CN116694651A - Method for improving nitrogen utilization efficiency of tea trees - Google Patents
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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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)
- C12N15/8241—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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- C12Y—ENZYMES
- C12Y603/00—Ligases forming carbon-nitrogen bonds (6.3)
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- C12Y603/01002—Glutamate-ammonia ligase (6.3.1.2)
Abstract
The invention provides a method for improving the utilization efficiency of nitrogen in tea trees, which comprises a cytoplasmic glutamine synthetase coding gene CsGS1.1, and belongs to the technical fields of plant molecular biology and genetic engineering. The invention discloses that CsGS1.1 of tea tree can improve the assimilation capability of plants to nitrogen for the first time. Through expression pattern analysis in tea tree varieties with high and low nitrogen utilization rate and overexpression of the gene in arabidopsis, the CsGS1.1 is proved to be related to the nitrogen utilization rate of tea trees, and the tolerance of plants under nitrogen deficiency can be improved. The invention provides gene resources for cultivating new nitrogen-efficient plant varieties and has wide application value.
Description
Technical Field
The invention relates to the technical fields of plant molecular biology and genetic engineering, in particular to a method for improving the utilization efficiency of nitrogen in tea trees.
Background
Tea trees are leaf crops, and mineral nutrition, especially nitrogen nutrition, plays a vital role in growth and development, physiological and biochemical metabolic processes and tea quality formation. For a long time, in order to improve the tea yield, the nitrogenous fertilizer application amount in tea gardens is increased year by year, so that the problems of excessive fertilization, serious ecological environment, agricultural non-point source pollution and the like are caused. The research on the nutrient utilization mechanism of the tea tree nitrogen is a scientific basis for nitrogenous fertilizer application in tea gardens, but the research on the molecular mechanism of the nitrogenous fertilizer utilization of the tea tree is still in depth. Therefore, the research of molecular mechanism of tea tree nitrogen is important to the production of tea in China.
Ammonium nitrogen and nitrate nitrogen are the two main inorganic nitrogen species that tea trees absorb and utilize nitrogen. After the tea tree absorbs the two inorganic nitrogen states, the tea tree needs to be circulated by glutamine synthetase/glutamine-alpha-ketoglutarate aminotransferase (GS/GOGAT), the circulation is a key link of the nitrogen utilization of the tea tree, and glutamine synthetase GS is the speed-limiting enzyme in the process. There are two types of isozymes for GS, including cytoplasmic glutamine synthetase GS1 located in the cytoplasm and plastid glutamine synthetase GS2 located in the chloroplast or mitochondria, the former being encoded by multiple genes, the latter encoding genes typically being only 1. The cytoplasmic glutamine synthetase gene family members related to tea trees and the role of the cytoplasmic glutamine synthetase gene family members in the utilization of tea tree nitrogen are not clear, so that further excavation and identification of the gene members, particularly excavation of members related to the utilization of tea tree nitrogen are needed, and the utilization efficiency of tea tree nitrogen can be regulated and controlled at a molecular level, and different field nitrogen conditions can be adapted, so that the quality of tea leaves can be improved through cultivation measures.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for improving the utilization efficiency of nitrogen in tea trees, which comprises a cytoplasmic glutamine synthetase coding gene CsGS1.1 and a new gene resource for improving the utilization efficiency of nitrogen in tea trees, improving the quality of tea trees and cultivating nitrogen-efficient tea tree varieties.
The invention provides a tea tree CsGS1.1 gene, in particular to a cytoplasmic glutamine synthetase coding gene CsGS1.1, which is derived from Zhejiang province main cultivated tea tree variety Longjing 43, and the nucleotide sequence of the gene is shown as SEQ ID No. 1.
The invention also provides application of the tea tree CsGS1.1 gene in improving the nitrogen utilization rate of plants, and a forward primer CsGS1.1F and a reverse primer CsGS1.1R of the gene are designed and obtained, and the nucleotide sequence of the primer CsGS1.1F is as follows: 5' -GGGGTACCATGTCTTTGCTATCAGA-3' as shown in SEQ ID No. 2; nucleotide sequence of the primer csgs1.1 r: 5' -GCTCTAGATGGCTTCCACAGCAGAGTGG-3' as shown in SEQ ID No. 3. The first two bases are protective base sequences, the underlined sequences are cleavage site sequences, and the first 8 bases are not gene sequences but are necessary sequences for constructing recombinant plasmids.
The invention also provides an application method of the tea tree CsGS1.1 gene in improving the nitrogen utilization rate of plants, which comprises the following steps:
1) Allowing the plant to contain the gene csgs1.1; or (b)
2) The plant over-expression gene CsGS1.1 is used for constructing a plant recombinant expression plasmid pCAMBIA1300-CsGS1.1-sGFP,
the acquisition mode is as follows:
extracting total RNA of the dragon well 43 leaves, carrying out reverse transcription to obtain cDNA, carrying out PCR (polymerase chain reaction) amplification by using the obtained cDNA as a template to obtain an amplified product CsGS1.1 gene, and connecting the amplified product to a modified pCAMBIA1300 plant expression vector containing a 35S promoter to obtain a plant recombinant expression plasmid pCAMBIA1300-CsGS1.1-sGFP;
the recombinant plasmid is introduced into agrobacterium and the transgenic arabidopsis strain is obtained by using a flocculation vacuum infiltration method.
Compared with the prior art, the invention has the advantages that:
a tea plant cytoplasmic glutamine synthetase gene CsGS1.1 was first cloned from Longjing 43. The CsGS1.1 gene is transferred into the Arabidopsis by utilizing a molecular biological means, and the nitrogen deficiency tolerance of the Arabidopsis strain which overexpresses the CsGS1.1 is proved to be extremely obviously enhanced for the first time.
Therefore, the gene related to the invention is related to plant nitrogen utilization, can be applied to regulating the growth and development of tea trees and improving the tea quality through cultivation measures such as nitrogen fertilizer application, and simultaneously provides new gene resources for cultivating plants with high-efficiency utilization of nitrogen, and has potential application value.
Drawings
FIG. 1 is a diagram showing the recombinant plasmid pCAMBIA1300-CsGS1.1-sGFP of the present invention;
FIG. 2 is a diagram showing the phenotype analysis of the overexpression CsGS1.1 Arabidopsis transgenic line under nitrogen deficiency, wherein A-B is obtained by the overexpression line; c, the growth phenotype of the over-expression strain under nitrogen deficiency; D-E, over-expressing plant biomass and root length of the strain; F-G, over-expressed strain ground and underground nitrogen content.
Description of the embodiments
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Referring to FIGS. 1-2, the invention provides a cytoplasmic glutamine synthetase coding gene CsGS1.1, which is derived from Zhejiang province main cultivated tea tree variety Longjing 43, and the nucleotide sequence is shown as SEQ ID No. 1.
Example 1 cloning of CsGS1.1
Experimental materials: tea tree cultivar "Longjing 43" [Camellia sinensis (L.) O. Kuntze. cv. Longjing43]Cutting seedlings.
The experimental method comprises the following steps:
1) Extracting total RNA from 0.1 g 'Longjing 43' tea leaves by adopting an RNAprep pure polysaccharide polyphenol plant total RNA extraction kit;
2) Using Prime Script RT The reagent Kit reverse transcribes total RNA into cDNA for gene cloning.
3) Cloning of csgs1.1 was performed using manual according to SMARTer RACE cDNA Amplification Kit (Clontech), and the cloned primer was designed as csgs1.1 gene specific primer: the 5' RACE primer is 5'-CTAGATCCGTCATAATTCCACTTTGGAA-3', and the nucleotide sequence is shown as SEQ ID No. 4; the 3' RACE primer is 5'-GGACGACATGAGACCGCCGACATCAA-3', and the nucleotide sequence is shown as SEQ ID No. 5.
4) Performing PCR amplification by using a KOD Fx NeO system, detecting a PCR product by using 1.2% agarose gel electrophoresis, purifying and recovering the PCR product by using MiniBEST Agarose Gel DNA Extraction Kit, connecting the recovered product to a pMD18-T carrier, transferring the recovered product into escherichia coli DH5 alpha by using a heat shock conversion method, performing PCR verification on bacterial liquid, selecting bacterial liquid of positive clones, and sending the bacterial liquid of positive clones to Shanghai platinum Shang Biotechnology Co, and finally obtaining the CsGS1.1 nucleotide sequence.
EXAMPLE 2 construction of pCAMBIA1300-CsGS1.1-sGFP recombinant vector
The experimental method comprises the following steps:
1) Amplifying a coding region of CsGS1.1 gene containing an enzyme cutting site by using CsGS1.1F and CsGS1.1R primer pairs mentioned in the above summary of the invention and using a Longjing 43 leaf cDNA as a template, connecting a PCR product into a vector pMD18-T, performing heat shock at 42 ℃, transferring into an escherichia coli competent cell (DH 5 alpha), and determining a correct gene sequence through colony PCR and sequencing verification;
2) The above plasmid with correct sequence was digested with KpnI and XbaI restriction enzymes, and then ligated with the vector of the same digested relative expression amount to construct a plant recombinant expression plasmid pCAMBIA1300-CsGS1.1-sGFP (FIG. 1).
EXAMPLE 3 construction and screening of overexpression CsGS1.1 Arabidopsis transgenic lines
1) Adding the recombinant vector obtained in example 2 to 200. Mu.l of Agrobacterium competent cells, ice-bathing for 30 min, freezing with liquid nitrogen for 5min, water-bathing at 37deg.C for 5min, ice-bathing for 2 min, adding 800. Mu.l of liquid LB medium, shaking-culturing at 28deg.C constant temperature shaker 220 r/min for 4-5 h, and plating 200. Mu.l of bacterial liquid on a plate (containing 50 mg. L) -1 Rif and 50 mg.L -1 Kana), culturing the plate in a constant temperature incubator at 28 deg.c for over 36 hr until monoclonal strain grows out, selecting the transformed strain, and performing colony PCR identification to selectAgrobacterium successfully transformed into the vector.
2) And (3) taking 20 mu 1 of agrobacterium tumefaciens bacterial solution containing pCAMBIA1300-CsGS1.1-sGFP recombinant plasmid, adding 5ml of LB liquid culture medium, and shaking at a constant temperature of 28 ℃ for 200 rpm overnight to activate the bacterial solution. This 5m 1 bacterial solution was poured into 250 ml of LB (kanamycin added) and shaken overnight at 28℃under constant temperature, at which time the concentration of Agrobacterium was around OD600 = 1.0, and the supernatant was discarded by centrifugation at 4000 rpm for 15 minutes in a 50 mL centrifuge tube. The buffer (1/10 MS, sucrose 100 g.L) was transformed with about 100 ml Arabidopsis thaliana -1 ，Silwet-L77 200 μl·L -1 Alternatively, a freshly prepared 5% sucrose solution may be used instead), an OD600 of about 1.0 is measured. Selecting wild arabidopsis seedlings growing for about 5 weeks, watering enough water to cut flowers and pods of the wild arabidopsis seedlings, leaving the flowers and pods to bloom, inverting the whole pot, dip-dyeing the whole pot in a conversion solution for 2 minutes, wiping off residues to obtain the conversion solution, sealing the whole plant by using a preservative film, keeping a high-humidity environment, culturing in the dark for 1 day, and culturing in a normal photoperiod to obtain transgenic T0 generation seeds.
3) The T0 generation Arabidopsis seeds are soaked in 75% alcohol for 5min (uniformly mixed), then the mixture is washed for 3 to 4 times by using sterilized water, then the mixture is sown on a 1/2MS culture medium plate containing hygromycin, the mixture is spring-treated for 2 days by a refrigerator at a temperature of 4 ℃, the mixture is taken out from an incubator until resistant seedlings grow for T1 generation, the resistant seedlings grown from the seeds harvested in the T1 generation after sowing are T2 generation until the T3 generation is used for the subsequent test (figures 2A-B).
Example 4 functional identification of overexpressing CsGS1.1 Arabidopsis transgenic lines in nitrogen utilization
After the screened overexpression CsGS1.1 arabidopsis homozygous strain OE1 and OE2 and wild type are used as materials and cultured for one week by 1/2MS, the arabidopsis overexpression strain with the root length of about 1cm and wild type seedlings are transferred to a 1/2MS culture plate without nitrogen for 10 days (see figure 2C), the biomass and the root length of the two overexpression strain plants are obviously superior to those of the wild type plants (figures 2D-E), and meanwhile, the content of the leaf and the root nitrogen of the overexpression strain OE1/2 is higher than that of the wild type plants (figures 2F-G), so that the nitrogen utilization efficiency of the overexpression strain is improved, and the nitrogen deficiency tolerance of the plants is enhanced.
The embodiment provides a teaCoding gene of tree cytoplasmic glutamine synthetaseCsGS1.1And the application of CsGS1.1 in nitrogen utilization is that the expression quantity of CsGS1.1 in tea tree varieties with high nitrogen utilization efficiency is obviously higher than that of tea tree varieties with low nitrogen utilization efficiency, and the CsGS1.1 is related to the reutilization efficiency of mature leaf storage nitrogen in the process of young shoot development of tea trees; meanwhile, the tolerance of the arabidopsis strain which overexpresses CsGS1.1 to nitrogen deficiency is remarkably enhanced. Therefore, the gene related to the invention is related to the utilization of the nitrogen of the tea tree, and can be applied to the regulation of the growth and development of the tea tree and the improvement of the tea quality through cultivation measures such as nitrogenous fertilizer application.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (5)
1. A method for improving the utilization efficiency of nitrogen in tea trees is characterized by comprising a cytoplasmic glutamine synthetase coding gene CsGS1.1, wherein the nucleotide sequence of the CsGS1.1 gene is shown as SEQ ID No. 1.
2. The method for improving the nitrogen utilization efficiency of tea trees according to claim 1, wherein a forward primer CsGS1.1F and a reverse primer CsGS1.1R for obtaining the gene are designed, the nucleotide sequence of the primer CsGS1.1F is shown as SEQ ID No.2, and the nucleotide sequence of the primer CsGS1.1R is shown as SEQ ID No. 3.
3. A method for increasing the nitrogen utilization efficiency of tea trees according to claim 1 or 2, comprising:
1) Allowing the tea tree to contain the gene CsGS1.1; or (b)
2) Tea tree over-expressed gene csgs1.1.
4. A method for improving nitrogen utilization efficiency of tea trees according to claim 3, wherein the tea tree overexpression gene csgs1.1 is used for constructing a tea tree recombinant expression plasmid pCAMBIA1300-csgs1.1-sGFP, and the obtaining mode is as follows:
extracting total RNA of the dragon well 43 leaves, carrying out reverse transcription to obtain cDNA, carrying out PCR amplification by using the obtained cDNA as a template to obtain an amplified product CsGS1.1 gene, and connecting the amplified product to a modified pCAMBIA1300 plant expression vector containing a 35S promoter to obtain a plant recombinant expression plasmid pCAMBIA1300-CsGS1.1-sGFP.
5. The method for improving nitrogen utilization efficiency of tea trees according to claim 4, wherein the recombinant plasmid is introduced into agrobacterium and a transgenic arabidopsis strain is obtained by using a flocculation vacuum infiltration method.
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