Gene ptoxth30 for improving drought resistance and salt tolerance of poplar and application thereof

Abstract

The application discloses a gene PtoXTH30 for improving drought resistance and salt tolerance of poplar and application of the gene, a nucleotide sequence of the gene is shown in SEQ ID NO. 2, the gene can accurately regulate drought resistance and salt tolerance of the poplar, and provides a key candidate gene for poplar stress resistance molecular breeding. The application also discloses application of the PtoXTH30 gene for improving drought resistance and salt tolerance of the poplar, and the drought resistance and salt tolerance of the poplar can be significantly enhanced. The application also discloses a method for regulating drought resistance and salt tolerance of the poplar, the drought resistance and salt tolerance of the poplar can be accurately regulated according to needs, a breeding cycle is effectively shortened, and a new drought-resistant and salt-tolerant transgenic variety is helpful to be cultivated.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to poplar. XTH Genes, particularly one that enhances the drought resistance and salt tolerance of poplar trees. PtoXTH30 And its applications. Background Technology

[0002] In nature, environmental stressors affecting plant growth and development are mainly divided into two categories: biotic stresses, including diseases and pests; and abiotic stresses, including drought, salinity, high temperature, heavy metal pollution, ozone, and radiation. As major abiotic stresses, drought and salinity stress have a significant impact on the growth, development, and physiological functions of plants in arid and semi-arid salinized areas. To maintain the health of my country's ecological environment and build an ecological civilization, discovering more drought- and salt-tolerant plants is a crucial technology for controlling the expansion of arid regions and the increase in soil salinization. Poplar is an important economic tree species in my country, characterized by rapid growth, high quality, and strong adaptability, and is widely distributed. Developing new drought- and salt-tolerant poplar varieties through genetic engineering can broaden its planting range, increase forestry productivity, and improve both economic and ecological benefits.

[0003] XTH (xyglucan endosyltransferase / hydrolase) belongs to the GH16 family of glycoside hydrolases and possesses dual activities as both an endosyltransferase and a hydrolase. It is a key enzyme regulating dynamic cell wall remodeling and growth in plants. This gene family is rich in members with significant functional differentiation, and has been identified in various plants: 33 in Arabidopsis thaliana, 29 in rice, 41 in poplar, 23 in cotton, 25 in tomato, 56 in tobacco, 61 in soybean, and 24 each in barley and pineapple. Current research on herbaceous plants shows that XTH family members in Arabidopsis thaliana exhibit stress-resistance specificity, for example: AtXTH19 Mutations can reduce cold resistance and freeze resistance. AtXTH17 and AtXTH31 The mutant exhibits enhanced aluminum tolerance. However, research on the function and regulatory mechanisms of XTH homologous genes in abiotic stress responses in poplar, a woody model plant, remains scarce. Therefore, exploring the stress resistance role of poplar XTH homologous genes is of significant scientific value for enriching research on stress resistance mechanisms in this plant species and is crucial for accelerating the genetic improvement of stress resistance in forest trees. Summary of the Invention

[0004] To overcome the above problems, the inventors conducted extensive research and discovered... PtoXTH30 Genes are associated with the drought and salt tolerance of poplar trees, and can be overexpressed in poplar trees. PtoXTH30The transgenic plants overexpressing the gene showed significantly better growth under salt and drought stress than wild-type and silent-expressing plants, demonstrating that this gene can precisely regulate the drought and salt tolerance of poplar. This enriches the gene regulatory network for drought and salt tolerance in poplar, lays a theoretical and genetic resource foundation for molecular breeding of poplar stress resistance, and shortens the breeding cycle, thus completing this invention.

[0005] Specifically, the object of the present invention is to provide the following aspects:

[0006] In a first aspect, a protein is provided to improve the drought resistance and salt tolerance of poplar trees, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] Secondly, a gene is provided to improve the drought resistance and salt tolerance of poplar trees, wherein the gene is... PtoXTH30 The gene is the gene encoding the protein described in the first aspect, and its nucleotide sequence is shown in SEQ ID NO.2.

[0008] Thirdly, the application of the protein described in the first aspect or the gene described in the second aspect in improving the drought resistance and salt tolerance of poplar trees is provided.

[0009] Fourthly, the application of the protein described in the first aspect or the gene described in the second aspect in poplar breeding, wherein the breeding aims to improve the drought resistance and salt tolerance of poplar.

[0010] Fifthly, providing the content described in the second aspect. PtoXTH30 Application of recombinant gene expression vectors in improving the drought resistance and salt tolerance of poplar trees.

[0011] In a sixth aspect, a method for regulating the drought resistance and salt tolerance of poplar trees is provided, the method comprising the steps of increasing or decreasing the content of the protein described in the first aspect in poplar trees.

[0012] The beneficial effects of this invention include:

[0013] (1) The present invention provides a method to improve the drought resistance and salt tolerance of poplar trees. PtoXTH30 Genes can precisely regulate the drought resistance and salt tolerance of poplar trees, providing key candidate genes for molecular breeding of poplar trees to resist stress and enriching the gene regulatory network for drought resistance and salt tolerance in poplar trees.

[0014] (2) The present invention provides a method to improve the drought resistance and salt tolerance of poplar trees. PtoXTH30 The application of genes can significantly enhance the drought resistance and salt tolerance of poplar trees, improve their ability to grow and survive in arid areas, lay a theoretical and genetic resource foundation for molecular breeding of poplar trees to resist stress, and promote the sustainable development of forestry.

[0015] (3) The method for regulating the drought resistance and salt tolerance of poplar provided by the present invention can accurately regulate the drought resistance and salt tolerance of poplar as needed, effectively shorten the breeding cycle, help to cultivate new drought-resistant and salt-tolerant transgenic varieties, and also provide new ideas for further research on plant stress resistance mechanisms. Attached Figure Description

[0016] Figure 1 This illustrates the RT-qPCR detection of Populus tomentosa in Example 1. PtoXTH30 A comparison of the relative expression levels of genes under different salt treatment times (0-48h); Figure 2 This illustrates the RT-qPCR detection of Populus tomentosa in Example 1. PtoXTH30 A comparison of the relative expression levels of genes under different drought treatment durations (0-3d); Figure 3 Showing RT-qPCR detection PtoXTH30 Transcriptional levels in overexpressing transgenic plants; error bars represent standard deviation. Figure 4 The RT-qPCR detection is shown in the figure. PtoXTH30 Transcriptional levels in silent transgenic plants; error bars represent standard deviation. Figure 5 Showing WT, PtoXTH30 Morphological phenotypes of overexpressing plants (OE-1, OE-7) and silent transgenic plants (RNAi-4, RNAi-10) before and after salt stress and drought stress; Figure 6 Showing WT, PtoXTH30 Analysis of malondialdehyde (MDA), hydrogen peroxide (H2O2), and peroxidase (POD) content in overexpressing transgenic plants (OE-1, OE-7) and silent transgenic plants (RNAi-4, RNAi-10) before and after treatment with 200 mM NaCl; Figure 7 Showing WT, PtoXTH30 Analysis of MDA, H2O2, and POD contents in overexpressing transgenic plants (OE-1, OE-7) and silent transgenic plants (RNAi-4, RNAi-10) before and after treatment with 20% PEG6000; Figure 8 Showing WT, PtoXTH30 Schematic diagram of NBT staining of leaves of overexpressing transgenic plants (OE-1, OE-7) and silent transgenic plants (RNAi-4, RNAi-10) before and after treatment with 200mM NaCl; Figure 9 Show WT, PtoXTH30 Schematic diagram of DAB staining of leaves of overexpressing transgenic plants (OE-1, OE-7) and silent transgenic plants (RNAi-4, RNAi-10) before and after treatment with 20% PEG6000. Detailed Implementation

[0017] The present invention will be further described in detail below through preferred embodiments and examples. Through these descriptions, the features and advantages of the present invention will become clearer and more apparent.

[0018] The term “exemplary” as used herein means “serving as an example, embodiment, or illustration.” Any embodiment illustrated herein as “exemplary” is not necessarily to be construed as superior to or better than other embodiments.

[0019] In a first aspect, the present invention provides a protein that improves the drought resistance and salt tolerance of poplar trees, the amino acid sequence of which is shown in SEQ ID NO.1.

[0020] In this invention, the poplar is preferably white poplar, and more preferably hairy white poplar.

[0021] In a second aspect, the present invention provides a gene for improving the drought resistance and salt tolerance of poplar trees, said gene being PtoXTH30 Genes, specifically the genes encoding the proteins described in the first aspect.

[0022] Preferably, the PtoXTH30 The nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0023] In this invention, PtoXTH30 The gene is located on chromosome 3 of the Populus tomentosa genome (http: / / db.cngb.org / cnsa / project / CNP0004290_40760cfc / reviewlink / ), with a start position of 10684237 and an end position of 10687578.

[0024] PtoXTH30 Gene sequence optimization information can be found in the following document: National Gene Bank: Genome Sequencing of Populus tomentosa Project number: CNP0004290, Sample number: CNS0752799, Assembly number: CNA0069009.

[0025] More preferably, the PtoXTH30 The nucleotide sequence of the coding region of the gene is shown in SEQ ID NO.3.

[0026] PtoXTH30 Preferred coding region sequences of genes can be found in the following file: National Gene Bank: Genome Sequencing of Populus tomentosa Project number: CNP0004290, Sample number: CNS0752799, Assembly number: CNA0069009; PtoXTH30Location: Chromosome 3, start position 10687377, end position 10687578; start position 10687163, end position 10687263; start position 10686179, end position 10686387; start position 10684237, end position 10684771; see http: / / db.cngb.org / cnsa / project / CNP0004290_40760cfc / reviewlink / for details.

[0027] In this invention, the PtoXTH30 Genes positively regulate the drought resistance and salt tolerance of poplar trees.

[0028] In a preferred embodiment, the poplar contains a high content of the protein described in the first aspect or the protein described in the second aspect. PtoXTH30 High gene expression levels indicate stronger drought resistance and salt tolerance in poplars; low levels of the protein described in the first aspect or the protein described in the second aspect in poplars... PtoXTH30 When gene expression levels are low, poplar trees are less drought-resistant and less salt-tolerant.

[0029] The present invention provides PtoXTH30 Genes can precisely regulate the drought resistance and salt tolerance of poplar trees, providing key candidate genes for molecular breeding of poplar trees to resist stress and enriching the gene regulatory network for drought resistance and salt tolerance in poplar trees.

[0030] A third aspect of the present invention provides the application of the protein described in the first aspect or the gene described in the second aspect in improving the drought resistance and salt tolerance of poplar trees.

[0031] The poplar tree is preferably Populus tomentosa, and more preferably Populus pubescens.

[0032] Preferably, the application increases the protein content described in the first aspect or the protein content described in the second aspect in poplar. PtoXTH30 It is achieved through the expression level of genes.

[0033] More preferably, the application will... PtoXTH30 This was achieved by overexpressing the gene in poplar trees.

[0034] A fourth aspect of the present invention provides the application of the protein described in the first aspect or the gene described in the second aspect in poplar breeding, wherein the breeding is for improving the drought resistance and salt tolerance of poplar.

[0035] The poplar tree is preferably Populus tomentosa, and more preferably Populus pubescens.

[0036] Preferably, the application is performed by overexpressing [a specific substance] in poplar trees. PtoXTH30 It is achieved through genes.

[0037] More preferably, the overexpression in poplarPtoXTH30 Genes include PtoXTH30 The steps for introducing gene overexpression recombinant vectors into poplar trees.

[0038] In a preferred embodiment, the PtoXTH30 The backbone vector for the gene overexpression recombinant vector is the pBI121 vector.

[0039] Preferably, the PtoXTH30 Gene overexpression recombinant vectors, through PtoXTH30 The coding region sequence of a gene is constructed onto a backbone vector to obtain the gene. PtoXTH30 The coding region sequence of the gene is shown in SEQ ID NO.3.

[0040] The present invention provides PtoXTH30 The application of genes, through overexpression in poplar trees PtoXTH30 The gene can significantly enhance the drought resistance and salt tolerance of poplar, laying a theoretical and genetic resource foundation for molecular breeding of poplar stress resistance.

[0041] A fifth aspect of the invention provides a system comprising the components described in the second aspect. PtoXTH30 Application of recombinant gene expression vectors in improving the drought resistance and / or salt tolerance of poplar trees.

[0042] Preferably, the recombinant expression vector is used to... PtoXTH30 The coding region sequence of a gene is constructed onto a backbone vector.

[0043] In this invention, plant expression vectors from the prior art can be used to construct expression vectors containing... PtoXTH30 Recombinant gene expression vectors.

[0044] Preferably, the backbone vector of the recombinant expression vector is the pBI121 vector.

[0045] In a preferred embodiment, the application includes the step of introducing a recombinant expression vector into poplar trees to obtain transgenic poplar trees.

[0046] Preferably, the recombinant expression vector is transformed into poplar trees using an Agrobacterium-mediated transformation method.

[0047] More preferably, the poplar is a white poplar, and more preferably a hairy white poplar.

[0048] In a sixth aspect, the present invention provides a method for regulating the drought resistance and / or salt tolerance of poplar trees, the method comprising the steps of increasing or decreasing the content of the protein described in the first aspect in poplar trees.

[0049] Preferably, the increase or decrease in the content of the protein described in the first aspect in poplar is achieved by increasing or decreasing the expression level of the gene described in the second aspect in poplar.

[0050] More preferably, the increase in the expression level of the gene described in the second aspect in poplar is achieved by... PtoXTH30 This was achieved by overexpressing the gene in poplar trees.

[0051] The reduction of gene expression in poplar trees in the second aspect is achieved by using gene silencing technology or gene editing technology to reduce gene expression in poplar trees.

[0052] Wherein, the overexpression utilizes PtoXTH30 Gene overexpression recombinant vectors are used;

[0053] The reduction of gene expression levels in poplar trees, as described in the second aspect, is achieved using gene silencing technology, preferably utilizing... PtoXTH30 Gene silencing and recombination are carried out using a gene silencing vector.

[0054] In a preferred embodiment, the PtoXTH30 Gene silencing recombination vectors, through PtoXTH30 The interference fragment of the gene was constructed onto a backbone vector.

[0055] The PtoXTH30 The interference fragment of the gene is PtoXTH30 -RNAi, whose nucleotide sequence is shown in SEQ ID NO.4;

[0056] The backbone vector of the silenced recombination vector is the pBI121 vector.

[0057] In a preferred embodiment, the method for regulating the drought resistance and / or salt tolerance of poplar trees includes the following steps:

[0058] Step 1, Build PtoXTH30 Gene overexpression recombinant vectors and silencing recombinant vectors.

[0059] Step 1 includes the following sub-steps:

[0060] Step 1-1, obtain PtoXTH30 CDS fragments and interference fragments of genes.

[0061] Preferably, the PtoXTH30 The sequence of the CDS fragment of the gene is shown in SEQ ID NO.3, and the sequence of the interference fragment is shown in SEQ ID NO.4.

[0062] More preferably, PtoXTH30 The primers for amplifying the CDS fragment of the gene are: PtoXTH30 -OE-F and PtoXTH30-OE-R, whose nucleotide sequences are shown in SEQ ID NO.5 (agaacacgggggactATGGATTGCTTATGTTGGT) and SEQ ID NO.6 (acccccggggatcctCATATCCGGATCATATTCTAAAGTA);

[0063] PtoXTH30 The primers for amplifying the interference fragment of the gene are: PtoXTH30 -RNAi-F and PtoXTH30 -RNAi-R, whose nucleotide sequences are shown in SEQ ID NO.7 (acccccggggatcctTGGCCAAATTTCTGAAACAAATGTC) and SEQ ID NO.8 (agaacacgggggactGTCAGGTTGGCTGTCTTCT).

[0064] Steps 1-2, will PtoXTH30 The CDS fragment and interference fragment of the gene were ligated to the enzyme-digested backbone vector, respectively.

[0065] Preferably, the backbone vector is digested with XbaI enzyme.

[0066] Step 2, PtoXTH30 Gene overexpression and silencing recombinant vectors were used to transform Agrobacterium, which was then used to infect poplar materials to obtain transgenic poplars.

[0067] Preferably, the poplar material is white poplar.

[0068] Step 3: Identify and perform phenotypic analysis on the transgenic poplar trees.

[0069] Preferably, the identification includes identification at the DNA level and identification at the transcription level.

[0070] More preferably, the DNA level identification is performed by PCR amplification using gDNA from transgenic poplar as a template, with the following reaction program: 95℃ for 2 min; (94℃ for 30 s; 55℃ for 30 s; 72℃ for 60 s) for 35 cycles; 72℃ for 5 min; 4℃ for ∞;

[0071] The transcription level was identified by RT-PCR amplification using cDNA from transgenic poplar as a template. The reaction program was as follows: 95℃ for 30 s; (95℃ for 5 s; 60℃ for 35 s) for 40 cycles; 95℃ for 15 s; 60℃ for 1 min; 95℃ for 15 s.

[0072] In a preferred embodiment, the phenotypic analysis includes analysis of salt stress treatment, analysis of drought stress treatment, physiological index analysis, NBT and DAB analysis.

[0073] The physiological index analysis includes the analysis of H2O2 (hydrogen peroxide), MDA (malondialdehyde), and POD (peroxidase).

[0074] Preferably, the salt stress treatment is as follows: the plants are cultured in a constant temperature culture room at 25°C under the same light conditions for 20 days, and then irrigated with 200mM NaCl solution for 17 days;

[0075] The drought stress treatment was as follows: the plants were cultured in a constant temperature incubator at 25℃ under the same light conditions for 20 days, and then irrigated with 20% PEG 6000 solution for 13 days.

[0076] In a preferred embodiment, under salt stress, the leaves of wild-type plants and silent plants exhibited greater... PtoXTH30 Overexpression leads to more severe wilting and yellowing of plants;

[0077] After 13 days of treatment with 20% PEG 6000 simulating drought stress, PtoXTH30 Overexpressing plants showed better growth and exhibited less leaf wrinkling and chlorosis compared to wild-type and silent plants.

[0078] Physiological parameters measured under salt and drought stress showed that, compared to wild-type plants and silent plants, PtoXTH30 Under both stress conditions, the accumulation of hydrogen peroxide (H2O2) and malondialdehyde (MDA) in gene-overexpressing plants was significantly reduced, while the activity of peroxidase (POD) was significantly increased.

[0079] After salt stress treatment, the leaves of wild-type plants and silent plants showed a deeper blue staining effect when stained with NBT, while PtoXTH30 The overexpression resulted in lighter coloration in the plants;

[0080] After drought stress treatment, PtoXTH30 The leaves of overexpressing plants showed significantly less brown precipitate (indicating H2O2) than those of wild-type and silent plants.

[0081] The method for regulating the drought resistance and salt tolerance of poplar trees provided by this invention can accurately regulate the drought resistance and salt tolerance of poplar trees as needed, effectively shorten the breeding cycle, help to cultivate new drought-resistant and salt-tolerant transgenic varieties, and also provide new ideas for further research on plant stress resistance mechanisms. Example

[0082] The present invention is further described below through specific examples; however, these examples are merely exemplary and do not constitute any limitation on the scope of protection of the present invention.

[0083] Unless otherwise specified, the reagents involved in the following examples are all commercially available conventional reagents, and the methods used are all methods commonly used in this technical field.

[0084] Example 1 Poplar PtoXTH30 Quantitative fluorescence detection of genes

[0085] (1) Two-month-old Populus tomentosa were subjected to salt stress and drought stress treatment with 150mM NaCl and 20% PGE 6000, respectively. Leaves of Populus tomentosa treated with 150mM NaCl at 0h, 6h, 12h, 24h, and 48h (5 time points) and treated with 20% PGE 6000 at 0d, 1d, and 3d (3 time points) were collected and RNA was extracted. The RNA was extracted using the Plant RNA Extraction Kit of Beijing TransGen Biotech Co., Ltd.

[0086] (2) The RNA extracted in step (1) was reverse transcribed to obtain cDNA, which was then processed using HiScript IV All-in-One Ultra RT SuperMix for qPCR-R433 (Nanjing Novizan Biotechnology Co., Ltd.).

[0087] The reaction system is shown in Table 1:

[0088] Table 1

[0089]

[0090] The reaction procedure was: 50℃ for 5 min; 85℃ for 5 sec.

[0091] (3) According to the white poplar PtoXTH30 Primers for quantitative real-time PCR were designed based on the gene CDS sequence (as shown in SEQ ID NO3). PtoXTH30 -qPCR-F and PtoXTH30 The qPCR-R primers are shown in SEQ ID NO. 9 and SEQ ID NO. 10, respectively. The internal control primers are Actin-F and Actin-R, with sequences shown in SEQ ID NO. 11 and SEQ ID NO. 12, respectively. Real-time PCR was used to determine the effects of salt and drought stress at different time points. PtoXTH30 Gene expression levels.

[0092] RT-qPCR experiments were performed using 2×ChamQ SYBR Color qPCR Master Mix. The reaction volume (20 μL) is shown in Table 2.

[0093] Table 2

[0094]

[0095] The reaction program was as follows: 95℃ for 30 s; (95℃ for 5 s; 60℃ for 35 s) for 40 cycles; 95℃ for 15 s; 60℃ for 1 min; 95℃ for 15 s.

[0096] The results of the real-time PCR reaction are as follows Figure 1 and Figure 2 As shown, it can be seen that regardless of whether it is salt treatment or drought treatment, as the duration of stress increases, Populus tomentosa... PtoXTH30 The expression levels of genes underwent significant and regular changes, indicating that... PtoXTH30 The gene is highly likely involved in the molecular regulatory network of Populus tomentosa and drought stress, playing a functional role in the poplar's resistance to abiotic stresses.

[0097] Example 2 PtoXTH30 Obtaining overexpressed and silenced transgenic poplar trees

[0098] (1) PtoXTH30 Construction of overexpression recombinant vectors and silencing recombinant vectors (RNAi recombinant vectors)

[0099] (1.1) PtoXTH30 Obtaining the target gene fragment

[0100] RNA was extracted from LM50 Populus tomentosa using the Plant RNA Extraction Kit from Beijing TransGen Biotechnology Co., Ltd.; the RNA was reverse transcribed into cDNA using the HiScript III 1st Strand cDNA Synthesis Kit (Nanjing Novizan Biotechnology Co., Ltd.).

[0101] Using cDNA as a template and referring to the Populus tomentosa genome file (http: / / db.cngb.org / cnsa / project / CNP0004290_40760cfc / reviewlink / ), primers for amplifying the target fragment were designed using the Primer BLAST tool (NCBI, https: / / blast.ncbi.nlm.nih.gov), taking into account various primer design principles. The target fragment included the fragment used to construct the overexpression vector. PtoXTH30 The CDS sequence of the gene and the target fragment for constructing the RNAi recombinant vector ( PtoXTH30 Gene CDS partially reversed sequence).

[0102] PtoXTH30 The primers for amplifying the CDS sequence of the gene are: PtoXTH30-OE-F and PtoXTH30 -OE-R, whose nucleotide sequences are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively; PtoXTH30 Interference fragments PtoXTH30 -RNAi ( PtoXTH30 Gene The amplification primers for the CDS partial reverse sequence are: PtoXTH30 -RNAi-F and PtoXTH30 -RNAi-R, whose nucleotide sequences are shown in SEQ ID NO.7 and SEQ ID NO.8, respectively. Among them, the gene... PtoXTH30 Located on chromosome 3 of the Populus tomentosa genome (http: / / db.cngb.org / cnsa / project / CNP0004290_40760cfc / reviewlink / ), with start position 10684237 and end position 10687578.

[0103] PCR amplification was performed using 2x Phanta Max Master Mix (Dye Plus) from Nanjing Vazyme Company. The PCR reaction system (50 μL) is shown in Table 3.

[0104] Table 3

[0105]

[0106] The reaction program was as follows: 95℃ for 3 min; (95℃ for 25 s; 55℃ for 30 s; 72℃ for 60 s (amplification efficiency 45 s / kb)) 36 cycles; 72℃ for 5 min; 4℃ to infinity.

[0107] The PCR products were subjected to agarose gel electrophoresis. After verification, the DNA was recovered and purified using the Kangwei Century DNA Clean-up Kit. Following purification, the purity and concentration of the purified DNA product were determined using an instrument.

[0108] get PtoXTH30 The CDS sequence of the gene is shown in SEQ ID NO.3, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.1. Interference fragments... PtoXTH30 The nucleotide sequence of -RNAi is shown in SEQ ID NO.4.

[0109] (1.2) Vector digestion

[0110] The pBI121 vector enzyme digestion reaction system is shown in Table 4:

[0111] Table 4

[0112]

[0113] The enzyme digestion reaction conditions are: 37℃ water bath for 1-2 hours.

[0114] The enzyme digestion products were purified by gel extraction using the Kangwei Century Gel Extraction Kit and purified using the Kangwei Century DNA Clean-up Kit. The purified products were then used as vector backbones and stored at -20°C.

[0115] (1.3) Connection transformation

[0116] Using the Uniclone One Step Seamless Cloning Kit from Beijing Jinsha Biotechnology Co., Ltd., respectively... PtoXTH30 The CDS and CDS partial reverse sequences were constructed into the pBI121 vector to obtain the overexpression recombinant vector and the RNAi recombinant vector, respectively. Ligation was performed at 50℃ for 10 min.

[0117] Take 50 μl of DH5α competent cells, add 5 μl of ligation product, gently tap to mix, and incubate on ice for 30 minutes. Heat shock in a 42°C water bath for 45 seconds, then quickly transfer to ice to cool for 2 minutes. Add 700 μl of antibiotic-free sterile LB broth to a clean bench, mix well, and incubate at 37°C and 220 rpm for 45 minutes to allow the cells to recover. After recovery, centrifuge at 6000 rpm for 1 minute, collect the cells, and resuspend the cells in 100 μl of supernatant. Spread the resuspended cells onto LB agar plates containing kanamycin (100 mg / ml) and incubate upside down at 37°C for 14-16 hours.

[0118] (1.4) Identification of positive clones by bacterial culture PCR

[0119] Single colonies from the plate were picked sequentially using sterilized pipette tips and added to 250 μl of LB broth containing kanamycin. The culture was incubated at 37°C with shaking at 200 rpm for approximately 3 hours to serve as amplification templates. PCR amplification was performed using Taq Plus Master Mix from Nanjing Vazyme. The PCR reaction system is shown in Table 5.

[0120] Table 5

[0121]

[0122] The sequence of the pBI121-R primer is shown in SEQ ID NO.13.

[0123] PCR products were detected by 1% agarose gel electrophoresis.

[0124] (1.5) Extraction of positive clone plasmids

[0125] Clones that tested positive for PCR were aspirated into 6 ml of LB liquid medium containing kanamycin and incubated overnight at 37°C with shaking at 200 rpm. Plasmids were extracted using a plasmid miniprep kit from Jiangsu Kangwei Reagent Co., Ltd., and sequenced by Beijing Ruiboxingke Biotechnology Co., Ltd. After the sequences were confirmed to be correct, the construction of the overexpression recombinant vector and the RNAi recombinant vector was completed.

[0126] (2) PtoXTH30 Genetic transformation

[0127] (2.1) Transformation of Agrobacterium with recombinant plasmids

[0128] Take about 1μg PtoXTH30 Overexpression recombinant vector plasmids and PtoXTH30- The RNAi recombinant vector plasmid was added to 100 μL of Agrobacterium GV3101 competent cells and gently mixed. The cells were then incubated sequentially on ice for 5 min, flash-frozen in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and in an ice bath for 5 min. 700 μL of antibiotic-free YEP liquid medium was added to the bacterial culture, and the mixture was thoroughly mixed. The culture was then incubated at 28°C and 200 rpm with shaking for 2–3 hours. After the incubation period, the cells were centrifuged at 6000 rpm for 1 minute, and a portion of the supernatant was discarded. 100 μL of the supernatant was mixed with the bacterial culture and evenly spread onto the surface of YEP solid medium containing 50 mg / L rifampin and 50 mg / L kanamycin. The culture was then incubated upside down at 28°C for 72–90 hours.

[0129] After single colonies have grown, a few monoclonal plaques are picked up with a sterile pipette tip and placed in a 2 mL centrifuge tube containing 250 μL of YEP liquid medium (1:1000 with added Rif and Kana). The tubes are then incubated on a shaker at 30°C and 200 rpm for 2 hours. After incubation, the bacterial culture is aspirated for PCR identification. Positive bacterial cultures that have been correctly identified are added to 50% glycerol, flash-frozen in liquid nitrogen, and stored at -80°C for subsequent genetic transformation experiments.

[0130] (2.2) Activation of Agrobacterium

[0131] Take out the contents from the -80℃ freezer PtoXTH30 Agrobacterium tumefaciens overexpressing recombinant vector plasmids and RNAi recombinant vector plasmids were streaked onto YEP solid medium (1:1000 with added Rif and Kana), sealed, and incubated upside down in a dark incubator at 28°C for 2-3 days.

[0132] Using a sterile pipette tip, pick a single colony from the plate and inoculate it into 3 mL of YEP liquid medium (1:1000 with added Rif and Kana). Incubate overnight at 30°C and 200 rpm with shaking. Transfer 1 mL of the bacterial culture to a 250 mL sterile Erlenmeyer flask containing 100 mL of YEP liquid medium (1:1000 with added Rif and Kana). Incubate at 30°C and 200 rpm with shaking for 4-5 hours, until the OD600 reaches 0.3-0.5.

[0133] Take 100 mL of bacterial suspension, centrifuge at 2560 g for 20 min at 4 °C, discard the supernatant, and resuspend the bacterial cells in 100 mL of WPMB resuspension solution in a sterile wide-mouth bottle. This bacterial suspension will be used for subsequent infection experiments.

[0134] (2.3) Callus Infection

[0135] Select leaves from healthy, sterile poplar seedlings (dark green leaves with thicker texture). Using a sterile scalpel, make 2-3 horizontal incisions along the midrib of the leaf. Lay the leaves face down on callus medium (CIM) and incubate in the dark at 25°C. After 20-30 days of leaf growth, loose white callus will form at the incision sites. Peel the callus off the leaf, divide it into soybean-sized pieces, and place them in a new callus medium. Transfer all the prepared callus to a sterile wide-mouth bottle containing bacterial solution and incubate on a shaker at 28°C and 160 rpm for 15-20 minutes.

[0136] In a clean bench, use filter paper to absorb excess bacterial solution from the callus and spread it evenly on a co-culture plate (WPMC). Incubate in the dark at 25°C for 2 days with the plate upside down.

[0137] After dark culture, the callus was transferred to differentiation medium (WPMD). The medium was changed approximately every 20 days for the first time, and then every 10 days thereafter. During this period, the callus will turn green, harden, and then partially turn red. This stage takes about two months, and the entire process is carried out in a 25°C light incubator.

[0138] After the adventitious buds grow to about half a centimeter, use sterile tweezers or a scalpel to cut them off and place them in a bud elongation medium to grow. When the adventitious buds grow to 1-2 centimeters, cut them off individually and place them on a rooting medium for rooting culture. The adventitious buds will grow roots in about 10 days and grow into complete plants.

[0139] The formulation of the culture medium is shown in Table 6:

[0140] Table 6

[0141]

[0142] After preparation, sterilize at 121℃ for 20 minutes under high temperature and pressure.

[0143] Example 3 PtoXTH30 Identification of overexpressing and silent transgenic poplar trees

[0144] (1) Wild-type white poplar, PtoXTH30 Crude extraction of DNA from gene-overexpressing and gene-silencing plants:

[0145] (i) Take a leaf sample to be extracted, place it in a 2mL centrifuge tube and add a grinding bead. After quick freezing with liquid nitrogen, use a tissue homogenizer to homogenize for 5 minutes.

[0146] (ii) After the mixture is broken into powder, add 500 μL of TBS buffer to the centrifuge tube, mix well, and then heat in a 65°C water bath for 10 min.

[0147] (iii) Centrifuge at 12000 rpm for 15 min, transfer the supernatant to a new centrifuge tube, add an equal volume of isopropanol, and allow to settle at room temperature for 30 min. A white flocculent precipitate will be visible.

[0148] (iv) Centrifuge at 12000 rpm for 15 min, slowly discard the supernatant, and add 1 ml of 75% ethanol to wash the precipitate.

[0149] (v) Centrifuge at 7500 rpm for 10 min, slowly pour off the supernatant and remove excess liquid with a pipette.

[0150] (vi) Place in a 40℃ oven for 2 hours.

[0151] (vii) Add 50 μL of ddH2O to the centrifuge tube to dissolve the precipitate and store it in a -20°C freezer.

[0152] (2) PtoXTH30 Identification of gene overexpression and silencing plants

[0153] (2.1) Identification at the DNA level

[0154] Using crudely extracted transgenic plant gDNA as a template, PCR identification was performed using Taq Plus Master Mix, with the reaction system as shown in Table 5 of Example 2.

[0155] The PCR reaction program was as follows: 95℃ for 2 min; (94℃ for 30 s; 55℃ for 30 s; 72℃ for 60 s) for 35 cycles; 72℃ for 5 min; 4℃ for ∞.

[0156] The PCR products were detected by agarose gel electrophoresis to check if the bands were correct. If correct, the plant was considered a healthy plant. PtoXTH30 Plants with overexpressed genes or plants with silenced genes.

[0157] PCR testing revealed that plants numbered OE-1, OE-2, OE-3, OE-4, OE-5, OE-6, and OE-7 were... PtoXTH30 The overexpressing transgenic plants, numbered RNAi-1, RNAi-2, RNAi-3, RNAi-4, RNAi-5, RNAi-6, RNAi-7, RNAi-8, RNAi-9, RNAi-10, RNAi-11, RNAi-12, RNAi-13, and RNAi-14, are... PtoXTH30 Silent genetically modified plants.

[0158] (2.2) Identification of transcription level

[0159] RT-qPCR detection PtoXTH30 Transcriptional levels in overexpressed and silenced plants:

[0160] (i) RNA was extracted from the leaves of the plant to be tested and reverse transcribed to obtain template cDNA. RNA extraction and reverse transcription of cDNA were performed according to the method described in Example 1.

[0161] (ii) RT-qPCR reaction was performed using 2×ChamQ SYBR Color qPCR Master Mix. The reaction system was as described in Table 2 of Example 1, and the primers were as follows. PtoXTH30 -qPCR-F and PtoXTH30 -qPCR-R, with Actin-F and Actin-R as internal reference primers.

[0162] The reaction program was as follows: 95℃ for 30 s; (95℃ for 5 s; 60℃ for 35 s) for 40 cycles; 95℃ for 15 s; 60℃ for 1 min; 95℃ for 15 s.

[0163] The detection results of overexpressing plants are as follows Figure 3 As shown: PtoXTH30 The expression levels of OE-1 and OE-7 were higher in the overexpressing transgenic plants, which were 47.26 times and 46.52 times higher than those in wild-type plants (WT), respectively.

[0164] The test results of silent plants are as follows Figure 4 As shown: PtoXTH30 The expression levels of RNAi-4 and RNAi-10 in the silenced transgenic plants were lower, at 0.146 times and 0.126 times that of the wild-type plants (WT), respectively.

[0165] Example 4 PtoXTH30 Stress phenotype analysis of overexpressing and silent transgenic plants

[0166] (1) Salt stress treatment

[0167] Poplar plants with uniform growth after 25 days of tissue culture were washed with warm water to remove the agar from their roots and transferred to a soil culture medium containing a 1:1.5 mixture of substrate and vermiculite. The light conditions were 8 hours of darkness followed by 16 hours of light. The plants were then cultured for 20 days in a 25°C constant temperature incubator under the same light conditions, and then irrigated with 200 mM NaCl solution for 17 days.

[0168] The growth of plants after salt stress treatment is as follows Figure 5 As shown, after treatment with 200mM NaCl for 17 days, the wild-type (WT) and PtoXTH30 The leaves of the silenced transgenic plants (numbered RNAi-4 and RNAi-10) showed more... PtoXTH30 The overexpression transgenic plants (numbered OE-1 and OE-7) exhibited more severe wilting and yellowing, indicating that overexpression... PtoXTH30 Genes can significantly enhance a plant's salt tolerance.

[0169] (2) Drought stress treatment

[0170] Plants with uniform growth after 25 days of tissue culture were washed with warm water to remove the agar from their roots and transferred to a soil culture medium containing a 1:1.5 mixture of substrate and vermiculite. The light conditions were 8 hours of darkness followed by 16 hours of light. The plants were then cultured for 20 days under the same light conditions in a 25°C constant temperature incubator, and then irrigated with a 20% PEG 6000 solution for 13 days.

[0171] The plant is growing well Figure 5 As shown, it can be seen that: PtoXTH30 The overexpressing transgenic plants (numbered OE-1 and OE-7) were slightly better than the wild type, and their growth was better under drought stress simulated by 20% PEG 6000. Compared with the wild type and the silent transgenic plants (numbered RNAi-4 and RNAi-10), the leaves showed less wrinkling and chlorosis.

[0172] (3) PtoXTH30 Physiological parameters of overexpressing and silencing transgenic plants were measured.

[0173] H2O2 (hydrogen peroxide), MDA (malondialdehyde), and POD (peroxidase) in the plants were detected using an ELISA reader, with kits from Keming Biotechnology used for all tests.

[0174] The measurement results under salt stress and drought stress are as follows: Figure 6 and 7 As shown, it can be seen that under both stress conditions, compared with wild-type poplar and PtoXTH30 Silent genetically modified plants PtoXTH30 The accumulation of H2O2 and MDA was significantly reduced in overexpressing transgenic plants, while the activity of POD was significantly increased. These results indicate, from a physiological and biochemical perspective...PtoXTH30 Gene overexpression can increase the activity of antioxidant enzymes, thereby reducing the peroxidation level of membrane lipids and thus improving the drought and salt tolerance of transgenic plants.

[0175] (4) PtoXTH30 NBT and DAB analyses of overexpressing and silent transgenic plants

[0176] Both NBT and DAB analyses used Solarbio reagent kits for staining.

[0177] NBT staining results are as follows Figure 8 As shown, it can be seen that after salt stress treatment, wild-type plants and PtoXTH30 The leaves of silent transgenic plants showed a deeper blue staining effect when stained with NBT, while PtoXTH30 The overexpression transgenic plants showed lighter staining, because NBT staining depth directly reflects intracellular superoxide anion (O2) levels. — The accumulation of ) indicates that the above results show: PtoXTH30 Gene overexpression can significantly reduce intracellular O2 in poplar cells under salt stress. — The accumulation level indicates PtoXTH30 Genes may improve the salt tolerance of poplar trees by reducing oxidative damage to cells caused by reactive oxygen species.

[0178] DAB staining results are as follows Figure 9 As shown, it can be seen that after drought stress, PtoXTH30 The leaves of overexpressing transgenic plants showed significantly less brown precipitate (indicating H2O2) than those of wild-type plants and PtoXTH30 Silent transgenic lines indicate PtoXTH30 Gene overexpression can reduce H2O2 accumulation under drought stress, which may improve the drought resistance of poplar trees by alleviating oxidative damage.

[0179] The present invention has been described in detail above with reference to specific embodiments and exemplary examples. However, these descriptions should not be construed as limiting the present invention. Those skilled in the art will understand that various equivalent substitutions, modifications, or improvements can be made to the technical solutions and embodiments of the present invention without departing from the spirit and scope of the present invention, and all such modifications and improvements fall within the scope of the present invention.

Claims

1. The application of proteins or genes that enhance the drought resistance and salt tolerance of poplar trees, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.1, and the gene is... PtoXTH30 The gene, whose nucleotide sequence is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, The application is achieved by increasing the content of the protein or the expression level of the gene described in claim 1 in poplar.

3. The application of proteins or genes that enhance the drought resistance and salt tolerance of poplar trees in poplar breeding, characterized by: The breeding program aims to improve the drought resistance and salt tolerance of poplar trees. The amino acid sequence of the protein is shown in SEQ ID NO.1, and the gene is... PtoXTH30 The gene, whose nucleotide sequence is shown in SEQ ID NO.

2.

4. The application according to claim 3, characterized in that, The application involves overexpression in poplar trees. PtoXTH30 It is achieved through genes.

5. Containing the contents of claim 1 PtoXTH30 Application of recombinant gene expression vectors in improving the drought resistance and salt tolerance of poplar trees.

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