CN116640198A - Cold-resistant gene EjGLK1 of loquat, protein encoded by gene and application of gene - Google Patents
Cold-resistant gene EjGLK1 of loquat, protein encoded by gene and application of gene Download PDFInfo
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Abstract
The invention relates to the field of plant molecular biology, in particular to a loquat leaf cold-resistant related gene EjGLK1, and a coded protein and application thereof. The full length of cDNA sequence of the gene is shown as SEQ ID No.1, and the amino acid sequence of the coded protein is shown as SEQ ID No. 2. The EjGLK1 gene provided by the invention can increase the cold resistance of plants by influencing the synthesis of chloroplast related genes and influencing the expression of related genes in a low-temperature stress pathway ICE-CBF-COR. The plant expression vector pFGC5941-35S containing the target gene was transformed into wild type Arabidopsis thaliana by Agrobacterium tumefaciens mediated transformation with EjGLK1 using the floral dip method. The result shows that the over-expression of EjGKL1 enhances the cold resistance of arabidopsis thaliana. Compared with the transgenic arabidopsis plant material obtained by deleting GLK gene from arabidopsis, the transgenic arabidopsis plant material obtained by utilizing the loquat EjGLK1 gene has the advantages that the chlorophyll content and the expression quantity of chloroplast development synthesis related genes are obviously improved, the expression of low-temperature pathway related genes and the content of stress-resistant related enzymes are influenced, the cold resistance of plants is further improved, and the transgenic arabidopsis plant material has a good application prospect.
Description
Technical Field
The invention belongs to the technical field of plant genetic engineering, and particularly relates to a loquat cold-resistant gene EjGLK1, and a coded protein and application thereof.
Background
Loquat is a fruit tree of the genus Eriobotrya in the family Rosaceae, which is native to China, has a unique flavor because its fruit is ripe in spring and summer and is light in season. Loquat flower bud differentiation in summer and autumn, autumn and winter flowering and fruit setting, thus being extremely easy to be influenced by low temperature and even freezing injury so as to influence the loquat yield (Jiang Jimou, chen Xiuping, deng Chaojun, xu Jizhi, zheng Shaoquan (2018). Major areas of China such as Zhejiang, fujian, chongqing and the like have been reported to show that low temperature and even freeze injury seriously affect loquat yield and even cause loquat death (Cai Lihong, (2012). Loquat science).
Compared with conventional crossbreeding, the loquat ploidy breeding is rapid and efficient, and has the characteristics of seedless fruits and vigorous nutrition growth compared with diploid loquat, such as triploid loquat (Peng Saiwei, wang Yongqing, yan Juan, tao Lian. (2011), the research progress of triploid loquat breeding, national loquat academy of sciences). As the polyploid obtained by cultivation has stronger stress resistance, the high-yield high-quality loquat variety gradually becomes one of effective measures for solving the problem of low-temperature freeze injury of the loquat. As proved by the research results of the loquat cold resistance, compared with the two-tetraploid loquat, the triploid loquat has obvious freezing stress resistance, and the photosynthetic efficiency and the like of the triploid loquat are obviously improved (Liu Mingxiu, 2021).
Loquat is easy to be frozen at low temperature disasters, so that photosynthesis is seriously affected, and the output and even the survival rate of the loquat are finally affected. The analysis of the gene regulation of the Eriobotrya japonica EjGLK1 is beneficial to further analysis of functions of the Eriobotrya japonica, plays an important role in resisting freezing stress of the Eriobotrya japonica, and lays a theoretical foundation for molecular markers and the like.
Disclosure of Invention
The invention aims to provide loquat EjGLK1 (Golden 2-like 1) and a coded protein and application thereof.
The invention provides a gene EjGLK1 for maintaining cold resistance of loquat, wherein the cDNA nucleotide sequence of EjGLK1 is shown as SEQ ID No. 1.
The invention provides the protein for maintaining the Eriobotrya japonica EjGLK1, and the amino acid sequence of the protein is shown as SEQ ID No. 2.
The invention provides an amplification primer pair for maintaining the loquat gene EjGLK1, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID No. 3; the nucleotide sequence of the downstream primer is shown as SEQ ID No. 4.
The invention provides an over-expression vector containing the gene, a host cell and engineering bacteria.
The invention also provides application of the gene in regulating and controlling the cold resistance of the loquat.
The present invention provides a transformant obtained by transforming a host with the above-mentioned expression vector.
The invention provides application of loquat EjGLK1 and protein, expression vector or transformant thereof in breeding for improving cold resistance of loquat.
The invention clones 1 related gene EjGLK1 from triploid varieties with cold resistance, and discovers that the gene EjGLK1 is positioned in cell nuclei. The real-time fluorescence quantitative PCR proves that the expression quantity of the EjGLK1 gene is obviously increased after the low-temperature treatment of the loquat, and the expression quantity is highest in the triploid with cold resistance, and also shows that the EjGLK1 gene has the effect of improving the low-temperature stress resistance of the loquat. The plant over-expression vector of EjGLK1 gene is constructed by utilizing a genetic engineering means, and is over-expressed in the GLK1 double-mutant Arabidopsis thaliana GLK1GLK2, so that the expression of chloroplast development related genes and the chlorophyll content are influenced, and the GLK1GLK2 double-mutant is recovered from yellow-white to normal green. Meanwhile, the change of related genes in a key regulatory pathway ICE-CBF-CORs of a low temperature stress pathway and the expression of ROS related enzymes are obviously influenced, and finally the cold resistance of arabidopsis is improved. The invention provides good application prospect for low-temperature stress regulation of angiosperm.
Drawings
FIG. 1 shows a clone electrophoresis photograph of the Eriobotrya japonica EjGLK1 gene. Wherein M is DL2000 DNA marker,1 is PCR product of EjGLK1 gene ORF, and the length is 1620bp.
FIG. 2 shows that the amino acid sequence of the loquat EjGLK1 encoded protein has highly conserved TEA domains and GCT-box transmembrane domains in comparison with the sequences of apple, pear, arabidopsis, peach, poplar, TEA, castor, grape, banana, jute, tobacco, millet and lotus.
FIG. 3 shows subcellular localization of the Eriobotrya japonica EjGLK1 gene transiently expressed in tobacco leaves, indicating that the EjGLK1 protein is localized to the nucleus. GFP: green fluorescent protein; DAPI: the nuclear dye stains the fluorescent signal; bright Field: bright field imaging; mered: combined image of GFP, DAPI and Bright field.
FIG. 4 shows the positive identification of EjGLK1 transgenic Arabidopsis. (A) PCR amplification and identification; (B) RT-qPCR identification. DL2000 DNA marker; positive control; negative control; glk1glk2 Arabidopsis double mutant.
FIG. 5 shows the identification of the GLK1 gene-deleted Arabidopsis double mutant GLK1GLK2 and wild type phenotype and expression level.
FIG. 6 shows the measurement of the differential phenotype and the expression level of related genes of EjGLK1 transgenic Arabidopsis, mutant and wild type freezing stress treatment. (A) EjGLK1 transgenic Arabidopsis and mutant and wild type, normal temperature and difference phenotype before and after freezing stress treatment; (B) Chlorophyll content of EjGLK1 transgenic Arabidopsis, mutant and wild type; (C) Survival rate statistics of EjGLK1 transgenic arabidopsis, mutant and wild type at normal temperature and before and after freezing stress treatment; (D) conductivity measurement; (E-H) measurement of expression level of chloroplast synthesis-related gene.
FIG. 7 shows the measurement of the expression level of EjGLK1 transgenic Arabidopsis, mutant and wild type low temperature pathway ICE-CBF-CORs related genes.
FIG. 8 shows the determination of enzyme activity associated with mutant and wild-type ROS in EjGLK1 transgenic Arabidopsis.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the examples were under routine experimental conditions or under conditions recommended by the manufacturer's instructions.
EXAMPLE 1 molecular cloning of the Eriobotrya japonica EjGLK1 Gene sequence
Extraction of triploid loquat leaf total RNA
Extracting cold-resistant triploid loquat leaf RNA by using a polysaccharide polyphenol plant total RNA extraction kit, obtaining corresponding cDNA by using a reverse transcription kit, and storing the corresponding RNA in an ultralow temperature refrigerator at-80 ℃.
According to the early loquat re-sequencing data of the team, homologous recombination primers are designed at two ends of the full-length sequence coded by the loquat EjGLK1 gene, and the forward primer sequence is EjGLK-F aattaccatggggcgcgccATGCTTCTTTTATCACCTTTGAGG; the reverse primer sequence is EjGLK-R tagactcacctaggatccTCAAGCACAGGAGGGTGGAAATTT.
The PCR amplification of the loquat EjGLK1 gene was performed using the cDNA obtained by reverse transcription as a template and the primers EjGLK-F and EjGLK-R. The PCR amplification system and the reaction conditions are as follows: the total volume of amplification was 20. Mu.L, the high fidelity enzyme PrimerSTAR 10. Mu.L, ddH2O 7. Mu.L, template (total cDNA of the leaf) 1. Mu.L, the upstream primer EjGLK-F1. Mu.L, and the downstream primer EjGLK-R1. Mu.L. The reaction conditions are as follows: 95-5 min; 95-30 s, 57-30 s, 72-30 s, and circulating for 35 times; 72-5 min. After the reaction is finished, taking out the amplified product, adding 2 mu L of 10×loading buffer, uniformly mixing, detecting by using 1% agarose gel electrophoresis, obtaining correct specific bands as shown in figure 1, performing gel recovery by using a gel recovery kit to obtain a purified product, and performing concentration identification on the obtained product and storing in a refrigerator at-20 ℃.
Using DNAMAN software, the CDS of EjGLK1 gene was 1620bp (SEQ ID No. 1), protein translation was performed, and 539 amino acid sequences (SEQ ID No. 2) were obtained after removal of the stop codon. The resulting sequences were aligned specifically for amino acid sequences with GLK proteins of apple, pear, arabidopsis, peach, poplar, TEA, castor, grape, banana, jute, tobacco, millet, lotus, and were found to have conserved TEA domains and GCT-box domains (fig. 2).
Example 2 construction of plant expression vector pFGC5941-35S of Eriobotrya japonica EjGLK1-eGFP
The restriction enzyme SmaI is used for carrying out enzyme digestion on plant expression vector pFGC5941-35S, wherein the enzyme digestion system is as follows: the total volume of the enzyme digestion is 20 mu L, wherein SmaI is 1 mu L,10 times color buffer is 10 mu L, and plant expression vector Pfgc5941-35S is 9 mu L of eGFP plasmid and 37-30 min. After the reaction is finished, taking out the product, using a correct specific strip obtained by 1% agarose gel electrophoresis, carrying out gel recovery through a gel recovery kit, and measuring the concentration of the obtained product and storing the product in a refrigerator at the temperature of minus 20 ℃. The purified EjGLK1 target gene fragment and the plant expression vector after enzyme digestion and recovery are connected through a ligase Cloning mix to transform escherichia coli. The obtained bacterial liquid is amplified and verified by using an EjGLK1 upstream primer and an EjGLK1 downstream primer, and a plant expression vector with the EjGLK1 gene is obtained by sequencing and comparison. Extracting plasmid from the correct bacterial liquid with plasmid extraction kit, measuring concentration, storing in refrigerator at-20deg.C, and storing activated coliform bacterial liquid in refrigerator at-80deg.C.
EXAMPLE 3 subcellular localization analysis of Eriobotrya japonica EjGLK1 Gene
10mL of positive Agrobacterium containing the pFGC5941-35S:: ejGLK1-eGFP plasmid was shaken at 28℃to an OD value between 0.8-1.2. Centrifugation at 6000rpm for 20min, discarding the supernatant to leave a precipitate, and adding 10mL of the heavy suspension (10 mM MgCl) 2 10mM MES, 150. Mu.M acetosyringone). Subjecting the resuspension to tobaccoLeaf transformation, fluorescent observation of GFP was performed after 48 hours of dark culture, and after GFP signal was observed, observation was performed after 10 minutes of staining with DAPI, and the results are shown in FIG. 3. The results indicate that EjGLK1 is localized to the nucleus.
Example 4 transfer of the transgenic expression vector pFGC5941-35S:: ejGLK1-eGFP into Arabidopsis thaliana
Firstly, the obtained arabidopsis mutant material glk1glk2 is identified, and the expression primers AtGLK1-F: TTGGGTCTCCGATTCTCCCT, atGLK-R: GGCGGTGCTCTAAATCTCGT are respectively used for the arabidopsis AtGLK1 and AtGLK2 genes through phenotypic observation; and AtGLK2-F TTCTCCGGCTCCAGTACTCA, atGLK-R AATCCCCGACCGTCGTAAAG, and the result shows that the obtained mutant Arabidopsis thaliana glk1glk2 has a yellowish white color compared with the wild type Col-0.
EjGLK1-eGFP was pre-chilled on ice to obtain vector plasmid pFGC5941-35S, after 1. Mu.g was pre-chilled on ice, 30. Mu.L of freeze-thawed Agrobacterium competent GV3101 was mixed well, placed on ice for 5min, transferred into liquid nitrogen for 5min, placed in a water bath at 37℃for 5min, added with 700. Mu.L of YEB medium, placed in a shaking table at 28℃ (220 rpm) for 2h, and the bacterial solution was spread on YRK medium with rifampicin and kanamycin resistance, placed in a 28℃incubator for inversion culture for 48h. PCR verification is carried out by using an amplification primer of EjGLK1 gene, and a PCR amplification system and reaction conditions are as follows: the total volume of amplification was 20. Mu.L, the low fidelity enzyme 2 XTaq was 10. Mu.L, and ddH 2 O was 7. Mu.L, the template (Agrobacterium to be verified) was 1. Mu.L, the upstream primer EjGLK-F was 1. Mu.L, and the downstream primer EjGLK-R was 1. Mu.L. The reaction conditions are as follows: 95-5 min; 95-30 s, 57-30 s, 72-30 s, and circulating for 30 times; and (3) obtaining the agrobacterium with the target fragment after 72-5 min.
Positive agrobacterium containing pFGC5941-35S:: ejGLK1-eGFP plasmid was activated in 2mL of YRK medium, placed on a shaker at 28℃until OD was between 0.8-1.2, and transferred into 100mL of YRK medium until OD was between 0.8-1.2. After centrifugation at 6000rpm for 20min, the supernatant was discarded to give a precipitate, which was resuspended in a heavy suspension (100 ml of system containing sucrose: 5g, surfactant Silwet L-77: 50. Mu.L).
Sowing the mutant arabidopsis thaliana into humus soil: vermiculite: the perlite is placed in nutrient soil with the ratio of 3:1:1, and then the nutrient soil is placed into a light incubator with the temperature of 23 ℃ for dark culture for 2 days (the humidity is 80 percent, and 16 hours of light is carried out), the culture is carried out until the flowers and the pods are subtracted and flower buds are left when the culture is to be infected, the prepared heavy suspension is used for infection for 10-30 seconds, and then the moist dark culture is kept for 24 hours, and the culture is carried out until the culture is moved to the light incubator with the temperature of 23 ℃. The method is repeated for 2 times at 7-day intervals a week, and cultured until seed collection.
Example 5 transgenic Arabidopsis screening of Eriobotrya japonica EjGLK1 Gene
And (3) collecting the obtained arabidopsis seeds to be screened, then dibbling the arabidopsis seeds on nutrient soil, and culturing the arabidopsis seeds in a dark state at 4 ℃ for 2 days, and then culturing the arabidopsis seeds in an illumination incubator normally. After leaves grow out, herbicide glufosinate is taken for spraying, the seedlings which grow normally are put into a 23 ℃ illumination incubator for verification to obtain positive seedlings, and the positive seedlings are transferred into arabidopsis nutrient soil for cultivation.
Example 6 transgenic Arabidopsis thaliana positive identification of Eriobotrya japonica EjGLK1 Gene (DNA amplification identification, real-time fluorescent quantitative PCR)
The amplification (DNA) identification procedure was as follows: extracting DNA of a strain to be detected by using a plant DNA extraction kit according to steps, taking agrobacterium containing EjGLK1 genes as a positive template, taking DNA of mutant arabidopsis thaliana glk1glk2 as a negative template, adding Taq enzyme and primers EjGLK-F and EjGLK-R, and carrying out PCR amplification identification.
Designing a specific expression primer of the EjGLK1 gene by DNAMAN to obtain an upstream primer eEjGLK-F CTACACCTCCTCCCATGCAC and a downstream primer eEjGLK-R CAGTACGAAGCGTCTGGAGG; and synthesizing an upstream primer of an Arabidopsis internal reference gene, namely an action-F CTCAAGAGGTTCTCAGCAGTA and a downstream primer of an action-R TCACCTTCTTCATCCGCAGTT, and performing real-time fluorescence quantitative PCR. The PCR reaction procedure was 95℃for 5min; dissolution curves were collected for 95℃30s,56℃30s,72℃30s,40 cycles: pre-dissolving at 60 ℃ for 90 seconds; then, the temperature was raised at a rate of 1.0℃per second, and the temperature was kept at 1℃for 5 seconds until 95℃was reached, and 3 biological replicates were performed for each reaction. The results are shown in FIG. 5, and transgenic Arabidopsis L3 and L4 are selected as positive plants for subsequent observation.
EXAMPLE 7 phenotypic identification of transgenic Arabidopsis thaliana of the Eriobotrya japonica EjGLK1 Gene
To further analyze the function of the heterologous over-expressed EjGLK1 gene in arabidopsis, two T3 generation lines of transgenic arabidopsis L3, L4 and mutant arabidopsis glk1glk2 were subjected to treatment observation and phenotypic analysis. Wild type Arabidopsis thaliana, mutant and transgenic T3 generation seeds were placed on an ultra clean bench, sterilized, placed on a 1/2MS culture plate, vertically cultured in an illumination incubator at 23℃for 10 days, and then subjected to a freezing stress treatment (-4 ℃) for 6 hours (FIG. 6).
The results show that: the mutant glk1glk2 is yellow-white compared with Col-0 and transgenic lines L3 and L4, and the expression level of chlorophyll content and chlorophyll synthesis related genes (HEMA 1-F TGCTTTGAACAGAATGTACGGT, HEMA1-R ATATGTAACGATACTGGGACC, GUN4-F GACGTGACTCTTCTCTCTCTTCC, GUN4-R GGTGGTGGAGGAGGAGGTGG, CHLH-F CGATTCATCTCTTCTCCTATC, CHLH-R TGGTTGATCTGTGTTTCTTC, CAO-F CACATCTTCATCAACTCCATG, CAO-R TTGTTGTCCTAAAACTAGCC) is also obviously lower than that of the mutant and transgenic lines (FIG. 6E-H). After 6 hours of freezing treatment, the results of statistical survival showed that the survival rate of the transgenic lines was significantly higher than that of the mutant lines. The result shows that the transgenic strain reverts the chlorophyll content of the mutant strain and the synthesis of related genes, and the transgenic strain obtains better resistance.
Example 8 expression level of Low temperature stress-related Gene in transgenic Arabidopsis lines
Designing a specific expression primer of a low-temperature stress related gene by utilizing NCBI website, wherein the upstream primer AtCBF1-F is AATGTTTGGCTCCGATTACG, and the downstream primer AtCBF1-R is CCCACTTACCGGAGTTTCTT; atCBF2 upstream expression primer AtCBF2-F GACCTTGGTGGAGGCTATTT and downstream primer AtCBF2-R ATCCCTTCGGCCATGTTATC; the AtCBF3 upstream primer AtCBF3-F TTATATGCACGATGAGGCGA and the AtCBF3-R ATGATTCCACTGTACGGACG; atICE1 upstream expression primer AtICE1-F GTTCGGGAATGAGGAGGTTT and downstream primer AtICE1-R AACACTCTCAGCCGCTTTAC; atRD29A upstream expression primer AtRD29A-F CTTGTCGACGAGAAGCAAAGAA, and downstream primer AtRD29A-R TCTTGATGGAGAATTCGTGTCC; atCOR47 upstream primer AtCOR47-F TGTCATCGAAAAGCTTCACCGA; atCOR47 downstream primer AtCOR47-R ACCGGGATGGTAGTGGAAACTG; atCOR15A upstream primer AtCOR15A-F GTCGTCGTTTCTCAACGCAAGA and AtCOR15A-R GCTTTCTCAGCTTCTTTACCCA. The results were shown by fluorescent quantitative PCR (fig. 7): the expression level of each low temperature stress related gene in the transgenic line after the freezing stress treatment is obviously increased compared with that of the mutant. The result shows that the heterologously expressed EjGLK1 gene significantly affects the change of related genes in a key regulatory pathway ICE-CBF-CORs of a low-temperature forced pathway.
Example 9 determination of ROS-related enzyme Activity in transgenic Arabidopsis lines
To analyze the effect of the EjGLK1 gene on cold tolerance of Arabidopsis thaliana, the activities of mutant Arabidopsis thaliana glk1glk2, wild type and transgenic lines L3, L4 leaf, superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate Peroxidase (APX) before and after freezing stress at-4℃were determined using a kit, as shown in FIG. 8. Under normal conditions, SOD, POD, CAT, APX enzyme activity was not significantly different in each strain; SOD, POD, CAT, APX enzyme activity increased in each strain after freeze stress at-4 ℃, but the mutant strain had a lower upward trend than the wild-type and transgenic strains. The result shows that the activity of the mutant glk1glk2 relative to active oxygen clearance is inhibited under the condition of freezing stress, the EjGLK1 heterologous transformation of the mutant glk1glk2 arabidopsis thaliana can further improve the activity of the enzymes of the mutant glk1glk2 under the condition of freezing stress, and the physiological index of active oxygen clearance shows that the heterologous transformation of the EjGLK1 improves the cold resistance of the mutant glk1glk2 arabidopsis thaliana.
The deletion of the mutant arabidopsis thaliana glk1 seriously affects the cold resistance and even the survival rate of plants, the loquat EjGLK1 gene can restore the cold resistance and the survival rate, and the result shows that the gene has the effect of increasing the cold resistance.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (10)
1. Loquat EjGLK1 protein which is:
1) A protein consisting of the amino acids shown in SEQ ID No. 2; or (b)
2) A protein derived from 1) which has equivalent activity and is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID No. 2.
2. A gene encoding the loquats EjGLK1 protein of claim 1.
3. The gene of claim 2, wherein the sequence is set forth in SEQ ID No. 1.
4. A vector comprising the gene of claim 2 or 3.
5. A host cell comprising the vector of claim 4.
6. An engineered bacterium comprising the gene of claim 2 or 3.
7. Use of the gene of claim 2 or 3 to affect the expression of a gene related to the low temperature regulatory pathway ICE-CBF-CORs under low temperature stress, thereby increasing the cold resistance of a plant.
8. The use according to claim 7, wherein the gene according to claim 2 or 3 is transferred into the genome of a plant and is heterologously expressed in the arabidopsis double mutant glk1gkl, whereby the mutant leaves are restored from yellow to green, the chlorophyll content is increased and the photosynthesis of the plant is enhanced.
9. The use of the gene of claim 2 or 3 to influence photosynthesis and expression of low temperature related pathway ICE-CBF-CORs related genes by increasing expression of chloroplast development related genes at low temperature, while significantly improving stress-resistance related enzyme activity, thereby enhancing cold resistance of plants.
10. Use according to claim 9, wherein the gene of claim 2 or 3 is transferred into the genome of a plant and overexpressed in the transgenic plant, and the cold resistance of the plant is enhanced by increasing the expression of a gene associated with a low temperature regulatory pathway and increasing the stress-resistance-associated enzyme activity at low temperature.
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