Cloning and application of salt-tolerant gene lpbhlh94 of lilium longiflorum

Abstract

The present application relates to the cloning of salt-tolerant gene LpbHLH94 of Lilium pumilum and its application, comprising the following steps: (1) cloning of the target gene; (2) construction of the expression vector; (3) detection of the physiological indexes of the transgenic plants. In this experiment, Lilium pumilum is taken as the research object, and the LpbHLH94 gene is cloned by using the RT-PCR technology; the overexpression vector is constructed, and the tobacco is transformed by using the leaf disc method mediated by Agrobacterium; the function of the overexpression of LpbHLH94 in improving the salt stress of tobacco is identified through the transgenic tobacco, the function of the bHLH gene of Lilium pumilum under the stress of adversity is revealed, the foundation for the further application of Lilium pumilum in lily breeding is laid, and the theoretical basis for the function and role of the bHLH transcription factor is provided.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of genetic technology, in particular to a Lilium pumilum LpbHLH94 gene cloning and application of the gene in improving salt stress resistance of transgenic tobacco. BACKGROUND

[0002] bHLH family transcription factors are one of the largest transcription factor families, have a variety of biological functions, and regulate the transcriptional expression of target genes by specifically binding to the cis-acting elements in the promoter region of related genes, thereby regulating the adaptive response of plants and playing a role in regulating the growth and development of plants, non-biological stress and metabolic network response. Research has found that bHLH transcription factors can regulate the tolerance of plants to salt stress by affecting the osmotic balance of plants, and play an important role in responding to salt stress.

[0003] Lilium pumilum, also known as mountain orchid, is a perennial herb of Liliaceae. Its unique flower shape is bell-shaped and involuted, and the flower color is bright red, which has high ornamental value. At the same time, it is a high-quality lily germplasm resource with strong disease resistance and excellent traits such as salt and alkali tolerance and drought tolerance, and has high application value in landscaping. It is a good parent material for breeding resistant lily varieties.

[0004] In this experiment, Lilium pumilum was used as the research object, and the gene LpbHLH94 was screened from the transcriptome of Lilium pumilum. It was cloned from the cDNA of Lilium pumilum by homologous recombination technology. Further, a tobacco overexpression vector was constructed, and a tobacco leaf disc was infected by Agrobacterium-mediated method to obtain an overexpression tobacco plant. Further, the related physiological indexes were measured to verify the salt tolerance function, which laid a foundation for further exploring the molecular mechanism of responding to high salt stress, and provided a reference for revealing the stress resistance mechanism of Lilium pumilum. SUMMARY

[0005] One of the purposes of the present application is to provide a salt-tolerant gene derived from Lilium pumilum, LpbHLH94, the nucleotide sequence of which is shown in SEQ ID NO. 1, and the amino acid sequence encoded by which is shown in SEQ ID NO. 2.

[0006] The second purpose of the present application is to provide a recombinant expression vector composed of the above-mentioned salt-tolerant gene LpbHLH94 and a plant expression vector.

[0007] As a preferred solution, the plant expression vector is GV1300.

[0008] The third purpose of the present application is to provide a recombinant Agrobacterium cell containing the above-mentioned salt-tolerant gene LpbHLH94 or the above-mentioned recombinant expression vector.

[0009] The fourth object of the present application is to provide a transgenic tobacco containing the salt-tolerant gene LpbHLH94, or the recombinant expression vector, or the recombinant Agrobacterium cell. The transgenic tobacco can be obtained by the following method: cloning the partial nucleotide sequence of the LpbHLH94 gene of Lilium paradoxum containing the ORF open reading frame by RT-PCR, obtaining the LpbHLH94-GV1300 recombinant plant overexpression vector by homologous recombination, transforming the recombinant plasmid into Agrobacterium EHA105 by freeze-thaw method, infecting tobacco by Agrobacterium-mediated leaf disc method, screening positive plants by hygromycin, collecting leaf of the positive plants to extract DNA and RNA, and performing molecular level identification; and detecting the transgenic tobacco with overexpression of LpbHLH94.

[0010] The fifth object of the present application is to provide the application of the salt-tolerant gene LpbHLH94 of Lilium paradoxum in improving the salt-tolerance of plants.

[0011] As a preferred solution, the plant is tobacco.

[0012] As a preferred solution, the salt-tolerance of tobacco to salt stress is enhanced by overexpression of the salt-tolerant gene LpbHLH94.

[0013] The present application has the following advantages and positive effects:

[0014] The present application provides a salt-tolerant gene LpbHLH94, and the full-length coding sequence of the gene is cloned. By determining the changes of physiological and biochemical indexes of heterologous transgenic tobacco and wild tobacco before and after salt stress treatment, it is found that the gene can endow tobacco with salt stress tolerance. It is shown that the LpbHLH94 gene has the function of improving the salt-tolerance of plants, and has important significance for revealing the molecular mechanism of Lilium paradoxum adapting to high-salt habitats and cultivating salt-tolerant crops. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 Lilium paradoxum RNA extraction electrophoresis result map.

[0016] Figure 2 Lilium paradoxum LpbHLH94 target fragment amplification electrophoresis map.

[0017] Figure 3 Lilium paradoxum LpbHLH94 bacterial liquid PCR electrophoresis map after being introduced into Escherichia coli.

[0018] Figure 4 Construction of Lilium paradoxum LpbHLH94 gene expression vector Figure 4-1 : Plant expression vector GV1300 double enzyme digestion product electrophoresis map Figure 4-2 : Recombinant plasmid PCR electrophoresis map

[0019] Figure 5 Figure 1 is a diagram of PCR identification of LpbHLH94 gene after transformation of Agrobacterium.

[0020] Figure 6 Figure 2 is a diagram of the transformation process of transgenic tobacco.

[0021] Figure 7 Figure 3 is a diagram of identification results of transgenic tobacco plants; A: electrophoresis of PCR identification of DNA of transgenic tobacco T0 plants; B: electrophoresis of PCR identification of RNA of transgenic tobacco T0 plants.

[0022] Figure 8 Figure 4 is a diagram of relative expression amount of transgenic tobacco.

[0023] Figure 9 Figure 5 is a diagram of phenotype changes of wild-type tobacco and transgenic tobacco after salt stress treatment.

[0024] Figure 10 Figure 6 is a diagram of physiological index detection results of wild-type tobacco and transgenic tobacco after salt stress treatment; 10-1 is a detection result of chlorophyll content after salt stress; 10-2 is a result of influence of salt stress on relative conductivity of transgenic tobacco; 10-3 is a result of influence of salt stress on antioxidant enzyme activity of transgenic tobacco; 10-4 is a result of influence of salt stress on malondialdehyde of transgenic tobacco; and 10-5 is a result of influence of salt stress on hydrogen peroxide of transgenic tobacco.

[0025] The application will be described in further detail below in combination with the drawings and specific embodiments. DETAILED DESCRIPTION

[0026] Example 1: Extraction of total RNA of L. pumilum and synthesis of cDNA

[0027] Total RNA of L. pumilum was extracted according to the instructions of Plant RNA Kit (OMEGA) kit, and the concentration and purity of the RNA were detected by a spectrophotometer, and the electrophoresis result is shown in Figure 1. Figure 1

[0028] The RNA was reversely transcribed into cDNA according to the instructions in the reverse transcription kit (CWBIO), and the reaction system was as follows:

[0029]

[0030] After 2 min at 42℃, the reaction was cooled on ice, and the steps were as shown in Table 1.

[0031]

[0032] The reaction procedure was 15 min at 37℃ and 5 s at 85℃, and the reaction product was stored in a refrigerator at -20℃ for standby.

[0033] ​Example 2: Primer design and PCR amplification of the LpbHLH94 gene of Lilium pulvinata.

[0034] Based on the coding region sequence of the *Lilium pulvinata* gene, specific primers were designed using Primer Premier5:

[0035]

[0036] Cloning of the coding region sequence: Using *Lilium pulvinata* cDNA as a template, PCR amplification was performed according to the instructions for the high-fidelity enzyme KOD-Plus-Neo (TOYOBO). The total reaction volume was 50 µL, in which:

[0037]

[0038] The PCR amplification procedure is as follows:

[0039]

[0040] After PCR, agarose gel electrophoresis was performed (Beijing, Saizhi Venture). The target band, accurately positioned, was cut and placed in a 1.5 mL centrifuge tube. The PCR product was purified and recovered according to the Gel Extraction Kit (OMEGA) instructions. 3 µL of the recovered product was then subjected to 1.5% agarose gel electrophoresis to check the band position. The qualified recovered product was stored at -20°C for later use. The electrophoresis results of the target fragment amplification are shown below. Figure 2 As shown.

[0041] Example 3: Construction of cloning vector and transformation of Escherichia coli.

[0042] Mix 4 µL of the recovered product with 1 µL of the 5 min TA / Blunt-ZeroCloning Kit vector in a PCR tube, and transform *E. coli* according to the Trans-T1 instructions. Incubate at 25 °C for 5 minutes. Add 5 µL of the ligation product to 50 µL of DH5α competent cells and incubate on ice for 30 minutes; heat shock at 42 °C for 45 seconds, then incubate on ice for 2 minutes; add 500 µL of LB medium and incubate at 37 °C with shaking at 200 rpm for 1 hour. Centrifuge at 5000 rpm for 1 minute, discard 300 µL of supernatant, resuspend the cells, plate them on Kana plates, and incubate upside down at 37 °C overnight.

[0043] Positive clone screening and identification: PCR verification was performed according to the 2×DiFAST Taq PCR Master Mix instructions.

[0044] The reaction system is shown in the table:

[0045]

[0046] The reaction procedure is shown in the table:

[0047]

[0048] After the PCR reaction, select bacterial solutions with bright and correctly positioned bands, add 10 mL of LB liquid medium containing 50 mg / L lambda, and incubate with shaking. The next day, take 500 µL of the bacterial solution for sequencing, and store the remaining bacterial solutions at 4°C. The PCR electrophoresis image of the successfully transformed *E. coli* is shown below. Figure 3 Sequencing results showed consistency with the transcriptome sequence, confirming successful cloning of the LpbHLH94 gene. The correctly sequenced bacterial culture was stored in 50% glycerol at -80°C for later use. Plasmids were extracted from the remaining bacterial culture according to the EZNAplasmid mini kit I (OMEGA) instructions. The concentration and purity were measured and recorded using a spectrophotometer, and the extracted plasmids were stored at -20°C for later use.

[0049] Example 4: Construction of the plant expression vector LpbHLH94 of Lilium pulcherrima.

[0050] Based on the GV1300 map of the plant expression vector and the ORF sequence of the LpbHLH94 gene, a single-fragment cloning URL was used ( https: / / crm.vazyme.com / cetool / singlefragment.html Design homologous recombination primer sequences,

[0051] The target fragment with homologous arms was amplified, and the primer sequences are shown in the table below:

[0052]

[0053] Using plasmids with correct cloning and sequencing results as templates, PCR amplification was performed using LpbHLH94-TY primers and KOD. After amplification, the target fragment was recovered from the gel and stored at -20°C for later use.

[0054] The GV1300 empty vector plasmid was linearized using two restriction endonucleases, KpnⅠ and BamHI. The PCR reaction was performed at 37℃ for 20 min. After the reaction, the products were subjected to electrophoresis on a 1.5% agarose gel. The electrophoresis image is shown below. Figure 4-1 The double-enzyme digested vector product was recovered via gel extraction. The recovered gel product, after passing testing, was stored at -20°C for later use. The reaction system is shown in the table below.

[0055]

[0056] Following the instructions of the Clon ExpressII One Step Cloning Kit (Novizan), the linearized vector and the insert plasmid were subjected to homologous recombination reaction at 37°C for 30 min. The reaction system is shown in the table below.

[0057]

[0058] After the recombination reaction was completed, the recombinant product was transformed into competent E. coli DH5α cells. The next day, single colonies were picked for colony PCR verification. The electrophoresis results are as follows: Figure 4-2 The bands indicated that the target gene fragment was successfully ligated into the vector, named GV1300-LpbHLH94-GFP. The positive bacterial culture with the correct bands was added to 10 mL of LB liquid medium containing 50 mg / L Kana and incubated overnight at 37°C with shaking at 200 rpm. The plasmid was extracted from the positive bacterial culture with the correct bands and stored at -20°C for later use.

[0059] Example 5: Transformation of Agrobacterium with plant expression vector.

[0060] The recombinant plasmid successfully constructed in the previous step was transferred into EHA105 Agrobacterium competent cells (Weidi Biotechnology) using the freeze-thaw method. The EHA105 Agrobacterium competent cells were then removed and placed at room temperature. When they partially thawed and were in an ice-water mixture, they were immediately inserted into ice. Add 5 µL of recombinant plasmid to 100 µL of EHA105 Agrobacterium competent cells, and sequentially incubate on ice for 5 min, flash freeze in liquid nitrogen for 5 min, heat shock at 37°C for 5 min, and incubate on ice for 5 min. Add 700 µL of antibiotic-free YEP liquid medium to a centrifuge tube and incubate at 28°C and 200 rpm for 2-3 h with shaking. Spread the bacterial culture onto YEP solid medium (containing 50 mg / L Kana and 20 mg / L Rif) and incubate upside down at 28°C for 2-3 days. After incubation, accurately pick single colonies and place them in 10 mL of YEP liquid medium (containing 50 mg / L Kana and 20 mg / L Rif) and incubate at 28°C and 200 rpm for 12-16 h with shaking. Then, use 1 µL of the bacterial culture as a template for PCR verification using GVTY-LpbHLH94. After identifying a positive monoclonal bacterial strain, 500 μL of the correctly banded bacterial solution was mixed thoroughly with an equal volume of 50% glycerol and stored at -80°C for later use. The PCR identification image of the bacterial solution after Agrobacterium transfection is shown below. Figure 5 .

[0061] Example 6: Transformation and infection of tobacco and identification of transgenic positive tobacco.

[0062] Select robust, sterile tobacco seedlings. In a laminar flow hood, slice tobacco leaves, avoiding the midrib. Lay the sliced ​​leaves face up on the pre-culture medium and incubate at 28°C in the dark for 2 days. Take the GV1300-LpbHLH94-GFP Agrobacterium suspension and streak it on YEP solid medium containing antibiotics for activation. The verified suspension is added to 10 mL of YEP liquid medium containing 50 mg / L Kana and 20 mg / L Rif and cultured until OD600 = 0.6-0.8. Resuspend the collected cells in 50 mL of 1 / 2 MS liquid medium. In a laminar flow hood, place the pre-cultured tobacco leaves into the invasive staining solution. After 5 minutes, remove the leaves, air dry them, and co-culture them on co-culture medium in the dark for 2 days. After co-culture, the leaves were transferred to selection medium, which was changed after 7 days, and then every 14 days until adventitious buds differentiated from the leaves. Rooting culture: When the adventitious buds grew to about 2 cm, they were cut off and transferred to rooting selection medium to induce rooting. When they grew to 3-4 leaves, DNA and RNA could be extracted from the leaves for detection of positive plants. Transgenic tobacco transformation process. Figure 6 ;

[0063] DNA was extracted from leaves of positive-positive and wild-type tobacco plants according to the Plant DNA Kit (OMEGA) instructions, and DNA quality was assessed. PCR was performed using GYTY-LpbHLH94 primers, with wild-type tobacco DNA and sterile water as controls. Detection was performed by 1% agarose gel electrophoresis. PCR identification and electrophoresis of DNA from T0 generation transgenic tobacco plants were also performed. Figure 7 A; Following the instructions of the Plant RNA Kit (OMEGA), RNA was extracted from positive plants and wild-type tobacco plants that passed DNA testing in the previous step and reverse transcribed into cDNA. Using cDNA as a template, PCR was performed using GYTY-LpbHLH94 primers. Wild-type tobacco DNA and sterile water were used as controls, and the results were detected by 1% agarose gel electrophoresis. PCR identification electrophoresis of RNA from transgenic tobacco T0 generation plants. Figure 7 B;

[0064] qRT-PCR primers for the LpbHLH94 gene were designed using the NCBI website. Wild-type tobacco cDNA was used as a control, and NtActin was used as an internal reference gene. Quantitative PCR was performed, and the relative expression levels of transgenic tobacco were as follows: Figure 8 .

[0065] The table shows the primer sequences for LpbHLH94 fluorescence quantitative PCR:

[0066]

[0067] To further investigate the role of the LpbHLH94 gene in salt stress, the two lines with the highest expression levels, OE1 and OE3, were selected for subsequent experiments. Transgenic tobacco seedlings that had been identified were transferred to flowerpots after two days of hardening off. The substrate was peat moss:vermiculite:perlite = 3:1:1, and the plants were cultured in a plant culture room with a photoperiod of 14h light / 10h darkness until seed harvest.

[0068] Example 7 verifies the salt tolerance of transgenic tobacco plants.

[0069] Seeds from wild-type tobacco, tobacco plants transgenic with the GV1300 empty vector, and transgenic tobacco plants overexpressing *Lilium filamentosa* LpbHLH94 (T1 generation, lines OE1 and OE3) were planted on 1 / 2 MS medium (containing 25 mg / L hygromycin). After 10 days, they were transferred to MS medium and cultured in wide-mouth bottles for 15 days. Tobacco plants with 3-4 cotyledons were then transferred to uniformly sized pots in a peat moss:vermiculite:perlite = 3:1:1 medium and cultured for another 15 days in a plant culture room with 16 hours of light daily and a temperature of 22-25℃. Between 9:00 and 10:30 AM, 100 mL of 300 mM NaCl solution was slowly injected into the pots without touching the plants. Tobacco plants watered with plain water during the same period served as a control. This process was repeated every other day. Photographs were taken and samples were collected at 0, 6, and 15 days of salt treatment, and the relevant parameters were measured. The measured indicators included phenotypic characteristics, chlorophyll content, relative electrical conductivity, antioxidant enzyme activity, hydrogen peroxide content, and malondialdehyde (MDA) content. Healthy leaves from the same location were selected for measurement. A sufficient number of healthy leaves were wrapped in aluminum foil and stored in an ultra-low temperature freezer at -80°C for later use. The results are as follows: Figure 9 and Figure 10 As shown.

[0070] The results showed that the transgenic tobacco plants had a strong salt tolerance.

Claims

1. A salt-tolerant gene for lily of the narrow leaf, LpbHLH94, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.1, or its encoded amino acid sequence is shown in SEQ ID NO.

2.

2. A recombinant overexpression vector, characterized in that, It contains the LpbHLH94 salt tolerance gene for Lilium pulvinata as described in claim 1.

3. A recombinant Agrobacterium cell, characterized in that, It contains the gene of claim 1 or the recombinant overexpression vector of claim 2.

4. The application of the LpbHLH94 salt tolerance gene from *Lilium pulvinata* in improving salt tolerance in tobacco, characterized by: The nucleotide sequence of the LpbHLH94 gene is shown in SEQ ID NO.1, or the amino acid sequence it encodes is shown in SEQ ID NO.

2.

5. The application as described in claim 4, characterized in that, Overexpression of the salt tolerance gene LpbHLH94 improved the salt tolerance of tobacco.

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