CN109694874B - Cloning and application of coding sequence of wheat gene TaCPSF30 - Google Patents

Abstract

The invention discloses cloning and application of a wheat gene TaCPSF30 coding sequence, belonging to the technical field of plant molecular biology and gene cloning, firstly obtaining cloning of a wheat gene TaCPSF30, constructing a TaCPSF30 overexpression recombinant vector pMDC83-TaCPSF30 by utilizing a coding region sequence (CDS) of the cloned TaCPSF30 gene and a Gateway-compatible 35S promoter-driven band GFP-labeled pMDC83 vector, then transforming an arabidopsis cpsf30 mutant by an agrobacterium-mediated floral dip method, and screening a homozygous transgenic plant to construct a heterologous complementary strain of transforming arabidopsis thaliana by TaCPSF 30.

Description

Cloning and application of coding sequence of wheat gene TaCPSF30

technical field

The invention belongs to the technical field of plant molecular biology and gene cloning, in particular to the cloning and application of a coding sequence of a wheat gene TaCPSF30.

Background technique

Wheat is an important food crop in my country, and increasing wheat yield and improving its quality has become an urgent strategic problem to be solved in my country's wheat production. Wheat is a nitrate-loving (NO ₃ ^- ) crop. Due to the low nitrogen utilization rate of crops, especially different varieties of wheat have different nitrogen utilization rates, large differences in yield are caused, which in turn affects farmers' income. The key to solving the above problems is to study the metabolic processes of NO ₃ ^- absorption, transport, assimilation and utilization in wheat to improve the nitrogen use efficiency of wheat.

A large number of studies have shown that the Arabidopsis polyadenylation factor CPSF30 plays an important role in the processing of target gene pre-mRNA (pre-mRNA), and is widely involved in gene expression regulation, oxidative stress, growth and development. We have recently discovered that recent studies have identified that Arabidopsis CPSF30 ^- L is an important gene regulating NO ₃ -metabolism. The gene can change the plant's absorption efficiency of NO ₃ ^- ; at the same time, the gene also affects the transport of NO ₃ ^- in roots and shoots. In addition, AtCPSF30 regulates the expression of NO ₃ ^- assimilation genes NIA and NiR, thereby affecting the assimilation and accumulation of NO ₃ ^- in plants. The above results indicate that AtCPSF30 gene is a key factor in the regulation ^of NO ₃ -metabolic pathways in plants.

The wheat variety Chinese Spring has a complex hexaploid genome, and it is very difficult to clone and identify genes involved in improving nitrogen use efficiency in wheat. In the previous work, the inventors discovered the homologous gene TaCPSF30 of Arabidopsis thaliana CPSF30 in Chinese spring, located in the A, B and D genomes, respectively. The results of protein secondary structure analysis showed that TaCPSF30 was highly consistent with AtCPSF30. The above results show that TaCPSF30 is very likely to regulate the metabolic processes of NO ₃ ^-absorption , transport and assimilation in wheat, which is worthy of further study.

SUMMARY OF THE INVENTION

The invention provides the cloning of the coding sequence of wheat gene TaCPSF30 and its application, obtaining the cloning of the coding sequence of the wheat gene TaCPSF30, and providing the cloning of the coding sequence of the wheat gene TaCPSF30 in the study of the mechanism of improving nitrogen utilization rate and genetic improvement of plants. application.

In order to achieve the above object, the technical scheme adopted in the present invention is:

The cloning of the coding sequence of the wheat gene TaCPSF30 of the present invention, the cloning of the coding sequence of the wheat gene TaCPSF30 is obtained according to the following method:

(1) Extracting total wheat RNA, reverse transcribing to synthesize cDNA;

(2) with cDNA as template, design specific primer, by PCR amplification gene TaCPSF30 coding region sequence, wherein, PCR amplification used primer sequence is as follows:

TaCPSF30 forward primer (F):

5'-CGGGGTACCATGGACGACGGCGACGGC-3';

TaCPSF30 reverse primer (R):

5'-CCGCTCGAGTCGCTTCCTTGACCGCCT-3';

(3) Recovering and purifying the target fragment in the amplification product of (2) and performing sequencing, the correct sequence is the clone of the coding sequence of the wheat gene TaCPSF30.

The application of the cloning of the coding sequence of the wheat gene TaCPSF30 of the present invention is the application of the cloning of the coding sequence of the wheat gene TaCPSF30 in the study of the mechanism of improving the nitrogen utilization rate of plants and the genetic improvement.

First, a recombinant plant expression vector compatible with the Gateway system was constructed. The expression vector contained the clone of the TaCPSF30 coding sequence driven by the 35S promoter and the GFP gene. Then the recombinant expression vector was transformed into the Arabidopsis cpsf30 mutant, and the pure cpsf30 mutant was obtained by screening. The combined transgenic heterologous complementary line was used to study the mechanism of the wheat gene TaCPSF30 in the complementary line in improving nitrogen use efficiency and its application in genetic improvement.

The application of the cloning of the coding sequence of the wheat gene TaCPSF30, the specific steps are as follows: S1, the construction of the pENTR3C-TaCPSF30 recombinant plasmid: the cloning of the coding sequence of the wheat gene TaCPSF30 is connected with the cloning vector pENTR3C by enzyme digestion: for the purified wheat gene TaCPSF30 The coding sequence is cloned by adding restriction sites such as KpnI and XhoI at the 5' end and the 3' end, respectively, and after double restriction digestion, the target gene fragment with sticky ends is obtained; Recombinant plasmid pENTR3C-TaCPSF30; S2, use Gateway technology to construct recombinant plant expression vector pMDC83-TaCPSF30: Perform LR reaction between the correctly sequenced recombinant plasmid pENTR3C-TaCPSF30 and Gateway system-compatible plant expression vector pMDC83 to obtain recombinant plant expression vector pMDC83-TaCPSF30 . S3, Agrobacterium-infected Arabidopsis: plant Arabidopsis cpsf30 mutants; prepare Agrobacterium-competent cells; then transform Agrobacterium-competent cells with recombinant expression vector pMDC83-TaCPSF30; pick the transformed A. Arabidopsis cpsf30 mutant; a transgenic plant capable of effectively cloning the coding sequence of the wheat gene TaCPSF30 was obtained.

The Agrobacterium is Agrobacterium GV3101.

The plant body is an Arabidopsis cpsf30 mutant or a wheat variety Chinese spring. ,

After picking the positive cloned transformed Agrobacterium in S3 to infect the Arabidopsis cpsf30 mutant, after three generations of selfing, the heterologous complementary line containing the coding sequence of wheat gene TaCPSF30 was screened out, which was used to study the coding sequence of wheat gene TaCPSF30 in To study the mechanism of improving nitrogen use efficiency in plants and the application in genetic improvement.

The beneficial effects of the present invention are as follows: the present invention obtains the clone of the wheat gene TaCPSF30 coding sequence, and uses the cloned wheat gene TaCPSF30 coding sequence and the Gateway system compatible 35S-driven plant expression vector pMDC83 with GFP tag to construct TaCPSF30 overexpression recombinant The vector pMDC83-TaCPSF30 was then used to transform the Arabidopsis cpsf30 mutant through Agrobacterium tumefaciens by dipping the flowers, and the homozygous transgenic heterologous complementary lines were screened to study the mechanism of the wheat gene TaCPSF30 in the complementary lines in improving nitrogen utilization. and applications in genetic improvement.

The wheat variety Chinese Spring has a complex hexaploid genome, and it is very difficult to clone and identify the genes involved in improving nitrogen use efficiency in wheat, while the Arabidopsis plant has a small genome and is easy to transform. Therefore, the present invention passes

Bioinformatics software predicts the TaCPSF30 gene, and then uses the plant expression vector to express the wheat TaCPSF30 gene in the Arabidopsis cpsf30 mutant, and identifies the function of the wheat TaCPSF30 gene by studying the physiological and biochemical phenotypes of the heterologous complementary lines. The tissue localization in transgenic Arabidopsis lays the foundation for the cloning of the wheat gene TaCPSF30 in the study of improving nitrogen use efficiency in crops and its application in genetic improvement.

Description of drawings

Fig. 1 is the clone of the coding sequence of the target wheat gene TaCPSF30, wherein the M swimming lane is the DL2000Marker; the 1-4 swimming lanes are all the coding region sequence clones of the wheat gene TaCPSF30;

Fig. 2 constructs the recombinant plant expression vector process of TaCPSF30 overexpression;

Figure 3 shows the identification results of positive colonies after the recombinant cloning vector pENTR3C-TaCPSF30 was transformed into E. coli DH5α competent cells; wherein M lane is DL2000Marker, lanes 1-4 are positive colonies, and lane 5 is a negative control;

Figure 4 shows the identification results of positive colonies after the recombinant expression vector pMDC83-TaCPSF30 was transformed into E. coli DH5α competent cells; wherein M lane is DL2000Marker, lanes 1-4 are positive colonies, and lane 5 is a negative control;

Figure 5 shows the identification results of positive colonies after the recombinant expression vector pMDC8383-TaCPSF30 was transformed into Agrobacterium GV3101 competent cells; wherein M lane is DL2000Marker, lanes 1-4 are positive colonies, and lane 5 is a negative control;

Fig. 6 is the detection result of TaCPSF30 expression in transgenic heterologous complementary plants;

Figure 7 Growth of transgenic heterologous complementary plants on 1/2MS solid medium at seedling stage, where A is Arabidopsis wild type; B is Arabidopsis cpsf30 mutant; C is TaCPSF30 transfected Arabidopsis cpsf30 mutant heterozygous Source complementation strain 1; D is TaCPSF30 transfected Arabidopsis cpsf30 mutant heterologous complementation strain 2;

Figure 8. Detection results of nitrate nitrogen assimilation genes and transport genes in heterologous complementation plants of Arabidopsis thaliana cpsf30 mutants transfected with TaCPSF30.

Detailed ways

The present invention will be described in detail below in conjunction with specific embodiments, but should not be construed as a limitation of the present invention. In the following examples, the test methods without specific conditions are usually operated under normal conditions, and the steps are not described in detail because they do not involve the invention.

The cloning and application of a common wheat gene TaCPSF30 coding sequence provided by the present invention include the following examples.

The materials and reagents used in the following examples are as follows:

Materials: Arabidopsis Col-0 wild type, Arabidopsis CPSF30 mutant cpsf30, Gateway system cloning vector pENTR3C and plant expression vector pMDC83 (invitrogen), Escherichia coli DH5α (invitrogen), Agrobacterium GV3101 (invitrogen).

Reagents: Phusion high-fidelity enzyme kit, Taq DNA polymerase kit (CWBIO), DNA-Marker (CWBIO), KpnI restriction endonuclease XhoI restriction endonuclease, DNA purification and recovery kit (Tiangen), plasmid mini Volume preparation kit (CWBIO), T4 DNA ligase (invitrogen), LR clonase (invitrogen), RNA purification kit (CWBIO), First Strand cDNA Synthesis Kit (Thermo Fisher), agarose (Promega), other reagents such as Trizol , chloroform, isopropanol, anhydrous ethanol, sodium hypochlorite, MES, sucrose, agar, etc., are all domestic analytical grades.

Example 1

A kind of clone of wheat gene TaCPSF30 coding sequence, the clone of described common wheat gene TaCPSF30 coding sequence is obtained according to the following method:

1. Extraction of total RNA from Arabidopsis (kit method)

Use the kit method (column total RNA extraction kit) to extract the total RNA of the corresponding Arabidopsis plants, the specific method is as follows:

(1) Weigh about 100mg Chinese spring wheat leaves in a 1.5mL Eppendorf tube, and fully grind them in liquid nitrogen;

(2) Add 1 mL of Buffer RLT (the volume of the material is less than or equal to 10% of the volume of Buffer RLT), fully shake and mix, and let it stand for 5 minutes at room temperature;

(3) In order to fully remove cell wall residues, proteins, fats, polysaccharides, etc., centrifuge at 4°C and 12000rpm for 10min, and transfer the supernatant to a new centrifuge tube;

(4) Phase separation:

①Add 0.2mL of chloroform, shake vigorously for 15s, and let stand for 2min at room temperature;

② Centrifugation (4℃, 12000rpm, no more than 12000rpm) for 10min;

(5) Precipitation and removal of polysaccharides:

① Take the colorless aqueous phase (about 50% of the original Buffer RLT volume, about 500 μL) into a new Eppendorf tube, do not suck the middle layer;

②Add 0.25mL of 70% ethanol, invert and mix;

(6) Add the solution obtained in the previous step to the collection tube that has been loaded into the adsorption column (if the liquid volume exceeds 700 μL, transfer it in two times), centrifuge at 4°C and 12000 rpm for 20 s, pour out the waste liquid in the collection tube, and put the Put the adsorption column back into the collection tube;

(7) Add 700 μL of Buffer RW1 to the adsorption column, centrifuge at 12000rpm for 20s at 4°C, pour off the waste liquid in the collection tube, and put the adsorption column back into the collection tube;

(8) Add 500 μL of Buffer RW2 to the adsorption column, centrifuge at 12000rpm for 20s at 4°C, pour off the waste liquid in the collection tube, and put the adsorption column back into the collection tube;

(9) Repeat step (8)

(10) 4°C, 12000rpm for 2min;

(11) place the empty adsorption column at room temperature for several minutes to dry thoroughly;

(12) Put the adsorption column in a new RNase-free centrifuge tube, add 30 μL of RNase-free water, place at room temperature for 1 min, centrifuge at 12000 rpm for 1 min at 4°C, collect the RNA solution, and store at -70°C.

2. Reverse transcription

Take 1 μg of total RNA, add 0.5 μL of Oligd(T), and supplement RNase-free water to a total volume of 6 μL.

Place in a 65°C water bath for 5 minutes, and then prepare the following reverse transcription reaction solution in the tube after a little centrifugation.

After incubating at 42 °C for 1 h, treat at 70 °C for 15 min, and then cool on ice.

3. PCR amplification using Phusion high-fidelity DNA polymerase

(1) Take a 0.2mL PCR tube and add the following components in sequence:

Forward primer (F): 5'-CGGGGTACCATGGACGACGGCGACGGC-3';

Reverse primer (R): 5'-CCGCTCGAG TCGCTTCCTTGACCGCCT-3';

(2) Close the PCR tube and put it into the PCR machine. The reaction conditions were as follows: pre-denaturation at 98 °C for 2 min, denaturation at 98 °C for 20 s, annealing for 20 s according to the annealing temperature of the primer, extension at 72 °C for 30 s/Kb, 30 cycles, and extension at 72 °C for 10 min.

(3) After the reaction is completed, electrophoresis in a 1% agarose gel to check the amplification effect. The detection result of PCR product agarose gel electrophoresis is shown in Figure 1, and a clear target band can be observed at 1953bp.

The invention also provides the application of the clone of the coding sequence of the wheat gene TaCPSF30 in the study of the mechanism of improving the nitrogen utilization rate of plants and the genetic improvement. First, a recombinant plant expression vector was constructed using the Gateway technology. The recombinant plant expression vector had a clone of the wheat gene TaCPSF30 coding sequence and a GFP gene connected with the 35S promoter on the recombinant plant expression vector (Fig. 2), and then the recombinant expression vector was transformed into Arabidopsis thaliana. In the cpsf30 mutant, a heterologous complementary transgenic plant was obtained, and the transgenic plant was used to study the mechanism of improving nitrogen use efficiency of wheat gene TaCPSF30 and its application in genetic improvement. Specific steps are as follows:

Construction of cloning vector pENTR 3C-TaCPSF30

1. PCR amplification of TaCPSF30 gene CDS and ligation with pENTR 3C

Forward primer (F): 5'-CGGGGTACCATGGACGACGGCGACGGC-3';

Reverse primer (R): 5'-CCGCTCGAG TCGCTTCCTTGACCGCCT-3';

Using the extracted cDNA as a template, PCR amplification was performed with specific primers F and R, and Kpn I and Xho I restriction sites were introduced upstream and downstream of the target gene, respectively. The PCR product was recovered with Tiangen (Beijing) gel recovery kit, and the PCR product was digested and ligated with Gateway-compatible pENTR 3C. The operation steps were carried out in the following table.

Element Dosage Target fragment recovery product 0.5-10ng pENT3C 0.5μL Salt solution 0.5μL H₂O Up to 3μL

After mixing, the reaction was performed at room temperature (25°C) for 30 minutes, followed by an ice bath for 1 minute to transform Escherichia coli DH5α. The surface was coated with an LB culture plate containing kanamycin, and cultured at 37°C for 12 hours. Then, a single white clone was picked and spread on a new LB culture plate containing kanamycin, and cultured at 37° C. for 12 hours for colony PCR amplification, as shown in FIG. 3 . At the same time, the correct strains were sent to Shanghai Sangon Company for DNA sequencing. DNAMAN software aligns the sequenced sequence with the predicted TaCPSF30 gene coding region sequence. After the alignment is correct, it indicates that the cloning vector pENTR 3C-TaCPSF30 has been successfully constructed, and the positive plasmid is extracted for use. Plasmid extraction was carried out using Beijing Kangwei company kit, according to the instructions.

(1) Take 1-5 mL of overnight cultured bacterial solution, add it to a centrifuge tube, centrifuge at 12,000 rpm for 1 min, and aspirate the supernatant as much as possible.

(2) Add 250 μL of Buffer P1 to the centrifuge tube with the bacterial pellet left, and use a pipette or a vortex shaker to thoroughly suspend the bacterial pellet. Note: If the bacterial block is not thoroughly mixed, the lysis effect will be affected, resulting in lower extraction yield and purity.

(3) Add 250 μL of Buffer P2 to the centrifuge tube, and gently invert and mix for 4-6 times to fully lyse the cells. At this time, the bacterial liquid should become clear and viscous. The time used should not exceed 5min, so as not to damage the plasmid.

(4) Add 350 μL of Buffer N3 to the centrifuge tube, and then gently invert and mix for 4-6 times. At this time, a white flocculent precipitate will appear. Centrifuge at 12,000 rpm for 10 minutes. At this time, a precipitate forms at the bottom of the centrifuge tube.

(5) Column equilibration: add 200 μL of Buffer PS to the collection tube loaded with the Spin Column, centrifuge at 12,000 rpm for 2 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

(6) Add the supernatant obtained in step 4 to the collection tube that has been loaded into the adsorption column, be careful not to suck out the precipitate, centrifuge at 12,000 rpm for 1 min, pour off the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

(7) Add 600 μL of Buffer PW to the collection tube, centrifuge at 12,000 rpm for 1 min, and discard the waste liquid in the collection tube.

(8) Repeat step 7.

(9) Put the adsorption column back into the collection tube, centrifuge at 12,000 rpm for 1 min, and pour out the waste liquid in the collection tube. Allow the cartridge to dry at room temperature for several minutes.

(10) Place the adsorption column in a new centrifuge tube, add 50-100 μL of Buffer EB dropwise to the middle of the adsorption membrane, leave at room temperature for several minutes, centrifuge at 12,000 rpm for 1 min, and collect the plasmid solution into the centrifuge tube. Store plasmids at -20°C.

2. Construction of plant expression vector pMDC83-TaCPSF30

The correctly sequenced cloning vector pENTR 3C-TaCPSF30 was subjected to LR reaction with the 35S promoter and the Gateway system-compatible plant expression vector pMDC83 to obtain a plant expression vector with the 35S promoter-driven TaCPSF30 gene fused with the GFP reporter gene. The LR reaction system is as follows: cloning vector 1 μL (about 100 ng) vector, pMDC83 vector 1 μL (about 100 ng), LR clonase 0.5 μL. The above substances were mixed well and then placed at room temperature (25°C) for 1-2 hours to transform E. coli DH5α. The transformed strains were coated on LB plates containing kanamycin and cultured at 37°C for 12 hours. Then, pick a single white clone and spread it on a new LB culture plate containing kanamycin, cultivate at 37° C. for 12 hours, and perform colony PCR amplification, as shown in FIG. 4 . The expression vector pMDC 83-TaCPSF30 was successfully constructed, and the positive plasmid was extracted for use. The extraction method was as described above.

Construction of heterologous complementation lines of Arabidopsis thaliana cpsf30 mutants complemented by wheat gene TaCPSF30

1. Preparation and transformation of Agrobacterium tumefaciens GV3101 competent cells

The preparation method of Agrobacterium competent cells is as follows:

(1) Pick a single colony of GV3101, inoculate it in 5 mL of YEP liquid medium, and cultivate overnight at 28°C and 200 rpm.

(2) Take 2 mL of culture into 50 mL of liquid YEP, and continue to culture until the OD600 is about 0.5.

(3) The culture was ice-bathed for 30 min, 4° C., 5000 rpm, and centrifuged for 5 min.

(4) Discard the supernatant, suspend the bacterial cells in 10 mL of 0.1 mol/L cold NaCl; centrifuge for 5 min at 4°C, 5000 rpm.

(5) Discard the supernatant, suspend the precipitate with 1 mL of 20 mmol/L cold CaCl2, aliquot into 200 μL/tube, freeze in liquid nitrogen and store at -70°C.

The expression vector transforms Agrobacterium by freeze-thaw method, and the transformation process is as follows:

(1) Take out GV3101 competent cells (250 μL) from -80°C, thaw on ice, and immediately add 1-2 μL of plasmid DNA.

(2) Stand on ice for 5 min.

(3) Freeze in liquid nitrogen for 5 min.

(4) Heat shock in a water bath at 37°C for 5 minutes.

(5) Add 1 mL of YEP liquid medium without antibiotics, and incubate at 28°C for 2-4h on a shaker.

(6) Centrifuge at 4000 rpm for 5 min at room temperature, collect the cells, and resuspend the cells with 80-100 μL of YEP solution.

(7) The bacterial liquid was evenly spread on YEP solid selective medium, and cultured at 28°C for 2-3 days.

(8) After the colonies were grown, a single colony was picked and re-streaked on a YEP solid plate containing antibiotics, and colony PCR identification was performed 36 hours later. The results are shown in Figure 5 .

2. Transformation of Arabidopsis

The genetic transformation of Arabidopsis thaliana adopts the inflorescence dip method as follows:

(1) Cut off the main inflorescence of Arabidopsis thaliana to induce the formation of side inflorescences, and the side inflorescences bloom at the same time, which is convenient for transformation. Before transformation, the material should be drenched with nutrient solution, and after transformation, watering or nutrient solution should be controlled so that the seeds can mature in time.

(2) Prepare the Agrobacterium required for transformation one day in advance, and add 2-5 mL of the overnight cultured bacterial solution to 250 mL of medium for shaking culture, so that it can be used to transform Arabidopsis the next day.

(3) Collect overnight cultured Agrobacterium cells, and resuspend in 50-100 mL of dip solution.

(4) After cutting off all the elongated seed pods and opened flowers of Arabidopsis thaliana, immerse the twigs in the dip solution, and shake the twigs up and down to facilitate the entry of the dip solution. The impregnation time generally lasts about 1h. The immersed Arabidopsis thaliana was placed in a large pot, covered with a film to keep moisture, and covered with a black film to protect from light. After dark treatment, Arabidopsis was taken out and placed under light, and seeds were harvested after maturity.

The recipe for Arabidopsis dip is as follows:

The pH was adjusted to 5.7 with 1M KOH.

3. Screening of transgenic lines

The infected Arabidopsis T0 generation seeds were collected and disinfected as follows:

(1) Take an appropriate amount of seeds in a 1.5mL Eppendorf tube, and indicate the relevant information;

(2) Prepare 70% ethanol in advance, use a pipette to take 1 mL of ethanol into the tube, suck and mix evenly, so that the seeds can fully contact the ethanol solution, and let stand for 5 minutes (note that the seeds on the tube wall and the tube cover are all Rinse down to ensure complete sterilization), and then use a 1mL pipette to aspirate the ethanol in the centrifuge tube (be careful not to aspirate the seeds);

(3) Rinse with Tween water;

(4) Take 1 mL of 2.6% sodium hypochlorite solution into the tube with a pipette, invert up and down to mix, and let stand for 10 minutes;

(5) Rinse five times with Tween water;

(6) Store at 4°C for later use.

Screening of transgenic plants

The sterilized T0 generation seeds were sown on MS solid medium containing 50 μg/mL hygromycin, and grown in a special plant culture room for 7 days under the culture conditions of 22°C and long day light (16h light/8h dark). In the T1 generation, the plants that can germinate and grow on the hygromycin MS plate were selected, and then transferred to vermiculite for culture, and the culture conditions were the same as above.

The T1 generation seeds were collected, sterilized according to the above method, sown on MS solid medium containing 50 μg/mL hygromycin, grown for 7 days in a special plant culture room, and the culture conditions were 22°C, long day (16h light/8h dark) . In the T2 generation, the plants that can germinate and grow on the hygromycin MS plate were selected, and then transferred to vermiculite for culture, and the culture conditions were the same as above.

The seeds of the T2 generation were collected, sterilized according to the above method, sown on MS solid medium containing 50 μg/mL hygromycin, and grown for 7 days in a special cultivation room for plants. . Plants that can germinate and grow on hygromycin MS plates without segregation were selected in the T3 generation to obtain heterocomplementary lines TaCPSF30/cpsf30-1 and TaCPSF30/cpsf30-2.

In order to verify the feasibility of the method of the present invention, we prepared a heterologous complementary line of the homozygous wheat gene TaCPSF30 complementary to the Arabidopsis cpsf30 mutant with reference to the method of Example 1, and studied the cloning of the wheat gene TaCPSF30 in crops to increase nitrogen The research on the utilization rate of nutrient and the application of genetic improvement are as follows:

1. Determination of TaCPSF30 expression in heterologous complementary lines

(1) Cultivation of heterologous complementary strains in 1/2MS medium

In order to detect the expression level of TaCPSF30 in the heterocomplementation lines, the seeds of the heterocomplementation lines TaCPSF30/cpsf30-1 and TaCPSF30/cpsf30-2 were first sterilized and then transferred to solid 1/2 liquid medium in a plant light incubator Cultured at 22°C and long-day (16h light/8h dark) for 7 days.

(2) Determination of the expression level of TaCPSF30

Real-time quantitative PCR (qRT-PCR) was used to detect the expression of TaCPSF30 in the heterocomplementary strains TaCPSF30/cpsf30-1 and TaCPSF30/cpsf30-2.

The cDNA samples obtained after reverse transcription of the RNA extracted in TaCPSF30/cpsf30-1 and TaCPSF30/cpsf30-2 were diluted to 0.5-2ng/μL, and the reaction system was shown in the following table:

qRT-PCR forward primer: 5'-GACTACGGCGAGTGCCGCGAG-3';

qRT-PCR reverse primer: 5'-AAGCCTGAACCAGGTGG-3'

The PCR tube was closed and placed in a real-time PCR instrument (ABI7500FAST). The reaction conditions were as follows: pre-denaturation at 50 °C for 20 s, then pre-denaturation at 95 °C for 10 s, then denaturation at 95 °C for 15 s, annealing at 60 °C for 1 min, followed by plate read, cycle 40 times from step 3, and the dissolution curve was 95 °C for 15 s, 60 ℃1min, 95℃30s, 60℃15s.

The reaction used tublin2 as an internal reference gene to ensure the accuracy of the results. Each treatment had three biological replicates and three systematic replicates. The expression levels of TaCPSF30 in the heterologous complementation lines TaCPSF30/cpsf30-1 and TaCPSF30/cpsf30-2 are shown in Figure 6, and the results show that the wheat gene TaCPSF30 can be highly expressed in the heterologous complementation lines.

2. Growth status of heterologous complementary lines under 1/2MS culture conditions at seedling stage

Nitrogen is the most important nutrient element affecting plant growth and development, and abnormal nitrogen metabolism can lead to a decrease in chlorophyll in leaves and a shortened taproot length. We observed the growth status of the heterocomplemented lines under the 1/2 MS culture conditions at the seedling stage, and the results are shown in Figure 7. After 7 days of growth in 1/2MS solid culture, the wild-type Arabidopsis thaliana grew well, and the leaves were green; the main root grew normally and was about 4 cm in length (Fig. 7A). However, the Arabidopsis cpsf30 mutant had poor growth, and the leaves were obviously chlorotic; the main root length was shortened, about 3 cm in length (Fig. 7B). The heterocomplementary lines TaCPSF30/cpsf30-1 and TaCPSF30/cpsf30-2 grew similarly to the wild type, with green leaves; the main root grew normally and was approximately 4 cm in length (Figure 7C and D). The above results show that the wheat gene TaCPSF30 can restore the Arabidopsis cpsf30 mutant to the wild-type phenotype under 1/2MS culture conditions, and can improve the nitrogen use efficiency of plants to maintain higher chlorophyll content and tap root growth.

3. TaCPSF30 can regulate the expression ^of NO ₃ -responsive genes in Arabidopsis

Plants utilize and metabolize nitrogen NO ₃ ^- , and the first step is to sense whether NO ₃ ^- exists in the external environment. More and more research is showing that it is not only a nutrient but also a signaling molecule. When plant roots sense the presence of NO ₃ ^- in the outside world, it will cause changes in the expression of many genes in the body. Among these genes, the NO ₃ ^{-assimilation} genes NIA and NiR, and the transport gene ^NRT2.1 were the most sensitive to NO ₃ -response. Therefore, the expression of these three genes is often used to measure the response of plants to nitrate nitrogen signals. We examined the effect of TaCPSF30 on Arabidopsis NIA, NiR and NRT2.1 expression in heterocomplemented lines (Figure 8). The results showed that the expression levels of NIA, NiR and NRT2.1 in Arabidopsis decreased after AtCPSF30 mutation, indicating that AtCPSF30 affects the NO ₃ ^-sensing of plants to the outside world and thus affects nitrogen utilization. TaCPSF30 can restore the expression of NIA, NiR and NRT2.1 in the cpsf30 mutant to wild-type levels, indicating that TaCPSF30 can regulate the expression of NIA, NiR ^and NRT2.1, three important NO ₃ -responsive genes that are involved in increasing nitrogen It plays a vital role in factor utilization.

Claims (5)

1. the application of a wheat gene TaCPSF30 coding sequence, is characterized in that, described wheat gene TaCPSF30 coding sequence is obtained according to the following method: (1) Extract the total RNA from wheat and reverse transcription to synthesize cDNA; (2) Using cDNA as a template, design specific primers to amplify the coding region sequence of the gene TaCPSF30 by PCR. The primer sequences used for PCR amplification are as follows: TaCPSF30 forward primer F: 5'-CGGGGTACCATGGACGACGGCGACGGC-3'; TaCPSF30 reverse primer R: 5'-CCGCTCGAGTCGCTTCCTTGACCGCCT-3'; (3) Recover and purify the target fragment in the amplification product of (2) and sequence it, and the correct sequence is the coding sequence of the wheat gene TaCPSF30; First, a recombinant plant expression vector compatible with the Gateway system was constructed. The expression vector contained the clone of the TaCPSF30 coding sequence driven by the 35S promoter and the GFP gene. Then the recombinant expression vector was transformed into the Arabidopsis cpsf30 mutant, and the pure cpsf30 mutant was obtained by screening. The combined transgenic heterologous complementary lines were used for the mechanism and genetic improvement of the wheat gene TaCPSF30 in the complementary lines in improving nitrogen use efficiency. 2. the application of wheat gene TaCPSF30 coding sequence according to claim 1, is characterized in that, concrete steps are as follows: S1, construction of pENTR3C-TaCPSF30 recombinant plasmid: Enzymatic digestion and connection of the coding sequence of the wheat gene TaCPSF30 and the cloning vector pENTR3C: KpnI and XhoI restriction sites were added to the purified wheat gene TaCPSF30 coding sequence at the 5' and 3' ends respectively. The target gene fragment at the end of the sex is then connected with the cloning vector pENTR3C to obtain the recombinant plasmid pENTR3C-TaCPSF30; S2, using Gateway technology to construct recombinant plant expression vector pMDC83-TaCPSF30: Perform LR reaction with the correctly sequenced recombinant plasmid pENTR3C-TaCPSF30 and the Gateway system compatible plant expression vector pMDC83 to obtain the recombinant plant expression vector pMDC83-TaCPSF30; S3, Agrobacterium-infected Arabidopsis: Plant the Arabidopsis cpsf30 mutant; prepare Agrobacterium competent cells; then transform the Agrobacterium competent cells with the recombinant expression vector pMDC83-TaCPSF30; pick the transformed Agrobacterium of the positive clone to infect the Arabidopsis cpsf30 mutant; Transgenic plants cloned from the coding sequence of the wheat gene TaCPSF30. 3. The application of the wheat gene TaCPSF30 coding sequence according to claim 2, wherein the Agrobacterium is Agrobacterium GV3101. 4. The application of the wheat gene TaCPSF30 coding sequence according to claim 2, wherein the plant body is an Arabidopsis cpsf30 mutant or a wheat variety Chinese spring. 5. the application of wheat gene TaCPSF30 coding sequence according to claim 2, is characterized in that, after the transformed Agrobacterium of picking positive clone in S3 infects Arabidopsis thaliana cpsf30 mutant, through 3 generations of self-crossing, screened out containing The heterologous complementary line of the coding sequence of wheat gene TaCPSF30 is used for improving nitrogen utilization mechanism and genetic improvement of the coding sequence of wheat gene TaCPSF30 in plants.

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