CN116515853B - A ryegrass salt-tolerance gene LpNAC022 and its application - Google Patents

Abstract

The present invention discloses a ryegrass salt-tolerance gene LpNAC022 and an application thereof. The gene LpNAC022 is expressed in a plant to improve the salt tolerance of the plant. The gene LpNAC022 is a nucleotide sequence shown in the sequence table Seq ID NO.1 or a nucleotide sequence complementary thereto. The gene LpNAC022 can be used to cultivate new plant varieties such as salt- and alkali-tolerant ryegrass. Based on whole genome association analysis combined with transcriptome sequencing results, the present invention discovered a gene LpNAC022 with significant resistance to salt stress. The ryegrass salt-tolerance gene LpNAC022 provided by the present invention can improve the salt tolerance of the plant by regulating the expression of salt stress-related genes, and is helpful for cultivating ryegrass varieties suitable for growth in saline-alkali lands, while reducing the workload of breeding, reducing the scale of breeding, shortening the breeding period, improving the breeding efficiency, and accelerating the selection of salt- and alkali-tolerant ryegrass varieties.

Description

A ryegrass salt-tolerance gene LpNAC022 and its application

Technical Field

The invention belongs to the technical field of plant genetic engineering and relates to a salt-tolerant gene, in particular to a ryegrass salt-tolerant gene and application of the salt-tolerant gene.

Background technique

Perennial ryegrass (Lolium perenne L.) is a world-renowned high-quality forage and lawn grass. It has the characteristics of rapid growth, strong disease resistance, trampling resistance, fast growth recovery, rich nutrition, and good palatability. It is an important pioneer grass species for lawn establishment and one of the important high-quality forage components of artificial grassland. It is also often used as the best pioneer grass species for coastal tidal flat improvement, ecological management and soil salinization improvement.

Ryegrass germplasm resources from different sources have great differences in salt tolerance. Preliminary gene expression studies have shown that some of these genes may be strongly associated with ryegrass salt tolerance, and further gene function studies are currently underway.

Strengthening the screening, evaluation and breeding of perennial ryegrass germplasm resources, exploring the key salt-tolerant genes of beneficial variations, breeding new varieties of salt-tolerant perennial ryegrass and promoting their use in actual production has important reference value for establishing a circular agricultural economic model of "improving saline-alkali land - planting forage - developing animal husbandry - returning organic fertilizer to the field - improving saline-alkali land". Perennial ryegrass has a strong salt-alkali tolerance, and a large number of studies have shown that its salt tolerance is positively correlated with yield and grass quality under saline soil conditions. Strengthening the research on the salt-tolerant mechanism of perennial ryegrass and accelerating the breeding of salt-tolerant varieties are of great significance to improving and rationally utilizing saline-alkali land, ensuring food security, and improving the ecological environment.

Ryegrass is a cross-pollinating forage grass, and its genetic transformation is difficult, its growth cycle is long, and the verification of gene function is relatively lagging. At present, the molecular mechanism of salt tolerance in perennial ryegrass is still unclear, and only a few studies have conducted preliminary explorations on the changes in its physiological and biochemical indicators under salt stress, and breeding research has progressed slowly.

Plant salt stress is a trait controlled by a complex regulatory network. The molecular mechanism of ryegrass salt tolerance is not very clear, and it is difficult to apply related genes and loci in ryegrass molecular assisted breeding. So far, no gene that can regulate ryegrass salt tolerance has been verified through functional analysis.

Summary of the invention

In view of this, one of the objects of the present invention is to provide a gene fragment capable of improving the salt tolerance of perennial ryegrass.

A second object of the present invention is to provide a protein encoded by a ryegrass salt-tolerance gene.

A third object of the present invention is to provide a primer pair for cloning the salt-tolerance gene of ryegrass.

A fourth object of the present invention is to provide an overexpression vector containing the ryegrass salt-tolerance gene.

A fifth object of the present invention is to provide a host cell containing a ryegrass salt-tolerance gene.

A sixth object of the present invention is to provide a use of a ryegrass salt-tolerance gene.

The inventors have continuously innovated and reformed through long-term exploration and attempts, as well as multiple experiments and efforts, to solve the above technical problems. The technical solution provided by the present invention is to provide a ryegrass salt-tolerance gene LpNAC022, which is expressed in plants to improve the salt tolerance of plants. The gene LpNAC022 is a group of the following nucleotide sequences:

A, nucleotide sequence shown in Seq ID NO.1 in the sequence listing;

B. A nucleotide sequence complementary to the sequence described in A.

Furthermore, the plant is ryegrass.

The present invention also provides a protein encoded by the aforementioned ryegrass salt-tolerance gene LpNAC022, wherein the amino acid sequence of the protein is the amino acid sequence shown in Seq ID NO.2 in the sequence table.

The present invention also provides a primer pair for cloning the aforementioned ryegrass salt tolerance gene LpNAC022, the base sequence of the primer pair is as follows:

Upstream primer F: 5′-ATGATCATGTCCGATCCGGC-3′;

Downstream primer R: 5’-TTAGAAAGGGAGCAGCGTGTG-3’.

The present invention also provides an overexpression vector containing the above-mentioned ryegrass salt-tolerance gene LpNAC022. The full-length plant salt-tolerance gene LpNAC022 is inserted into a vector pCambia1301, which contains a CaMV 35S promoter, to obtain an overexpression vector pCambia1301-35S-LpNAC022.

The present invention also provides a host cell containing the above-mentioned overexpression vector, and the overexpression vector pCambia1301-35S-LpNAC022 is transferred into Agrobacterium tumefaciens strain to obtain the host cell.

The present invention also provides a use of the aforementioned ryegrass salt-tolerance gene LpNAC022 for cultivating new salt- and alkali-tolerant plant varieties.

Compared with the prior art, one of the above technical solutions has the following advantages:

1. Based on whole genome association analysis combined with transcriptome sequencing results, the present invention discovered a gene LpNAC022 with significant resistance to salt stress. The ryegrass salt tolerance gene LpNAC022 provided by the present invention can improve the salt tolerance of ryegrass by regulating the expression of salt stress-related genes, which is helpful for cultivating ryegrass varieties suitable for growth in saline-alkali land, while reducing the breeding workload, reducing the breeding scale, shortening the breeding years, improving the breeding efficiency, and accelerating the selection of salt-alkali tolerant ryegrass varieties.

2. The present invention only requires a pair of primers and one round of amplification to clone the salt-tolerant gene LpNAC022, which greatly shortens the time required.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

FIG1 is a diagram showing the prediction of the hydrophilicity/hydrophobicity of the protein encoded by the ryegrass salt-tolerance gene LpNAC022 of the present invention.

FIG. 2 is a diagram showing the predicted secondary structure of the protein encoded by the ryegrass salt-tolerance gene LpNAC022 of the present invention.

FIG3 is a diagram showing the predicted tertiary structure of the protein encoded by the ryegrass salt tolerance gene LpNAC022 of the present invention.

Figure 4 is a diagram showing the functional verification results of Saccharomyces cerevisiae under salt stress. In Figure 4, CK represents the control group, 1.4M NaCl represents the treatment group with 1.4M NaCl added; pYES II-LpNAC022 is yeast connected with the target gene LpNAC022, and pYES II is an empty yeast without exogenous gene.

Figure 5 is a diagram of the Arabidopsis germination salt stress experiment and analysis results. In Figure 5, (A) is the result of the Arabidopsis germination salt stress experiment, and (B) is the statistical result of the germination rate on the 2nd, 8th and 14th days of the Arabidopsis germination salt stress experiment; CK is the control group and 150mM NaCl is the treatment group.

Figure 6 is a salt stress experiment of Arabidopsis thaliana seedlings. In Figure 6, 1/2MS is the control group, and 1/2MS+150mM NaCl is the treatment group.

Figure 7 shows the growth index data of Arabidopsis thaliana seedlings under salt stress for 7 days. In Figure 7, (A) is fresh weight, (B) is root length, and (C) is lateral root number.

Detailed ways

The following is a description with reference to the accompanying drawings and a specific embodiment.

In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention.

It should be noted that similar reference numerals and letters denote similar items in the following drawings, and thus, once an item is defined in one drawing, it may not be further defined or explained in the subsequent drawings.

The experimental material was the ryegrass variety ‘Gentleman’, which was planted in the Wenjiang campus of Sichuan Agricultural University. The mature leaves were used as materials to extract total RNA. The plant total RNA extraction kit of Tiangen (Beijing) Biochemical Technology Co., Ltd. was used for RNA extraction, and the operation was carried out according to the instructions attached. The extracted RNA plasmid was used for bioinformatics analysis and functional analysis.

Cloning and bioinformatics analysis of the salt-tolerance gene LpNAC022 from ryegrass

The integrity of RNA plasmid was tested by 1% agarose gel electrophoresis, and the concentration and purity of RNA were determined by ultra-micro spectrophotometer. ABM's 5X All-In-One RT MasterMix with AccuRT was used for reverse transcription, and the operation procedure was referred to the attached instruction manual.

Using the ryegrass reference genome as a template, primers were designed using the full-length cds sequence:

Upstream primer F1: 5'-ATGATCATGTCCGATCCGGC-3', as shown in Seq ID NO.3 in the sequence list;

Downstream primer R1: 5’-TTAGAAAGGGAGCAGCGTGTG-3’, as shown in the sequence table Seq ID NO.4.

cDNA was used as a template for amplification. The PrimeSTAR Max DNA Polymerase Kit from TaKaRa was used for amplification. The operation procedure was referred to the instruction manual. The PCR amplification system is shown in Table 1.

Table 1 PCR amplification system

The reaction conditions were as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 1 min, annealing at 55°C for 30 s, extension at 72°C for 1 min, 35 cycles, and finally running at 72°C for 5 min. PCR products were detected by 0.8% agarose gel electrophoresis.

In this embodiment, only one pair of primers and one amplification are needed to clone the LpNAC022 gene, and the sequence of the LpNAC022 gene is shown in Seq ID NO. 1 in the sequence list. The protein sequence encoded by the LpNAC022 gene is shown in Seq ID NO. 2 in the sequence list.

Cut the gel under UV light, and use TaKaRa's MiniBESTAgaroseGelDNAExtractionKit to recover and purify the target fragment. For specific operations, refer to the attached instructions. Use TaKaRa's DNAA-TailingKit to add an "A" tail to the 3' end of the target fragment DNA. After completion, take 4μl of the above DNA solution, add 1μl pMD18-T vector and 5μl Solution (containing ligase) and mix well, and react at 16℃ for 30min. After the reaction is completed, add 100μl DH5α competent cells to the above solution, ice bath for 30 minutes, heat at 42℃ for 45s, and then place on ice for 1min. Add 890μl SOC medium to the transformed competent cells, culture at 37℃ for 60 minutes, apply to LB medium containing ampicillin (Amp) and invert overnight culture. After culture, select single colonies for culture and use bacterial liquid PCR to verify whether the target fragment is successfully inserted. The target band can be amplified by bacterial liquid PCR for double-end sequencing, sequencing primer M13, to verify whether the cloning is successful.

The full length of the Lolium perenne gene LpNAC022 gene fragment is 1050 bp, and the sequence is shown in SEQ ID NO.1. The encoded protein contains 349 amino acids, and the sequence is shown in SEQ ID NO.2. Positions 10 to 136 are the conserved domains of the NAM (No apical meristem protein) protein. Analysis of the encoded amino acid sequence using ProtParam online software showed that the protein molecular weight was 38.19402 kD, the theoretical isoelectric point (pI) was 8.88, the total average hydrophilicity was -0.549, and the instability coefficient was 39.28, which was a stable alkaline protein.

The prediction results of the hydrophilicity/hydrophobicity of the protein encoded by the LpNAC022 gene are shown in Figure 1. The secondary structure of a protein is of great significance in the rapid folding of a protein peptide chain into a conformation with a specific function. The prediction of the secondary structure of a protein not only helps to understand the function of the protein and its mechanism of action, but also has a very important significance for correctly predicting the spatial structure of the protein. In addition, protein secondary structure information is widely used in protein molecular visualization, protein alignment, and protein structure prediction. SOPMA (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html) was used to predict the secondary structure of the perennial ryegrass LpNAC022 protein, as shown in Figure 2. The results showed that the protein contained about 17.48% α-helix, 13.47% extended main chain, 3.15% β-turn structure, and 65.90% random coil. FIG3 shows the predicted tertiary structure of the protein encoded by the LpNAC022 gene. Subcellular prediction shows that the protein encoded by the LpNAC022 gene is localized in the cell nucleus.

Functional analysis of LpNAC022 gene expressed in yeast

To preliminarily identify the function of LpNAC022, the previously cloned LpNAC022 fragment was selected and digested with Hind III and Xba I as restriction sites. The digested fragment was connected to the linearized pYES II yeast expression plasmid using the ClonExpress Ultra One Step Cloningkit kit from Novozyme (Nanjing). The recombinant pYES II-LpNAC022 plasmid was transferred into the Saccharomyces cerevisiae strain (INVScI) via Carrier DNA (Coollybo, China), with the blank pYESII plasmid as a control. The transformed yeast was cultured at 28°C for 48 hours in yeast-deficient medium (SD-Ura) containing 20 mg/mL glucose, and single clones were selected for PCR confirmation.

The primers are:

Forward primer F2: 5'-ttggtaccgagctcggatccatgatcatgtccgatccggc-3'; as shown in the sequence list SeqID NO.5;

Reverse primer R2: 5’-acatgatgcggccctctagattagaaagggagcagcgtgtg-3’; as shown in the sequence table Seq ID NO.6.

The forward primer F2 and the reverse primer R2 are based on the forward primer F1 and the reverse primer R1, and the homology arms for connecting the vector are added.

Positive transformed yeast was selected and cultured in liquid yeast extract peptone glucose medium (YPG) containing 2 mg/mL galactose, centrifuged at 150 rpm, and diluted by a multiple of 10. The diluted yeast suspension was spread on YPG medium containing 1.4 M NaCl and cultured at 28°C.

The yeast culture results are shown in Figure 4. In Figure 4, CK represents the control group, and 1.4M NaCl represents the treatment group with 1.4M NaCl added. pYES II-LpNAC022 is yeast connected with the target gene LpNAC022, and pYES II is empty yeast without foreign gene.

The results showed that the introduction of LpNAC022 made yeast cells grow faster under salt stress, indicating that the LpNAC022 gene has salt tolerance and can improve the tolerance of yeast to salt stress.

Functional analysis of LpNAC022 gene transferred into Arabidopsis

1. Overexpression vector construction

Using pCAMBIA1301-35SN as a vector, two restriction sites XbaI and SalI were selected to design LpNAC022 specific primers (Table 2), and the aforementioned RNA plasmid was used as a template. The target gene was amplified using a high-fidelity enzyme and the product was purified and recovered. Then, pCAMBIA1301-35SN was linearized using XbaI and SalI restriction endonucleases (NEB), and the product was purified and recovered. The purified target fragments were connected with the linearized pCAMBIA1301-35SN using ClonExpress II One Step Cloning Kit (Nanjing Novogene Biotech Co., Ltd.). The ligation products were transformed into Trelief ^TM 5α Chemically Competent Cell competent cells and spread on LB agar solid culture medium containing kanamycin (Kan, 50 mg/L). Positive single colonies were selected and verified by PCR and sequencing using universal primer M13 (Table 4-2). The correct bacterial liquid was used for plasmid extraction to obtain the overexpression vector pCambia1301-35S-LpNAC022.

Primer design for constructing overexpression Arabidopsis plants:

LpNAC022(1301)F: 5′-aagcttatcgataccgtcgacatgatcatgtccgatccggc-3′, as shown in Seq ID NO.7,

LpNAC022(1301)R: 5′-gggggatccactagttctagattagaaagggagcagcgtgtg-3′, as shown in Seq ID NO.8,

M13F: 5′-cgccagggttttcccagtcacgac-3′, as shown in Seq ID NO.9 in the sequence table,

M13R: 5′-agcggataacaatttcacacagga-3′, as shown in Seq ID NO.10 in the sequence listing,

2. Transformation of Agrobacterium with Overexpression Vector

The recombinant plasmid pCambia1301-35S-LpNAC022 in 1 was transferred into GV3101 Chemically Competent Cell (Shanghai Weidi Biotechnology Co., Ltd.) according to the instructions, and added to LB liquid medium without antibiotics for 4 hours. After centrifugation and resuspension, it was spread on LB solid medium containing Kan (50 mg/L) and rifampicin (50 mg/L), and inverted in a 28°C incubator for 2-3 days. Monoclonal colonies were selected, placed in liquid LB medium containing Kan and rifampicin, and shaken and cultured in a shaker at 28°C and 200r/min for 24 hours, and then PCR detection of bacterial liquid was performed. The positive monoclonal bacteria were host cells. The host cells were stored at -80°C with glycerol.

3. Arabidopsis transformation and functional verification

Arabidopsis planting: Wild-type seeds were planted in 1/2MS medium. After two days of vernalization, they were placed in a plant growth room with a light intensity of 14h/day and a humidity of 40-60% for about 2 weeks. They were then transplanted into pots (9cm×9cm×11cm), with 4 plants planted in each pot. After sowing, they were watered and covered with plastic wrap and cultured in a plant growth room.

Topping: Water every two days after transplanting, water with Hoagland's nutrient solution every week, and cut off the flower buds when the Arabidopsis first blooms;

Prepare the infection solution: Resuspend Agrobacterium in 5% sucrose solution to OD = 0.8. Add surfactant silwet-77 to a concentration of 0.02% (200uL/L) before infection, mix well and place at room temperature for 1 hour;

Dipping: After removing the pod-bearing flowers of Arabidopsis thaliana at the flowering stage, immerse the aerial part of the plant in the host cell suspension for 20-30 seconds;

Dark culture: After infection, the plants are bagged and cultured in the dark for 48 hours;

Cultivation after infection: watering method is the same as before infection;

Seed collection: Collect seeds when the siliques open naturally;

Screening of transgenic seeds: Cultivate the seeds after immersion in 1/2MS medium containing 50mg/L hygromycin, and culture them normally for 7-10 days after vernalization. Determine whether they are transgenic seeds based on their growth conditions: seeds that have been successfully transferred with the recombinant plasmid can grow normally on the resistant medium, while non-transgenic seeds cannot grow normally;

Transplantation of transgenic plants and positive testing: After the transgenic seeds germinate on the plate for 2 weeks, the positive plants are transferred to the soil for further cultivation. When the plants grow well, the leaves of the positive plants are taken for DNA extraction and PCR verification is performed using the target gene sequence primers.

The germination salt stress experiment was carried out on T3 transgenic plant seeds. The salt stress concentration was 150 mM NaCl, and the germination rate was counted on the 2nd, 8th and 14th days.

The experimental results are shown in Figure 5, in which (A) is the result of the Arabidopsis germination salt stress experiment, and (B) is the statistical results of the germination rate of Arabidopsis germination salt stress experiment on the 2nd, 8th and 14th days; CK is the control group, 150mM NaCl is the treatment group; WT is the non-transgenic wild-type Arabidopsis plant, and OE1 and OE2 are transgenic Arabidopsis plants. The results show that the germination rate of Arabidopsis transgenic with LpNAC022 gene is significantly better than that of wild-type Arabidopsis under salt stress conditions.

When the T3 transgenic plants grew to the 2-pair leaf stage, some transgenic Arabidopsis plants (OE1 and OE2) and non-transgenic wild-type Arabidopsis (WT) plants were placed in 1/2MS medium as a control, and some transgenic Arabidopsis plants (OE1 and OE2) and non-transgenic wild-type Arabidopsis (WT) plants were placed in 1/2MS+150mM NaCl medium to simulate high salt stress. After 7 days, fresh weight, root length and lateral root number were measured, and the results are shown in Figures 6 and 7.

Referring to Figure 6, in the control treatment, WT, OE1 and OE2 plants all grew normally. Under salt stress conditions, the growth of WT, OE1 and OE2 plants was inhibited, but the root system and aerial part growth of OE1 and OE2 were better than WT. According to the 7-day index data of Arabidopsis seedlings under salt stress shown in Figure 7, the fresh weight, root length and number of lateral roots of OE1 and OE2 after 7 days of 150mM NaCl treatment were significantly greater than WT, and the difference reached a significant level, while there was no difference in the control group (1/2MS).

The research results showed that the salt tolerance of Arabidopsis thaliana with the LpNAC022 gene was significantly better than that of wild-type Arabidopsis thaliana.

Based on the results of this example, the LpNAC022 gene can be used to breed salt-alkali tolerant perennial ryegrass varieties.

The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims (2)

1. A use of a ryegrass salt tolerance gene LpNAC022, characterized in that it is expressed in plants to improve the salt tolerance of plants; the gene LpNAC022 is the nucleotide sequence shown in the sequence table Seq ID NO.1. 2. The use of the ryegrass salt-tolerance gene LpNAC022 according to claim 1, characterized in that it is used to cultivate new salt-tolerant ryegrass varieties.