Gene for degrading chlorophyll as well as encoding protein and application thereof

Abstract

The invention discloses a gene for degrading chlorophyll as well as an encoding protein and application thereof, and belongs to the fields of molecular biology, plant genetic engineering and horticulture. Protein coded by the gene is located in chloroplast, and chlorophyll degradation can be efficiently promoted under various stress conditions. By performing different treatments on Huangkui tea trees and performing gene function verification in model plants such as nicotiana benthamiana and arabidopsis thaliana, it is found that the CsSGRs gene is subjected to induced expression by factors such as natural aging, abscisic acid, illumination intensity and low temperature, and the gene participates in a chlorophyll degradation approach, so that the functions are redundant. In addition, a bZIP family transcription factor CsABF4 is screened out, and CsSGRs gene expression can be regulated and controlled. The invention provides gene resources and theoretical basis for regulating and controlling the chlorophyll content of plants and improving related characters of plant leaf color, and has potential application value in the fields of plant breeding and gardening.

Description

TECHNICAL FIELD

[0001] The present application relates to the fields of molecular biology, plant genetic engineering and horticulture, and in particular to a gene for efficiently degrading chlorophyll isolated from a tea plant, the encoded protein thereof and the application thereof in degrading chlorophyll and regulating plant leaf color. BACKGROUND

[0002] Plant leaf color is closely related to chlorophyll content, and changes in chlorophyll content not only affect photosynthesis but also have an important impact on the appearance quality of plants. In the fields of agriculture and horticulture, plant varieties with special leaf color are often of great interest due to their unique appearance or physiological characteristics. For example, yellow tea varieties are favored by consumers because of their high umami taste and low astringency due to the characteristics of high amino acids and low catechins. Chlorophyll degradation is a complex physiological process regulated by multiple factors. Although the chlorophyll degradation pathway in plants has been partially understood, the key genes for efficiently degrading chlorophyll and their mechanisms in some plants still need further research. Existing studies have shown that the SGR gene family plays an important role in chlorophyll degradation, but the functions and regulatory mechanisms of SGR genes differ in different plants. Therefore, it is of great significance to identify new genes for efficiently degrading chlorophyll and elucidate their functions and regulatory mechanisms for cultivating new plant varieties with excellent leaf color traits.

[0003] Existing studies have shown that chlorophyll degradation mainly occurs through enzymatic and non-enzymatic pathways. The enzymatic pathway includes chlorophyllase (CLH), pheophytinase (PPH), pheophytinase (PAO), peroxidase (POD) and lipoxygenase (LOX). First, chlorophyllase (CLH) catalyzes the dephyllization of chlorophyll to produce dephyllized chlorophyll. Studies have shown that the activity of CLH differs significantly among different tea varieties, affecting the green tea fixation effect after fixation. Second, pheophytinase (PPH) and pheophytinase (PAO) catalyze the ring opening of pheophytin to produce colorless products, indirectly affecting the color of tea leaves during fermentation. Finally, peroxidase (POD) and lipoxygenase (LOX) promote the degradation of chlorophyll through oxidation during wilting. The non-enzymatic pathway is caused by high temperature, light or acidic environment, leading to the destruction of chlorophyll structure, such as the dephyllization of chlorophyll due to heat during fixation, forming brown dephyllized chlorophyll.

[0004] In tea plants, the formation of yellow tea varieties is closely related to chlorophyll metabolism, but the key genes regulating chlorophyll degradation and their mechanisms in tea plants have not been fully elucidated. In-depth study of chlorophyll degradation genes in tea plants not only helps to reveal the molecular mechanism of tea yellowing but also provides a protein for degrading chlorophyll, which can provide theoretical support for improving tea varieties after purification. SUMMARY

[0005] In order to efficiently degrade chlorophyll in plants, regulate the color of leaves of plants, a specific chlorophyll-degrading gene is isolated from a local yellowing tea plant, specifically named as CsSGRs gene, including CsSGR1 or CsSGR2, and the nucleotide sequence is specifically as follows:

[0006] The nucleotide sequence of the CsSGR1 gene is as follows:

[0007] ATGGGTACTTTGACTGCTTCTCTTGTGCTTCCATCTGAGCTAAAGCCTTCCTCACTCTCTCAACTTCACCAACATAGCTCTCTTTTCATCCACAGAAGAAGATCACCCAAGAAGCACCAATCTATTGTCCCTGTTGCAAGGTTGTTTGGACCAGCAATCTTTGAAGCTTCAAAGCTGCAGGTTCTGTTCCTAGGAGTTGATGAAAAGAAGCATCCAGGGCAGCTTCCAAGAACTTATACACTCACACATAGTGATATAACCTCTAAGCTCACTCTGGCCATCTCTCAATCAATAAACAACTCTCAGTTACAGGGGTGGTACAATAAGTTTCAGAGAGATGAAGTTGTAGCAGAGTGGAAGAAAGTCCAGGAGAAAATGTCTCTCCATGTTCACTGTCACATAAGTGGTGGCCACTTTCTATTAGATCTCTGTGCTAGGCTCAGATTCTTCATCTTCTCCAAGGAACTCCCTGTGGTTTTGAAGGCCTTTGTACATGGAGATGGGAATTTGTTCAACAACTATCCAGAATTAGAGGAAGCTCTGGTTTGGGTATATTTTCACTCCAACATTCCAGAATACAACAAGGTGGAGTGTTGGGGGCCACTCAAGGATGCTGCAGCACCTTCTAAGGTTAGTAGTTGTGGGTCCTACAAAGACAAAGGAGATGAAACCCCATCAATAAGCAACTGGGAGATGCCAAAGCCATGCCAAGAGAACTGCACATGTTGCTTTCCACCAATCAGCTTGTTGATCCCTTGGTCACCAAGATCTATCGGGTCAGGATCGGACTCAATGGACCCACCAAACCCAACTCCAACAAACAACAACTACAAAGATGGTCTTACACAACTCTATGATTGA;

[0008] The nucleotide sequence of the CsSGR2 gene is as follows:

[0009] ATGGGTACTCTGACCACTTCTCTTGTGCTTCCATCAGGTCTAAAGCCTTTCTCAATCTCTCAACAACAAAGCTCTCTTTTCATCTACACAAGAACACTTCCCAAGAAGCACCAATCTATTGTCCCTGTTGCAAGGTTATTTGGGCCAGCTATATTTGAAGCTTCAAAGCTGAAGGTTTTGTTCCTAGGAACTGATGAAAAGAAGCATCCAGGGAAGCTTCCTAGAACTTATACACTTACACACTGTGATTTAACCTCTAAGCTCACTCTTGCCATCTCTCAAACCATTAACAATTCCCAGTTACAGGGATGGTATCATAGATTACAGAGAGATGAAGTGATCGCAGAGTGGAAGAAGGTTAGAGGAAAAATGTCTCTCCATGTTCACTGTCACATAAGTGGTGGTCACTTTCTATTAGATTTGTGTGCCAGCCTAAGATTCTTCATCTTCTCCAAAGAACTCCCTGTGGTTTTGAAGGCCTTTGTTCATGGAGATGGGAATTTGTTCAATAACTATCCAGAATTAGAGGAATCTTTGGTTTGGGTTTATTTTCACTCCACTTTTCCAGAATTCAACAAGGTGGAATGTTGGGGACCACTCAAGGCTGCTGCAGCTCCTTCTGGTGGGGTCAATAGGGCTCACCAGGAAAGAAGTCATGAAATTCAATCAAGCAGCTGTGATCCTCTGGTGCCACATCCATGCCAAGAAGACTGCACATGCTGCTTTCCTCCAATGAACATGCATGTGCATGATTCATCCTTGCAGCAGAAGGAACTCTAG.

[0010] Based on the above-mentioned gene, a protein capable of degrading chlorophyll can be obtained, specifically, a protein encoded by CsSGR1 gene or a protein encoded by CsSGR2 gene, wherein CsSGR1 encodes 286 amino acids and CsSGR2 encodes 259 amino acids, and the specific amino acid sequences are as follows:

[0011] Amino acid sequence of CsSGR1 gene:

[0012] MGTLTASLVLPSELKPSSLSQLHQHSSLFIHRRRSPKKHQSIVPVARLFGPAIFEASKLQVLFLGVDEKKHPGQLPRTYTLTHSDITSKLTLAISQSINNSQLQGWYNKFQRDEVVAEWKKVQEKMSLHVHCHISGGHFLLDLCARLRFFIFSKELPVVLKAFVHGDGNLFNNYPELEEALVWVYFHSNIPEYNKVECWGPLKDAAAPSKVSSCGSYKDKGDETPSISNWEMPKPCQENCTCCFPPISLLIPWSPRSIGSGSDSMDPPNPTPTNNNYKDGLTQLYD;

[0013] Amino acid sequence of CsSGR2 gene:

[0014] MGTLTTSLVLPSGLKPFSISQQQSSLFIYTRTLPKKHQSIVPVARLFGPAIFEASKLKVLFLGTDEKKHPGKLPRTYTLTHCDLTSKLTLAISQTINNSQLQGWYHRLQRDEVIAEWKKVRGKMSLHVHCHISGGHFLLDLCASLRFFIFSKELPVVLKAFVHGDGNLFNNYPELEESLVWVYFHSTFPEFNKVECWGPLKAAAAPSGGVNRAHQERSHEIQSSSCDPLVPHPCQEDCTCCFPPMNMHVHDSSLQQKEL.

[0015] wherein the gene degrading chlorophyll is transgenically introduced into a plant for regulating the color of the leaves of the plant.

[0016] wherein the plant comprises Arabidopsis thaliana.

[0017] wherein the encoded protein is used to prepare a proteinase degrading chlorophyll. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Electrophoresis map of CsSGR gene cloning. The bands of CsSGR1 and CsSGR2 genes amplified by PCR using tea plant leaf cDNA as template are shown, verifying the success of gene cloning.

[0019] Figure 2Figure 6: Subcellular localization of CsSGRs protein. The CsSGRs plasmid with pUC19 expression vector was transformed into tea tree protoplast, and laser confocal analysis showed that the fluorescence signal appeared in chloroplast, indicating that the CsSGRs protein was located in chloroplast.

[0020] Figure 3 Figure 7: Yeast two-hybrid verification of the interaction between CsSGRs and CsPAO. The results showed that CsSGR1 and CsPAO produced weak interaction, while CsSGR2 and CsPAO did not produce interaction, suggesting that CsSGR1 is a key regulatory gene of the PAO pathway.

[0021] Figure 4 Figure 8: Dual luciferase interaction analysis of ProCsSGRs and CsABF4. The fluorescence signal of ProCsSGRs and CsABF4 co-transformation was significantly enhanced compared with the control group, indicating that CsABF4 and the promoter of CsSGRs can interact in vivo, activating the expression of CsSGRs.

[0022] Beneficial effects

[0023] The protein encoded by CsSGRs gene is located in chloroplast. By transforming the CsSGRs plasmid with pUC19 expression vector into tea tree protoplast, laser confocal analysis showed that the fluorescence signal appeared in chloroplast, indicating that it functions in chloroplast.

[0024] Functional characteristics: After transient overexpression in N. benthamiana and stable overexpression in Arabidopsis, the leaves of the plants turned yellow and the chlorophyll content decreased. The detached leaves of Arabidopsis SGR mutant nye1 and nye1 nye2 remained green after dark treatment, while the complementary mutant nye1 of tea tree CsSGRs lost the stay-green trait, indicating that tea tree CsSGRs and Arabidopsis AtSGR1 have similar functions, both involved in chlorophyll degradation and susceptible to dark induction, and the two genes are functionally redundant. In addition, yeast two-hybrid experiment found that CsSGR1 and CsPAO, a key gene of PAO pathway, produced weak interaction, suggesting that CsSGR1 is a key regulatory gene of PAO pathway, which can regulate chlorophyll degradation and reduce the toxic effect of degradation products on cells.

[0025] Transcriptional regulation mechanism: The bZIP family transcription factor CsABF4 selected can bind to the ABRE element on the promoter of CsSGRs gene, regulating the chlorophyll degradation mechanism of CsSGRs gene under abscisic acid treatment and 4°C low temperature treatment. This finding provides important clues for further study of the transcriptional regulation network of plant chlorophyll degradation, and helps to further reveal the molecular mechanism of plant response to environmental stress.

[0026] The application provides application of a CsSGRs gene and a protein coded by the CsSGRs gene, which can be used for regulating plant leaf color, changing plant chlorophyll content by regulating expression level of the gene, realizing accurate regulation of plant leaf color, and providing an effective means for cultivating special leaf color plant varieties. The application changes leaf color by regulating chlorophyll content through the CsSGRs gene; meanwhile, the gene can also be used for regulating plant aging process, and adjusting expression can delay or accelerate plant aging, so that crop aging time can be regulated as needed in agricultural production to improve yield and quality. DETAILED DESCRIPTION

[0027] The application will be further illustrated below in combination with specific examples. It should be understood that the examples are only used for illustrating the application, and are not used for limiting the scope of the application, and after reading the application, various equivalent modifications of the application by those skilled in the art fall within the scope defined by the appended claims.

[0028] The specific conditions not mentioned in the examples are carried out according to conventional conditions or conditions suggested by manufacturers. The reagents or instruments not mentioned by manufacturers are conventional products which can be obtained by market purchase.

[0029] Plant material: The 'Huangkui' tea seeds used in the experiment are all taken from the 'Huangkui' planting base in Langxi County, Xuancheng City, Anhui Province. The tea seeds are sowed in soil after treatment and placed in the climate room of the Biological Science and Technology Building of Anhui Agricultural University for cultivation, which is used for tea seedling treatment experiment. Arabidopsis thaliana and Nicotiana benthamiana are cultured under long-day conditions (16h light, 8h darkness, ambient temperature 25±1℃, light intensity 150-200μmol / m 2 / s).

[0030] Experimental instrument: The instrument used in the experiment is high-speed centrifuge U.S BECKMAN, intelligent PCR instrument, fluorescence quantitative PCR instrument is Bio-Rad, laser confocal is Leica AG, frozen mixing ball mill is MW400Retsch GmbH, KQ-100 type ultrasonic instrument is Kunshan Ultrasonic Instrument Co., Ltd., TDI-60B centrifuge Shanghai Anting Scientific Instrument Factory.

[0031] The experimental reagents are as follows.

[0032] ①Antibiotics

[0033] 50mg / mL of antibiotics: kanamycin (Kan), ampicillin (Amp), gentamicin (Gen), glufosinate-ammonium, dissolved in sterilized water, then filtered by a high-pressure sterilized water system filter head (pore size 0.22μm), and stored at -20℃;

[0034] 300 mg / mL of antibiotic: Timentin, Hygromycin, dissolved with sterilized water, filtered with sterilized water system filter head (pore size 0.22 μm), stored at -20 °C;

[0035] 20 mg / mL of antibiotic: Rif, dissolved with methanol, stored at -20 °C.

[0036] ②Culture medium

[0037] LB medium (1 L): 10 g of tryptone + 5 g of yeast extract + 10 g of NaCl; MS blank medium (1 L): 4.47 g of MS powder + 30 g of C12H22O11+ 0.01 g of Vc; solid medium, add 1.2%-1.5% agar powder. 121 °C high pressure steam sterilization for 21 min.

[0038] ③Experimental reagents and carriers

[0039] RNAiso Plus, Rnase Free water, RNAiso-mate cDNA synthesis Kit, LA Taq enzyme, rTaq enzyme, T4 DNA ligase, restriction enzyme, SYBR Green Realtime PCR Master Mix TAKARA, small plasmid extraction kit, high-purity DNA gel recovery kit, small DNA extraction kit Aidlab Biotechnologies, high-fidelity DNA high-fidelity polymerase Thermo Fisher Scientific, 40% acrylamide condensing agent, N,N,N',N'-tetramethyl-1,2-ethylene diamine

[0040] Amresco, maltose, Tris, NaCl, EDTA, DTT Sangon Biotech, Novablue (DE3) competent E. coli, Trans-T1 competent cells, BL21 competent cells TransGen Biotech, pESY-Blunt simple vector Institute of Botany, pdonr2007 vector, pCB2004 vector, GV3101 Agrobacterium Lab of Xiangchengbin, USTC.

[0041] Example 1

[0042] According to the previous experiments, the research group will be yellow tea tree shade treatment, most of the leaves in the shade of green, a few leaves still show yellow, the research group will be yellow leaves and green leaves of the gene comparative analysis of chlorophyll degradation gene CsSGRs.

[0043] The gene CsSGRs provided by the embodiment has the function of degrading chlorophyll efficiently. The CsSGR1 gene has a specific nucleotide sequence, and its expression is induced by natural aging, abscisic acid, light intensity and 4℃ low temperature; the CsSGR2 gene has a corresponding nucleotide sequence, and its expression is induced by natural aging, abscisic acid and light intensity. According to the induction and expression analysis, it is speculated that CsSGR1 is a major gene.

[0044] The protein encoded by the CsSGRs gene is located in the chloroplast and has the amino acid sequence shown in SEQ ID NO:X and SEQ ID NO:Y. The protein can participate in the chlorophyll degradation pathway under various stress conditions, remove the central magnesium ion of chlorophyll a to form de-magnesium chlorophyll a, thereby degrading chlorophyll efficiently and making the leaves yellow.

[0045] The transgenic engineering bacteria contain the recombinant plasmid or the exogenous CsSGRs gene nucleotide sequence integrated in the genome. The transgenic engineering bacteria can be used for gene expression and protein production, and provide materials for further research on gene function and application.

[0046] ①Gene characteristics

[0047] Sequence characteristics: A CsSGRs gene with the function of degrading chlorophyll efficiently is isolated from tea tree ‘Huanggui’, and contains CsSGR1 or CsSGR2, which has a unique nucleotide sequence, as follows.

[0048] The nucleotide sequence of the CsSGR1 gene is as follows:

[0049] ATGGGTACTTTGACTGCTTCTCTTGTGCTTCCATCTGAGCTAAAGCCTTCCTCACTCTCTCAACTTCACCAACATAGCTCTCTTTTCATCCACAGAAGAAGATCACCCAAGAAGCACCAATCTATTGTCCCTGTTGCAAGGTTGTTTGGACCAGCAATCTTTGAAGCTTCAAAGCTGCAGGTTCTGTTCCTAGGAGTTGATGAAAAGAAGCATCCAGGGCAGCTTCCAAGAACTTATACACTCACACATAGTGATATAACCTCTAAGCTCACTCTGGCCATCTCTCAATCAATAAACAACTCTCAGTTACAGGGGTGGTACAATAAGTTTCAGAGAGATGAAGTTGTAGCAGAGTGGAAGAAAGTCCAGGAGAAAATGTCTCTCCATGTTCACTGTCACATAAGTGGTGGCCACTTTCTATTAGATCTCTGTGCTAGGCTCAGATTCTTCATCTTCTCCAAGGAACTCCCTGTGGTTTTGAAGGCCTTTGTACATGGAGATGGGAATTTGTTCAACAACTATCCAGAATTAGAGGAAGCTCTGGTTTGGGTATATTTTCACTCCAACATTCCAGAATACAACAAGGTGGAGTGTTGGGGGCCACTCAAGGATGCTGCAGCACCTTCTAAGGTTAGTAGTTGTGGGTCCTACAAAGACAAAGGAGATGAAACCCCATCAATAAGCAACTGGGAGATGCCAAAGCCATGCCAAGAGAACTGCACATGTTGCTTTCCACCAATCAGCTTGTTGATCCCTTGGTCACCAAGATCTATCGGGTCAGGATCGGACTCAATGGACCCACCAAACCCAACTCCAACAAACAACAACTACAAAGATGGTCTTACACAACTCTATGATTGA.

[0050] The nucleotide sequence of the CsSGR2 gene is as follows:

[0051] ATGGGTACTCTGACCACTTCTCTTGTGCTTCCATCAGGTCTAAAGCCTTTCTCAATCTCTCAACAACAAAGCTCTCTTTTCATCTACACAAGAACACTTCCCAAGAAGCACCAATCTATTGTCCCTGTTGCAAGGTTATTTGGGCCAGCTATATTTGAAGCTTCAAAGCTGAAGGTTTTGTTCCTAGGAACTGATGAAAAGAAGCATCCAGGGAAGCTTCCTAGAACTTATACACTTACACACTGTGATTTAACCTCTAAGCTCACTCTTGCCATCTCTCAAACCATTAACAATTCCCAGTTACAGGGATGGTATCATAGATTACAGAGAGATGAAGTGATCGCAGAGTGGAAGAAGGTTAGAGGAAAAATGTCTCTCCATGTTCACTGTCACATAAGTGGTGGTCACTTTCTATTAGATTTGTGTGCCAGCCTAAGATTCTTCATCTTCTCCAAAGAACTCCCTGTGGTTTTGAAGGCCTTTGTTCATGGAGATGGGAATTTGTTCAATAACTATCCAGAATTAGAGGAATCTTTGGTTTGGGTTTATTTTCACTCCACTTTTCCAGAATTCAACAAGGTGGAATGTTGGGGACCACTCAAGGCTGCTGCAGCTCCTTCTGGTGGGGTCAATAGGGCTCACCAGGAAAGAAGTCATGAAATTCAATCAAGCAGCTGTGATCCTCTGGTGCCACATCCATGCCAAGAAGACTGCACATGCTGCTTTCCTCCAATGAACATGCATGTGCATGATTCATCCTTGCAGCAGAAGGAACTCTAG.

[0052] The open reading frame of CsSGR1 gene is 861 bp, and the open reading frame of CsSGR2 gene is 780 bp. Bioinformatics analysis shows that the CsSGRs are closely related to VvSGRs, and have a highly conserved domain, a chloroplast transit peptide and a cysteine-rich core motif, which is essential for the demagnification activity of the CsSGRs.

[0053] Expression regulation characteristics: The expression of CsSGR1 is induced by natural aging, abscisic acid, light intensity and low temperature at 4℃, and presents different expression patterns under different stresses. For example, the expression level is up-regulated by about 4 times under cold stress for 7 days, about 5 times of the expression level at 0h under drought stress for 72h, up-regulated by 17 times under salt stress for 72h, and up-regulated by about 3.5 times under MeJA stress for 24h. CsSGR2 is induced by natural aging, abscisic acid and light intensity, and the expression level is up-regulated by about 4 times of the expression level at 0h under salt stress for 72h, and up-regulated by about 5.5 times of the expression level at 0h after MeJA stress for 12h. And it is speculated that CsSGR1 is the main gene through expression analysis. In terms of tissue expression, CsSGRs are expressed in all tissues of the plant, and the expression level is positively correlated with maturity, with the highest content in old leaves.

[0054] ②Coding protein characteristic sequence: A protein encoded by CsSGRs gene has a specific amino acid sequence, CsSGR1 encodes 286 amino acids, and CsSGR2 encodes 259 amino acids, and there are highly conserved domains, chloroplast transit peptides and cysteine-rich core motifs and other structural characteristics, as follows.

[0055] Amino acid sequence of CsSGR1 gene:

[0056] MGTLTASLVLPSELKPSSLSQLHQHSSLFIHRRRSPKKHQSIVPVARLFGPAIFEASKLQVLFLGVDEKKHPGQLPRTYTLTHSDITSKLTLAISQSINNSQLQGWYNKFQRDEVVAEWKKVQEKMSLHVHCHISGGHFLLDLCARLRFFIFSKELPVVLKAFVHGDGNLFNNYPELEEALVWVYFHSNIPEYNKVECWGPLKDAAAPSKVSSCGSYKDKGDETPSISNWEMPKPCQENCTCCFPPISLLIPWSPRSIGSGSDSMDPPNPTPTNNNYKDGLTQLYD;

[0057] Amino acid sequence of CsSGR2 gene:

[0058] MGTLTTSLVLPSGLKPFSISQQQSSLFIYTRTLPKKHQSIVPVARLFGPAIFEASKLKVLFLGTDEKKHPGKLPRTYTLTHCDLTSKLTLAISQTINNSQLQGWYHRLQRDEVIAEWKKVRGKMSLHVHCHISGGHFLLDLCASLRFFIFSKELPVVLKAFVHGDGNLFNNYPELEESLVWVYFHSTFPEFNKVECWGPLKAAAAPSGGVNRAHQERSHEIQSSSCDPLVPHPCQEDCTCCFPPMNMHVHDSSLQQKEL.

[0059] Positioning characteristics:

[0060] ③ Transcriptional regulation characteristics

[0061] A bZIP family transcription factor CsABF4 involved in the ABA-dependent pathway, the expression amount of which is up-regulated under abscisic acid and 4℃ low temperature treatment. Through yeast single hybridization and dual luciferase experiments, it is proved that CsABF4 can bind to the ABRE element on the CsSGRs promoter, regulate the chlorophyll degradation mechanism of CsSGRs gene under abscisic acid treatment, and participate in the regulation of the chlorophyll degradation mechanism of CsSGR1 under 4℃ low temperature. Analysis of CsSGRs promoter elements found that it has ABRE elements related to abscisic acid response, which provides a binding site basis for the action of transcription factors.

[0062] Example 2

[0063] In this embodiment, according to the extracted chlorophyll degradation gene CsSGRs, the encoded protein thereof is verified to be able to degrade chlorophyll and regulate plant leaf color in Arabidopsis thaliana, and the specific experimental method is as follows.

[0064] Tea seedling treatment: Select seedlings of 'Huangkui' with consistent growth state, and perform light, abscisic acid and low temperature treatment. Light treatment sets up shading (50 μmol / m 2 / s), normal light (150 μmol / m 2 / s) and high light (300 μmol / m 2 / s) three intensities, and the treatment lasts for 15 days; abscisic acid treatment uses 100 mg / L abscisic acid solution, which is sprayed every three days, and the treatment lasts for 15 days; low temperature treatment sets the experimental group temperature to 4℃, and the control group to 20℃, and the treatment lasts for 15 days. After treatment, the chlorophyll of 1-3 leaves is detected and the gene expression amount is analyzed.

[0065] Chlorophyll content determination: 95% ethanol extraction method was used to determine the chlorophyll content. 0.10 g of fresh leaf tissue was weighed, 10 mL of extraction solution was added, and it was placed in the dark at room temperature for 48 h. After centrifugation, the supernatant was measured for absorbance at OD665nm, OD649nm and OD470nm. The contents of chlorophyll a, chlorophyll b and carotenoids were calculated.

[0066] RNA extraction and cDNA synthesis: Tea tree RNA extraction was performed using a specific method. Reagents were added to RNase-free centrifuge tubes, and the plant tissue was ground and subjected to lysis, centrifugation, and precipitation. The extracted RNA was measured for content and stored at -80°C. The RNA was reverse transcribed into cDNA using a kit from TAKARA, and the diluted cDNA was stored at -20°C for future use.

[0067] Gene cloning and vector construction: Primer Premier 5.0 was used to design primers, and tea tree leaf cDNA was used as a template for PCR amplification. The amplified products were verified by agarose gel electrophoresis, and the gel was recovered and ligated to the corresponding vector. The transformed E. coli was sequenced to verify the correctness and the plasmid was extracted. When constructing the vector, the gel recovery product was ligated to the enzyme-digested vector, the competent cells were transformed, and the positive colonies were sent for sequencing.

[0068] Gene expression analysis: Fluorescent quantitative PCR technology was used to analyze the expression of CsSGRs genes in different tissues and organs under different stress treatments. Fluorescent quantitative primers were designed, and the amplification efficiency of the primers was verified. The cDNA from each treatment was used as a template for quantitative analysis, and tea tree CsGAPDH was used as an internal reference to calculate the relative expression of the genes.

[0069] Subcellular localization experiment: A vector linker primer was designed to construct CsSGRs gene into PUC19 plasmid vector, and E. coli competent Trans-T1 was transformed. Tea tree mesophyll protoplasts were extracted, and the constructed plasmid was transformed into protoplasts. After culture, the fluorescence signal was observed under a laser confocal microscope to determine the protein localization.

[0070] Nicotiana benthamiana transient overexpression: attb linker primers were designed to construct CsSGRs gene into pCAMBIA1305-GFP plasmid, and Agrobacterium GV3101 was transformed. After screening positive strains, Nicotiana benthamiana was injected, and the leaf phenotype was observed after dark culture. Western Blot was used to verify protein expression.

[0071] Arabidopsis thaliana genetic transformation and stress treatment: CsSGRs gene was transformed into wild-type Arabidopsis thaliana by inflorescence soaking method, and positive plants were selected for subsequent experiments. Phenotype observation and pigment content determination were performed on transgenic Arabidopsis thaliana at different growth stages, and in vitro leaf dark stress treatment was performed to observe leaf yellowing.

[0072] Arabidopsis mutant complementation experiment: Arabidopsis DNA is extracted, and tea tree CsSGRs gene is complemented to AtSGR Arabidopsis mutant, and positive plants are screened. The complementary plants are subjected to dark stress treatment, and the leaf trait changes are observed.

[0073] Interaction verification experiment: The interaction relationship between CsSGRs and CsPAO is verified by yeast two-hybrid experiment; the interaction relationship between ProCsSGRs and CsABF4 is verified by yeast single-hybrid and dual-luciferase experiments, and the transcriptional regulation mechanism of the gene is explored.

[0074] Experimental results

[0075] Gene screening and bioinformatics analysis: Through the shading transcriptome analysis of 'Huangkui', the chlorophyll degradation gene CsSGRs is screened. Phylogenetic tree analysis shows that tea tree CsSGRs has a closer genetic relationship with grape VvSGRs, and may have similar functions. Amino acid sequence analysis shows that CsSGRs, like other related plant SGR proteins, has a highly conserved domain, a chloroplast transit peptide and a cysteine-rich core motif.

[0076] Gene expression analysis: CsSGRs expression analysis under different stresses shows that CsSGR1 is up-regulated under cold, drought, salt and MeJA stresses, and responds more obviously to cold, drought and MeJA; CsSGR2 is only up-regulated under salt and MeJA induction. Tissue expression analysis shows that CsSGRs are expressed in all plant tissues, and the expression amount is positively correlated with maturity. Abscisic acid, light and low temperature induction expression analysis shows that the expression amount of CsSGRs is negatively correlated with chlorophyll content under corresponding treatment.

[0077] Gene function research: CsSGRs gene is successfully cloned, and subcellular localization shows that its protein is located in chloroplast. Nicotiana benthamiana transient overexpression and Arabidopsis stable overexpression experiments show that CsSGRs gene overexpression leads to yellowing of plant leaves and reduction of chlorophyll content. Dark stress response experiment of transgenic Arabidopsis and dark stress treatment experiment of AtSGRs mutant and CsSGRs complemented Arabidopsis show that CsSGRs gene can be induced by darkness, and has similar function with AtSGR1 of Arabidopsis. Yeast two-hybrid experiment shows that CsSGR1 and CsPAO produce weak interaction, and it is speculated that CsSGR1 is a key regulatory gene of PAO pathway.

[0078] Transcriptional regulation mechanism research: analysis of CsSGRs promoter elements found that it has ABRE elements associated with abscisic acid response. Through phylogenetic analysis, amino acid sequence analysis and induced expression difference analysis, the bZIP family transcription factor CsABF4 was screened. Yeast single hybrid and dual luciferase experiments proved that CsABF4 can bind to the ABRE element on the CsSGRs promoter, regulate the chlorophyll degradation mechanism of CsSGRs gene under abscisic acid treatment, and regulate the chlorophyll degradation mechanism of CsSGR1 under 4℃ low temperature.

[0079] Example 3

[0080] The transgenic method of example 2, the CsSGRs gene is transformed into other plants by inflorescence soaking method, and the CsSGRs gene can be applied to regulate plant leaf color. By regulating the expression level of the gene, the chlorophyll content of the plant can be changed, so as to realize the precise regulation of the leaf color of the plant, and provide an effective means for cultivating plant varieties with special leaf color.

[0081] For example, in tea tree breeding, more yellow or other leaf color variation tea varieties can be cultivated by using the gene, and the quality and ornamental value of tea leaves can be improved.

[0082] Second, plant senescence regulation: since chlorophyll degradation is closely related to plant senescence, the CsSGRs gene can be used to regulate plant senescence. By regulating the expression of the gene, the plant senescence can be delayed or accelerated. In agricultural production, the senescence time of crops can be regulated according to actual needs, and the yield and quality of crops can be improved.

[0083] Example 4

[0084] In this example, the cloned CsSGR is connected with pMD19-T vector overnight, and then transformed into Escherichia coli DH5α by CaCl2-mediated heat shock transformation method. Single colony colonies in overnight culture are placed in EP tubes containing 600 μL LB (containing the corresponding antibiotic), and cultured at 37℃ for 6-8h. After the bacterial solution is turbid (OD value is 0.5-0.6), colony PCR analysis is performed for identification.

[0085] After the single colony with correct sequence was selected and expanded, the plasmid was extracted. The pMD19-T-Cs-SGR and pCAMBIA2300 expression vector were digested by BamHI and XbaI respectively, and the products were recovered by agarose gel electrophoresis using a purification recovery kit. The small fragment of pMD19-T-Cs-SGR and the digested pCAMBIA2300 expression vector were recovered and ligated using T4 ligase to construct the pCAMBIA2300-Cs-SGR recombinant vector. Subsequently, the recombinant plasmid vector was successfully transformed into Agrobacterium GV3101 strain by liquid nitrogen freezing and thawing method. Finally, the Agrobacterium strain carrying the Cs-SGR gene was stored in an ultra-low temperature refrigerator at -80℃.

[0086] Tomato seeds were pretreated by rubbing the surface with hands, then washed with distilled water for 3 times, surface sterilized with 75% alcohol for 30-60s, washed with sterilized distilled water for 3 times, and finally sterilized with 10% NaClO for 10 min, washed with sterilized distilled water for 3 times, and then planted on 1 / 2MS solid medium. After 3 days of dark culture and 4 days of light culture, the plant grew to 8-10 cm, and the aseptic seedlings were obtained. The hypocotyls of the seedlings were cut to about 1.0 cm long and dark cultured in the pre-culture medium for 2 days. The Agrobacterium strain was suspended in MS liquid medium to prepare a recombinant bacterial solution. The dark cultured hypocotyls were co-cultured with the activated pCAMBIA2300-Cs-SGR bacterial solution (OD 0.6-0.8) for 15 min, and then the immersion solution was discarded. Then the surface of the explants was dried with filter paper. The explants were transferred to MS medium with filter paper. After 48 h of dark culture, they were transferred to selection medium MS with the addition of 6-BA (1.0 mg·L -1 ), IAA (0.3 mg·L -1 ), Kan (80 mg·L -1 ), and Tm (timentin: 100 mg·L -1 ). After 60 days of normal light culture, shoots were induced. The medium was replaced every 15 days. The regenerated shoots induced were separated from the callus using a scalpel and then inoculated on 1 / 2MS rooting medium containing IAA (0.6 mg·L -1 ), Kan (80 mg·L -1 ), and Tm (100 mg·L -1 ) to induce rooting. About 30 days later, strong roots were induced. Subsequently, the air-permeable membrane was opened, some sterile water was added to the medium, and the plants were transferred to a cultivation substrate of humus:vermiculite = 1:1 with an air-permeable bag attached to control humidity and allow regular ventilation. After the tissue culture seedlings fully adapted to the external environment, the air-permeable bag was removed. The growth conditions of the tissue culture seedlings were as follows: day and night temperatures of 23℃ / 25℃, relative humidity of (70±5)%, light cycle of 16h / 8h, and light intensity of 73 μmol·m-2 ·s -1 ; Growth conditions of the cultivated plants: temperature (28 ± 2) °C, relative humidity (70 ± 5) %, photoperiod 16 h / 8 h, light intensity 116 μmol·m -2 ·s -1 .

[0087] The identification of the transgenic positive lines, first, the DNA of the wild type tomato and the transformed tomato plants was extracted by CTAB method, and the untransformed wild type (WT) tomato was used as negative control, and the recombinant plasmid was used as positive control, and the DNA-PCR identification was carried out; secondly, the total RNA of the tomato was extracted by using EasyPure plant RNA extraction kit, and the specific method was carried out according to the operation instruction of the kit. The first strand cDNA was generated from the RNA by using reverse transcription system. With cDNA as template, pCAMBIA2300-CsSGR recombinant plasmid as positive control, WT as negative control, the expression level of CsSGR in T1 generation transgenic lines was evaluated. Roche 480 system was used for qPCR detection. The reaction system was as follows: 5 μL 2×SYBR Green Real MasterMix (SYBR Green, Applied Bio-systems), 0.5 μL cDNA, and 0.5 μL of CsSGR gene specific upstream and downstream primers, and the reaction volume was supplemented with ddH 2O to 10 μL. The GAPDH gene was used as an internal reference gene. Three biological replicates were performed. Finally, two independent T1 generation homologous lines of CsSGR transgenic tomato were selected for subsequent study.

[0088] Compared with WT, the leaves of CsSGR transgenic tomato are yellow.

[0089] Example 5

[0090] This example first purifies the CsSGRs protein, which specifically includes the following steps.

[0091] 1. Recombinant protein expression:

[0092] The CsSGRs gene was cloned into a prokaryotic expression vector (such as pET28a), transformed into E. coli BL21 (DE3), and induced to express by IPTG (0.5 mM, 16 °C overnight).

[0093] 2. Protein purification:

[0094] After breaking the bacterial cells and centrifuging to take the supernatant, the His-tagged protein was purified by Ni-NTA affinity chromatography, and eluted with a buffer containing 300 mM imidazole.

[0095] Ultrafiltration centrifugation desalination, replacement to reaction buffer.

[0096] SDS-PAGE and Western blot to verify the purity and concentration of protein (Bradford method).

[0097] Second step, preparation of chlorophyll substrate, including the following steps.

[0098] 1. Plant chloroplast extraction:

[0099] Fresh plant leaves (such as spinach) were ground in liquid nitrogen and added to extraction buffer (0.4 M sucrose, 50 mM Tris-HCl pH 8.0, 5 mM MgCl2). The chloroplasts were collected by centrifugation (3000 x g, 10 min).

[0100] 2. Chlorophyll extraction:

[0101] The chloroplast pellet was extracted with 80% acetone for 24 hours in the dark. The supernatant was collected by centrifugation (10,000 x g, 10 min) and the chlorophyll concentration was determined (formula: Chl a+b = 17.76 A 645 + 7.34 A 663 ).

[0102] The reaction system (200 μL) was mixed: 50 μL CsSGRs protein (final concentration 1 μg / μL); 100 μL chlorophyll substrate (final concentration 0.1 mg / mL) and 50 μL reaction buffer (buffer containing cofactor ATP / Mg 2+ ).

[0103] The results show that the CsSGRs encoded protein can rapidly degrade chlorophyll.

Claims

1. A gene for degrading chlorophyll, characterized by: The chlorophyll-degrading gene is a CsSGRs gene, including CsSGR1 or CsSGR2, and its nucleotide sequence is as follows: The nucleotide sequence of the CsSGR1 gene is as follows: ; The nucleotide sequence of the CsSGR2 gene is as follows: .

2. A protein encoding a chlorophyll-degrading protein, characterized in that: The chlorophyll-degrading protein is the protein encoded by the CsSGR1 gene or the protein encoded by the CsSGR2 gene, wherein CsSGR1 encodes 286 amino acids and CsSGR2 encodes 259 amino acids. The specific amino acid sequences are as follows: Amino acid sequence of the CsSGR1 gene: MGTLTASLVLPSELKPSSLSQLHQHSSLFIHRRRSPKKHQSIVPVARLFGPAIFEASKLQVLFLGVDEKKHPGQLPRTYTLTHSDITSKLTLAISQSINNSQLQGWYNKFQRDEVVAEWKKVQEKMSLHVHCHISGGHFLLDL CARLRFFIFSKELPVVLKAFVHGDGNLFNNYPELEEALVWVYFHSNIPEYNKVECWGPLKDAAAPSKVSSCGSYKDKGDETPSISNWEMPKPCQENCTCCFPPISLLIPWSPRSIGSGSDSMDPPNPTPTNNNYKDGLTQLYD; Amino acid sequence of the CsSGR2 gene: MGTLTTSLVLPSGLKPFSISQQQSSLFIYTRTLPKKHQSIVPVARLFGPAIFEASKLKVLFLGTDEKKHPGKLPRTYTLTHCDLTSKLTLAISQTINNSQLQGWYHRLQRDEVIAEWKKVRGKMSLHVHC HISGGHFLLDLCASLRFFIFSKELPVVLKAFVHGDGNLFNNYPELEESLVWVYFHSTFPEFNKVECWGPLKAAAAPSGGVNRAHQERSHEIQSSSCDPLVPHPCQEDCTCCFPPMNMHVHDSSLQQKEL.

3. The use of the chlorophyll-degrading gene according to claim 1, characterized in that: The chlorophyll-degrading gene is genetically modified into plants to regulate the color of plant leaves.

4. The use of the chlorophyll-degrading gene according to claim 3, characterized in that: The plants include Arabidopsis thaliana.

5. The chlorophyll-degrading protein according to claim 2, characterized in that: The encoded protein is used for preparing a protease for degrading chlorophyll.

6. A method for degrading chlorophyll, characterized in that: The method for degrading chlorophyll is to clone CsSGRs genes in plants.

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