FtLEA14, a late embryogenesis abundant protein, from fritillaria thunbergii miq, its coding gene and use

Abstract

The application discloses a late embryogenesis abundant protein FtLEA14 of Fritillaria thunbergii, a coding gene and application, an amino acid sequence of the late embryogenesis abundant protein LEA14 is shown as SEQ ID NO.2, a nucleotide sequence of the coding gene is shown as SEQ ID NO.1, and the application further provides application of the coding gene of the late embryogenesis abundant protein FtLEA14 of Fritillaria thunbergii in improving drought resistance of Fritillaria thunbergii. The application provides a theoretical basis for promoting the drought stress resistance of Fritillaria thunbergii by using genetic engineering technology, and has important application value.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering application technology, and relates to a protein abundant in the late embryonic development of Fritillaria thunbergii with the core function of improving plant drought tolerance. Specifically, it relates to the protein FtLEA14, the encoding gene, and its application in the late embryonic development of Fritillaria thunbergii. Background Technology

[0002] Fritillaria thunbergii ( Fritillaria thunbergii Fritillaria thunbergii (Miq.) is a perennial herb belonging to the genus Fritillaria in the family Liliaceae. Its bulbs, after being sliced ​​and dried, are used as the medicinal material "Zhejiang Fritillaria," which has the effects of clearing heat, resolving phlegm, relieving cough, detoxifying, dispersing nodules, and reducing swelling. The main medicinal components of Zhejiang Fritillaria are zebulin A and zebulin B, among others. Modern pharmacological studies have shown that Zhejiang Fritillaria also has certain effects in anti-inflammatory, anti-tumor, antioxidant, and neuroprotective properties. Zhejiang Fritillaria prefers humid and cool climates and requires moist soil during its growing season, disliking drought. Especially during the reproductive growth and bulb enlargement stages, it often encounters seasonal drought, severely affecting the quality and yield of the medicinal material.

[0003] Late-embryonic abundant proteins (LEA proteins) are key regulators of plant responses to abiotic stresses, accumulating in large quantities as early as late embryonic development. Studies have shown that LEA proteins are closely related to seed dehydration tolerance, embryonic maturation, and responses to various environmental stresses such as drought, high salinity, and low temperature. However, their specific functions in the response of Fritillaria thunbergii remain unclear. Summary of the Invention

[0004] This invention provides the nucleotide and amino acid sequences of FtLEA14, a protein abundant in the late embryonic development of Fritillaria thunbergii, as well as its subcellular localization and function in resisting drought stress, paving the way for future regulation using genetic engineering techniques. FtLEA14 This study provides a theoretical basis for improving the drought stress resistance of Fritillaria thunbergii by gene expression.

[0005] On the one hand, the present invention provides FtLEA14, a late-stage embryonic abundant protein of Fritillaria thunbergii, which reduces leaf water loss and thus improves the plant's resistance to drought stress. The aforementioned late-stage embryonic abundant protein FtLEA14 includes a polypeptide (protein) having the amino acid sequence shown in SEQ ID NO. 2; or a protein having the characteristics of FtLEA14, formed by substitution, deletion, or addition of one or more amino acids to the amino acid sequence shown in SEQ ID NO. 2.

[0006] On the other hand, the present invention provides a coding gene for FtLEA14, a protein abundant in the late embryonic development of Fritillaria thunbergii, the nucleotide sequence of which is shown in positions 1 to 456 of SEQ ID NO.1.

[0007] In this invention, "isolated DNA" and "purified DNA" refer to DNA or fragments that have been isolated from sequences flanking them in their natural state, and also to DNA or fragments that have been separated from components that accompany nucleic acids in their natural state, and from proteins that accompany them in the cell.

[0008] In this invention, the gene encoding FtLEA14, a abundant protein in the late embryonic development of Fritillaria thunbergii, refers to the nucleotide sequence encoding a polypeptide with Fritillaria thunbergii protein activity, such as the nucleotide sequence from position 1 to 456 of SEQ ID NO.1 and its degenerate sequence. This degenerate sequence refers to a sequence in which one or more codons in positions 1 to 456 of SEQ ID NO.1 are replaced by degenerate codons encoding the same amino acid. Due to codon degeneracy, a degenerate sequence with less than 70% homology to the nucleotide sequence from position 1 to 456 of SEQ ID NO.1 can also encode the sequence shown in SEQ ID NO.2. The aforementioned encoding gene can also refer to a nucleotide sequence with at least 70% homology to the nucleotide sequence shown in SEQ ID NO.1.

[0009] Fritillaria thunbergii of the present invention FtLEA14 Gene-related nucleotide full-length sequences or fragments thereof can usually be obtained by PCR amplification, recombination, or artificial synthesis.

[0010] The present invention also provides a recombinant expression vector containing the encoding gene of FtLEA14, a protein abundant in the late embryonic development of Fritillaria thunbergii. The recombinant expression vector is pHB-FtLEA14-YFP.

[0011] The present invention also provides an application of the above-mentioned encoding gene of Fritillaria thunbergii FtLEA14 in improving the drought resistance of Fritillaria thunbergii, by overexpressing the above-mentioned encoding gene to reduce the rate of water loss from leaves.

[0012] Beneficial Effects: Fritillaria thunbergii, a traditional and precious Chinese medicinal herb, has a large market demand. Currently, the entire genome of Fritillaria thunbergii has not been sequenced. This invention is the first to clone the coding sequence of FtLEA14, a protein abundant in late embryonic development of Fritillaria thunbergii, and analyzes the subcellular localization of FtLEA14 protein using transient expression in tobacco leaf epidermal cells; a water loss rate experiment on detached tobacco leaves shows that after 210 min of water loss, overexpression... FtLEA1 The gene can significantly reduce the rate of water loss from leaves, effectively improving the drought resistance of plants. The above research demonstrates the application of genetic engineering techniques to regulate... FtLEA14 This provides a theoretical basis and technical support for enhancing the drought resistance of Fritillaria thunbergii, ensuring yield and quality, and breeding new varieties with high stress resistance, and has important application prospects. Attached Figure Description

[0013] Figure 1 For the present invention FtLEA14 Image showing the PCR amplification results of the gene; Figure 2 This is a comparison of FtLEA14, a protein abundant in the late embryonic development of Fritillaria thunbergii, with its homologous protein (DNAMAN). Figure 3 Phylogenetic tree analysis of FtLEA14, a protein abundant in the late embryonic development of Fritillaria thunbergii, and its homologous proteins; Figure 4 A map showing the localization of FtLEA14, a protein abundant in the late embryonic development of Fritillaria thunbergii, in the epidermal cells of tobacco leaves. Figure 5 For overexpression FtLEA14 A diagram showing the effect of genes on the rate of water loss from plant leaves. Detailed Implementation

[0014] The present invention will be further illustrated below with reference to the accompanying drawings and specific embodiments. However, these embodiments are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0015] Experimental methods in the following examples that do not specify specific conditions are generally used under conventional conditions, such as those described in Molecular Cloning: A Laboratory Manual (4th Edition), or as recommended in the reagent instructions.

[0016] Example 1: Fritillaria thunbergii FtLEA14 Cloning of genes 1. Obtaining plant materials Total RNA was extracted from the stems, leaves, buds, and flowers of normally growing Fritillaria thunbergii.

[0017] 2. RNA extraction Using Beijing Quanshijin Biotechnology Co., Ltd. TransZol The "UP Plant Total RNA Extraction Kit" extracts total RNA, and the integrity of the RNA is identified by gel electrophoresis. The purity and concentration of the RNA are determined by a spectrophotometer (Nanodrop 2000).

[0018] 3. Full-length cloning of genes Based on the nucleotide sequence and protein function annotation results provided by the laboratory's previous full-length transcriptome analysis, the *Fritillaria thunbergii* was obtained. FtLEA14 Full-length gene.

[0019] The extracted RNA was reverse transcribed (TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix) to obtain cDNA. Using the first-strand cDNA as a template, primer FtLEA14 was used... -PCR was performed using F(5'-ATGTCCGAGCTACTAAATAAGGC-3') and FtLEA14-R(5'-TCAGAAGATATCAGACAGACTAGGC-3') to amplify a 456 bp fragment (e.g., Figure 1 (As shown), the pMD18-T vector was recovered and ligated, and M13-47 and RV-M were used as universal primers. The sample was then sent to Hangzhou Youkang Biotechnology Co., Ltd. for sequencing.

[0020] Combining the sequencing results with NCBI's ORF Finding (http: / / www.ncbi.nlm.nih.gov / gorf) predictions, the Zhejiang Fritillaria was obtained. FtLEA14 The open reading frame of the gene was amplified by PCR to obtain a 456 bp sample of Fritillaria thunbergii. FtLEA14 Nucleotide sequence (SEQ ID NO.1). The sequencing results were compared with the BLAST database on the NCBI website (GenBank, http: / / blast.ncbi.nlm.nih.gov / ), and its nucleotide sequence and encoded protein showed high homology with the known Arabidopsis thaliana AtLEA14 gene.

[0021] Example 2: Fritillaria thunbergii FtLEA14 Gene sequence information and homology analysis Fritillaria thunbergii of the present invention FtLEA14 The full-length coding sequence of the gene is 456 bp, and the detailed sequence is shown in SEQ ID NO.1. Based on the open reading frame sequence, the amino acid sequence of FtLEA14, a protein abundant in the late embryonic development of Fritillaria thunbergii, was deduced. It consists of 151 amino acids, has a molecular weight of 16.54 kDa, and an isoelectric point (pI) of 5.68. The detailed sequence is shown in SEQ ID NO.2.

[0022] Fritillaria thunbergii FtLEA14 The open reading frame sequence of the gene and the amino acid sequence of its encoded protein were subjected to nucleotide and protein homology searches in NCBI using the BLAST program. The results showed that it was similar to Arabidopsis thaliana. AtLEA14 Genes exhibit high similarity at the amino acid level and contain conserved WHy domains (such as... Figure 2 (As shown). Phylogenetic analysis showed that the abundant protein FtLEA14 in the late embryonic development of Fritillaria thunbergii has high homology with LEA14 proteins in other known species (e.g., Figure 3 (As shown).

[0023] Example 3: Construction of recombinant expression vector FtLEA14-YFP and subcellular localization analysis of FtLEA14, a protein abundant in late embryonic development, in tobacco leaves. based on FtLEA14Design specific primers F (5'-TCCTCTCTCAAGCTTGGATCCATGTCCGAGCTACTAAATAAGGC-3') and R (5'-CCCTTGCTCACCATACTAGTGAAGATATCAGACAGACTAGGC-3') to introduce the gene's coding sequence. BamH I and Spe I. Restriction site. Using homologous recombination, the target fragment with the restriction site was ligated into the pHB-YFP vector to construct the pHB-FtLEA14-YFP recombinant expression vector.

[0024] The correct recombinant expression vector was screened using PCR and sequencing. The recombinant plasmid was then transformed into Agrobacterium competent cells GV3101 using the freeze-thaw method to obtain recombinant Agrobacterium GV3101 containing the recombinant expression vector. Simultaneously, the empty vector (35S: YFP, vector) was transformed as a positive control.

[0025] The identified GV3101 strain was inoculated into 5 mL of YEP (containing 50 mg / L Kan) and cultured at 28°C and 180 rpm until OD. 600 The value is approximately 0.6; take 1 mL of bacterial culture and add it to 25 mL of YEP liquid medium, and incubate at 28°C until the OD value reaches 0.6. 600 The OD value was approximately 0.6; 10 mL of bacterial culture was centrifuged at 4500 rpm for 15 min; the bacterial cells were resuspended in MS liquid medium until the OD value reached approximately 0.6. 600 The concentration was approximately 0.6, and AS and MES were added. The mixture was then placed at room temperature in the dark for at least 3 hours. The bacterial solution was injected into tobacco leaves using a syringe, and after 48 hours of dark incubation, it was observed using a laser confocal microscope (excitation wavelength 514 nm). The results are as follows: Figure 4 As shown, the empty vector (35S:YFP) is localized to the cell nucleus and cell membrane, while FtLEA14-YFP is localized to the cell nucleus and cell membrane.

[0026] Example 4: Overexpression of Fritillaria thunbergii FtLEA14 Effects of genes on leaf water loss rate The method for obtaining positive Agrobacterium GV3101 and transiently infecting tobacco leaves was as described in Example 3, with the empty vector (35S: YFP, vector) from Example 3 used as a control. After 48 h of dark incubation, the relative water loss (RWL) of tobacco leaves was measured under room temperature and 50% relative humidity. RWL = 100 × (W1 - W2) / W1, where W1 is the fresh weight of the separated leaves before dehydration, and W2 is the leaf weight after dehydration for 20 min, 40 min, 60 min, 90 min, 120 min, 150 min, 210 min, 270 min, 330 min, and 390 min. The water loss rate was calculated and a line graph was plotted, as shown below.Figure 5 As shown. By Figure 5 It can be seen that the expression FtLEA14 The water loss rate of detached leaves expressing the gene was significantly lower than that of leaves expressing the empty vector, especially after 210 min of water loss. FtLEA1 The gene can significantly reduce the rate of water loss from leaves, effectively improving the drought resistance of plants, thus demonstrating... FtLEA14 It can reduce the water loss rate of plants and improve their tolerance to osmotic stress.

[0027] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A protein FtLEA14 abundant in the late embryonic development of Fritillaria thunbergii, characterized in that, The amino acid sequence of FtLEA14, a protein abundant in late embryonic development, is shown in SEQ ID NO.

2.

2. A gene encoding FtLEA14, a protein abundant in the late embryonic development of Fritillaria thunbergii as described in claim 1, characterized in that, The encoding gene is a nucleotide sequence as shown in SEQ ID NO.

1.

3. A recombinant expression vector, characterized in that, It includes the gene encoding FtLEA14, a protein abundant in the late embryonic development of Fritillaria thunbergii as described in claim 2.

4. The recombinant expression vector according to claim 3, characterized in that, The recombinant expression vector is pHB-FtLEA14-YFP.

5. The application of the gene encoding FtLEA14, a protein abundant in the late embryonic development of Fritillaria thunbergii as described in claim 2, in improving the drought resistance of Fritillaria thunbergii, characterized in that, Overexpression of the encoded gene reduces the rate of water loss from leaves.

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