Regulation of key gene cbLTP63 in response to salt stress of catalpa bungei and application thereof

Abstract

The present application relates to a kind of regulation Catalpa bungei salt stress response key gene CbLTP63 and its application, belong to plant genetic engineering and biotechnology field.CbLTP63 gene nucleotide sequence is as described in Seq 1, coding amino acid sequence is as described in Seq 2.The present application relates to the cloning of Catalpa bungei salt stress response key gene CbLTP63;CbLTP63-GFP subcellular localization is positioned on cell membrane by identifying agrobacterium injection method;By qRT-PCR analysis, it is identified that the gene responds to salt stress at transcription level, is down-regulated by H2O2, ABA, SA;By transgenic identification, the gene is Catalpa bungei salt stress response negative regulation gene, and the salt tolerance of Catalpa bungei callus after gene silencing is significantly enhanced;Also related to the use of the gene regulation Catalpa bungei stress response.The present application discloses a kind of Catalpa bungei salt stress response key gene CbLTP63, and has important application value in the field of Catalpa bungei genetic engineering and clone forestry.

Description

Technical Field

[0001] This invention relates to a key gene CbLTP63 that regulates the salt stress response of Catalpa bungei and its application, particularly to the application of the CbLTP63 gene in improving the salt stress resistance of Catalpa bungei, belonging to the fields of plant genetic engineering and biotechnology. Background Technology

[0002] my country is a major consumer of high-grade timber, but domestic supply has consistently been insufficient, resulting in a timber import dependency rate exceeding 50%. my country's more than 35 million mu of coastal mudflats and 1.5 billion mu of inland saline-alkali land represent important forest resources. Simultaneously, with global warming, soil salinization and secondary salinization are increasing year by year, gradually eroding existing forest resources and posing a global ecological problem. Catalpa bungei is a precious deciduous hardwood species unique to my country and traditionally cultivated. It possesses advantages such as rapid growth, salt and alkali tolerance, and excellent wood quality, making it an excellent species for soil remediation and protection in coastal mudflats and inland saline-alkali lands. Salt stress caused by high soil salinity has become one of the main abiotic stress factors affecting Catalpa bungei production. Therefore, in-depth research into the molecular mechanisms of salt tolerance in Catalpa bungei, the discovery of new salt-tolerant gene resources, and genetic improvement of Catalpa bungei at the gene level are of significant scientific value for improving stress resistance and compensating for the shortcomings of conventional breeding methods.

[0003] Research over the past decade has shown that small peptides, as signaling molecules, are widely involved in the regulation of plant growth, development, and environmental adaptation, and have become one of the cutting-edge research topics in plant science. Non-specific lipid transfer proteins (nsLTPs) are a class of basic secreted proteins unique to plants, belonging to the prolysin superfamily. The molecular weight of plant nsLTPs is generally around 7-9 kDa. A typical nsLTP amino acid sequence contains an N-signal peptide and eight highly conserved cysteine ​​residues (C-Xn-C-Xn-CC-Xn-CXC-Xn-C-Xn-C), and some nsLTPs also contain a motif (glycosyl anchoring site) at their C-terminus. nsLTPs can bind and transport lipids such as glycolipids, phospholipids, and fatty acids, and lipids play an important role in growth, development, and abiotic stress. Recent studies have found that nsLTP is also involved in abscisic acid (ABA) and salicylic acid (SA) signal transduction, stomatal movement, and salt stress response, but not in Catalpa bungei.

[0004] During the rooting process of softwood cuttings, the high temperature and humidity environment can cause stress to the cuttings, potentially leading to the discovery of genes related to stress. Therefore, RNA-seq technology was used to enrich differentially expressed genes at different developmental stages of softwood cuttings of the superior Catalpa cultivar 'Yuqiu No. 1'. Differential expression of the comp51934_c0 gene, homologous to the Arabidopsis thaliana At5G05960 (AtLTPV.3), was identified. Based on Catalpa genomic data provided by the Chinese Academy of Forestry, this gene was named CbLTP63. Summary of the Invention

[0005] In view of the difficulties and shortcomings of the existing technology, the main purpose of this invention is to provide a key gene CbLTP63 for improving the salt stress resistance of Catalpa trees. Another purpose of this invention is to provide an application of the key gene CbLTP63 for regulating the salt stress response of Catalpa trees.

[0006] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0007] A key gene, CbLTP63, that regulates the salt stress response of Catalpa trees has its nucleotide sequence shown in Seq 1.

[0008] A translational protein of CbLTP63, a key gene regulating the salt stress response of Catalpa bungei, has its amino acid sequence shown in Seq 2.

[0009] A plant expression vector, pCAMBIA2300-CbLTP63, containing the key gene CbLTP63 regulating the salt stress response of Catalpa bungei, is described. The vector assembles a constitutive strong expression promoter CaMV35S at the 5' end of the CbLTP63 gene and a strong terminator NOS at the 3' end of the CbLTP63 gene. The vector diagram is shown below. Figure 3 As shown.

[0010] The GUS gene in the above vectors can serve as a reporter gene for screening transgenic catalpa trees, and the NPTⅡ gene can serve as a marker gene for screening transgenic catalpa trees. The vectors contain LB and RB sequences, which facilitate the integration of the vector fragment, including the CbLTP63 gene, into the catalpa tree genome.

[0011] A pTRV2 vector containing CbLTP63, a key gene regulating the salt stress response of Catalpa bungei. (See diagram of the pTRV2 vector.) Figure 4 As shown. The vector is started by the constitutive strong expression promoter CaMV35S and contains the ppk2 RNA2 sequence of TRV and the interference sequence of the CbLTP63 gene. TRV2:CbLTP63 is mixed with the helper vector TRV1 and then infects Catalpa callus to achieve CbLTP63 gene interference.

[0012] Advantages of this invention: This invention isolates a novel nsLTP gene from 'Yuqiu No. 1', homologous to the Arabidopsis thaliana At5G05960 (AtLTPV.3) gene, and names it CbLTP63. Plant overexpression vector pCAMBIA2300-CbLTP63 and VIGS vector TRV2:CbLTP63 were constructed to obtain CbLTP63 overexpression and silencing lines, thereby studying the function of the CbLTP63 gene in the salt stress response of Catalpa trees. This invention is the first to clone and elucidate the function of the nsLTP gene in the salt stress response of Catalpa trees, and also provides the first application of CbLTP63, a key gene regulating the salt stress response of Catalpa trees.

[0013] Compared with existing technologies, this invention introduces the CbLTP63 gene into the genome of *Catalpa bungei*. Transgenic *Catalpa bungei* trees overexpressing the CbLTP63 gene exhibit downregulated salt tolerance compared to wild-type trees. Furthermore, VIGS silencing of CbLTP63 transcriptional expression in *Catalpa bungei* callus tissue significantly increases the survival rate of silenced callus after high-salt treatment. This demonstrates that CbLTP63 is a key salt-sensitive gene with significant application value in forest tree genetic engineering, salt-tolerant breeding of forest trees, and forestry production. Attached Figure Description

[0014] The invention will now be described in further detail with reference to the accompanying drawings, but this is not intended to limit the invention.

[0015] Figure 1 This is a CbLTP63 gene sequence analysis.

[0016] A: Phylogenetic analysis of CbLTP63 and Arabidopsis LTPs genes; B: 8CM motif analysis of Type V LTPs; C: N signal peptide analysis of CbLTP63; D: Transmembrane domain analysis of CbLTP63.

[0017] Figure 2 This is an analysis of CbLTP63 gene expression.

[0018] qRT-PCR was used to analyze the transcriptional level of the CbLTP63 gene in different tissues; with water treatment as a control, the expression of the CbLTP63 gene under NaCl, H2O2, ABA and SA treatment conditions was analyzed.

[0019] Figure 3 This is a schematic diagram of the structure of the plant expression vector pCAMBIA2300-CbLTP63.

[0020] Figure 4 This is a diagram of the pTRV2 vector.

[0021] Figure 5 CbLTP63 is a negative regulatory gene for salt tolerance in catalpa trees.

[0022] A: qRT-PCR analysis of CbLTP63 expression in five randomly selected transgenic lines (OE1, OE3, OE4, OE5, OE7); B: Salt tolerance analysis of wild-type (WT) and transgenic lines (OE1, OE4) seedlings; C: Statistical analysis of survival rate of WT and transgenic lines; D: Statistical analysis of maximum fluorescence efficiency (Fv / Fm) of WT and transgenic lines.

[0023] Figure 6 Silencing the CbLTP63 gene improves the salt tolerance of the catalpa tree.

[0024] Silencing the CbLTP63 gene in Catalpa bungei using VIGS enhanced the salt tolerance of the silenced callus. *p<0.05 indicates a significant difference (Student's t-test). Detailed implementation method:

[0025] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0026] The reagents used in the implementation examples mainly include molecular biology experimental reagents and kits, all of which are commercially available. Unless otherwise specified, the methods provided in the embodiments of this invention are conventional methods.

[0027] Implementation Case 1: Cloning of the CbLTP63 gene in Catalpa bungei

[0028] 1. Extraction and reverse processing of total RNA from Catalpa roots

[0029] Stem segments or roots of the easily rooting catalpa cultivar 'Yuqiu No. 1' (0, 1, 15, 35DAC) were collected, rapidly frozen in liquid nitrogen, and then stored at -80℃ for later use. Total RNA was extracted using the FastPure Plant Total RNA Isolation Kit (Novizan, RC401), following the kit's instructions. Total RNA was quantified using NanoDrop, and integrity was assessed by 1% agarose gel electrophoresis.

[0030] cDNA synthesis was performed according to the instructions of the HiScript III 1st Strand cDNA Synthesis Kit (+gDNAwiper) (Novazia, R312), and the reverse transcription product was stored at -20°C.

[0031] 2. Cloning and sequencing of the CbLTP63 gene

[0032] Based on the genome information of Catalpa macrocarpa from the Chinese Academy of Forestry, primers (CbLTP63-F: GAAAGAACAGTGGGGCAAAATG; CbLTP63-R: AAGGTCGGAAAGAGAAGGGGTA) were designed in the 5' and 3' uncoding regions of the CbLTP63 gene, and the CbLTP63 gene was cloned by PCR.

[0033] PCR amplification was performed using a high-fidelity enzyme (Novizan, P505).

[0034] PCR reaction system:

[0035] 2×Phanta Max Buffer, 25μL; dNTP Mix (10mM each), 1μL; 100ng / L cDNA, 2μL; CbLTP63-F (10μM), 2μL; CbLTP63-R (10μM), 2μL; Phanta Max Super-Fidelity DNAPolymerase, 1μL; ddH20, up to 50μL.

[0036] Pre-denaturation at 95℃ for 3 min, denaturation at 95℃ for 15 s, annealing at 58℃ for 15 s, extension at 72℃ for 1 min, 35 cycles, and final extension at 72℃ for 10 min.

[0037] After the reaction, the target fragment was excised by 1% agarose gel electrophoresis and the PCR product was recovered using an agarose gel DNA recovery kit according to the manufacturer's instructions. The fragment was then ligated into the pMD19-T vector (Takara, D102A) as per the manufacturer's instructions. The ligation product was then transformed into top10 E. coli competent cells.

[0038] E. coli transformation:

[0039] (a) Add 5 μL of plasmid to 100 μL of competent E. coli cells, incubate on ice for 30 min, heat shock at 42°C for 90 s, and then rapidly cool on ice for 1-2 min.

[0040] (b) Add 800 μL of blank LB liquid medium and incubate at 37°C and 150 rpm for 1.5-2 h.

[0041] (c) Centrifuge at 5000 rpm for 3 min, discard 700 μL of supernatant, mix the remaining culture medium and bacterial cells, take 100 μL and spread it on LB solid medium containing Amp, and incubate at 37°C upside down until colonies grow.

[0042] (d) Pick a single colony from the plate and place it in 1 mL of LB liquid containing antibiotics, and incubate overnight on a shaker at 37°C.

[0043] PCR detection of E. coli transformants:

[0044] PCR amplification was performed using cultured E. coli as a template. The primers were: CbLTP63-F / R.

[0045] The specific steps are as follows:

[0046] Add the following to a 0.2 mL PCR reaction tube: 10×PCR Buffer, 2 μL; DNTPs (10 mmol / L), 0.4 μL; MgCl2 (25 mmol / L), 1.5 μL; Taq enzyme, 0.2 μL; Forward (10 μmol / L), 1 μL; Reverse (10 μmol / L), 1 μL; bacterial culture, 2 μL; dd H2O, up to 20 μL.

[0047] PCR amplification procedure:

[0048] Denaturation at 94℃ for 3 min, denaturation at 94℃ for 30 s, annealing at 56℃ for 1 min, extension at 72℃ for 1 min, 35 cycles, total extension at 72℃ for 10 min.

[0049] Half of the PCR-positive clones were added to 0.3 volumes of 50% glycerol and stored at -80°C. The other half were sequenced (Nanjing Sipujin Biotechnology Co., Ltd.). Sequencing analysis showed that the coding region of the gene, as shown in Seq 1, is 351 bp (including the stop codon).

[0050] Implementation Case 2: Protein sequence of the CbLTP63 gene in Catalpa bungei

[0051] The full-length cDNA sequence of the *Catella macrantha* CbLTP63 gene was translated using Primer 5 software. This gene encodes a protein sequence containing 116 amino acids, as shown in Seq 2. The amino acid sequence of the CbLTP63 gene was aligned with that of the *Arabidopsis thaliana* LTP family for sequence comparison and evolutionary analysis. The results are as follows: Figure 1 As shown. It has the highest sequence identity with Arabidopsis thaliana At5G05960 (AtLTPV.3) gene and belongs to group V; the 8CM motif is C-X14-C-X14-CC-X11-CXC-X24-C-X10-C; the signal peptide is located at the N-terminus and has a transmembrane domain.

[0052] Implementation Case 3: Expression Pattern Analysis of CbLTP63 Gene in Catalpa Tree

[0053] Quantitative primers were designed based on the cDNA sequence of the CbLTP63 gene: qRT-F: TGCTGCGGTTTCAAGGAGTT; qRT-R: CATCGCTTGGGAATGGTCAA. The *Catalpa bungei* actin gene was used as an internal control gene, with primers: Actin-qRT-F: GATGATGATGCTCCAAGAGCTGT; Actin-qRT-R: TCCATATCATCCCAGTTGCT. The transcriptional level of CbLTP63 in different tissues of the root, stem, and leaf of the cultivated *Catalpa bungei* cultivar 'Yuqiu No. 1' was analyzed using qRT-PCR. The expression of the CbLTP63 gene under NaCl, H2O2, ABA, and SA treatments was also analyzed, with water treatment as a control. The reagents used for qRT-PCR were SYBR Premix Ex TaqII (Takara, RR820), and the instrument was an Applied Biosystems Step One Plus™ Real-Time PCR System (Applied Biosystems, USA). The procedure was performed according to the manufacturer's instructions. Results are as follows: Figure 2 As shown, CbLTP63 is predominantly expressed in roots. Furthermore, after 6 hours of salt treatment, the CbLTP63 gene begins to respond to salt treatment, and its expression level gradually decreases with increasing salt treatment time, indicating that salt inhibits its expression. After H2O2 treatment, the relative expression level of the CbLTP63 gene first decreases and then slightly increases, reaching its lowest level at 6 hours. After ABA treatment, the relative expression level of the CbLTP63 gene consistently decreases, reaching only 6.55% of the control level after 24 hours. After SA treatment, the relative expression level of the CbLTP63 gene first increases and then rapidly decreases, reaching only 4.67% of the control level after 24 hours. These results suggest that the CbLTP63 gene may play an important role in the salt stress response of *Catalpa bungei*.

[0054] Implementation Case 4: Construction of the plant expression vector pCAMBIA2300-CbLTP63 for the CbLTP63 gene of Catalpa bungei

[0055] The CbLTP63 gene was amplified by PCR, with the 5' end of the gene sequence introduced into a SalI restriction site and the 3' end introduced into an XbaI restriction site. The amplification primers were pCAMBIA2300-CbLTP63-F:ACGC. GTCGAC ATGGCATCAGTATCAGTGAA ;pCAMBIA2300-CbLTP63-R:GC TCTAGATTA AGGCAACGTGTAAGCTCCAC. PCR was performed using a high-fidelity enzyme, following the procedures described in Example 1. The amplified fragment was double-digested with SalⅠ (New England BioLabs, R3138) and XbaⅠ (New England BioLabs, R0145) according to the manufacturer's instructions. The fragment was then ligated to the pCAMBIA2300 vector, which had been digested with the same enzymes, using T4 DNA ligase (Takara, K314BA). The T4 ligation reaction was performed according to the manufacturer's instructions to construct the pCAMBIA2300-CbLTP63 plant expression vector. A schematic diagram of the vector is shown below. Figure 3 As shown.

[0056] In this vector, the CbLTP63 gene is initiated by the constitutive strong expression promoter CaMV35S, the GUS gene serves as a reporter gene for screening transgenic catalpa trees, and the NPTⅡ gene serves as a marker gene for screening transgenic catalpa trees.

[0057] Implementation Case 5: Construction of the VIGS vector TRV2:CbLTP63 for the CbLTP63 gene of Catalpa bungei

[0058] The CbLTP63 gene was amplified by PCR, with the 5' end of the gene sequence introduced into an EcoRI restriction site and the 3' end introduced into a SacI restriction site. The amplification primers were TRV2:CbLTP63-F: TCTGTGAGTAAGGTTACCGAATTCGTGGTTTTGCGTTGTGG; TRV2:CbLTP63-R: CCGGGCCTCGAGACGCGTGAGCTCATCGCTTGGGAATGGTC. High-fidelity enzymes were used for PCR, and the operation steps were as described in Example 1. The pTRV2 vector was double-digested with EcoRI (New England BioLabs, M0211S) and SacI (New England BioLabs, R3156) according to the manufacturer's instructions. Recombination was then performed with the amplified fragment according to the instructions of the recombination kit (Beijing Jinsha, SC612) to construct the TRV2:CbLTP63 vector. A schematic diagram of the pTRV2 vector is shown below. Figure 4 As shown.

[0059] The vector is initiated by the constitutive strong expression promoter CaMV35S and contains the ppk2 RNA2 sequence of TRV and the interference sequence of the CbLTP63 gene. TRV2:CbLTP63 is mixed with the helper vector TRV1 and then infects Catalpa callus to achieve CbLTP63 gene interference.

[0060] Implementation Case 6: Obtaining and Phenotypic Identification of CbLTP63 Transgenic Catalpa Trees

[0061] 1. Obtaining CbLTP63 transgenic catalpa trees

[0062] The plant expression vector pCAMBIA2300-CbLTP63 and the VIGS vector TRV2:CbLTP63 constructed in Case 4 were transformed into Agrobacterium tumefaciens EHA105 for subsequent genetic transformation of Catalpa trees.

[0063] Agrobacterium-mediated transformation:

[0064] The transformation of Agrobacterium was performed using the freeze-thaw method in a clean bench.

[0065] (a) Add 1 μg of recombinant plasmid DNA to 200 μL of EHA105 competent cells and place on ice for 30 min;

[0066] (b) Quick-freeze in liquid nitrogen for 1 minute; remove and immediately place in a 37°C water bath until thawed;

[0067] (c) Add 1 mL of antibiotic-free liquid LB to a microcentrifuge tube and gently shake on a shaker at 28°C for 2 hours;

[0068] (d) Centrifuge at 5,000 rpm for 1 min and resuspend the precipitate in 100 μL of LB liquid without antibiotics;

[0069] (e) Take 10 μL of bacterial culture and spread it evenly on LB solid medium containing antibiotics, and incubate at 28°C for 2–3 days.

[0070] (f) Pick a single colony from the plate and place it in 1 mL of LB liquid containing antibiotics, and incubate on a shaker at 28°C for 1–2 days.

[0071] PCR detection of Agrobacterium transformants was performed using primers pCAMBIA2300-CbLTP63-F / R, following the procedures described in Example 1 (PCR detection of E. coli transformants). For clones that showed positive PCR amplification, 0.5 times the volume of 50% glycerol was added, and the clones were stored at -80°C.

[0072] The target gene CbLTP63 was introduced into the *Catella asiatica* genome or the *Catella asiatica* CbLTP63 gene was silenced using Agrobacterium tumefaciens (EHA105)-mediated transformation. Transgenic plants were identified by GUS staining and PCR. GUS staining was performed using a GUS staining kit (Solepro, G3060) according to the manufacturer's instructions. PCR detection identified positive plants by detecting the GUS gene. The GUS gene detection primers were: GUS-F: TGAATCCGCACCTCTGG, GUS-R: TTCATTGTTTGCCTCCCT. The PCR procedure was the same as in Case Study 1. After GUS staining and PCR amplification, a total of 30 CbLTP63 overexpressing transgenic plants were obtained.

[0073] 2. Phenotypic identification of CbLTP63-overexpressing transgenic Catalpa bungei

[0074] Five positive transgenic lines (OE1, OE3, OE4, OE5, and OE7) were randomly selected, and the overexpression of the CbLTP63 gene was analyzed using qRT-PCR. Figure 5 A) qRT-PCR was performed according to Case 3. Two transgenic lines with relatively high expression levels, OE1 and OE4, were selected for subsequent experiments.

[0075] All catalpa wood materials were cultured at 25-28℃, with a light intensity of 1500 lx and a light intensity of 12 h / d.

[0076] Transgenic and wild-type seedlings, aged 4 weeks, were irrigated with 300 mM NaCl. Survival rates of wild-type and transgenic seedlings were recorded at 10, 15, 20, 25, and 30 days post-treatment. Results showed that the survival rate of both transgenic and wild-type seedlings gradually decreased with increasing treatment time, and the survival rate of transgenic seedlings was consistently lower than that of the control. Figure 5 B). After 30 days of salt treatment, the survival rate of transgenic seedlings was significantly lower than that of the control ( Figure 5 C). After salt treatment, the Fv / Fm value of transgenic seedlings was also significantly lower than that of the control. Figure 5 D). The above results indicate that the CbLTP63 gene is a negative regulator of the salt stress response in Catalpa bungei.

[0077] 3. Silent CbLTP63 gene enhances salt tolerance in Catalpa bungei callus.

[0078] Catalpa embryogenic callus was transformed using Agrobacterium tumefaciens (EHA105)-mediated transformation to silence the Catalpa CbLTP63 gene, and the expression of the CbLTP63 gene in the silenced callus was analyzed by qRT-PCR. Figure 6 ), qRT-PCR was performed according to Case 3.

[0079] Callus silencing the CbLTP63 gene after high-salt treatment (300 mM NaCl) showed a significantly increased survival rate compared to the control. Figure 6 The above results indicate that silencing the negative regulator of salt stress response in Catalpa bungei significantly improves its salt tolerance, suggesting that the CbLTP63 gene is a key regulatory gene in the salt stress response of Catalpa bungei.

[0080] Appendix to the Instruction Manual

[0081] Seq 1:

[0082] ATGGCATCAGTATCAGTGAAGTGGTTTTGCATTGTGGGATTTTTAGCAATCG

[0083] TTAGCTTCAACTACGCATATGCAGTCGGTGAGTGCCCCAAGTCCACACCCG

[0084] ACGCGGAGGCCATGAAGCTGTTCCCTTGCGCGTCGGCAGCACAAGACCCG

[0085] AATGCTGCGGTTTCAAGGAGTTGCTGTGCTCAGGTGAAGAAACTTGGACA

[0086] GAACCCCAACTGCTTGTGCGCTGTTATGCTGTCTGATACGGCTAAGAGCGC

[0087] CGGCGTGAAGCCTGAGATTGCCTTGACCATTCCCAAGCGATGCAACTTTGC

[0088] TGACAGGCCTGTGGGTTACAAGTGTGGAGCTTACACGTTGCCTTAA

[0089] Seq 2:

[0090] MASVSVKWFCIVGFLAIVSFNYAYAVGECPKSTPDAEAMKLFPCASAAQDPN

[0091] AAVSRSCCAQVKKLGQNPNCLCAVMLSDTAKSAGVKPEIALTIPKRCNFADRP VGYKCGAYTLP*。

Claims

1. A key gene CbLTP63 regulating the salt stress response of Catalpa bungei, characterized in that, The nucleotide sequence of the CbLTP63 gene is as described in SEQ ID NO.

1.

2. The key gene CbLTP63 for regulating salt stress response in Catalpa bungei according to claim 1, characterized in that, The amino acid sequence encoded by the CbLTP63 gene is as described in SEQ ID NO.

2.

3. The application of the key gene CbLTP63 for regulating the salt stress response of Catalpa bungei according to claim 1 or 2, characterized in that, The CbLTP63 gene is a negative regulator of the salt stress response of Catalpa trees. Silencing the CbLTP63 gene significantly enhances the salt tolerance of Catalpa callus.

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