Larix gmelinii drought resistance improving gene LgERF4 and application thereof

Abstract

This invention belongs to the field of genetic engineering technology, specifically relating to improving the drought resistance of larch trees. LgERF4 Genes and their applications. (The following is a separate section...) LgERF4 The nucleotide sequence of the gene is shown in SEQ ID NO.1. This invention is the first to experimentally demonstrate... LgERF4 Gene overexpression can enhance the drought resistance of larch by significantly increasing the activity of antioxidant enzymes and the content of 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, specifically relating to improving the drought resistance of larch trees. LgERF4 Genes and their applications. Background Technology

[0002] Drought is a natural phenomenon; severe drought can limit plant growth and development, and even lead to plant death. Larch belongs to the genus *Larch* of the family Pinaceae. Larix The larch is a tall deciduous tree. As one of the representative cold-humid coniferous forest species in the north, it has the characteristics of excellent wood quality, rapid early growth and good ecological benefits. It is a major timber forest and greening tree species in northern my country and has important value in forestry production.

[0003] ERF4 Ethylene-Responsive Factor 4 (EREBP) is a member of the AP2 / EREBP transcription factor family. AtERF4 The gene encodes an ethylene response factor and is also involved in the regulation of ABA, which plays an important role in plant development, stress response, and hormone signal transduction. ERF4 It acts as an interaction factor between ethylene and jasmonic acid signaling, affecting processes such as fruit ripening and stress response; ERF4 Isoenzymes can act as both activators and inhibitors in the regulation of senescence in Arabidopsis thaliana, for example, ERF4-A Activable CATALASE3 (CAT3) gene expression, while ERF4-R It can inhibit the expression of this gene; ERF4 It is also related to cell growth and cell growth regulation. ERF4 and TCP15 (Teosinte branched1 / Cycloidea / Proliferating cell factors 15) interact and antagonize each other's activities to regulate cell growth and intracellular replication; in studies of Brassica oleracea, the Brassica oleracea... BrERF4 Overexpression of the gene enhances Arabidopsis' tolerance to salt and drought.

[0004] However, no reports have been made on conifers, especially Dahurian larch. ERF4 The drought resistance function of genes. Summary of the Invention

[0005] Existing technologies lack research on the function of drought-resistant genes in Dahurian larch. To fill this gap, this invention provides a method for improving the drought resistance of larch. LgERF4 Genes and their applications.

[0006] The first objective of this invention is to provide a method for improving the drought resistance of larch.LgERF4 A gene, a nucleotide sequence of which is shown as SEQ ID NO. 1, and an amino acid sequence of a protein encoded by the gene is shown as SEQ ID NO. 2.

[0007] The present application proves for the first time that LgERF4 The gene is a drought stress response regulating gene of Larix gmelinii, and the overexpression of the gene can significantly improve the antioxidant enzyme activity and the content of osmotic adjustment substances, thereby significantly enhancing the drought resistance of Larix gmelinii. The finding provides important gene resources and theoretical basis for breeding new Larix gmelinii varieties with strong drought resistance.

[0008] The third object of the present application provides a vector containing the gene.

[0009] The fourth object of the present application provides a genetically engineered bacterium containing the vector.

[0010] The fifth object of the present application provides application of the protein, the gene, the vector or the genetically engineered bacterium in improving the drought resistance of Larix gmelinii.

[0011] Further, improving the drought resistance of Larix gmelinii includes:

[0012] Improving the growth amount of Larix gmelinii cells under drought stress;

[0013] Improving the water content of Larix gmelinii cells under drought stress;

[0014] Improving the content of osmotic adjustment substances of Larix gmelinii cells under drought stress;

[0015] Improving the antioxidant enzyme activity of Larix gmelinii cells under drought stress;

[0016] Reducing the relative conductivity of Larix gmelinii cells under drought stress;

[0017] Reducing the H2O2 content of Larix gmelinii cells under drought stress

[0018] Reducing the malondialdehyde content of Larix gmelinii cells under drought stress.

[0019] Further, the osmotic adjustment substances include soluble protein, proline and soluble sugar.

[0020] Further, the antioxidant enzyme includes superoxide dismutase, catalase and peroxidase.

[0021] The sixth object of the present application provides a method for constructing a transgenic Larix gmelinii, comprising the following steps:

[0022] Overexpressing the gene in Larix gmelinii, and then obtaining a transgenic Larix gmelinii with improved drought resistance.

[0023] The present application has the following beneficial effects:

[0024] The present application first discloses that the gene in Larix gmelinii can significantly enhance the drought resistance of Larix gmelinii. LgERF4 Through a series of scientific experiments, the present application proves that the overexpression of the gene can significantly improve the antioxidant enzyme activity and the content of osmotic adjustment substances, thereby significantly enhancing the drought resistance of Larix gmelinii. LgERF4

[0025] Under drought stress, the levels of reactive oxygen species (ROS), malondialdehyde (MDA) and hydrogen peroxide (H2O2) in plant cells will increase sharply, causing oxidative damage to cells. Antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) can effectively scavenge ROS and protect cells from oxidative damage. The present application found through experiments that the activities of these antioxidant enzymes in Larix gmelinii cells with overexpression of the gene are significantly higher than those in the control group (wild type), indicating that the overexpression of the gene enhances the antioxidant capacity of the plant. LgERF4 LgERF4

[0026] Meanwhile, osmotic adjustment substances such as proline, soluble sugar and soluble protein play an important role in maintaining cell osmotic pressure balance, protecting cell structure and function. Experimental results show that in Larix gmelinii with overexpression of the gene, the contents of these osmotic adjustment substances increase significantly, which helps the plant to maintain water balance and normal physiological functions of cells under drought conditions. LgERF4 BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 To identify the overexpression of the gene in Larix gmelinii cell lines at the DNA level. LgERF4 To identify the overexpression of the gene in Larix gmelinii cell lines at the RNA level.

[0028] Figure 2 To identify the overexpression of the gene in Larix gmelinii cell lines at the RNA level. LgERF4 To identify the overexpression of the gene in Larix gmelinii cell lines at the RNA level.

[0029] Figure 3 To identify the overexpression of the gene in Larix gmelinii cell lines at the RNA level. LgERF4 Growth phenotype of overexpression of the gene in Larix gmelinii cell lines and wild type Larix gmelinii cell lines under 9% PEG4000 treatment.

[0030] Figure 4 Growth amount of overexpression of the gene in Larix gmelinii cell lines and wild type cell lines after 21 days of PEG treatment. LgERF4 Growth amount of overexpression of the gene in Larix gmelinii cell lines and wild type cell lines after 21 days of PEG treatment.

[0031] Figure 5 Relative water content of overexpression of the gene in Larix gmelinii cell lines and wild type cell lines after 21 days of PEG treatment. LgERF4 Relative water content of overexpression of the gene in Larix gmelinii cell lines and wild type cell lines after 21 days of PEG treatment. ​​​​

[0032] Figure 6 PEG treatment for 21 days to overexpress LgERF4 Relative electrical conductivity in Japanese larch cell lines and wild-type cell lines.

[0033] Figure 7 PEG treatment for 21 days to overexpress LgERF4 Soluble protein content in Japanese larch cell lines and wild-type cell lines.

[0034] Figure 8 PEG treatment for 21 days to overexpress LgERF4 Proline content in Japanese larch cell lines and wild-type cell lines.

[0035] Figure 9 PEG treatment for 21 days to overexpress LgERF4 Soluble sugar content in Japanese larch cell lines and wild-type cell lines.

[0036] Figure 10 PEG treatment for 21 days to overexpress LgERF4 Malondialdehyde (MDA) content in Japanese larch cell lines and wild-type cell lines.

[0037] Figure 11 PEG treatment for 21 days to overexpress LgERF4 SOD enzyme activity in Japanese larch cell lines and wild-type cell lines.

[0038] Figure 12 PEG treatment for 21 days to overexpress LgERF4 POD enzyme activity in Japanese larch cell lines and wild-type cell lines.

[0039] Figure 13 PEG treatment for 21 days to overexpress LgERF4 H2O2 content in Japanese larch cell lines and wild-type cell lines. Detailed Implementation

[0040] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this 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 are commercially available unless otherwise specified.

[0041] The following examples involve experimental materials:

[0042] 1) Plant materials

[0043] The Japanese larch cell line used in the present application is a C6 cell line provided by Qiliwang Research Group of China Forestry Science Research Institute, which is disclosed in the literature: Sun Haitao, Yang Ling, Qiliwang, et al. The effect of Japanese larch embryoid dry treatment on germination [J]. Forestry Science Research, 2024, 37(02): 90-95. DOI: CNKI: SUN: LYKX.0.2024-02-010. The public can obtain the test in the repeated examples from the applicant.

[0044] 2) Strains and vectors

[0045] 21) Strains

[0046] The agrobacterium strain is GV3101.

[0047] 22) Vectors

[0048] The plant overexpression vector pCAMBIA1300 can be obtained from commercial channels.

[0049] 3) Reagent preparation:

[0050] LB medium (solid, 1L): tryptone 10 g + yeast extract 5 g + sodium chloride 10 g + agar 15 g + 950 mL ultrapure water, pH 5.8, 121℃ high pressure sterilization for 15 min.

[0051] LB medium (liquid, 1L): tryptone 10 g + yeast extract 5 g + sodium chloride 10 g + agar 15 g + 950 mL ultrapure water, pH 5.8, 121℃ high pressure sterilization for 15 min.

[0052] Larch subculture medium (1L): b80 basic medium + sucrose 30 g + plant gel powder 3 g, pH 5.8, 121℃ high temperature and high pressure sterilization for 20 min.

[0053] Larch co-culture medium (1L): b80 basic medium + sucrose 30 g + plant gel powder 3 g, pH 5.8, 121℃ high temperature and high pressure sterilization for 20 min, then add 40 mg acetyl cinnamaldehyde.

[0054] Larch selection medium (1L): b80 basic medium + sucrose 30 g + plant gel powder 3 g, pH 5.8, 121℃ high temperature and high pressure sterilization for 20 min, then add 5 mg cefotaxime and 5 mg hygromycin.

[0055] Example 1: Xing'an larch LgERF4 Cloning of genes and construction of overexpression plasmid and recombinant agrobacterium

[0056] 1. Larix gmelinii LgERF4 Cloning of the gene

[0057] The RNA of Larix gmelinii was extracted by CTAB method, and then the cDNA was obtained by reverse transcription of the RNA after DNase enzyme digestion using Primescript ™ 1st strand cDNAsynthesis kit kit, and used as a template. The specific primers PcNRAMP1-F (SEQ ID NO. 3) and PcNRAMP1-R (SEQ ID NO. 4) were designed for gene cloning. The amplified band of about 555 bp was recovered by gel, and the positive plasmid after ligation with pMD19-T vector was sent for sequencing. The sequence after sequencing was compared with the sequence in the genome. The obtained LgERF4 The coding region length of the gene was 555 bp (shown in SEQ ID NO. 1), encoded amino acid 154 aa (shown in SEQ ID NO. 2), and the molecular weight was 21.48 kDa. The positive plasmid after sequencing was named as pM- LgERF4-1 .

[0058] SEQ ID NO. 1: ATGGCGGTCGAAACCATGCGCATGGCGAGGGTGGAGTTAAGAGGTTGCGAGGACGAATCCCGCGCTGTCAAAGAAACCCATTTCAGGGGCGTCAGAAAACGGCCGTGGGGAAGATTCGCAGCGGAAATCAGAGATCCATGGAAGAAGACTCGAGTGTGGCTGGGTACCTTCGACACTGCCGAGGAAGCCGCCCGTGCATACGATAATGCCGCTAGGAGATTGCGCGGTCATAAAGCTAAGACCAATTTTTCTCTTGCTGTTGATGATCATAACGCTAATTCCGCCGGCGCACTCTCCTGGACTCGAACGCTGCACCCTCAGCAGCAGCCCGATCTGAACGCCACCGCTTTTGCTTCCGTTTTCGTATCAAACAAGAGACGCGAAGTTTCCTTTGGAAGCGACCGGCTGGAGTTCGAATCTGGTAACAATTCTGTCCGTGCTGCGCCGTCTGAACAGAAGGCCTCTTCCTCCCCGCGGAAGGCTCCTTTGTTGCTGGATCTTAATTTTCCACCCGTCGCTGATCCGGAGAATGAAAGCGCAGCTCCATGGAATTAA.

[0059] SEQ ID NO. 2: MAVETMRMARVELRGCEDESRAVKETHFRGVRKRPWGRFAAEIRDPWKKTRVWLGTFDTAEEAARAYDNAARRLRGHKAKTNFSLAVDDHNANSAGALSWTRTLHPQQQPDLNATAFASVFVSNKRREVSFGSDRLEFESGNNSVRAAPSEQKASSSPRKAPLLLDLNFPPVADPENESAAPWN.

[0060] SEQ ID NO. 3:ATGGCGGTCGAAACCATGCG.

[0061] SEQ ID NO. 4: TTAATTCCATGGAGCTGCGC.

[0062] SEQ ID NO. 5: CAGCAACAAAGGAGCCTTCC.

[0063] SEQ ID NO. 6: CAGGTAACGTGTTCTTCGTC.

[0064] 2. Construction of overexpression plasmids and transformation of Agrobacterium

[0065] Will LgERF4 The pCAMBIA1300 gene and plant expression vector pCAMBIA1300 were used to obtain the pCAMBIA1300-LgERF4 plasmid through homologous recombination. After the pCAMBIA1300-LgERF4 plasmid was transformed into DH5α Escherichia coli competent cells, single colonies with normal growth were selected for sequencing. The positive plasmid was then transformed into Agrobacterium to obtain recombinant Agrobacterium.

[0066] Example 2: LgERF4 Functional analysis of genes in Japanese larch

[0067] 1 Experimental Methods

[0068] 1.1 Genetic transformation steps of Japanese larch

[0069] (1) The recombinant Agrobacterium obtained in Example 1 was activated, and single clones were selected and cultured to OD. 600 = Between 0.6 and 0.8.

[0070] (2) The Japanese larch suspension cell line cultured in the dark for 1 week was used as the experimental material.

[0071] (3) Collect the cultured Agrobacterium by centrifugation, add 40 mg / L acetylsuccione for resuspending, so that its OD=0.1, then transfer all suspended cell lines to the resuspended solution and then filter.

[0072] (4) Transfer the cell line to b80 co-culture medium and co-culture in the dark for 2 days.

[0073] (5) After the co-culture stage, the explants are cleaned.

[0074] (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.

[0075] 1.2 Identification and screening of larch strains overexpressing the gene

[0076] Total DNA and RNA were extracted from overexpressing and wild-type Japanese larch cell lines using the CTAB method.

[0077] Using cell line DNA as a template, pCAMBIA1300- LgERF4 -F (SEQ ID NO.5) and pCAMBIA1300- LgERF4 PCR was performed using primers -R (SEQ ID NO. 6) to identify transgenic plants. The positive control was the recombinant vector plasmid, and the negative control was the wild-type Japanese larch cell line.

[0078] Using cDNA from transgenic and wild-type cell lines as templates, LgERF4- 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 It is an internal reference gene.

[0079] 1.3 PEG treatment of Japanese larch cell lines

[0080] 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.

[0081] 1.4 Measurement of growth

[0082] 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.

[0083] 1.4 Measurement of physiological and biochemical indicators

[0084] 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%.

[0085] 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.

[0086] 2. Experimental Results

[0087] 2.1 LgERF4 Identification and Phenotypic Analysis of Cell Lines Overexpressing Japanese Larch

[0088] exist Figure 1 and Figure 2 In our study, we identified six transgenic cell lines at the DNA and RNA molecular levels. The results showed that no corresponding DNA band was detected in the wild-type cells, while the overexpression cell lines exhibited a band consistent with the recombinant plasmid at 1000 bp. Compared to the wild-type cell lines, the overexpression cell lines showed… LgERF4 The expression level of was significantly upregulated, among which LgERF4 -OE4 and OE5 showed the highest fold increases. The above conclusions prove... LgERF4 The gene has been successfully transferred into a Japanese larch cell line. Figure 3 It can be seen that on day 20, overexpression LgERF4 The transgenic cell lines and wild-type Japanese larch cell lines showed better growth under PEG stress treatment, with larger cell clusters.

[0089] 2.2 LgERF4 Growth analysis of overexpression of Japanese larch cell lines

[0090] Depend on Figure 4It 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. LgERF4 The growth of transgenic cell lines significantly increased under PEG treatment, among which... LgERF4- OE4, OE5, and OE6 had the highest growth rates, at 1111.67%, 619.89%, and 912.87%, respectively.

[0091] 2.3 LgERF4 Analysis of relative water content of overexpressing Japanese larch cell lines

[0092] Depend on Figure 5 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. LgERF4 The relative water content of the transgenic cell lines significantly increased under PEG treatment, among which... LgERF4 -OE1, OE2, and OE5 have the highest relative moisture content, at 87.48%, 87.45%, and 86.83%, respectively.

[0093] 2.4 LgERF4 Analysis of relative conductivity of overexpressing Japanese larch cell lines

[0094] 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. LgERF4 The relative conductivity of the transgenic cell lines treated with PEG was significantly reduced, among which... LgERF4 -OE4, OE5, and OE6 have the lowest relative conductivity, at 35.87%, 34.73%, and 42.17%, respectively.

[0095] 2.5 LgERF4 Analysis of soluble protein content in overexpressed Japanese larch cell lines

[0096] 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. LgERF4 The content of soluble proteins in the transgenic cell lines treated with PEG increased significantly, among which... LgERF4 -OE2, OE4, and OE1 had the highest soluble protein content, at 0.47 μg / g, 0.43 μg / g, and 0.38 μg / g, respectively.

[0097] 2.6 LgERF4 Analysis of proline content in overexpressed Japanese larch cell lines

[0098] 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. LgERF4 The proline content in the transgenic cell lines treated with PEG was significantly increased, among which...LgERF4 The highest proline content was found in OE2, OE5, and OE4, at 39.14 μg / g, 33.64 μg / g, and 28.84 μg / g, respectively.

[0099] 2.7 LgERF4 Analysis of soluble sugar content in overexpressed Japanese larch cell lines

[0100] 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. LgERF4 The soluble sugar content of the transgenic cell lines significantly increased under PEG treatment, among which... LgERF4 -OE5, OE2, and OE6 had the highest soluble sugar content, at 1.97 mg / g, 1.94 mg / g, and 1.61 mg / g, respectively.

[0101] 2.8 LgERF4 Analysis of MDA content in overexpressing Japanese larch cell lines

[0102] 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. LgERF4 The MDA content in the transgenic cell lines treated with PEG was significantly reduced, among which... LgERF4 -OE4, OE5, and OE2 had the lowest MDA content, at 3.58 nmol / g, 4.93 nmol / g, and 5.29 nmol / g, respectively.

[0103] 2.9 LgERF4 Analysis of SOD activity in overexpression of Japanese larch cell lines

[0104] 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. LgERF4 The transgenic cell lines showed significantly increased SOD activity under PEG treatment, among which... LgERF4 - OE5, OE2, and OE6 had the highest SOD activity, at 158.38 U / g, 137.31 U / g, and 131.67 U / g, respectively.

[0105] 2.10 LgERF4 Analysis of POD activity overexpression in Japanese larch cell lines

[0106] 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. LgERF4 The transgenic cell lines showed significantly increased POD activity under PEG treatment, among which... LgERF4-OE2, OE5, and OE1 showed the highest POD activity, at 639.27 U / g, 616.47 U / g, and 601.33 U / g, respectively.

[0107] 2.11 LgERF4 Analysis of H2O2 content in overexpressing Japanese larch cell lines

[0108] 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. LgERF4 The H2O2 content in the transgenic cell lines treated with PEG was significantly reduced, among which... LgERF4 -OE1, OE5, and OE2 had the lowest H2O2 content, at 2.88 Umol / g, 3.02 Umol / g, and 3.17 Umol / g, respectively.

[0109] 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.

Claims

1. A method of increasing drought tolerance in larch comprising LgERF4 introducing into the larch a gene The nucleotide sequence is shown as SEQ ID NO.

1.

2. A vector containing the gene of claim 1.

3. A genetically engineered bacterium containing the vector of claim 2.

4. Use of the gene of claim 1, the vector of claim 2 or the genetically engineered bacterium of claim 3 in improving drought resistance of larch.

5. Use according to claim 4, characterized in that, Improving drought resistance of larch includes: increasing the growth amount of larch cells under drought stress; increasing the water content of larch cells under drought stress; increasing the content of osmotic adjustment substances of larch cells under drought stress; the osmotic adjustment substances include soluble protein, proline and soluble sugar; increasing the antioxidant enzyme activity of larch cells under drought stress; the antioxidant enzymes include superoxide dismutase, catalase and peroxidase; decreasing the relative electrical conductivity of larch cells under drought stress; decreasing the H2O2 content of larch cells under drought stress; and / or decreasing the malondialdehyde content of larch cells under drought stress.

6. A method of constructing a transgenic larch, comprising, The method comprises the following steps: overexpressing the gene of claim 1 in larch to obtain transgenic larch with improved drought resistance.

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