Encoding protein of psEcd1 of populus simonii and application of the encoding protein in improving drought tolerance of plants

Abstract

The application discloses a leaflet poplar PsEcd1 coding protein and application thereof in improving plant drought resistance. By cloning a leaflet poplar PsEcd1 gene, a plant expression vector is constructed and transformed into Arabidopsis and poplar, and it is found that the transgenic plants exhibit significant drought resistance enhancement under drought stress. Experimental results show that overexpression of the PsEcd1 gene significantly reduces the cell damage degree of the transgenic plants, improves the antioxidant enzyme activity, and enhances the water retention capacity and ROS clearance capacity of the plants. The transgenic Arabidopsis and poplar are superior to the wild type plants in physiological indexes such as root length, fresh weight, relative conductivity and relative water content under drought conditions. The application discloses an important role of the PsEcd1 gene in improving plant drought resistance, and provides important theoretical and practical basis for cultivating new drought-resistant transgenic varieties in the field of forest molecular breeding.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to Populus simonii PsEcd1 and its application in improving plant drought resistance. Background Technology

[0002] Populus simonii, a species of poplar endemic to my country, is widely distributed in northern my country and is an important forest germplasm resource. It is characterized by drought resistance, tolerance to poor soil, and strong adaptability, and is one of the main tree species used in the Three-North Shelterbelt Project. It plays a vital ecological and environmental protection role in the Yellow River Basin and possesses high ecological and economic value. Studying the physiological and ecological characteristics and molecular mechanisms of Populus simonii under drought conditions, and exploring its drought resistance mechanisms, is crucial for improving and utilizing arid regions, constructing and restoring the ecological environment, and enhancing economic benefits.

[0003] Ecdysoneless (Ecd) protein is an evolutionarily conserved protein, also known as Ecd1 and hEcd in humans. Its function is crucial for Drosophila embryonic development and yeast cell growth, but there is no research on Ecd in plants. Currently, Ecd functional research mainly focuses on mammals, including: (1) the biochemical function of hEcd protein, which is related to the regulation of the cell cycle and the control of the level and function of key tumor suppressor proteins. As a new promoter of mammalian cell cycle progression, Ecd's function is related to its ability to remove the inhibitory effect of Rb family tumor suppressor factors on E2F transcription factors; (2) Ecd is a new pre-mRNA splicing factor; (3) Ecd is a new tumor promoting factor, differentially expressed in pancreatic cancer, and may regulate glucose metabolism in cancer cells. Due to the conserved structural domain of Ecd, and its important oncogenic role in the development of breast tumors by promoting cell survival, it is speculated that the Ecd gene also plays an important role in regulating plant cell survival during drought resistance. Summary of the Invention

[0004] The technical problem to be solved by this invention is: how to use PsEcd1 derived from Populus simonii to improve the drought resistance of plants.

[0005] The technical solution of the present invention is: the use of the Populus simonii PsEcd1 gene in improving plant drought resistance, wherein the nucleotide sequence of the Populus simonii PsEcd1 gene is as shown in SEQ ID No.1 or a degenerate sequence encoding the same protein as SEQ ID No.1.

[0006] Furthermore, overexpression of the Populus simonii PsEcd1 gene in plants enhances their drought resistance.

[0007] Furthermore, the plants mentioned are Arabidopsis thaliana and poplar.

[0008] A method for cultivating drought-resistant plants includes the following steps:

[0009] (1) The gene fragment encoding the protein shown in SEQ ID No.2 was introduced into a plant expression vector to construct an overexpression vector;

[0010] (2) Genetically transform plants with the overexpression vector obtained in step (1);

[0011] (3) Select positive plants to obtain drought-resistant plants.

[0012] Furthermore, the nucleotide sequence of the gene fragment is shown in SEQ ID No. 1.

[0013] Furthermore, the genetic transformation method is Agrobacterium-mediated leaf disc method.

[0014] Furthermore, the plant is Arabidopsis thaliana or poplar.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] This invention, by observing cell damage and physiological indicators in PsEcd1 transgenic plants and wild-type plants under drought stress, found that the transgenic lines showed significantly less cell damage than the wild-type lines under drought stress. Simultaneously, the physiological indicators also confirmed that the PsEcd1 overexpressing lines exhibited significantly better stress resistance than the wild-type plants. This discovery reveals that PsEcd1 enhances plant resistance to drought stress, providing important theoretical and practical significance for cultivating drought-resistant transgenic varieties in the field of forestry molecular breeding. Attached Figure Description

[0017] Figure 1 The real-time quantitative PCR analysis provided in Example 2 of this invention was used to analyze the expression level of the PsEcd1 gene in Populus tomentosa under drought stress.

[0018] Figure 2 This is a schematic diagram of quantitative data on transgenic Arabidopsis expression provided in Example 4 of the present invention.

[0019] Figure 3 This is a schematic diagram showing the fresh weight and root length of transgenic Arabidopsis and wild-type Arabidopsis after drought treatment, as provided in Example 5 of the present invention. A shows the growth of WT and PsEcd1 transgenic Arabidopsis seedlings in 1 / 2 MS medium, B shows the growth of WT and PsEcd1 transgenic Arabidopsis seedlings in 1 / 2 MS medium containing 150 mM mannitol, C shows the effect of drought stress on the fresh weight of WT and PsEcd1 transgenic Arabidopsis, and D shows the effect of drought stress on the root length of WT and PsEcd1 transgenic Arabidopsis.

[0020] Figure 4 This is a phenotypic diagram of transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana in soil provided in Example 5 of the present invention.

[0021] Figure 5 This is a schematic diagram showing the relative electrical conductivity of transgenic Arabidopsis and wild-type Arabidopsis after drought treatment, as provided in Example 5 of the present invention.

[0022] Figure 6 This is a schematic diagram showing the relative water content of transgenic Arabidopsis and wild-type Arabidopsis after drought treatment, as provided in Example 5 of the present invention.

[0023] Figure 7 This is a schematic diagram of DAB and NBT staining on leaves of transgenic Arabidopsis and wild-type Arabidopsis after drought treatment, as provided in Example 5 of the present invention.

[0024] Figure 8 This is a schematic diagram showing the SOD content of leaves of transgenic Arabidopsis and wild-type Arabidopsis after drought treatment, as provided in Example 5 of the invention.

[0025] Figure 9 This is a schematic diagram of the POD content of leaves of transgenic Arabidopsis and wild-type Arabidopsis after drought treatment, as provided in Example 5 of the invention.

[0026] Figure 10 This is a schematic diagram of the CAT content in the leaves of transgenic Arabidopsis and wild-type Arabidopsis after drought treatment, as provided in Example 5 of the invention.

[0027] Figure 11 This is a schematic diagram of the quantitative data of transgenic poplar expression provided in Example 6 of the present invention.

[0028] Figure 12 The phenotypes of transgenic poplar and 84K poplar after drought treatment provided in Example 7 of this invention.

[0029] Figure 13 This is a comparison of the root lengths of transgenic poplar and 84K poplar after drought treatment, as provided in Example 7 of this invention.

[0030] Figure 14 This is a comparison of the number of main roots of transgenic poplar and 84K poplar after drought treatment, as provided in Example 7 of the present invention.

[0031] Figure 15 This is a comparison of the number of lateral roots of transgenic poplar and 84K poplar after drought treatment, as provided in Example 7 of the present invention. Detailed Implementation

[0032] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from commercial sources.

[0033] Example 1: Cloning of the PsEcd1 gene in Populus simonii

[0034] 1.1 Obtaining the target gene sequence

[0035] Based on the sequence numbers in *Populus hairy-fruited* from the Phytozome database, sequences corresponding to the coding region of the PsEcd1 gene in *Populus simonii* were selected. Primers were then designed using Primer 5 software based on the selected sequences, and the full-length gene was amplified by PCR. The primers are shown in Table 1.

[0036] Table 1 Cloning primers

[0037]

[0038] Using cDNA derived from total RNA extracted from Populus simonii leaves as a template, gene cloning was performed according to a high-fidelity enzyme PCR reaction system. The specific reaction system (20 μl) was as follows: PrimeSTAR, 10 μl; clone F, 1 μl; clone R, 1 μl; Populus simonii cDNA, 2 μl; ddH2O, 6 μl.

[0039] The PCR cloning reaction program was as follows: 95℃ for 5 minutes; 95℃ for 10 seconds, 57℃ for 30 seconds, 72℃ for 50 seconds, for 40 cycles; 72℃ for 2 minutes. DNA fragments were recovered using the Novizan DNA Product Purification Kit.

[0040] 1.2 Target gene ligation into T vector

[0041] The recovered target fragment was ligated to the T vector. The reaction system consisted of: T vector, 1 μl; the above DNA product, 30 ng; Solution I, 5 μl; and finally, ddH2O was added to a final volume of 10 μl. The ligation was carried out overnight at 16°C. After the ligation reaction was completed, the product was transformed into *E. coli*, and single colonies were selected for colony PCR verification. Positive colonies were then confirmed by sequencing. After sequencing verification, the nucleotide sequence is shown in SEQ ID No. 1. This gene fragment was named PsEcd1, consisting of 1929 bp bases, encoding 642 amino acids (SEQ ID No. 2).

[0042] Example 2: Analysis of the expression characteristics of the PsEcd1 gene in Populus simonii

[0043] 2.1 Primer Design

[0044] Primers for PsEcd1 quantitative analysis were designed using Primer3 software. The primers are shown in Table 2.

[0045] Table 2 Quantitative Primers

[0046]

[0047] 2.2 Drought stress treatment on Populus simonii tissue culture seedlings

[0048] Normally growing Populus simonii tissue culture seedlings were transferred to a medium containing 100 mM Mannitol for treatment. Total RNA was extracted from the roots and reverse transcribed into cDNA. A qRT-PCR reaction was then performed with the following reaction mixture (20 μl): SYBR qPCR Master Mix, 10 μl; Quantitative F, 0.4 μl; Quantitative R, 0.4 μl; Populus simonii cDNA, 2 μl; ddH2O, 7.2 μl.

[0049] The PCR reaction program was set as follows: 95℃ for 30 seconds, 95℃ for 10 seconds, 60℃ for 30 seconds, for 39 cycles, using the instrument's default melting curve acquisition program.

[0050] Analyze the obtained data, such as Figure 1 As shown in the figure. The results indicate that the expression level of the PsEcd1 gene in Populus simonii increases under drought stress, suggesting that this gene may be induced by drought stress.

[0051] Example 3: Construction of the Populus simonii PsEcd1 gene expression vector

[0052] 3.1 Primer Design

[0053] Plant overexpression vectors were constructed, and primers containing the pCAMBIA1302 homologous arm were designed. The primers are shown in Table 3.

[0054] Table 3 Overexpression Primers

[0055]

[0056] 3.2 Construction of pCAMBIA1302-PsEcd1 overexpression vector

[0057] The PsEcd1 gene was amplified using overexpression F and overexpression R primers. The product was recovered, and the pCAMBIA1302 vector was double-digested and linearized. The target gene was then constructed into the vector using Novigotase recombinase. The reaction mixture (10 μl) consisted of: target gene, 15 ng; vector, 105 ng; 5×CE II Buffer, 2 μl; Exnase II, 1 μl; and finally, ddH2O was used to bring the volume to 10 μl.

[0058] The reaction procedure is as follows: 37°C for 30 min, then cool to 4°C or immediately place on ice to cool.

[0059] The ligated vector was transformed into E. coli, and single-clone bacterial cultures were selected for colony PCR verification. Positive colonies were sequenced for testing, and finally, the pCAMBIA1302-PsEcd1 overexpression vector was constructed.

[0060] Example 4: Genetic transformation of the Arabidopsis thaliana PsEcd1 gene

[0061] The constructed pCAMBIA1302-PsEcd1 overexpression vector was transformed into Agrobacterium GV3101 using a heat shock method. The PsEcd1 gene was then transferred into Arabidopsis thaliana via Agrobacterium-mediated transformation, following these steps: Robust Arabidopsis thaliana plants, 4-6 cm tall, with numerous semi-open flower buds and a few pods, were selected after one month of cultivation. The cultivation temperature was 23-25℃, and the light intensity was 16 / 8h (day / night). The flower buds were inoculated with bacterial solution containing the pCAMBIA1302-PsEcd1 expression vector for 45-60 seconds, watered, and cultured in the dark for 12 hours. Afterward, the plants were placed under normal conditions. A second inoculation was performed using the same method after 7 days. Once the transformed pods matured, the seeds were collected and evenly spread on 1 / 2 MS solid medium containing antibiotics to screen for positive lines. Approximately 10 days later, seedlings growing normally on the screening medium were considered positive lines. After two true leaves emerged, the seedlings were transferred to soil for further cultivation. cDNA was extracted from leaves of rooted plants using the CTAB method and verified by PCR. RNA was extracted from 15 positive seedlings selected and transgene expression levels were verified by qRT-PCR. The results are as follows. Figure 2 As shown, OE-5 and OE-15 plants with appropriate expression levels were selected for subsequent phenotypic identification and analysis of drought resistance.

[0062] Example 5: Drought resistance analysis of Arabidopsis thaliana PsEcd1 transgenic plants

[0063] 5.1 Root length and fresh weight of transgenic Arabidopsis thaliana

[0064] Transgenic Arabidopsis thaliana seedlings from two lines, wild-type Arabidopsis thaliana WT and OE-5, OE-15, were cultured for 7 days on MS medium plates containing and without 150 mM mannitol (simulating drought). The results were as follows: Figure 3 As shown in the figure. The results showed that the root growth and fresh weight of transgenic Arabidopsis thaliana under Mannitol stress were much stronger than those of wild-type Arabidopsis thaliana. The root length and fresh weight of transgenic Arabidopsis thaliana had a significant effect on the difference between wild-type and transgenic Arabidopsis thaliana, indicating that the introduction of this gene into Arabidopsis thaliana can make the plants more drought-resistant and have a stronger growth advantage than wild-type plants.

[0065] 5.2 Phenotypic Analysis of Transgenic Arabidopsis

[0066] Two transgenic homozygous lines, OE-5 and OE-15, from the wild-type Arabidopsis thaliana WT and T4 generations, were cultured in normal MS medium for 10-13 days before being transferred to soil for growth. When the Arabidopsis seedlings reached 6-8 cotyledons, plants with similar growth vigor were selected. Figure 4 After 13 days of normal watering, the phenotypic characteristics of the Arabidopsis seedlings were observed, and no significant difference was found between the WT and OE-5 and OE-15 lines. At this point, the seedlings were subjected to drought treatment, watering was stopped, and all other conditions remained normal. Ten days later, the phenotypic characteristics of the Arabidopsis seedlings were observed, and it was found that the growth of the wild-type Arabidopsis plant WT was significantly weaker than that of the transgenic lines OE-5 and OE-15, and the yellowing of WT leaves was also more pronounced. After 10 days of drought, watering was resumed, and the phenotypic characteristics of the Arabidopsis plants were observed again after 3 days. It was found that the transgenic lines OE-5 and OE-15 had resumed growth, and the yellowing had lessened, while the WT plants showed no obvious signs of growth. This indicates that transgenic Arabidopsis has stronger drought tolerance than wild-type Arabidopsis. Figure 4 ).

[0067] 5.3 Determination of relative conductivity of transgenic Arabidopsis thaliana

[0068] Leaves from Arabidopsis thaliana under normal conditions and drought stress (section 5.2) were collected. The fresh leaves were cleaned and dried with filter paper. A perforator was then used to punch holes in the leaves, avoiding the main vein. Twenty leaf discs were randomly generated from each treatment. These discs were placed in a small beaker, and 50 mL of pure water was added. A vacuum pump was used to evacuate the beaker for 20 minutes. The conductivity of the liquid in the beaker was then measured and recorded as R1. The beaker was then heated in a boiling water bath for 20 minutes. After cooling to room temperature, the conductivity was measured again and recorded as R2. The formula for calculating the relative conductivity is as follows: Relative conductivity (%) = R1 / R2 × ​​100%. Results are as follows... Figure 5 As shown: Under normal treatment, there was no difference in relative electrical conductivity between the WT line and the PsEcd1 gene-transformed line. Under drought stress, the relative electrical conductivity of the PsEcd1 gene-overexpressing line was significantly lower than that of the WT line, indicating that these two lines were far more resistant to drought than the WT line. The experimental results demonstrate that the PsEcd1 gene plays a crucial role in drought resistance in Arabidopsis thaliana plants.

[0069] 5.4 Determination of relative water content in transgenic Arabidopsis thaliana

[0070] The relative moisture content of Arabidopsis thaliana leaves was determined using the drying and weighing method. First, leaves from each group of Arabidopsis thaliana samples in section 5.2 were collected. These fresh leaves were cleaned, and the moisture was absorbed with filter paper. The fresh weight of the leaves was recorded as FW. Then, the leaves were divided into two equal portions. One portion was placed in a paper bag and placed in an electrically heated drying oven at 100-105℃ for 15 minutes to achieve a constant moisture content. The oven temperature was then adjusted to 70-80℃ until the leaves reached constant weight, and this dry weight was recorded as DW. The other portion of leaves was transferred to pure water and left for 60 minutes until constant weight was achieved. The leaves were then removed, and the moisture was absorbed with filter paper. The saturated moisture content of the leaves was recorded as SFW. At this point, the formula for calculating the relative water content of Arabidopsis leaves can be derived as follows: Relative water content of leaves (%) = (FW - DW) / (SFW - DW) × 100%. The results show... Figure 6 Under normal conditions, the relative water content of wild-type Arabidopsis thaliana and transgenic Arabidopsis thaliana is not much different and remains at a high level. However, under drought conditions, the relative water content of plant leaves decreases, and the relative water content of wild-type Arabidopsis thaliana is even lower. This indicates that transgenic PsEcd1 enhances the water retention capacity of Arabidopsis thaliana, thus making transgenic Arabidopsis thaliana more drought-resistant.

[0071] 5.5 Analysis of transgenic Arabidopsis NBT and DAB

[0072] First, accurately weigh 1.97 g of Tris-HCl, dissolve and dilute to 250 mL, adjust the pH to 5.5, then add 0.25 g of 3,3'-diaminobenzidine (DAB) powder and mix well to prepare DAB staining solution (1 mg / mL). Next, mix 91.5 mL of PBS buffer A and 85 mL of PBS buffer B thoroughly, dilute to 400 mL, and add 0.2 g of Nitroblue Tetrazolium (NBT) powder and mix well to prepare NBT staining solution (0.5 mg / mL). Finally, add acetic acid, glycerol, and anhydrous ethanol in a 1:1:3 volume ratio to an Erlenmeyer flask, mix and shake well to prepare destaining solution.

[0073] Take a 50 ml test tube, place the leaf to be tested inside, and then add the staining solution to ensure the leaf is completely submerged. Vacuum the test tube to allow the leaf to sink to the bottom. Wrap the test tube with aluminum foil and place it in a constant temperature incubator set to 37 degrees Celsius for 6 to 8 hours for staining. After staining, remove the leaf with tweezers and transfer it to a destaining solution. Boil in a 100 degrees Celsius water bath for 5 minutes until the chlorophyll in the leaf is completely decolorized. After the leaf cools to room temperature, clean it with anhydrous ethanol and gently blot away excess moisture with filter paper to observe the color development of reactive oxygen species on the leaf. The results are as follows: Figure 7 As shown, under drought stress, DAB and NBT showed deeper coloring in 84K poplar, while PsEcd1 transgenic plants showed lighter coloring; under normal conditions, there was no difference.

[0074] 5.6 Enzyme Activity Analysis

[0075] Superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were all detected using a Solarbio reagent kit under UV spectrophotometry. Results are as follows: Figure 8 , 9 As shown in Figure 10, under normal conditions, the POD and SOD activities of PsEcd1 transgenic plants differed significantly from those of wild-type plants, while the CAT activity did not differ significantly. However, after drought stress, the POD, SOD, and CAT activities of transgenic plants were significantly higher than those of wild-type plants.

[0076] Example 6: Genetic transformation of the PsEcd1 gene in poplar trees

[0077] PsEcd1 was used to transform 84K poplar leaf discs via Agrobacterium-mediated transformation. PsEcd1-infected leaf discs were placed in adventitious bud induction medium (4.43 g / L MS + 0.5 mg / L 6-BA and 0.05 mg / L NAA) and co-cultured for 3 days in the dark at 22 ± 2℃. The co-cultured leaf discs were then transferred to a co-culture medium containing 3 mg / L Hygromycin B and 200 mg / L Timentin, and resistant adventitious buds were induced and screened under conditions of 23-25℃ and 16 / 8 h light / dark (day / night). After approximately 30 days of induction culture, the resistant adventitious buds were transferred to a rooting medium (1 / 2 MS basal medium supplemented with 0.05 mg / L IBA and 0.02 mg / L NAA) containing 3 mg / L Hygromycin B and 200 mg / L Timentin, until adventitious roots were induced. DNA was extracted from the leaves of rooted plants using the CTAB method and verified by PCR. RNA was extracted from the 20 positive seedlings selected, and the transgene expression level was verified by qRT-PCR. The results are as follows: Figure 11 As shown, OE-5, OE-6, and OE-7 plants with appropriate expression levels were selected for subsequent phenotypic identification and analysis of drought resistance.

[0078] Example 7: Drought resistance analysis of PsEcd1 transgenic poplar trees

[0079] Poplar trees with 84K rootstock and those transgenic with PsEcd1 were cultured in media containing 0 mM, 150 mM, and 200 mM Mannitol (simulating drought). Root length, number of taproots, and number of lateral roots were observed and recorded. Figure 12 , 13 As shown in Figures 14 and 15, under normal conditions, there were no significant differences in root length and taproot number between 84K poplar and PsEcd1 transgenic poplar. However, there were significant differences in the number of lateral roots between the PsEcd1 transgenic OE-5 plants and 84K poplar. At 150 mM Manntiol, the root length, taproot number, and lateral root number of the PsEcd1 transgenic poplar were significantly greater than those of 84K poplar; these differences were even more pronounced at 200 mM Manntiol. Compared to 84K poplar, the PsEcd1 transgenic poplar showed a significant increase in root length and taproot number under drought stress, indicating that the PsEcd1 transgenic poplar is more likely to survive drought conditions and is more drought-resistant.

[0080] In conclusion, plants transgenic with the PsEcd1 gene can participate in drought stress by maintaining a stable state of reactive oxygen species (ROS), thereby improving the plant's drought resistance.

Claims

1. Populus parvifolia PsEcd1 use of the gene in improving drought tolerance of plants, said Populus parvifolia PsEcd1 the nucleotide sequence of the gene is shown as SEQ ID No. 1 or a degenerate sequence encoding the same protein as SEQ ID No. 1; and the plant is Arabidopsis thaliana or Populus.

2. Use according to claim 1, characterized in that, By overexpressing Populus parvifolia PsEcd1 gene in plants, thereby increasing drought tolerance in plants.

3. A method for breeding drought tolerant plants, characterized in that, The method comprises the following steps: (1) introducing a gene fragment encoding the protein shown in SEQ ID No. 2 into a plant expression vector to construct an overexpression vector; (2) genetically transforming a plant with the overexpression vector obtained in step (1); (3) screening positive plants, thereby obtaining a drought-tolerant plant; The plant is Arabidopsis thaliana or a poplar.

4. The method of claim 3, wherein, The nucleotide sequence of the gene fragment is shown in SEQ ID No.

1.

5. The method of claim 3, wherein, The genetic transformation method is an Agrobacterium-mediated leaf disc method.

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