CN114085854B - A rice drought-resistant and salt-tolerant gene OsSKL2 and its application - Google Patents

Abstract

The application discloses a drought-resistant and salt-tolerant gene OsSKL2 of rice and application thereof, and relates to the technical field of genetic engineering, wherein the gene has a nucleotide sequence shown as SEQ ID NO. 1. The gene can regulate and control the survival rate of rice under drought and high salt, and has positive regulation and control effects on rice drought and high salt; the gene provides direct evidence for the regulation and control effect of shikimic acid in abiotic stress, provides a new research approach for improving abiotic stress of crops, and has important theoretical and practical significance for cultivating drought-resistant and salt-resistant materials.

Description

A rice drought-resistant and salt-tolerant gene OsSKL2 and its application

Technical field

The invention relates to the field of genetic engineering technology, and specifically relates to a rice drought-resistant and salt-tolerant gene OsSKL2 and its application.

Background technique

Rice is the world's main food crop, providing food for more than half of the world's population. However, with the growth of population, deterioration of environmental conditions and reduction of cultivated land area, the contradiction between supply and demand between population increase and food demand has become increasingly prominent. At present, the main environmental factors causing the decline in rice yield are drought and soil salinization. According to statistics, the rice planting area and output relying on traditional flood irrigation methods account for approximately 55% and 75% of the world's total rice area and output. In recent years, the reduction in grain production caused by drought and soil salinization has shown an aggravating trend, threatening food security production. Therefore, discovering drought-resistant and salt-tolerant genes and analyzing their mechanisms of action is an important goal of biotechnology breeding, which is of great significance to ensuring high and stable rice yields and food safety production.

In order to adapt to changes in the external environment, plants have evolved a variety of pathways and regulatory mechanisms during their long-term evolution to cope with environmental stimuli and ensure normal growth and development. Studies have shown that stress sensors located on the surface of plant cells (such as cell wall, plasma membrane) and organelles (such as cytoplasm, nucleus) capture external stress signals and quickly transform and transmit the stimulation signals into the cells to influence second messengers (such as Ca ²⁺ , ROS, NO, phospholipids, etc.) levels, thereby changing the expression of regulatory genes and transcription factors. Transcription factors induce plant protection mechanisms such as detoxification and repair stress responses by binding and regulating the expression of downstream functional genes to ensure the normal growth of plants. The maize transcription factor ZmbHLH124 directly binds to and activates the expression of ZmDREB2A, thereby improving the drought tolerance of maize. In rice, OsMYB3R-2 reduces the sensitivity of ABA and enhances rice's resistance to cold, high salt and drought by regulating the expression of cell cycle protein genes such as OsCycB1;1 and OsKNOLLE2. Although there have been many reports on drought and salt tolerance genes in plants, little is known about the main functional genes that directly involve primary metabolism and secondary metabolism pathways in drought and high salt stress.

The shikimate cycle widely exists in plants and microorganisms and is an important hub connecting carbohydrate metabolism and secondary metabolism, and provides substrates for other secondary metabolisms. The shikimate pathway is a biochemical reaction catalyzed by multiple metabolic enzymes, and its products are phenylalanine, tyrosine, and tryptophan. Among them, shikimate kinase (SK) catalyzes the fifth step of the shikimate pathway. The genes encoding SK in rice are SK-1, SK-2, SK-3, SKL1, and SKL2. The study found that SK1 and SK2 are closely related to the formation and development of chloroplasts. Although studies have shown that shikimic acid content changes significantly under drought and high-salt conditions, there is no direct evidence on how to regulate the response to drought and high-salt stress, and its functional genes are also unclear. Therefore, a rice drought-resistant and salt-tolerant gene OsSKL2 and its application were proposed.

Contents of the invention

The purpose of the present invention is to overcome the shortcomings of the existing technology and provide a rice drought-resistant and salt-tolerant gene OsSKL2 and its application.

The present invention achieves the above objects through the following technical solutions:

The invention provides a rice drought-resistant and salt-tolerant gene OsSKL2, which has a nucleotide sequence shown in SEQ ID NO. 1 and a full length of 1128 bp.

The invention also provides an application of the above-mentioned rice drought resistance and salt tolerance gene OsSKL2 in improving rice drought resistance and salt tolerance.

A further improvement is that the gene OsSKL2 has a positive regulatory effect on the drought resistance and salt tolerance of rice. Overexpression of transgenic rice plants can improve the survival rate of rice under drought and high salt conditions, while RNAi silencing of transgenic rice plants can improve the survival rate of rice under drought and high salt conditions. The survival rate is significantly reduced under

The present invention also provides a protein encoded by the above-mentioned rice drought resistance and salt tolerance gene OsSKL2, which has the amino acid sequence shown in SEQ ID NO.2.

A further improvement is that the protein is located in chloroplasts and is induced by drought and high salt expression, and the shikimate kinase encoded by it is a key metabolic enzyme that catalyzes the shikimate pathway.

The present invention also provides a plasmid vector, which is obtained by inserting the above-mentioned rice drought resistance and salt tolerance gene OsSKL2 into the pEASY-T1 vector.

The present invention also provides a genetically engineered host cell, which is an Escherichia coli competent Trans5α cell with the above plasmid vector.

The invention also provides a method for obtaining the rice drought-resistant and salt-tolerant gene OsSKL2. The nucleotide sequence of the OsSKL2 gene is used as a template to design specific amplification primers, and the rice Nipponbare variety is used as the material, RNA is extracted and reverse transcribed into cDNA. , the gene OsSKL2 was obtained through PCR amplification technology.

A further improvement is that the specific amplification primer for PCR amplification is:

SKL2–F-1: ATGAGGATAGGGGCAGGGGCGAACA;

SKL2–R-1: TCAGATATTGGTCGGTGGGGCGTCG.

The present invention also provides an over-expression vector, which is a pCAMBIA1301 plant expression vector in which a multiple cloning site region is sequentially connected with the 35S promoter, the above-mentioned gene OsSKL2 and the NOS terminator.

The present invention also provides a subcellular localization vector. The subcellular fusion vector is obtained by cutting the above-mentioned gene OsSKL2 and the pCAMBIA1305 vector simultaneously with SpeI and BamHI and then ligating them. Specifically, it is pCAMBIA1305-SKL2. The upstream and downstream primers used for ligation are as follows:

OsSKL2-F:ATGTTGGCCTCCACTTGCTTCTCCG;

OsSKL2-R:TATGTTGGTGGGTGGTGCGTCGGA.

The invention provides the following beneficial effects:

Compared with the existing technology, the present invention has the following advantages: the present invention provides a rice drought-resistant and salt-tolerant gene OsSKL2 and its application. Positive regulatory effect; the discovery of this gene provides direct evidence for the regulatory role of shikimic acid in abiotic stress, provides a new research approach for improving abiotic stress in crops, and has important theoretical and theoretical implications for cultivating drought-resistant and salt-tolerant materials. Practical significance.

Description of the drawings

Figure 1 shows the map of RNAi silencing vector pEASY-T1;

Figure 2 shows the overexpression pCAMBIA1301 vector map;

Figure 3 shows the high-salt and drought-induced expression pattern of OsSKL2; 100mM mannitol solution and 200mM NaCl solution were used to treat four-week-old wild-type rice Zhonghua 11 respectively, and sampling was performed after 12 hours;

Figure 4 shows the subcellular localization analysis of OsSKL2;

Figure 5 shows the molecular identification diagram of OsSKL2 overexpression and RNAi silencing lines; WT is wild type, OE3 and OE6 are overexpression transgenic plants, and Ri6 and Ri9 are silenced transgenic plants;

Figure 6 shows the phenotype and survival rate statistics of OsSKL2 transgenic rice under normal and salt treatments; Figure 6a shows the phenotypes of wild-type and transgenic plants under high salt treatment, and Figure 6b shows the germination rate statistics. Four-week-old wild-type and transgenic rice plants were treated with 120mM and 140mM NaCl respectively. WT was a wild-type plant, OE3 and OE6 were overexpression transgenic plants, and Ri6 and Ri9 were silent transgenic plants;

Figure 7 shows the phenotype and survival rate statistics of OsSKL2 transgenic rice under normal and drought treatments; Figure 7a shows the phenotypes of wild-type and transgenic plants under drought treatment, and Figure 7b shows the germination rate statistics. Four-week-old wild-type and transgenic rice plants were treated with 20% PEG respectively. WT was a wild-type plant, OE3 and OE6 were overexpression transgenic plants, and Ri6 and Ri9 were silent transgenic plants.

Detailed ways

The present application will be described in further detail below in conjunction with the accompanying drawings. It is necessary to point out here that the following specific embodiments are only used to further illustrate the present application and cannot be understood as limiting the protection scope of the present application. Those skilled in the field can refer to The above application contents make some non-essential improvements and adjustments to this application.

1. Material

Unless otherwise stated, the methods used in this example are conventional methods known to those skilled in the art. The reagents and other materials used, unless otherwise stated, are all commercially available products.

2. Method

2.1OsSKL2 gene acquisition

Select the rice Nipponbare variety, extract RNA and reverse-transcribe it into cDNA. Using rice cDNA as a template, primers SKL2–F-1 and SKL2–R- were designed based on the OsSKL2 gene sequence and the rice genome database, combined with the multiple cloning sites of the plant expression vector. 1. Perform PCR amplification and obtain PCR amplification products.

The primer sequence is:

SKL2–F-1: ATGAGGATAGGGGCAGGGGCGAACA;

SKL2–R-1:TCAGATATTGGTCGGTGGGGCGTCG;

The PCR reaction program was: 98°C, pre-denaturation for 10 min; 98°C, denaturation for 20 s; 65°C, annealing for 20 s; 72°C, extension for 2 min, 30 cycles; 72°C, renaturation for 10 min; and stored at 10°C.

The PCR amplification product was detected by agarose gel electrophoresis with a mass ratio of 2%, and the target fragment was recovered and connected to the pEASY-T1 vector (purchased from Quanshijin Biotechnology Co., Ltd., the vector map is shown in Figure 1), and obtained Connection product. The ligation product was transformed into E. coli competent Trans5α cells, and the plasmid was extracted. Use the extracted plasmid as a template, use SKL2-F-1 and SKL2-R-1 as primers for PCR amplification verification, and double-digest the plasmid with Kpn I and Pst I for detection, screen positive clones, and divide the positive clones into It was sent to BGI for sequencing, and the sequencing results were compared using Sequencher software. The results were consistent with predictions. The obtained recombinant plasmid was named T-SKL2.

2.2 Construction of OsSKL2 gene overexpression vector and silencing expression vector

Using pCAMBIA1301 (purchased from Shanghai Jielan Biotechnology Co., Ltd., the vector map is shown in Figure 2) as the original vector, a 35S promoter was connected between the EcoRI and SacI restriction sites of its multiple cloning site, and SphI A NOS terminator was added between the HindIII restriction site and the modified vector p1. Double-digest T-SKL2 with KpnI and PstI to obtain the target gene fragment, and double-digest p1 with KpnI and PstI to obtain the vector fragment. The above two fragments are connected using T ₄ ligase to construct the vector p1-OsSKL2, which is used as a transgenic rice OsSKL2 gene overexpression vector. Rice cDNA was used as a template, and OsSKL2 specific primers were used to amplify part of the OsSKL2 gene fragment. The products were double digested with BamHI and HindIII, cloned into the pRNAi-Ubi vector digested with the same enzyme, and transformed into TOP10 competent cells. namycin) and PCR screening and identification, and an intermediate recombinant plasmid with successful forward assembly of the target fragment was obtained. The vector-specific universal primer RNAi-Pst/RNAi-Mlu was used to amplify the intermediate recombinant plasmid. The product was double digested with PstI and MluI and cloned into the intermediate recombinant plasmid digested with the same enzyme. Then it was transformed into DH10B competent cells and treated with kanamycin LB. Plate screening\PCR\enzyme digestion and sequencing identification completed the construction of pRNAi-ubiSKL2 transformation vector. pRNAi-ubiSKL2 was digested with BamHI single enzyme, cloned into pRNAi-IP digested with the same enzyme (the pRNAi-IP vector after single enzyme digestion was treated with alkaline phosphatase), and transformed into DH10B competent cells. LB plate screening, enzyme digestion detection and sequencing identification completed the construction of pRNAi-IP-SKL2 vector.

2.3 Analysis of the induced expression pattern of OsSKL2

Four-week-old wild-type rice flower 11 was treated with 100mM mannitol solution and 200mM NaCl solution respectively, and then the roots and leaves of the rice treated for 12 hours were selected, their RNA was extracted, and their cDNA was obtained by reverse transcription, and then qRT-PCR was performed. Determine the expression levels in roots and leaves under treatment.

Design quantitative primers as follows:

SKL2-F:TTGGCTTCGGAAGGAGTGGATT

SKL2-R:TTCCCGAGCTCACACTTCTACG

The qPCR reaction program was: 94°C, pre-denaturation for 5 min; 94°C, denaturation for 20 s; 60°C, annealing for 20 s; 72°C, extension for 1 min, 30 cycles. Analyze the results graphically.

The results are shown in Figure 3. The expression of gene OsSKL2 was significantly induced by drought and high salt.

2.4 Subcellular localization analysis of OsSKL2

First, the vector of pCAMBIA1305-SKL2 was constructed. The SKL2 and p1305 vectors were cut with SpeI and BamHI at the same time, and then the target band was recovered by gel electrophoresis, and then the pCAMBIA1305-SKL2 vector was ligated with T4 ligase overnight to recover the pCAMBIA1305-SKL2 vector. The above vector was transferred into rice leaf protoplasts using a PEG transient transformation system. The transformation system was: 100 μL protoplasts, 100 μL pCAMBIA1305-SKL2 vector plasmid and 200 μL 45% PEG. Induce transformation for 30 minutes, dilute and centrifuge with 440 μL of W5 solution, then add 200 μL of WI solution, incubate in the dark for 12-18 hours, and then observe with a laser confocal microscope.

The primers used in subcellular localization are as follows:

OsSKL2-F:ATGTTGGCCTCCACTTGCTTCTCCG

OsSKL2-R:TATGTTGGTGGGTGGTGCGTCGGA

The results are shown in Figure 4, and the gene OsSKL2 was found to be located in chloroplasts.

2.5 Obtaining and identifying plants with Agrobacterium-mediated overexpression and silent expression of OsSKL2 gene

For the method of obtaining Agrobacterium-mediated overexpression plants and silent expression plants and the medium formula used in the culture process, please refer to the literature: Methods in Molecular Biology, vol.343: AgrobacteriumProtocols, 2/e, volume 1, specifically including the following steps:

(1) Obtaining induced callus from mature embryos of rice

Select mature and plump rice seeds, peel them and set aside. Prepare 1-2L of sterilized water, 75% (mass fraction) alcohol, hypochlorous acid and Tween (specific ratio: sterilized water: hypochlorous acid = 1:1, total volume: Tween = 1mL :1ul). First wash with 75% alcohol for about 5 minutes, then disinfect with sterilized water for 15 minutes, divided into two periods of 7-8 minutes each, then defoam with 75% alcohol 3 times, and finally wash with sterilized water several times until When the water becomes clear, put the mature seeds on sterilized filter paper to absorb the water, then place them on the callus induction medium and culture them at 26±1°C; after 10-15 days, transfer the induced milky yellow callus into Subculture on subculture medium. Subculture once every two weeks. After two subcultures, select subcultures for 5-7 days, and use calli with light yellow color for co-culture.

(2) Preparation of Agrobacterium bacteria for transformation of rice

The p1-OsSKL2 and pRNAi-IP-SKL2 vectors were transformed into Agrobacterium tumefaciens LBA4404, and recombinant Agrobacterium tumefaciens LBA4404/p1-OsSKL2 and LBA4404/pRNAi-IP-SKL2 were obtained. Recombinant Agrobacterium LBA4404/p1-OsSKL2 and LBA4404/pRNAi-IP-SKL2 were inoculated into YEP liquid medium (containing 50 μg/mL kanamycin and 50 μg/mL rifampicin), and cultured with shaking at 28°C until OD ₆₀₀ = 0.6-1.0; centrifuge at 5000 rpm for 5 minutes at room temperature to collect the cells, and then suspend them in a liquid co-culture medium. Adjust the cell concentration to OD ₆₀₀ = 0.4, which is the Agrobacterium suspension for co-culture transformation of rice.

(3) Agrobacterium infects rice vegetative organs

Select fresh, tender, milky yellow calluses, put them into a 100mL sterile conical flask, and add the Agrobacterium suspension prepared above; culture on a shaker at 28°C and 220rpm for 20-30 minutes; pour out the bacterial solution and remove the infection. The resulting callus was placed on a petri dish containing sterile filter paper to absorb excess bacterial fluid, then transferred to a solid co-culture medium and cultured in the dark at 22°C for 2-3 days.

(4) Selective training

Wash the co-cultivated calli with sterile water until the water becomes clear. Discard the cleaning solution, transfer it to a solution containing carbenicillin (100 mg/ml), shake and wash it for more than 30 minutes, blot it dry with sterile filter paper, and place it on the Dry the callus in a petri dish containing sterile filter paper; then select the callus to a screening medium containing carbenicillin (500mg/ml) and hygromycin (50mg/L), and culture it under light at 28°C for about 30 days until it grows out New resistance to healing.

(5) Differentiation of resistant callus

Select the new calli that grow well and are milky yellow from the screening medium and place them on the differentiation medium containing 50 mg/L hygromycin. They are first cultured in the dark at 28°C for 3 days, and then transferred to 30°C for full light culture for 15 days. -30d, green spots will appear; seedlings will differentiate after 30-40d.

(6) Rooting, seedling strengthening and transplanting

When the differentiated seedlings are 3cm high, transfer them to the rooting medium and cultivate them for 2-3 weeks; select seedlings 15cm tall and with well-developed root systems, add water and harden the seedlings at room temperature for 2-3 days, then wash away the medium with warm water and move them into a medium containing Cultivation of rice in buckets of culture soil. After the seedlings grow strong, they are moved to paddy fields for growth. Among the 32 transgenic rice seedlings with independent transformation events, 21 plants simultaneously amplified the target gene OsSKL2 fragment and hygromycin gene, and 8 plants were silenced by RNAi. After the T ₀ generation transgenic plants mature, the T ₁ generation seeds are harvested, and the T ₁ generation seeds are self-propagated to obtain T ₂ generation seeds.

2.6 Identification of transgenic rice

In order to detect transgenic rice, the extracted total DNA of transgenic rice was used as a template, the target gene OsSKL2 fragment and the hygromycin gene were used as detection objects, primers were designed, and PCR amplification was performed to preliminarily identify the transgenic plants.

Primers for detecting the OsSKL2 fragment of the target gene:

SKL2–F-2:ATGAGGATAGGGGCAGGGGCGAACA

SKL2–R-2:TCAGATATTGGTCGGTGGGGCGTCG

PCR reaction conditions were: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s; annealing at 60°C for 30 s; extension at 72°C for 1 min, 35 cycles; total extension at 72°C for 5 min.

Primers for detecting hygromycin gene:

Hyg-F: 5’-TAGGAGGGCGTGGATATGGC-3’

Hyg-R: 5’-TACACAGCCATCGGTCCAGA-3’

The PCR reaction program was: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 62°C for 30 s, extension at 72°C for 1 min, 34 cycles; total extension at 72°C for 5 min.

Primers for detecting pRNAi-IP-SKL2:

SKL2-Ri-N-F: TCCAGTCTGCACAAGTGAGC

SKL2-Ri-N-R:TCCAGCACAACACATTCAGC

Pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds; annealing at 60°C for 30 seconds; extension at 72°C for 1 minute, 35 cycles; total extension at 72°C for 5 minutes.

The results are shown in Figure 5. Overexpression (OE3 and OE6) and RNAi silencing (Ri6 and Ri9) transgenic rice plants were obtained.

2.7 Phenotypic identification and survival statistics of OsSKL2 transgenic rice under high salt and drought treatments

The above-identified OsSKL2 transgene overexpression and silent expression rice were selected for study, and wild-type Zhonghua 11 was used as a control in each group of treatments. Four-week-old wild-type and transgenic plants were treated with 120mM NaCl, 140mM NaCl and 20% PEG respectively and rehydrated for 1 day after drought treatment. After processing, photos were taken and survival rate statistics were taken.

The results are shown in Figures 6 and 7. After treatment with 120mM NaCl and 140mM NaCl, compared with the wild type, the survival rate of overexpression plants was significantly higher, while the survival rate of silent plants was significantly lower; similarly, rehydration after drought treatment The experimental results also showed that the survival rate of overexpressed plants was significantly higher, while the survival rate of silent plants was significantly lower; these results indicate that the OsSKL2 gene has salt and drought resistance.

The above-mentioned embodiments only express several implementation modes of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the patent scope of the present invention. It should be noted that, for those of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention.

sequence list

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<120> A rice drought-resistant and salt-tolerant gene OsSKL2 and its application

<141> 2021-12-16

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Ser Val His Ile Val Gly Ala Ser Thr Ala Ile Ala Gly Ala Val Ala

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Claims (2)

1. Drought-resistant and salt-resistant gene of riceOsSKL2The application of the gene in improving drought resistance and salt tolerance of rice is characterized in that the geneOsSKL2The nucleotide sequence of (2) is shown as SEQ ID NO. 1.

2. According to claim 1The use of (2) is characterized in that the geneOsSKL2The over-expression has positive regulation and control effect on drought resistance and salt tolerance of rice.