Gene PtoMYB20 for regulating and controlling salt tolerance and drought resistance of poplar and application of gene PtoMYB20

Abstract

The invention discloses a gene PtoMYB20 for regulating and controlling salt tolerance and drought resistance of poplars. A nucleotide sequence of the gene comprises a sequence as shown in SEQ ID NO. 1. The gene interference fragment is introduced into a plant to obtain a silent transgenic plant, under the conditions of salt stress, drought stress and salt-drought composite stress, the H2O2 and MDA contents of the silent transgenic plant are lower than those of a wild plant, and the SOD and POD activities of the silent transgenic plant are higher than those of the wild plant, so that the damage degree of the plant is effectively reduced; the method is of great significance in improving salt tolerance and drought resistance of the poplar and relieving damage of adversity stress to forestry yield. The invention also discloses a breeding method of the transgenic poplar with strong salt tolerance and drought resistance, provides a new poplar germplasm which not only improves the stress resistance of the poplar but also does not influence the wood yield, and lays a theoretical and gene resource foundation for the stress-resistant molecular breeding of the poplar.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and particularly relates to a gene PtoMYB20 for regulating salt tolerance and drought resistance of poplar and an application thereof. Background Art

[0002] Transcription factors are widely involved in regulating physiological processes such as plant growth and development, metabolism, hormone responses, and biotic and abiotic stresses. MYB is the most abundant and widely distributed transcription factor family in plants, comprising highly conserved DNA sequences. It plays an important role in plant stress resistance, anthocyanin regulation, and flavonoid biosynthesis and metabolism. Related studies have reported that MYB transcription factors participate in plant defense processes, stress responses, and plant hormone signaling in species such as Arabidopsis, Populus trichocarpa, Helianthus annuus, and Rice. However, in poplar, little research has been conducted on the functions and regulatory mechanisms of MYB transcription factors under combined salt and drought stress. Therefore, in-depth research on the role of MYB transcription factors in poplar stress resistance is necessary to lay a theoretical and genetic resource foundation for molecular breeding of poplar stress resistance. Summary of the Invention

[0003] In order to overcome the above problems, the present invention provides a gene PtoMYB20 for regulating poplar's salt and drought resistance. By constructing an overexpression recombinant vector and an RNAi recombinant vector of PtoMYB20 and introducing them into poplar, PtoMYB20 overexpression transgenic plants and silenced transgenic plants were obtained, respectively. Through stress phenotype analysis of the plants, it was found that under salt stress, drought stress and salt-drought combined stress, the growth of the silenced transgenic plants was significantly better than that of the wild-type and overexpression transgenic plants, indicating that the gene plays an important regulatory role in regulating the process of poplar's resistance to adversity. Downregulating or inhibiting the expression of the gene PtoMYB20 can effectively improve the ability of poplar to resist salt and drought stress, laying a theoretical and genetic resource foundation for molecular breeding of poplar stress resistance, thereby completing the present invention.

[0004] Specifically, the purpose of the present invention is to provide the following aspects:

[0005] In a first aspect, a gene PtoMYB20 for regulating salt and drought tolerance in poplar is provided, wherein the nucleotide sequence of the gene comprises the sequence shown in SEQ ID NO.1.

[0006] In a second aspect, a protein encoding the gene PtoMYB20 that regulates salt and drought tolerance in poplar is provided, wherein the amino acid sequence of the encoded protein includes the sequence shown in SEQ ID NO.3.

[0007] In a third aspect, an interference fragment PtoMYB20-RNAi for regulating the salt and drought tolerance gene PtoMYB20 of poplar is provided, wherein the nucleotide sequence of the interference fragment includes the sequence shown in SEQ ID NO.4.

[0008] In a fourth aspect, a recombinant vector for overexpression of the gene PtoMYB20 described in the first aspect is provided, wherein the basic vector is a pBI121 vector.

[0009] The overexpression vector is obtained by inserting the CDS sequence of the gene PtoMYB20 into the pBI121 vector.

[0010] In a fifth aspect, an RNAi recombinant vector of the gene PtoMYB20 described in the first aspect is provided, wherein the RNAi recombinant vector is obtained by constructing an interference fragment of the gene PtoMYB20 onto a basic vector.

[0011] The interference fragment of the gene PtoMYB20 is PtoMYB20-RNAi, and the basic vector of the RNAi recombinant vector is the pBI121 vector.

[0012] In a sixth aspect, a method for breeding transgenic poplars with strong salt and drought resistance is provided, the method comprising the steps of introducing the interference fragment PtoMYB20-RNAi of PtoMYB20 into poplars to obtain PtoMYB20 gene-silenced plants.

[0013] In a seventh aspect, the invention provides the use of the gene PtoMYB20 for regulating salt and drought tolerance of poplar described in the first aspect in enhancing the salt and drought tolerance of poplar.

[0014] The beneficial effects of the present invention include:

[0015] (1) The gene PtoMYB20 for regulating salt and drought tolerance in poplars provided by the present invention can effectively improve the ability of poplars to resist salt and drought stress by introducing an interference fragment into poplar plants to downregulate or inhibit the expression of the gene PtoMYB20, thereby helping to reduce the damage caused by adverse stress to forestry production;

[0016] (2) The present invention provides a method for breeding transgenic poplars with strong salt and drought resistance. The transgenic poplar plants obtained therefrom have good growth and low plant damage under salt and drought stress conditions. The breeding method can provide new poplar germplasm that improves the stress resistance of poplars without affecting wood yield, laying a theoretical and genetic resource foundation for molecular breeding of poplars with strong stress resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1A comparison chart showing the relative expression levels of the PtoMYB20 gene of Populus tomentosa detected by RT-qPCR under different salt treatment times (0-48 h) in Example 1 is shown;

[0018] Figure 2 A comparison chart showing the relative expression levels of the PtoMYB20 gene of Populus tomentosa detected by RT-qPCR under normal and drought stress conditions in Example 1 is shown;

[0019] Figure 3 The transcription level of PtoMYB20 in the overexpression plants detected by RT-qPCR in Example 5 is shown, and the error bars represent the standard deviation;

[0020] Figure 4 The transcription level of PtoMYB20 in silenced plants detected by RT-qPCR in Example 5 is shown, and the error bars represent standard deviations;

[0021] Figure 5 The morphological phenotypes of WT, PtoMYB20 overexpression (OE-1, OE-9) and silenced (RNAi-1, RNAi-5) transgenic plants before and after salt stress, drought stress, and salt-drought combined stress in Example 6 are shown;

[0022] Figure 6 a to d show the analysis of malondialdehyde (MDA), hydrogen peroxide (H2O2), peroxidase (POD), and superoxide dismutase (SOD) content in WT, PtoMYB20 overexpression (OE-1, OE-9), and silenced (RNAi-1, RNAi-5) transgenic plants before and after treatment with 150 mM NaCl in Example 6, respectively;

[0023] Figure 7 a to d show the MDA, H2O2, POD, and SOD content analysis of WT, PtoMYB20 overexpression (OE-1, OE-9), and silenced (RNAi-1, RNAi-5) transgenic plants before and after 20% PEG6000 treatment in Example 6, respectively;

[0024] Figure 8 a to d show the MDA, H2O2, POD, and SOD content analysis of WT, PtoMYB20 overexpression (OE-1, OE-9), and silenced (RNAi-1, RNAi-5) transgenic plants in Example 6 before and after treatment with 150 mM NaCl + 20% PEG6000. DETAILED DESCRIPTION

[0025] The present invention will be further described in detail below through preferred embodiments and examples. Through these descriptions, the characteristics and advantages of the present invention will become more clear and distinct.

[0026] The word “exemplary” is used exclusively herein to mean “serving as an example, example, or illustration.” Any embodiment described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments.

[0027] The first aspect of the present invention provides a gene PtoMYB20 that regulates salt and drought tolerance in poplars. ， The nucleotide sequence thereof includes the sequence shown in SEQ ID NO.1, and preferably the nucleotide sequence thereof is shown in SEQ ID NO.1.

[0028] Preferably, the poplar is Populus tomentosa.

[0029] According to a preferred embodiment of the present invention, the gene PtoMYB20 regulating poplar salt and drought tolerance is located on chromosome 9 of the Populus tomentosa genome (http: / / db.cngb.org / cnsa / project / CNP0004290_40760cfc / reviewlink / ), with a starting position of 5388256 and an ending position of 5390921.

[0030] Preferably, the nucleotide sequence of the gene PtoMYB20 regulating poplar salt and drought tolerance can be found in the following files: National Gene Bank: Genome sequencing of Populus tomentosa; Project Number: CNP0004290, Sample Number: CNS0752799, Assembly Number: CNA0069009.

[0031] According to a preferred embodiment of the present invention, the coding region of the gene PtoMYB20 regulating salt and drought tolerance in poplar includes the sequence shown in SEQ ID NO.2, and preferably the nucleotide sequence of the coding region is shown in SEQ ID NO.2.

[0032] Preferably, the coding sequence of the gene PtoMYB20 regulating salt and drought tolerance in poplar can be found in the following files: National Gene Bank: Genome sequencing of Populus tomentosa; Project No.: CNP0004290, Sample No.: CNS0752799, Assembly No.: CNA0069009; PtoMYB20 location: chromosome 9, starting position 5390626, ending position 5390921; starting position 5388857, ending position 5389550; see for details. http: / / db.cngb.org / cnsa / project / CNP0004290_40760cfc / reviewlink / .

[0033] In a second aspect, the present invention provides a protein encoding the gene PtoMYB20 that regulates salt and drought tolerance in poplars. The amino acid sequence of the encoded protein includes the sequence shown in SEQ ID NO.3, and preferably the nucleotide sequence is shown in SEQ ID NO.3.

[0034] The third aspect of the present invention provides an interference fragment PtoMYB20-RNAi for regulating the salt and drought tolerance gene PtoMYB20 in poplar, whose nucleotide sequence includes the sequence shown in SEQ ID NO.4, preferably the nucleotide sequence is shown in SEQ ID NO.4.

[0035] The interference fragment can be used to downregulate or inhibit the expression of PtoMYB20.

[0036] The fourth aspect of the present invention provides an overexpression recombinant vector comprising the gene PtoMYB20 described in the first aspect, wherein the basic vector is the pBI121 vector.

[0037] The overexpression vector is obtained by inserting the CDS sequence of the gene PtoMYB20 into the pBI121 vector.

[0038] In a fifth aspect, the present invention provides an RNAi recombinant vector of the gene PtoMYB20 described in the first aspect, wherein the RNAi recombinant vector is obtained by constructing an interference fragment of the gene PtoMYB20 onto a basic vector.

[0039] The interference fragment of the gene PtoMYB20 is PtoMYB20-RNAi, and the basic vector of the RNAi recombinant vector is the pBI121 vector.

[0040] In a sixth aspect, the present invention provides a method for breeding transgenic poplars with strong salt and drought tolerance, comprising the steps of introducing the interference fragment PtoMYB20-RNAi of PtoMYB20 into poplars to obtain PtoMYB20 gene-silenced plants.

[0041] Preferably, the method for breeding transgenic poplars with strong salt and drought tolerance comprises the following steps:

[0042] Step 1: construct a PtoMYB20 overexpression recombinant vector and an RNAi recombinant vector.

[0043] Preferably, the overexpression recombinant vector and the RNAi recombinant vector are constructed according to a method comprising the following steps:

[0044] Step 1-1, digest the vector and amplify the target fragment;

[0045] Step 1-2, recovery and purification of the vector backbone and target fragment;

[0046] Steps 1-3, ligating the vector to the target fragment;

[0047] Steps 1-4: Identify the connected recombinant vector.

[0048] In step 1-1, the target fragment includes the CDS sequence of the gene PtoMYB20 and the reverse sequence of the CDS portion (interference fragment PtoMYB20-RNAi), the CDS sequence of the gene PtoMYB20 is shown as SEQ ID NO.2, and the sequence of the interference fragment is shown as SEQ ID NO.4.

[0049] According to a preferred embodiment of the present invention, in step 1-1, the basic vectors used by the overexpression recombinant vector and the RNAi recombinant vector are both pBI121 vectors, totaling 13629 bp, containing a strong 35S promoter (CaMV35S), resistant to kanamycin (Kana), and can be digested with XbaI and BamHI.

[0050] In a further preferred embodiment, the overexpression recombinant vector is obtained by constructing the CDS sequence of the gene PtoMYB20 into the pBI121 vector;

[0051] The RNAi recombinant vector is obtained by constructing the interference fragment PtoMYB20-RNAi of PtoMYB20 into the pBI121 vector.

[0052] In a further preferred embodiment, the CDS sequence of the gene PtoMYB20 is amplified by primers P1 (PtoMYB20-OE-F) and P2 (PtoMYB20-OE-R), the nucleotide sequence of primer P1 includes the sequence shown in SEQ ID NO.5, and the nucleotide sequence of primer P2 includes the sequence shown in SEQ ID NO.6;

[0053] The amplification primers for the PtoMYB20 interference fragment PtoMYB20-RNAi are P3 (PtoMYB20-RNAi-F) and P4 (PtoMYB20-RNAi-R), the nucleotide sequence of primer P3 includes the sequence shown in SEQ ID NO.7, and the nucleotide sequence of primer P4 includes the sequence shown in SEQ ID NO.8.

[0054] Preferably, the nucleotide sequence of primer P1 is shown as SEQ ID NO.5, and the nucleotide sequence of primer P2 is shown as SEQ ID NO.6;

[0055] The nucleotide sequence of primer P3 is shown in SEQ ID NO.7, and the nucleotide sequence of primer P4 is shown in SEQ ID NO.8.

[0056] According to a preferred embodiment of the present invention, in steps 1-4, the recombinant vector is transformed into competent E. coli cells to obtain a single colony, which is then amplified and identified by PCR.

[0057] In a further preferred embodiment, the PCR amplification program for identifying a single colony is: 95°C for 3 min; 35 cycles of (94°C for 30 s; 55°C for 30 s; 72°C for 60 s); 72°C for 5 min; 4°C∞.

[0058] Step 2: Genetic transformation of the PtoMYB20 gene was performed, and overexpression plants and silenced plants were identified.

[0059] Preferably, step 2 includes the following sub-steps:

[0060] Step 2.1: Transform the recombinant vector into Agrobacterium to obtain Agrobacterium culture solution.

[0061] Wherein, the Agrobacterium is GV3101.

[0062] Step 2.2: Infect poplar callus with Agrobacterium tumefaciens solution and culture the callus into a complete plant.

[0063] Wherein, the poplar is Populus tomentosa

[0064] Step 2.3: Identify the obtained complete plants.

[0065] Preferably, the identification of overexpressing plants and silenced plants includes identification at the DNA level and identification at the transcription level.

[0066] Step 3: subject the overexpression plants and silenced plants to stress treatment and conduct phenotypic analysis to obtain transgenic poplar plants with strong salt and drought tolerance.

[0067] The stress treatment includes salt stress treatment, drought stress treatment and salt-drought combined stress treatment.

[0068] In this study, stress phenotype analysis of overexpressing and silenced plants revealed that, under normal growth conditions, poplar plants overexpressing PtoMYB20 showed taller heights than wild-type plants, while silenced poplar plants showed no significant growth differences compared to wild-type plants. However, under salt stress, drought stress, and combined salt and drought stress, silenced poplar plants showed significantly better growth than wild-type and overexpressing plants.

[0069] Under salt stress, drought stress, and combined salt and drought stress, PtoMYB20-RNAi plants (silenced plants) showed lower levels of reactive oxygen species (such as H2O2 and MDA) and higher antioxidant enzyme activities (such as SOD and POD) compared to wild-type plants. This indicates that under stress conditions, antioxidant enzyme activity increased in silenced plants, effectively slowing the excessive accumulation of reactive oxygen species and reducing plant damage. However, PtoMYB20-overexpressing poplar plants showed the opposite trend.

[0070] The breeding method of transgenic poplars with strong salt and drought resistance provided by the present invention can provide new poplar germplasm that improves the stress resistance of poplars without affecting wood yield, and helps to alleviate the damage caused by adverse stress to forestry yield.

[0071] The seventh aspect of the present invention provides the use of the gene PtoMYB20 that regulates salt and drought tolerance in poplars in enhancing the salt and drought tolerance of poplars.

[0072] Example

[0073] The present invention is further described below through specific examples. However, these examples are merely exemplary and do not constitute any limitation to the scope of protection of the present invention.

[0074] Unless otherwise specified, the reagents involved in the following examples are all conventional reagents available on the market, and the methods used are all commonly used methods in this technical field.

[0075] Example 1 Analysis of the expression level of the poplar PtoMYB20 gene

[0076] (1) Two-month-old white poplars were subjected to salt stress with 150 mM NaCl. Leaves were collected at five time points (0 h, 6 h, 12 h, 24 h, and 48 h) after treatment and RNA was extracted. RNA was extracted using a plant RNA extraction kit from Beijing Quanshijin Biotechnology Co., Ltd.

[0077] (2) The stem xylem of three-month-old Populus tomentosa plants under normal growth (soil moisture content ≥ 70%) and drought stress (soil moisture content 20-25% as a drought environment, 40 days of long-term drought treatment) were collected and RNA was extracted.

[0078] (3) The RNA extracted in steps (1) and (2) was reverse transcribed to obtain cDNA using HiScript IV All-in-One Ultra RT SuperMix for qPCR-R433 (Nanjing Novozymes Biotech Co., Ltd.).

[0079] The reaction system (20 μL) is shown in Table 1:

[0080] Table 1

[0081]

[0082] The reaction procedure was: 50°C for 5 min; 85°C for 5 sec.

[0083] (4) Based on the CDS sequence of the PtoMYB20 gene of Populus tomentosa (shown in SEQ ID NO. 2), primers P5 (PtoMYB20-qPCR-F) and P6 (PtoMYB20-qPCR-R) were designed for fluorescence quantitative PCR reaction. The expression level of the PtoMYB20 gene under different stress times was determined by fluorescence quantitative PCR. The nucleotide sequence of primer P5 is shown in SEQ ID NO. 9, and the nucleotide sequence of primer P6 is shown in SEQ ID NO. 10. The internal reference primers were P7 (Actin-F) and P8 (Actin-R), and their nucleotide sequences are shown in SEQ ID NO. 11 and SEQ ID NO. 12, respectively.

[0084] RT-qPCR experiments were performed using 2×ChamQ SYBR Color qPCR Master Mix. The reaction system (20 μL) is shown in Table 2:

[0085] Table 2

[0086]

[0087]

[0088] The reaction program was: 95°C for 30 s; (95°C for 5 s; 60°C for 35 s) for 40 cycles; 95°C for 15 s; 60°C for 1 min; 95°C for 15 s.

[0089] The results of the fluorescence quantitative PCR reaction are as follows Figure 1 and Figure 2 As shown in the data, it can be seen that: with the extension of salt treatment time, the expression level of PtoMYB20 gene showed an overall trend of first decreasing and then increasing; after drought treatment, the expression level of PtoMYB20 gene decreased significantly.

[0090] Therefore, it is speculated that this gene plays an important regulatory role in the growth resistance of poplar to stress (salt and drought stress).

[0091] Example 2 Obtaining the target fragment of the PtoMYB20 gene

[0092] RNA was extracted from LM50 white poplar using a plant RNA extraction kit from Beijing Quanshijin Biotechnology Co., Ltd., and reverse transcribed into cDNA using a HiScript III 1st Strand cDNA Synthesis Kit (Nanjing Novozymes Biotechnology Co., Ltd.).

[0093] Using cDNA as a template, referring to the Populus tomentosa genome file (http: / / db.cngb.org / cnsa / project / CNP0004290_40760cfc / reviewlink / ), and considering various principles of primer design, primers for amplifying target fragments were designed using the primer blast tool (NCBI, https: / / blast.ncbi.nlm.nih.gov). The target fragments included the target fragment for constructing an overexpression vector (the CDS sequence of the gene PtoMYB20) and the target fragment for constructing an RNAi recombinant vector (a partial reverse sequence of the CDS of the gene PtoMYB20).

[0094] The primers for amplifying the CDS sequence of the gene PtoMYB20 are P1 (PtoMYB20-OE-F) and P2 (PtoMYB20-OE-R). The nucleotide sequence of primer P1 is shown in SEQ ID NO. 5, and the nucleotide sequence of primer P2 is shown in SEQ ID NO. 6.

[0095] The amplification primers for the interference fragment PtoMYB20-RNAi of PtoMYB20 are P3 (PtoMYB20-RNAi-F) and P4 (PtoMYB20-RNAi-R). The nucleotide sequence of primer P3 is shown in SEQ ID NO.7, and the nucleotide sequence of primer P4 is shown in SEQ ID NO.8.

[0096] Among them, the gene PtoMYB20 is located on chromosome 9 of the Populus tomentosa genome (http: / / db.cngb.org / cnsa / project / CNP0004290_40760cfc / reviewlink / ), with the starting position 5388256 and the ending position 5390921.

[0097] PCR amplification was performed using 2x Phanta Max Master Mix (Dye Plus) from Nanjing Vazyme. The PCR reaction system (50 μL) is shown in Table 3:

[0098] Table 3

[0099]

[0100] The reaction program was as follows: 95°C for 3 min; 36 cycles of (95°C for 25 s; 55°C for 30 s; 72°C for 60 s (amplification efficiency 45 s / kb)); 72°C for 5 min; and 4°C∞.

[0101] The PCR products were run on agarose gel electrophoresis, recovered after detection, and then purified using the Kangwei Century DNA Clean-up Kit. After purification, the purity and concentration of the purified DNA product were measured using an instrument.

[0102] Finally, the CDS sequence of the PtoMYB20 gene was obtained, as shown in SEQ ID NO.2, the amino acid sequence of the encoded protein was shown in SEQ ID NO.3, and the nucleotide sequence of the interference fragment PtoMYB20-RNAi was shown in SEQ ID NO.4.

[0103] Example 3 Construction of PtoMYB20 gene overexpression vector and RNAi recombinant vector

[0104] (1) Enzyme digestion of vector

[0105] The selected overexpression and RNAi vectors are both pBI121 vectors, totaling 13629 bp, which contain a strong 35S promoter (CaMV35S), are resistant to kanamycin (Kana), and can be digested with XbaI and BamHI.

[0106] The enzyme digestion reaction system is shown in Table 4:

[0107] Table 4

[0108]

[0109] The enzyme digestion reaction conditions are: 37°C water bath for 1-2 hours.

[0110] The digested product was purified by gel recovery using the Kangwei Gel Extraction Kit and purified using Kangwei DNA Clean-up Kit. After purification, it was used as a vector backbone and stored at -20°C.

[0111] (2) Connection conversion

[0112] (2.1) Ligation of expression vector

[0113] The CDS and partial reverse-transcript of PtoMYB20 were constructed into the pBI121 vector using the Uniclone One-Step Seamless Cloning Kit from Beijing Jinsha Biotechnology Co., Ltd., generating overexpression and RNAi recombinant vectors, respectively. Ligation was performed at 50°C for 10 minutes, and the ligation products were used to transform competent E. coli DH5α cells.

[0114] (2.2) Transformation of E. coli DH5α competent cells

[0115] Take 50μl of DH5α competent cells, add 5μl of ligation product, flick to mix, and let it stand on ice for 30 minutes. Subsequently, place the mixture in a 42°C water bath for heat shock for 45 seconds, quickly transfer to ice and cool for 2 minutes, and avoid shaking during the process to ensure transformation efficiency. Next, add 700μl of sterile LB liquid culture medium without antibiotics, blow to mix evenly, and place it on a shaker at 37°C and 220rpm for 45 minutes to revive the bacteria. After recovery, collect the bacteria by centrifugation at 6000rpm for 1 minute, retain 100μl of supernatant to resuspend the bacteria, spread it on LB solid plate culture medium containing kanamycin (100mg / ml), and culture it upside down at 37°C for 14-16 hours.

[0116] (2.3) Identification of positive clones by PCR

[0117] Use a sterile pipette tip to pick individual colonies from the plate and place them in 250 μl of LB liquid medium containing kanamycin. Incubate at 37°C with shaking at 200 rpm / min for approximately 3 hours to serve as a template for amplification. Separately, use the gene PCR purified product (CDS sequence or partial reverse CDS sequence) and ddH2O (double-distilled water) as templates, setting up positive and negative controls, respectively. PCR amplification was performed using Taq Plus Master Mix from Nanjing Vazyme. The PCR reaction system is shown in Table 5:

[0118] Table 5

[0119]

[0120] The nucleotide sequence of pBI121-R (primer P9) is shown in SEQ ID NO.13.

[0121] The reaction procedure was as follows: 95°C for 3 min; 35 cycles of (94°C for 30 s; 55°C for 30 s; 72°C for 60 s); 72°C for 5 min; and 4°C∞.

[0122] The PCR products were detected by 1% agarose gel electrophoresis, and the colonies that could amplify the band with the same size as the positive control were considered positive clones.

[0123] (2.4) Extraction of positive clone plasmids

[0124] PCR-positive clones were transferred to 6 ml of LB liquid medium containing kanamycin and cultured overnight at 37°C with shaking at 200 rpm. Plasmids were extracted using a plasmid extraction kit from Jiangsu Kangwei Reagent Co., Ltd. and sequenced by Beijing Ruibo Xingke Biotechnology Co., Ltd. After sequence alignment, the construction of the overexpression and RNAi recombinant vectors was completed.

[0125] Example 4 Genetic transformation of PtoMYB20 gene

[0126] (1) Transformation of Agrobacterium with recombinant plasmid

[0127] (1.1) Add approximately 1 μg of PtoMYB20 overexpression vector plasmid and RNAi vector plasmid to 100 μL of Agrobacterium GV3101 competent cells and mix gently.

[0128] (1.2) Place on ice for 5 minutes, quick-freeze in liquid nitrogen for 5 minutes, immediately place in a 37°C water bath for 5 minutes, and then place in an ice bath for 5 minutes.

[0129] (1.3) Add 700 μL of antibiotic-free YEP liquid medium to the bacterial suspension, mix thoroughly, and incubate with shaking at 28°C and 200 rpm for 2-3 hours. After the recovery, centrifuge the suspension at 6000 rpm for 1 minute, discard a portion of the supernatant, and retain 100 μL of the supernatant to mix with the bacterial suspension. Using sterilized and cooled glass beads, evenly spread the bacterial suspension on the surface of YEP solid medium containing 50 mg / L rifampicin and 50 mg / L kanamycin. Then, incubate the suspension in an inverted position at 28°C in an incubator for 72-90 hours.

[0130] (1.4) After single colonies have grown, use a sterile pipette to pick several monoclonal plaques and place them in a 2-mL centrifuge tube containing 250 μL of YEP liquid medium (supplemented with Rif and Kana at a 1:1000 dilution). Incubate on a shaker at 30°C, 200 rpm, for 2 hours. After incubation, aspirate the culture medium for PCR identification. Positive bacterial cultures identified were added to 50% glycerol, snap-frozen in liquid nitrogen, and stored at -80°C for subsequent genetic transformation experiments.

[0131] (2) Activation of Agrobacterium

[0132] (2.1) Remove the Agrobacterium solution containing the overexpression vector plasmid and the RNAi vector plasmid from the -80°C freezer, streak inoculate each onto YEP solid medium (1:1000 supplemented with Rif and Kana), seal the plate, and culture inverted in a dark incubator at 28°C for 2–3 days.

[0133] (2.2) Use a sterile pipette to pick a single colony from the plate and inoculate it into 3 mL of YEP liquid medium (1:1000 supplemented with Rif and Kana). Incubate overnight on a shaker at 30°C, 200 rpm. Transfer 1 mL of the bacterial suspension to a 250 mL sterile Erlenmeyer flask containing 100 mL of YEP liquid medium (1:1000 supplemented with Rif and Kana). Incubate on a shaker at 30°C, 200 rpm, for 4-5 hours, until the OD600 reaches 0.3-0.5.

[0134] (2.3) In a laminar flow hood, transfer 100 mL of bacterial suspension to two 50 mL sterile centrifuge tubes and centrifuge at 2560 g at 4°C for 20 min to collect the cells. In a laminar flow hood, discard the supernatant and resuspend the cells in 100 mL of resuspension buffer (WPMB) in a sterile wide-mouth bottle. This suspension will be used for subsequent infection experiments.

[0135] The composition of the resuspension (WPMB) is: WPM + 0.9 ml / L VB1 + 0.5 g MES + 20 g sucrose + 1 mg / L 2,4-D + 0.1 ml / L KT + 200 mM AS (added as needed), pH = 5.6. After preparation, it is sterilized at 121°C for 20 min under high temperature and high pressure.

[0136] (3) Infection by wound healing method

[0137] Select leaves from healthy and sterile Populus tomentosa seedlings (dark green leaves with a thick texture). Use a sterile scalpel to remove the petiole, leaving a growing callus. Make 2-3 horizontal cuts on the main vein of the leaf and spread the leaf face down on callus culture medium (CIM) for incubation in the dark at 25°C. After the leaf has grown for 20 to 30 days, loose white callus will grow on the wound. Peel the callus off the leaf, cut it into pieces the size of soybeans, and place it in a new callus culture medium. Transfer all the prepared callus to a sterile wide-mouth bottle containing the bacterial solution and place it in a shaker at 28°C, 160 rpm, and infect for 15-20 minutes.

[0138] The composition of callus culture medium (CIM) is: WPM + 1 mg / L 2,4-D + 0.1 ml / L KT + 0.5 g MES + 20 g sucrose + 5 g gelatin, pH = 5.9. After preparation, it is sterilized at 121°C for 20 min under high temperature and high pressure.

[0139] (4) Co-culture

[0140] In a clean bench, calli were removed from the bacterial solution with tweezers, excess bacterial solution was absorbed with filter paper, and the infected calli were spread onto co-culture plates (WPMC) and cultured upside down at 25°C in the dark for 2 days.

[0141] The composition of the co-culture medium (WPMC) in the co-culture plate is: WPM+0.9 ml / L VB1+0.5 g MES+20 g sucrose+7 g agar+100 mM AS, pH=5.9. After preparation, it is sterilized by high temperature and high pressure at 121° C. for 20 min.

[0142] (5) Differentiation culture

[0143] After dark culture, appropriate plant resistance is selected according to the carrier and differentiation medium containing corresponding antibiotics is prepared.

[0144] The calli are transferred to differentiation medium (WPMD) and the medium is changed every 20 days for the first time, and every 10 days thereafter. During this period, the calli will turn green, harden, and then partially turn red. This stage lasts for about two months and is maintained in a 25°C light incubator.

[0145] The composition of the differentiation medium (WPMD) is: WPM + 0.9 ml / L VB1 + 0.5 g MES + 20 g sucrose + 0.5 mg / L 6BA + 0.1 mg / L NAA + 0.002 mg / LTDZ + 7 g agar + 250 mg / L TMT + 250 mg / L cephalosporin + 20 mg / L kana, pH = 5.9. After preparation, it is sterilized at 121°C for 20 min under high temperature and high pressure.

[0146] (6) Root induction of clustered shoots

[0147] After the adventitious buds grow to about half a centimeter, use sterile tweezers or a scalpel to cut off the buds (pay attention to the growth point of the buds to avoid selecting buds that differentiate from the same cell) and place them in a bud elongation medium for growth. After a period of elongation culture, when the adventitious buds grow to 1-2 cm, use sterile tweezers or a scalpel to cut off the buds individually and place them in a rooting medium for rooting culture. After about 10 days, the buds will grow roots and the rooting culture will grow into complete plants.

[0148] Among them, the composition of the elongation medium is: WPM+20g sucrose+0.3mg / L6BA+0.2mg / LIBA+7g agar+250mg / lTMT+250MG / L cephalosporin. After preparation, it is sterilized at 121°C for 20 minutes.

[0149] The composition of the rooting medium is: 1 / 2MS + 20g sucrose + 0.05mg / LIBA + 0.02mg / L NAA + 7g agar + 250mg / l TMT + 250MG / L cephalosporin + 20mg / L Kana. After preparation, it is sterilized at 121°C for 20min under high temperature and high pressure.

[0150] Example 5 Identification of PtoMYB20 gene overexpression and silenced plants

[0151] (1) Crude DNA extraction of wild-type Populus tomentosa, PtoMYB20 gene overexpressing plants, and silenced plants (RNAi plants):

[0152] (i) Place a leaf sample to be extracted in a 2 mL centrifuge tube and add one grinding bead. Quickly freeze in liquid nitrogen and then crush using a tissue crusher for 5 min.

[0153] (ii) After the powder is crushed, add 500 μL of TBS buffer to the centrifuge tube, mix well, and heat in a 65°C water bath for 10 min.

[0154] (iii) Centrifuge at 12,000 rpm for 15 min, transfer the supernatant to a new centrifuge tube, add an equal volume of isopropanol, and let it settle at room temperature for 30 min until a white flocculent precipitate is visible.

[0155] (iv) Centrifuge at 12000 rpm for 15 min, slowly discard the supernatant, and add 1 ml of 75% ethanol to wash the precipitate.

[0156] (v) Centrifuge at 7500 rpm for 10 min, slowly discard the supernatant and remove excess liquid with a pipette.

[0157] (vi) Place in a 40°C oven for 2 h.

[0158] (vii) Add 50 μL of ddH2O to the centrifuge tube to dissolve the precipitate and store in a -20°C refrigerator.

[0159] (2) Identification of PtoMYB20 gene overexpression and silenced plants

[0160] (2.1) DNA level identification

[0161] The crudely extracted transgenic plant gDNA was used as a template and PCR identification was performed using Taq Plus Master Mix. The reaction system was as shown in Table 5 of Example 3.

[0162] The PCR reaction program was as follows: 95°C for 2 min; 35 cycles of (94°C for 30 s; 55°C for 30 s; 72°C for 60 s); 72°C for 5 min; and 4°C∞.

[0163] The PCR product was detected by agarose gel electrophoresis to check whether the bands were correct. If correct, the plant was a PtoMYB20 gene overexpression plant or silenced plant.

[0164] PCR detection showed that the plants numbered OE-1, OE-2, OE-4, OE-6, OE-9, OE-14, OE-17, and OE-18 were PtoMYB20 overexpressing transgenic plants, and the plants numbered RNAi-1, RNAi-2, RNAi-3, RNAi-4, RNAi-5, RNAi-10, and RNAi-11 were PtoMYB20 silenced transgenic plants.

[0165] (2.2) Identification of transcriptional levels

[0166] RT-qPCR was used to detect the transcription level of PtoMYB20 in overexpression and silenced plants:

[0167] (i) RNA was extracted from leaves of the plants 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.

[0168] (ii) RT-qPCR was performed using 2× ChamQ SYBR Color qPCR Master Mix. The reaction system was based on Table 2 of Example 1. Primers were P5 (PtoMYB20-qPCR-F) and P6 (PtoMYB20-qPCR-R), and internal control primers were P7 (Actin-F) and P8 (Actin-R).

[0169] The reaction program was: 95°C for 30 s; (95°C for 5 s; 60°C for 35 s) for 40 cycles; 95°C for 15 s; 60°C for 1 min; 95°C for 15 s.

[0170] The results of the overexpression plant test were as follows Figure 3 As shown by Figure 3 It can be seen that the PtoMYB20 overexpression plants numbered OE-1 and OE-9 had higher expression levels, which were 6.21 times and 9.74 times that of the wild-type plant (WT), respectively;

[0171] The results of the silent plant test are as follows Figure 4 As shown by Figure 4 It can be seen that the expression levels of PtoMYB20 silenced plants numbered RNAi-1 and RNAi-5 were low, 0.059 times and 0.047 times that of the wild-type plant (WT), respectively.

[0172] Example 6 Analysis of stress phenotypes of PtoMYB20 gene overexpression and RNAi plants

[0173] (1) Salt stress treatment

[0174] Poplar plants grown under tissue culture for 25 days with consistent growth were washed with warm water to remove the root agar. The plants were then transplanted into a soil culture medium consisting of a 1:1.5 mixture of substrate and vermiculite. The light cycle was 8 hours in the dark and 16 hours in the light. The plants were cultured in a 25°C constant temperature incubator under the same light conditions for 30 days, followed by irrigation with 150 mM NaCl solution for 25 days.

[0175] Plant growth Figure 5 As shown in the figure, after 25 days of treatment with 150 mM NaCl, the PtoMYB20-RNAi plants (silenced plants, numbered RNAi-1 and RNAi-5) showed good growth under salt stress, with slightly yellowed leaves. In contrast, most leaves of the wild-type and PtoMYB20-overexpressing plants (numbered OE-1 and OE-9) had turned yellow and dried, and some had fallen off. Furthermore, before salt stress, the height of the PtoMYB20-overexpressing plants was taller than that of the wild-type (WT) and PtoMYB20-RNAi plants (silenced plants). After salt stress, the height of the PtoMYB20-overexpressing plants gradually became comparable to that of the WT and PtoMYB20-RNAi plants.

[0176] The physiological indicators of the plants were measured, and the results were as follows Figure 6 As shown in the figure, under salt stress, the reactive oxygen species (ROS) such as H2O2 (hydrogen peroxide) and MDA (malondialdehyde) in PtoMYB20-RNAi plants (silenced plants) were lower than those in WT plants, while the activities of antioxidant enzymes such as SOD (superoxide dismutase) and POD (peroxidase) were higher than those in WT plants. This indicates that the antioxidant enzyme activities of silenced plants increased under salt stress, effectively slowing down the excessive accumulation of ROS and reducing the degree of plant damage. However, PtoMYB20 overexpression plants showed the opposite trend.

[0177] (2) Drought stress treatment

[0178] Plants that had grown uniformly for 25 days under tissue culture conditions were washed with warm water to remove the root agar. The plants were then transplanted into a soil culture medium consisting of a 1:1.5 mixture of substrate and vermiculite. The light cycle was 8 hours in the dark and 16 hours in the light. The plants were cultured in a 25°C constant temperature incubator under the same light conditions for 30 days. The plants were then watered with a 20% PEG 6000 solution for 22 days.

[0179] Plant growth after drought stress treatment Figure 5 As shown in Figure 2, after 22 days of simulated drought stress treatment with 20% PEG 6000, PtoMYB20-RNAi plants (silenced plants) maintained better growth compared to wild-type (WT) and PtoMYB20-overexpressing plants. Leaves of PtoMYB20-overexpressing plants withered and wilted more severely than under salt stress, while leaves of PtoMYB20-RNAi plants did not wither and showed significantly less wilting and damage.

[0180] The physiological indicators of the plants were measured, and the results were as follows Figure 7 As shown in the figure, under drought stress, the reactive oxygen species (ROS) such as H2O2 and MDA in PtoMYB20-RNAi plants (silenced plants) were lower than those in WT plants, while the activities of antioxidant enzymes such as SOD and POD were higher than those in WT plants. This indicates that under drought stress, the antioxidant enzyme activities of silenced plants increased, effectively slowing down the excessive accumulation of ROS and reducing the degree of plant damage. However, PtoMYB20 overexpression plants showed the opposite trend.

[0181] (3) Salt-drought combined stress treatment

[0182] Plants that had grown uniformly for 25 days under tissue culture conditions were washed with warm water to remove the root agar and transplanted into a soil culture medium consisting of a 1:1.5 mixture of substrate and vermiculite. The medium was cultured under a constant light intensity of 8 hours in the dark and 16 hours in the light. The plants were cultured in a 25°C constant temperature incubator for 30 days under the same light intensity, and then irrigated with a mixture of 20% PEG 6000 and 150 mM NaCl for 15 days.

[0183] Plant growth after salt-drought combined stress treatment Figure 5 As shown in the figure, after 15 days of combined salt and drought stress, the leaves of the PtoMYB20 overexpressing plants withered and wilted severely, with most of the leaves falling off and showing significant damage. In sharp contrast, the wild-type (WT) plants and the PtoMYB20-RNAi plants (silenced plants) showed relatively good growth. Among them, the leaves of the PtoMYB20-RNAi plants were particularly slightly damaged, with almost no obvious signs of withering, showing strong tolerance to the combined stress.

[0184] The physiological indicators of the plants were measured, and the results were as follows Figure 8 As shown in the figure, under combined salt-drought stress, the contents of reactive oxygen species such as H2O2 and MDA in PtoMYB20-RNAi plants were lower than those in WT, while the activities of antioxidant enzymes such as SOD and POD were higher than those in WT. This indicates that under combined salt-drought stress, the antioxidant enzyme activities of silenced plants increased, effectively slowing down the excessive accumulation of reactive oxygen species and reducing the degree of plant damage. However, the opposite trend was observed in PtoMYB20-overexpressing plants.

[0185] The present invention has been described in detail above with reference to specific embodiments and exemplary examples. However, these descriptions are not to be construed as limiting the present invention. Those skilled in the art will appreciate that, without departing from the spirit and scope of the present invention, various equivalent substitutions, modifications, or improvements may be made to the technical solutions and implementations of the present invention, all of which fall within the scope of the present invention.

Claims

1. A gene PtoMYB20 that regulates salt and drought tolerance in poplars, characterized in that: The nucleotide sequence of the gene includes the sequence shown in SEQ ID NO.

1.

2. A protein encoding a gene PtoMYB20 that regulates salt and drought tolerance in poplars, characterized in that: The amino acid sequence of the encoded protein includes the sequence shown in SEQ ID NO.

3.

3. An interference fragment PtoMYB20-RNAi for regulating the salt and drought tolerance gene PtoMYB20 in poplar, characterized in that: The nucleotide sequence of the interference fragment includes the sequence shown in SEQ ID NO.

4.

4. An overexpression recombinant vector comprising the gene PtoMYB20 according to claim 1, characterized in that: Its basic vector is pBI121 vector, The overexpression vector is obtained by inserting the CDS sequence of the gene PtoMYB20 into the pBI121 vector.

5. An RNAi recombinant vector of the gene PtoMYB20 according to claim 1, characterized in that: The RNAi recombinant vector is obtained by constructing the interference fragment of the gene PtoMYB20 into the basic vector. The interference fragment of the gene PtoMYB20 is PtoMYB20-RNAi, and the basic vector of the RNAi recombinant vector is the pBI121 vector.

6. A method for breeding transgenic poplars with strong salt and drought tolerance, characterized in that: The method comprises the steps of introducing the interference fragment PtoMYB20-RNAi of PtoMYB20 into poplar to obtain PtoMYB20 gene silenced plants.

7. The method for breeding transgenic poplars with strong salt and drought tolerance according to claim 6, characterized in that: The method comprises the following steps: Step 1, constructing a PtoMYB20 overexpression recombinant vector and an RNAi recombinant vector; Step 2, genetic transformation of the PtoMYB20 gene and identification of overexpressing plants and silenced plants; Step 3: subject the overexpression plants and silenced plants to stress treatment and conduct phenotypic analysis to obtain transgenic poplar plants with strong salt and drought tolerance.

8. Use of the gene PtoMYB20 for regulating salt and drought tolerance in poplar according to claim 1 in enhancing the salt and drought tolerance in poplar.

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