Larix gmelinii drought response gene LgLEA10, protein and application thereof

Abstract

This invention belongs to the field of genetic engineering technology, and more specifically, relates to a drought-responsive gene in larch trees. LgLEA10 Proteins and their applications. This invention provides a larch drought response gene. LgLEA10 The nucleotide sequence of the protein is shown in SEQ ID NO.1, and the amino acid sequence of the protein it expresses is shown in SEQ ID NO.2. This invention is the first to demonstrate that the Dahurian larch... LgLEA10 The gene is a regulatory gene for the larch's response to drought. This invention demonstrates through experiments that... LgLEA10 Gene overexpression can enhance the drought resistance of larch by significantly increasing the activity of antioxidant enzymes and osmotic regulators. This discovery provides important genetic resources and theoretical basis for breeding new larch varieties with strong drought resistance.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and more specifically, relates to a drought-responsive gene in larch trees. LgLEA10 Proteins and their applications. Background Technology

[0002] Drought is a natural phenomenon. Severe drought can limit the growth and development of plants and even lead to their death. This includes tree species with important ecological and economic value, such as the larch (Pinaceae), a representative tree species of northern cold-temperate coniferous forests, whose growth and development are also affected by drought.

[0003] To cope with drought and other adverse conditions, plants have evolved a series of molecular-level adaptation mechanisms, among which late embryogenesis abundant protein (LEA) plays a crucial role. LEA is highly hydrophilic and acts as a dehydration protectant. LEA proteins have three main biological functions. First, some LEA proteins can bind to calcium ions and other metal ions to isolate them from metal ion contamination. Second, some LEA proteins fold into α-helices during dehydration and insert laterally into the cell membrane, thereby maintaining cell structural stability. Finally, LEA proteins act as molecular chaperones, providing a protective protein structure, preventing protein aggregation, maintaining their three-dimensional structure, and stabilizing the glassy state of the intracellular matrix. However, no drought-resistant LEA genes have yet been reported to be cloned in larch. Summary of the Invention

[0004] The purpose of this invention is to provide a drought-responsive gene for larch. LgLEA10 Proteins and their applications are studied to address the aforementioned technical problems.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] This invention provides a drought response gene for larch. LgLEA10 The LgLEA10 The nucleotide sequence is shown in SEQ ID NO.1.

[0007] This invention is the first to reveal the properties of larch in the Xing'an Mountains. LgLEA10 The key role of the gene in regulating the response of larch to drought stress was identified by cloning a gene from Dahurian larch. LgLEA10 The gene was confirmed through experiments using an overexpression vector. LgLEA10 Gene overexpression can significantly increase the activity of antioxidant enzymes and the content of osmotic regulators, thereby significantly enhancing the drought resistance of larch.

[0008] The present invention also provides the larch drought response gene. LgLEA10 The encoded protein, the amino acid sequence of which is shown in SEQ ID NO.2.

[0009] The present invention also provides a recombinant expression vector containing the larch drought response gene. LgLEA10 .

[0010] Furthermore, the larch drought response gene is... LgLEA10 Insertion of pCAMBIA1300 overexpression vector plasmid Captain I and Sac Obtained between I sites.

[0011] The present invention provides recombinant engineered bacteria comprising the above-mentioned recombinant expression vector.

[0012] Further, the recombinant expression vector was transferred into Agrobacterium GV3101 to obtain the product.

[0013] This invention provides the larch drought response gene LgLEA10 and the larch drought response gene. LgLEA10 The application of the encoded protein, the recombinant expression vector, and the recombinant engineered bacteria in improving plant drought resistance, characterized in that improving plant drought resistance includes:

[0014] Increase plant cell growth under drought stress;

[0015] Increase the content of osmotic regulatory substances in plant cells under drought stress;

[0016] To enhance the activity of antioxidant enzymes in plant cells under drought stress;

[0017] The plants mentioned include larch.

[0018] Furthermore, the osmotic regulating substances include soluble proteins, proline, and soluble sugars.

[0019] Furthermore, the antioxidant enzymes include superoxide dismutase and peroxidase.

[0020] The present invention has at least the following beneficial effects:

[0021] Under drought stress, the level of reactive oxygen species (ROS) in plant cells increases sharply, causing oxidative damage to the cells. Antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) can effectively scavenge ROS and protect cells from oxidative damage. This invention has found through experiments that... LgLEA10 In larch plants with gene overexpression, the activities of these antioxidant enzymes were significantly higher than those in the control group, indicating that... LgLEA10 Gene overexpression enhances the plant's antioxidant capacity.

[0022] Meanwhile, osmotic regulators such as proline, soluble sugars, and soluble proteins play a crucial role in maintaining cellular osmotic pressure balance and protecting cell structure and function. Experimental results show that... LgLEA10 In larch plants with gene overexpression, the content of these osmotic regulators is significantly increased, which helps the plants maintain cellular water balance and normal physiological functions under drought conditions.

[0023] In summary, this invention not only demonstrates for the first time... LgLEA10 The gene is a regulatory gene for larch's response to drought stress. Experiments have also confirmed that its overexpression can significantly increase the activity of antioxidant enzymes and the content of osmotic regulatory substances, thereby significantly enhancing the drought resistance of larch. This discovery provides important genetic resources and theoretical basis for breeding new larch varieties with strong drought resistance. Attached Figure Description

[0024] Figure 1 For overexpression LgLEA10 Image showing DNA level identification of Japanese larch cell lines.

[0025] Figure 2 For overexpression LgLEA10 RNA level identification diagram of Japanese larch cell line.

[0026] Figure 3 For overexpression LgLEA10 Phenotypic representations of drought stress response of Japanese larch cell lines and wild-type Japanese larch cell lines under treatment with and without 9% PEG4000.

[0027] Figure 4 PEG treatment for 21 days to overexpress LgLEA10 A statistical chart showing the growth of Japanese larch cell lines and wild-type cell lines.

[0028] Figure 5 PEG treatment for 21 days to overexpress LgLEA10 Statistical chart of relative water content in Japanese larch cell lines and wild-type cell lines.

[0029] Figure 6 PEG treatment for 21 days to overexpress LgLEA10 Statistical graph of relative conductivity in Japanese larch cell lines and wild-type cell lines.

[0030] Figure 7 PEG treatment for 21 days to overexpress LgLEA10 Statistical chart of soluble protein content in Japanese larch cell lines and wild-type cell lines.

[0031] Figure 8 PEG treatment for 21 days to overexpress LgLEA10 Statistical chart of proline content in Japanese larch cell lines and wild-type cell lines.

[0032] Figure 9 PEG treatment for 21 days to overexpress LgLEA10 Statistical chart of soluble sugar content in Japanese larch cell lines and wild-type cell lines.

[0033] Figure 10 PEG treatment for 21 days to overexpress LgLEA10 Statistical chart of MDA content in Japanese larch cell lines and wild-type cell lines.

[0034] Figure 11 PEG treatment for 21 days to overexpress LgLEA10 Statistical graph of SOD enzyme activity in Japanese larch cell lines and wild-type cell lines.

[0035] Figure 12 PEG treatment for 21 days to overexpress LgLEA10 Statistical graph of POD enzyme activity in Japanese larch cell lines and wild-type cell lines.

[0036] Figure 13 PEG treatment for 21 days to overexpress LgLEA10 Statistical graph of H2O2 content in Japanese larch cell lines and wild-type cell lines. Different lowercase letters indicate significant differences (P<0.05). Detailed Implementation

[0037] The present invention will now be described in detail with reference to specific embodiments, but these should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments can be obtained commercially unless otherwise specified.

[0038] In the following examples: the Japanese larch cell line is disclosed in the literature “Sun Haitao, Yang Ling, Qi Liwang, et al. Effects of drying treatment of Japanese larch embryoids on germination [J]. Forestry Science Research, 2024, 37(02):90-95.”, the Agrobacterium strain is GV3101, and the plant overexpression vector pCAMBIA1300 is disclosed in the literature “Huang Yuqin, Zhang Ruxin, Zhang Xinyuan, et al. Cloning and expression analysis of larch LkTCTP promoter [J]. Forestry Science Research, 2025, 38(02):48-57.”

[0039] The preparation of 1L of b80 basal culture medium is shown in Table 1.

[0040] Table 1: B80 basal culture medium:

[0041]

[0042] LB medium (solid, 1L): 10g tryptone + 5g yeast extract + 10g sodium chloride + 15g agar + 950mL ultrapure water, pH 5.8, autoclaved at 121℃ for 15min.

[0043] LB medium (liquid, 1L): 10g tryptone + 5g yeast extract + 10g sodium chloride + 15g agar + 950mL ultrapure water, pH 5.8, autoclaved at 121℃ for 15min.

[0044] Larch subculture medium (1L): Table 1 shows b80 basal medium + 30g sucrose + 3g plant gel powder (Sigma), pH 5.8, autoclaved at 121℃ for 20min.

[0045] Larch co-culture medium (1L): Table 1 shows b80 basal medium + 30g sucrose + 3g plant gel powder, pH 5.8, autoclaved at 121℃ for 20min, then 40mg acetylsyl syringone was added.

[0046] Larch selective medium (1L): Table 1 shows b80 basal medium + 30g sucrose + 3g plant gel powder, pH 5.8, autoclaved at 121℃ for 20min, then add 5mg cephalosporin and 5mg hygromycin.

[0047] Example 1: Dahurian larch LgLEA10 Gene cloning and construction of overexpression plasmids and recombinant Agrobacterium.

[0048] 1. Dahurian larch LgLEA10 Cloning of genes

[0049] RNA was extracted from *Larix davidii* using the CTAB method. Subsequently, DNase-digested RNA was reverse transcribed into cDNA using the Primescript™ 1st strand cDNA synthesis kit, which served as a template. Specific primers PcNRAMP1-F and PcNRAMP1-R were designed for gene cloning. The amplified band of approximately 456 bp was recovered from the gel and sent to Qingke Biotechnology Co., Ltd. for sequencing. The sequenced sequence was compared with the genome sequence. The cloned... LgLEA10 The coding region of the gene is 456 bp in length. LgLEA10 The nucleotide sequence of the gene is shown in SEQ ID NO.1. LgLEA10The amino acid sequence of the gene-expressed protein is shown in SEQ ID NO.2, and its molecular weight is 21.48 kDa. The nucleotide sequence of PcNRAMP1-F is shown in SEQ ID NO.3, and the nucleotide sequence of PcNRAMP1-R is shown in SEQ ID NO.4.

[0050] SEQ ID NO.1: ATGGCGAGTTTGATGGACAAAGCGAAACAGTTCGTAGTAGACAAGATCGCCCATATAGAAAAACCGAGCGCAGATTGACAGACATCGATGTGAAAAATCTCACCATGCAAACAATCACTCTGGAGAGCGCTATTGACATAACCAATCCTTACAGCCATGACCTACCAATTGGGCAAATTTCTTTCCGACTCCGAAGCGCAGACCGAGAGATAGCGTCTGGAACAA TTCTAGATCCTGGGTCGGTGAAGGCCAATGACAGGACAGGCATGCTCGTTCCAGTGACAGTGCCTTACGACTTCTTGATCAGTTATTATGAAAGACCTTGGCAGGGATTGGGATATTGACTATGAGTGGGATATTGGACTTACCATGCACATTCCTGTTGTTGGCAAATTCACCCTTCCCCTCAACAAGAAGGGCACTATCAAGCTGCCCACTTTATCAGATGTTTTTTAA.

[0051] SEQ ID NO. 2: MASLMDKAKQFVVDKIAHIEKPSADVTDIDVKNLTMQTITLESAIDITNPYSHDLPIGQISFRLRSADREIASGTILDPGSVKANDRTGMLVPVTVPYDFLISIMKDLGRDWDIDYEWDIGLTMHIPVVGKFTLPLNKKGTIKLPTLSDVF.

[0052] SEQ ID NO. 3: ATGGCGAGTTTGATGGACAAAGCGAAA.

[0053] SEQ ID NO. 4: TTAAAAAACATCTGATAAAGTGGGCAGCTT.

[0054] 2. Construction of overexpression plasmids and transformation of Agrobacterium

[0055] Will LgLEA10 Gene and plant expression vector pCAMBIA1300 vector were digested with enzymes. Captain I and Sac pCAMBIA1300 was obtained via homologous recombination. LgLEA10 plasmid, pCAMBIA1300- LgLEA10 After the plasmid was transformed into competent DH5α Escherichia coli cells, single colonies with normal growth were selected for sequencing. The positive plasmid was then transformed into Agrobacterium to obtain recombinant Agrobacterium.

[0056] Example 2: LgLEA10 Functional analysis of genes in Japanese larch.

[0057] I. Experimental Methods.

[0058] 1. Genetic transformation steps of Japanese larch

[0059] (1) The recombinant Agrobacterium obtained in Example 1 was activated, and single clones were selected and cultured until OD600=0.7.

[0060] (2) The Japanese larch suspension cell line cultured in the dark for 1 week was used as the experimental material.

[0061] (3) Collect the cultured Agrobacterium by centrifugation, add 40 mg / L of acetylsuccinone for resuspending, so that its OD=0.1, then transfer all suspended cell lines to the resuspended solution and then filter.

[0062] (4) Transfer the cell line to b80 co-culture medium and co-culture in the dark for 2 days.

[0063] (5) After the co-culture stage, the explants are cleaned.

[0064] (6) After cleaning, the explants were transferred to a culture dish of b80 selective medium and cultured in the dark for about 1 month. Then the selected cell lines were transferred to b80 medium for subculture.

[0065] 2. Identification and screening of overexpressing larch strains

[0066] Total DNA and RNA were extracted from overexpressing and wild-type Japanese larch cell lines using the CTAB method.

[0067] Using cell line DNA as a template, pCAMBIA1300- LgLEA10- F and pCAMBIA1300- LgLEA10- PCR was performed using primers R to identify transgenic plants. The positive control was the recombinant vector plasmid, and the negative control was the wild-type Japanese larch cell line. pCAMBIA1300- LgLEA10-The nucleotide sequence of F is shown in SEQ ID NO.5, pCAMBIA1300- LgLEA10- The nucleotide sequence of R is shown in SEQ ID NO.6.

[0068] SEQ ID NO. 5: AGCCTGCTTGAGCAAAGAAA.

[0069] SEQ ID NO. 6: CAGGTAACGTGTTCTTCGTC.

[0070] Using cDNA from transgenic and wild-type cell lines as templates, LgLEA10- Semi-quantitative PCR was performed using RT-F / R to identify the relative expression levels of different transgenic lines of Japanese larch cell lines. Wild-type Japanese larch cell lines served as controls. LgActin was used as an internal reference gene.

[0071] 3. PEG treatment of Japanese larch cell lines

[0072] Wild-type Japanese larch cell lines and transgenic Japanese larch cell lines were cultured in a culture room for 4 weeks. Japanese larch cell lines with similar growth were selected and cultured on b80 medium supplemented with 9% PEG4000. After 20 days, the growth and physiological and biochemical parameters of the wild-type Japanese larch cell lines and six overexpressing Japanese larch cell lines were measured.

[0073] 4. Measurement of growth

[0074] At the beginning of the experiment, 0.5g of each transgenic line was weighed and cultured on b80 medium supplemented with 9% PEG4000. Each line was replicated in 3 times. The weight was measured again after 20 days. The difference between the weight on day 20 and the weight on day 0 was the growth amount.

[0075] 5. Measurement of physiological and biochemical indicators

[0076] Relative water content of callus (%) = (fresh weight of callus – dry weight of callus) / (saturated fresh weight of callus – dry weight of callus) × 100%; Relative electrical conductivity of callus (%) = (initial electrical conductivity of callus – blank electrical conductivity) / (electrical conductivity of callus after boiling – blank electrical conductivity) × 100%.

[0077] Using the SOD (superoxide dismutase) kit (BC0175), POD (peroxidase) kit (BC0090), MDA (malondialdehyde) kit (BC0020), hydrogen peroxide content kit (BC3595), soluble sugar content kit (BC0035), and CAT (catalase) kit (BC0200) from Beijing Solarbio Science & Technology Co., Ltd., the relevant indicators were measured using an enzyme-linked immunosorbent assay (ELISA) reader according to the instructions.

[0078] II. Experimental Results.

[0079] 1. LgLEA10 Identification and Phenotypic Analysis of Overexpression Cell Lines of Japanese Larch

[0080] exist Figure 1~2 In this study, DNA and RNA molecular-level identification was performed on six transgenic cell lines. The results showed that no corresponding DNA band was detected in the wild-type cells, while the overexpression cell lines exhibited a band at 1000 bp consistent with the recombinant plasmid. Compared to the wild-type cell lines, the overexpression cell lines showed... LgLEA10 The expression level of was significantly upregulated, among which LGLEA10-OE 4. OE The expression fold is highest at 5. The above conclusions prove... LgLEA10 The gene has been successfully transferred into a Japanese larch cell line. Figure 3 It can be seen that on day 20, overexpression LgLEA10 The transgenic cell lines and wild-type Japanese larch cell lines showed better growth under PEG treatment, with larger cell clusters.

[0081] 2. LgLEA10 Growth analysis of overexpression of Japanese larch cell lines

[0082] Depend on Figure 4 It can be seen that, on day 20, compared with the wild-type Japanese larch cell line, overexpression of [the specific cell line] was significantly higher. LgLEA10 The growth of transgenic cell lines was significantly increased under PEG treatment, among which... LGLEA10-OE 6. OE The growth rates of 2 were the highest, at 678.04% and 461.16% respectively.

[0083] 3. LgLEA10 Analysis of relative water content of overexpressing Japanese larch cell lines

[0084] Depend on Figure 5 It can be seen that, on day 20, compared with the wild-type Japanese larch cell line, overexpression LgLEA10 The relative water content of the transgenic cell lines significantly increased under PEG treatment, among which... LGLEA10-OE 8. OE The relative moisture content of 10 was the highest, at 87.8% and 87.73% respectively.

[0085] 4. LgLEA10 Analysis of relative conductivity of overexpressing Japanese larch cell lines

[0086] Depend on Figure 6 It can be seen that, on day 20, compared with the wild-type Japanese larch cell line, overexpression of [the specific cell line] was significantly higher. LgLEA10 The relative conductivity of the transgenic cell lines was significantly reduced under PEG treatment, among which... LGLEA10-OE 8. OE The relative conductivity of 6 was the lowest, at 25.8% and 32.13%, respectively.

[0087] 5. LgLEA10 Analysis of soluble protein content in overexpressed Japanese larch cell lines

[0088] Depend on Figure 7 It can be seen that, on day 20, compared with the wild-type Japanese larch cell line, overexpression of [the specific cell line] was significantly higher. LgLEA10 The content of soluble proteins in the transgenic cell lines treated with PEG increased significantly, among which... LgLEA10-OE1 0、 OE The highest soluble protein content was found in group 6, at 0.39 μg / g and 0.37 μg / g, respectively.

[0089] 6. LgLEA10 Analysis of proline content in overexpressed Japanese larch cell lines

[0090] Depend on Figure 8 It can be seen that, on day 20, compared with the wild-type Japanese larch cell line, overexpression of [the specific cell line] was significantly higher. LgLEA10 The proline content in the transgenic cell lines treated with PEG was significantly increased, among which... LGLEA10-OE 10. OE The highest proline content was found in group 3, at 58.38 μg / g and 38.58 μg / g, respectively.

[0091] 7. LgLEA10 Analysis of soluble sugar content in overexpressed Japanese larch cell lines

[0092] Depend on Figure 9 It can be seen that, on day 20, compared with the wild-type Japanese larch cell line, overexpression of [the specific cell line] was significantly higher. LgLEA10 The soluble sugar content of the transgenic cell lines treated with PEG increased significantly, among which... LGLEA10-OE 2. OE The highest soluble sugar content was found in group 8, at 2.41 mg / g and 2.28 mg / g, respectively.

[0093] 8. LgLEA10Analysis of MDA content in overexpressing Japanese larch cell lines

[0094] Depend on Figure 10 It can be seen that, on day 20, compared with the wild-type Japanese larch cell line, overexpression of [the specific cell line] was significantly higher. LgLEA10 The MDA content of the transgenic cell lines was significantly reduced under PEG treatment, among which... LgLEA10-OE2 , OE The MDA content of 8 was the lowest, at 2.42 nmol / g and 3.36 nmol / g, respectively.

[0095] 9. LgLEA10 Analysis of SOD activity in overexpression of Japanese larch cell lines

[0096] Depend on Figure 11 It can be seen that, on day 20, compared with the wild-type Japanese larch cell line, overexpression of [the specific cell line] was significantly higher. LgLEA10 The transgenic cell lines showed significantly increased SOD activity under PEG treatment, among which... LGLEA10-OE 8. OE The highest SOD activity was observed in 2, at 186.96 U / g and 162.82 U / g, respectively.

[0097] 10. LgLEA10 Analysis of POD activity overexpression in Japanese larch cell lines

[0098] Depend on Figure 12 It can be seen that, on day 20, compared with the wild-type Japanese larch cell line, overexpression of [the specific cell line] was significantly higher. LgLEA10 The transgenic cell lines showed significantly increased POD activity under PEG treatment, among which... LGLEA10-OE 8. OE10 The highest POD activities were observed in the two samples, at 850.47 U / g and 804.8 U / g, respectively.

[0099] 11. LgLEA10 Analysis of H2O2 content in overexpressing Japanese larch cell lines

[0100] Depend on Figure 13 It can be seen that, on day 20, compared with the wild-type Japanese larch cell line, overexpression of [the specific cell line] was significantly higher. LgLEA10 The H2O2 content of the transgenic cell lines was significantly reduced under PEG treatment, among which... LGLEA10-OE8、OE2 The H2O2 content was the lowest, at 2.1 Umol / g and 3.1 Umol / g, respectively.

[0101] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0102] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.

[0103] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A drought-responsive gene in larch LgLEA10 Its characteristics are, The LgLEA10 The nucleotide sequence is shown in SEQ ID NO.

1.

2. The larch drought response gene as described in claim 1 LgLEA10 The encoded protein is characterized by, The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. A recombinant expression vector, characterized in that, It contains the larch drought response gene as described in claim 1. LgLEA10 .

4. The recombinant expression vector according to claim 3, characterized in that, The larch drought response gene is described. LgLEA10 Insertion of pCAMBIA1300 overexpression vector plasmid Kpn I and Sac Obtained between I sites.

5. Recombinant engineered bacteria comprising the recombinant expression vector of claim 3.

6. The recombinant engineered bacteria according to claim 5, characterized in that, The recombinant expression vector was obtained by transforming it into Agrobacterium GV3101.

7. The larch drought response gene according to claim 1 LgLEA10, The larch drought response gene of claim 2 LgLEA10 The application of the encoded protein, the recombinant expression vector of claim 3, or the recombinant engineered bacteria of claim 5 in improving plant drought resistance, characterized in that... Improving plant drought resistance includes: Increase plant cell growth under drought stress; Increase the content of osmotic regulatory substances in plant cells under drought stress; To enhance the activity of antioxidant enzymes in plant cells under drought stress; The plant in question is a larch.

8. The application according to claim 7, characterized in that, The osmotic regulating substances include soluble proteins, proline, and soluble sugars.

9. The application according to claim 7, characterized in that, The antioxidant enzymes include superoxide dismutase and peroxidase.

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