CN116199756B - OsMYB44 gene from rice and application thereof - Google Patents
OsMYB44 gene from rice and application thereof Download PDFInfo
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- APJYDQYYACXCRM-UHFFFAOYSA-N tryptamine Chemical compound C1=CC=C2C(CCN)=CNC2=C1 APJYDQYYACXCRM-UHFFFAOYSA-N 0.000 claims abstract description 60
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
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- C12N15/09—Recombinant DNA-technology
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- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
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- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
Abstract
The invention discloses an OsMYB44 gene from rice and application thereof, wherein the nucleotide sequence of the OsMYB44 gene is shown as SEQ ID No.2, and the amino acid sequence of the encoded protein is shown as SEQ ID No. 3. The cloned OsMYB44 fragment can effectively improve the content of tryptamine and derivatives in rice and other crops, and enhance the tolerance of the crops to stress such as UV-B and the like.
Description
Technical Field
The invention relates to the technical field of plant engineering. In particular to cloning, functional verification and application research of a MYB type transcription factor OsMYB44 involved in rice tryptamine accumulation.
Background
Ultraviolet light-B (UV-B), a typical oxidative stress, poses serious hazards to plants from molecular responses such as DNA, signal peptides and proteins to cellular changes, ultimately resulting in morphological and physiological changes such as leaf chlorosis, rust spots, curls, dry out, and even death, severely affecting plant growth and development (dhania et al, 2017). In recent years, the ozone layer in the atmosphere has been continuously destroyed due to rapid and sustained climate change (Lesk et al 2016;Rortais et al, 2017;Zandalinas etal, 2021). UV-B is increasingly one of the most challenging threats affecting global agricultural production (Yin et al, 2017). Under the background that the influence of the UV-B on the crop yield is increasingly serious, the research on the response, regulation and tolerance mechanisms of the crop to the UV-B is an important measure for stabilizing the crop yield and increasing the crop yield. Rice is an important grain crop and plays a role in guaranteeing the critical and safe strategic demands of grains in China (Muthamilarasan and Prasad, 2021). Therefore, the research on the response, regulation and tolerance of the rice to the UV-B has important significance for stabilizing the yield of the rice under the stress of the UV-B and promoting the yield increase of the rice by breeding the rice variety with the tolerance of the UV-B.
The tryptamine substance is an important secondary metabolite derived from tryptophan metabolic pathway, has strong antioxidant activity, can directly and indirectly remove Reactive Oxygen Species (ROS), thereby inhibiting cell death or apoptosis, protecting lipid, protein, nucleic acid and the like in cells from participating in growth, development, biological and abiotic stress of plants (Cho and Lee,2015;Stefano et al, 2021). Studies have shown that UV-B significantly induces the accumulation of tryptamine synthesis genes and tryptamine species in multiple species (Ishihara et al, 2008; kaur et al, 2015). However, there are few reports on tryptamine metabolic regulation networks in plants that respond to UV-B, and the genetic basis for tryptamine responses and tolerance to UV-B remains unclear. Rice is used as an important crop, a mechanism for resolving tryptamine response and tolerance to UV-B in the rice, and theoretical support is provided for stable yield of the rice and promotion of yield increase of the rice by breeding of UV-B tolerance rice varieties under the background of continuous damage of the current global ozone layer.
Disclosure of Invention
The technical problems to be solved by the invention are as follows: how to provide a transcription factor of the MYB type involved in rice tryptamine accumulation.
The technical scheme of the invention is as follows: the nucleotide sequence of the OsMYB44 gene from rice is shown as SEQ ID No. 2.
The amino acid sequence of the protein encoded by the OsMYB44 gene is shown as SEQ ID No. 3.
An expression vector for expressing the protein of the amino acid sequence shown in SEQ ID No. 3.
Further, the expression vector contains a nucleic acid fragment shown in SEQ ID No. 2.
The application of the OsMYB44 gene in improving the UV-B tolerance of plants.
Further, the use refers to over-expression of the OsMYB44 gene in plants, thereby increasing UV-B tolerance in plants.
The application of the OsMYB44 gene in improving accumulation of plant tryptamine and derivatives thereof.
Further, the use refers to overexpression of the OsMYB44 gene in plants, thereby increasing plant tryptamine and its derivative accumulation.
Further, the plant is rice.
The applicant screens out a transcription factor of MYB type by analysis of rice transcriptome data under UV-B stress, and the transcription factor is homologous to AtMYB44 of Arabidopsis thaliana and is named as OsMYB44. The nucleotide sequence of the full-length genome of the OsMYB44 is 1950bp (see SEQ ID No.1 of the sequence table), the nucleotide sequence of the coding region is 960bp (see SEQ ID No.2 of the sequence table), the amino acid sequence of 319 proteins is encoded (see SEQ ID No.3 of the sequence table), and the gene does not contain introns.
The cDNA of rice variety ZH11 is used as a template, the full-length coding region (SEQ ID No. 2) of the OsMYB44 gene is amplified, an OsMYB44 related vector is constructed, and the accumulation of tryptamine and the tolerance of UV-B stress regulated by the OsMYB44 are verified through the phenotypic analysis of single yeast, transient expression of tobacco, gene expression quantity, tryptamine content in genetic material and UV-B stress resistance. Overexpression of OsMYB44 in rice significantly enhanced accumulation of tryptamine and tryptamine derivatives and significantly enhanced UV-B tolerance. Through the development and utilization of OsMYB44, the content of tryptamine in rice and other crops can be effectively improved, and the tolerance of the crops to abiotic stress such as UV-B is enhanced.
Compared with the prior art, the invention has the following beneficial effects:
the cloned OsMYB44 fragment can effectively improve the content of tryptamine and derivatives in rice and other crops, and enhance the tolerance of the crops to stress such as UV-B and the like.
Drawings
Fig. 1: UV-B induced expression levels of OsMYB44. Standard deviation was based on three biological replicates, each sample being a mix of 3 strains of material. * Representative of P <0.01 (t-test).
Fig. 2: subcellular localization of OsMYB44. Co-transformation of 35S: osMYB44-GFP with 35S: osGhd7-RFP as a nuclear marker vector in tobacco leaves was performed on a scale of 20. Mu.m.
Fig. 3: schematic structural diagram of genetic transformation over-expression OsMYB44 vector.
Fig. 4: CRISPR/Cas9 mediates mutations in OsMYB44.
Fig. 5: OMYB44 and tryptamine synthetic gene expression level in OsMYB44 rice genetic material. The wild rice plant is ZH11; the OsMYB44 over-expressed plants are OE-1 and OE-2; osMYB44 mutant plants CR-1 and CR-2. Standard deviation was based on three biological replicates, each sample being a mix of 3 strains of material.
Fig. 6: content of tryptamine and derivatives in the genetic material of OsMYB44 rice. The wild rice plant is ZH11; the over-expressed plants are OE-1 and OE-2; osMYB44 mutant plants CR-1 and CR-2.
Fig. 7: phenotypic analysis of OsMYB44 Rice genetic Material resistant to UV-B. Wild rice plant ZH11; the over-expressed plants are OE-1 and OE-2; osMYB44 mutant plants CR-1 and CR-2.
Fig. 8: survival rate and fresh weight of aerial parts of OsMYB44 rice genetic material after UV-B treatment. The wild rice plant is ZH11; the over-expressed plants are OE-1 and OE-2; osMYB44 mutant plants CR-1 and CR-2. Standard deviation was based on three biological replicates, each sample being a mix of 3 strains of material. * And represents P <0.05 and P <0.01, respectively (t-test).
Detailed Description
The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from commercial sources.
The inventors succeeded in cloning the MYB-type transcription factor OsMYB44 involved in tryptamine accumulation and enhancing UV-B tolerance from rice by analysis and screening of transcriptome data of leaves of seedlings of rice treated with UV-B stress.
Example 1 acquisition of OsMYB44 Gene
1. OsMYB44 gene determination and structural analysis
After the rice seedlings are subjected to UV-B stress treatment, the expression level of a MYB transcription factor located on the No. 9 chromosome of the rice is obviously up-regulated. Further, it was revealed by quantitative PCR detection that the transcription factor was induced by UV-B at UV-B treatment for 1h, 3h and 12h. Among them, the transcription factor induction was most pronounced at 1h of UV-B treatment (fig. 1). In the rice genome database TIGR (http:// rice. Plant biology. Msu. Edu /), this MYB transcription factor is homologous to Arabidopsis AtMYB44, which is numbered LOC_Os09g01960, and is designated OsMYB44. The full-length genome sequence of the OsMYB44 is 1950bp (SEQ ID No. 1), the coding sequence is 960bp (SEQ ID No. 2), 319 amino acids (SEQ ID No. 3) are coded, and the gene has no intron.
2. Amplification of OsMYB44 Gene
The applicant designs two PCR primers according to the position and structure of the OsMYB44 gene in the japonica rice 'Japanese sunny' genome (rice genome database TIGR) by taking genome sequences at two sides of the OsMYB44 gene as templates, namely: osMYB44-F (5'-AAAAAGCAGGCTTAGATGATGGCGTCTTGTCGG-3') (underlined for attB1 site) and OsMYB44-R (5'-AGAAAGCTGGGTATGCTTCTTGCTCTGCTTTCTTG-3') (underlined for attB2 site). In order to obtain the full-length cDNA sequence of the OsMYB44 gene, the applicant extracted total RNA of flower 11 (ZH 11) in rice varieties and reverse transcribed the total RNA into cDNA. PCR amplification of the target fragment was BP-reacted with pDONR207 with universal adaptors attB1 and attB2 vectors, and the resulting clone was cloned into the portal vector. The reaction product is transformed into escherichia coli DH5a, 1mL of LB is added for resuscitation for 1h, 150 mu L of resuscitated bacterial liquid is coated on a flat plate containing a resistant LA culture medium, and the mixture is cultured for 12h at a temperature of 37 ℃. The monoclonal is subjected to PCR detection, and plasmid sequencing is extracted. And (3) sequencing and analyzing the PCR product to obtain a cDNA sequence of the OsMYB44 gene, and amplifying the cDNA sequence from ZH11 to obtain a 960bp OsMYB44 gene sequence. After the sequence alignment is correct, the sequence is respectively subjected to LR reaction with the target vector.
Example 2, osMYB44 subcellular localization analysis
1. Construction of OsMYB44 subcellular localization transient expression vector
The 960bp OsMYB44 gene fragment obtained from ZH11 through amplification is subjected to LR reaction with a target vector. The reaction product is transformed into escherichia coli DH5a, 1mL of LB is added for resuscitation for 1h, 150 mu L of resuscitated bacterial liquid is coated on a flat plate containing a resistant LA culture medium, and the mixture is cultured for 12h at a temperature of 37 ℃. And (3) carrying out PCR detection on the monoclonal antibody, and extracting plasmids to obtain the target vector.
2. Transient transformation of tobacco
Planting Nicotiana benthamiana. The culture was performed at 25℃and 70% relative humidity for about 4-5 weeks under 14h light/10 h dark conditions. The vector containing the gene of interest is transferred into agrobacterium EHA 105. Picking agrobacterium containing target vector to 5mL LB culture containing corresponding antibioticsThe medium was incubated at 28℃and 200rpm for 2d. Transferring 1mL of the cultured agrobacterium solution to 20mL of LB medium containing the corresponding antibiotics for expansion culture. At 28℃and 200rpm, the agrobacteria were cultivated until they grew to the logarithmic phase (OD 600 = 0.5-0.6). Centrifugation at 5000rpm for 10min at room temperature to collect the cells, and the cells were washed with a dip dye (containing 10mM MgCl) 2 The agrobacterium cells were suspended to od600=1.0 in 10mm mes,150 μm acetosyringone, ph=5.6) and left to stand for 2-3h (at least 0.5h, up to no more than 3 h) at room temperature. The needle was removed with a 1mL syringe, and the bacterial liquid containing the desired plasmid was aspirated and gently injected into the leaf. Meanwhile, the marker marks the water-stained area of the tobacco leaves. The injected plants are placed at about 21 ℃ and cultured for 1d under dark condition, and then are placed under illumination condition and cultured for 1d, so that the expression condition of GFP can be observed under a laser confocal microscope. Specific results indicate that OsMYB44 co-localizes with OsGhd7 into the nuclei of tobacco leaves (fig. 2).
Analysis of tolerance of genetic Material of OsMYB44 to UV-B
1. Construction and genetic transformation of OsMYB44 genetic transformation vector
The 960bp OsMYB44 gene fragment obtained from ZH11 is amplified to carry out LR reaction with a target vector pJC034 (the vector modified by the experiment can be used for over-expression of genes) to obtain the target vector, and the vector map is shown in figure 3. The vector carries agrobacterium-mediated genetic transformation vectors with a maize ubiquitin gene promoter featuring constitutive and overexpression. Meanwhile, through CRISPR/Cas9 mediated gene mutation, a mutant vector of OsMYB44 is constructed, and specific mutation sites are shown in figure 4.
2. Genetic transformation of OsMYB44
The method comprises the steps of introducing correctly cloned plasmids into a rice variety ZH11 through agrobacterium EHA105 mediation and a rice genetic transformation system, and obtaining transgenic plants through the steps of preculture, infection, co-culture, screening of calli with hygromycin resistance, differentiation, rooting, seedling hardening, transplanting and the like.
3. Analysis of tryptamine gene expression level and tryptamine content in OsMYB44 genetic material
First, for T0 generation transgenic waterDetection of rice plant DNA level confirms construction of positive plants of OsMYB44. T1-generation positive rice plants and wild-type rice plants that were grown to a 4-week-old over-expression of OsMYB44 and knockout of mutant OsMYB44 were subjected to a stress of 12.8. Mu.W cm -2 UV-B stress treatment. When the plants are treated by UV-B for 12 hours, RNA samples and metabolic samples of rice leaves of the plants treated by UV-B and untreated are taken, and the expression quantity of tryptamine synthetic genes and the content of tryptamine substances in the plants are analyzed. The results showed that compared with the control wild-type ZH11, the expression level of the OsMYB44 gene in the over-expressed OsMYB44 positive transformed plant was greatly improved, and the expression level of the OsMYB44 gene in the mutant OsMYB44 positive transformed plant was not significantly different (fig. 5), which indicates that the constructed genetic material was not problematic and could be used for further research and analysis. Further quantitative PCR detection shows that the expression level of key genes OsTS alpha and OsTDC1 of the tryptamine synthesis pathway in the rice plant with the over-expression of OsMYB44 is obviously up-regulated, and the expression level of OsTS alpha and OsTDC1 after UV-B treatment is more obviously up-regulated; the expression levels of OsTS a and ostcd 1 were significantly down-regulated in the oryb 44 mutated rice plants after UV-B treatment relative to wild type (fig. 5), suggesting that the involvement of OsMYB44 in regulation of tryptamine in rice is UV-B dependent. Further analysis of the content of tryptamine and tryptamine derivatives showed that tryptophan, tryptamine, N-Benzoyltryptamine, N-Acetyltryptamine, N-p-coumaroyl tryptamine and N-cinnamoyl tryptamine were significantly upregulated in rice plants overexpressing OsMYB 44; trytophan, N-Benzoyltryptamine, N-Acetyltryptamine, N-p-coumaroyl tryptamine and N-Cinnamoyl tryptamine were significantly down-regulated in OsMYB44 mutated rice plants (FIG. 6).
4. Analysis of tolerance of OsMYB44 genetic Material to UV-B
Meanwhile, by combining the experimental conditions in the earlier stage, the strength of the T1 generation positive rice plants and the wild rice plants which grow to 4 weeks and over-express the OsMYB44 and knockout the OsMYB44 is 12.8 mu W cm -2 UV-B stress treatment. When UV-B was treated for 72h, it was transferred to a light incubator for normal growth and tolerance of OsMYB44 genetic material to UV-B was observed. After the rice plants grow normally to 10d, the survival rate and the overground of the rice plants are countedPartial fresh weight. The results show that rice plants over-expressing OsMYB44 exhibit tolerance to UV-B; knockout mutant OsMYB44 rice plants exhibited sensitivity to UV-B (fig. 7). Moreover, the survival rate and the fresh weight of the overrepresented OsMYB44 rice plants after the UV-B treatment are obviously higher than those of wild plants; the survival rate and fresh weight of the aerial parts of the knockout mutant OsMYB44 rice plants are obviously lower than those of wild plants (figure 8).
Claims (1)
1.OsMYB44Application of gene in improving rice UV-B tolerance or tryptamine and derivative accumulation thereofOsMYB44The nucleotide sequence of the gene is shown as SEQ ID No.2, and the application refers to the overexpression in riceOsMYB44The gene can improve the UV-B tolerance of rice or the accumulation of tryptamine and derivatives thereof.
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