Alfalfa MsNF-YB8 gene and its application

Abstract

The present invention relates to the technical field of plant genetic engineering, and in particular to alfalfa MsNF‑YB8 gene and its application. The nucleotide sequence of the alfalfa MsNF‑YB8 gene is shown in SEQ ID NO.1. The present invention cloned the MsNF‑YB8 gene related to salt tolerance from alfalfa, constructed a recombinant vector of the MsNF‑YB8 gene, and verified the function of the MsNF‑YB8 gene in improving plant salt tolerance in Arabidopsis thaliana. The present invention can be used to improve the salt tolerance of alfalfa and create alfalfa germplasm material with strong salt tolerance.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, and in particular to alfalfa MsNF-YB8 gene and application thereof. Background Art

[0002] Alfalfa (Medicago sativa L.) is the most widely planted perennial forage legume, mainly distributed in the north, northwest and northeast regions. It has a well-developed root system, is salt-tolerant and has strong stress resistance, and is a forage with extremely high nutritional value. In addition, alfalfa, as a biofuel raw material for the production of ethanol, has great potential in the future energy revolution. However, due to the increasing degree of land salinization, the productivity and growth persistence of many wild alfalfa species have been greatly affected. Therefore, in-depth research on the molecular mechanism of alfalfa's adaptation to salt stress and the discovery of stress-resistant genes are crucial to improving the local alfalfa breeding structure and increasing the utilization rate of salinized land.

[0003] Nuclear Factor Y (NF-Y), also known as heme-binding protein or CCAAT-box binding factor, is a trimer complex composed of three different subunits: NF-YA, NF-YB, and NF-YC. Among them, NF-YB is expressed in plants with spatiotemporal specificity and participates in the regulation of various biological processes, including embryonic development, photosynthesis, flowering time regulation, etc. It also plays a key role in plant response to abiotic stresses such as drought, salt stress, and temperature changes. In plants, Arabidopsis has 13 NF-YB subunits, rice has 11, and soybean has 39. Miao Yahui et al.'s research showed that PwNF-YB8 in Picea awilsonii is involved in pollen germination and pollen tube growth, and plays a role in drought and salt stress responses; Nan Jin's research found that in corn (Zea mays L.), overexpression of the ZmNF-YB13 gene will improve the drought and salt tolerance of the plant; Wang Mengyu's research showed that most genes of the NF-Y family in apple (Malus domestica) are involved in drought and salt stress responses, and it was found that there are many stress response-related elements and cis-acting elements of plant hormone response in the promoter region of MdNF-Ys, MBS is involved in drought stress response, TC-rich repeats are involved in stress response, and LTR is involved in low temperature response, which can regulate adverse stress. In recent years, the functional research of NF-YB family members has been continuously deepened, revealing their important role in plant life activities, and providing new gene resources and strategies for crop improvement and molecular breeding. Summary of the invention

[0004] The purpose of the present invention is to provide alfalfa MsNF-YB8 gene and application thereof.

[0005] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:

[0006] In a first aspect, a gene of MsNF-YB8 of alfalfa is provided, and the nucleotide sequence thereof is shown as SEQ ID NO.1.

[0007] In a second aspect, a transgenic vector containing the alfalfa MsNF-YB8 gene is provided.

[0008] In a third aspect, a recombinant strain or recombinant cell containing the alfalfa MsNF-YB8 gene is provided.

[0009] In a fourth aspect, the invention provides application of overexpression of MsNF-YB8 gene of alfalfa in improving salt tolerance of Arabidopsis thaliana.

[0010] The fifth aspect provides the application of the alfalfa MsNF-YB8 gene in improving the resistance quality of alfalfa and breeding of excellent salt-tolerant alfalfa varieties.

[0011] The beneficial effects of the present invention are:

[0012] The invention cloned the MsNF-YB8 gene related to salt tolerance from alfalfa, constructed a recombinant vector of the MsNF-YB8 gene, and verified the function of the MsNF-YB8 gene in improving plant salt tolerance in Arabidopsis thaliana. The invention can be used to improve the salt tolerance of alfalfa and create alfalfa germplasm material with strong salt tolerance. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Schematic diagram of the relative expression changes of MsNF-YB8 gene under control treatment and salt stress conditions in the example;

[0014] Figure 2 Schematic diagram of the change in relative expression of MsNF-YB8 gene with stress time in the embodiment;

[0015] Figure 3 Schematic diagram of the expression of MsNF-YB8 gene in transgenic Arabidopsis thaliana in the examples;

[0016] Figure 4 Schematic diagram of the effect of MsNF-YB8 gene on the phenotype of Arabidopsis strains in the embodiment;

[0017] Figure 5 This is a schematic diagram of the effect of the MsNF-YB8 gene on the salt tolerance of Arabidopsis seedlings in the examples. DETAILED DESCRIPTION

[0018] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0019] Example

[0020] 1. Cloning and sequence identification of MsNF-YB8 gene from alfalfa

[0021] According to the sequence information of MsNF-YB8 in the alfalfa genome database, the primers MsNF-YB8-F / R were designed using SnapGene 6.0.2 software for full-length amplification of the MsNF-YB8 gene. Total RNA of alfalfa was extracted according to the instructions of the RNA kit and reversely transcribed into cDNA. The CDS sequence of the MsNF-YB8 gene was cloned using cDNA as a template. The reaction system was: 50 μL, cDNA 2 μL, primers 2 μL each, 2×Taq Master Mix 25 μL, double distilled water 19 μL; reaction procedure: 98℃30s, 98℃10s, 57℃5s, 72℃5s (35 cycles), 72℃1min. The PCR product was then detected by agarose gel electrophoresis, and the electrophoresis fragment of the target gene was cut out, and the target fragment was recovered using a gel recovery kit. The target fragment was connected to the pTOPO vector and transformed into Escherichia coli DH5α. The positive monoclonal strain was selected and sent to Beijing BGI for sequencing by PCR identification. The sequence obtained by sequencing was aligned using SnapGene 6.0.2 software to obtain the correct sequence of MsNF-YB8 gene (SEQ ID NO.1).

[0022] 2. Analysis of the expression pattern of MsNF-YB8 gene in alfalfa

[0023] Select "Zhongmu No. 1" seeds with full grains, sow them in plug trays filled with rich soil, and cultivate them in a greenhouse with 16h light / 8h dark, temperature 25±2℃, humidity 60%~70%, and light intensity of 160μmol / (m2·s). After about 2 weeks of cultivation, the seedlings with consistent growth were transplanted into a culture box filled with Hoagland nutrient solution for hydroponics. After another 2 weeks of cultivation, 200mmol·L -1NaCl treatment, root, stem and leaf samples were collected at 0, 4 and 8 h of treatment, immediately frozen with liquid nitrogen, and stored in a -80℃ refrigerator for later use. Total RNA of each sample was extracted according to the instructions of the RNA kit, and reverse transcribed into cDNA. Real-time fluorescence quantitative PCR (qRT-PCR) was performed using quantitative primers MsNF-YB8-qPCR-F / R and alfalfa internal reference primers Msactin-F / R. Reaction system: 20μL, 2×ChamQUniversal SYBR qPCR Master Mix 10μL, 0.4μL of each primer, 2μL of cDNA, 7.2μL of double distilled water; reaction procedure: step 1: pre-denaturation at 95℃ for 30s; step 2: 95℃ for 10s, 60℃ for 30s (40 cycles); step 3: melting: 95℃ for 15s, 60℃ for 60s, 95℃ for 15s. Use The relative expression level of genes was calculated by the method, and three biological replicates and three technical replicates were set up in the experiment. All data were expressed as mean values ​​and statistically analyzed using SPSS27.0 software. One-way ANOVA was used to test the relative expression of MsNF-YB8, a differentially expressed gene related to salt stress, in different tissues of alfalfa.

[0024] 3. Construction of transgenic vector of MsNF-YB8 gene in alfalfa

[0025] Using "Zhongmu No. 1" alfalfa cDNA as a template, W37-MsNF-YB8-F / R containing NcoI and SpeI restriction sites as primers, and high-fidelity enzymes to amplify the full length of MsNF-YB8 gene. The pCAMBIA1300 vector was digested with restriction endonucleases NcoI and SpeI, and the amplified product was connected to the pCAMBIA1300 vector with homologous recombinases and transformed into Escherichia coli DH5α. The positive single colony was picked and shaken, and the recombinant expression vector plasmid was extracted after sequencing and alignment.

[0026] 4. Acquisition, screening, identification and salt tolerance analysis of transgenic Arabidopsis

[0027] The recombinant expression vector was transformed into Agrobacterium GV3101, and then transferred into wild-type Arabidopsis by the floral dipping method. The infected Arabidopsis T0 seeds were harvested, and positive seedlings were screened for two consecutive generations on 1 / 2MS solid medium containing hygromycin resistance to obtain T2 homozygous seeds. Homozygous plants were cultured, RNA was extracted and reverse transcribed to obtain cDNA, and the expression of MsNF-YB8 gene in transgenic lines was detected by semi-quantitative RT-PCR using RT-MsNF-YB8-F / R and Arabidopsis internal reference primers Atactin-F / R as specific primers. In the clean bench, wild-type Arabidopsis and transgenic line T2 homozygous Arabidopsis seeds were disinfected with 75% alcohol for 20 minutes, then rinsed with sterile water 5-6 times, and then evenly sown on 1 / 2MS solid medium, vernalized at 4°C, and cultured in a greenhouse vertically after 2 days. After 4 days of growth, seedlings with the same growth conditions and consistent growth were selected and transferred to 1 / 2MS solid culture medium containing 0, 75 and 100 mmol·L-1 NaCl. After further cultivation in the greenhouse for 3 days, photos were taken and the root length was counted using ImageJ software.

[0028] Table 1 Primers used in the experiment

[0029]

[0030] 5. Experimental results

[0031] Using cDNA from alfalfa tissue of "Zhongmu No. 1" as a template, qRT-PCR was used to analyze the expression of MsNF-YB8 gene in the control treatment and salt (200mmol·L -1 The relative expression changes under NaCl) stress conditions. Figure 1-2 As shown in the data, the MsNF-YB8 gene can be expressed in the roots, stems and leaves of alfalfa. Among them, under the control treatment, the expression level of MsNF-YB8 gene in leaves was significantly higher than that in stems and roots, which was 4.13 times that of roots (p<0.01). During the 8-h stress period, with the extension of stress time, the expression levels of MsNF-YB8 gene in roots and leaves showed an upward trend, among which the expression level in roots was 24.33 times that of the control, and in leaves was 4.56 times that of the control. The expression level in roots was significantly higher than that in leaves (p<0.01).

[0032] Agrobacterium (GV3101) containing the MsNF-YB8 gene was successfully transferred into wild-type Arabidopsis thaliana (WT) by Agrobacterium infection. Through screening by generations using hygromycin resistance medium, T2 generation homozygous seeds were finally obtained, and a total of 20 homozygous strains were screened. Three transgenic strains (L7, L19, and L20) were randomly selected for positive identification. The expression of the MsNF-YB8 gene in these transgenic strains was detected using semi-quantitative RT-PCR technology. The results are as follows Figure 3 As shown, no expression signal of MsNF-YB8 gene was found in WT, while bands of target gene expression were found in the three transgenic lines L7, L19 and L20, indicating that MsNF-YB8 gene was successfully overexpressed in Arabidopsis.

[0033] To detect the effect of MsNF-YB8 gene on Arabidopsis seedlings, seedlings grown on 1 / 2MS medium for 4 days were transferred to medium containing 0 and 75 mmol·L -1 The strains were treated on 1 / 2MS solid medium containing NaCl, and the phenotypic differences between different strains were observed every day. Figure 4-5 As shown, at 0mmol·L -1 Under NaCl conditions, there was no difference between transgenic lines L7, L19, L20 and WT seedlings, but under 75 mmol·L -1 Under NaCl treatment, the root length of L7, L19, and L20 seedlings was significantly longer than that of WT (P<0.01), indicating that the expression of MsNF-YB8 gene was induced by salt stress. The above results show that overexpression of MsNF-YB8 gene can improve the salt tolerance of Arabidopsis.

[0034] The invention cloned the MsNF-YB8 gene related to salt tolerance from alfalfa, constructed a recombinant vector of the MsNF-YB8 gene, and verified the function of the MsNF-YB8 gene in improving plant salt tolerance in Arabidopsis thaliana. The invention can be used to improve the salt tolerance of alfalfa and create alfalfa germplasm material with strong salt tolerance.

[0035] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0036] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. Alfalfa MsNF-YB8 A gene characterized by Its nucleotide sequence is shown in SEQ ID NO.

1.

2. Containing the alfalfa according to claim 1 MsNF-YB8 Genetic transgenic vector.

3. The alfalfa according to claim 1 MsNF-YB8 Application of gene overexpression in improving salt tolerance in Arabidopsis thaliana.

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