CN108795927A - The clone of common wheat gene TaSPX3 coded sequences and its application - Google Patents

Abstract

The invention belongs to the technical field of gene cloning, and in particular relates to the cloning and application of a common wheat gene TaSPX3. The present invention firstly obtains the clone of wheat gene TaSPX3, utilizes the full-length cDNA sequence of the TaSPX3 gene obtained by cloning and the pS1300-GFP vector with GFP tag after transformation, constructs TaSPX3 overexpression recombinant vector T-P, and then uses Agrobacterium-mediated The transgenic Arabidopsis thaliana was transformed by the tidbit infection method, and the positive mutant plants obtained by the detection were molecularly identified and self-homozygous through generations, and the homozygous transgenic plants of the T3 generation were obtained, and TaSPX3 Arabidopsis overexpression lines were constructed, which confirmed the The expression of TaSPX3 gene can improve the low phosphorus tolerance of Arabidopsis plants, and lay the foundation for the application of TaSPX3 gene in the genetic improvement of crop phosphorus efficiency.

Description

Cloning and Application of Common Wheat Gene TaSPX3 Coding Sequence

technical field

The invention belongs to the technical field of gene cloning, and in particular relates to the cloning and application of a common wheat gene TaSPX3 coding sequence.

Background technique

Phosphorus is an important macroelement in the process of plant growth and development, but because phosphorus is easy to form insoluble precipitates in the soil, it is difficult to be absorbed by crops, thereby reducing the bioavailability of phosphorus, and the available phosphorus in the soil is much lower than Phosphorus content required by crops. As wheat is one of the main food crops in my country, the lack of available phosphorus in the soil seriously restricts the increase of wheat yield, so finding the low phosphorus tolerance gene in wheat has become an effective method to solve this problem. A large number of studies have proved that the SPX domain is closely related to phosphorus signaling, and may play an important role in the mechanism of wheat's low phosphorus tolerance. In the early stage, the inventors used Affymatrix gene chip technology to screen out the gene TaSPX3, which was strongly induced and up-regulated by phosphorus stress under hydroponic conditions. This gene belongs to the plant SPX family closely related to the regulation of phosphorus homeostasis, and it is very likely to play a role in the mechanism of wheat tolerance to low phosphorus. important role. Therefore, studying the gene function of wheat TaSPX3 and improving the phosphorus utilization rate of crops can better lay the foundation for molecular breeding.

A large number of studies have proved that the SPX domain is closely related to phosphorus signaling, and may play an important role in the mechanism of wheat's low phosphorus tolerance. Therefore, studying the gene function of wheat TaSPX3 and improving the phosphorus utilization rate of crops can better lay the foundation for molecular breeding. The wheat TaSPX gene has dual regulatory roles, not only involved in phosphorus regulation, but also involved in the process of wheat disease resistance, which provides new resistance gene resources for the study of wheat disease resistance and immune regulation.

However, due to the complex hexaploid genome of common wheat, the functional analysis and application of phosphorus-regulated genes including the SPX gene are very difficult, and the prior art lacks the cloning and application of a wheat gene TaSPX3 coding sequence.

Contents of the invention

The invention provides the cloning and application of a common wheat gene TaSPX3 coding sequence, obtains the cloning of the wheat gene TaSPX3 coding sequence, and provides the application of the cloning of the wheat gene TaSPX3 coding sequence in the study of plant low phosphorus tolerance mechanism and genetic improvement .

The first object of the present invention is to provide a kind of cloning of common wheat gene TaSPX3 coding sequence, the cloning of described wheat gene TaSPX3 coding sequence obtains according to the following method:

(1) Extracting wheat total RNA and synthesizing wheat cDNA;

(2) Using wheat cDNA as a template PCR amplification gene TaSPX3 full-length cDNA, wherein the primer sequences used for PCR amplification are as follows:

TaSPX3 upstream primers:

5'-GCTCTAGAATGAAGTTTGGGAAGAGGCTCAAG-3';

TaSPX3 downstream primers:

5'-GGGTCGACTCAAACCACCCGGACAATACC-3';

(3) Recover and purify the target fragment in the amplified product of (2) and sequence and identify it, and the sequence that is correctly sequenced is the clone of the coding sequence of the wheat gene TaSPX3.

The second object of the present invention is to provide an application of the cloning of the coding sequence of the wheat gene TaSPX3 in the study of the mechanism of plant tolerance to low phosphorus and genetic improvement.

Preferably, according to the application of the cloning of the coding sequence of the wheat gene TaSPX3 in the study of the mechanism of plant tolerance to low phosphorus and genetic improvement, an improved recombinant plant expression vector is first constructed, and the recombinant plant expression vector is connected with the coding sequence of the wheat gene TaSPX3 and GFP gene, and then transform the recombinant expression vector into plants to obtain transformed plants, and use the transformed plants to study the application of wheat gene TaSPX3 in the mechanism of plant tolerance to low phosphorus and genetic improvement.

Preferably, according to the application of the cloning of the wheat gene TaSPX3 coding sequence in the study of plant tolerance to low phosphorus mechanism and genetic improvement, the specific steps are as follows:

S1, construction of T-TaSPX3 recombinant plasmid

Cloning and connecting the coding sequence of the wheat gene TaSPX3 -T vector: add an A tail to the clone of the purified wheat gene TaSPX3 coding sequence to obtain the target gene with the A tail; then combine the target gene with the A tail with -T vector connection to obtain the recombinant plasmid T-TaSPX3;

S2, construction of improved recombinant plant expression vector T-P

The recombinant plasmid T-TaSPX3 and p1300-GFP vector were double-digested with XbaI and SalI, respectively, to obtain the double-digested product of recombinant plasmid T-TaSPX3 and the double-digested product of p1300-GFP vector; -TaSPX3 double digestion product and p1300-GFP vector double digestion product were ligated to obtain an improved recombinant plant expression vector T-P;

S3, Agrobacterium infection of plants

Plant wild-type plants; prepare Agrobacterium competent cells; then use recombinant expression vector T-P to transform Agrobacterium competent cells; pick positive clones of transformed Agrobacterium to infect plants; obtain a transgene that can effectively clone the coding sequence of the wheat gene TaSPX3 plant body.

Preferably, according to the above application, the Agrobacterium is Agrobacterium GV3101.

Preferably, according to the above application, the plant body is Arabidopsis thaliana or wheat.

Preferably, according to the above-mentioned application, after the transformed Agrobacterium infecting the plant body with the positive clone picked in S3, after 3 generations of selfing, the plant body containing the coding sequence of the wheat gene TaSPX3 is screened out, which is used to study the presence of the coding sequence of the wheat gene TaSPX3. Plant tolerance to low phosphorus mechanism and application in genetic improvement.

Compared with the prior art, the cloning and application of the common wheat gene TaSPX3 coding sequence of the present invention have the following beneficial effects:

The present invention obtains the cloning of the coding sequence of the common wheat gene TaSPX3, utilizes the full-length cDNA sequence of the coding sequence of the wheat gene TaSPX3 obtained by cloning and the transformed pS1300-GFP vector with the GFP tag, constructs the recombinant vector T-P for overexpression of TaSPX3, and then uses Agrobacterium was used as a medium to transform Arabidopsis thaliana through the tidbit infection method, and the positive mutant plants obtained by the detection were molecularly identified and self-homozygous through generation by generation, and the homozygous transgenic plants of the T3 generation (plants after 3 generations of self-cross) were obtained , to construct a TaSPX3 Arabidopsis overexpression line.

Common wheat has a complex hexaploid genome, and the functional analysis of phosphorus-regulated genes including SPX genes is very difficult, while Arabidopsis plants have a small genome and are easy to transform. Therefore, the present invention firstly predicts the SPX gene on the basis of bioinformatics, then uses the plant expression vector to overexpress the wheat TaSPX3 gene in Arabidopsis thaliana, and preliminarily clarifies the function of the wheat TaSPX3 gene by studying the physiological phenotype and expression analysis. Observing the tissue localization of TaSPX3 in transgenic Arabidopsis will lay the foundation for the research on the mechanism of cloning of wheat gene TaSPX3 in plant (especially wheat) tolerance to low phosphorus and the application of genetic improvement.

Description of drawings

Fig. 1 is the electrophoresis detection figure of wheat total RNA;

Wherein all swimming lanes are wheat total RNA samples;

Fig. 2 is the full-length cDNA clone of the target wheat gene TaSPX3;

Among them, lane M is DL2000Marker; lanes 1 and 2 are full-length cDNA clones of wheat gene TaSPX3;

Fig. 3 is the positive colony identification result after transforming Escherichia coli DH5α competent cell;

Among them, lane M is DL2000Marker, and lanes 1-6 are all positive colonies;

Fig. 4 is the PCR verification electrophoresis result of recombinant plasmid T-TaSPX3;

Among them, lane M is DL2000Marker; lanes 1, 2, and 3 are recombinant plasmid T-TaSPX3;

Fig. 5 is the electrophoresis diagram of the double restriction product of recombinant plasmid T-TaSPX3 and the double restriction product of p1300-GFP vector;

Lane M is DL2000Marker; lane 1 is the product of p1300-GFP vector double digestion, and lane 2 is the product of recombinant plasmid T-TaSPX3 vector double digestion;

Fig. 6 is the result of enzyme digestion identification of recombinant expression vector T-P and the result of plasmid PCR identification;

Among them, lane M is DL2000Marker; lane 1 is the verification of single enzyme digestion of recombinant expression vector T-P; lane 2 is verification of double digestion of recombinant expression vector T-P with XbaI and SalI; lane 3 is verification of PCR of recombinant expression vector T-P; lane 5 is verification of 1 Parallel repeated experiment of swimming lanes; 6 lanes of parallel repeated experiments of 2 swimming lanes; 7 swimming lanes of parallel repeated experiments of 3 swimming lanes.

Fig. 7 is the antibiotic screening figure of transgenic plant;

Figure 8 is the molecular detection results of transgenic plants

Among them, lane M is DL2000Marker; lanes 1-2 are wild-type Arabidopsis (TaSPX3yg); lanes 3-4 are wild-type Arabidopsis (actin2); lanes 5-6 are transgenic Arabidopsis (TaSPX3yg); The lane is transgenic Arabidopsis (actin2).

Figure 9 is the growth status of transgenic Arabidopsis and wild-type Arabidopsis at the seedling stage under different phosphorus conditions;

Wherein, Fig. 9A is the result of phosphorus deficiency treatment, Fig. 9B is the result of low phosphorus treatment, and Fig. 9C is the result of high phosphorus treatment;

Figure 10 is the relative expression level of TaSPX3 gene in Arabidopsis roots in 14 days of MS;

Figure 11 is the relative expression level of TaSPX3 gene in Arabidopsis shoot in 14 days MS;

Figure 12 Growth status of transgenic Arabidopsis and wild-type Arabidopsis at adult plant stage under different phosphorus conditions;

Wherein, Fig. 12A is the plant growth result of transgenic Arabidopsis T3-7, and Fig. 12B is the plant growth result of wild-type Arabidopsis;

Fig. 13 is the relative expression level of TaSPX3 gene in Arabidopsis roots under 14-day hydroponic conditions;

Fig. 14 is the relative expression level of TaSPX3 gene in Arabidopsis shoots under hydroponic conditions for 14 days.

Detailed ways

The present invention will be described in detail below in conjunction with specific examples, but should not be construed as a limitation of the present invention. The test methods for which specific conditions are not indicated in the following examples are usually operated under conventional conditions, and the steps are not described in detail because they do not involve the invention point.

The cloning and application of a common wheat gene TaSPX3 coding sequence provided by the present invention include the following examples.

Materials and reagents used in the following examples are as follows:

Materials: Colombian wild-type Arabidopsis, pS1300-GFP vector (invitrogen), Escherichia coli DH5α (invitrogen), Agrobacterium GV3101 (invitrogen).

Reagents: KOD high-fidelity enzyme kit, Taq DNA polymerase kit (CWBIO), DNA-Marker (Takara), XbaI restriction endonuclease, SalI restriction endonuclease, DNA purification and recovery kit (GeneMark), -T vector (Promega), plasmid miniprep kit (GENERAYTM), T4 DNA ligase (invitrogen), Taq Master Mix (invitrogen), RNA purification kit (Thermo Fisher), HiScript II 1st Strand cDNA Synthesis Kit (Vazyme), agar Sugar (Promega), and other reagents such as Trizol, chloroform, isopropanol, absolute ethanol, sodium hypochlorite, MES, sucrose, agar, etc., were of domestic analytical grade.

Example 1

A kind of cloning of common wheat gene TaSPX3 coding sequence, the cloning of described common wheat gene TaSPX3 coding sequence is obtained according to the following method:

(1) Extracting wheat total RNA and synthesizing wheat cDNA;

A1, extraction of wheat total RNA: Methods Trizol method was used to extract wheat total RNA.

A2, quality detection of wheat total RNA

① Determination of concentration: Take 1 μL RNA solution and measure its concentration on NanoDrop2000, and the values of A260/A280 and A260/A230 represent its purity. After detection, the concentration of wheat total RNA was 1859ng/μL, A260/A280 was 1.93, and A260/A230 was 1.98.

②Agarose gel electrophoresis detection of RNA:

1% agarose electrophoresis to detect RNA, 125V voltage for 20min, then began to illuminate the gel, Figure 1 is the electrophoresis detection map of wheat total RNA, 28S bands can be seen, the results show that the quality of RNA extraction is good, and can be used for subsequent experiments .

A3. Removal of genomic DNA in wheat total RNA: Use RNA purification kit to remove genomic DNA in an RNase-free centrifuge tube, and add the following reagents according to Table 1.

Table 1 Reagent addition amount for removing DNA

A4, synthetic wheat cDNA

a. RNA template denaturation: Add the following reagents in Table 2 to the RNase-free centrifuge tube:

Table 2 Reagent addition amount for RNA template denaturation

substance Dosage Random hexamers (50ng/μL) 1μL Total RNA 1pg-5μg RNase free ddH2O to 8μL

Heat at 65°C for 5 minutes, quickly place on ice to quench, and let stand on ice for 2 minutes to obtain RNA denaturation solution.

b. Prepare the first-strand cDNA synthesis reaction solution:

RNA Denaturation Solution 8μL

2×RT Mix 10μL

HiScript II Enzyme Mix 2μL

c. Carry out the first-strand cDNA synthesis reaction according to the following conditions

25℃ 5min

50℃ 45min

85℃ 2min

The wheat cDNA was obtained and stored at -80°C.

(2) The full-length cDNA of TaSPX3 gene was amplified by PCR using wheat cDNA as a template.

Table 3 TaSPX3 full-length primer sequences

The full-length TaSPX3 gene was amplified by PCR using wheat cDNA as a template, and the system added to the PCR tube is shown in Table 4.

Table 4 TaSPX3 gene full-length PCR system

substance Dosage 10×PCR buffer for KOD-Plus-Neo 5μL dNTP Mix 5μL _MgSO4 3μL TaSPX3 upstream primers (see Table 3) 2μL TaSPX3 downstream primers (see Table 3) 2μL cDNA 1μL KOD-Plus-Neo 1μL ddH ₂ O To 50μL

Amplification was started according to the following procedure: denaturation at 94°C for 5 min, followed by denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, and 32 cycles. The final extension step was 5 min at 72°C and stored at 4°C. The amplified product is the full-length cDNA of the wheat gene TaSPX3, which is used as the target gene TaSPX3.

(3) recovering and purifying the target gene TaSPX3 (789bp) in the amplified product of (2), and the target gene TaSPX3 is the clone of the coding sequence of the wheat gene TaSPX3.

Take 50 μL of the PCR reaction product in S13, use DM2000 DNA-Mraker as a reference, electrophoresis on a 1% agarose gel, and scan and image the gel imaging system. The full-length cDNA clone of the target gene TaSPX3 is shown in Figure 2. The results in Figure 2 show that there is a single clear band at about 780bp in 1% agarose gel, which is similar in size to TaSPX3 and is the product of TaSPX3. used in the next purification step. The specific steps of purification were carried out according to the instructions of the DNA purification and recovery kit.

The present invention also provides an application of the cloning of the wheat gene TaSPX3 in the mechanism of plant tolerance to low phosphorus and genetic improvement. First, an improved recombinant plant expression vector is constructed, and the recombinant plant expression vector is connected with the coding sequence of the wheat gene TaSPX3. and the GFP gene, and then transform the recombinant expression vector into plants to obtain transformed plants, and use the transformed plants to study the low phosphorus tolerance mechanism of the wheat gene TaSPX3 and carry out genetic improvement. Specific steps are as follows:

S1, construction of T-TaSPX3 recombinant plasmid

Cloning and connecting the coding sequence of the wheat gene TaSPX3 -T vector: Since the gene amplified by the high-fidelity enzyme used in this example is blunt-ended, an A-tail was added to the clone of the purified wheat gene TaSPX3 coding sequence to obtain the target gene with an A-tail. Use the following method: 10 μL of the purified gene TaSPX3+1 μL of Taq Master Mix, incubate at 72°C for 30 min to obtain the target gene with an A tail.

Target gene TaSPX3 and -T carrier connection, the connection system is shown in Table 5:

Table 5TaSPX3 with -T carrier linkage system

The above system was mixed well at room temperature (preferably 25°C) for 1 hour, and then ligated overnight at 4°C to obtain the recombinant plasmid T-TaSPX3. The recombinant plasmid T-TaSPX3 is transformed into Escherichia coli DH5α competent cells for storage, and the recombinant plasmid T-TaSPX3 is extracted when used.

S11, preparation of Escherichia coli DH5α competent cells: Escherichia coli DH5α competent cells were carried out according to conventional molecular experiment methods, and stored at -80°C for future use.

S12, Transform Escherichia coli DH5α competent cells with T-TaSPX3 recombinant plasmid: Step S12 is carried out according to the conventional molecular experiment method

S13, colony PCR identification

The streaked colonies were picked for preliminary identification by colony PCR. The PCR reaction system is shown in Table 6:

Table 6 Colony PCR identification system

substance Dosage 2×Taq master Mix 10μL TaSPX3 upstream primer 1μL TaSPX3 upstream primer 1μL ddH ₂ O To 20μL

The PCR reaction program for colony PCR was: denaturation at 94°C for 5 min, followed by denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, and 32 cycles. The final extension step was 5 min at 72°C and stored at 4°C. Figure 3 is the result of positive colony identification after transformation of Escherichia coli DH5α competent cells.

S14, extraction of plasmids, plasmid PCR identification and sequencing

Pick the positive colonies of Stretchella spp. and shake them, culture them overnight in 10mL LB+10μL Amp at 37°C and 220rpm, then extract the plasmid according to the plasmid extraction instructions of the plasmid miniprep kit, and take 3μL for agarose gel electrophoresis detection. Afterwards, plasmid PCR identification was carried out, and the PCR system was as follows: 10 μL of 2×Taq master Mix, 1 μL of TaSPX3 upstream primer, 1 μL of TaSPX3 upstream primer, 1 μL of plasmid, and 20 μL of ddH ₂ O. The PCR reaction program for plasmid PCR was: denaturation at 94°C for 5 min, followed by denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, and 32 cycles. The final extension step was 5 min at 72°C, and finally stored at 4°C. At the same time, it will be sent to the company for sequencing. The PCR identification of the recombinant plasmid T-TaSPX3 is shown in Figure 4, and Figure 4 is the PCR verification electrophoresis result of the recombinant plasmid T-TaSPX3. The results showed that the product of TaSPX3 could be amplified using the recombinant plasmid T-TaSPX3 as a template, and the band was clear and single. The subsequent sequencing results also proved that the product had the same sequence as TaSPX3, so it could be used in the next experiment.

S2, construction of recombinant expression vector T-P

Select the recombinant plasmid T-TaSPX3 with correct sequencing, expand the recombinant plasmid T-TaSPX3 and p1300-GFP vectors, culture overnight at 37°C, 220rpm, and then extract the plasmids to obtain recombinant plasmids T-TaSPX3 and p1300-GFP plasmids respectively .

The recombinant plasmid T-TaSPX3 and p1300-GFP vector were double-digested with XbaI and SalI, respectively, to obtain the double-digested product of recombinant plasmid T-TaSPX3 and the double-digested product of p1300-GFP vector; -TaSPX3 double enzyme digestion product and p1300-GFP vector double enzyme digestion product were connected, and the recombinant expression vector T-P; the specific construction steps were as follows: (1) Use XbaI and SalI to double enzyme the recombinant plasmid T-TaSPX3 and p1300-GFP vector respectively Digestion, double digestion steps are as follows: a. Digest the plasmid with XbaI, and the restriction system of the recombinant plasmid T-TaSPX3 is shown in Table 7.

Table 7 Enzyme digestion system of recombinant plasmid T-TaSPX3

substance Dosage 10×Quick Cut Buffer 5μL QuickCut XbaI 1.5μL recombinant plasmid T-TaSPX3 30μL ddH ₂ O up to 50μL

Enzyme digestion conditions 37°C, 3h, purification after enzyme digestion: make up the enzyme digestion system to 100 μL, add 1/10 volume of NaAc (3M, PH-5.2), add double volume of absolute ethanol, precipitate at -20°C for 1h, Centrifuge at 12,000 rpm for 10 min at 4°C, take the supernatant to dry, and add 35 μL of ddH ₂ O to obtain the XbaI digested product.

b. Continue to digest the plasmid with SalI, the system is shown in Table 8:

Table 8 Enzyme digestion system of SalI

Enzyme digestion conditions were 37°C for 5 hours. After the enzyme digestion, electrophoresis detection and gel-tapping recovery were performed to obtain the double-digestion product of the recombinant plasmid T-TaSPX3.

The preparation process of the p1300-GFP vector double digestion product (about 10000bp) is the same as that of the recombinant plasmid T-TaSPX3 double digestion product (about 780bp), the difference is that the recombinant plasmid T-TaSPX3 is replaced by the p1300-GFP vector. The electrophoresis images of the double digestion product of the recombinant plasmid T-TaSPX3 and the double digestion product of the p1300-GFP vector are shown in FIG. 5 .

T4 DNA ligase was used to ligate the recombinant plasmid T-TaSPX3 double-digestion product recovered after double-digestion and the p1300-GFP vector double-digestion product. The ligation system is shown in Table 9:

Table 9 connection system

substance Dosage 5×T4DNA Ligase Buffer 4μL T4DNA Ligase 1μL Enzyme digestion and recovery of target fragments 12μL Enzyme digestion and recovery of GFP expression vector 3μL

Use a metal water bath to control the temperature at 16°C for connection, and the connection reaction is completed after 24 hours to obtain the recombinant expression vector T-P. The ligation product was transformed into Escherichia coli DH5α competent cells for transformation.

(2) Recombinant expression vector T-P colony PCR, plasmid PCR identification, enzyme digestion verification

The ligated product was identified by colony PCR and plasmid PCR, and the recombinant expression vector T-P was extracted from Escherichia coli DH5α competent cells for PCR, the method was the same as that of S13 and S14. At the same time, the double enzyme digestion identification was carried out. Figure 6 shows the results of the enzyme digestion identification of the recombinant expression vector T-P and the identification results of the plasmid PCR.

Then inoculate the single clone of the recombinant vector T-P into LB liquid medium containing Kan, shake the bacteria, culture overnight at 37°C and 220 rpm, extract the plasmid, and obtain the recombinant vector T-P for use.

S3, Agrobacterium infection of Arabidopsis

S31, growing wild-type Arabidopsis

Wild-type Arabidopsis was grown according to the conventional method and set aside.

S32, preparing Agrobacterium competent cells

Agrobacterium GV3101 competent cells were prepared according to conventional methods, and stored in a -80°C refrigerator for later use.

S33, transform Agrobacterium competent cells with recombinant expression vector T-P

①Take the Agrobacterium competent cells and place them on ice, add 2 μL of recombinant expression vector T-P, mix well, freeze them in liquid nitrogen, and then place them on ice for an ice bath. ② Heat shock at 42°C for 90s, add 800 μL LB liquid medium without antibiotics, and incubate at 28°C, 160rpm for 3h. ③Centrifuge at 5000r/min for 4min, absorb 700μL of supernatant, mix the remaining part, spread on LB plate containing Rif and Kan, and incubate at 28℃. ④Randomly select a single colony for streak growth and culture at 28°C. ⑤ Carry out PCR identification on the positive colonies, and pick out the transformed Agrobacterium with positive clones.

S34, using the tidbit infection method to infect Arabidopsis thaliana with the transformed Agrobacterium that has obtained positive clones

① Inoculate the transformed Agrobacterium containing positive clones into 10 mL of LB liquid culture medium containing rifampicin and kanamycin, and culture at 28°C with shaking at 180 rpm until OD600 = 0.6. ②Centrifuge at 5000rpm for 10min, discard the supernatant, and collect the bacteria. ③ Suspend the cells with 200 mL MS salt solution (1/2 MS, 5% sucrose, 200 μL/L Silwet L-77, pH 5.8). ④ Immerse the inflorescences of Arabidopsis thaliana that were watered one day in advance into the above suspension for 30 seconds, put them in a fresh-keeping bag and keep them moist in the dark for 1 day, then put them on the light culture rack for normal cultivation until the seeds are harvested. ⑤ After picking the transformed Agrobacterium of positive clones and infecting the plants, after 3 generations of selfing, the plants containing the TaSPX3 gene were screened out, as transgenic plants capable of effectively cloning the wheat TaSPX3 gene, the screening steps were as follows: a. Antibiotic screening : Harvest mature T1 generation seeds and screen transgenic plants on corresponding hygromycin-resistant MS medium; After germination, only the transgenic seeds can grow normally, and then these germinated seedlings are cultivated, and after generation-by-generation molecular identification and homozygous selfing, T3 generation homozygous transgenic plants are obtained. b. Detection of transgenic Arabidopsis at the molecular level: the transgenic plants screened above were moved to nutrient soil for culture, and after 14 days of culture, the leaves were taken to extract RNA, reverse-transcribed to synthesize cDNA, and then identified by PCR. The primers used were designed TaSPX3-specific molecular detection sequence primers (Table 10), and the successfully identified transgenic plants were harvested per plant, and then screened generation by generation. Figure 8 shows the molecular detection results of the transgenic plants.

Table 10TaSPX3-specific molecular detection sequence primers

In order to verify the feasibility of the method of the present invention, we prepared T3 generation transgenic Arabidopsis seedlings with reference to the method of Example 1, and studied the impact of phosphorus stress on the morphology of transgenic Arabidopsis thaliana seedlings, as follows:

1. Cultivation of transgenic Arabidopsis in MS medium

1.1 Cultivate transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana of the T3 generation under the conditions of phosphorus deficiency, low phosphorus and high phosphorus in MS medium, and observe and identify the function of TaSPX3 gene. First, the seeds of Arabidopsis thaliana were germinated on 1/2MS medium. See Table 11 for the recipe. After 5 days, the seedlings were transplanted and treated with phosphorus deficiency, low phosphorus (5 μM) and high phosphorus (500 μM) respectively. The recipe is shown in Table 11 and Table 12. and Table 13.

Table 11 1/2MS medium formula (1L)

substance Dosage MS powder 2.165g MES 0.43g sucrose 20g agar 10g

Table 12 Commonly used nutrient elements 100× mother liquor formula

100×Mother solution KNO ₃ 190(g/L) 100×Mother liquor NH ₄ NO ₃ 165(g/L) 100×Mother liquor KH ₂ PO ₄ 17(g/L) 100×Mother liquor K ₂ SO ₄ 10.88(g/L)

1/2MS medium formula (1L) of different phosphorus concentration of table 13

1.2 Growth status of transgenic Arabidopsis under different phosphorus conditions at seedling stage

After growing on MS medium under different phosphorus conditions for 14 days, observations were made, and the roots and shoots were taken from the roots and shoots, which were quick-frozen in liquid nitrogen and stored at -80°C. The results are shown in Figure 9, which shows the growth status of transgenic Arabidopsis and wild-type Arabidopsis at the seedling stage under different phosphorus conditions, wherein Figure 9A is the result of phosphorus deficiency treatment, and Figure 9B is the result of low phosphorus (5 μM) treatment , Figure 9C is the result of high phosphorus (500 μM) treatment, wherein, T3-2 represents transgenic Arabidopsis, wt represents wild type Arabidopsis. The growth status of the transgenic and wild-type Arabidopsis at the seedling stage under different phosphorus conditions showed that compared with the wild-type transgenic Arabidopsis under phosphorus-deficient and low-phosphorus conditions, the shoots of the transgenic Arabidopsis remained bright green, while the wild type was dark green. Green; the wild type has more lateral roots than the transgenic one, and there is little difference in length. But there was little difference between the transgenic and the wild type under high phosphorus conditions.

1.3 qRT-PCR detection of TaSPX3 gene in transgenic Arabidopsis

Transgenic Arabidopsis and wild-type Arabidopsis were cultured in MS medium for 14 days to extract root and shoot RNA, reverse transcribe it into cDNA, and then use it for fluorescence quantification. The integrity of the cDNA was detected with Arabidopsis actin2 primers, and the primer sequences are shown in Table 14. The results of agarose gel electrophoresis showed that there was a clear band around 108bp, which proved that the cDNA was complete and could be used in the next experiment.

Table 14 Primer sequences of Arabidopsis housekeeping gene actin2

Fluorescent quantitative detection of the relative expression of TaSPX3 gene. The kit used was the QuantiFast SYBR Green PCR kit from TIAGEENG Company, and qRT-PCR amplification was performed according to the instructions, and the primers used were the primer sequences in Table 10. Each mixed sample was repeated 3 times, and the relative expression of each gene was calculated, and the calculation formula was: target gene=2- ^ΔΔCt . Figure 10 is the relative expression level of TaSPX3 gene in Arabidopsis root in 14 days MS, and Figure 11 is the relative expression level of TaSPX3 gene in Arabidopsis shoot in 14 days MS, overexpressed Arabidopsis was cultured simultaneously under different phosphorus conditions and wild-type Arabidopsis thaliana, the fluorescent quantitative results of wheat TaSPX3 after 14 days showed that the expression of TaSPX3 could not be detected in the wild-type; while in the overexpression plants, the expression level of TaSPX3 was the same as that under the high-phosphorus condition Compared with the lower one, it was significantly increased, indicating that TaSPX3 may be a low phosphorus-induced gene in wheat.

2 Effects of phosphorus stress on the adult plant morphology of transgenic Arabidopsis

2.1 Hydroponics of transgenic Arabidopsis under different phosphorus conditions

Arabidopsis thaliana of generation T3 were cultured under different phosphorus conditions to observe and identify the function of TaSPX3 gene. First, the transgenic Arabidopsis thaliana was grown on MS medium under normal phosphorus conditions, until the four-leaf stage began to transplant seedlings, and hydroponics was carried out under conditions of phosphorus deficiency, low phosphorus (5 μM), and high phosphorus (500 μM). The formulation is shown in Table 15.

Table 15 Formula of nutrient solution for hydroponic Arabidopsis

2.2 Growth status of transgenic Arabidopsis under different phosphorus conditions at the adult plant stage

Observation was carried out after 14 days of hydroponic growth under different phosphorus conditions, and the roots and shoots were taken from liquid nitrogen and stored at -80°C. Figure 12 is the growth status of transgenic Arabidopsis and wild-type Arabidopsis under different phosphorus conditions at the adult stage, wherein Figure 12A is the plant growth result of transgenic Arabidopsis T3-7, and Figure 12B is the growth result of wild-type Arabidopsis Plant growth results. After 14 days of hydroponics, overexpressed Arabidopsis and wild-type Arabidopsis had a large difference in the growth process of the adult plant. Almost all of them are heading and flowering, but most of the wild types do not have this feature. Under phosphorus deficiency conditions, both transgenic and wild-type plants headed, but the growth of transgenic plants was better than that of wild-type plants, which indicated that overexpression of TaSPX3 could promote the growth of Arabidopsis under phosphorus-deficient stress, while under high-phosphorus and low-phosphorus conditions The effect of promoting biomass was not obvious, but they all promoted flowering earlier.

2.3 qRT-PCR detection of TaSPX3 gene in transgenic Arabidopsis

(1) RNA extraction from transgenic Arabidopsis and wild-type Arabidopsis 14-day roots and shoots was reverse-transcribed into cDNA, and the integrity of the cDNA was detected by the housekeeping gene actin2. Fluorescent quantitative detection of the relative expression of TaSPX3 gene. Figure 13 shows the relative expression level of TaSPX3 gene in the root of Arabidopsis thaliana after 14 days of hydroponic growth, and Figure 14 shows the relative expression level of TaSPX3 gene in the shoot of Arabidopsis thaliana after 14 days of hydroponic growth. The results of fluorescence quantification of wheat TaSPX3 in Arabidopsis and wild-type Arabidopsis after 14 days showed that the expression of TaSPX3 gene was not detected in wild-type wt, while the expression of transgenic Arabidopsis T3-2 existed under different phosphorus conditions. Certain differences, the expression level of TaSPX3 gene was higher under the condition of phosphorus deficiency and low phosphorus than that under the condition of phosphorus.

It should be noted that when the present invention gives a numerical range, it should be understood that, unless otherwise stated in the present invention, the two endpoints of each numerical range and any value between the two endpoints can be selected. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While preferred embodiments of the invention have been described, additional changes and modifications to these embodiments can be made by those skilled in the art once the basic inventive concept is appreciated. Therefore, it is intended that the appended claims be construed to cover the preferred embodiment as well as all changes and modifications which fall within the scope of the invention.

Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and equivalent technologies thereof, the present invention also intends to include these modifications and variations.

sequence listing

<110> Henan Agricultural University

<120> Cloning and application of common wheat gene TaSPX3 coding sequence

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 32

<212>DNA

<213> Artificial sequence

<400> 1

gctctagaat gaagtttggg aagaggctca ag 32

<210> 2

<211> 29

<212>DNA

<213> Artificial sequence

<400> 2

gggtcgactc aaaccacccg gacaatacc 29

<210> 3

<211> 20

<212>DNA

<213> Artificial sequence

<400> 3

gctcgtccga ctcgtctccg 20

<210> 4

<211> 21

<212>DNA

<213> Artificial sequence

<400> 4

cgccttcacc gtctccctta c 21

<210> 5

<211> 24

<212>DNA

<213> Artificial sequence

<400> 5

attcagatgc ccagaagtct tgtt 24

<210> 6

<211> 22

<212>DNA

<213> Artificial sequence

<400> 6

tttgctcata cggtcagcga ta 22

Claims (7)

1. a kind of cloning of common wheat gene TaSPX3 coding sequence is characterized in that, the cloning of described wheat gene TaSPX3 coding sequence obtains according to the following method: (1) Extracting wheat total RNA and synthesizing wheat cDNA; (2) Using wheat cDNA as a template PCR amplification gene TaSPX3 full-length cDNA, wherein the primer sequences used for PCR amplification are as follows: TaSPX3 upstream primers: 5'-GCTCTAGAATGAAGTTTGGGAAGAGGCTCAAG-3'; TaSPX3 downstream primers: 5'-GGGTCGACTCAAACCACCCGGACAATACC-3'; (3) Recovering and purifying the target fragment in the amplified product of (2) and identifying it by sequencing, and the one sequenced correctly is the clone of the coding sequence of the common wheat gene TaSPX3. 2. the application of the cloning of the wheat gene TaSPX3 coding sequence according to claim 1 in the study of plant tolerance to low phosphorus mechanism and genetic improvement. 3. the application of the cloning of the wheat gene TaSPX3 coding sequence according to claim 2 in the research plant tolerance low phosphorus mechanism and genetic improvement, it is characterized in that, construct the improved recombinant plant expression vector earlier, on the recombinant plant expression vector The clone with the coding sequence of the wheat gene TaSPX3 and the GFP gene are connected, and then the recombinant expression vector is transformed into a plant to obtain a transformed plant, and the transformed plant is used to study the application of the wheat gene TaSPX3 in plant low phosphorus tolerance mechanism and genetic improvement. 4. the application of the cloning of the wheat gene TaSPX3 coding sequence according to claim 3 in the study of plant tolerance to low phosphorus mechanism and genetic improvement, is characterized in that, concrete steps are as follows: S1, Construction of T-TaSPX3 recombinant plasmid Cloning and connecting the coding sequence of the wheat gene TaSPX3 Vector: add A tail to the clone of the purified wheat gene TaSPX3 coding sequence to obtain the target gene with A tail; then combine the target gene with A tail with The vector was ligated to obtain the recombinant plasmid T-TaSPX3; S2, construction of improved recombinant plant expression vector T-P The recombinant plasmid T-TaSPX3 and p1300-GFP vector were double-digested with XbaI and SalI, respectively, and the recombinant plasmid T-TaSPX3 double-digested product and the p1300-GFP vector double-digested product were respectively obtained; the recombinant plasmid was digested with T4 DNA ligase The T-TaSPX3 double digestion product and the p1300-GFP vector double digestion product were connected to obtain an improved recombinant plant expression vector T-P; S3, Agrobacterium infection of plants Plant wild-type plants; prepare Agrobacterium competent cells; then use recombinant expression vector T-P to transform Agrobacterium competent cells; pick positive clones of transformed Agrobacterium to infect plants; obtain a transgene that can effectively clone the coding sequence of the wheat gene TaSPX3 plant body. 5. The application according to claim 4, characterized in that the Agrobacterium is Agrobacterium GV3101. 6. The application according to claim 4, wherein the plant body is Arabidopsis thaliana or wheat. 7. application according to claim 4, it is characterized in that, after the transformed agrobacterium infection plant body that picks positive clone in S3, through 3 generations of selfing, screen out the plant body that contains wheat gene TaSPX3 coding sequence, use To study the application of the wheat gene TaSPX3 coding sequence in the mechanism of plant tolerance to low phosphorus and genetic improvement.