Plant salt tolerance-related protein PeRD19A and its application

Abstract

The present invention discloses a plant salt tolerance-related protein PeRD19A and its application. The nucleic acid sequence of the plant salt tolerance-related protein PeRD19A is shown in SEQ ID NO. 1, and the protein sequence of the plant salt tolerance-related protein PeRD19A is shown in SEQ ID NO. 2. The present invention relates to the field of plant proteins, and specifically to the plant salt tolerance-related protein PeRD19A and its application.

Description

Technical Field

[0001] The present invention relates to the field of plant proteins, and in particular to a plant salt tolerance-related protein PeRD19A and applications thereof. Background Art

[0002] Trees growing in natural environments often face a variety of abiotic stresses, such as drought and salt stress. These external stresses not only hinder the normal growth and development of plants but also cause significant economic losses to forestry production. Although traditional improvement methods, such as breeding for salt tolerance and soil improvement, can alleviate the damage caused by salt stress to a certain extent, they are difficult to fundamentally solve the problem of promoting poplars in saline-alkali soils. In this context, genetic engineering of poplars for stress resistance has become an important means to improve their salt stress tolerance. Therefore, the discovery of stress-resistant genes and the cultivation of new stress-resistant tree varieties have important theoretical and practical significance for forestry production. To cope with the damage caused by various adverse conditions, plants have developed a series of stress resistance mechanisms, such as delaying stress-induced cell death and degrading damaged proteins through proteases. Cysteine ​​proteases are a large family of proteases that are widely involved in protein hydrolysis, leaf senescence, and programmed cell death in plants. However, there are few studies on the role of poplar cysteine ​​proteases under salt stress. Therefore, studying their role in regulating poplar salt tolerance is of great significance for breeding new salt-tolerant poplar varieties.

[0003] Cysteine ​​proteases (CPs) in plants are mainly divided into papain, legumain, caspase, and the less studied calcium-dependent cysteine ​​protease (calpain) [1]. Cysteine ​​proteases play an important role in programmed cell death, cell aging, and coping with adverse stress. Papain is a type of plant cysteine ​​protease and is the largest in its class. It has also been studied relatively thoroughly. Papain was first isolated from papaya. Subsequently, various homologous cysteine ​​proteases were discovered in viruses, microorganisms, animals, and plants. To date, nearly 600 species have been studied. Since a significant common feature of these proteases is that they all contain a Cys-His-Asn catalytic ternary moiety in their active sites, it can be used as an important marker to distinguish them from other members of the cysteine ​​protease family [2]. Existing studies have shown that papain can be divided into nine subfamilies (RD21A, CEP1, XCP2, XBCP3, THI1, SAG12, RD19A, AALP, and CTB3) based on their protodomains. Except for the RD19A-like protease and CTBs subfamilies, the other seven subfamilies all carry the ERFNIN motif, while CTBs lack this motif, while RD19A-like proteases carry the conserved ERFNAQ motif. Furthermore, unlike other subfamilies, RD19A-like proteases carry two additional, highly conserved cysteine ​​residue pairs, forming stable α-helices and β-sheets, respectively.

[0004] Papain is the most closely related group of plant cysteine ​​proteases to PCD. It is widely involved in xylem vessel formation, tapetum disintegration, suspensor PCD, and stress-induced PCD [3]. PCD is an important component of plant development and defense mechanisms under stress. AtXCP1 and AtXCP2 are two xylem-specific papains that are abundantly expressed in the xylem during PCD of Arabidopsis hypocotyls [4]. Cathepsins belong to the papain C1A family and are important regulatory factors involved in leaf senescence and PCD in plants. Three cathepsin B proteins (AtCathB1-3) identified by tandem mass spectrometry in Arabidopsis showed a significant reduction in PCD induced by abiotic stress (such as oxidative stress) and endoplasmic reticulum stress in their triple mutants [5]. AtRD19C, discovered in Arabidopsis, was first described as an inducible protein that responds to low temperature and drought stress. Recent studies have shown that AtRD19C, after being activated by the vacuolar processing enzyme β-VPE, interacts with the selenium-binding protein SBP1 and participates in anther development through PCD[6]. CaCP34 is a member of the papain family of pepper (Capsicum annuum). Studies have shown that overexpression of CaCP34 in pepper leaves significantly increased proline content and antioxidant enzyme activity, and the instantaneous MDA and H2O2 levels in the leaves were lower than those in the control. Silencing CaCP34 increased the susceptibility of pepper to stress-induced senescence, demonstrating that CaCP34 enhances pepper's resistance to salt- and osmotic-induced leaf senescence by regulating the antioxidant system[7]. Existing studies have shown that cathepsin B-like protease 2 (CathB2) is involved in the PCD process. SlCathB2 is differentially expressed in different tissues of tomato (Solanum lycopersicum), and the expression levels of SlCathB2-1 and SlCathB2-2 are significantly increased under high temperature stress[8]. In Arabidopsis overexpressing AtGSK1, NaCl stress-induced genes AtCP1 and RD29A are significantly induced and upregulated, indicating that members of the papain family are involved in the regulatory network responding to NaCl stress[9].

[0005] These studies demonstrate that cysteine ​​protease gene members play important roles in regulating plant growth and development in response to abiotic stresses (drought, salt, and low temperature). However, studies investigating the role of cysteine ​​protease genes in regulating salt tolerance in poplars are rare. Currently, soil salinization remains a serious problem that needs to be addressed worldwide, posing new challenges for the breeding of stress-tolerant varieties in agriculture and forestry. Therefore, identifying salt-tolerance-related cysteine ​​protein genes in poplars and identifying their roles in regulating salt tolerance are crucial for the development of transgenic poplar varieties that are salt-tolerant. Summary of the Invention

[0006] The present invention explores poplar salt-tolerance-related cysteine ​​protein gene resources and identifies their functions in regulating salt tolerance, which is of great significance to salt-tolerant transgenic breeding of poplar.

[0007] In order to solve the above problems, the technical solution adopted by the present invention is as follows: The present invention is a plant salt tolerance related protein PeRD19A and its application.

[0008] In a first aspect, the nucleic acid sequence of the plant salt tolerance-related protein PeRD19A is shown as SEQ ID NO.1, and the protein sequence of the plant salt tolerance-related protein PeRD19A is shown as SEQ ID NO.2.

[0009] In a second aspect, the application of the plant salt tolerance-related protein PeRD19A comprises the following steps:

[0010] Step 1: Cloning of the PeRD19A gene and construction of an overexpression vector.

[0011] Using Primer 6.0 software, we designed specific primers (PeRD19A-F / R) based on the PeRD19A gene sequence (XM_002301968.3) to amplify the target gene from Populus euphorbiae 107 cDNA. The amplified product was ligated into the overexpression vector pNC-Cam2304-35S and transformed into competent Escherichia coli DH5α cells. Positive clones were sent to our company for sequencing. Single clones that were sequenced correctly were used to extract plasmids by shaking and then transformed into competent Agrobacterium tumefaciens EHA105 cells.

[0012] Step 2: Genetic transformation of "Beilin No. 1" poplar.

[0013] A single colony of Agrobacterium containing the PeRD19A gene was inoculated into LB liquid medium and shaken. The bacterial suspension was transferred to the LB liquid medium and continued to shake, after which the colonies were collected. The colonies were resuspended in sterile suspension and used for infection. Genetic transformation was performed using the leaf disc method in a clean bench. Two to three wounds were evenly scored on a leaf and then placed in the suspension for infection. A solution of acetosyringone (a surface adsorbent) at a final concentration of 100 μmol / L was added to the suspension and gently shaken for 10-15 minutes. The leaf that had fully absorbed the Agrobacterium was removed and blotted dry with sterile filter paper to remove any excess bacterial suspension. The leaf was then flattened and placed in co-cultivation medium and incubated in the dark at 25°C for 2-3 days. The co-cultivated leaf was blotted dry with sterile filter paper to remove any surface adhering liquid and then transferred to poplar selection differentiation medium for selection. After 7-10 days, the leaf was significantly thickened and transferred to selection stemming medium. When the height of the adventitious buds exceeds 1 cm, the resistant adventitious buds are transplanted into the rooting screening medium. After a 7-10 day induction period, adventitious roots are grown and then develop into complete plants, thus preliminarily confirming that the resistant strains have been obtained.

[0014] Step 3: Transplantation of resistant strains.

[0015] After initially identifying resistant strains, wait for adventitious roots to form in the tissue culture seedlings and select seedlings with consistent growth to harden in a suitable space for 3 days. Once the seedlings have gradually adapted to the external environment, plant them in pots containing a uniform mixture of garden soil, nutrient soil, vermiculite, and sand in a 1:1:1:1 ratio and cultivate them in an artificial climate chamber. After transplanting, cover with plastic wrap. Once tender leaves appear at the top of the seedlings, gradually remove the plastic wrap to allow them to acclimate. Water them promptly until the plastic wrap is completely removed.

[0016] Step 4: PCR identification of resistant strains and qRT-PCR detection of positive strains.

[0017] Young leaves of potted seedlings were cut and DNA of each strain was extracted. Plasmid was used as a positive control and wild type as a negative control. PCR identification was performed on the resistant strains to confirm whether they contained the PeRD19A target gene, so as to identify the PeRD19A overexpressing transgenic positive strains. Using the wild type 'Beilin No. 1' poplar as a control, qRT-PCR detection was performed on the PeRD19A overexpressing strains. The third fully expanded leaf of each plant was cut and total RNA was extracted from the PeRD19A overexpressing strains and the wild type 'Beilin No. 1' poplar, which was reverse transcribed into cDNA. The relative expression level of the PeRD19A gene was detected using qRT-PCR technology.

[0018] Step 5: Salt tolerance test of PeRD19A overexpression strains.

[0019] When potted seedlings reached approximately 20 cm in a climatic chamber (26°C, 49% humidity), salt stress testing was conducted. Based on qRT-PCR results, only strains OE1 and OE3 with relatively high expression levels were selected for subsequent salt tolerance testing. Salt and non-salt stress treatments were established, and seedlings with relatively consistent growth patterns were selected, with five biological replicates per strain. Under the non-salt stress treatment, five randomly selected plants from each of the WT, OE1, and OE3 strains served as a control group, for a total of 15 plants. Under the salt stress treatment, five randomly selected plants from each of the WT, OE1, and OE3 strains served as a control group, for a total of 15 plants. The soil dry weight per pot was 250 ± 5 g after drying and weighing. Each pot was watered with 200 mL of water per pot in the non-salt stress treatment and 200 mL of a 200 mmol / L NaCl solution per pot in the salt stress treatment. All treatments were watered every three days. The salt tolerance experiment was conducted until the seventh day, during which time, one supplemental irrigation was administered. The NaCl content in the salt-stressed soil accumulated to approximately 1.87%. At this point, the wild-type plants under salt stress showed severe wilting from bottom to top, significantly different from OE1 and OE3. Growth and physiological parameters were then measured.

[0020] Step 6: Growth and physiological index determination,

[0021] (1) Growth index determination,

[0022] The plant height before and after salt stress treatment was measured and recorded using a tape measure (accuracy of 0.1 cm), and the increase in plant height was calculated and recorded. After the measurement and sampling of each treatment were completed, the plants were removed from the soil, the aboveground parts were cut off to obtain the complete roots, and they were rinsed with clean water and placed in an oven to dry at 90°C. Finally, the root dry weight was weighed, and the taproot length was measured using a standard ruler (accuracy of 0.1 cm). The number of lateral roots of each plant was recorded, with the primary lateral root as the standard. Five biological replicates were performed.

[0023] (2) Determination of antioxidant enzyme activity and malondialdehyde content,

[0024] Cut the third fully expanded leaf from each plant and quickly freeze it in a mortar filled with liquid nitrogen and grind it into a fine powder. Transfer it to an electronic balance and accurately weigh 0.3g. Then quickly transfer it to a pre-cooled centrifuge tube. Then add 5mL of 50mmol / L pH=7.8 phosphate buffer stored at 4℃ and mix the powder until it becomes a homogenous slurry. Centrifuge at 4℃, 12,000rpm for 20min. After centrifugation, collect the supernatant in a new centrifuge tube as the crude extract required for the determination of CAT, SOD, POD and MDA content. Five biological replicates were performed.

[0025] (3) Determination of relative conductivity, photosynthetic pigment content and photosynthesis index,

[0026] After cutting off the third fully expanded leaf of each strain, the leaves were washed with deionized water to remove impurities and then chopped. 10 mL of deionized water was added and vacuumed until the material sank. The leaves were then soaked in a centrifuge tube at room temperature for 15 h. The conductivity before boiling (R1) was measured using a conductivity meter. The centrifuge tube was then placed in a boiling water bath for 15-20 min, removed and cooled to room temperature. The conductivity after boiling (R2) was measured and the relative conductivity was calculated.

[0027] The third fully expanded leaf of each plant was selected, and the net photosynthetic rate and stomatal conductance were measured using a Li-6400XT portable photosynthetic instrument. The third fully expanded leaf of each plant was cut, and 0.1 g was accurately weighed without cutting the midrib and soaked in a test tube containing 10 mL of 95% ethanol. The pigment was extracted for 24 hours in the dark. After mixing, 0.2 mL was taken from each tube and added to a 96-well plate. The absorbance of each sample extract was measured at 665 nm, 649 nm, and 470 nm (A665, A649, A470), respectively, with 5 biological replicates. The concentrations of chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids were calculated (mg·L-1).

[0028] (4) Determination of soluble sugar, starch and free proline content,

[0029] Five biological replicates were performed using the third fully expanded leaf of each PeRD19A-overexpressing strain and the wild-type 'Beilin No. 1' poplar line. Soluble sugar content assay kits (microplate assay, 96 samples), starch content assay kits (microplate assay, 96 samples), and proline content assay kits (microplate assay, 96 samples) manufactured by Suzhou Grace Biotechnology Co., Ltd. Specific procedures were described in the kit instructions.

[0030] The beneficial effects achieved by the present invention using the above structure are as follows: Poplar, as an important tree species with an extremely wide distribution range worldwide, has the characteristics of fast growth rate and strong adaptability. However, soil salinization is still a serious problem that needs to be solved worldwide, which poses a new challenge to the cultivation of stress-resistant varieties in agriculture and forestry. Cysteine ​​proteases have been fully explored and studied in terms of programmed cell death, cell aging and coping with adverse stress in plants. Poplar, as a model plant among woody plants, has also been widely explored for its stress resistance mechanism for coping with abiotic stress. However, research on the involvement of cysteine ​​proteases in salt resistance physiology is still quite lagging behind. The present invention focuses on the current problems, takes the poplar cysteine ​​protease PeRD19A gene as the starting point, and explores its function in salt stress response. The results show that transgenic plants overexpressing this gene can improve salt tolerance, which will provide genetic resources and theoretical basis for the cultivation of new salt-tolerant poplar varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The obtained map of the target gene;

[0032] Figure 2 This is the PCR detection diagram of Escherichia coli transformed with the recombinant overexpression vector;

[0033] Figure 3 This is the sequence alignment of PeRD19A sequencing results;

[0034] Figure 4 This is the PCR detection diagram of Agrobacterium transformed with the recombinant overexpression vector;

[0035] Figure 5 is the PCR detection diagram of the transgenic line;

[0036] Figure 6 The relative expression levels of target genes in the wild type and four PeRD19A overexpression lines are shown;

[0037] Figure 7 Figure 2 shows the wild type and PeRD19A overexpression lines at 7 days after salt stress;

[0038] Figure 8 This figure shows the effect of salt stress on the increase in plant height of PeRD19A overexpression lines;

[0039] Figure 9 Figure 2 is the effect of salt stress on the roots of PeRD19A overexpression lines;

[0040] Figure 10 This figure shows the effect of salt stress on the content of antioxidant enzymes in the PeRD19A overexpression line;

[0041] Figure 11 Figure 2 shows the effect of salt stress on the cell membrane stability of the PeRD19A overexpression line;

[0042] Figure 12 This is the effect of salt stress on the net photosynthetic rate and stomatal conductance of leaves of PeRD19A overexpression lines;

[0043] Figure 13 This figure shows the effect of salt stress on the content of photosynthetic pigments in leaves of PeRD19A overexpression lines;

[0044] Figure 14 This figure shows the effect of salt stress on the free proline, soluble sugar and starch content of the PeRD19A overexpression line. DETAILED DESCRIPTION

[0045] The nucleic acid sequence of the plant salt tolerance-related protein PeRD19A is shown in SEQ ID NO.1.

[0046] >PeRD19A nucleic acid sequence

[0047] ATGGAGCGCTTTTCTCTGCTCGCACTCCTCCTTCTAACACTCCTATGCTCAGCAGTTGCATCCACAGTATCTTCCAACGACCTCGACGATCCTTTAATCAGACAAGTCGTATCAGACGGCGAGGATGATCTCCTCAACGCAGAGCATCATTTCACTTCTTTCAAATCCAAATTCGGCAAAACATACGCGACTCAAGAAGAGCATGATTACCGATTTGGTGTTTTCAAAGCTAATCTTCGCCGTGCGAAGAAACACCAGATGATAGACCCCACCGCCGCCCACGGCGTCACCAAGTTCTCTGACCTGACACCGAAAGAATTCCGCCGCCAGTTCCTTGGATTGAAGCGGCGGCTACGGTTGCCTACGGACGCTAACAAGGCGCCGATCCTTCCTACTACTGATCTACCTACTGATTATGACTGGCGCGATCACGGTGCCGTTACAGAAGTCAAAGACCAGGGATCGTGTGGATCGTGCTGGTCGTTTAGTGCTACCGGGGCGTTAGAAGGAGCTCATTATTTAGCCACAGGAGAGCTTGCGAGCCTGAGTGAGCAGCAGCTTGTGGACTGCGATCATGAGTGTGATCCAGAAGAATATGGTGCTTGTGACTCTGGCTGCGATGGTGGGCTGATGAATAATGCCTTCGAGTACGCACTCAAGGCAGGTGGACTTGAACGTGAGGCAGACTATCCTTACACTGGAACTGACGGTGGCACTTGCAAATTTGACAAGAGCAAAGTTGTAGCATCCGTATCTAACTTCAGTGTTGTTTCAATCGATGAAGATCAAATTGCTGCGAATCTGGTGAAGCATGGTCCACTCTCAGTGGCGATCAATGCAGCTTTTATGCAGACATATGTAGGTGGAGTTTCATGCCCATACATTTGCTCGAAGCGTCAGGATCACGGGGTGCTACTGGTGGGGTATGGATCCGCTGGTTATGCTCCTATCCGGTTTAAGGAAAAGCCCTTCTGGATTATCAAGAATTCATGGGGACAGAATTGGGGAGAGAATGGCTATTACAAAATCTGCAGGGGTCGCAATATCTGTGGTGTAGATTCCATGGTTTCAACTGTTGCTGCAATTCACACTACAGCTCAGTAG

[0048] The protein sequence of the salt tolerance-related protein PeRD19A in plants is shown in SEQ ID NO.2.

[0049] >PeRD19A protein sequence

[0050] MERFSLLALLLLTLLCSAVASTVSSNDLDDPLIRQVVSDGEDDLLNAEHHFTSFKSKFGKTYATQEEHDYRFGVFKANLRRAKKHQMIDPTAAHGVTKFSDLTPKEFRRQFLGLKRRLRLPTDANKAPILPTTDLPTDYDWRDHGAVTEVKDQGSCGSCWSFSATGALEGAHYLATGELASLS EQQLVDCDHECDPEEYGACDSGCDGGLMNNAFEYALKAGGLEREADYPYTGTDGGTCKFDKSKVVASVSNFSVVSIDEDQIAANLVKHGPLSVAINAAFMQTYVGGVSCPYICSKRQDHGVLLVGYGSAGYAPIRFKEKPFWIIKNSWGQNWGENGYYKICRGRNICGVDSMVSTVAAIHTTAQ

[0051] Examples, such as Figures 1 to 14 As shown,

[0052] The application of the plant salt tolerance-related protein PeRD19A comprises the following steps:

[0053] Step 1: Cloning of the PeRD19A gene and construction of an overexpression vector.

[0054] Using Primer 6.0 software, we designed specific primers (PeRD19A-F / R) based on the PeRD19A gene sequence (XM_002301968.3) to amplify the target gene from Populus euphorbiae 107 cDNA. The amplified product was ligated into the overexpression vector pNC-Cam2304-35S and transformed into competent Escherichia coli DH5α cells. Positive clones were sent to our company for sequencing. Single clones that were sequenced correctly were used to extract plasmids by shaking and then transformed into competent Agrobacterium tumefaciens EHA105 cells.

[0055] Step 2: Genetic transformation of "Beilin No. 1" poplar.

[0056] A single colony of Agrobacterium containing the PeRD19A gene was inoculated into LB liquid medium and shaken. The bacterial suspension was transferred to the LB liquid medium and continued to shake, after which the colonies were collected. The colonies were resuspended in sterile suspension and used for infection. Genetic transformation was performed using the leaf disc method in a clean bench. Two to three wounds were evenly scored on a leaf and then placed in the suspension for infection. A solution of acetosyringone (a surface adsorbent) at a final concentration of 100 μmol / L was added to the suspension and gently shaken for 10-15 minutes. The leaf that had fully absorbed the Agrobacterium was removed and blotted dry with sterile filter paper to remove any excess bacterial suspension. The leaf was then flattened and placed in co-cultivation medium and incubated in the dark at 25°C for 2-3 days. The co-cultivated leaf was blotted dry with sterile filter paper to remove any surface adhering liquid and then transferred to poplar selection differentiation medium for selection. After 7-10 days, the leaf was significantly thickened and transferred to selection stemming medium. When the height of the adventitious buds exceeds 1 cm, the resistant adventitious buds are transplanted into the rooting screening medium. After a 7-10 day induction period, adventitious roots are grown and then develop into complete plants, thus preliminarily confirming that the resistant strains have been obtained.

[0057] Step 3: Transplantation of resistant strains.

[0058] After initially identifying resistant strains, wait for adventitious roots to form in the tissue culture seedlings and select seedlings with consistent growth to harden in a suitable space for 3 days. Once the seedlings have gradually adapted to the external environment, plant them in pots containing a uniform mixture of garden soil, nutrient soil, vermiculite, and sand in a 1:1:1:1 ratio and cultivate them in an artificial climate chamber. After transplanting, cover with plastic wrap. Once tender leaves appear at the top of the seedlings, gradually remove the plastic wrap to allow them to acclimate. Water them promptly until the plastic wrap is completely removed.

[0059] Step 4: PCR identification of resistant strains and qRT-PCR detection of positive strains.

[0060] Young leaves of potted seedlings were cut and DNA of each strain was extracted. Plasmid was used as a positive control and wild type as a negative control. PCR identification was performed on the resistant strains to confirm whether they contained the PeRD19A target gene, so as to identify the PeRD19A overexpressing transgenic positive strains. Using the wild type 'Beilin No. 1' poplar as a control, qRT-PCR detection was performed on the PeRD19A overexpressing strains. The third fully expanded leaf of each plant was cut and total RNA was extracted from the PeRD19A overexpressing strains and the wild type 'Beilin No. 1' poplar, which was reverse transcribed into cDNA. The relative expression level of the PeRD19A gene was detected using qRT-PCR technology.

[0061] Step 5: Salt tolerance test of PeRD19A overexpression strains.

[0062] When potted seedlings reached approximately 20 cm in a climatic chamber (26°C, 49% humidity), salt stress testing was conducted. Based on qRT-PCR results, only strains OE1 and OE3 with relatively high expression levels were selected for subsequent salt tolerance testing. Salt and non-salt stress treatments were established, and seedlings with relatively consistent growth patterns were selected, with five biological replicates per strain. Under the non-salt stress treatment, five randomly selected plants from each of the WT, OE1, and OE3 strains served as a control group, for a total of 15 plants. Under the salt stress treatment, five randomly selected plants from each of the WT, OE1, and OE3 strains served as a control group, for a total of 15 plants. The soil dry weight per pot was 250 ± 5 g after drying and weighing. Each pot was watered with 200 mL of water per pot in the non-salt stress treatment and 200 mL of a 200 mmol / L NaCl solution per pot in the salt stress treatment. All treatments were watered every three days. The salt tolerance experiment was conducted until the seventh day, during which time, one supplemental irrigation was administered. The NaCl content in the salt-stressed soil accumulated to approximately 1.87%. At this point, the wild-type plants under salt stress showed severe wilting from bottom to top, significantly different from OE1 and OE3. Growth and physiological parameters were then measured.

[0063] Step 6: Growth and physiological index determination,

[0064] (1) Growth index determination,

[0065] The plant height before and after salt stress treatment was measured and recorded using a tape measure (accuracy of 0.1 cm), and the increase in plant height was calculated and recorded. After the measurement and sampling of each treatment were completed, the plants were removed from the soil, the aboveground parts were cut off to obtain the complete roots, and they were rinsed with clean water and placed in an oven to dry at 90°C. Finally, the root dry weight was weighed, and the taproot length was measured using a standard ruler (accuracy of 0.1 cm). The number of lateral roots of each plant was recorded, with the primary lateral root as the standard. Five biological replicates were performed.

[0066] (2) Determination of antioxidant enzyme activity and malondialdehyde content,

[0067] Cut the third fully expanded leaf from each plant and quickly freeze it in a mortar filled with liquid nitrogen and grind it into a fine powder. Transfer it to an electronic balance and accurately weigh 0.3g. Then quickly transfer it to a pre-cooled centrifuge tube. Then add 5mL of 50mmol / L pH=7.8 phosphate buffer stored at 4℃ and mix the powder until it becomes a homogenous slurry. Centrifuge at 4℃, 12,000rpm for 20min. After centrifugation, collect the supernatant in a new centrifuge tube as the crude extract required for the determination of CAT, SOD, POD and MDA content. Five biological replicates were performed.

[0068] (3) Determination of relative conductivity, photosynthetic pigment content and photosynthesis index,

[0069] After cutting off the third fully expanded leaf of each strain, the leaves were washed with deionized water to remove impurities and then chopped. 10 mL of deionized water was added and vacuumed until the material sank. The leaves were then soaked in a centrifuge tube at room temperature for 15 h. The conductivity before boiling (R1) was measured using a conductivity meter. The centrifuge tube was then placed in a boiling water bath for 15-20 min, removed and cooled to room temperature. The conductivity after boiling (R2) was measured and the relative conductivity was calculated.

[0070] The third fully expanded leaf of each plant was selected, and the net photosynthetic rate and stomatal conductance were measured using a Li-6400XT portable photosynthetic instrument. The third fully expanded leaf of each plant was cut, and 0.1 g was accurately weighed without cutting the midrib and soaked in a test tube containing 10 mL of 95% ethanol. The pigment was extracted for 24 hours in the dark. After mixing, 0.2 mL was taken from each tube and added to a 96-well plate. The absorbance of each sample extract was measured at 665 nm, 649 nm, and 470 nm (A665, A649, A470), respectively, with 5 biological replicates. The concentrations of chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids were calculated (mg·L-1).

[0071] (4) Determination of soluble sugar, starch and free proline content,

[0072] Five biological replicates were performed using the third fully expanded leaf of each PeRD19A-overexpressing strain and the wild-type 'Beilin No. 1' poplar line. Soluble sugar content assay kits (microplate assay, 96 samples), starch content assay kits (microplate assay, 96 samples), and proline content assay kits (microplate assay, 96 samples) manufactured by Suzhou Grace Biotechnology Co., Ltd. Specific procedures were described in the kit instructions.

[0073] The verification test method is as follows:

[0074] First, cloning of the PeRD19A gene,

[0075] like Figure 1 The results showed that the extracted poplar RNA was not degraded. The RNA was tested to be free of contamination and met the experimental requirements. Figure 1 b shows that the product obtained by PCR amplification with primers PeRD19A F / R is consistent with the expected fragment, which is 1104 bp.

[0076] Figure 1 Note:

[0077] (a) is the RNA electrophoresis diagram;

[0078] (b) is the cloning of the PeRD19A gene;

[0079] M: DL2000 DNA Marker (2000, 1000, 750, 500, 250, 100 bp from top to bottom);

[0080] 1-2: Test sample.

[0081] 2. Construction of overexpression vector. The recombinant overexpression vector pNC-Cam2304-35S-PeRD19A was transformed into Escherichia coli, and the bacterial solution was tested by electrophoresis. Figure 2 The results showed that most of the target bands were clear and accurately located. The E. coli solution with the correct target bands was sent to BGI for sequencing. DNAMAN was used to perform Blast comparison on the sequencing results. Figure 3 The PeRD19A gene showed 10 base mutations between Populus occidentalis 107 and Populus trichocarpa, confirming that there are certain differences between different species. The bacterial suspension with the correct sequence was preserved, the plasmid was extracted, and the plasmid was transformed into Agrobacterium. A single Agrobacterium colony was selected for PCR verification of the target gene. Results Figure 4 The results showed that the target band was located accurately, proving that the recombinant plasmid pNC-Cam2304-35S-PeRD19A was successfully transformed into Agrobacterium.

[0082] Figure 4 Note: 1-8 are the amplified PeRD19A genes.

[0083] Third, the acquisition and detection of transgenic lines, the wild type "Beilin No. 1" poplar was transformed by Agrobacterium-mediated leaf disc method, and pNC-Cam2304-35S-PeRD19A was transformed into the leaves of tissue culture seedlings to obtain four resistant lines. Using potted seedlings as materials, DNA was extracted from the wild type "Beilin No. 1" poplar and the four transgenic poplar lines, and 35S+PeRD19A R was amplified by PCR for detection. The electrophoresis results are shown in Figure 2. Figure 5 The results showed that the 35S+PeRD19A R target gene band of about 750 bp was amplified in all four transgenic poplar lines, indicating that the target gene PeRD19A was successfully inserted into the target poplar genome.

[0084] Figure 5 Note: CK+ is the positive control (plasmid containing the target gene), WT is the wild-type 'Beilin No. 1' poplar, OE1, OE2, OE3, and OE4 are four transgenic poplar lines.

[0085] Using potted seedlings as materials, wild-type "Beilin No. 1" poplar and four PeRD19A overexpression lines were selected, with three biological replicates for each line. The relative expression levels of the PeRD19A gene in the transgenic lines were detected by qRT-PCR. Figure 6The results showed that among the four PeRD19A overexpression lines, only OE2 showed no significant difference from the wild type. The relative expression levels of the PeRD19A gene in OE1, OE3, and OE4 lines were significantly higher than that in the wild type, ranging from 37.34 to 155.95 times that of the wild type.

[0086] Figure 6 Note: Different lowercase letters in multiple comparisons indicate significant differences among different strains.

[0087] Fourth, analysis of the growth characteristics of PeRD19A overexpression lines under salt stress. Based on the results of qRT-PCR, the OE1 and OE3 lines with relatively high expression levels were selected for salt stress experiments. After 7 days of salt stress, the growth status of the wild type (WT) and the two PeRD19A overexpression lines OE1 and OE3 were as follows: Figure 7 As shown, the lower leaves of the two overexpression lines only showed slight wrinkling and yellowing, while the lower leaves of the WT showed severe wilt and partial necrosis; observing the top of the plant, the leaves of the overexpression lines OE1 and OE3 were bright green, while the leaves of the WT were yellow-green, and the growth potential of the WT was significantly weaker than that of OE1 and OE3.

[0088] Depend on Figure 8 The results showed that the increase in plant height of OE1 and OE3 under salt stress treatment was significantly higher than that of WT, which were 8.6 and 11.4 times that of WT, respectively. There was no significant difference in the growth of WT and OE1 and OE3 under non-salt stress treatment.

[0089] Figure 8 Note: Different lowercase letters in multiple comparisons indicate significant differences among different strains under the same treatment.

[0090] Under salt stress, the root system is the part of the plant most directly affected by salt stress. In order to resist adversity, plants usually change the morphology of the root system and increase the underground biomass to improve the root-to-shoot ratio. The measurement results of the root system are listed in Figure 9 Under salt stress treatment, only the first-level root number of OE3 increased significantly compared with the control group, while WT and OE1 showed no significant changes compared with the control group; under salt stress treatment, the root weight of OE1 and OE3 increased but not significantly, and the root weight of WT decreased significantly compared with the non-salt stress treatment. The root length of each plant line showed no significant change compared with the non-salt stress treatment.

[0091] Figure 9 Note:

[0092] In the t-test, “*” represents the significant difference between the same strain under different treatments.

[0093] “*” means p < 0.05, “**” means p < 0.01, “***” means p < 0.001, and “****” means p < 0.0001;

[0094] “ns” means no significant difference.

[0095] Comprehensive plant growth indicators showed that salt stress had a relatively weak inhibitory effect on the growth of the PeRD19A overexpression line, indicating that the PeRD19A overexpression line had stronger salt stress tolerance than the wild type.

[0096] 5. Analysis of membrane stability and antioxidant enzyme activity of PeRD19A overexpressing strain under salt stress.

[0097] The results of the antioxidant enzyme content determination showed that Figure 10 Under salt stress, the SOD, POD, and CAT contents of OE1 and OE3 were significantly higher than those of the WT. Under non-salt stress, the SOD, POD, and CAT contents of OE1 and OE3 were not significantly different from those of the WT. Under salt stress, the antioxidant enzyme activities of OE1 and OE3 were higher than those of the WT, indicating that the overexpression lines have a stronger ROS scavenging ability than the WT. Therefore, overexpression of the PeRD19A gene can enhance antioxidant enzyme activity in poplar.

[0098] Figure 10 Note: FW (Fresh weight) stands for fresh weight;

[0099] (a) is the SOD content; (b) is the POD content; (c) is the CAT content.

[0100] The results of MDA content determination were as follows Figure 11 As shown in a, the MDA content of OE1 and OE3 did not change significantly under salt stress, while the MDA content of WT was significantly higher than that under non-salt stress. Figure 11 As shown in Figure b, the relative conductivity of OE1 and OE3 did not change significantly under salt stress, while the relative conductivity of WT changed significantly compared to the non-salt stress treatment. These results indicate that salt stress causes oxidative damage to the cell membranes of all poplar strains, but the damage to the cell membranes of OE1 and OE3 is less severe, while the cell membrane of WT is severely damaged, indicating that overexpression of the PeRD19A gene enhances the cell membrane's ability to resist oxidative damage.

[0101] Figure 11 Note: (a) is MDA content; (b) is relative conductivity.

[0102] 6. Analysis of photosynthetic pigment content and photosynthesis parameters in PeRD19A overexpression lines under salt stress.

[0103] Stomatal conductance can affect the transpiration and photosynthetic rate of plants. Figure 12It can be seen that the stomatal conductance and net photosynthetic rate of OE1 and OE3 under salt stress treatment were not significantly different from those under non-salt stress treatment, while the stomatal conductance and net photosynthetic rate of WT were severely inhibited. Plants overexpressing salt-tolerant genes showed higher content of photosynthetic pigments. Figure 13 After 7 days of salt stress, the total chlorophyll (including chlorophyll a and chlorophyll b) and carotenoid contents of OE1 and OE3 in the salt-stressed and non-salt-stressed treatments were not significantly different, with OE3 outperforming OE1. The contents of both types of photosynthetic pigments in the WT were significantly lower than those in the non-salt-stressed treatment. These results suggest that salt stress inhibits photosynthesis less in the PeRD19A-overexpressing strain than in the wild-type strain.

[0104] 7. Analysis of soluble sugar, starch and free proline contents in PeRD19A overexpression lines under salt stress.

[0105] Depend on Figure 14 The results show that the soluble sugar content of OE1 and OE3 under salt stress was significantly higher than that under non-salt stress, while the starch and free proline contents increased compared to the non-salt stress treatment but were not significantly different. The soluble sugar, starch, and free proline contents of WT were not significantly different from those under non-salt stress. Based on these results, it is speculated that under salt stress, OE1 and OE3 are better able to accumulate soluble organic matter and store organic matter, balance osmotic pressure, and maintain normal life activities compared to WT.

[0106] It should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. Application of the PeRD19A gene in improving salt tolerance in poplars, characterized in that: The PeRD19A gene is overexpressed in poplar, and the protein sequence encoded by the PeRD19A gene is shown in SEQ ID NO.

2.

2. The use of the PeRD19A gene according to claim 1 in improving salt tolerance of poplar, characterized in that: The nucleic acid sequence of the PeRD19A gene is shown in SEQ ID NO.

1.

3. The use according to claim 1 or 2, characterized in that: The steps include: Step 1: cloning the PeRD19A gene and constructing an overexpression vector; Step 2: Genetic transformation of poplar; Step 3: Transplantation of resistant strains; Step 4: PCR identification of resistant strains and qRT-PCR detection of positive strains; Step 5: Salt tolerance test of overexpression strains. Step six: Determination of growth and physiological indicators.

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