CN116463363A - Cloning of corn sphingosine kinase ZmSphK1 gene and application thereof in salt stress - Google Patents

Abstract

The invention belongs to the field of corn gene cloning, and relates to research on cloning of corn sphingosine kinase ZmSphK1 gene and application of the corn sphingosine kinase ZmSphK1 gene in salt stress. The expression method comprises the steps of prokaryotic expression vector pET28a-ZmSphK1, plant expression vector pCambia1300-ZmZmSphK1 construction, bacterial liquid PCR and enzyme digestion identification, agrobacterium competent for plasmid transformation, arabidopsis plant transformation by plasmid transformation bacteria using a dip-in method and the like, and lays a foundation for researching the function of maize gene ZmZmSphK1 in salt stress by utilizing the transformation arabidopsis plant, wherein the method comprises the steps of extracting total RNA of maize leaves, reversely transcribing the total RNA into cDNA, carrying out real-time fluorescence quantitative PCR (RT-PCR), carrying out gene cloning, carrying out the expression method, carrying out the PCR and enzyme digestion identification, and the like.

Description

Cloning of corn sphingosine kinase ZmSphK1 gene and application thereof in salt stress

Technical Field

The invention belongs to the technical field of corn gene cloning, and in particular relates to application of corn sphingosine kinase ZmSphK1 gene cloning in salt stress

Background

Corn, one of the main food crops and feed sources for people, has been valued and favored by students worldwide because of its wide application value and low cost. In China, corn has become an important crop with highest total yield, largest planting area and single yield inferior to rice in three crops. However, tolerance of maize to salt stress is weak. When under salt stress, plant growth is inhibited, net photosynthetic rate, chlorophyll content and root system vigor are significantly reduced, and the effect on the overground part is greater than on the underground part. Too high a salt concentration may lead to retarded maize growth, reduced crop yield and even extensive wilting. Therefore, the research on the salt tolerance of corn is of great significance for improving the corn yield and cultivating excellent resistant varieties.

The important component of membrane lipids, sphingomyelin, the metabolite sphingomyelin-1-phosphate (S1P), is a very potent lipid mediator in animal cells, playing an important role in a number of biological processes in animal systems. Whereas sphingosine kinase (SphK) is a key enzyme required for S1P synthesis, sphingosine forms S1P under the catalysis of sphingosine kinase. Sphingosine kinase is a novel class of lipid kinase with high evolutionary conservation, expression in humans, mice, yeast and plants, and similar kinase in nematodes and drosophila. Two sphingosine kinases, sphK and SphK2, have been identified in mammals. According to recent studies, it has been found that S1P is also present in plants, and plays an essential role in adverse stress in addition to the growth and development processes of plants. It has recently been demonstrated that the enzyme, sphingosine kinase (SphK), which synthesizes S1P, is produced by stimulation of the plant hormone abscisic acid in the protected cells of arabidopsis thaliana (Arabidopsis thaliana); S1P can effectively regulate and control the opening and closing of the protective cells. Recent studies indicate that S1P may be involved in a variety of signal transduction pathways in plants: (1) stress resistance and adaptability of plants are mainly controlled by plant hormone abscisic acid (ABA) when the plants are stressed by abiotic, and S1P can participate in an ABA-mediated drought signal transduction pathway; (2) calcium ions act as secondary messengers of many adversity stresses and exert a very good effect of improving stress resistance in plants. And S1P can be involved in regulating the concentration of calcium ions in plants and possibly plays a role in the signal transduction process of guard cells, and can influence the opening and closing of stomata by causing the concentration change of the calcium ions, the closing degree of the stomata caused by S1P and the dosage of the stomata in the cells show specificity, and the effect caused by high concentration is faster. However, how S1P functions when plants resist salt stress is not clear so far. The research on various enzymes in a plant stress-resistant system provides a good breeding strategy for plants to resist stress, and is a hot spot of current research. For this, we cloned and identified the sphingosine kinase gene zmssphk 1 involved in S1P synthesis and metabolism in maize.

Disclosure of Invention

The invention provides a corn sphingosine kinase ZmSphK1 gene cloning method, which can effectively clone and express corn ZmSphK1 genes.

The first aim is to provide a method for cloning corn sphingosine kinase ZmSphK1 gene, which comprises the following specific steps:

(1) Preparation and treatment of materials: the maize inbred line variety "1104", variety "Mo17", is provided by molecular breeding laboratories. Mixing nutrient soil and vermiculite according to a proper proportion, selecting 10 seeds with full seeds for planting, and storing the sheared leaves in an ultralow temperature refrigerator for RNA extraction;

(2) Extraction of total RNA of corn leaves: in the experiment, RNA of the leaf blade is extracted by using a Takara RNA extraction kit, and DNA pollution in the extracted RNA is removed by DNase I reaction. The whole extraction process should be operated on ice and the mask is worn;

(3) Reverse transcription into cDNA and real-time fluorescent quantitative PCR (RT-PCR): in this experiment, cDNA was synthesized using Takara reverse transcription kit, and 4. Mu.l of RNA was added to 20. Mu.l of the reaction system. Performing reverse transcription according to a set reaction program, and then storing a reverse transcription product in a low-temperature refrigerator at-20 ℃ for later use; the expression of ZmSphK1 gene was analyzed by fluorescent quantitative Real-time RT-PCR as follows: the apparatus used for fluorescent quantitative PCR was the Qiagen fluorescent quantitative PCR apparatus model number Rotor-gene Q. Kit TB Green used ^TM Permix Ex taq ^TM II (Takara Cat#RR820A). 25. Mu.l of a PCR stock solution was prepared according to the instructions; the PCR reaction procedure was as follows: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 57℃for 30s, elongation at 72℃for 30s,35 cycles; preserving at 4 ℃. After the reaction is finished, a2 delta CT method is applied to data analysis ^[12] The method comprises the following steps:

Fold Change＝2 ^{- [ (Ct treatment-Ct treatment actin) - (Ct control-Ct control actin)]}

From this expression, it is possible to preliminarily judge the approximate change in the expression level of ZmSphK1 gene.

(4) Cloning of the genes:

and (3) synthesizing a primer: zm-ZmSphK1-F:5'-GATGCAAGAAACCAAGCATCGG-3'; zm-ZmSphK1-R:5'-ATTCAAGGCGAGCACTTCCC-3'

Performing PCR amplification by using the cDNA reverse transcribed in the step 3 as a template, wherein a PCR amplification system is as follows: 2 XGC Buffer II 25. Mu.l, dNTP 8. Mu.l, zm-ZmSphK1-F1 1. Mu.l, zm-ZmSphK1-R1 1. Mu.l, cDNA template 2. Mu.l, la Taq enzyme 0.5. Mu.l, ddH ₂ O 12.5μl；

And (3) after the PCR is finished, carrying out agarose gel electrophoresis, and recovering a band of about 1100bp to obtain the corn sphingosine kinase ZmSphK1 gene.

Application of corn sphingosine kinase ZmSphK1 gene in salt stress

The second purpose of the invention is to provide an application method of the corn sphingosine kinase ZmSphK1 gene, which comprises the following steps:

s1, construction of pMD19-ZmZmSphK1 recombinant plasmid: connecting the clone of the corn gene ZmZmSphK1 with a pMD19-T vector, and connecting the clone of the purified gene ZmZmSphK1 with the pMD19-T vector to obtain a recombinant plasmid pMD19-ZmZmSphK1;

s2, construction of a prokaryotic expression vector pET28a-ZmSphK 1: the ZmZmSphK1-XbaI-F and ZmZmSphK1-BamHI-R are used as primers, the PMD19-T-ZmZmSphK1 plasmid with correct sequence is used as a template for PCR amplification, and the target fragment is purified and recovered after gel cutting. The plasmid of the pET28a expression vector was digested with restriction enzymes Xba I and BamHI. And (3) carrying out agarose gel electrophoresis after double enzyme digestion is completed, cutting and recovering target fragments, and carrying out a ligation reaction on the PCR product and the product DNA recovered by the pET28a expression vector gel. Obtaining a plant expression vector pET28a-ZmZmSphK1;

s3, construction of a plant expression vector pCambia1300-ZmZmSphK 1: the ZmZmSphK1-XbaI-F and ZmZmSphK1-BamHI-R are used as primers, the PMD19-T-ZmZmSphK1 plasmid with correct sequence is used as a template for PCR amplification, and the target fragment is purified and recovered after gel cutting. The plasmid of the pCambia1300 expression vector was double digested with restriction enzymes Xba I and BamHI. And (3) carrying out agarose gel electrophoresis after double enzyme digestion is completed, cutting and recovering target fragments, and carrying out a ligation reaction on the PCR product and the product DNA recovered by the pCambia1300 expression vector gel. Obtaining a plant expression vector pCambia1300-ZmZmSphK1;

s4, infecting plants by agrobacterium: planting wild arabidopsis, preparing agrobacterium competent cells, then transforming the agrobacterium competent cells by using a plant expression vector pCambia1300-ZmZmSphK1, and infecting the arabidopsis by using agrobacterium to obtain a transgenic plant capable of effectively cloning a corn ZmZmSphK1 gene.

The agrobacterium in the invention is agrobacterium GV3101.

After infecting the plant body with the agrobacterium described in S4, three generations of selfing were performed to screen plant bodies containing the zmssphk 1 gene.

The invention provides a method for cloning and applying a corn sphingosine kinase ZmSphK1 gene, which can effectively clone and express the corn sphingosine kinase ZmSphK1 gene, and the invention also discovers that the capability of the ZmSphK1 gene in resisting salt stress is enhanced in prokaryotic expression products and eukaryotic expression products.

The ZmSphK1 gene of the invention has the sequence length of 1044bp, the coding region length of 1029bp and total coding of 342 amino acids.

The molecular weight of the protein coded by the ZmSphK1 gene is 37192.41kDa in http:// web. Expasy. Org/ProtParam/website through ProtParam prediction, and the theoretical isoelectric point is 5.58; the total number of negatively charged residues (Asp+Glu) is 42 and the total number of positively charged residues (Arg+Lys) is 35; the instability coefficient was 38.25, which was shown to be relatively stable.

The ZmSphK1 gene of the invention uses BioXM2.6 software to carry out sequence comparison on a sequencing result and a ZmSphK1 target gene (LOC 100383275), wherein the sequence_0 is the ZmSphK1 target gene, and the sequence_1 is the sequencing result, such as the sequence 1 in a sequence table.

The ZmSphK1 gene of the invention is logged in NCBI website, blast is used for searching homologous sequences among plant species, MEGA5.0 software is used for aligning protein sequences, genetic evolution analysis is carried out, and the result is shown in figure 1 and is consistent with the corn sequences queried by NCBI.

The invention amplifies full-length cDNA of ZmSphK1 gene from corn, clones the gene ZmSphK1 on the basis, connects the gene ZmSphK1 to a plant expression vector, and converts Arabidopsis thaliana by using an agrobacterium infection method, and data show that the gene can obviously improve salt tolerance of transgenic Arabidopsis thaliana in seed germination stage and seedling strengthening stage. The gene of the invention can provide theoretical basis and gene source for cultivating new varieties of crops, and is helpful for fundamentally improving the salt tolerance of corn crops.

Drawings

FIG. 1 is a genetic evolutionary analysis of the ZmSphK1 gene sequence;

FIG. 2 shows the relative expression level of ZmSphK1 (left, 1104 varieties; right, mo17 varieties);

FIG. 3 shows the total RNA electrophoresis results of maize leaves from 1 to 2.1104; 3-4.Mo17 total RNA;

FIG. 4 shows the result of electrophoresis of PCR amplification of ZmSphK1 gene, wherein lane M is DNA;

a marker; 1. lane 2 is amplification product;

FIG. 5 is a map of the pMD19-T vector;

FIG. 6 shows PCR identification of bacterial liquid of pMD19-T-ZmSphK1 recombinant E.coli, wherein M lanes are DNA markers; 1. lanes 2 and 3 are bacterial solutions of pMD19-T-ZmSphK 1;

FIG. 7 shows the identification of M.DL2000 DNA markers by cleavage of the pMD19-T-ZmSphK1 plasmid; 1-4, enzyme digestion identification results of recombinant plasmids;

FIG. 8 is a diagram of a prokaryotic expression vector pET28a (+);

FIG. 9 shows the result of PCR amplification of pMD19-ZmSphK1 as a template, wherein lane M is a DNA marker; 1. lanes 2 and 3 are PCR results using pMD19-ZmSphK1 as a template;

FIG. 10 shows the PCR result of prokaryotic vector pET28-ZmSphK1 bacterial liquid, wherein the M lane is a DNA marker; 2. 3 is a negative plasmid; 1. 4, 5 are positive plasmids;

FIG. 11 is a graph showing the growth of cells after salt stress treatment (A, 0.6mol/L NaCl; B,0.8mol/L NaCl; C,1.0mol/L NaCl);

FIG. 12 is a p1300 vector diagram;

FIG. 13 shows PCR of the bacterial liquid of Agrobacterium tumefaciens of pCambia1300-ZmZmSphK1, wherein lane M is a DNA marker; 1. lanes 2, 3 and 4 are bacterial solutions of pCambia1300-ZmZmSphK1;

FIG. 14 is an electrophoretogram of the plant expression vector pCambia1300-ZmZmSphK1 double cleavage product: wherein lanes M1 and M2 are DNA markers; 1. lane 2 is the double cleavage product of the plant expression vector pCambia1300-ZmZmSphK1;

FIG. 15 is a dip-flower method of infecting Arabidopsis thaliana;

FIG. 16 shows the PCR results of ZmSphK 1-transformed Arabidopsis;

FIG. 17 shows the ZmZmSphK1 gene electrophoresis band M of corn; 1: negative water control; 2. 3, 4, 5: four overexpressing lines;

FIG. 18 shows the relative expression of ZmSphK1 gene in transgenic and wild type Arabidopsis by fluorescent quantitative PCR;

FIG. 19 is a root length assay for wild-type and ZmZmSphK1 gene over-expressed plants;

FIG. 20 shows seedling vigor five days after transplanting;

FIG. 21 shows the dry fresh weight measurement of ZmZmSphK1 gene over-expressed plants and wild type plants;

FIG. 22 shows the culture medium salt treated plants, A, observed after three days; observing after five days; observing after eight days;

fig. 23 is a salt-treated plant in soil, a: a week later, clear water control group; b: one week later, salt treatment experimental group; c: two weeks later, clear water control group; d: salt treatment experimental groups after two weeks;

FIG. 24 is a graph showing the measured content of plant peroxidase;

Detailed Description

The invention provides a corn sphingosine kinase ZmSphK1 gene cloning and cloning method and an expression method, which can effectively clone and express a corn ZmSphK1 gene and enable the corn ZmSphK1 gene to be effectively expressed in a bacterial strain.

For the purpose of making the technical solutions and advantages of the present invention more apparent, the present invention will be further described in detail by means of the accompanying drawings and specific embodiments.

The experimental methods used in the following examples are all conventional methods unless otherwise specified; materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The strain was purchased from Bokang Biolimited using E.coli competent cells DH 5. Alpha. And Agrobacterium competent cells GV3101. Plant expression vector pCAMBIA1300, RNA extraction kit, reverse transcription kit, agarose gel DNA recovery kit, etc. are all purchased from Takara company. Sequencing and primer synthesis were performed at Qingdao Rui Boxing Corp and Shanghai Pair Senno Biotech Co.

Example 1 the invention provides a cloning method of a corn sphingosine kinase ZmSphK1 gene, which comprises the following specific steps:

(1) Preparation and treatment of materials

The maize inbred line variety salt tolerant "1104" and the salt intolerant variety "Mo17" are provided by a molecular breeding laboratory. Mixing nutrient soil and vermiculite according to a proper proportion, selecting 10 seeds with full seeds, planting, carrying out five treatments and two repetition, wherein each treatment is carried out on 1 seed, preparing 200mmol/L NaCl solution when corn grows to a trefoil period, carrying out salt stress treatment by using the solution irrigation material, respectively cutting leaves after 0 hour, 6 hours, 12 hours, 24 hours and 48 hours for RNA extraction, and storing the cut leaves in an ultralow temperature refrigerator for RNA extraction.

(2) Extraction of total RNA from corn leaves

Transferring the ultralow-temperature frozen corn leaves into a foam box containing liquid nitrogen, freezing the mortar by the liquid nitrogen in advance, taking out the sample, grinding the sample by using a pestle until the sample is ground into white fine powder, and adding the liquid nitrogen from time to prevent tissue from melting; the subsequent steps are described with reference to the RNA kit instructions, and it should be noted that the whole process of RNA extraction should be performed on ice and that wearing a mask reduces conversations to prevent the introduction of RNase; in addition, DNase I reactions are required to remove genomic DNA.

After total RNA is extracted, agarose gel electrophoresis results are shown in figure 2, the extracted RNA can be seen to contain RNA with two different sizes through two clearer bands, the brightness proportion shows that the extracted RNA has good integrity and purity, and the extracted RNA can be used for amplifying ZmSphK1 genes in the next step.

(3) Reverse transcription and real-time fluorescence quantitative PCR (RT-PCR)

Since RNA is easily degraded, it is generally necessary to reverse transcribe into cDNA in time for preservation. The RNA was removed from the freezer at-80℃and a reaction solution was prepared by taking 0.2ml PCR reaction tube on ice.

In this experiment, cDNA was synthesized using Takara reverse transcription kit, and 4. Mu.l of RNA was added to 20. Mu.l of the reaction system. Performing reverse transcription according to a set reaction program, and then storing a reverse transcription product in a low-temperature refrigerator at-20 ℃ for later use; quantifying Real with fluorescence-time RT-PCR analysis of the expression of zmssphk 1 gene, by the following method: the apparatus used for fluorescent quantitative PCR was the Qiagen fluorescent quantitative PCR apparatus model number Rotor-gene Q. Kit TB Green used ^TM Permix Ex taq ^TM II (Takara Cat#RR820A). 25. Mu.l of a PCR stock solution was prepared according to the instructions; the PCR reaction procedure was as follows: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 57℃for 30s, elongation at 72℃for 30s,40 cycles; preserving at 4 ℃. After the reaction is finished, a2 delta CT method is applied to data analysis ^[12] The method comprises the following steps: fold Change = 2 ^{- [ (Ct treatment-Ct treatment actin) - (Ct control-Ct control actin)]}

From this expression, it is possible to preliminarily judge the approximate change in the expression level of ZmSphK1 gene.

For the variety 1104, after the treatment of salt stress for 6 to 12 hours, the expression quantity of the ZmSphK1 gene is slightly improved compared with the control, and the expression quantity is maximum after the treatment for 24 hours; for the variety Mo17, the expression level of ZmSphK1 gene is greatly different from that of the variety 1104, and the expression level of the ZmSphK1 gene is always in a low state in 6-24 hours, and only slightly rises in 48 hours. The reason for this is presumed to be that the expression of the gene exhibits salt inducibility. As shown in fig. 2.

(4) And (3) RNA quality detection: the concentration of the extracted RNA was measured by an ultra-micro spectrophotometer, and the purity and concentration of the RNA were measured, followed by 0.8% agarose gel electrophoresis (0.12 g agarose in 15ml1 XTAE). As shown in fig. 3.

(5) cDNA template synthesis: adding the reaction solution according to the reaction system, and fully and uniformly mixing. The reaction is carried out in a PCR instrument, and the reaction procedure is as follows: firstly, the reaction is carried out for 15min at 37 ℃, then the reaction is carried out for 5s at 85 ℃, and finally the preservation is carried out at 4 ℃. The reaction system is as follows: master mix 2. Mu.l; total RNA 2. Mu.l; RNase Free water 6. Mu.l; total 10. Mu.l.

(6) Amplification of the maize sphingosine kinase Gene ZmZmSphK1

And (3) synthesizing a primer: zm-ZmSphK1-F:5'-GATGCAAGAAACCAAGCATCGG-3'; zm-ZmSphK1-R:5'-ATTCAAGGCGAGCACTTCCC-3'

PCR amplification was performed using the reverse transcribed cDNA as a template, and the PCR amplification system was: 2 XGC Buffer II25 μl, dNTP 8 μl, zm-ZmSphK1-F1 μl, zm-ZmSphK1-R1 μl, cDNA template 2 μl, la Taq enzyme 0.5 μl, ddH ₂ O12.5. Mu.l; PCR reaction procedure: pre-denaturation at 94℃for 1min; denaturation at 94℃for 30s, annealing at 57℃for 45s, elongation at 72℃for 1min,35 cycles; extending at 72 ℃ for 5min; preserving at 4 ℃.

Using cDNA obtained after completion of the reverse transcription PCR reaction as a template, zmSphK1 gene was amplified with a designed primer, and agarose gel electrophoresis was performed at 5. Mu.l of the reaction solution after completion of the amplification to obtain a single band of about 1100bp in size, as shown in FIG. 4. The bands are clear and bright, the product is single, and the amplification conditions adopted are proper. And (5) carrying out gel recovery on the amplified product.

Example 2a method for expressing the maize sphingosine kinase zmssphk 1 gene, comprising the steps of:

construction of pMD19-ZmZmSphK1 recombinant plasmid

Because the La taq has good fidelity, and the PCR product amplified by La taq can automatically attach an A base tail at the 3' end, the DNA fragment after gel cutting and recovery can directly construct a recombinant plasmid with a pMD19-T linear vector, the map is shown in figure 5, and the A tail is not required to be added. The ligation reaction solution was prepared as follows:

T-Vector pMD19 (Simple) 1 μl; PCR product 1. Mu.l; ddH2O3 μl. Adding Solution I with equal volume into the prepared DNA ligation reaction Solution, fully and uniformly mixing the reaction Solution, setting a program to react for 30min at 16 ℃, and preserving at-20 ℃ for later use after the reaction is finished;

the clone of ZmZmSphK1 recovered by the gel is connected with a pMD19-T vector, the clone of the purified gene ZmZmSphK1 is connected with the pMD19-T vector to obtain a recombinant plasmid pMD19-ZmZmSphK1, DH5 alpha is transformed, bacterial liquid PCR verification is carried out, the bacterial liquid PCR verification is carried out, and the double enzyme digestion verification is carried out, and the sequencing is carried out after the step of FIG. 7.

Construction of prokaryotic expression vector pET28-ZmSphK1

Primer design: as shown in FIG. 8, by combining the information shown on the pET28a (+) map of the prokaryotic expression vector with the cleavage site analysis of BioXM2.6 software, the cleavage site was judged to be selected as a point, and the primers containing the XbaI cleavage site TCTAGA and the BamHI cleavage site GGATCC were designed based on the sequencing result as follows:

XbaⅠ-ZmSphK1-F(5’-GCTCTAGAGCCATTGAAGCTTCTGGCGTGC-3’)

BamHⅠ-ZmSphK1-R(5’-CGGGATCCCGGGGCTAACGAAGCAAAGTCC-3’)

the positive clone plasmid was extracted, the ZmSphK1 coding region was obtained from the plasmid pMD19-ZmSphK1 by PCR amplification as shown in FIG. 9, the amplified product and pET28a (+) vector were digested with XbaI and BamHI enzymes to cut out complementary cohesive ends, the reaction conditions were 37℃for 3 hours, agarose gel electrophoresis was performed after the completion of the digestion reaction, and then the digested gel was recovered. The enzyme digestion reaction is 10KBuffer 2.5 μl; xbaI 1.0 μl; bamHI 1.0 μl; 15.0 μl of Template; ddH2O30.5 μl.

Constructing pET28-ZmSphK1 by a connection reaction system: buffer 3.0. Mu.l; t4 ligase 1.0 μl; T-Vector pET28a (+) 1.0 μl; cDNA 2.0. Mu.l; ddH2O 3.0 μl.

Adding the equal volume of Solution I into the prepared DNA ligation reaction Solution, and fully and uniformly mixing. The reaction was carried out at 16℃for 30min. The temperature is required appropriately, and if the temperature is too high, the cyclization of DNA may be inhibited.

Adding 10 mu l of the ligation reaction solution into 100 mu l of competent cells, performing transformation by a heat shock method, adding 500 mu l of LB liquid medium without antibiotics into the solution after transformation, uniformly mixing, and putting the solution into a shaking table incubator at 37 ℃ for recovery culture at 200rpm for 1 hour; and centrifuging the transformed bacteria after recovery culture at 7000rpm for 2min, sucking 400-500 μl of redundant supernatant, uniformly mixing the residual liquid and thalli, transferring the precipitate to a previously prepared kanamycin-containing flat plate, uniformly coating, sealing, and culturing in an incubator at 37 ℃ for 12-16 hours.

Detection of recombinant vector: single colony is picked up and amplified, and bacterial liquid PCR is performed by taking bacterial liquid as a template, as shown in FIG. 10. Positive clones were sent to the company for sequencing.

Salt resistance analysis of recombinant E.coli BL21 (pET 28a-ZmSphK 1)

Recombinant plasmid pET28a-ZmSphK1 is extracted from E.coli, transferred into an expression strain BL21, inoculated into a liquid LB culture medium containing kanamycin sulfate, and shake-cultivated at 200rpm for 12-16 hours.

600. Mu.l of BL21 (pET 28a-ZmSphK 1) and BL21 (empty vector) of the control group were inoculated into LB liquid medium with NaCl concentration of 0.6mol/L, 0.8mol/L and 1.0mol/L, respectively, cultured at 37℃and 200rpm, OD600 values were measured every one hour, and then a growth curve was drawn.

Average OD is measured ₆₀₀ As shown in FIG. 11, the recombinant plasmid pET28a-ZmSphK1 was transferred to a higher strain than the empty vector pET28 a.

Construction of pCambia1300-ZmZmSphK1 recombinant vector

And (3) synthesizing a primer: as shown in FIG. 12, the restriction site analysis in combination with BioXM2.6 software determined that XbaI restriction site and BamHI restriction site were selected, and primers containing XbaI restriction site TCTAGA and BamHI restriction site GGATCC were designed based on the sequencing result as follows:

XbaⅠ-ZmSphK1-F(5’-GCTCTAGAGCCATTGAAGCTTCTGGCGTGC-3’)

BamHⅠ-ZmSphK1-R(5’-CGGGATCCCGGGGCTAACGAAGCAAAGTCC-3’)

and (3) carrying out a ligation reaction on the PCR product and the product DNA recovered by the pCambia1300 expression vector gel, wherein the ligation reaction system is as follows:

T-Vector pCambia1300 3μl；PCR product 12μl；DNA Ligation Kit 1μl；10×Buffer 2μl；RNase free H ₂ O 2μl；Total 20μl。

transformation of ligation products into competent E.coli

E.coli competent cells DH5 alpha were transformed by heat shock method, after the transformation was completed, the bacterial liquid was uniformly coated on a preheated LB+Kan (LB final concentration: 50 mg/L) plate with a sterile coating rod. The plates were incubated upside down in a constant temperature incubator at 37℃and after 10-16h the transformation efficiency was observed.

Bacterial liquid PCR detection

The single colony in the flat plate is picked by a sterilized toothpick in an ultra-clean workbench, added into a 10mL centrifuge tube filled with 1mL LB liquid culture medium, cultured in a constant temperature incubator at 37 ℃ and 200rpm until bacterial liquid is turbid, and the bacterial liquid is taken for PCR detection reaction.

PCR reaction system: microobial 1μl；10×PCR Buffer 2.5μl；dNTP Mixture 2μl；Primer F(10μM)1μl；Primer R(10μM)1μl；rTaq(5U/μL)0.2μl；ddH ₂ O 17.3μl；Total 25μl。

PCR reaction procedure: pre-denaturation at 94℃for 1min; denaturation at 94℃for 30s, annealing at 60℃for 30s, elongation at 72℃for 1min,35 cycles; extending at 72 ℃ for 5min; preserving at 4 ℃.

Detection of recombinant vector: and (3) sequencing positive clones obtained by PCR detection of bacterial liquid by a company, and simultaneously carrying out double enzyme digestion verification on plasmids.

Extraction of plasmid DNA: the plasmid DNA was extracted from the bacterial liquid with correct sequence, and the extraction method was referred to as a kit for plasmid extraction from Takara. The extracted DNA is preserved at-20 ℃.

The agrobacterium GV3101 was transformed by freeze thawing: the agrobacterium GV3101 is transformed by freeze thawing method, after the transformation is completed, about 100 mu l of supernatant is taken to lightly blow and resuspension fungus blocks, the resuspension fungus blocks are coated on LB plates containing corresponding antibiotics, and the LB plates are placed in a 28 ℃ incubator for inversion culture for 2-3 days. Single colonies were picked and amplified for bacterial liquid PCR verification as shown in FIG. 13. And (5) detecting the same bacterial liquid by PCR. The cleavage assay is shown in FIG. 14.

Example 3 a method for using the maize sphingosine kinase zmssphk 1 gene, comprising the steps of:

preparing infection buffer solution

The formulation of the infection buffer is as follows: 1/2MS 2.47g;1/2BS 0.5g;5% sucrose 50g; 0.5g of MES; 0.044. Mu. Mol/l6-BA 400. Mu.l; total 1000mL.

6-BA mother liquor: 0.01g of 100ml of H is added ₂ In O, 10. Mu.l was added to 100ml of the infection buffer. The pH was adjusted to 5.7 with KOH and 100. Mu.l of 0.01% silwet was added prior to infestation.

Cultivation of wild-type homozygous Arabidopsis thaliana

Washing wild Arabidopsis seeds with sodium hypochlorite and 70% alcohol at an ultra-clean workbench (4 ℃ refrigerator is used for low-temperature vernalization in advance), washing and airing the seeds, then lightly scraping the seeds with a sterilized toothpick, uniformly spreading the seeds on an MS culture medium, placing the MS culture medium in an illumination incubator for culture, and transferring the Arabidopsis seeds into a flowerpot for culture when the Arabidopsis grows to a trefoil period.

Transformation of Arabidopsis thaliana

When the wild arabidopsis grows to flower most quickly, the bud which is already opened is cut off by scissors, the prepared invasion solution is poured into a wide-mouth bottle which is sterilized in advance, the arabidopsis is immersed into the invasion solution for more than 45s, as shown in fig. 15, the arabidopsis is wrapped by a preservative film to prevent the invasion solution from volatilizing, the arabidopsis is placed in a tray horizontally, the arabidopsis is placed in the dark for 24 hours, and then the arabidopsis is cultivated by standing under illumination. After one week, the arabidopsis thaliana is infected for the second time, and seeds are harvested when the fructus corni turns yellow.

Screening of Arabidopsis thaliana

After kan resistance is subjected to preliminary screening, most plants begin to yellow after a period of cultivation, a small number of plants still keep green, presumably the plants kept green are arabidopsis plants transferred with ZmSphK1 genes, seeds transferred with ZmSphK1 genes are harvested, seeds transferred with ZmSphK1 genes of T3 generation are harvested, the seeds are washed by sodium hypochlorite and 70% alcohol (4 ℃ refrigerator is used for low-temperature vernalization in advance) on an ultra-clean workbench, the seeds are washed and dried, then the seeds are lightly scraped by sterilizing toothpick, evenly spread on MS culture medium added with kan resistance, placed in a lighting incubator for cultivation, and transferred into a flowerpot for cultivation when arabidopsis grows to the three-leaf period.

Extraction of transgenic Arabidopsis group DNA

When the plants are slightly bigger, part of leaves are cut (not all of the leaves can be cut off, and the death of the arabidopsis is prevented), and the transgenic arabidopsis group DNA is extracted by a CTAB method, as shown in figure 16, the method needs to be noted that a water bath of 65 ℃ is firstly used for preheating 2% CTAB extracting solution before the experiment, 70% ethanol used for washing is put into a refrigerator for precooling, and the washing is not too hard, so that the experiment failure caused by flushing down DNA sediment is prevented.

PCR identification of transgenic Arabidopsis thaliana

The extracted arabidopsis leaf DNA is used as a template, and PCR reaction is carried out according to the overexpression primer of the ZmSphK1 gene designed before, so that the transgenic arabidopsis is further identified, and the reaction system and the reaction conditions are the same.

As shown in FIG. 17, the electrophoresis results show that No. 1-No. 5 have bright bands at 1100bp, and the transgenic plants are preliminarily deduced.

Gene expression analysis of ZmZmSphK1 Arabidopsis line

And (3) performing RNA quality analysis, and performing fluorescence quantitative analysis. Taking wild type Arabidopsis thaliana as a reference substance, taking the endogenous gene beta-acting of Arabidopsis thaliana as an internal reference, and taking 2X ChamQ Universal SYBR qPCR Master Mix mu l; forward primer 0.4 μl; downstream primer 0.4.4 μl; cDNA 2.0. Mu.l; RNase free water was added to 20. Mu.l. The procedure is pre-denaturation for 95 min; denaturation at 95℃for 10s; annealing at 55-60 ℃ for 30s; extending for 30s72 ℃;40 cycles. The system was identified by qRT-PCR mass analysis, and the relative expression level of ZmZmSphK1 gene in Arabidopsis was detected as shown in FIG. 18. Three strains with high expression level are selected for abiotic stress treatment.

Example 4 salt stress phenotypic analysis of transgenic Arabidopsis thaliana containing the maize sphingosine kinase ZmSphK1 Gene

In order to observe the character generated by the over-expression of the ZmZmSphK1 gene in the plant, the plant is respectively compared with the wild plant by measuring the root length, the plant height and the fresh-to-dry ratio.

Root length: MS culture medium was arranged to divide the region by strain classification. Taking out from the greenhouse about two weeks after sowing, carefully taking out the well-developed plants with tweezers, putting the well-developed plants in a corresponding area of the culture medium, and measuring root length.

Plant height: the ruler is used for measuring the plant height of the overground part of the plant, and three groups of data are measured for each plant line

Fresh-to-dry ratio: firstly, cutting off leaves on the overground part of the plant, removing residual soil of the plant, weighing and recording data, and then respectively filling the measured plant into net bags with the same size and weight, and marking. And (5) after all the strains are measured, putting the mesh bag into a dryer for drying. And taking out the dried mesh bag, placing the mesh bag on a balance for weighing, removing the self weight of the mesh bag as a result, and recording data. Three sets of data were measured for each strain.

The root length average values of WT, OE1, OE2 and OE3 were 1.8mm, 2.7mm, 2.8mm and 2.7mm, respectively. It is also evident from the observation of FIG. 19 that the plant root length over-expressed by the ZmZmSphK1 gene is longer than that of the wild type Arabidopsis plant, and thus it can be derived that the ZmZmSphK1 gene expression contributes to root length growth. After five days of seedling transplanting, the plant growth vigor is continuously observed, as shown in the following figure 20, the leaves of the transgenic plant seedling are larger than those of the wild plant, and the growth and development are faster. After the plant grows to 1 month old, the ratio of the plant height to the dry fresh weight is measured, and as shown in figure 21, the ZmZmSphK1 gene over-expression plant is observed to be slightly higher than the wild type plant in the measurement result of the dry fresh weight.

Determination of germination rate and greening rate under salt stress

Preparing an MS culture medium containing 200mmol/L NaCl, equally dividing the plate into four parts, cleaning the seeds of four strains, then respectively putting the seeds into corresponding culture areas one by using a gun head, arranging the seeds in order, airing the seeds in an ultra-clean workbench after sowing, sealing the seeds, placing the seeds in a constant temperature box at 22 ℃ for growing for about four days, observing germination rate, continuously placing the plate in the constant temperature box for culturing and growing for one week, observing plant greening rate, and recording.

The germination rate of the seeds observed after three days in the constant temperature chamber is shown as figure 22A, and the germination rate of the wild seeds is obviously smaller than that of the plants with the over-expressed ZmZmSphK1 genes under the salt stress treatment environment observed after three days. The greening rate of the wild type seeds obtained after five days is shown as figure 22B, which is obviously smaller than that of plants over-expressed by the ZmZmSphK1 gene. Therefore, the ZmZmSphK1 gene overexpression can be obtained to promote the germination and greening of plants. After 8 days of growth in a 22 ℃ incubator, the growth vigor of four strains is observed, as shown in the following figure 22℃, the OE1, OE2 and OE3 are stronger and stronger in blades under the environment and less affected by salt stress, while the wild plants grow slower in the salt environment, the blades are smaller and more affected by salt stress. Thus, the ZmZmSphK1 gene can play a role in salt tolerance to a certain extent.

Soil inner salt treatment ZmZmSphK1 gene over-expression Arabidopsis thaliana

When seedlings are transplanted into soil and grow to a trefoil period, two groups of experiments of NaCl solution and clear water control are set, each group of experiments is set with three repetitions, each group of plants is put into a big basin, 200ml of salt solution is irrigated each time by a salt treatment experiment group, the clear water control group irrigates equal amount of water simultaneously, irrigation is carried out twice a day, and the difference of plant growth vigor is observed after one week.

As shown in FIG. 23, the plant height and the leaf size of the ZmZmSphK1 gene over-expression plant and the wild plant on the overground part are not obviously different, the wild plant is greatly influenced by salt stress after salt treatment, the plant is short and small, the growth and development are obviously inhibited, and compared with the plant system of the ZmZmSphK1 gene over-expression, the plant system is more robust, which shows that the influence of the salt stress is less. And continuing to subject the plant to salt stress, and observing the plant to start withering after two weeks, wherein the plant under the salt stress is shown in figure 23, and the ZmZmSphK1 gene over-expressed plant is more obviously compared with a wild type, so that the expression of the ZmZmSphK1 gene can resist the salt stress to a certain extent.

Determination of biochemical indexes of ZmZmSphK1 gene over-expression Arabidopsis thaliana

Four strains of about two weeks are cultivated under the conditions of salt treatment and clear water treatment, three plants with better growth vigor are respectively taken, blades are sheared, the cleaned plants are respectively wrapped by filter paper and put into liquid nitrogen, after soaking for a period of time, samples are taken out by tweezers, poured into a mortar and ground into powder, and the powder is put into a centrifuge tube and transferred into an ultralow temperature refrigerator at the temperature of minus 80 ℃ for storage for standby.

Peroxidase (POD) Activity assay

Peroxidases are prevalent in animal plants, microorganisms, and the like, and hydrogen peroxide oxidizes a specific substrate under the catalysis of the peroxidase. This material has a maximum light absorption at 470nm, so that the peroxidase activity can be determined at an absorbance value of 470 nm. The specific operation steps are as follows:

preparing reagents in the kit: adding 5ml of the second reagent into the first reagent for full dissolution, and preserving the incomplete reagent at 4 ℃; adding 1.5ml of the second reagent into the third reagent for full dissolution, and preserving at the temperature of 4 ℃ which is not used up; the other two reagents are in common use.

About 0.1g of tissue in each sample was weighed into two ml centrifuge tubes, 1ml of each extract was added, and after shaking and shaking, ice bath homogenization was performed. Centrifuging at 12000rpm for 10 min at 4deg.C, sucking supernatant to a new centrifuge tube, and placing on ice for testing without sucking precipitate.

The microplate reader is turned on to preheat for more than 30min, and the measurement wavelength is 4700nm. Reagents were added to the well plate as a sample 10 reagent one 40 reagent two 140 reagent three 10 system. And (3) calculating: immediately after reagent addition, absorbance at 470nm, A2 was read once after waiting for one minute to give Δa=a2-A1, pod=100×Δa/W (W is sample mass)

As shown in FIG. 24, the measurement of peroxidase in plants revealed that POD values of wild-type plants and plants overexpressed by ZmZmSphK1 gene increased generally after salt stress treatment, but the most increase in the wild-type POD value indicated that the wild-type plants were most damaged, and thus it was also demonstrated that overexpression of ZmZmSphK1 gene reduced damage to plants due to salt stress.

The above description of the specific embodiments of the present invention has been given by way of example only, and the present invention is not limited to the above described specific embodiments. Any equivalent modifications and substitutions for the present invention will occur to those skilled in the art, and are also within the scope of the present invention. Accordingly, equivalent changes and modifications are intended to be included within the scope of the present invention without departing from the spirit and scope thereof.

Claims (10)

1. A method for cloning corn gene ZmSphK1 is characterized in that: the method comprises the following steps:

(1) Extracting total RNA of corn leaves, and reversely transcribing the total RNA into cDNA:

(2) Designing a specific primer by taking cDNA as a template, and amplifying a coding region sequence of a gene ZmSphK1 by PCR, wherein the primers used for PCR amplification are as follows:

Zm-ZmSphK1-F：5’-CATTGAAGCTTCTGGCGTGC-3’；

Zm-ZmSphK1-R：5’-GGGCTAACGAAGCAAAGTCC-3’

(3) Performing PCR amplification by using the cDNA reverse transcribed in the step 2 as a template, wherein a PCR amplification system is as follows: 2 XGC Buffer II 25. Mu.l, dNTP 8. Mu.l, zm-ZmSphK1-F1 1. Mu.l, zm-ZmSphK1-R1 1. Mu.l, cDNA template 2. Mu.l, la Taq enzyme 0.5. Mu.l, ddH ₂ O12.5. Mu.l; and (3) after the PCR is finished, carrying out agarose gel electrophoresis, and recovering 1100bp bands to obtain the corn sphingosine kinase ZmSphK1 gene.

2. The method for cloning of maize sphingosine kinase zmssphk 1 according to claim 1, wherein the recovered 1100bp band-linked pMD19-T intermediate vector is transformed into competent cells of escherichia coli and kept ready for use.

3. The cloning method according to claim 1, wherein the maize sphingosine kinase ZmSphK1 is obtained by cloning and expressed in the prokaryotic expression vector pET28a and the eukaryotic expression vector pCambia 1300.

4. The prokaryotic expression vector pET28a-ZmSphK1 of the corn sphingosine kinase ZmSphK1 according to claim 3, which comprises the following operation steps:

(1) Constructing a pET28a-ZmSphK1 recombinant vector;

(2) Ligation product transformation competent E.coli DH 5. Alpha;

(3) Extracting plasmid and then converting competent escherichia coli BL21;

(4) Performing bacterial liquid PCR detection;

(5) Detecting a recombinant vector;

(6) Salt resistance analysis of recombinant E.coli BL21 (pET 28a-ZmSphK 1).

5. The method for expressing corn sphingosine kinase ZmSphK1 in eukaryotic expression vector according to claim 3, wherein the method comprises the following steps:

(1) Constructing a pCambia1300-ZmZmSphK1 recombinant vector;

(2) Ligation product transformation competent E.coli DH 5. Alpha;

(3) Performing bacterial liquid PCR detection;

(4) Detecting a recombinant vector;

(5) Transforming agrobacterium GV3101 by freeze thawing;

(6) The recombinant plasmid-transformed bacteria transformed plant bodies and ZmSphK1 was expressed in Arabidopsis thaliana. After three generations of selfing, plants containing ZmSphK1 gene are selected.

6. The method for cloning and expressing a maize sphingosine kinase ZmSphK1 according to claim 1, wherein the sequence of the ZmSphK1 gene is 1044bp, the coding region is 1029bp, and 342 amino acids are totally coded.

7. The method for cloning and expressing a maize sphingosine kinase ZmSphK1 according to claim 1, wherein the molecular weight of the protein encoded by the ZmSphK1 gene is predicted to be 37192.41kDa by ProtParam at http:// web. Expasy. Org/protParam/website, and the theoretical isoelectric point is 5.58; the total number of negatively charged residues (Asp+Glu) is 42 and the total number of positively charged residues (Arg+Lys) is 35; the instability coefficient was 38.25, which was shown to be relatively stable.

8. The gene clone of zein kinase zmssphk 1 according to claim 1, wherein the alignment of protein sequences using Blast and MEGA5.0 software was performed on the NCBI website, and the results of genetic evolution analysis were shown in FIG. 1, indicating that our cloned zmssphk 1 was identical to the sequence queried in NCBI.

9. A method for constructing an Arabidopsis plant expression vector, which is characterized in that the corn ammonia alcohol kinase ZmSphK1 is inserted into a pCambia1300 vector to enable the gene to be effectively expressed in the vector.

10. The expression vector pCambia1300-ZmZmSphK1 of claim 9 having salt tolerance.