Salt and heat resistant gene and application thereof

Abstract

The present application provides a gene, characterized in that the nucleotide sequence of the gene is shown in the sequence table SEQ ID No. 1. The gene provided by the present application can be used to improve the salt and heat tolerance of transgenic plants.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a salt- and heat-resistant gene and its applications. Background Technology

[0002] Salt stress affects seed germination, crop growth, and yield. Salined land covers over 800 million hectares worldwide, approximately 6% of the world's total land area, and this area is expanding. my country is also affected by soil salinization, with about 500 million mu (approximately 33 million hectares) of saline land nationwide. Furthermore, the area of ​​saline-alkali land is rapidly increasing over time, with about 100,000 mu (approximately 6,667 hectares) of new saline-alkali land appearing annually in the Yellow River Delta region alone.

[0003] Temperature is crucial for crop growth and development, and numerous studies have shown that temperature changes significantly impact crop yield. Developing stress-resistant crop varieties using molecular breeding techniques is an important approach to addressing the harmful effects of these environmental factors on agricultural production.

[0004] In our previous experiments, we analyzed the transcriptome of rapeseed after abiotic stress treatment and discovered a gene that was induced by both salt and high temperature. This gene is a member of the NAC transcription factor family. NAC is a large family of plant-specific transcription factors, with 105 family members in Arabidopsis thaliana and, based on genome sequencing information, we estimate that there are more than 390 family members in rapeseed.

[0005] Members of the NAC transcription factor family are widely involved in regulating plant growth and development. They are involved in the regulation of numerous plant physiological processes, including apical meristem development, floral development, root development, hormone signal transduction, nutrient metabolism, and responses to biotic and abiotic stresses. The first NAC transcription factor gene isolated from petunia, NAM (no apical meristem), acts in apical meristem formation and floral development. In Arabidopsis, among NAC transcription factors, the NAP gene is involved in floral morphogenesis, AtNAC1 and AtNAC2 are involved in lateral root development, CUC1 participates in the formation of cup-shaped cotyledons, ATAF1 / 2 and CUC2 regulate cell division, and RD26 and ANAC036 participate in the signal transduction of plant hormones such as ABA. In Arabidopsis, the expression of ANAC019, ANAC055, and ANAC072 is induced by drought, high salt, and ABA, and their overexpression significantly enhances the drought tolerance of transgenic plants. Overexpression of RD26 (encoding a NAC transcription factor) significantly enhanced Arabidopsis thaliana's tolerance to abiotic stresses. The pathogen-associated protein ATAF2 (an NAC transcription factor) negatively regulates Arabidopsis's defense response. Overexpression of NTL8 (an NAC transcription factor) delayed flowering and enhanced salt tolerance in Arabidopsis under salt stress. ATAF1 in Arabidopsis acts as a negative regulator in plant stress responses. Heterologous expression of GmNTL1 / GmNTL11 (NAC transcription factors) proteins led to increased plant tolerance to abiotic stressors. In wheat, the NAC transcription factor NAM-B1 is involved in nutrient storage. In rice, the NAC transcription factor OsNAC5 is associated with the reactivation of nutrients from green tissues to seeds. Overexpression of rice NAC transcription factors SNAC1 and SNAC2 enhanced drought and salt tolerance during growth stages. Overexpression of rice OsNAC6 improved the salt, drought, and rice blast resistance of transgenic rice. In chili peppers, inhibition of CaNAC2 increases seedling sensitivity to cold stress. In buckwheat, FtNAC4 / 6 / 7 / 8 / 9 are associated with various abiotic stresses, including drought, high salinity, cold, SA, and JA. NAC transcription factors are involved in secondary cell wall (SCW) biosynthesis in Arabidopsis, alfalfa, and cotton.

[0006] Although there are over 390 potential NAC transcription factors in rapeseed, related research is limited. In 2003, Hegedus et al. subjected rapeseed to mechanical damage, beetle feeding, or low-temperature stress. Through cDNA library analysis and screening of reduced expression sequence markers, they identified eight NAC transcription factor genes. Their expression in Arabidopsis thaliana caused developmental abnormalities in transgenic Arabidopsis. In 2012, Zhong et al. cloned two rapeseed NAC transcription factors, BnaNAC2 and BnaNAC5, and conducted complementation experiments in yeast and Arabidopsis mutants. Both increased the sensitivity of yeast mutants to high salt and osmotic stress, while BnaNAC5 decreased the salt sensitivity of the Arabidopsis vni2 mutant. Four years ago, BnaNAC485, a homolog of the Arabidopsis stress-related NAC gene RD26, was cloned. Overexpression of this gene increased stomatal sensitivity to ABA, thereby enhancing the tolerance of Arabidopsis and rapeseed to various abiotic stresses. In 2015, Wang et al. cloned 41 rapeseed NAC transcription factors and found that expression of two of them, BnaNAC19 and BnaNAC82, in tobacco caused ROS accumulation and cell death. Subsequently, this research group successively discovered that BnaNAC103, BnaNAC55, BnaNAC56, and BnaNAC87 can all lead to ROS accumulation and are involved in cell death. This indicates that the functions of most rapeseed NAC genes have not yet been confirmed, and research on their transcriptional regulation is only just beginning. Summary of the Invention

[0007] The purpose of this invention is to provide a salt- and heat-resistant gene, its recombinant vector, and its applications.

[0008] To achieve the objectives of this invention, the present invention first provides a gene, the nucleotide sequence of which is shown in SEQ ID NO: 1, and the nucleotide sequence of its protein-coding region is shown in SEQ ID NO: 2. Preferably, the gene is derived from rapeseed.

[0009] In the process of studying the stress resistance mechanism of rapeseed, this invention obtained the nucleotide sequence shown in SEQ ID NO: 1 by extracting rapeseed DNA and performing PCR. The nucleotide sequence shown in SEQ ID NO: 2 was obtained by extracting RNA from rapeseed roots and performing PCR on the cDNA obtained through reverse transcription.

[0010] Specifically, the gene is obtained in the following manner:

[0011] DNA was extracted from leaves of the rapeseed variety Zhongshuang 11 21 days after emergence. The gene sequence was retrieved from the BARD database, and primers were designed at its 5'UTR and 3'UTR for PCR amplification. The upstream primer was fl-F (SEQ ID NO: 4), and the downstream primer was fl-R (SEQ ID NO: 5). The PCR amplification system consisted of 10 μL of 2X TransStartFastPfuPCR SuperMix, diluted to 10 μM fl-F and fl-R (0.5 μL each), 8 μL of PCR water, and 1 μL of DNA.

[0012] PCR reaction program: 94℃ for 5 min; 94℃ for 30 sec, 50℃ for 30 sec, 72℃ for 2 min, 35 cycles; final extension at 72℃ for 7 min. After agarose gel electrophoresis, the target band was excised from the PCR products. The extracted bands were then ligated into the T vector (using pMD) using the EZNAGel Extraction Kit (Omega Bio-tek, Inc., USA). TM The 18-T Vector Cloning Kit (TaKaRa) was used to transform E. coli DH5α competent cells. Positive single colonies were picked and sent to Xi'an Qingke Company for sequencing to obtain the gene sequence (SEQ ID NO: 1).

[0013] RNA was extracted from roots of rapeseed seedlings 21 days after emergence (Double 11). The concentration and quality of RNA were determined using a Nanodrop 2000c spectrophotometer, and RNA integrity was further assessed using agarose gel electrophoresis. PrimeScript was used to analyze the RNA. TM Reverse transcription was performed using an RT reagent kit (TaKaRa Bio Inc, Japan) to obtain cDNA.

[0014] Based on the DNA sequence obtained in Example 1, primers were designed at the 5' and 3' ends for PCR amplification (the primer pair contains adapter sequences for homologous recombination with the PROK2 vector). The upstream primer was PR-F (SEQ ID NO: 6), and the downstream primer was PR-R (SEQ ID NO: 7). The PCR amplification system consisted of: 10 μL of 2X TransStartFastPfuPCRSuperMix, 0.5 μL each of PR-F and PR-R diluted to 10 μM, 8 μL of PCR water, and 1 μL of cDNA.

[0015] PCR reaction program: 94℃ for 5 min; 94℃ for 30 sec, 60℃ for 30 sec, 72℃ for 2 min, 35 cycles; final extension at 72℃ for 7 min. After agarose gel electrophoresis, the target band was excised from the PCR products. The extracted bands were then ligated into the T vector (using pMD) using the EZNAGel Extraction Kit (Omega Bio-tek, Inc., USA). TM The 18-T Vector Cloning Kit (TaKaRa) was used to transform E. coli DH5α competent cells. Positive single colonies were picked and sent to Xi'an Qingke Company for sequencing to obtain the nucleotide sequence encoding the protein (SEQ ID NO: 2).

[0016] The present invention also provides a recombinant expression vector pROK2-N3 for the gene.

[0017] The present invention also provides engineered bacteria containing the gene, including a host cell and a target gene transferred into the host cell, wherein the nucleotide sequence of the target gene is shown in SEQ ID NO: 2.

[0018] This invention also provides the application of this gene in transformed plants.

[0019] Furthermore, the plants include cruciferous plants such as rapeseed, Arabidopsis thaliana, and linden. When the plant is Arabidopsis thaliana, it can achieve a significant improvement in salt and heat tolerance.

[0020] Furthermore, the application involves transforming the gene within Arabidopsis thaliana to enhance its salt and heat tolerance.

[0021] The beneficial effects obtained by the present invention are as follows: it provides a gene that can be expressed in plants and its recombinant vector. The gene provided by the present invention can be used to improve the salt and heat tolerance of transgenic plants. Attached Figure Description

[0022] Figure 1 This is a map of the recombinant plant expression vector pROK2-N3, where N3 represents the coding region sequence of the rapeseed gene (SEQ ID NO: 2).

[0023] Figure 2 The results are PCR identification of transgenic Arabidopsis thaliana. 1-9 are different transgenic lines; - is the negative control (Col-0); P is the positive control (plasmid containing the target gene); M is M5 DL2000 plus DNA Maker; the amplification product size is 648bp.

[0024] Figure 3This section compares the growth of transgenic and wild-type Arabidopsis thaliana after treatment with 200 mM NaCl. Control represents wild-type Arabidopsis thaliana Col-0; VE represents the control line transformed with the pROK2 empty vector; OE7 and OE9 represent transgenic Arabidopsis thaliana transformed with pROK2-N3. A shows the plant growth status. The top row shows the growth status under irrigation, and the bottom row shows the growth status under 200 mM NaCl irrigation. B shows the biomass of Arabidopsis thaliana after stress treatment.

[0025] Figure 4 This section compares the growth status of transgenic and wild-type materials after high-temperature treatment. Control represents wild-type Arabidopsis thaliana Col-0; VE represents the control line transformed with the pROK2 empty vector; OE7 and OE9 represent transgenic Arabidopsis thaliana transformed with pROK2-N3. A shows the plant growth status. The top row shows the growth status without stress, and the bottom row shows the growth status after high-temperature stress. B shows the statistical results of the number of surviving leaves in Arabidopsis thaliana after high-temperature treatment. Detailed Implementation

[0026] Example 1: Obtaining the gene sequence

[0027] DNA was extracted from leaves of the rapeseed variety Zhongshuang 11 21 days after emergence. The gene sequence was retrieved from the BARD database, and primers were designed at its 5'UTR and 3'UTR for PCR amplification. The upstream primer was fl-F (SEQ ID NO: 4), and the downstream primer was fl-R (SEQ ID NO: 5). The PCR amplification system consisted of 10 μL of 2X TransStartFastPfuPCR SuperMix, diluted to 10 μM fl-F and fl-R (0.5 μL each), 8 μL of PCR water, and 1 μL of DNA.

[0028] PCR reaction program: 94℃ for 5 min; 94℃ for 30 sec, 50℃ for 30 sec, 72℃ for 2 min, 35 cycles; final extension at 72℃ for 7 min. After agarose gel electrophoresis, the target band was excised from the PCR products. The extracted bands were then ligated into the T vector (using pMD) using the EZNAGel Extraction Kit (Omega Bio-tek, Inc., USA). TM The 18-T Vector Cloning Kit (TaKaRa) was used to transform E. coli DH5α competent cells. Positive single colonies were picked and sent to Xi'an Qingke Company for sequencing to obtain the gene sequence (SEQ ID NO: 1).

[0029] Example 2: Obtaining the sequence of a gene-encoding protein region

[0030] RNA was extracted from roots of rapeseed seedlings 21 days after emergence (Double 11). The concentration and quality of RNA were determined using a Nanodrop 2000c spectrophotometer, and RNA integrity was further assessed using agarose gel electrophoresis. PrimeScript was used to analyze the RNA. TM Reverse transcription was performed using an RT reagent kit (TaKaRa Bio Inc, Japan) to obtain cDNA.

[0031] Based on the DNA sequence obtained in Example 1, primers were designed at the 5' and 3' ends for PCR amplification (the primer pair contains adapter sequences for homologous recombination with the PROK2 vector). The upstream primer was PR-F (SEQ ID NO: 6), and the downstream primer was PR-R (SEQ ID NO: 7). The PCR amplification system consisted of: 10 μL of 2X TransStartFastPfuPCRSuperMix, 0.5 μL each of PR-F and PR-R diluted to 10 μM, 8 μL of PCR water, and 1 μL of cDNA.

[0032] PCR reaction program: 94℃ for 5 min; 94℃ for 30 sec, 60℃ for 30 sec, 72℃ for 2 min, 35 cycles; final extension at 72℃ for 7 min. After agarose gel electrophoresis, the target band was excised from the PCR products. The extracted bands were then ligated into the T vector (using pMD) using the EZNAGel Extraction Kit (Omega Bio-tek, Inc., USA). TM The 18-T Vector Cloning Kit (TaKaRa) was used to transform *E. coli* DH5α competent cells. Positive single colonies were picked and sent to Xi'an Qingke Company for sequencing, yielding the nucleotide sequence encoding the protein (SEQ ID NO: 2). Based on this sequence, the protein sequence it encodes can be deduced (SEQ ID NO: 3).

[0033] Example 3 Construction of plant expression vector

[0034] The pROK2 vector was extracted and double-digested with XbaI and SmaI. The digestion system consisted of: 2 μL 10X buffer, 1 μL XbaI, 1 μL SmaI, 4 μL vector, and 12 μL water. The mixture was incubated at 37°C for 2 hours. The linearized vector was purified using the EZNAGel Extraction Kit (Omega Bio-tek, Inc., USA). Next, the PCR product from Example 2 was incubated with the digested and purified pROK2 vector using an In-fusion HD reaction. The 5 μL reaction mixture consisted of: 1 μL 5X In-fusion HD Enzyme premix, 2 μL PCR product, and 2 μL of the digested and purified pROK2 vector. The reaction was incubated at 50°C for 20 minutes. The reaction product was transformed into DH5α competent cells, and positive plasmids were extracted to obtain the desired results. Figure 1 The plant expression vector pROK2-N3 with the structure shown was sequenced and verified, and the nucleotide sequence shown in SEQ ID NO: 2 was obtained.

[0035] Example 4: Obtaining and Molecular Identification of Transgenic Arabidopsis

[0036] The constructed plant expression vector pROK2-N3 was transformed into wild-type Arabidopsis thaliana Col-0 using the Arabidopsis inflorescence infection method, with the empty pROK2 vector as a control. Transgenic Arabidopsis plants (Kan resistant) were obtained through resistance selection, and positive plants were detected by RT-PCR. Figure 2 The detection primers were 35S (SEQ ID NO: 8) for the upstream primer and N3R (SEQ ID NO: 9) for the downstream primer. Homozygous seeds were obtained after three generations of screening and used for phenotypic analysis.

[0037] Example 5: Salt tolerance analysis of transgenic Arabidopsis thaliana

[0038] The control group (wild-type Col-0 and transgenic lines transformed with the pROK2 empty vector) and two transgenic lines, OE7 and OE9, were sown in pots filled with peat moss:vermiculite:perlite (3:1:1) and cultured for 2 weeks in a 22°C light incubator. Afterward, they were treated with 200 mM NaCl, and phenotypic observations were performed. The results showed that after 2 weeks of salt stress treatment, the transgenic plants exhibited better growth than the control group. The transgenic plants only showed yellowing at the leaf margins, while the control group showed yellowing of the entire leaf. Figure 3 A), and the biomass of the transgenic plants was greater than that of the control ( Figure 3 B). This indicates that the rapeseed gene can improve the salt tolerance of Arabidopsis thaliana.

[0039] Example 6: Heat resistance analysis of transgenic Arabidopsis thaliana

[0040] The control group (wild-type Col-0 and transgenic lines transformed with the pROK2 empty vector) and two transgenic lines, OE7 and OE9, were sown in pots filled with peat moss:vermiculite:perlite (3:1:1). After two weeks of cultivation in a 22℃ light-controlled incubator, they were transferred to a 33℃ incubator for six days, then returned to a 22℃ light-controlled incubator for eight days of recovery before phenotypic observation. The results showed that the transgenic plants exhibited better growth after high-temperature stress treatment than the control group. Figure 4 A) In the control plant, multiple leaves withered, while the transgenic plant retained more leaves than the control. Figure 4 B) indicates that this rapeseed gene can improve the heat resistance of Arabidopsis thaliana.

[0041] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of a recombinant expression vector containing a gene with a nucleotide sequence as shown in SEQ ID NO.2 in improving the salt and heat tolerance of Arabidopsis thaliana, characterized in that, The application involves introducing the recombinant expression vector into Arabidopsis thaliana.

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