CN111411124A - Application of peanut hairy root strain in improving low-nitrogen and high-salt resistance of peanuts - Google Patents
Application of peanut hairy root strain in improving low-nitrogen and high-salt resistance of peanuts Download PDFInfo
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Abstract
The invention provides an application of a peanut hairy root strain in improving low-nitrogen resistance and high-salt resistance of peanuts, belonging to the technical field of plant genetic engineering. The technical scheme of the invention comprises the following steps: cloning AhCEP1 gene from peanut, constructing plant gene expression vector, obtaining peanut hairy root containing peanut AhCEP1 gene by agrobacterium rhizogenes mediated method, identifying whether peanut AhCEP1 gene is expressed in transgenic peanut hairy root, determining expression quantity, and screening out transgenic peanut hairy root positive strain. The method provided by the invention can effectively improve the low-nitrogen and high-salt resistance of the peanut hairy root, can improve the content of the peanut hairy root in a low-nitrogen and high-salt stress environment, and has a very wide application prospect in the field of peanut stress resistance.
Description
Technical Field
The invention belongs to the technical field of plant genetic engineering, and particularly relates to an application of a peanut hairy root strain in improving low-nitrogen resistance and high-salt resistance of peanuts.
Background
Peanuts are important oil crops in China, but with social development and continuous improvement of the living standard of people, the edible oil yield in China is seriously insufficient, so that the treatment and utilization of saline-alkali soil are important ways for solving the contradiction between shortage of cultivated land and conflict between grain and oil in China, and low nitrogen and high salt are two main hazards for the growth of peanuts in saline-alkali soil at present. Wherein, nitrogen is one of the essential main elements for the growth of the peanuts, the nitrogen supply is insufficient, the synthesis of protein, chlorophyll and nucleic acid of the peanuts is blocked, so that the peanut plants are short and small, the leaf surfaces are yellow and thin, the photosynthetic strength of the leaves is reduced, and the yield of the peanuts is seriously influenced.
At present, the genetic transformation approach is one of important biological approaches for improving the stress resistance of peanut varieties and improving the low-nitrogen resistance and high-salt resistance of peanuts. Meanwhile, researches show that the C-tertiary peptides (CEPs) gene plays a very important and unique role in the growth and development of plants and the stress response process of the plants, but the researches are mainly concentrated in arabidopsis thaliana and alfalfa, and the researches on the CEP gene of peanuts are very few, so that the application of the hairy root strain of the peanuts in improving the low-nitrogen and high-salt resistance of the peanuts is an effective solution for solving the problem of shortage of cultivated land in China, relieving the conflict of land competition between grains and oil and greatly improving the yield of the peanuts.
Disclosure of Invention
The invention provides an application of a peanut hairy root strain with directional high efficiency and short breeding period in improving the low nitrogen resistance and high salt resistance of peanuts, aiming at the technical problem that the research of a peanut CEP gene in the aspects of peanut growth and development and stress response is less in the prior art.
In order to achieve the purpose, the invention adopts the technical scheme that:
the application of the peanut hairy root strain in improving the low-nitrogen resistance and high-salt resistance of peanuts is characterized in that the peanut hairy root strain is obtained by the following method, and the specific steps are as follows:
after cloning AhCEP1 gene from peanut cDNA, connecting the AhCEP1 gene to an expression vector to construct a plant expression vector containing AhCEP1 gene;
transforming the plant expression vector containing the AhCEP1 gene into agrobacterium rhizogenes to obtain agrobacterium rhizogenes containing the AhCEP1 gene;
infecting peanut leaves by utilizing agrobacterium rhizogenes liquid containing AhCEP1 gene to obtain peanut leaves infected by the agrobacterium rhizogenes liquid containing AhCEP1 gene, inducing the peanut leaves to generate transgenic peanut hairy roots, and transferring the peanut leaves to an MS solid culture medium containing kanamycin for culture;
and (3) determining the expression quantity of the AhCEP1 gene in the transgenic peanut hairy root after identifying whether the AhCEP1 gene in the transgenic peanut hairy root is expressed, and screening a transgenic peanut hairy root positive strain to obtain a peanut hairy root strain.
In the method, the method for extracting the peanut RNA is a Trizol extraction method or is obtained by extraction according to the instruction of an RNA extraction kit; the reverse transcription process is prepared by using a FastKing gDNA dispering RT SuperMix reverse transcription kit for instruction; the peanut leaves are pretreated before the peanut secretory peptide gene-containing agrobacterium rhizogenes liquid is used for infecting the leaves, namely 2-3 scratches are added on the leaves, so that the aim of ensuring the agrobacterium rhizogenes liquid to enter the leaves to complete the induction process is fulfilled;
in addition, the expression level of AhCEP1 gene in the transgenic peanut hairy root is measured by qRT or RT-PCR method, and a control group and an experimental group are respectively arranged. Among them, the control group was hairy roots transformed with an empty vector (i.e., background expression level of CEP1 gene). As long as the expression level of the experimental group is higher than the background level, the overexpression strain is obtained. Since the expression level of different genes transformed into plants varies, no specific numerical limitation is made. A plurality of over-expression strains can be obtained by carrying out a genetic transformation experiment, and a transgenic strain with the highest expression quantity is further selected for carrying out a later stage stress experiment.
Preferably, the method for constructing the plant expression vector is an enzyme digestion ligation method or a gateway system construction method.
Preferably, the method for infecting the peanut leaves by using the agrobacterium rhizogenes liquid containing the AhCEP1 gene is a soaking method or a smearing method.
Preferably, the concentration OD of the liquid of the Agrobacterium rhizogenes containing AhCEP1 gene6001.0 to 2.0; the culture time of the transgenic peanut hairy roots is 2-8 weeks, and fresh culture solution is replaced every 2 weeks.
Preferably, the method for identifying the AhCEP1 gene expression in the transgenic peanut hairy roots is an RT-PCR method or a qRT-PCR method.
Preferably, the determination of the expression level of the AhCEP1 gene is carried out by the following method:
identifying AhCEP1 gene expression in the transgenic peanut hairy roots by using an RT-PCR method or a qRT-PCR method to obtain different PCR products;
and respectively carrying out agarose gel electrophoresis analysis on the PCR products, and judging the expression quantity of the AhCEP1 gene in the hairy roots of the transgenic peanuts according to the brightness of the bands.
In the above optimization, the judgment of the expression level of the AhCEP1 gene in the transgenic peanut hairy root according to the brightness of the band is mainly realized by the following method:
and (3) increasing and decreasing the content of the sample template by using the primer of the internal reference gene AhACTin11 through multiple PCR reactions and agarose gel electrophoresis, so that the brightness of the target strip obtained by amplifying the internal reference gene in different final samples is kept consistent, the sample loading amount is the final experimental template amount, the AhCEP1 gene primer is used for PCR amplification, and the brightness of the strip in different obtained samples is the expression level of the AhCEP1 gene of different transgenic hairy roots.
Preferably, the method further comprises determining the biomass of the transgenic peanut hairy root positive line by the steps of:
selecting branches from the same transgenic peanut hairy root positive strain, weighing, transferring to a liquid culture medium, and continuously culturing;
replacing a fresh liquid culture medium every 1-2 weeks, finally washing and drying the liquid culture medium by using sterile water, and respectively weighing the fresh weights of the hairy roots of the transgenic peanuts to obtain the biomass of the hairy roots of the transgenic peanuts, wherein the formula is as follows:
fresh weight of transgenic peanut hairy root-weight of each bottle of transgenic peanut hairy root-inoculation weight.
Preferably, the liquid culture medium is a MS liquid culture medium, a low nitrogen liquid culture medium and a high salt liquid culture medium.
Preferably, the culture conditions of the transgenic peanut hairy root branch are 23-26 ℃ and 150-180 rpm.
In the above-mentioned preferred embodiments, the temperature condition of the cultivation condition of the transgenic peanut hairy root branch can be 23, 24, 25, 26 ℃ or any value within the above-mentioned limited range, and the rotation speed condition can be 150, 160, 170, 180rpm or any value within the above-mentioned limited range, and the invention is within the protection scope of the present invention.
Application of peanut hairy root strain in improving low-nitrogen and high-salt resistance of peanuts, wherein low-nitrogen refers to KNO (potassium dihydrogen phosphate) in liquid culture medium3The concentration of (b) is less than or equal to 1mM, and the high salt means that the concentration of NaCl in the liquid culture medium is more than or equal to 200 mM.
Compared with the prior art, the invention has the advantages and positive effects that:
1. the application of the peanut hairy root strain in improving the low-nitrogen and high-salt resistance of peanuts can effectively improve the low-nitrogen and high-salt resistance of peanuts, experiments show that the full-length cDNA of the AhCEP1 gene is connected to an expression vector started by a 35S promoter, agrobacterium rhizogenes is used for infecting peanut leaves to induce and generate transgenic peanut hairy roots, and the gene is found to be capable of enhancing the low-nitrogen and high-salt resistance of the peanut hairy roots and improving the biomass of the peanut hairy roots in a low-nitrogen and high-salt environment.
2. The application of the peanut hairy root strain in improving the low-nitrogen and high-salt resistance of peanuts is realized by adopting a genetic transformation method, the method has the advantages of high directional efficiency, short period, simplicity and convenience in operation and the like, and the defects of long breeding period, low efficiency, slow development, difficulty in controlling mutation direction, high toxicity of chemical mutagens, easiness in generating residues and the like of the traditional stress-resistant breeding method are overcome.
Drawings
Fig. 1 is a schematic diagram of a result of constructing an expression vector of peanut AhCEP1-pCAMBIA1300 provided by an embodiment of the present invention, wherein, a diagram is a schematic diagram of an AhCEP1 conserved protein domain, a diagram B is a schematic diagram of an electrophoresis result of a band cloned to a target gene AhCEP1, a diagram C is a schematic diagram of an enzyme digestion result of a plasmid in which the target gene AhCEP1 is connected to a cloning vector, a diagram C is a schematic diagram of an enzyme digestion result of a plasmid in which an original expression vector is connected, a diagram D is a positive clone obtained by connecting the target gene AhCEP1 to an expression vector, and a diagram E is a schematic diagram of an enzyme digestion result of a plasmid in which an expression vector with an AhCEP 1;
FIG. 2 is a schematic diagram of the morphology of transgenic peanut hairy roots provided by the embodiments of the present invention;
FIG. 3 is a diagram showing the result of identifying a positive strain of transgenic peanut hairy roots, provided in an example of the present invention, wherein lane 1 is transgenic peanut hairy roots (CK) transformed with an empty vector, the remaining lanes are transgenic peanut hairy roots (35S: AhCEP1), AhACTin11 is an internal reference gene;
FIG. 4 is a graph of the biomass statistics of transgenic peanut hairy roots under high salt and low nitrogen stress provided by embodiments of the present invention;
FIG. 5 shows Na in transgenic peanut hairy roots (35S: AhCEP1) and Control (CK) provided by an embodiment of the present invention+And NO3 -qRT-PCR detection result diagram of the expression quantity of the transport related gene.
Detailed Description
In order to more clearly and specifically describe the method for improving the low-nitrogen resistance and high-salt resistance of the peanuts and the application thereof provided by the embodiment of the invention, the technical scheme in the embodiment of the invention will be clearly and completely described below, and it is obvious that the described embodiment is only a part of the embodiment of the invention, but not all of the embodiment. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention, and will be described below with reference to specific embodiments.
Example 1: extraction and reverse transcription of peanut RNA
(1) Experimental materials:
culture medium:
escherichia coli L B liquid culture medium comprises Tryptone (10 g), yeast extract (Yeast) 5g, sodium chloride (NaCl)10g, distilled water 1000m L, pH adjusted to 7.0, and autoclaving at 121 deg.C for 20 min.
An Agrobacterium rhizogenes YEP liquid culture medium comprises 10g of Tryptone (Tryptone), 10g of yeast extract (Yeast), 5g of sodium chloride (NaCl), 1000m of distilled water L, adjusting pH to 7.0, and sterilizing with high pressure steam at 121 deg.C for 20 min.
Hairy root induction and propagation MS culture medium with 25m L20 × MS mass (3.8g KNO)3，3.3g NH4NO3，0.34g KH2PO4Distilled water 100m L), 5m L100 × MS trace (0.223g MnSO)4·4H2O，0.086g ZnSO4·7H2O，0.062g H3BO3，0.0083g KI，2.5mg Na2MoO4·2H2O，0.25mg CuSO4·5H2O，0.25mgCoCl2·6H2O, distilled water 100m L), 5m L100 × iron salt (0.374g EDTA-Na2，0.278g FeSO4·7H2O), 10m L100 × VB (inositol 1g, VB)10.1g，VB30.01g，VB60.01g of water, 100m L% of water), 5m L100 × Ca2+，5mL 100×Mg2+15g of sucrose and 1000m of L distilled water, the pH value is adjusted to 5.8, and the mixture is sterilized by high-pressure steam at 121 ℃ for 20 min.
Low nitrogen medium (0.2mM KNO)3)：10mL 1M KPi，0.2mL 1M KNO3，1mL 1M CaCl2，2mL 1MMgSO4，2mL 500×MN solution(1.55g H3BO3，68mg ZnCl2，CuSO4·5H2O 125mg，MnSO4·H2O1.01g，Na2MoO4·2H2O 24mg)，5mL200×FeNa2EDTA(FeSO4·7H2O 5.56g，Na2EDTA·2H2O7.46g), sucrose 10g, distilled water 1000m L, adjusting pH to 5.6, and autoclaving at 121 deg.C for 20 min.
Normal nitrogen medium (5mM KNO)3)：10mL 1M KPi，5mL 1M KNO3，1mL 1M CaCl2，2mL 1MMgSO4，2mL 500×MN solution(1.55g H3BO3，68mg ZnCl2，CuSO4·5H2O 125mg，MnSO4·H2O1.01g，Na2MoO4·2H2O 24mg)，5mL 200×FeNa2EDTA(FeSO4·7H2O 5.56g，Na2EDTA·2H2O7.46g), sucrose 10g, distilled water 1000m L, adjusting pH to 5.6, and autoclaving at 121 deg.C for 20 min.
(2) The experimental method comprises the following steps:
the invention uses the peanut genome database website PeanutBase (https:// www.peanutbase.org) to find the gene AhCEP1, uses Pfam (Protein family: http:// Pfam. sanger. ac. uk /) and SMART (Simple modulated Architecture Research Tool: http:// SMART. embl. heidelberg. de /) analysis to find that the gene encodes a typical CEP small peptide (CDS315bp is shown in SEQ ID NO.1, amino acid 104aa is shown in SEQ ID NO:2), and has an N-terminal signal peptide and a C-terminal conserved CEP structural domain (FIG. 1A).
Primers are designed according to the CDS sequence of the AhCEP1 gene of the peanut and cloned by the following method:
s1, peanut RNA extraction
The method comprises the following steps of extracting total RNA of peanuts by using a total RNA extraction kit (DP432) of a Tiangen plant:
(S1-1) fully grinding peanut seedlings growing for about 15 days in water culture in liquid nitrogen, adding an R L reagent in a 400 mu L kit into every 50-100mg of tissue, uniformly mixing by vortex oscillation, and incubating at 56 ℃ for 1-3min to completely crack plant tissues;
(S1-2) transferring the solution in the step one to a filter column CS (the filter column CS is placed in a collecting tube), centrifuging for 5min at 12,000rpm, carefully sucking the supernatant in the collecting tube to a new RNase-Free 1.5m L centrifuge tube by using a suction head with a cut part of the tail end, and avoiding the contact of the suction head with cell debris sediment in the collecting tube as much as possible;
(S1-3) slowly adding absolute ethyl alcohol with the volume of 0.5 time of that of the supernatant, gently mixing uniformly, transferring the solution into an adsorption column CR3, centrifuging at 12,000rpm for 1min, pouring off waste liquid in the collection tube, and putting the adsorption column CR3 back into the collection tube;
(S1-4) adding 350 mu L deproteinizing solution RW1 into adsorption column CR3, centrifuging at 12,000rpm for 1min, pouring off waste liquid in the collection tube, and putting adsorption column CR3 back into the collection tube;
(S1-5) adding 80 μ L of DNase I working solution (10 μ L DNase I stock solution and 70 μ L RDD buffer solution, gently mixing them uniformly) to the center of an adsorption column CR3, and standing at room temperature for 15 min;
(S1-6) adding 350 mu L deproteinizing solution RW1 into adsorption column CR3 again, centrifuging at 12,000rpm for 1min, pouring off waste liquid in the collection tube, and putting adsorption column CR3 back into the collection tube;
(S1-7) adding 500 mu L rinsing liquid RW into the adsorption column CR3, standing at room temperature for 2min, centrifuging at 12,000rpm for 1min, pouring off waste liquid in the collection tube, and repeating the step (S1-7) again;
(S1-8) idling at 12,000rpm for 2min, and dumping waste liquid. Placing the adsorption column CR3 at room temperature for several minutes to completely dry the residual rinsing liquid;
(S1-9) placing the adsorption column CR3 into a new RNase-Free centrifuge tube, and suspending 50 mu L RNase-Free ddH dropwise into the middle part of the adsorption membrane2O, standing at room temperature for 2min, centrifuging at 12,000rpm for 2min to obtain RNA solution, and storing in a refrigerator at-80 deg.C for a long time.
S2 Synthesis of reverse transcription first Strand cDNA
(S2-1) dissolving the extracted RNA, determining the concentration of the RNA, and then carrying out reverse transcription by using a Tiangen FastKinggDNAdispering RT SuperMix reverse transcription kit;
(S2-2) taking 2 mu g of total RNA, adding 4 mu L5 × FastKing-RT SuperMix, replenishing water to 20 mu L, incubating at 42 ℃ for 15min, and inactivating enzyme at 95 ℃ for 3min to obtain peanut cDNA.
Example 2: cloning of AhCEP1 gene and construction of expression vector
(1) Experimental materials:
AhCEP1 gene primer sequence:
upstream primer 5-GGATCCATGGCCAATTCCAAACTTGTGTTG-3’；
Downstream primer 5-GAGCTCCTAGTTTGGCGTGGGGGAGG-3’；
Wherein the underlined part is the restriction enzyme cutting site, the restriction enzyme cutting site of the upstream primer is BamHI, and the restriction enzyme cutting site of the downstream primer is SacI.
(2) The experimental method comprises the following steps:
cloning of Q1 and AhCEP1 genes
(Q1-1) peanut cDNA is taken as a template, AhCEP1 is amplified by utilizing Novozan high fidelity enzyme (2 × Phanta Max Master Mix, p515), and the reaction system is 2 × Phanta Max Master Mix 12.5 mu L, an upstream primer 1 mu L, a downstream primer 1 mu L template 1 mu L, and water is supplemented to 25 mu L;
the PCR reaction system is as follows: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 25s, annealing at 55 ℃ for 25s, extension at 72 ℃ for 20s, 35 cycles, and extension at 72 ℃ for 5 min;
(Q1-2) after completion of PCR reaction, agarose gel electrophoresis was carried out to observe a band of interest as shown in FIG. 1B, and then gel cutting and gel recovery were carried out according to the procedure of Tiangen ordinary agarose gel DNA recovery kit (DP 209).
Q2 expression vector construction
(Q2-1) construction of cloning vector PCR product was ligated overnight with pMD19-T simple cloning vector in the reaction system pMD19-T simple 0.5. mu. L, Solution I5.0. mu. L and gel recovery product 4.5. mu. L the ligation product was transformed into E.coli DH-5 α strain by heat shock method and grown overnight on L B plate containing ampicillin;
(Q2-2) selecting a white single colony to perform colony PCR, wherein a reaction system is 2 × Taq Master Mix 10 mu L, an upstream primer is 1 mu L, a downstream primer is 1 mu L, a bacterial liquid template is 1 mu L, water is supplemented to 20 mu L, and a positive colony is selected and transferred to a 5m L bacteria shaking tube for amplification culture;
(Q2-3) extraction of plasmid DNA: extracting plasmid DNA by using a small and medium-volume Tiangen plasmid kit (DP 106);
(Q2-4) sequence determination: the sequencing work is completed by the Biotechnology engineering Co., Ltd;
(Q2-5) construction of expression vector: the correct pMD19-T simple plasmid carrying AhCEP1 gene and pCAMBIA1300 empty vector carrying 35S were digested with BamHI and SacI, after one hour at 37 ℃ they were subjected to agarose gel electrophoresis, the correct band was excised and recovered (FIG. 1C), and the recovered gel product was used T from Thermo4DNA ligase connection, the connection product is transformed into DH-5 α strain, the strain grows on L B plate containing kanamycin overnight, white single colony is selected and streaked on L B plate, colony PCR (figure 1D) is carried out, positive colony is selected and cultured in L B liquid culture medium overnight for enlargement, plasmid is extracted by utilizing a Tiangen plasmid miniprep kit and enzyme digestion identification is carried out, as shown in figure 1E, an overexpression vector of 35S AhCEP1 is constructed;
(Q2-6) taking 2.0 mu L plasmid freeze-thaw method to transform agrobacterium rhizogenes R1601, and preparing to infect peanut leaves.
Example 3: transgenic peanut hairy root induction
P1, peanut explant harvesting
Planting Luhua No. 11 peanut seeds in a flowerpot (the bottom diameter is 36cm, the height of the flowerpot is 26cm) of an artificial climate chamber, planting peanuts in single grains, wherein 4 grains are planted in each pot, the culture temperature is 28 ℃ in the daytime, 25 ℃ at night, and 16h of light/8 h of dark. When the peanut seedlings grow to 3-6 leaf-compound periods, 2-4 leaf-compound plants are selected from the bottom upwards as experimental materials. Washing peanut leaf with tap water for 10min, washing with sterile water for 2min, and soaking in 4% sodium hypochlorite solution for 2min until the cut of leaf stalk is slightly white. Then, washing the mixture for 3 to 4 times by using sterile water, then soaking the mixture for 10min by using the sterile water, and airing the water for later use;
activation of P2, AhCEP1-R1601 Strain
100 μ L of AhCEP1-R1601 Agrobacterium stored at-80 ℃ was pipetted into 100m L of YEP liquid medium containing kanamycin (100 μ g/m L) and hygromycin (200 μ g/m L), and shake-cultured at 28 ℃ and 180rpm18h, inoculating 100 μ L bacteria solution in 100m L YEP liquid culture medium the next day, shake culturing at 28 deg.C, continuously activating for 2-3 times, and culturing to OD600The value is 1.5 for use. Meanwhile, the pCAMBIA1300 empty vector is used for transforming the Agrobacterium rhizogenes R1601 strain as a control strain.
P3 and AhCEP1-R1601 infected peanut leaf
The method comprises the following steps of cutting disinfected peanut leaves into about 1cm, adding 2-3 scratches on the leaves, infecting the leaves with AhCEP1-R1601, soaking the treated peanut leaves in AhCEP1-R1601 bacterial solution for 5min, gently shaking the leaves during the soaking to enable the surfaces of the leaves to be fully soaked with the bacterial solution, taking out the leaves, sucking the bacterial solution with sterile filter paper, flatly paving the leaves on the surface of an MS solid culture medium, wherein 8-12 peanut leaves are placed in each dish, transferring the leaves to the MS solid culture medium added with 100 mg/L cefotaxime sodium after 24h (inhibiting the growth of agrobacterium rhizogenes), transferring the leaves to the MS solid culture medium after 24h, sequentially transferring the leaves on the two culture media in turn until the agrobacterium rhizogenes do not grow any more, then culturing the leaves on the MS solid culture medium containing kanamycin, and inducing and differentiating to generate transgenic peanut hairy roots, wherein the transgenic agrobacterium rhizogenes R1601 is used as a reference, and the method is consistent with the formed transgenic infected hairy roots (the shape of the peanut roots as shown in a picture 2).
Example 4: screening and expression quantity identification of transgenic peanut hairy root positive strain
Selecting hairy roots with the length of more than 2cm, transferring the hairy roots to an MS solid culture medium for subculture for 2 weeks, intercepting part of the hairy roots to extract RNA, and specifically operating as follows:
(1) respectively taking different transgenic peanut hairy root strains (the hairy root transformed by the no-load pCAMBIA1300 is taken as a reference, namely CK), washing with sterile water for 3 times, and airing for later use;
(2) grinding the mixture into powder by liquid nitrogen, and extracting the RNA of the peanut hairy roots by using a total RNA extraction kit (DP432) of the Tiangen plants. Dissolving the extracted RNA, determining the concentration of the RNA, and performing reverse transcription by using a Tiangen FastKing gDNA dispensing RTSuperMix reverse transcription kit to obtain peanut hairy root cDNA;
(3) diluting the reverse transcription cDNA by 25 times to be used as an RT-PCR template, and respectively carrying out RT-PCR amplification by using primers AhCEP1 and an internal reference gene AhACTin11, wherein the reaction system comprises 2 × Phanta Max Master Mix 12.5 mu L, an upstream primer 1 mu L, a downstream primer 1 mu L template 5 mu L and water which is supplemented to 25 mu L;
the PCR reaction system is as follows: pre-denaturing at 95 ℃ for 5min, denaturing at 95 ℃ for 25s, annealing at 55 ℃ for 25s, extending at 72 ℃ for 25s, and selecting PCR products of 25-35 cycles to perform agarose gel electrophoresis analysis respectively to obtain PCR cycle numbers of amplified AhCEP1 and reference gene AhACTin11 which are relatively proper;
(4) and (3) increasing or decreasing the template amount of the sample by using the AhActin11 primer through multiple PCR reactions and agarose gel electrophoresis, so that the brightness of the target bands obtained by amplifying the reference genes in different samples is consistent. Using this sample loading as the final assay template, RT-PCR amplification was performed using AhCEP1 primer, and the brightness of the bands in the different samples obtained at this time was the expression level of AhCEP1 in the different transgenic hairy roots (FIG. 3). The expression level of the positive transgenic peanut hairy root strain AhCEP1 is obviously higher than that of a control, a transgenic strain (35S: AhCEP1) with higher expression level of AhCEP1 is selected for phenotype identification, and the identification result is shown in figure 3.
Example 5: phenotypic identification of transgenic peanut hairy roots under high-salt and low-nitrogen stress
Experimental methods:
selecting branches of transgenic peanut hairy roots subjected to subculture 35S, AhCEP1 and Control (CK), weighing, transferring the branches to MS and MS liquid culture media containing 150mM NaCl respectively, subculturing at 23-26 ℃ and 160rpm, continuously culturing for 6 weeks, replacing fresh liquid culture media containing NaCl or normal MS liquid culture media every 2 weeks, observing morphological characteristics of different transgenic hairy roots, washing with sterile water, and drying in the air to respectively weigh the fresh weights of the different transgenic peanut hairy roots, namely the biomass (fresh weight is the weight of the hairy roots in each bottle-inoculation weight) (figure 4);
selecting the branches of the 35S AhCEP1 and CK peanut hairy roots in subculture, weighing, transferring to a normal nitrogen liquid culture medium (5mM potassium nitrate) and a low nitrogen liquid culture medium (0.2mM potassium nitrate) respectively, subculturing at 23-26 ℃ and 160rpm, continuously culturing for 6 weeks, replacing fresh normal nitrogen or low nitrogen liquid culture medium every 2 weeks, observing the morphological characteristics of different transgenic hairy roots, washing with sterile water, and airing to respectively weigh the biomass of the different transgenic peanut hairy roots (figure 4).
Analysis of experimental results:
as shown in FIG. 4A, the growth conditions of the transgenic peanut hairy root 35S: AhCEP1 under the conditions of high salt and low nitrogen stress are better than that of the blank control group CK, and then the biomass of the transgenic peanut hairy root 35S: AhCEP1 and the blank control group CK is measured (FIG. 4B), and the results show that: under the condition of high salt stress, the biomass of the CK in the blank control group is 7.2g, and the biomass of the 35S of the hairy root of the transgenic peanut is 9.5g of AhCEP 1; under the condition of low nitrogen stress, the biomass of the CK in the blank control group is 6.9g, and the biomass of the 35S/AhCEP 1 in the transgenic peanut hairy roots is 9.8 g; in the control group, the biomass of CK in the blank control group is 14.5g, and the biomass of 35S: AhCEP1 in the transgenic peanut hairy root is 13.2 g. Therefore, the biomass of the transgenic peanut hairy root 35S, AhCEP1, induced by the method provided by the embodiment of the invention is obviously improved under the conditions of high salt and low nitrogen stress, wherein the biomass is improved by 31.9% under the condition of high salt stress and 42.0% under the condition of low nitrogen stress, and the biomass is obviously higher than that of a control group.
Example 6: overexpression of AhCEP1 transgenic peanut hairy root 35S, detection of salt stress and nitrogen transport related gene expression level in AhCEP1
Experimental procedure:
gene specific primers were designed according to the qRT-PCR experimental requirements using software such as Beacon Designer 7 and Primer Premier 5.0, and primers were synthesized by Shanghai Bioengineering services Ltd and purified by PAGE.
qRT-PCR analysis was performed using qRT-PCR 96-well plates (Axygen, USA) and high transmittance sealing membrane (Axygen, USA), Icycler real-time PCR system (Bio-Rad, USA) with a fluorescent quantitative PCR instrument. The reaction system is referred to SYBR Green Realtime PCR Master Mix (QPK-201) instructions, and the reaction conditions are as follows:
60s at 95.0 ℃; (2)95.0 ℃ for 10 s; (3)58.0 plus or minus 5.0 ℃ for 10 s; (4)72.0 ℃ for 15 s; (5) plate Read; (6) incubate at 65 ℃ for 20 s; (7) the melt from 65 ℃ to 95 ℃, the read energy is 0.5 ℃, and the hold is 1 s; (8) end; wherein (2), (3), (4)50-60 cycles.
Mixing all samples, carrying out qRT-PCR amplification, analyzing a melting curve to verify the specificity of primer amplification, and designing a primer if a single peak indicates that the primer can carry out specific amplification, if the double peaks exist, properly adjusting the annealing temperature and the primer dosage, and if the primer cannot carry out specific amplification. Sequentially diluting the mixed template according to the concentration of 10 times, diluting for 3 times totally, and constructing a relative standard curve by using samples with 4 concentrations;
peanut AhActin11 is used as internal reference, and the concentration of each template is adjusted to make the difference of Ct value of the internal reference less than 2. Simultaneously amplifying internal references of each gene amplification, reading Ct value under default condition, calculating average expression amount and relative deviation by adopting a dual-standard curve method in data analysis, and simultaneously utilizing 2-ΔΔCtRoughly calculating relative expression quantity with an internal reference, and determining the expression abundance of the gene;
analysis of experimental results:
the results of the experiment are shown in FIG. 5, and FIG. 5A shows that critical Na is present after salt stress treatment+Transporter High-affinity K+Transporter 1(HKT1) and Sodium hydrogen exchange 1-like were significantly up-regulated in 35S AhCEP1 transgenic peanut hairy roots, the remaining two Na' S+The transporter expression did not change significantly. As shown in FIG. 5B, NO under low nitrogen stress3 -The expression of the key transporters NRT1.1 and NRT2.1 in 35S AhCEP1 transgenic peanut hairy roots was higher than that of the control, and there was no significant difference in NRT 3.1. Taken together, it shows that AhCEP1 regulates Na+Transporter High-affinity K+transporter 1(HKT1) and Sodium hydrogen exchanger 1-like for increasing Na content+Clearing, thereby reducing the toxic action of salt stress; by up-regulating NO3 -Expression of transporters NRT1.1 and NRT2.1, increasing NO3 -The absorption rate and the low nitrogen stress tolerance of the peanuts are enhanced.
Sequence listing
<110> institute for peanut research in Shandong province
Application of peanut hairy root strain in improving low-nitrogen and high-salt resistance of peanuts
<160>4
<170>PatentIn version 3.5
<210>1
<211>315
<212>DNA
<213>CEP NO.1
<400>1
1 ATGGCCAATT CCAAACTTGT GTTCATTGTC AGTTCTATCC TCCTAGCCTT GGTGATTTTG
61 AATGGTACCT TTTCAGTTCT AGGAAGGCCA CTCAAAACGG AAAACAATAA TAATGTAAAA
121 GTGCCAACCG CCTATGAAGA CGAGGACAAT ATAGACAAGA TGGCTATAGC GGCGGAAAAA
181 ACTGTCGTGT GGCGCCGTTA CACCTCACAG AATTCACCGC CAGCCGATGG GGTCGGAAAT
241 TGGACCGATG ATTTCCGACC CACCGATCCA GGCCACAGTC CCGGAGCGGG CCATTCCTCC
301 CCCACGCCAA ACTAG 315
<210>2
<211>104
<212>Protein
<213>CEP NO.2
<400>2
1 MANSKLVFIV SSILLALVIL NGTFSVLGRP LKTENNNNVK VPTAYEDEDN IDKMAIAAEK
61 TVVWRRYTSQ NSPPADGVGN WTDDFRPTDP GHSPGAGHSS PTPN 104
<210>3
<211>30
<212>DNA
<213>AhCEP1-F
<400>3
1 GGATCCATGG CCAATTCCAA ACTTGTGTTG 30
<210>4
<211>26
<212>DNA
<213>AhCEP1-R
<400>4
1 GAGCTCCTAG TTTGGCGTGG GGGAGG 26
Claims (10)
1. The application of the peanut hairy root strain in improving the low-nitrogen resistance and high-salt resistance of peanuts is characterized in that the peanut hairy root strain is obtained by the following method, and the specific steps are as follows:
after cloning AhCEP1 gene from peanut cDNA, connecting the AhCEP1 gene to an expression vector to construct a plant expression vector containing AhCEP1 gene;
transforming the plant expression vector containing the AhCEP1 gene into agrobacterium rhizogenes to obtain agrobacterium rhizogenes containing the AhCEP1 gene;
infecting peanut leaves by utilizing agrobacterium rhizogenes liquid containing AhCEP1 gene to obtain peanut leaves infected by the agrobacterium rhizogenes liquid containing AhCEP1 gene, inducing the peanut leaves to generate transgenic peanut hairy roots, and transferring the peanut leaves to an MS solid culture medium containing kanamycin for culture;
and (3) after the AhCEP1 gene in the transgenic peanut hairy root is identified to be expressed, determining the expression quantity of the AhCEP1 gene in the transgenic peanut hairy root, and screening a transgenic peanut hairy root positive strain to obtain a peanut hairy root strain.
2. The use of claim 1, wherein the plant expression vector is constructed by enzymatic ligation or gateway system construction.
3. The use of claim 1, wherein the method for infecting peanut leaves with the agrobacterium rhizogenes solution containing AhCEP1 gene is soaking method or smearing method.
4. The use of claim 1, wherein the concentration OD of the Agrobacterium rhizogenes solution containing AhCEP1 gene6001.0 to 2.0; the culture time of the transgenic peanut hairy roots is 2-8 weeks, and fresh culture solution is replaced every 2 weeks.
5. The use according to claim 1, wherein the method for identifying the AhCEP1 gene expression in the transgenic peanut hairy roots is RT-PCR method or qRT-PCR method.
6. The use according to claim 5, wherein the determination of the expression level of AhCEP1 gene is carried out by the following method:
identifying AhCEP1 gene expression in the transgenic peanut hairy roots by using an RT-PCR method or a qRT-PCR method to obtain different PCR products;
and respectively carrying out agarose gel electrophoresis analysis on the PCR products, and judging the expression quantity of the AhCEP1 gene in the hairy roots of the transgenic peanuts according to the brightness of the bands.
7. The use of claim 1, wherein the method further comprises determining the biomass of the transgenic peanut hairy root positive line by:
selecting branches from the same transgenic peanut hairy root positive strain, weighing, transferring to a liquid culture medium, and continuously culturing;
replacing a fresh liquid culture medium every 1-2 weeks, finally washing and drying the liquid culture medium by using sterile water, and respectively weighing the fresh weights of the hairy roots of the transgenic peanuts to obtain the biomass of the hairy roots of the transgenic peanuts, wherein the formula is as follows:
fresh weight of transgenic peanut hairy root-weight of each bottle of transgenic peanut hairy root-inoculation weight.
8. The use according to claim 7, wherein the liquid medium is MS liquid medium, low nitrogen liquid medium and high salt liquid medium.
9. The use of claim 7, wherein the transgenic peanut hairy root branches are cultured under conditions of 23-26 ℃ and 150-180 rpm.
10. The use according to any one of claims 1 to 9, low nitrogen being KNO in said liquid medium3Is less than or equal to 1mM and is highSalt means that the concentration of NaCl in the liquid medium is 200mM or more.
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| CN107245489A (en) * | 2017-06-08 | 2017-10-13 | 山东农业大学 | A kind of apple polypeptide hormone gene M dCEP7 of regulation and control root system development and its application |
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| CN113621038A (en) * | 2021-08-02 | 2021-11-09 | 山东省花生研究所 | Oligopeptide capable of improving germination rate of plant seeds under salt stress and application thereof |
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