Application of Japanese eelgrass ZjPIP1 gene in improving plant stress resistance

Abstract

The present invention discloses Japanese eelgrass ZjPIP1 The application of genes in improving plant stress resistance belongs to the field of genetic engineering technology. ZjPIP1 The nucleic acid sequence of the gene is shown in SEQ ID No. 1. ZjPIP1 The plant expression vector of the gene and its transformation into Arabidopsis thaliana can significantly improve the resistance of Arabidopsis thaliana to high temperature stress, drought stress and high salt stress. ZjPIP1 Genes have important application prospects in improving plant stress resistance.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology, and specifically relates to Japanese eelgrass ZjPIP1 Application of genes in improving plant stress resistance. Background Art

[0002] Organisms in their natural environments often face various adverse conditions (such as drought, high salinity, and high temperatures) that can inhibit their growth or even lead to their death. With the continuous deterioration of the environment, adverse stresses such as high salinity have become a global problem.

[0003] Rapidly advancing genetic engineering technologies are providing new avenues for genetic improvement. Japanese eelgrass, a key support species for seagrass beds, is widely distributed in coastal intertidal zones. It is susceptible to abiotic stress fluctuations such as water loss, increased salinity, and rising temperatures, making it an excellent candidate for identifying genes that confer resistance to abiotic stresses. Cloning genes associated with stress resistance from Japanese eelgrass and genetically transforming them into plants could pave the way for breeding new species with diverse resistance traits. Summary of the Invention

[0004] In view of the problems existing in the prior art, the present invention aims to provide Japanese eelgrass ZjPIP1 Application of genes in improving plant stress resistance.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] Japanese eelgrass ZjPIP1 Application of genes in improving plant stress resistance, Japanese eelgrass ZjPIP1 The nucleic acid sequence of the gene is shown in SEQ ID No.1.

[0007] On the basis of the above scheme, by increasing the Japanese eel grass in plants ZjPIP1 The expression level of genes can improve the resistance of plants to adverse stress.

[0008] On the basis of the above scheme, by constructing Japanese eel grass ZjPIP1 Plant expression vectors of genes, transforming plants to express them in plants, and improving the expression of genes in plants ZjPIP1 Gene expression level.

[0009] On the basis of the above scheme, the method of transforming plants is one of the following methods: Agrobacterium-mediated method, gene gun method, electric shock method, PEG method, and liposome method.

[0010] Based on the above scheme, the Agrobacterium is Agrobacterium tumefaciens or Agrobacterium rhizogenes.

[0011] Based on the above scheme, the stress resistance is the ability to resist high temperature stress, high salt stress and / or drought stress.

[0012] Based on the above scheme, the plant is Japanese eelgrass or Arabidopsis thaliana.

[0013] Advantages of the technical solution of the present invention:

[0014] The present invention cloned a gene related to plant stress resistance from Japanese eelgrass ZjPIP1 , build ZjPIP1 The plant expression vector of the gene was transformed into Arabidopsis; the results showed that overexpression ZjPIP1 The morphology and development of Arabidopsis plants expressing the gene are normal. Under adverse stress, overexpression of ZjPIP1 The growth of Arabidopsis thaliana with overexpression of the gene was significantly better than that of wild-type Arabidopsis; ZjPIP1 Arabidopsis seedlings with the gene can at least withstand high temperature stress of 30°C, high salt stress of 150 mM NaCl, or mannitol stress of 200 mM. ZjPIP1 Genes have important application prospects in improving plant stress resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Japanese eelgrass after high salt and high temperature stress ZjPIP1 Changes in gene expression (the left figure shows high temperature stress, and the right figure shows high salt stress);

[0016] Figure 2 Columbia wild-type Arabidopsis thaliana under normal conditions and overexpression ZjPIP1 The growth of Arabidopsis seedlings with the gene;

[0017] Figure 3 After high temperature stress treatment, wild-type Arabidopsis thaliana and overexpression ZjPIP1 The growth of Arabidopsis seedlings with the gene;

[0018] Figure 4 After drought stress treatment, wild-type Arabidopsis thaliana and overexpressing ZjPIP1 The growth of Arabidopsis thaliana seedlings with the gene;

[0019] Figure 5 After high salt stress treatment, wild-type Arabidopsis thaliana and overexpressing ZjPIP1 The growth of Arabidopsis seedlings with the gene;

[0020] Figure 6 Under stress conditions, wild-type Arabidopsis thaliana and overexpressing ZjPIP1 Observation of Arabidopsis plant phenotypes of the gene;

[0021] Figure 7Under stress conditions, wild-type Arabidopsis thaliana and overexpressing ZjPIP1 The water content of Arabidopsis leaf tissue with the gene is the percentage of its saturated water content;

[0022] Figure 8 Under stress conditions, wild-type Arabidopsis thaliana and overexpressing ZjPIP1 NBT and DAB staining results of Arabidopsis leaves of genes;

[0023] Figure 9 Under stress conditions, wild-type Arabidopsis thaliana and overexpressing ZjPIP1 H2O2 accumulation in Arabidopsis leaves of the gene;

[0024] Figure 10 Under stress conditions, wild-type Arabidopsis thaliana and overexpressing ZjPIP1 Catalase activity in Arabidopsis leaves of the gene;

[0025] Figure 11 Under stress conditions, wild-type Arabidopsis thaliana and overexpressing ZjPIP1 Genes for Arabidopsis leaf MDA content. DETAILED DESCRIPTION

[0026] The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. Below, in conjunction with specific examples, the present invention will be further described in detail with reference to data. The following examples are merely for illustration of the present invention and are not intended to limit the scope of the present invention in any way.

[0027] The experimental methods in the following examples, unless otherwise specified, are all conventional methods and are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. The experimental materials, reagents, and drugs used in the following examples, unless otherwise specified, can all be purchased through general channels.

[0028] In the following embodiments:

[0029] Japanese eelgrass was provided by the Marine Ecology Laboratory, College of Marine Life Sciences, Ocean University of China;

[0030] Escherichia coli DH5α was purchased from Shanghai Ruixing Gene Technology Co., Ltd.;

[0031] The plant expression vector Super1300 was purchased from Weike (Qingdao) Biotechnology Co., Ltd.;

[0032] Agrobacterium strain GV3101 was purchased from Weike (Qingdao) Biotechnology Co., Ltd.;

[0033] Columbia wild-type Arabidopsis thaliana was purchased from Weike (Qingdao) Biotechnology Co., Ltd.

[0034] Example 1

[0035] ZjPIP1 Gene cloning

[0036] Total RNA was extracted from Japanese eelgrass and reverse transcribed into cDNA as a template. ZjPIP1 -F and ZjPIP1 -R) for PCR amplification; cloned ZjPIP1 The nucleic acid sequence of the gene is shown in SEQ ID No. 1, and the amino acid sequence encoded by the gene is shown in SEQ ID No. 2.

[0037] The PCR reaction system is as follows: 2 μL cDNA, 25 μL 2× Buffer, 4 μL 10 pM dNTPs, 2 μL each of 10 μM forward and reverse primers, 0.5 μL 5 U / μL PrimerSTAR HS DNA Polymerase, and 14.5 μL ddH2O. Add the sample and mix thoroughly on ice.

[0038] PCR reaction conditions were as follows: 98°C for 3 min; 98°C for 5 sec, 56°C for 15 sec; 72°C for 30 sec, 35 cycles; 72°C for 5 min.

[0039] SEQ ID No.1 (5'→3'):

[0040] ATGGACACCAAACTCGAGAGTTCATCGGCGCCGGAGATGGAGAGAGAACATGGAGCGGAAAAAGAGTACACCGATCCCGAAGCAACTCCATGGATCGATGTGAGGGAACTCAATTGTTGGCCCTTGTACCGTGCCGTGATCACCGAGTTTGTGGCAACAATGGTTTTCCTTTACATCGCCGTGACTGCTTTAGTCGGGTACAAAAATGAATCAAGCAAACACAACAACATAGGTCTGCTTGGCATCGCATGGGTGTTCGGAGGCATGATCTTCGTGTTAGTCTATTGCACCGCCGGTATCTCGGGAGGTCATCTCAACCCAGCAGTTACCTTCGGTATGCTTATCGCACGGAAGTGCTCTCTTCCGCGCGCCTTGATGTACGTGATAGCGCAGTGTTTGGGTGCTGTTTGCGGGGTTGGTTTCGTCAAAGTCACCTCCGGAGTAGACATCTACCGTTCCGTCGGAGGGGGTGCCAATTCAGTGTCAGATGGGTACACTAATGGTTCTGCCCTCGGAGCGGAGATTCTGGGGACTTTCGTTCTAATTTTCACCATTTTCTCGGCGACGGATCCCAAGAGAAAAGACAGAGACTCTCACGTCCCGACTCTCGCACCGCTGGTGATAGGGTTCGCTGTCTTCATGGTGCACTTAGGTACGATTAGTATCGGAGGAACGGGTATTAATCCGGCGAGGAGCTTTGGAGCGGCTGTTGTTTACAATAATCACCAGGCTTGGAGTGAACTTTGGATCTTTGTTGTCGGACCGTTGCTTGGATCGCTTATAGCTACTTCATTCCACCAGCTTGTCATCCGGGCATCCAATACCAAAAAGATCGTCAATTCTTTTGCGAGTCCATGA

[0041] SEQ ID No.2:

[0042] MDTKLESSSAPEMEREHGAEKEYTDPEATPWIDVRELNCWPLYRAVITEFVATMVFLYIAVTALVGYKNESSKHNNIGLLGIAWVFGGMIFVLVYCTAGISGGHLNPAVTFGMLIARKCSLPRALMYVIAQCLGAVCGVGFV KVTSGVDIYRSVGGGANSVSDGYTNGSALGAEILGTFVLIFTIFSATDPKRKDRDSHVPTLAPLVIGFAVFMVHLGTISIGTGINPARSFGAAVVYNNHQAWSELWIFVVGPLLGSLIATSFHQLVIRASNTKKIVNSFASP

[0043] described ZjPIP1 -F and ZjPIP1 -R primer sequences are shown in SEQ ID No. 3 and SEQ ID No. 4.

[0044] ZjPIP1 -F: 5'-ATGGACACCAAACTCGAGAGT-3' (SEQ ID No. 3);

[0045] ZjPIP1 -R: 5'-TCATGGACTCGCAAAAGAATT-3' (SEQ ID No. 4).

[0046] Example 2

[0047] Japanese eelgrass after high salt and high temperature stress ZjPIP1 Changes in gene expression

[0048] High-salinity stress treatment: Natural seawater was used as the substrate, and either purified water or different concentrations of NaCl (99.5% analytical grade, Sinopharm, China) were added to artificially adjust the salinity of the culture environment. Three salinities were set for the stress experiment: 30‰ (CK30), 50‰ (HS50), and 70‰ (HS70). Because the salinity of natural seawater ranges from 29‰ to 32‰, the 30‰ (CK30) group served as the control. Japanese eelgrass was cultured under the high-salinity stress test system for 5 days at a temperature of 20±2°C.

[0049] High temperature stress treatment: Using natural seawater as the base, Japanese eelgrass was cultured at 20℃, 30℃, and 30℃ environments, with 20℃ set as the control group. Japanese eelgrass was cultured for 7 hours and the culture salinity was 30‰.

[0050] The roots and rhizomes of Japanese eelgrass were covered with cleaned and disinfected gravel and planted in transparent PVC pots. The pots were evenly spaced within glass containers containing seawater of varying salinity and temperature. Ten plants of similar growth were placed in each pot. The gravel used for planting was washed twice with seawater of the appropriate salinity and temperature before use. The high-salinity stress treatment group required the replacement of seawater and pots every two days, and the cleaning of the gravel and culture equipment to prevent nutrient deficiencies or excessive growth of epiphytes that could affect normal seagrass growth.

[0051] Total RNA was extracted from Japanese eelgrass leaves after high salt and high temperature stress using a kit, and the sample quality and integrity were assessed using an Agilent 2100 bioanalyzer and agarose gel electrophoresis. mRNA in Japanese eelgrass samples was enriched using Oligo (dT) magnetic beads, fragmented, and after adding primers, the enriched mRNA was reverse transcribed into cDNA using the NEB #7530 kit (New England Biolabs, USA). The cDNA was purified using AMPure XP Beads to obtain a cDNA library. The cDNA library was sequenced by Illumina Novaseq6000, and the raw data was filtered by FASTQ to obtain high-quality Clean Reads. The Clean Reads were aligned with the Japanese eelgrass genome assembled earlier by our research group using alignment software, and StringTie and RSEM software were used to reconstruct transcripts and calculate the FPKM value (fragment per kilobase of transcript per million mapped reads) of the transcripts. Statistics ZjPIP1 The FPKM value of the gene is shown in Figure 1 .

[0052] Depend on Figure 1 It can be seen that ZjPIP1 The expression level of the gene in the high salt and high temperature stress treatment groups was significantly higher than that in the control group, indicating that its expression is related to abiotic stress resistance.

[0053] Example 3

[0054] ZjPIP1 Construction of gene plant expression vector and acquisition of recombinant strains

[0055] Using the cDNA of Japanese eelgrass as a template, SacI and XbaI restriction sites (P1 and P2) were added to the upstream and downstream primers respectively. The coding region of the ZjPIP1 gene with SacI and XbaI restriction sites at both ends was amplified by PCR. The PCR product was recovered and ligated with the cloning vector pMD18-T (purchased from TaKaRa) under the action of T4 DNA ligase. The ligation product was transformed into Escherichia coli DH5α to obtain ampicillin-resistant colonies. The recombinant plasmid was extracted and double-digested with SacI and XbaI to recover the recombinant plasmid containing ZjPIP1 The enzyme-cut fragment of the gene coding sequence was cloned into the corresponding enzyme cutting site of the plant expression vector Super1300 to obtain the plant expression vector Super1300- ZjPIP1 .

[0056] The primers used were.

[0057] P1: 5'-ctagaggatccccggATGGACACCAAACTCGAGAGTTC-3' (SEQ ID No. 5);

[0058] P2: 5'-gatcggggaaattcgTCATGGACTCGCAAAAGAATTG-3' (SEQ ID No. 6).

[0059] Super1300- ZjPIP1 Transformation of recombinant plasmid into Agrobacterium strain using liquid nitrogen freeze-thaw method GV3101 Competent cells are used to screen out recombinant strains containing recombinant plasmids.

[0060] Example 4

[0061] Construct overexpression ZjPIP1 Arabidopsis thaliana

[0062] (1) A single colony of the recombinant strain constructed in Example 3 was selected and inoculated into LB (rifampicin 50 mg / L, kanamycin 50 mg / L) liquid medium. The culture was cultured at 28°C and 180 rpm until OD600 = 0.5-0.8. Then, 2 mL of the bacterial solution was transferred to 50 mL of LB (rifampicin 50 mg / L, kanamycin 50 mg / L) medium and cultured until OD600 = 0.6-0.8. The bacterial solution was centrifuged at 5000 rpm for 15 min and then suspended in the same volume of liquid 1 / 2 MS (0.02% Silwet L-77) for later use.

[0063] (2) Soak Arabidopsis seeds in 1% NaClO for 5 minutes, rinse with sterile water 4-6 times, and plant them on the substrate soil.

[0064] (3) Select healthy Arabidopsis plants in the early fruiting stage and place them upside down with their pots on top of a container containing the Agrobacterium suspension prepared in step (1). Immerse the entire inflorescence in the Agrobacterium suspension for 20-30 seconds, taking care to minimize contact of the leaves with the suspension. Remove the pots and place them horizontally in a dark box for approximately 24 hours, maintaining a constant humidity (relative humidity 60%). After 24 hours, place the treated Arabidopsis plants under light conditions of 22-25°C to allow normal growth and harvest the seeds.

[0065] (4) Inoculate the harvested transgenic Arabidopsis seeds into 20 mL MS (hygromycin 30 mg / L) culture medium and culture at 22°C for about 1 week. Select the bright green and healthy Arabidopsis seedlings and transplant them into the matrix soil. Extract the genomic DNA of the transgenic plants and design primers using the above vector sequence for PCR amplification. The correct identification is the overexpression ZjPIP1 genes of Arabidopsis thaliana.

[0066] The PCR reaction program was as follows: 95°C, 5 min; 95°C, 50 s, 55°C, 50 s, 72°C, 1 min, 32 cycles; 72°C, 10 min.

[0067] The primers for identifying transgenic plants were

[0068] P3: 5'-TTTCCTTTACATCGCCGTGAC-3' (SEQ ID No. 7);

[0069] P4: 5'-GCAAACAGCACCCAAACACT-3' (SEQ ID No. 8).

[0070] (5) Seeds of T1 plants identified as positive by PCR were harvested and inoculated into 20 mL MS (30 mg / L hygromycin) culture medium. The ratio of positive plants to negative plants in each line was approximately 3:1. Arabidopsis seedlings that could grow normally and robustly on the screening culture medium were selected, transplanted into substrate soil, and seeds were harvested from each plant to obtain T2 transgenic plants. Transgenic Arabidopsis seeds harvested from each plant were inoculated into 20 mL MS (30 mg / L hygromycin) culture medium. Transgenic plants whose seeds could germinate and grow on the screening culture medium were selected as homozygous lines, transplanted into substrate soil, and seeds were harvested from each plant to obtain homozygous T3 transgenic plants.

[0071] Example 5

[0072] Overexpression ZjPIP1 Stress resistance detection of Arabidopsis thaliana

[0073] The overexpression obtained by the method of Example 3 ZjPIP1The seeds of Arabidopsis thaliana with the gene and the wild-type Arabidopsis thaliana were inoculated in 1 / 2MS medium for germination. One week later, the seedlings were transferred to 1 / 2MS medium containing 150 mM NaCl (simulating high salt stress) or 200 mM mannitol (simulating drought stress) at 22°C. The growth of the seedlings was observed for 2 weeks.

[0074] The overexpression obtained by the method of Example 3 ZjPIP1 The seeds of Arabidopsis thaliana with the gene and the wild-type Arabidopsis thaliana were inoculated in 1 / 2MS medium for germination. After 1 week, the seedlings were transferred to 1 / 2MS medium at a culture temperature of 30°C (simulating high temperature stress) and cultured for 2 weeks, and the growth of the seedlings was observed.

[0075] The results are as follows Figures 2 to 5 As shown, overexpression under high temperature, high salt or drought stress conditions ZjPIP1 The growth of Arabidopsis seedlings (OE1, OE2, OE3) with the overexpressed gene was significantly better than that of the wild-type Arabidopsis seedlings (WT). ZjPIP1 Arabidopsis seedlings expressing the gene can tolerate at least 150 mM NaCl, 200 mM mannitol and a high temperature of 30°C.

[0076] Arabidopsis seedlings of various strains germinated on MS medium were transplanted into a mixture of nutrient soil and vermiculite (3:1) for further cultivation. Arabidopsis plants of various strains were cultured normally (watered), subjected to salt stress (irrigated with 150 mM NaCl), high temperature stress (30°C), or drought stress (without watering). Phenotypic observations were performed after 7 days, and leaves were collected for testing of physiological and biochemical indicators. Figure 6 As shown, overexpression under adverse stress conditions ZjPIP1 The growth of Arabidopsis plants with the genes (OE1, OE2, OE3) was also significantly better than that of wild-type Arabidopsis seedlings (WT).

[0077] Overexpression ZjPIP1 Water content detection in genetically modified Arabidopsis thaliana

[0078] Overexpression of the above-mentioned high salt stress, high temperature stress and drought stress treatment ZjPIP1 The leaves of Arabidopsis thaliana plants expressing the gene were used as materials to determine the overexpression of ZjPIP1 The dry and fresh weights and relative water content (RWC) of Arabidopsis thaliana with the gene and the wild-type Arabidopsis thaliana of Columbia were measured. The fresh weight of the leaves or aerial parts of Arabidopsis thaliana cultured normally or after stress treatment was weighed and recorded as W F Immerse the leaves completely in distilled water for 36-48 hours until they fully absorb water, wipe the surface moisture of the leaves dry and weigh their mass, record it as W T; Transfer the leaves or above-ground parts into a forced air drying oven and dry them at 80℃ until constant weight is reached. Weigh the dry mass and record it as W D According to the formula: RWC = (W F -W D ) / (W T -W D ) × 100%, calculate the percentage of leaf tissue water content to its saturated water content. The result is as follows Figure 7 As shown in the figure, after high temperature stress, high salt stress, and drought stress, the water content of the transgenic Arabidopsis lines was 1.36 times, 1.43 times, and 1.42 times that of the non-transgenic control, respectively.

[0079] Overexpression ZjPIP1 Physiological index detection of genetic Arabidopsis

[0080] To detect overexpression after stress treatment ZjPIP1 The accumulation of reactive oxygen species in leaves of the genetic Arabidopsis lines and the Columbia wild-type Arabidopsis control was determined by staining with NBT and DAB. Figure 8 As shown in the NBT staining results, the more blue the leaves are, the more O2 - The more the accumulation, the less blue part of the leaves of the transgenic lines than the non-transgenic control (WT), indicating that O2 - The accumulation amount is small; in the DAB staining results, the more brown part of the leaf, the more H2O2 accumulation; the brown part of the leaf of the overexpression line is less than that of the wild-type control (WT), indicating that the H2O2 accumulation amount is small. The H2O2 accumulation amount, H2O2 scavenging ability and MDA content of the leaves of the transgenic Arabidopsis lines and non-transgenic controls were further measured. The results are as follows Figures 9 to 11 As shown; after high salt stress, drought stress, and high temperature stress, the H2O2 accumulation of the transgenic lines decreased by 60.88%, 75.90%, and 65.51%, respectively, compared with the non-transgenic control ( Figure 9 ); The catalase activities of the transgenic lines increased by 1.81 times, 4.96 times, and 2.06 times, respectively ( Figure 10 ); MDA content decreased by 59.74%, 71.79%, and 49.98% respectively ( Figure 11 ).

[0081] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other manner. Any person skilled in the art may utilize the above-disclosed technical content to modify or modify the present invention into equivalent embodiments. However, any simple modifications, equivalent variations, and modifications to the above embodiments that do not depart from the technical content of the present invention and are based on the technical essence of the present invention remain within the scope of protection of the present invention.

Claims

1. Japanese eelgrass ZjPIP1 The application of a gene in improving plant stress resistance is characterized in that: Japanese eelgrass ZjPIP1 The nucleic acid sequence of the gene is shown in SEQ ID No. 1; by increasing the expression of Japanese eelgrass in plants ZjPIP1 The expression level of the gene improves the plant's resistance to adverse stress, wherein the stress resistance is resistance to high temperature stress, high salt stress and / or drought stress; The plant is Japanese eelgrass or Arabidopsis thaliana.

2. Japanese eelgrass according to claim 1 ZjPIP1 The application of a gene in improving plant stress resistance is characterized in that: By constructing Japanese eelgrass ZjPIP1 Plant expression vectors of genes, transforming plants to express them in plants, and improving the expression of genes in plants ZjPIP1 Gene expression level.

3. Japanese eelgrass according to claim 2 ZjPIP1 The application of a gene in improving plant stress resistance is characterized in that: The method of transforming plants is one of Agrobacterium-mediated method, gene gun method, electric shock method, PEG method and liposome method.

4. Japanese eelgrass according to claim 3 ZjPIP1 The application of a gene in improving plant stress resistance is characterized in that: The Agrobacterium is Agrobacterium tumefaciens or Agrobacterium rhizogenes.

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