calculated.
Relative conductivity (%) - (S1-S0)/(S2-S0). times.100%
1.4 MDA assay
Referring to the method of leaf Shang hong [109], with slight modification, 0.20g of each of the roots, stems and leaves was weighed and put in a mortar, a small amount of quartz sand and 2mL of distilled water were added and ground to a homogenate, and the homogenate was transferred to a 20mL graduated tube, and the mortar was washed twice with 3mL of distilled water and poured with the extract. Then another graduated tube was taken and 5mL of distilled water was added as a blank control. Respectively adding 5mL of 0.50% thiobarbituric acid solution into the extractive solution and blank control, heating in boiling water bath for 15min, taking out the test tube, cooling, and recording the liquid volume. Transferring the liquid into a centrifuge tube, centrifuging at 3000rpm for 10min by a centrifuge, taking the supernatant into a 1cm cuvette, measuring the light absorption values at the wavelengths of 600nm, 532nm and 450nm, and calculating the MDA content.
The extract had MDA concentration (μmol · L-1) ═ a 532-a 600)/(0.155 × L) -0.56 × a450
Fresh tissue MDA content (nmol. g-1) ═ MDA concentration of extract solution × V (mL)/W (g)
Wherein A600, A532 and A450 respectively represent absorbance values of a solution to be detected at wavelengths of 600nm, 532nm and 450nm, L is a cuvette thickness (cm), V is a total volume (mL) of an extracting solution, and W is a sample weight (g).
1.5 extraction and reverse transcription of Total RNA from roots, stems and leaves
According to
The method of the Super total RNA extraction kit comprises the steps of extracting the total RNA of roots, stems and leaves after treatment at different stress temperatures and time respectively, running 1% agarose gel electrophoresis (0.27g agarose and 27ml 1 XTAE), and observing the RNA quality. The two RNA bands are obvious and do not disperse, which indicates that the RNA is not obviously degraded, and the next step of total RNA reverse transcription can be carried out
The RT Master Mix Kit reverse transcription synthesized cDNA.
1.6 P4Fluorescent primer design and relative expression quantity determination of type ATPase gene
Using alfalfa Actin (Actin) as internal reference, according to the registered P in NBCI4The sequence of the type ATPase gene is referenced[8]Primers were designed using Primer design tool online software (https:// www.ncbi.nlm.nih.gov/tools/Primer-blast/index. cgilink _ LOC ═ BlastHome) (table 1) and synthesized by TSINGKE bio-corporation. By using
Method for measuring P in alfalfa roots, stems and leaves by using qPCR Master Mix (2X) kit4Relative expression level of the type ATPase gene. The apparatus used isQuantstrudio 5.0, reaction system is
qPCR Master Mix,2x 10uL,10uM Primer F 0.4uL,10uM Primer R 0.4uL,CXR 100x★0.2uL,Template DNA2 uL,ddH2O7 uL. The reaction program is Hold Stage at 50 ℃ for 2min and 95 ℃ for 10 min; PCR Stage95 deg.C for 5s, 60 deg.C for 15s, 72 deg.C for 34 s; melt Curve Stage95 ℃ 15s, 60 ℃ 1min, 95 ℃ 15 s. P4The relative expression level of the type ATPase gene was calculated by 2-ΔΔCtThe method is carried out.
TABLE 1 Gene primers selected for testing
1.5 data processing and analysis
Data analysis was performed using Excel, SPSS 17.0, DPS 2006 software. The membership function value (R (Xi)) is calculated by referring to Liuwenyu, etc., and the membership function value (R (Xi)) is (Xi-X)min)/(Xmax-Xmin) Wherein X represents a measurement of a sample, XminIs the minimum value of the measured value of the sample, XmaxIs the maximum value of the measurement value of the sample; degree of membership ═ Σ (R (X)1)+R(X2)+…+R(Xi) N, where n is the number of samples.
2 results and analysis
2.1 Effect of different stress temperatures and times on the relative conductivities of different parts of Medicago sativa
As can be seen from FIG. 1, different stress temperatures and times affect the relative conductivities of different parts of alfalfa.
The relative conductivity of roots after being stressed for 2d at 35 ℃ is 36.33% at the lowest (P < 0.05) compared with the roots at different stress temperatures and time, and the difference is significant compared with a control (P < 0.05); after the stem is stressed for 2 days at 30 ℃, the relative conductivity is 13.86 percent at the lowest (P is less than 0.05), and the difference is not significant compared with a control; the relative conductivity of the leaves after being stressed for 2 days at 30 ℃ is 12.22 percent at the lowest, and the difference is not significant compared with the control. Under the same temperature, the relative conductivity of roots continuously rises along with the increase of stress time after the roots are stressed for 0-4 d; after the stem and the leaf are stressed at 35 ℃ for 0-4 d respectively, the relative conductivity is firstly increased and then decreased along with the increase of the stress time, and after the stem and the leaf are stressed at other temperatures for 0-4 d, the relative conductivity is continuously increased.
2.2 Effect of different stress temperatures and times on the MDA content in different parts of Medicago sativa
As can be seen from FIG. 2, different stress temperatures and times affect the MDA content in different parts of alfalfa. Compared with roots and stems stressed for 2d at 30 ℃ under different stress temperatures and time, the MDA content is at least 2.54nmol g-1、25.00nmol·g-1(ii) a After the leaves are stressed for 2 days at 35 ℃, the minimum MDA content is 93.28nmol g-1All were significantly different compared to the control (P < 0.05). And (3) at the same temperature, after roots, stems and leaves are stressed for 0-4 d respectively, the MDA content continuously rises along with the increase of the stress time.
2.3 extraction of Total RNA samples after different temperature and time stresses
As can be seen from FIG. 3, the total RNA of different samples extracted by agarose gel electrophoresis detection has two distinct bands and no dispersion, which indicates that the RNA is not significantly degraded and can be used for the next step P4And measuring the relative expression quantity of the type ATPase gene.
2.4 different stress temperatures and times on root P4Influence of the relative expression amount of the type ATPase Gene
As can be seen from Table 1, different stress temperatures and times affect root P4Relative expression level of the type ATPase gene. Compared with the same gene at different stress temperatures and times, ALA 1-ALA 4, ALA 6-ALA 10 and ALA12 are highest after being stressed for 4 days at 35 ℃, and are respectively 2.05, 38.37(P is less than 0.05), 6.65, 12.59(P is less than 0.05), 408.50(P is less than 0.05), 10.98(P is less than 0.05), 5.95(P is less than 0.05), 3.00(P is less than 0.05), 1.88 and 0.89; ALA5 and ALA11 were highest in the control, 2.66, 1.02 respectively (P < 0.05). Analysis of the results4The relative expression quantity of the ATP enzyme gene can be seen, the number of roots which are stressed at 35 ℃ for 4 days is the most, and 10 genes exist; the different genes were compared with each other with ALA6 being highest 408.50 after 35 ℃ stress for 4d, and secondly ALA2 being lowest after 35 ℃ stress for 4dThe height is 38.37. ALA6 shows a continuous increase after being stressed for 0-4 days at 35 ℃ and 40 ℃ respectively; stress is carried out for 0-4 d at 30 ℃, and the stress is shown to be increased firstly and then reduced. ALA2 shows a continuous increase after being stressed for 0-4 days at 30 ℃ and 35 ℃ respectively; stress is carried out for 0-4 d at 40 ℃, and the stress is firstly reduced and then increased.
TABLE 1 different stress temperatures and times on alfalfa root P4Effect of the relative expression levels of the Gene of the type ATPase
Note: under different stress temperatures and times, different lower case letters of the same gene show that the difference is obvious (P is less than 0.05), and the same is shown below.
2.5 different stress temperature and time on Stem P4Influence of the relative expression amount of the type ATPase Gene
As can be seen from Table 2, different stress temperatures and times both affect the stem P4Relative expression level of the type ATPase gene. Compared with the same gene at different stress temperatures and times, the highest stress values of ALA1, ALA 3-ALA 7, ALA9 and ALA10 are 1.20, 1.40(P is less than 0.05), 4.91(P is less than 0.05), 2.21(P is less than 0.05), 51.50(P is less than 0.05), 6.40(P is less than 0.05), 2.34(P is less than 0.05) and 4.03(P is less than 0.05) after the stress of the same gene at 35 ℃ for 4 days; ALA2 and ALA11 were highest after stress at 30 ℃ for 2 days, 18.99(P < 0.05), and 1.52(P < 0.05), respectively; ALA8 was up to 1.45(P < 0.05) after stress at 30 ℃ for 4 d; ALA12 was up to 1.01 after stress for 4 days at 40 ℃. Analysis of the results4The relative expression quantity of the ATP enzyme gene can be seen, the number of the stems which are relatively high after being stressed for 4 days at 35 ℃ is the most, and 8 genes exist; the different genes were compared with each other with ALA6 being highest 51.50 after stress at 35 ℃ for 4d and secondly ALA2 being highest 18.99 after stress at 30 ℃ for 2 d. ALA6 shows a continuous increase after being stressed for 0-4 days at 35 ℃ and 40 ℃ respectively; stress is carried out for 0-4 d at 30 ℃, and the stress is shown to be increased firstly and then reduced. ALA2 is stressed at 35 ℃ and 40 ℃ for 0-4 days respectively, and then is reduced and increased; stress is carried out for 0-4 d at 30 ℃, and the stress is shown to be increased firstly and then reduced.
TABLE 2 different stress temperatures and times on alfalfa stem P4Type ATInfluence of the relative expression levels of the P enzyme Gene
2.6 different stress temperatures and times on the leaf P4Influence of the relative expression amount of the type ATPase Gene
As can be seen from Table 3, different stress temperatures and times both affect the leaf P4Relative expression level of the type ATPase gene. Compared with the same gene at different stress temperatures and time, the highest stress values of ALA 2-ALA 3, ALA 6-ALA 7, ALA 9-ALA 10 and ALA12 are respectively 9.69, 1.26, 17.66(P is less than 0.05), 2.94(P is less than 0.05), 1.88(P is less than 0.05), 1.27(P is less than 0.05) and 0.95(P is less than 0.05) after the stress of the same gene is carried out for 4 days at 30 ℃; ALA1 was up to 1.81 after stress for 2 days at 40 ℃; ALA4 was up to 5.18(P < 0.05) after stress for 2d at 35 ℃; ALA5 is at most 3.29 at normal temperature, and is reduced at stress temperature and time; ALA8 and ALA11 were highest after 2 days of stress at 30 ℃ and were 1.01(P < 0.05) and 1.00(P < 0.05), respectively. Analysis of the results4The relative expression quantity of the ATP enzyme gene can be seen, the number of leaves which are stressed at 30 ℃ for 4 days is the most, and 7 genes exist; compared with each other, the relative expression level of ALA6 is 17.66 at the highest after stress of 4d at 30 ℃, and the relative expression level of ALA2 is 9.69 at the highest after stress of 4d at 30 ℃. ALA6 shows a continuous increase after being stressed for 0-4 days at 30 ℃ and 40 ℃ respectively; stress is carried out for 0-4 d at 35 ℃, and the stress is firstly reduced and then increased. ALA2 is stressed at 35 ℃ and 40 ℃ for 0-4 days respectively, and then is increased and then reduced; the stress is 0-4 days at 30 ℃, and the increase is shown to be continuous.
TABLE 3 different stress temperatures and times for alfalfa leaf P4Effect of the relative expression levels of the Gene of the type ATPase
2.7 P4Membership functions and cluster analysis of heat-resistant correlation of ATP enzyme gene
Subjecting 3 indexes of alfalfa root, stem and leaf to membership function analysis (Table)4) The 3 indexes of ALA6 have the highest membership function value and the highest membership degree which are all equal to 1.00, and are ranked as 1 st, which shows that ALA6 has the highest heat resistance correlation. Secondly, the 3 index membership function values and the membership degrees of ALA2 belong to the 2 nd, are respectively 0.19, 0.33 and 0.96, and are ranked on the 2 nd, which shows that ALA2 has high heat-resistant correlation. ALA12 was at the end of the ranking in the thermotolerant correlation, indicating that the gene has the worst thermotolerant correlation. As can be seen from FIG. 4, P can be clustered4The heat-resistant correlation of the ATP enzyme gene is divided into 4 categories, the 1 st category is ALA6, and the heat-resistant correlation is highest; the 2 nd type is ALA2, and the heat resistance correlation is higher; the 3 rd group is ALA1, ALA4 and ALA5, and has low heat resistance correlation; the 4 th group is ALA3 and ALA 7-ALA 12, and has the lowest correlation of heat resistance.
TABLE 4 alfalfa P4Membership function analysis of heat-resistant correlation of ATP enzyme gene
Root and stem of alfalfa at 35 deg.C for 4d, leaf at 30 deg.C for 4d, P4The number of the type ATPase genes with high relative expression level is the most, and the genes respectively comprise 10 genes, 8 genes and 7 genes, wherein the relative expression level of ALA6 is the highest and is respectively 408.50, 51.50 and 17.66, and the heat-resistant correlation of ALA6 is the highest through membership functions and cluster analysis.
The above description is only an embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that are not thought of through the inventive work should be included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope defined by the claims.
Claims (5)
1. A method for identifying the heat resistance of alfalfa by using ALA6 is characterized by comprising the following steps:
step one, material selection treatment
Selecting alfalfa; sowing seeds in a flowerpot, placing the flowerpot in an illumination incubator with a substrate of sterilized peat soil, and pouring Hoagland nutrient solution;
step two, high temperature stress method
Subjecting alfalfa of certain seedling age to different air high temperature and time stresses, and measuring 12P of root, stem and leaf4Type ATPase Gene: relative expression levels of ALA 1-ALA 12;
step three, measuring the relative conductivity
Referring to the method of leaf good red and slightly improving, 0.20g of roots, stems and leaves are respectively weighed and put into 3 test tubes, 15mL of distilled water is added into the test tubes, the test tubes are placed for 30min at room temperature, a conductivity instrument is used for measuring the conductivity S1, then the test tubes are placed in a boiling water bath for 20min, after cooling, the conductivity instrument is used for measuring the conductivity S2 and the conductivity S0 of the distilled water, and the relative conductivity is calculated;
relative conductivity (%) - (S1-S0)/(S2-S0). times.100%
Step four, measuring the content of MDA
Respectively weighing 0.20g of roots, stems and leaves, putting the roots, the stems and the leaves into a mortar, adding a small amount of quartz sand and 2mL of distilled water, grinding the mixture until the mixture is homogenized, transferring the mixture into a 20mL graduated test tube, washing the mortar twice by using 3mL of distilled water, and pouring the extract; then another graduated test tube is taken, and 5mL of distilled water is added as a blank control; respectively adding 5mL of 0.50% thiobarbituric acid solution into the extracting solution and the blank control, heating for 15min on a boiling water bath, taking out the test tube, cooling, and recording the volume of the liquid; transferring the liquid into a centrifuge tube, centrifuging at 3000rpm for 10min by a centrifuge, taking the supernatant into a 1cm cuvette, measuring the light absorption values at the wavelengths of 600nm, 532nm and 450nm, and calculating the content of MDA;
step six, extraction and reverse transcription of total RNA of roots, stems and leaves
According to
The method of the Super total RNA extraction kit comprises the steps of respectively extracting the total RNA of roots, stems and leaves after treatment at different stress temperatures and time,and run through a 1% agarose gel electrophoresis (0.27g agarose and 27ml 1 XTAE) to observe its RNA quality; the two RNA bands are obvious and do not disperse, which indicates that the RNA is not obviously degraded, and the next step of total RNA reverse transcription can be carried out
Synthesizing cDNA by reverse transcription of an RT Master Mix Kit;
step seven, P4Fluorescent primer design and relative expression quantity determination of type ATPase gene
Using alfalfa Actin (Actin) as internal reference, according to the registered P in NBCI4The sequence of the type ATPase gene is taken as a reference [8]Primers were designed using Primer design tool online software (https:// www.ncbi.nlm.nih.gov/tools/Primer-blast/index. cgilink _ LOC ═ BlastHome) (table 1), and synthesized by TSINGKE bio-corporation; by using
Method for measuring P in alfalfa roots, stems and leaves by using qPCR Master Mix (2X) kit4Relative expression level of type ATPase gene; the used instrument is Quantstudio 5.0, and the reaction system is
qPCR Master Mix,2x 10uL,10uM Primer F 0.4uL,10uM Primer R 0.4uL,CXR 100x★0.2uL,Template DNA2 uL,ddH2O7 uL; the reaction program is Hold Stage at 50 ℃ for 2min and 95 ℃ for 10 min; PCR Stage95 ℃ for 5s, 60 ℃ for 15s, and 72 ℃ for 34 s; melt dark Stage95 ℃ for 15s, 60 ℃ for 1min, 95 ℃ for 15 s; p4The relative expression level of the type ATPase gene was calculated by 2—△△CtA method;
TABLE 1 Gene primers selected for testing
Step eight, data processing and analysis
Performing data analysis by using Excel, SPSS 17.0 and DPS 2006 software; and calculating membership function values.
2. The method according to claim 1, wherein in the first step, specifically: the test material was alfalfa of 'decheng'; sowing seeds in a flowerpot (the substrate is sterilized peat soil), placing in an illumination incubator at 23 deg.C/20 deg.C (white/night), with a photoperiod of 16h/8h (white/night), relative humidity of 65%, and light intensity of 400 μmol · m-2·s-1200mL of Hoagland nutrient solution is poured every 2 days.
3. The method as claimed in claim 1, wherein in the second step, the seedling age of 130d is selected by respectively controlling the stress temperature at 23 ℃/20 ℃ (Control), the stress temperature at 30 ℃/25 ℃, the stress temperature at 35 ℃/25 ℃, the stress temperature at 40 ℃/25 ℃, the stress time at 2d and 4d, the photoperiod at 16h/8h (white/night), the relative humidity at 65%, and the light intensity at 400 μmol · m-2·s-1200mL of Hoagland nutrient solution is poured every 2 days.
4. The method according to claim 1, wherein in the fourth step, the specific calculation method of the MDA concentration of the extract and the MDA content of the fresh tissue is as follows:
the extract had MDA concentration (μmol · L-1) ═ a 532-a 600)/(0.155 × L) -0.56 × a450
Fresh tissue MDA content (nmol. g-1) ═ MDA concentration of extract solution × V (mL)/W (g)
Wherein A600, A532 and A450 respectively represent absorbance values of a solution to be detected at wavelengths of 600nm, 532nm and 450nm, L is a cuvette thickness (cm), V is a total volume (mL) of an extracting solution, and W is a sample weight (g).
5. The method according to claim 1, wherein in the eighth step, the membership function values are calculated by: membership function value (r (Xi)) of (Xi-Xmin)/(Xmax-Xmin), where X represents a sample measurement value, Xmin is the minimum value of the sample measurement value, and Xmax is the maximum value of the sample measurement value; the degree of membership ═ Σ (R (X1) + R (X2) + … + R (xi))/n, where n is the number of samples.
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| CN105154518A (en) * | 2015-08-05 | 2015-12-16 | 云南农业大学 | Method for identifying heat resistance of medicago sativa by utilizing SOD enzyme |
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| CN105154518A (en) * | 2015-08-05 | 2015-12-16 | 云南农业大学 | Method for identifying heat resistance of medicago sativa by utilizing SOD enzyme |
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