Gene ptoMYB20 for regulating poplar salt tolerance and drought resistance and application thereof

Abstract

The application discloses a gene PtoMYB20 for regulating salt tolerance and drought resistance of poplar, and the nucleotide sequence of the gene comprises a sequence as shown in SEQ ID NO.1. The interference fragment of the gene is introduced into a plant to obtain a silenced transgenic plant, under the conditions of salt stress, drought stress and salt-drought combined stress, the H2O2 and MDA contents of the transgenic plant are lower than those of a wild type plant, and the SOD and POD activities are higher than those of the wild type plant, so that the damage degree of the plant is effectively reduced, and the application has important significance for improving the salt tolerance and drought resistance of the poplar and reducing the damage of adversity stress to forestry yield. The application further discloses a breeding method of the transgenic poplar with strong salt tolerance and drought resistance, and provides a new poplar germplasm which improves the stress resistance of the poplar and does not affect the wood yield, and lays a theoretical and gene resource foundation for poplar stress resistance molecular breeding.

Description

TECHNICAL FIELD

[0001] The application 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 application thereof. BACKGROUND

[0002] Transcription factors are widely involved in the regulation of physiological processes such as plant growth and development, metabolism, hormone response, biological stress and abiotic stress. MYB is the most abundant and widely distributed transcription factor family in plants, which contains a highly conserved DNA sequence, and plays an important role in biological processes such as plant stress resistance, anthocyanin regulation, flavonoid synthesis and metabolism. Related research reports that in Arabidopsis, Populus trichocarpa, Helianthus annuus, rice and other species, MYB transcription factors are involved in plant defense processes, stress response processes and plant hormone signal transduction processes. However, in poplar, the function and regulation mechanism of MYB transcription factors under salt and drought combined stress are less studied, so it is necessary to further study the role of MYB transcription factors in poplar stress resistance, so as to lay a theoretical and gene resource foundation for poplar stress resistance molecular breeding. SUMMARY

[0003] In order to overcome the above problems, the application provides a gene PtoMYB20 for regulating salt tolerance and drought resistance of poplar. By constructing a PtoMYB20 overexpression recombinant vector and a RNAi recombinant vector and introducing them into poplar, PtoMYB20 overexpression transgenic plants and silencing transgenic plants are obtained, respectively. Through stress phenotype analysis of the plants, it is found that the growth of the silencing transgenic plants is obviously better than that of the wild type and the overexpression transgenic plants under salt stress, drought stress and salt and drought combined stress, which indicates that the gene plays an important regulatory role in regulating the resistance of poplar to adversity. Down-regulation or inhibition of the expression of the gene PtoMYB20 can effectively improve the ability of poplar to resist salt and drought stress, thereby laying a theoretical and gene resource foundation for poplar stress resistance molecular breeding, and thus the application is completed.

[0004] Specifically, the application aims to provide the following aspects:

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

[0006] In a second aspect, a coding protein of the gene PtoMYB20 for regulating salt tolerance and drought resistance of poplar is provided, and the amino acid of the coding protein comprises a sequence as shown in SEQ ID NO. 3.

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

[0008] In a fourth aspect, a recombinant overexpression vector comprising the gene PtoMYB20 of 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 of the first aspect is provided, wherein the RNAi recombinant vector is obtained by constructing the interference fragment of the gene PtoMYB20 into a basic vector.

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

[0012] In a sixth aspect, a breeding method for a transgenic poplar with strong salt and drought tolerance is provided, and the method comprises the step of introducing the interference fragment PtoMYB20-RNAi of PtoMYB20 into the poplar to obtain a PtoMYB20 gene silencing plant.

[0013] In a seventh aspect, the gene PtoMYB20 for regulating salt and drought tolerance of poplar of the first aspect is applied to enhance the salt and drought tolerance of poplar.

[0014] The present application has the following beneficial effects:

[0015] (1) The gene PtoMYB20 for regulating salt and drought tolerance of poplar provided by the present application can effectively improve the ability of poplar to resist salt and drought stress by introducing the interference fragment into the poplar plant to down-regulate or inhibit the expression of the gene PtoMYB20, which helps to alleviate the damage caused by adverse stress to forestry yield.

[0016] (2) The breeding method for a transgenic poplar with strong salt and drought tolerance provided by the present application can provide a new germplasm of poplar which improves the stress resistance of poplar without affecting the yield of wood, and lays a theoretical and genetic resource foundation for molecular breeding of poplar. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1Figure 6 shows the relative expression of PtoMYB20 in Populus tomentosa under different salt treatment time (0-48h) detected by RT-qPCR in Example 1.

[0018] Figure 2 Figure 7 shows the relative expression of PtoMYB20 in Populus tomentosa under normal and drought stress detected by RT-qPCR in Example 1.

[0019] Figure 3 Figure 8 shows the transcription level of PtoMYB20 in overexpression plants detected by RT-qPCR in Example 5, and the error bar represents the standard deviation.

[0020] Figure 4 Figure 9 shows the transcription level of PtoMYB20 in silencing plants detected by RT-qPCR in Example 5, and the error bar represents the standard deviation.

[0021] Figure 5 Figure 10 shows the morphological phenotype of WT, PtoMYB20 overexpression (OE-1, OE-9) and silencing (RNAi-1, RNAi-5) transgenic plants before and after salt stress, drought stress, and salt-drought combined stress in Example 6.

[0022] Figure 6 Figures 11a-11d show the content analysis of MDA, H2O2, POD, and SOD of WT, PtoMYB20 overexpression (OE-1, OE-9) and silencing (RNAi-1, RNAi-5) transgenic plants before and after 150mM NaCl treatment in Example 6.

[0023] Figure 7 Figures 12a-12d show the content analysis of MDA, H2O2, POD, and SOD of WT, PtoMYB20 overexpression (OE-1, OE-9) and silencing (RNAi-1, RNAi-5) transgenic plants before and after 20% PEG6000 treatment in Example 6.

[0024] Figure 8 Figures 13a-13d show the content analysis of MDA, H2O2, POD, and SOD of WT, PtoMYB20 overexpression (OE-1, OE-9) and silencing (RNAi-1, RNAi-5) transgenic plants before and after 150mM NaCl+20% PEG6000 treatment in Example 6. DETAILED DESCRIPTION

[0025] The present application will be further described in details by preferred embodiments and examples. The features and advantages of the present application will become more apparent through these descriptions.

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

[0027] In a first aspect, the application provides a Populus salt and drought tolerance regulating gene PtoMYB20 ， The nucleotide sequence of the Populus salt and drought tolerance regulating gene PtoMYB20 comprises the sequence shown as SEQ ID NO. 1, preferably the nucleotide sequence of the Populus salt and drought tolerance regulating gene PtoMYB20 is shown as SEQ ID NO. 1.

[0028] Preferably, the Populus is Populus tomentosa.

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

[0030] Preferably, the nucleotide sequence of the Populus salt and drought tolerance regulating gene PtoMYB20 can be found in the following file: 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 application, the coding region of the Populus salt and drought tolerance regulating gene PtoMYB20 comprises the sequence shown as SEQ ID NO. 2, preferably the nucleotide sequence of the coding region of the Populus salt and drought tolerance regulating gene PtoMYB20 is shown as SEQ ID NO. 2.

[0032] Preferably, the coding region sequence of the Populus salt and drought tolerance regulating gene PtoMYB20 can be found in the following file: National Gene Bank: Genome sequencing of Populus tomentosa; Project number: CNP0004290, Sample number: CNS0752799, Assembly number: CNA0069009; PtoMYB20 location: chromosome 9, start position 5390626, end position 5390921; start position 5388857, end position 5389550; see http: / / db.cngb.org / cnsa / project / CNP0004290_40760cfc / reviewlink / .

[0033] The application provides a coding protein of a poplar salt-tolerant and drought-resistant gene PtoMYB20, and the amino acid sequence of the coding protein comprises a sequence as shown in SEQ ID NO. 3, preferably the nucleotide sequence of the coding protein comprises a sequence as shown in SEQ ID NO. 3.

[0034] The third aspect of the application provides an interference fragment PtoMYB20-RNAi of the poplar salt-tolerant and drought-resistant gene PtoMYB20, and the nucleotide sequence of the interference fragment comprises a sequence as shown in SEQ ID NO. 4, preferably the nucleotide sequence of the interference fragment comprises a sequence as shown in SEQ ID NO. 4.

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

[0036] The fourth aspect of the application provides a PtoMYB20 overexpression recombinant vector comprising the gene PtoMYB20, and the basic vector of the PtoMYB20 overexpression recombinant vector is a pBI121 vector.

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

[0038] The fifth aspect of the application provides an RNAi recombinant vector of the gene PtoMYB20, and the RNAi recombinant vector is obtained by constructing the interference fragment of the gene PtoMYB20 into a basic vector,

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

[0040] The sixth aspect of the application provides a breeding method of a transgenic poplar with strong salt-tolerant and drought-resistant capability, and the breeding method comprises the following steps: introducing the interference fragment PtoMYB20-RNAi of PtoMYB20 into the poplar to obtain a PtoMYB20 gene silencing plant.

[0041] Preferably, the breeding method of the transgenic poplar with strong salt-tolerant and drought-resistant capability comprises the following steps:

[0042] Step 1, constructing a PtoMYB20 overexpression recombinant vector and an RNAi recombinant vector.

[0043] Preferably, the construction of the PtoMYB20 overexpression recombinant vector and the RNAi recombinant vector is obtained by a method comprising the following steps:

[0044] Step 1-1, enzyme digestion of a vector and amplification of a target fragment;

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

[0046] Step 1-3, connecting the vector with the target fragment;

[0047] Step 1-4, identifying the connected recombinant vector.

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

[0049] According to a preferred embodiment of the present application, in step 1-1, the basic vectors of the overexpression recombinant vector and the RNAi recombinant vector are both pBI121 vectors, 13629 bp in length, containing a 35S strong promoter (CaMV35S), and the resistance is kanamycin (Kana), which can be cut by XbaI and BamHI.

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

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

[0052] In a further preferred embodiment, the CDS sequence of the gene PtoMYB20 is obtained by amplification of 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 of the interference fragment PtoMYB20-RNAi of PtoMYB20 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 in SEQ ID NO. 5, and the nucleotide sequence of primer P2 is shown in SEQ ID NO. 6.

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

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

[0057] In a further preferred embodiment, the PCR amplification procedure for identifying the single colonies is as follows: 95℃ for 3 min; (94℃ for 30 s; 55℃ for 30 s; 72℃ for 60 s) for 35 cycles; 72℃ for 5 min; 4℃ for ∞.

[0058] Step 2, genetic transformation of PtoMYB20 gene is performed, and overexpression plants and silenced plants are identified.

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

[0060] Step 2.1, the recombinant vector is transformed into Agrobacterium to obtain Agrobacterium bacterial liquid.

[0061] In the application, the Agrobacterium is GV3101.

[0062] Step 2.2, the Agrobacterium bacterial liquid is used to infect poplar callus, and the callus is cultured into a complete plant.

[0063] In the application, the poplar is Populus tomentosa

[0064] Step 2.3, the obtained complete plant is identified.

[0065] Preferably, the identification of the overexpression plants and the silenced plants comprises DNA level identification and transcription level identification.

[0066] Step 3, the overexpression plants and the silenced plants are subjected to stress treatment, and phenotype analysis is performed to obtain transgenic poplar plants with strong salt and drought resistance.

[0067] In the application, the stress treatment comprises salt stress treatment, drought stress treatment and salt-drought combined stress treatment.

[0068] In the application, through stress phenotype analysis of the overexpression plants and the silenced plants, it is found that under normal growth conditions, the PtoMYB20 overexpression poplar plants have higher plant height compared with the wild type; but the growth of the silenced poplar plants has no obvious difference compared with the wild type. Under salt stress, drought stress and salt-drought combined stress, the growth of the silenced poplar plants is obviously better than that of the wild type and the overexpression plants.

[0069] Under salt stress, drought stress and salt-drought combined stress, the active oxygen (such as H2O2, MDA) content of the PtoMYB20-RNAi plant (silencing plant) is lower than that of the wild type plant, and the antioxidant enzyme activity (such as SOD, POD) is higher, that is, under stress conditions, the antioxidant enzyme activity of the silencing plant increases, effectively slows down the excessive accumulation of active oxygen, and reduces the degree of plant damage. The PtoMYB20 overexpression poplar plant shows the opposite trend to the silencing plant.

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

[0071] In a seventh aspect, the present application provides an application of the salt and drought resistance regulating gene PtoMYB20 in enhancing the salt and drought resistance of poplar.

[0072] Examples

[0073] The present application is further described below by specific examples, but these examples are merely exemplary and do not constitute any limitation on the scope of protection of the present application.

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

[0075] Example 1 Analysis of expression amount of Populus PtoMYB20 gene

[0076] (1) Two-month-old Populus tomentosa were subjected to salt stress treatment with 150mM NaCl, and the leaves of Populus tomentosa at 0h, 6h, 12h, 24h and 48h after treatment were collected and RNA was extracted. Among them, the RNA was extracted by the plant RNA extraction kit of Beijing Quanshi Gold Biotechnology Co., Ltd.

[0077] (2) The stems of three-month-old Populus tomentosa plants under normal growth (soil water content ≥70%) and drought stress (soil water 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, which was subjected to qPCR by using HiScript IV All-in-One Ultra RT SuperMix for qPCR-R433 (Nanjing Novogene Bioinformatics Technology Co., Ltd.).

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

[0080] Table 1

[0081]

[0082] Reaction procedure: 50℃ 5min; 85℃ 5sec.

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

[0084] RT-qPCR experiment was performed using 2xChamQ SYBR Color qPCR Master Mix, and the reaction system (20 μL) is as shown in Table 2:

[0085] Table 2

[0086]

[0087]

[0088] Reaction procedure: 95℃ 30s; (95℃ 5s; 60℃ 35s) 40 cycles; 95℃ 15s; 60℃ 1min; 95℃ 15s.

[0089] The results of fluorescence quantitative PCR reaction are shown in Figure 1 and Figure 2 It can be seen that: with the extension of salt treatment time, the expression amount of PtoMYB20 gene presents a trend of first decreasing and then increasing; the expression amount of PtoMYB20 gene after drought treatment decreases obviously.

[0090] Therefore, it is speculated that the gene plays an important regulatory role in the growth of Populus tomentosa under stress (salt and drought stress).

[0091] Example 2 Obtaining of PtoMYB20 gene target fragment

[0092] The RNA of LM50 Populus tomentosa was extracted by using the plant RNA extraction kit of Beijing ZhenGong Jin Bio-technology Co., Ltd., and the RNA was reversely transcribed into cDNA by using HiScript III 1st Strand cDNA Synthesis Kit (Nanjing Novogene Bio-technology Co., Ltd.).

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

[0094] The amplification primers of the CDS sequence of gene PtoMYB20 were P1 (PtoMYB20-OE-F) and P2 (PtoMYB20-OE-R), the nucleotide sequence of primer P1 was as shown in SEQ ID NO. 5, and the nucleotide sequence of primer P2 was as shown in SEQ ID NO. 6;

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

[0096] Wherein, the gene PtoMYB20 is located on chromosome 9 of the Populus tomentosa genome (http: / / db.cngb.org / cnsa / project / CNP0004290_40760cfc / reviewlink / ) with a start position of 5388256 and an end position of 5390921.

[0097] The 2x Phanta Max Master Mix (Dye Plus) of Nanjing Vazyme Co., Ltd. was used for PCR amplification, and the PCR experimental reaction system (50 μL) was as shown in Table 3:

[0098] Table 3

[0099]

[0100] Reaction procedure: 95℃ 3min; (95℃ 25s; 55℃ 30s; 72℃ 60s (45s / kb for amplification efficiency)) 36 cycles; 72℃ 5min; 4℃ ∞.

[0101] The product of the PCR reaction was subjected to agarose gel electrophoresis, and after detection, it was recovered and then purified using the DNA Clean-up Kit from Kangwei Century. After purification, the purity and concentration of the DNA purification product were determined using the instrument.

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

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

[0104] (1) Enzymatic digestion of the vector

[0105] The selected overexpression and RNAi vectors are pBI121 vectors, which are 13629 bp in total, containing a 35S strong promoter (CaMV35S) and resistance 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℃ water bath for 1-2h.

[0110] The product after enzyme digestion was subjected to gel recovery and purification, and the Gel Extraction Kit from Kangwei Century was used for recovery, and the DNA Clean-up Kit from Kangwei Century was used for purification. After purification, it was used as a vector skeleton and stored at -20℃.

[0111] (2) Ligation and transformation

[0112] (2.1) Ligation of the expression vector

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

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

[0115] Take 50 μl of DH5α competent cells, add 5 μl of ligation product, mix gently and stand on ice for 30 min. Then, place the mixture in a 42°C water bath for 45 seconds, quickly transfer to ice for 2 min, avoid shaking during the period to ensure transformation efficiency. Then, add 700 μl of sterile LB liquid medium without antibiotics, mix and beat evenly, then place in a 37°C, 220 rpm shaker for 45 min to recover the bacteria. After recovery, centrifuge at 6000 rpm for 1 min to collect the bacteria, resuspend the bacteria with 100 μl of supernatant, and spread on LB solid plate medium containing kanamycin (100 mg / ml), and incubate at 37°C for 14-16 hours.

[0116] (2.3) Bacterial liquid PCR identification of positive clones

[0117] Use a sterilized gun head to pick single colonies from the plate in 250 μl of LB liquid medium containing kanamycin, and incubate at 37°C, 200 rpm / min for about 3 h as amplification template. Another gene PCR purification product (CDS sequence or CDS partial reverse sequence and ddH2O (double distilled water) as template, set as positive and negative controls, respectively, use Taq Plus Master Mix of Nanjing Vazyme Company for PCR amplification, and 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 program is: 95°C for 3 min; (94°C for 30 s; 55°C for 30 s; 72°C for 60 s) for 35 cycles; 72°C for 5 min; 4°C for ∞.

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

[0123] (2.4) Positive clone plasmid extraction

[0124] The PCR positive clones were taken to 6 ml LB liquid medium containing kanamycin and cultured at 37°C with 200 rpm shaking overnight. Plasmid extraction was performed using the plasmid extraction kit from Jiangsu Kangwei Reagent Co., Ltd. and sequencing was performed by Beijing Ruibo Xingke Biotechnology Co., Ltd. After the sequence determination was compared and no error was found, the construction of the overexpression recombinant vector and the RNAi recombinant vector was completed.

[0125] Example 4 Genetic transformation of PtoMYB20 gene

[0126] (1) Recombinant plasmid transformation of Agrobacterium

[0127] (1.1) About 1 μg of PtoMYB20 overexpression vector plasmid and RNAi vector plasmid was added to 100 μL of Agrobacterium GV3101 competent cells, and mixed gently.

[0128] (1.2) Successively stand on ice for 5 min, freeze in liquid nitrogen for 5 min, immediately put into 37°C water bath for 5 min, and ice bath for 5 min.

[0129] (1.3) Add 700 μL of YEP liquid medium without antibiotics to the bacterial solution, mix thoroughly, and then incubate at 28°C with 200 rpm shaking for 2-3 hours. After recovery culture, centrifuge the bacterial solution at 6000 rpm for 1 minute, discard part of the supernatant, and reserve 100 μL of supernatant mixed with the bacterial solution. Use sterilized and cooled glass beads to evenly spread the bacterial solution on the surface of YEP solid medium containing 50 mg / L rifampicin and 50 mg / L kanamycin, and then place it in a 28°C incubator for inverted culture for 72-90 hours.

[0130] (1.4) After single colonies grow, use a sterile gun head to pick several single colony spots and place them in a 2 mL centrifuge tube containing 250 μL of YEP liquid medium (1:1000 addition of Rif, Kana), and incubate at 30°C with 200 rpm shaking for 2 hours. After the culture is completed, the bacterial solution is taken for PCR identification. The correct positive bacterial solution is added with 50% glycerol and frozen in liquid nitrogen, and stored in a -80°C refrigerator for subsequent genetic transformation experiments.

[0131] (2) Activation of Agrobacterium

[0132] (2.1) Take the Agrobacterium containing the overexpression vector plasmid and the RNAi vector plasmid from the -80°C refrigerator, and streak inoculate on YEP solid medium (1:1000 addition of Rif, Kana), seal the plate, and incubate in a 28°C dark incubator for 2-3 days.

[0133] (2.2) Use a sterile gun head to pick a single colony from the plate and inoculate into 3 mL YEP liquid medium (1:1000 add Rif, Kana), and place in a shaker at 30°C, 200 rpm overnight. Take 1 mL of bacterial solution and transfer it to a 250 mL sterile conical flask containing 100 mL YEP liquid medium (1:1000 add Rif, Kana), and place in a shaker at 30°C, 200 rpm for 4-5 h, until the OD600 is 0.3-0.5.

[0134] (2.3) In the clean bench, transfer 100 mL of bacterial solution to two 50 mL sterile centrifuge tubes, 2560 g, 4°C centrifuge for 20 min. Collect the bacterial cells. In the clean bench, discard the supernatant and resuspend the bacterial cells in a sterile jar with 100 mL of resuspension solution (WPMB), which is used for subsequent infection experiments.

[0135] The composition of the resuspension solution (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 (freshly added) PH = 5.6, and after preparation, perform 121°C, 20 min high temperature and high pressure sterilization.

[0136] (3) Callus method infection

[0137] Select healthy and sterile poplar seedlings (leaves are dark green and thick), use a sterile scalpel to remove the petiole, and leave the petiole to grow callus. Make 2-3 horizontal cuts on the main vein of the leaf, and place the leaf upside down on the callus culture medium (CIM) in a 25°C environment. After 20-30 days of leaf growth, white and loose callus will grow at the wound site. The callus is peeled off from the leaf and divided into soybean-sized pieces and placed in new callus culture medium. The prepared callus is transferred to a sterile jar containing bacterial solution and placed in a shaker at 28°C, 160 rpm for 15-20 min.

[0138] The composition of the 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 gel PH = 5.9, and after preparation, perform 121°C, 20 min high temperature and high pressure sterilization.

[0139] (4) Co-culture

[0140] In the clean bench, use tweezers to remove the callus from the bacterial solution, use filter paper to absorb the excess bacterial solution, and place the infected callus on the co-culture plate (WPMC) and incubate 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, and after preparation, high-temperature and high-pressure sterilization at 121°C for 20 min is performed.

[0142] (5) Differentiation culture

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

[0144] The callus is transferred to the differentiation culture medium (WPMD), and the medium is replaced once every 20 days for the first time, and then once every 10 days. During this period, the callus will turn green, harden, and then partially turn red. This stage is about two months, and the whole process is cultured in a 25°C light incubator.

[0145] The composition of the differentiation culture 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 / L TDZ + 7 g agar + 250 mg / L TMT + 250 mg / L cefotaxime + 20 mg / L kana PH = 5.9, and after preparation, high-temperature and high-pressure sterilization at 121°C for 20 min is performed.

[0146] (6) Cluster bud induction rooting

[0147] After the adventitious bud grows to about half a centimeter, the adventitious bud is cut off using sterile tweezers or a surgical knife (note that the growth point of the adventitious bud is observed to avoid selecting adventitious buds differentiated from the same cell), and is placed in the bud elongation medium for growth. After a period of elongation culture, when the adventitious bud grows to 1-2 cm, the adventitious bud is cut off individually using sterile tweezers or a surgical knife, and is placed in the rooting culture medium for rooting culture. After about 10 days, the adventitious bud will grow roots, and the rooting culture will become a complete plant.

[0148] The composition of the elongation culture medium is: WPM + 20 g sucrose + 0.3 mg / L 6BA + 0.2 mg / L IBA + 7 g agar + 250 mg / L TMT + 250 mg / L cefotaxime, and after preparation, high-temperature and high-pressure sterilization at 121°C for 20 min is performed.

[0149] The composition of the rooting culture medium is: 1 / 2MS + 20 g sucrose + 0.05 mg / L IBA + 0.02 mg / L NAA + 7 g agar + 250 mg / L TMT + 250 mg / L cefotaxime + 20 mg / L Kana, and after preparation, high-temperature and high-pressure sterilization at 121°C for 20 min is performed.

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

[0151] (1) Wild type, PtoMYB20 gene overexpression plants and silencing plants (RNAi plants) DNA extraction:

[0152] (i) Take a piece of the sample leaf to be extracted and place it in a 2 mL centrifuge tube and add 1 grinding bead, freeze in liquid nitrogen and crush with a tissue crusher for 5 min.

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

[0154] (iii) Centrifuge at 12000 rpm for 15 min, transfer the supernatant to a new centrifuge tube, add an equal volume of isopropanol, and let it stand at room temperature for 30 min. A white flocculent precipitate can be seen.

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

[0156] (v) Centrifuge at 7500 rpm for 10 min, slowly pour off the supernatant and clean up the remaining liquid with a pipette.

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

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

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

[0160] (2.1) DNA level identification

[0161] Use the crude transgenic plant gDNA as a template, and use Taq Plus Master Mix for PCR identification. The reaction system is referred to Table 5 in Example 3.

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

[0163] Detect the PCR product by agarose gel electrophoresis to check if the band is correct. If correct, the plant is a PtoMYB20 gene overexpression plant or a silencing plant.

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

[0165] (2.2) Identification of transcription level

[0166] The transcriptional level of PtoMYB20 in overexpressed and silenced plants was detected by RT-qPCR:

[0167] (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.

[0168] (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. The primers were P5 (PtoMYB20-qPCR-F) and P6 (PtoMYB20-qPCR-R), and the internal control primers were P7 (Actin-F) and P8 (Actin-R).

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

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

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

[0172] Example 6 Stress phenotype analysis of PtoMYB20 gene overexpression and RNAi plants

[0173] (1) Salt stress treatment

[0174] The plantlets with consistent growth vigor under tissue culture conditions for 25 days were washed with warm water to remove the root agar, and then were transplanted into soil culture medium mixed with substrate and vermiculite at a ratio of 1:1.5. The light condition was 8 h dark culture and 16 h light culture. After 30 days of culture under the same light condition at 25°C in a constant temperature culture room, the plantlets were irrigated with 150 mM NaCl solution for 25 days.

[0175] The growth vigor of the plantlets is shown in Figure 5 It can be seen that after 25 days of treatment with 150 mM NaCl, the PtoMYB20-RNAi plantlets (silenced plantlets, numbered as RNAi-1, RNAi-5) grew better under salt stress, and the leaves were slightly yellow. Most of the leaves of the wild type and PtoMYB20 overexpression plantlets (numbered as OE-1, OE-9) had already withered and dried, and some leaves had fallen off. In addition, before salt stress, the height of the PtoMYB20 overexpression plantlets was higher than that of the wild type (WT) and PtoMYB20-RNAi plantlets (silenced plantlets), and after salt stress, the height of the PtoMYB20 overexpression plantlets gradually leveled off with that of the WT and PtoMYB20-RNAi plantlets.

[0176] The physiological indexes of the plantlets were determined, and the results are shown in Figure 6 It can be seen that under salt stress, the contents of active oxygen such as H2O2 (hydrogen peroxide) and MDA (malondialdehyde) in the PtoMYB20-RNAi plantlets (silenced plantlets) were lower than those in the WT, and the activities of antioxidant enzymes such as SOD (superoxide dismutase) and POD (peroxidase) were higher than those in the WT, that is, the activities of antioxidant enzymes in the silenced plantlets increased under salt stress, effectively slowing down the excessive accumulation of active oxygen and reducing the degree of plant damage. The PtoMYB20 overexpression plantlets showed the opposite trend.

[0177] (2) Drought stress treatment

[0178] The plantlets with consistent growth vigor under tissue culture conditions for 25 days were washed with warm water to remove the root agar, and then were transplanted into soil culture medium mixed with substrate and vermiculite at a ratio of 1:1.5. The light condition was 8 h dark culture and 16 h light culture. After 30 days of culture under the same light condition at 25°C in a constant temperature culture room, the plantlets were irrigated with 20% PEG 6000 solution for 22 days.

[0179] The growth vigor of the plantlets after drought stress treatment is shown in Figure 5 It can be seen that after 22 days of treatment with 20% PEG 6000 to simulate drought stress, the PtoMYB20-RNAi plantlets (silenced plantlets) grew better than the wild type (WT) and PtoMYB20 overexpression plantlets. The leaves of the PtoMYB20 overexpression plantlets withered and wilted more severely than under salt stress, while the leaves of the PtoMYB20-RNAi plantlets did not wither, and the degree of wilting and damage was significantly lighter.

[0180] The physiological indexes of the plants were determined, and the results are shown in Table 3. Figure 7 As can be seen from Table 3, under drought stress, the contents of active oxygen such as H2O2 and MDA of PtoMYB20-RNAi plants (silenced plants) were lower than those of WT, and the activities of antioxidant enzymes such as SOD and POD were higher than those of WT, that is, under drought stress, the activities of antioxidant enzymes of the silenced plants were increased, which effectively slowed down the excessive accumulation of active oxygen and reduced the damage degree of the plants. However, the PtoMYB20 overexpression plants showed the opposite trend.

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

[0182] The plants grown under tissue culture conditions for 25 days and having the same growth trend were washed with warm water to remove the root agar, and then were transplanted into a soil culture medium mixed with substrate and vermiculite at a ratio of 1:1.5, and were cultured under light conditions of 8 h darkness and 16 h light. Then, the plants were cultured under the same light conditions at 25°C in a constant temperature culture room for 30 days, and then were irrigated with a mixed solution of 20% PEG 6000 and 150 mM NaCl for 15 days.

[0183] The growth trend of the plants after salt and drought combined stress treatment is shown in Table 4. Figure 5 As can be seen from Table 4, after 15 days of salt and drought combined stress treatment, the leaves of the PtoMYB20 overexpression plants were extremely serious in wilting and drooping, most of the leaves fell off, and the damage was significant. In sharp contrast, the wild type (WT) plants and the PtoMYB20-RNAi plants (silenced plants) showed relatively good growth trend. Among them, the damage degree of the leaves of the PtoMYB20-RNAi plants was particularly slight, and no obvious wilting was observed, which showed strong tolerance under combined stress.

[0184] The physiological indexes of the plants were determined, and the results are shown in Table 5. Figure 8 As can be seen from Table 5, under salt and drought combined stress, the contents of active oxygen such as H2O2 and MDA of the PtoMYB20-RNAi plants were lower than those of WT, and the activities of antioxidant enzymes such as SOD and POD were higher than those of WT, that is, under salt and drought combined stress, the activities of antioxidant enzymes of the silenced plants were increased, which effectively slowed down the excessive accumulation of active oxygen and reduced the damage degree of the plants. However, the PtoMYB20 overexpression plants showed the opposite trend.

[0185] The above detailed description of the present application is made in combination with the specific embodiments and exemplary examples, but these descriptions cannot be understood as limitations of the present application. Those skilled in the art understand that various equivalent substitutions, modifications or improvements can be made to the technical solutions and embodiments of the present application without departing from the spirit and scope of the present application, and these all fall within the scope of the present application.

Claims

1. A breeding method for enhancing the salt and drought tolerance of poplar trees, characterized in that, The method includes the following steps: Step 1, Build PtoMYB20 Overexpression recombinant vectors and RNAi recombinant vectors; The RNAi recombinant vector transmits genes PtoMYB20 The interference fragment was constructed into a basic vector to obtain the gene, which is the gene regulating salt tolerance and drought resistance in poplar. PtoMYB20, Its nucleotide sequence is shown in SEQ ID NO.1, and the poplar is Populus tomentosa; the interfering fragment is PtoMYB20-RNAi, and its nucleotide sequence is shown in SEQ ID NO.

4. The base vector of the RNAi recombinant vector is the pBI121 vector. Step 2, proceed PtoMYB20 Genetic transformation and identification of overexpressing and silent plants; Step 3: The overexpressing plants and the silent plants were subjected to stress treatment and phenotypic analysis was performed to obtain transgenic poplar plants with strong salt and drought resistance. The stress treatments include salt stress treatment, drought stress treatment, and combined salt and drought stress treatment; Under salt stress, drought stress, and combined salt and drought stress, the growth of silent poplar plants was significantly better than that of wild-type and overexpression plants. After 25 days of treatment with 150 mM NaCl, PtoMYB20-RNAi plants showed better growth, with slightly yellowed leaves, while wild-type and... PtoMYB20 Most of the leaves of the overexpressing plant have turned yellow and dried out, and some leaves have fallen off. After 22 days of treatment with 20% PEG 6000 simulating drought stress, PtoMYB20 The leaves of overexpressing plants showed more severe drying and wilting than those under salt stress, while the leaves of PtoMYB20-RNAi plants did not dry out, and the degree of wilting and damage was significantly less. After 15 days of combined salt and drought stress treatment PtoMYB20 The leaves of the overexpressing plants were severely withered and wilted, with most leaves falling off and showing significant damage; the leaves of the PtoMYB20-RNAi plants were particularly slightly damaged, with almost no obvious signs of drying.

2. The method according to claim 1, characterized in that, The gene PtoMYB20 The amino acid sequence encoding the protein is shown in SEQ ID NO.

3.

3. The method according to claim 1, characterized in that, In step 1, the PtoMYB20 The base vector for the overexpression recombinant vector is the pBI121 vector. The overexpression recombinant vector transmits genes PtoMYB20 The CDS sequence was obtained by inserting it into the pBI121 vector.

4. Gene knockout PtoMYB20 The poplar tree mentioned in the application of enhancing the salt and drought resistance of poplar trees is the white poplar.

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