PpbZIP1 and PpbZIP18 genes of phytoneum parvum and application of PpbZIP1 and PpbZIP18 genes
By cloning the PpbZIP1 and PpbZIP18 genes from Xiaolibao moss and verifying their function in Arabidopsis, the problem of insufficient drought tolerance in plants was solved, and the effect of significantly improving drought tolerance in plants was achieved.
Patent Information
- Application Number
- CN202510016559.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-06
- Publication Date
- 2025-04-04
AI Technical Summary
The prior art is difficult to effectively improve the drought tolerance of plants, especially in non-seed plants, where relevant molecular function knowledge is lacking.
The function of these genes was verified in Arabidopsis by cloning the PpbZIP1 and PpbZIP18 genes from the Xiaoliban moss, and the weighted gene co-expression network analysis (WGCNA) and heterologous overexpression techniques were used.
Overexpressing the PpbZIP1 and PpbZIP18 genes can significantly improve the tolerance of plants to drought stress and enhance their viability under drought conditions.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of genetic engineering technology, and in particular to Physcomitrella patens PpbZIP1 Genes and PpbZIP18 Genes and their applications. Background Art
[0002] Water is the source of life. The current global water shortage has seriously threatened food supply and food security. In order to cope with this situation, it is urgent to enhance plant drought tolerance and develop new varieties of highly drought-tolerant crops.
[0003] Physcomitrella patens Physcomitrium patens ) is the first bryophyte to have its genome sequenced completely, and is an ideal material for studying the evolution of plants from aquatic to terrestrial. Xiao, L. et al.'s research showed that the gametophyte of Physcomitrella patens can survive in dry conditions below -100 MPa in the absence of exogenous ABA after gradual drying in an open system ( Physcomitrella patens' Desiccation Tolerance: Rate of Dehydration and the Involvement of Endogenous Abscisic Acid (ABA). Plant Cell Environ, 2018.41(1): 275-284.). As a representative of early landing plants, Physcomitrella patens has evolved a series of survival mechanisms to cope with drought stress. By analyzing histone modifications, Widiez et al. found that the protonema tissue is epigenetically prepared to adapt to drought environments ( The Chromatin Landscape of the Moss Physcomitrella patens and Its Dynamics during Development and Drought Stress. The Plant Journal, 2014. 79(1): 67-81.). Due to the lack of long-distance water regulation vascular system and epidermal system, mosses adopt a "water change" strategy to deal with dehydration stress, that is, when there is a lack of water, they lose most of the water in their tissues, and once the environment is full of water, they quickly absorb water without causing harm to themselves. This water regulation strategy may be an ancient characteristic of ancient plants to adapt to terrestrial environments, but it has been gradually lost in the evolution of most vascular plants ( Desiccation Tolerance in Bryophytes: A Reflection of the Primitive Strategy for Plant Survival in Dehydrating Habitats. Integrative & Comparative Biology, 2005. 45(5): 788-799.). Therefore, studying dehydration tolerance in mosses helps us understand the adaptation of plants to terrestrial environments.
[0004] The basic leucine zipper (bZIP) transcription factor family is one of the most numerous and relatively conservative transcription factor families in higher plants. It has been identified in many plants, such as 78 in Arabidopsis thaliana ( The Arabidopsis bZIP Transcription Factor Family - An Update.Curr Opin Plant Biol, 2018. 45(PtA): 36-49.), 86 were identified in rice, 352 in soybean (Glycinemax), and 216 in corn (Zea mays). Mini Review Roles of the bZIP Gene Family in Rice. Genet Mol Res, 2014. 13(2): 3025- 3036.). It is widely distributed in eukaryotes and plays an important role in growth and stress response. Although some bZIP members have been widely studied in plants, there is still a lack of knowledge about the evolution of bZIP in land plant lineages and their molecular functions in drought-resistant plants, especially non-seed plants. Summary of the invention
[0005] The purpose of the present invention is to clone a gene with drought stress resistance from Physcomitrella patens, so as to provide a theoretical basis and candidate gene resources for the creation of new germplasm of drought-resistant crops.
[0006] To achieve the above object, the present invention adopts the following technical solution: Based on the analysis of the dehydration-rehydration transcriptome data of Physcomitrella patens, the present invention identified key modules related to dehydration treatment and key genes belonging to the bZIP family through weighted gene co-expression network analysis (WGCNA), and screened out two candidate dehydration tolerance key genes in Physcomitrella patens that were strongly induced by dehydration and whose expression gradually decreased after rehydration by analyzing the expression pattern of bZIP family genes in response to dehydration-rehydration: PpbZIP18 ABI5 homologous gene PpbZIP1 / PpABI5 , the mRNA expression levels of these two genes were up-regulated during dehydration treatment and down-regulated after rehydration.
[0007] PpbZIP1 and PpbZIP18 Gene cloning and analysis: The full-length CDS sequence of the target gene was cloned using the cDNA of the gametophyte of Physcomitrella patens after drought treatment. PpbZIP1 The CDS sequence is shown in SEQ ID NO. 1, and its length is 426 bp; PpbZIP18 The CDS sequence is shown in SEQ ID NO.2, and its length is 1584 bp.
[0008] Subcellular localization of PpbZIP1 and PpbZIP18: PpbZIP1 and PpbZIP18The gene fragments were cloned into the plant expression vector pCAMBIA1300-GFP and transformed into tobacco by Agrobacterium-mediated genetic transformation. Laser confocal microscopy showed that PpbZIP1 and PpbZIP18 were localized in the nucleus, which was consistent with the localization of their transcription factors.
[0009] PpbZIP1 and PpbZIP18 Functional verification of genes: by heterologous overexpression PpbZIP1 and PpbZIP18 The gene function was verified in the model plant Arabidopsis. The results showed that after 13 days of drought treatment, the leaves of each strain of Arabidopsis turned yellow and purple, but the growth of wild-type Arabidopsis was more significantly inhibited. The inhibition of PpbZIP1 overexpression strains and PpbZIP18 overexpression strains was much lighter than that of the wild type. Five days after rehydration, the wild-type Arabidopsis died, and the growth of PpbZIP1 overexpression strains and PpbZIP18 overexpression strains partially recovered. This shows that overexpression of PpbZIP1 and PpbZIP18 can enhance the drought tolerance of Arabidopsis.
[0010] Therefore, the present invention provides a small physodon PpbZIP1 Gene or PpbZIP18 Application of genes in regulating plant tolerance to drought stress.
[0011] Mechanistic studies have shown that the PpbZIP1 gene or PpbZIP18 gene positively regulates the plant's tolerance to drought stress.
[0012] Furthermore, the plant may be, but is not limited to, Physcomitrella patens and Arabidopsis thaliana.
[0013] Based on the above PpbZIP1 Gene or PpbZIP18 The positive regulatory effect of genes on plant drought stress resistance can be achieved by using genetic engineering technology and other means to upregulate the PpbZIP1 Gene or PpbZIP18 Specifically, the application includes: using biological technology to make the plant PpbZIP1 Gene or PpbZIP18 Gene expression is upregulated to improve the tolerance of plants to drought stress. This method can be used to create drought-tolerant crops with significantly improved drought resistance.
[0014] The present invention provides a breeding method for improving plant tolerance to drought stress, the method comprising: modifying the nucleotide sequence as shown in SEQ ID NO. PpbZIP1 The gene fragment or nucleotide sequence is shown in SEQ ID NO.2 PpbZIP18The gene fragment is cloned into a plant overexpression vector to construct a recombinant plasmid, and the target fragment in the recombinant plasmid is transferred into the recipient plant species using Agrobacterium-mediated technology to cultivate transgenic plants with enhanced tolerance to drought stress.
[0015] The plant overexpression vector in the present invention can be a vector known in the art for Agrobacterium-mediated genetic transformation technology, and can be but not limited to pCAMBIA1300 vector.
[0016] In the present invention, the Agrobacterium can be any agrobacterium-mediated genetic transformation technique known in the art, and may be but not limited to GV3101.
[0017] Furthermore, the recipient plant may be, but is not limited to, Arabidopsis, tobacco, rice, soybean, tomato, lettuce, poplar, etc.
[0018] By overexpression in Arabidopsis PpbZIP1 Gene or PpbZIP18 The gene significantly improves Arabidopsis' tolerance to drought stress.
[0019] The present invention has the following beneficial effects: The present invention is to treat Physcomitrella patens PpbZIP1 and PpbZIP18 The cloning and analysis of the gene, combined with heterologous overexpression technology, verified the function of the gene in Arabidopsis thaliana. PpbZIP1 or PpbZIP18 The gene can significantly improve the tolerance of plants to drought stress. The invention provides a theoretical basis and candidate gene resources for the development of drought-resistant crops. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 These are the results of WGCNA analysis of dehydration and rehydration of Physcomitrella patens.
[0021] Figure 2 Statistics of the number of TFs in each family of the MEturquoise module.
[0022] Figure 3 These are the construction diagrams, linearization diagrams and vector construction verification diagrams of pCAMBIA1300-GFP::PpbZIP1 and pCAMBIA1300-GFP::PpbZIP18 vectors, where A is the linearization diagram of pCAMBIA1300-GFP, B is the construction verification diagram of pCAMBIA1300-GFP::PpbZIP1 vector, and C is the construction verification diagram of pCAMBIA1300-GFP::PpbZIP18 vector.
[0023] Figure 4Schematic diagram for the construction of pCAMBIA1300-GFP::PpbZIP1 and pCAMBIA1300-GFP::PpbZIP18 vectors.
[0024] Figure 5 This is the subcellular localization map of PpbZIP1 and PpbZIP18.
[0025] Figure 6 This is the molecular identification of the T3 generation homozygous transgenic Arabidopsis lines, where A is the genome level identification and B is the transcription level identification.
[0026] Figure 7 Phenotypes of Arabidopsis thaliana Col-0, PpbZIP1 OE-1, PpbZIP1 OE-4, PpbZIP18 OE-1, and PpbZIP18 OE-2 before and after drought-rewatering treatment. DETAILED DESCRIPTION
[0027] The technical solution of the present invention is further described in detail below through specific embodiments. It should be understood that the implementation of the present invention is not limited to the following embodiments, and any form of modification and / or change made to the present invention will fall within the protection scope of the present invention.
[0028] In the present invention, unless otherwise specified, all parts and percentages are by weight, and the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are all conventional methods in the art unless otherwise specified.
[0029] The present invention uses the Gransden 2004 strain of Physcomitrella patens ( The Physcomitrella patens Chromosome - scale Assembly Reveals Moss Genome Structure and Evolution. The Plant Journal, 93(3), 515–533. ) was used as the material; based on the analysis of the dehydration-rehydration transcriptome data of Physcomitrella patens, the key modules related to dehydration treatment and the key genes belonging to the bZIP family were identified through weighted gene co-expression network analysis (WGCNA); and by analyzing the functional differences of bZIP family genes in dehydration-rehydration response between Physcomitrella patens and Arabidopsis thaliana, two key dehydration tolerance genes in Physcomitrella patens were screened: PpbZIP1 (with Arabidopsis ABI5 Gene homology, also known as PpABI5 )and PpbZIP18 Furthermore, the cloned and analyzed PpbZIP1 / PpABI5 and PpbZIP18 The effect of genes on drought stress provides a theoretical basis and candidate gene resources for the development of drought-tolerant crops.
[0030] Example 1: WGCNA analysis of dehydration-rehydration transcriptome data of Physcomitrella patens, construction of co-expression regulatory network, and analysis of expression patterns of bZIP family genes in response to dehydration-rehydration 1. WGCNA analysis To study the gene expression during dehydration and rehydration of Physcomitrella patens, the genes expressed during dehydration and rehydration were compared and their expression relationship was analyzed. The differentially expressed genes in the transcriptome data were analyzed by WGCNA.
[0031] like Figure 1 As shown, a total of 12 modules were obtained using the dynamic cutting tree method, among which the module MEturquoise contained the largest number of genes, with 3437 genes. This module was significantly enriched under severe dehydration treatment, indicating that the genes in this module were highly correlated with severe dehydration.
[0032] 2. Analysis of TF quantity of module MEturquoise Based on the WGCNA analysis results, modules with significant differences were selected, and the co-expression regulatory network was analyzed using GENIE3. A list of genes with weight values greater than 0.1 was output, and the number of transcription factors in each gene family was counted.
[0033] like Figure 2 As shown, the results showed that the bZIP transcription factor family accounted for a large proportion, so the bZIP family was analyzed subsequently.
[0034] 3. Expression patterns of bZIP family genes in response to dehydration-rehydration In order to more intuitively display the expression level of each Physcomitrella patens bZIP gene after dehydration-rehydration treatment, an expression heat map was drawn using Tbtools.
[0035] From the heat map, we can see that two bZIPs of Physcomitrella patens ( PpbZIP1 and PpbZIP2 ) was upregulated during dehydration and downregulated after rehydration. PpbZIP18 The dehydration-rehydration response pattern of PpbZIP1 and PpbZIP2 In general, the dehydration-rehydration response patterns of bZIP genes in the same branch on the evolutionary tree are similar.
[0036] Therefore, we screened into Physcomitrella patens PpbZIP1 / PpABI5 and PpbZIP18 Two key genes for dehydration tolerance.
[0037] Embodiment 2: PpbZIP1 and PpbZIP18 Cloning and analysis of CDS regions 1. Growth conditions of Physcomitrella patens The samples were cultured aseptically in an artificial climate box with cold light at 25°C and an optical density of 55 µmol / m 2 / s.
[0038] 2. PpbZIP1 and PpbZIP18 CDS region cloning The gametophytes of Physcomitrella patens that grew well on the culture medium were dehydrated. The specific method was as follows: 2 to 3 sterilized dry filter papers were placed in a culture dish in a clean bench, and then the gametophytes of Physcomitrella patens were placed on the filter paper. After about 48 hours, samples were taken. Total RNA was extracted using Trizol reagent, and RNA was first treated with DNase I to remove genomic DNA, and then cDNA was synthesized using the Evo M-MLV reverse transcription reagent premix kit. Refer to the Phytozome database PpbZIP1 and PpbZIP18 Primers were designed based on the CDS sequence of the gene.
[0039] PpbZIP1-F: 5'-AAGGTACCATGGGGCTGGCGCCGAATT-3'; PpbZIP1-R: 5'-AACTGCAGCTACCACGTTGCCGTCTTCGT-3'; PpbZIP18-F: 5'-GGGGTACCATGGATAGCGTCTCCACA-3'; PpbZIP18-R: 5'-ACGCGTCGACTGAATTGTGCACCATAAAGG-3'.
[0040] The target gene was amplified using the cDNA of Physcomitrella patens after drought treatment as a template. They were successfully cloned. It is worth noting that in the Phytozome database, there are 4 types of variable splicing predicted for the PpABI5 / bZIP1 gene. In the end, only the pair of primers designed based on the transcript annotated as primary transcript successfully cloned the target gene. However, the sequencing results showed that the cloned fragment was 18 bp longer than the CDS sequence predicted in the database, which may be a new variable splicing of the gene.
[0041] 3. Overexpression vector construction The cloned CDS sequence was connected to the pCAMBIA1300-GFP vector using T4 DNA ligase and transformed into E. coli DH5α. The positive clones were selected and sent to the company for sequencing. The correct clones were extracted from plasmids and stored in glycerol. PCR primer synthesis and gene sequencing were completed by Beijing Qingke Biotechnology Co., Ltd. PpbZIP1 and PpbZIP18The nucleotide sequences of the CDS regions of the genes are shown in SEQ ID No. 1 and SEQ ID No. 2, respectively.
[0042] 4. Subcellular localization analysis Transform the constructed 35S::PpbZIP1-GFP, 35S::PpbZIP18-GFP and pCAMBIA1300-GFP into Agrobacterium GV3101 competent cells, pick the positive single clones and inoculate them into 1 mL LB liquid medium containing kanamycin and rifampicin, and culture them at 28℃ for about 2 days until they turn orange-yellow. Take 200 µL of the bacterial solution and inoculate it into 20 mL LB liquid medium with corresponding resistance, and culture them at 28℃ for about 8 h. Centrifuge at 4000 rpm for 11 min to collect the bacteria. Prepare the resuspension solution: 1 mM MgCl2, 10 mM MES, 0.15 mM acetosyringone. Resuspend the bacteria to OD 600 When the concentration reaches about 0.6, the infection solution is obtained. Let it stand at room temperature for about 2 hours. Use a 1 mL syringe to inject the infection solution into the leaves of Nicotiana benthamiana that have grown for about 1 month. After about 12 hours of weak light recovery after injection, culture normally for 2 days and observe with a laser confocal microscope.
[0043] like Figure 5 As shown, PpbZIP1 and PpbZIP18 are localized in the nucleus, which is consistent with the localization of their transcription factors.
[0044] Example 3: Validation of the heterologous overexpression method in wild-type Arabidopsis PpbZIP1 and PpbZIP18 Gene function 1. Identification of T3 homozygous transgenic Arabidopsis lines The overexpression vector constructed in Example 2 was transformed into Col-0 Arabidopsis by Agrobacterium-mediated genetic transformation. The seeds were screened with hygromycin, and some seeds turned white before growing true leaves. They were considered negative, and the plants that could grow four leaves were considered transgenic positive. Positive plants were planted, screened with hygromycin, and the segregation ratio of T1 offspring was counted. T2 generation positive plants with a segregation ratio of 3:1 were planted. The T3 generation homozygous transgenic Arabidopsis strains PpbZIP1 OE-1, PpbZIP1 OE-4, PpbZIP1 OE-5, PpbZIP18 OE-1, and PpbZIP18 OE-2 obtained by hygromycin screening were extracted. The DNA of these homozygous transgenic Arabidopsis leaves was extracted, and PCR was used to verify whether the transgenic was successful.
[0045] PCR validation primers: PpbZIP1-F: 5'-ATGGGGCTGGCGCCGAATT-3'; PpbZIP1-R: 5'-CTACCACGTTGCCGTCTTCGT-3'; PpbZIP18-F: 5'-ATGGATAGCGTCTCCACA-3'; PpbZIP18-R: 5'-CGACTGAATTGTGCACCATAAAGG-3'.
[0046] like Figure 6 As shown in A, the target bands were amplified in these homozygous transgenic Arabidopsis, indicating that the transformation was successful.
[0047] 2. Use semi-quantitative PCR to identify the expression level of the transferred gene in each homozygous transgenic plant. Extract RNA from the T3 generation homozygous transgenic and wild-type Arabidopsis plants that are positive for PCR verification, remove genomic DNA with DNase I, and then reverse transcribe it into cDNA. Use semi-quantitative detection to detect the expression level of the transferred gene.
[0048] Semi-quantitative PCR validation primers: PpbZIP1-F: 5'-ATGGGGCTGGCGCCGAATT-3'; PpbZIP1-R: 5'-CTACCACGTTGCCGTCTTCGT-3'; PpbZIP18-qF: 5'-AACAGTGGAGTATTCAGCCTTC-3'; PpbZIP18-qR: 5'-CTCGCAGGTAACTTGTATGGAA-3'; ACTIN2-F: 5'-CTAAGCTCTCAAGATCAAAGGC-3'; ACTIN2-R: 5'-AACATTCAAAGAGTTTCAAGG-3'.
[0049] like Figure 6 As shown in B, PpbZIP1 OE-1, PpbZIP1 OE-4, and PpbZIP1 OE-5 transgenic Arabidopsis were successfully transcribed. PpbZIP1 Gene, among which PpbZIP1 OE-1 and PpbZIP1 OE-4 had the highest expression level. PpbZIP18OE-1 and OE-2 were also successfully transcribed. PpbZIP18 .
[0050] The strains with higher expression levels, PpbZIP1 OE-1, PpbZIP1 OE-4, PpbZIP18 OE-1, and PpbZIP18OE-2, were selected for the next functional analysis.
[0051] 3. Analysis of drought resistance function of PpbZIP1 and PpbZIP18 overexpression in Arabidopsis The 3-week-old homozygous transgenic lines Col-0, PpbZIP1 OE-1, PpbZIP1 OE-4, PpbZIP18 OE-1, and PpbZIP18 OE-2 Arabidopsis were subjected to drought treatment in a natural state, i.e., without watering. All lines were subjected to drought for 10 to 15 days (the specific number of days depended on the state of the plant), and then rewatered for 3 to 5 days after drought. The results were observed and photographed.
[0052] The results are as follows Figure 7 As shown in the figure, after 13 days of drought treatment, the leaves of Arabidopsis thaliana in each strain turned yellow and purple, but the growth of wild-type Arabidopsis thaliana was more significantly inhibited. The inhibition of PpbZIP1 OE-1, PpbZIP1 OE-4, and PpbZIP18 OE-2 was much lighter than that of the wild type. After 5 days of rehydration, the wild-type Arabidopsis died, the growth of PpbZIP1 OE-1, PpbZIP1 OE-4, and PpbZIP18 OE-2 partially recovered, and PpbZIP18 OE-1 began to grow new green shoots. This shows that overexpression of PpbZIP1 and PpbZIP18 can enhance the drought tolerance of Arabidopsis thaliana.
[0053] In summary, through the PpbZIP1 and PpbZIP18 The authors cloned and analyzed the genes and verified their functions in col-0 Arabidopsis using heterologous overexpression technology. They found that overexpression of PpbZIP1 and PpbZIP18 could enhance drought tolerance in Arabidopsis without affecting the normal growth of the plants.
[0054] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above examples, and there are many other variations, such as using Agrobacterium-mediated genetic transformation to transform Physcomitrella patens PpbZIP1 Gene or PpbZIP18 Genes are transferred into other crops such as tomatoes, lettuce, poplars, rice and soybeans to create drought-tolerant crops. All variations that can be directly derived or associated with the disclosure of the present invention by a person of ordinary skill in the art should be considered as within the scope of protection of the present invention.
Claims
1. Physcomitrella patens PpbZIP1 Gene or PpbZIP18 The use of a gene in regulating plant drought stress tolerance is characterized in that: Said PpbZIP1 The CDS sequence of the gene is shown in SEQ ID NO.1; PpbZIP18 The CDS sequence of the gene is shown as SEQ ID NO.
2.
2. The use according to claim 1, characterized in that Said PpbZIP1 Gene or PpbZIP18 The gene positively regulates the plant's tolerance to drought stress.
3. The use according to claim 2, characterized in that: The plants are Physcomitrella patens and Arabidopsis thaliana.
4. The use according to claim 1, characterized in that The application includes: using biological technology to make the PpbZIP1 Gene or PpbZIP18 Gene expression is upregulated to improve plant tolerance to drought stress.
5. A method for improving plant tolerance to drought stress, characterized in that: The method comprises: PpbZIP1 The gene fragment or nucleotide sequence is shown in SEQ ID NO.2 PpbZIP18 The gene fragment is cloned into a plant overexpression vector to construct a recombinant plasmid, and the target fragment in the recombinant plasmid is transferred into the recipient plant using Agrobacterium-mediated technology to cultivate transgenic plants with enhanced tolerance to drought stress.
6. The method according to claim 5, characterized in that The plant overexpression vector is a pCAMBIA1300 vector.
7. The method according to claim 5, characterized in that The Agrobacterium used was GV3101.
8. The method according to claim 5, characterized in that The recipient plant is Arabidopsis, tobacco, rice, soybean, tomato, lettuce or poplar.