PsEcd1 encoded protein of populus simonii and application of PsEcd1 encoded protein in improvement of drought tolerance of plants

Abstract

The invention discloses populus simonii PsEcd1 encoding protein and application thereof in improving drought tolerance of plants. By cloning the populus simonii PsEcd1 gene, constructing a plant expression vector and transforming arabidopsis thaliana and poplar, it is found that a transgenic plant shows significant drought tolerance enhancement under drought stress. Experimental results show that overexpression of the PsEcd1 gene significantly reduces the cell damage degree of the transgenic plant, improves the antioxidant enzyme activity, and enhances the water retention capacity and ROS removal capacity of the plant. Physiological indexes such as root length, fresh weight, relative conductivity and relative water content of transgenic arabidopsis thaliana and poplar under drought conditions are superior to those of wild plants. The invention discloses an important role of the PsEcd1 gene in improving the drought resistance of plants, and provides an important theoretical and practical basis for cultivating new drought-resistant transgenic varieties in the field of forest molecular breeding.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and relates to Populus simonii PsEcd1 and application thereof in improving plant drought resistance. Background Art

[0002] Populus simonii is a species of the Populus genus endemic to my country. Widely distributed across northern my country, it is a key forest resource. Characterized by drought resistance, tolerance to infertility, and strong adaptability, it is a key tree species in the construction of my country's "Three Norths" shelterbelt system. It plays a crucial role in ecological and environmental protection 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 tolerance mechanisms, is crucial for improving and utilizing drought-prone areas, renovating and restoring ecological environments, and improving economic efficiency.

[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 Ecd has not been studied in plants. Currently, research on Ecd function is mainly focused on mammals, including: (1) the biochemical function of hEcd protein, which is related to cell cycle regulation and the control of the levels and functions of key tumor suppressor proteins. Ecd is a new promoter of mammalian cell cycle progression, and its function is related to its ability to remove the inhibitory effects of Rb family tumor suppressors on E2F transcription factors; (2) Ecd is a new pre-mRNA splicing factor; (3) Ecd is a new tumor-promoting factor that is differentially expressed in pancreatic cancer and may regulate glucose metabolism in cancer cells. Because Ecd has a conserved structural domain, Ecd plays an important oncogenic role in breast tumorigenesis 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 the present invention is: how to utilize PsEcd1 derived from Populus simonii to improve the drought resistance of plants.

[0005] The technical solution of the present invention is: 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, the drought tolerance of plants is improved by overexpressing the PsEcd1 gene of Populus simonii in plants.

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

[0008] A method for cultivating drought-tolerant plants comprises the following steps:

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

[0010] (2) genetically transforming the overexpression vector obtained in step (1) into plants;

[0011] (3) Screening positive plants to obtain drought-tolerant plants.

[0012] Furthermore, the nucleotide sequence of the gene fragment is shown as 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] By observing the cell damage and physiological indicators of PsEcd1 transgenic and wild-type plants under drought stress, the present study found that the transgenic lines experienced significantly less cell damage under drought stress than the wild-type lines. Physiological indicators also confirmed that the PsEcd1-overexpressing lines exhibited significantly better stress resistance than the wild-type plants. This finding, which reveals that PsEcd1 enhances plant resistance to drought stress, provides important theoretical and practical implications for the development of new drought-resistant transgenic varieties in the field of molecular forest breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The real-time fluorescence quantitative PCR provided in Example 2 of the present invention analyzed the expression level of the PsEcd1 gene in Populus simonii under drought stress.

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

[0019] Figure 3 Schematic diagram of the fresh weight and root length of transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana after drought treatment 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 thaliana, and D shows the effect of drought stress on the root length of WT and PsEcd1 transgenic Arabidopsis thaliana.

[0020] Figure 4 Schematic diagram of the phenotypes 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 of the relative electrical conductivity of transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana after drought treatment provided in Example 5 of the present invention.

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

[0023] Figure 7 Schematic diagram of DAB and NBT staining of leaves of transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana after drought treatment provided in Example 5 of the present invention.

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

[0025] Figure 9 This is a schematic diagram of the POD content in the leaves of transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana after drought treatment 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 thaliana and wild-type Arabidopsis thaliana 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 This is the phenotype of the transgenic poplar and 84K poplar after drought treatment provided in Example 7 of the present invention.

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

[0030] Figure 14 Comparison of the taproot numbers of the transgenic poplar and 84K poplar after drought treatment provided in Example 7 of the present invention.

[0031] Figure 15 Comparison of the number of lateral roots of the transgenic poplar and 84K poplar after drought treatment provided in Example 7 of the present invention. DETAILED DESCRIPTION

[0032] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

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

[0034] 1.1 Obtaining the target gene sequence

[0035] Based on the sequence numbers in the Phytozome database of Populus trichocarpa, we screened out the coding region sequences of genes homologous to Populus simonii PsEcd1. Then, we used Primer5 software to design primers based on the screened sequences and amplified the full length of the gene using PCR. The primers are shown in Table 1:

[0036] Table 1 Cloning primers

[0037]

[0038] Using cDNA reverse-transcribed from total RNA extracted from Populus simonii leaves as a template, gene cloning was performed using a high-fidelity 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°C for 5 minutes, 95°C for 10 seconds, 57°C for 30 seconds, and 72°C for 50 seconds, for 40 cycles, and 72°C for 2 minutes. DNA fragments were recovered using the Novezan DNA Product Purification Kit.

[0040] 1.2 Target gene connected to T vector

[0041] The recovered target fragment was ligated with a T-vector. The reaction system consisted of: 1 μl of T-vector; 30 ng of the aforementioned DNA product; and 5 μl of Solution I. Finally, the volume was adjusted to 10 μl with ddH₂O and the ligation was incubated overnight at 16°C. After the ligation reaction, the product was transformed into E. coli, and individual colonies were selected for PCR verification. Positive colonies were then confirmed by sequencing. Sequencing confirmed the nucleotide sequence shown in SEQ ID No. 1. The gene fragment was named PsEcd1, consisting of 1929 base pairs (bp) encoding 642 amino acids (SEQ ID No. 2).

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

[0043] 2.1 Primer design

[0044] Primer3 software was used to design PsEcd1 quantitative primers for expression pattern analysis. The primers are shown in Table 2:

[0045] Table 2 Quantitative primers

[0046]

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

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

[0049] The PCR reaction program was set as follows: 95°C for 30 seconds, 95°C for 10 seconds, and 60°C for 30 seconds, for 39 cycles, and the instrument default melting curve acquisition program was used.

[0050] The obtained data were analyzed, such as Figure 1 The results showed that under drought stress, the expression level of PsEcd1 gene in Populus simonii increased, so it was speculated that this gene might be induced by drought stress.

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

[0052] 3.1 Primer design

[0053] To construct a plant overexpression vector, primers with homology arms to pCAMBIA1302 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 primers Overexpression F and Overexpression R. The product was recovered, and the pCAMBIA1302 vector was double-digested and linearized. The target gene was then constructed into the vector using Novion's native recombinase. The reaction system (10 μl) was as follows: target gene, 15 ng; vector, 105 ng; 5× CE II Buffer, 2 μl; Exnase II, 1 μl; and finally, ddH2O was added to bring the volume to 10 μl.

[0058] The reaction procedure was: 37°C for 30 min and then cooled to 4°C or immediately placed on ice for cooling.

[0059] The ligated vector was transformed into Escherichia coli, and the single clone bacterial solution was picked for bacterial solution PCR verification and positive colony sequencing test, and finally the pCAMBIA1302-PsEcd1 overexpression vector was constructed.

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

[0061] The constructed pCAMBIA1302-PsEcd1 overexpression vector was transformed into Agrobacterium GV3101 via heat shock. The PsEcd1 gene was transformed into Arabidopsis thaliana via Agrobacterium-mediated transformation as follows: One-month-old robust Arabidopsis thaliana plants, 4-6 cm tall, with numerous partially opened flower buds and very few fruit pods, were selected. The culture temperature was 23-25°C and the light intensity was 16 / 8 hours (day / night). The flower buds were infected with a bacterial solution containing the pCAMBIA1302-PsEcd1 expression vector for 45-60 seconds, watered using a watering can, and incubated under shade for 12 hours. The plants were then cultured under normal conditions. Seven days later, the plants were infected again using the same method. After the transformed fruit pods matured, the seeds were collected and evenly plated on 1 / 2 MS solid medium containing antibiotics to select positive strains. Approximately 10 days later, seedlings that grew normally on the culture medium were identified as positive strains. After growing two true leaves, the seedlings were transferred to soil for culture. cDNA was extracted from the leaves of the rooted plants using the CTAB method and verified by PCR. RNA was extracted from the 15 positive seedlings screened and the transgenic expression was verified by qRT-PCR. 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 Analysis of drought resistance of Arabidopsis PsEcd1 transgenic plants

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

[0064] The wild-type Arabidopsis WT and transgenic Arabidopsis thaliana strains OE-5 and OE-15 were cultured on MS medium plates without or with 150 mM mannitol (simulating drought) for 7 days. Figure 3 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. There was a significant difference in root length and fresh weight between transgenic and wild-type Arabidopsis thaliana, indicating that the transfer of this gene into Arabidopsis thaliana can make the plant more drought-resistant and have a stronger growth advantage than wild-type plants.

[0065] 5.2 Phenotypic analysis of transgenic Arabidopsis

[0066] The wild-type Arabidopsis thaliana WT and two transgenic homozygous lines OE-5 and OE-15 of the T4 generation were cultured in normal MS medium for 10-13 days and then transferred to soil for growth. When the Arabidopsis seedlings grew to 6-8 cotyledons, plants with basically the same growth were selected. Figure 4 . After growing for 13 days under normal watering conditions, the phenotype of the Arabidopsis seedlings was observed after 13 days, and it was found that there was no significant difference between the WT and OE-5 and OE-15 lines at this time. At this time, the Arabidopsis seedlings were drought treated, watering was stopped, and the other conditions were normal. After 10 days, the phenotype of the Arabidopsis seedlings was 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 degree of yellowing of the WT leaves was also stronger. After 10 days of drought, watering was resumed, and the phenotype of the Arabidopsis plants was continued to be observed after 3 days. It was found that the transgenic lines OE-5 and OE-15 had resumed growth, and the yellowing phenomenon was weakened, while the WT plants showed no obvious signs of growth. This shows that transgenic Arabidopsis has stronger drought resistance than wild-type Arabidopsis ( Figure 4 ).

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

[0068] Take the plant leaves under normal conditions and drought stress in 5.2, clean these collected fresh leaves, and use filter paper to absorb the moisture on the Arabidopsis leaves, then take out the puncher to punch holes in the Arabidopsis leaves, and be careful to avoid the main veins when punching. Randomly punch out twenty leaf discs for each treatment, put these leaf discs into a small beaker, and add 50mL of pure water to the small beaker, take out the vacuum pump, and put the beaker in for vacuuming. After a total of 20 minutes, measure the conductivity of the liquid in the beaker at this time, and record it as R1. Put the small beaker into a boiling water bath and heat it for 20 minutes. After it cools to room temperature, measure its conductivity again and record it as R2. The calculation formula for relative conductivity can be obtained as follows: Relative conductivity (%) = R1 / R2×100%. The results are as follows. Figure 5 As shown in the results, under normal conditions, there was no difference in relative conductivity between the WT and PsEcd1-transgenic lines. However, under drought stress, the PsEcd1-overexpressing lines had significantly lower relative conductivity than the WT, indicating that these two lines are much more resistant to stress than the WT. These results suggest that the PsEcd1 gene plays a crucial role in drought resistance in Arabidopsis plants.

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

[0070] The relative water content of Arabidopsis leaves was determined using the oven-drying weighing method. First, collect leaves from each group of Arabidopsis samples described in 5.2. These fresh leaves were cleaned and dried with filter paper. The fresh weight of the leaves was measured using an electronic scale and recorded as FW. The leaves were then divided into two equal portions. One portion was placed in a paper bag and placed in an electric forced-air drying oven for 15 minutes at a temperature of 100-105°C. The oven temperature was then adjusted to 70-80°C, and the leaves were dried to a constant weight. The dry weight at this point was measured using an electronic scale and recorded as DW. The other portion of leaves was placed in pure water and allowed to stand for 60 minutes. After the leaves reached a constant weight, the leaves were removed and dried with filter paper. The saturated water content (SFW) was measured using an electronic scale. At this point, the calculation formula for the relative water content of Arabidopsis leaves can be obtained as follows: relative water content of leaves (%) = (FW-DW) / (SFW-DW)×100%. The results show that Figure 6 Under normal conditions, the relative water content of wild-type Arabidopsis and transgenic Arabidopsis is not much different and remains at a high level. However, under drought conditions, the relative water content of the plant leaves decreases, and the relative water content of the wild-type Arabidopsis is even lower, indicating that the transgenic PsEcd1 enhances the water retention capacity of Arabidopsis, thereby making the transgenic Arabidopsis more drought-resistant.

[0071] 5.5 NBT and DAB analysis of transgenic Arabidopsis

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

[0073] Take a 50 ml test tube and place the leaves to be tested in it, then add the staining solution to ensure that the leaves are completely submerged. Vacuum the test tube to make the leaves sink to the bottom. Wrap the test tube with tin foil and place it in a constant temperature incubator set at 37 degrees Celsius for 6 to 8 hours of staining. After the staining process is completed, use tweezers to remove the leaves and move them to the decolorizing solution. Boil in a water bath at 100 degrees Celsius for 5 minutes until the chlorophyll in the leaves is completely decolorized. After the leaves have cooled to room temperature, clean them with anhydrous ethanol and gently absorb excess water from the surface of the leaves with filter paper to observe the color development of active oxygen on the leaves. The results are as follows Figure 7 As shown in the figure, under drought stress, the coloring effect of DAB and NBT on 84K poplar was darker, while the coloring effect on PsEcd1 transgenic plants was lighter, and there was no difference under normal conditions.

[0074] 5.6 Enzyme activity analysis

[0075] Superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were detected using a Solebol kit under a UV spectrophotometer. Figure 8 、 9 As shown in Figures 10 and 11, under normal conditions, the POD and SOD activities of PsEcd1 transgenic plants were significantly different from those of wild-type plants, while the CAT activity was not significantly different from that of wild-type plants. 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 Populus simonii PsEcd1 gene in poplar

[0077] PsEcd1 was transformed into 84K poplar leaf discs via Agrobacterium-mediated transfection. PsEcd1-infected leaf discs were placed in adventitious shoot induction medium (4.43 g / L MS + 0.5 mg / L 6-BA and 0.05 mg / L NAA) and co-cultured at 22 ± 2°C in the dark for 3 days. The co-cultured leaf discs were then transferred to co-culture medium containing 3 mg / L Hygromycin B and 200 mg / L Timentin. Resistant adventitious shoots were induced and selected at 23–25°C under a 16 / 8 h light cycle (day / night). After approximately 30 days of induction, resistant shoots were transferred to rooting medium (1 / 2 MS minimal 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 from the leaves of rooted plants was extracted using the CTAB method and verified by PCR. RNA was extracted from the 20 positive seedlings screened, and the transgenic expression level was verified by qRT-PCR. 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 Analysis of Drought Resistance of Populus simonii PsEcd1 Transgenic Poplars

[0079] 84K poplar and PsEcd1-transfected poplar plants were cultured in medium containing 0mM, 150mM, and 200mM Mannitol (mannitol simulates drought), and the root length, main root number, and lateral root number 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 transgenic PsEcd1 poplars. However, there was a significant difference in lateral root number between PsEcd1 transgenic OE-5 plants and 84K poplars. At 150 mM Manntiol, the root length, taproot number, and lateral root number of transgenic PsEcd1 poplars were significantly greater than those of 84K poplars. The differences were even more pronounced at 200 mM Manntiol. Compared to 84K poplar, transgenic PsEcd1 poplars showed a significant increase in root length and taproot number under drought stress, indicating that transgenic PsEcd1 poplars are more likely to survive drought and are more drought-resistant.

[0080] In summary, plants transgenic for PsEcd1 can participate in drought stress by maintaining a stable state of reactive oxygen species (ROS), thereby improving the drought tolerance of plants.

Claims

1. Use of the Populus simonii PsEcd1 gene in improving plant drought tolerance, 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.

2. The use according to claim 1, characterized in that By overexpressing the PsEcd1 gene in plants, the drought tolerance of plants is improved.

3. The use according to claim 1 or 2, characterized in that The plants are Arabidopsis thaliana and poplar.

4. A method for cultivating drought-tolerant plants, characterized in that: The steps include: (1) introducing the gene fragment encoding the protein shown in SEQ ID No. 2 into a plant expression vector to construct an overexpression vector; (2) genetically transforming the overexpression vector obtained in step (1) into plants; (3) Screening positive plants to obtain drought-tolerant plants.

5. The method according to claim 4, characterized in that The nucleotide sequence of the gene fragment is shown in SEQ ID No.

1.

6. The method according to claim 4, characterized in that The genetic transformation method is: Agrobacterium-mediated leaf disc method.

7. The method according to any one of claims 4 to 6, characterized in that The plant is Arabidopsis thaliana or poplar.

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