CN113444161A

CN113444161A - Radish annexin RsANN1a and coding gene and application thereof

Info

Publication number: CN113444161A
Application number: CN202110730694.4A
Authority: CN
Inventors: 柳李旺; 沈峰; 徐良; 王燕; 应佳丽; 何晴
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2021-06-29
Filing date: 2021-06-29
Publication date: 2021-09-28

Abstract

The invention discloses radish annexin RsANN1a and a coding gene and application thereof, wherein the coding gene comprises the following components: (1) the nucleotide and amino acid sequences of the radish annexin RsANN1a are respectively shown in SEQ ID NO: 1 and SEQ ID NO: 2 is shown in the specification; (2) cloning RsANN1a by using radish cDNA as a template; (3) the expression characteristic of the RsANN1a gene under high-temperature stress is analyzed, and the result shows that the RsANN1a gene can respond to the high-temperature stress and the expression level thereof is obviously increased under the high-temperature treatment; (4) the invention also identifies the function of the RsANN1a gene, and the over-expression of the RsANN1a in wild arabidopsis thaliana can obviously improve the heat resistance of the wild arabidopsis thaliana. The invention provides a theoretical basis for cultivating high-temperature resistant radish by utilizing a genetic engineering technology in the future, and has high application value.

Description

Radish annexin RsANN1a and coding gene and application thereof

Technical Field

The invention relates to radish annexin RsANN1a, and a coding gene and application thereof, and belongs to the technical field of biology.

Background

In recent years, with the development of economy, global temperature is increasing year by year, and temperature plays a crucial role in the growth and development of plants. High temperature stress has a significant effect on the normal growth and development of plants and has become one of the important factors causing the reduction of the nutritional quality and yield of vegetable crops.

The annexin ANN1 gene can participate in the transduction of ion signals and the regulation of the content of active oxygen in plants, so that the resistance of the plants to the adversity stress is improved. After arabidopsis seedlings are subjected to high-temperature treatment for 5 minutes, the AtANN1 is identified by means of proteomics, and then the research on the biological functions of the seedlings shows that the high-temperature resistance of arabidopsis can be remarkably improved by over-expressing the AtANN 1. Through pre-transcriptome-proteome combined analysis, the RsANN1a gene was found to be significantly up-regulated in expression after the radish was subjected to heat treatment. The research results provide a theoretical basis for separating and cloning the radish RsANN1a gene and carrying out biological function verification on the radish RsANN1a gene.

Radish (Raphanus sativus L.) belongs to the genus Raphanus of the family Brassicaceae, a primary and a secondary herbaceous plant. The radish is cool and pleased to be cultivated in autumn and winter, and the high temperature is a main factor for limiting the production and supply of the radish in summer. No report about RsANN1a gene in the radish heat stress response process is found at present. Therefore, the function of the RsANN1a gene in a heat-resistant genetic physiological mechanism of radish is clarified, and a radish excellent variety with strong heat resistance is cultivated, so that the method has important theoretical and practical significance for ensuring high-quality production of radish in summer and realizing annual supply.

Disclosure of Invention

The invention aims to fill the blank in the aspects of cloning, expression pattern analysis and application of the radish RsANN1a gene. The invention provides a radish annexin RsANN1a, a coding gene and application thereof, and specifically relates to a cDNA and an amino acid sequence of radish annexin RsANN1 a; furthermore, the invention provides expression patterns of the RsANN1a gene after different high-temperature treatments. The invention also provides evidence that RsANN1a can improve the stress tolerance of radish to high-temperature stress.

The purpose of the invention is realized by the following technical scheme:

the invention provides radish annexin RsANN1a, the nucleotide sequence of which is shown as SEQ ID NO: 1, and the amino acid sequence is shown as SEQ ID NO: 2, respectively.

The invention provides a primer pair for amplifying the gene coding the RsANN1a protein, wherein the base sequence of the primer pair is shown as SEQ ID NO: 3. SEQ ID NO: 4, respectively.

The invention provides a primer pair for fluorescent quantitative PCR analysis of the gene coding RsANN1a protein, wherein the base sequence of the primer pair is shown as SEQ ID NO: 5-SEQ D NO: shown in fig. 8.

The invention provides an artificial miRNA (artificial microRNA, amiR) primer pair for constructing and inhibiting the gene expression of the RsANN1a protein, wherein the base sequence of the primer pair is shown as SEQ D NO: 9-SEQ ID NO: as shown at 14.

The invention provides application of radish RsANN1a protein in regulation and control of the stress tolerance of radish to a high-temperature stress environment.

The invention provides a method for verifying the gene function of the RsANN1a protein, which comprises the step of introducing a gene coding the RsANN1a protein into wild arabidopsis thaliana to obtain a corresponding transgenic arabidopsis thaliana strain.

The invention also provides application of the RsANN1a protein gene in breeding, and particularly relates to application in cultivation of radishes with strong heat resistance.

The full-length sequence of the radish RsANN1a related nucleotide can be obtained by a PCR amplification method. In view of this, primers can be designed based on the nucleotide sequences disclosed herein, especially open reading frame sequences, and the cDNA library prepared by conventional methods known to those skilled in the art can be used as a template to amplify the sequences. After obtaining the sequence, cloning the sequence into a target expression vector by using a method of enzyme digestion and connection, and then transforming an agrobacterium strain by a heat shock method. Finally, the wild arabidopsis thaliana is transformed by utilizing an agrobacterium-mediated method.

Compared with the prior art, the invention has the following outstanding effects:

the invention finds a key gene RsANN1a related to radish heat resistance, and experiments prove that the expression level of RsANN1a after heat stress is obviously higher than that before heat treatment. The key role of the RsANN1a gene in improving the high temperature resistance of radish is also confirmed by transgenic technology. The invention has higher application value in crop molecular improvement and has important theoretical significance and practical significance for cultivating high-temperature resistant radish.

Drawings

FIG. 1 shows RsANN1a cDNA amplification. Wherein, lane 1 is DL2000 molecular weight marker, and lanes 2-3 are RsANN1a amplified fragment;

FIG. 2 shows the expression characteristics of RsANN1a gene in radish leaves under high temperature stress at different times;

FIG. 3 is a transgenic plant validation electrophoretogram. Wherein, lane 1 is DL2000 molecular weight marker, lane 2 is wild type Arabidopsis thaliana plant, lanes 3-8 are amiR-RsANN1a transgenic Arabidopsis thaliana plant (lane 7 is false positive plant), and lanes 9-14 are transgenic Arabidopsis thaliana plant overexpressing RsANN1 a;

FIG. 4 shows the growth of WT, over-expressing RsANN1a and amiR-RsANN1a transgenic plants after hyperthermia treatment.

Detailed Description

The present invention is described in detail below with reference to specific examples, which will assist those skilled in the art in further understanding the present invention, but are not intended to limit the present invention in any way. It should be noted that the described embodiments are only a part of the present invention, and all other similar embodiments obtained by a person skilled in the art without making other inventive efforts shall fall within the scope of the present invention.

The embodiment of the invention discloses radish annexin RsANN1a, and a coding gene and application thereof. The methods used in this example are all conventional experimental methods, and are not described herein.

Example 1 cloning method of RsANN1a Gene

1. Obtaining plant material

The plant material used in the experiment was radish, which was planted in the university of Nanjing university of agriculture, the school district of the health guard.

2. Total RNA extraction and cDNA Synthesis

Total RNA of the sample was extracted using RNA simple Total RNA Kit (DP419, TIANGEN, Beijing) Kit from Tiangen Biochemical technology Ltd. 0.1 percent of DEPC-H is used for plastic articles such as gun heads, centrifuge tubes and the like used in the experiment₂Soaking in chloroform solution overnight in a mortar and a pestle, sterilizing at high temperature and high pressure, and oven drying.

The cDNA synthesis was performed by reverse transcription using a reverse transcription kit (TaKaRa Biotechnology Co., Ltd.) manufactured by Baozi medical technology (Beijing) Co., Ltd.

3. Cloning of the Gene of interest

Using primers

RsANN1a-F1(SEQ ID NO：3)：5′-TGCTCTAGACCAGAGAGAGTGAAAGAA-3′

RsANN1a-R1(SEQ ID NO：4)：5′-TCCCCCGGGAAAACACAAACAAACGAA-3′

And carrying out PCR, amplifying to obtain a fragment with an expected length, recovering and connecting the fragment to a pMD-19T (Baozi medical science and bioengineering (Dalian) Co., Ltd.), connecting for 30min, converting the escherichia coli competence DH5 alpha, selecting a bacterial solution (shown in figure 1) with a correct strip after detection, carrying out shake propagation on the bacterial solution, and then, sending the bacterial solution to Sipu's biotechnology Co., Ltd (Nanjing) for sequencing to obtain a cDNA sequence.

Example 2 analysis of expression characteristics of RsANN1a Gene under high temperature stress

1. Obtaining plant material

The plant material used in the experiment was radish, which was planted in the university of Nanjing university of agriculture, the school district of the health guard. When the plants grow to 4-5 true leaves, radish seedlings with consistent growth vigor are selected and are respectively treated at the high temperature of 40 ℃ for 0, 3, 6, 12, 24 and 48 hours. After the treatment, leaf samples were collected and put into liquid nitrogen for quick freezing, and then stored in a refrigerator at-80 ℃ for later use.

2. Total RNA extraction and cDNA Synthesis

The cDNA was synthesized by reverse transcription using a reverse transcription kit Takara Biotechnology Co., Ltd, manufactured by Baori physician's technology (Beijing) Ltd.

3. Specific primers are designed to carry out real-time fluorescent quantitative PCR analysis on the expression quantity of the gene after high-temperature treatment for different time. Designing specific primers for quantitative analysis of RsANN1a gene in fluorescent quantitative PCR by using primer design software according to the obtained cDNA sequence of radish RsANN1a,

RsANN1a-F2(SEQ ID NO：5)：5′-AATCTTGCTCTGGACTATG-3′

RsANN1a-R2(SEQ ID NO：6)：5′-AACATCCTCTTCAATAGACTT-3′

the reference gene primer is as follows:

RsACTIN-F(SEQ ID NO：7)：5′-GCATCACACTTTCTACAAC-3′

RsACTIN-R(SEQ ID NO：8)：5′-CCTGGATAGCAACATACAT-3′

real-time fluorescent quantitative PCR analysis of RsANN1a

The first strand of the synthesized cDNA was used as a template, and the first strand was amplified with primers specific to RsANN1a and the reference gene, respectively, to perform quantitative fluorescence analysis, and then subjected to quantitative fluorescence PCR using a Roche Light Cycler 480 quantitative fluorescence PCR instrument. By using 2^-ΔΔCtThe method is used for relative quantitative analysis. The results show that the expression level of the RsANN1a gene is increased along with the increase of the exposure time at the high temperature of 40 ℃ and reaches a peak after 3h, and the expression level of the RsANN1a gene is greatly reduced after 6h of treatment and is only 8.8 times of that of CK. Then, the expression level was significantly increased and gradually decreased after 12h of treatment, and was 18.9 times that of CK after 48h of treatment (FIG. 1). As can be seen from the above results, the RsANN1a gene was able to respond to high temperature stress, andis strongly induced under high temperature treatment.

Example 3 functional verification of RsANN1a gene in radish thermostable process.

Construction of RsANN1a overexpression vector

And (3) carrying out amplification on the cloned escherichia coli liquid of the RsANN1a, extracting plasmids, carrying out double enzyme digestion on the pMD-RsANN1a and the pCAMBIA2301 expression vector by using restriction endonucleases Xba I and Sma I, detecting through agarose gel electrophoresis, and cutting off a fragment with a correct length to recover and purify. And respectively carrying out overnight connection on the recovered target fragments and corresponding expression vectors at 16 ℃ by using T4 ligase, then transforming the T4 ligation product into escherichia coli competent DH5 alpha, respectively extracting bacterial liquid plasmids with correct bands, and further carrying out double enzyme digestion verification. The plasmid which is verified to be correct by double enzyme digestion is the RsANN1a overexpression vector which is successfully constructed.

Construction of amiR-RsANN1a vector

The pMD-pre-miR319a plasmid preserved in the laboratory is used as a template, 6 designed specific primers (SEQ ID NO: 5-SEQ ID NO: 10) are utilized, amplification products are obtained by respectively reacting a, b and c, and then the amplification products of the three times of reaction are respectively recovered and purified. Finally, d reaction was performed to obtain amiR-RsANN1a fragment. And (d) recovering and purifying the reaction fragment, cloning and sequencing, and finally extracting the plasmid in the monoclonal bacteria liquid with correct sequencing. The intermediate vector pMD-amiR-RsANN1a and the modified pCAMBIA2301 expression vector were double digested with restriction enzymes Xba I and Sac I, respectively. After the digestion, the amiR-RsANN1a fragment and the pCAMBLA2301 large fragment were recovered and ligated with T4 ligase at 16 ℃ overnight. And then transferring the ligation product into escherichia coli competence, then picking out a single clone for verification, extracting a bacterial liquid plasmid which is successfully verified, and further verifying (double enzyme digestion verification). The plasmid which is verified to be correct by double enzyme digestion is the amiR-RsANN1a expression vector which is successfully constructed.

The sequence of the amiR-RsANN1a specific primer is as follows:

miR-A(SEQ ID NO：9)：5′-CGTCTAGACAAACACACGCTCGGACGCAT-3′

miR-B(SEQ ID NO：10)：5′-ACGAGCTCCATGGCGATGCCTTAAATAAA-3′

miR-I(SEQ ID NO：11)：

5′-GATATTAGCCAATAAAGCCTCTCTCTCTCTTTTGTATTCC-3′

miR-II(SEQ ID NO：12)：

5′-GAGAGAGGCTTTATTGGCTAATATCAAAGAGAATCAATGA-3′

miR-III(SEQ ID NO：13)：

5′-GAGAAAGGCTTTATTCGCTAATTTCACAGGTCGTGATATG-3′

miR-IV(SEQ ID NO：14)：

5′-GAAATTAGCGAATAAAGCCTTTCTCTACATATATATTCCT-3′

3. genetic transformation of Arabidopsis thaliana by floral dip method

(1) Transferring the constructed plasmid into agrobacterium tumefaciens by a freeze-thaw method;

(2) carrying out enlarged culture on the agrobacterium tumefaciens positive bacterial liquid;

(3) centrifuging the enlarged culture bacterial solution at room temperature at 5000rpm for 15min, and discarding the supernatant;

(4) resuspending and precipitating with prepared staining solution (1/2MS culture medium +50g L-1 sucrose +200 μ L L-1 Silwet L-77), continuously adjusting pH to 5.8, and adjusting OD600 to 0.3-0.6;

(5) selecting arabidopsis thaliana at the initial flowering stage, and watering the arabidopsis thaliana thoroughly before infection;

(6) placing inflorescences of arabidopsis thaliana in the invasion dye solution for about 1min, wrapping and moisturizing the inflorescences with a preservative film after the infection is finished, shading for 24h, and then switching to normal growth;

(7) carrying out secondary infection after about one week, and referring to the steps (2) to (6);

(8) and (5) collecting the seeds after the seeds are mature.

4. Screening and identifying transgenic arabidopsis positive plants

(1) Screening of transgenic Arabidopsis positive plants

T to be harvested₀The seeds were allowed to dry in a seed cabinet for 2 weeks. Before screening transgenic positive plants, T is used₀The seeds are placed in a refrigerator at 4 deg.C for 2-3 days for low-temperature vernalization, and then sterilized and sown in the container100mg/L Kan 1/2MS solid medium, placed in the culture room for culture. After 2-3 weeks, it can be seen that wild type and the Arabidopsis thaliana which is not successfully transferred into the recombinant plasmid show yellowing or death, while the transgenic positive plant successfully transferred into the recombinant plasmid shows normal growth and developed root system.

(2) Identification of transgenic Arabidopsis positive plants

In order to verify whether the target gene is successfully transferred into arabidopsis thaliana, 7 strains and 6 strains are respectively selected to be subjected to Kan screening to over-express RsANN1a and amiRANN1a T₂Transgenic plants are generated, and the genome DNA of the plants is respectively extracted. WT, over-expressing RsANN1a and amiR-RsANN1a transgenic plants were PCR amplified using the universal primer M13 and WT was used as a negative control. The results showed that a band of about 2200bp could be amplified from the genomic DNA of all 7 RsANN1a transgenic plants using the universal primer M13, and that about 1600bp could be amplified from 5 of 6 amiR-RsANN1a transgenic plants, but no band was amplified from the genomic DNA of the WT plant (FIG. 3). In addition, the sequence information of the pCAMBIA2301 plasmid vector after being combined with the modification shows that the size of the amplified band is consistent with that of the band after the target fragment is inserted. The RsANN1a gene was shown to be successfully integrated into the Arabidopsis genome, while the amiR-RsANN1a gene was also successfully introduced into the genome of 5 Arabidopsis strains.

5. Phenotypic identification of transgenic plants overexpressing RsANN1a and amiR-RsANN1a

Wild type Arabidopsis thaliana with the age of 15d seedlings and transgenic plants with overexpression RsANN1a and amiR-RsANN1a are respectively subjected to high temperature treatment at 37 ℃ for 8h, and then transferred to normal conditions. After 5 days of restoration of growth, plants were photographed and the survival rate was measured and expressed as a percentage of the total number of seeds inoculated. The results show that after the high temperature treatment, partial plants are hindered from growing, and whitening and even death phenomena are shown, and then the survival rate of the WT plants is counted to find that the survival rate of the WT plants is 30.7 percent. Compared with the amiR-RsANN1a transgenic plant, most of the plants are very sensitive to high temperature performance, most of the plants show whitening and death phenomena after high temperature treatment, only a few of the plants can grow normally, and the survival rate of the plants is only 20.8% of that of the amiR-RsANN1a transgenic plant (figure 4). The above results indicate that the RsANN1a gene can play an important role in responding to heat stress.

The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the invention is not limited to the particular embodiments described above, but that modifications and adaptations may occur to one skilled in the art, within the scope of the appended claims. Accordingly, the invention is not to be limited to the embodiments shown herein.

Claims

1. A radish annexin RsANN1a, a coding gene and an application thereof are characterized in that a nucleotide sequence is shown as SEQ ID NO: 1 and the amino acid sequence is shown as SEQ ID NO: 2, respectively.

2.A primer pair for amplifying the RsANN1a gene of claim 1, wherein the base sequence of the primer pair is as shown in SEQ ID NO: 3. SEQ ID NO: 4, respectively.

3. A primer pair for the fluorescent quantitative PCR analysis of the gene encoding the RsANN1a protein according to claim 1, wherein the base sequences of the primer pair are as shown in SEQ ID NO: 5-SEQ ID NO: shown in fig. 8.

4. An artificial miRNA (amiR) primer pair for constructing the gene encoding the RsANN1a protein of claim 1, wherein the base sequence of the primer pair is as shown in SEQ ID NO: 9-SEQ ID NO: as shown at 14.

5. The RsANN1a protein gene function verification method according to claim 1, wherein the constructed overexpression and amiR-RsANN1a vector are transferred into Arabidopsis thaliana by a floral dip method to obtain a corresponding Arabidopsis thaliana transgenic line, and the Arabidopsis thaliana transgenic line meets the following conditions:

(1) the growth potential of the over-expression transgenic strain under high-temperature stress is obviously higher than that of the wild type and the amiR-RsANN1a strain;

(2) the survival rate of the over-expression transgenic line under high-temperature stress is obviously higher than that of the wild type and the amiR-RsANN1a line.

6. Use of the RsANN1a protein gene as claimed in claim 1, in breeding, particularly in breeding plants with high temperature tolerance.

7. Use of the RsANN1a protein gene of claim 1, wherein the plant is radish.