Differential expression analysis method of tannin synthesis related genes in eucalyptus grandis

Abstract

The invention relates to the technical field of biology, and discloses a method for differential expression analysis of tannin synthesis related genes in eucalyptus grandis, which comprises the following steps: dividing eucalyptus grandis into four groups according to the tannin content condition according to a preliminary survey result, sampling, taking 200g of leaves from each single plant, wrapping the leaves with tin foil paper, and putting the wrapped leaves into a liquid nitrogen box; extracting tannin of the eucalyptus grandis; tannin is used for carrying out an antibacterial test on each group; qPCR (quantitative polymerase chain reaction) experiments are carried out on the basis of known gene sequences of CHS, CH I, F3H and LAR families and published Eucalyptus grandis genomes, and transcription expression conditions of the gene families are verified; based on qPCR experimental results of CHS, CH I, F3H and LAR families, quantitative expression conditions of tannin synthesis related genes in eucalyptus grandis are analyzed, and the relationship between the quantitative expression conditions and tannin characters is discussed. The method has the advantage that the improvement process of tannin synthesis related genes in eucalyptus grandis is accelerated, so that the tannin content of eucalyptus grandis is increased.

Description

Technical Field

[0001] The invention relates to the technical field of biology, and in particular to a method for differential expression analysis of tannin synthesis-related genes in Eucalyptus grandis. Background Art

[0002] Tannins are important secondary metabolites in plants, with significant biological activity and economic value, and are widely used in medicine, food, cosmetics and other fields. Eucalyptus grandis has become an important economic forest due to its rapid growth and strong adaptability, but its low tannin content limits its application potential in related industries. Research and improvement of the tannin synthesis ability of Eucalyptus grandis has important theoretical and practical significance.

[0003] Current research mainly focuses on the functional analysis and expression regulation mechanism of tannin synthesis-related genes, and multiple genes have been found to be involved in tannin synthesis. However, the differential expression analysis of tannin synthesis-related genes in Eucalyptus grandis is still relatively lacking and lacks systematic data support. This leads to our insufficient understanding of the key genes in the tannin synthesis process of Eucalyptus grandis, which in turn affects the effectiveness of its improvement work. Therefore, it is necessary to design a method for differential expression analysis of tannin synthesis-related genes in Eucalyptus grandis to accelerate the improvement process of tannin synthesis-related genes in Eucalyptus grandis, thereby increasing its tannin content. Summary of the invention

[0004] 1. Technical issues to be solved

[0005] In view of the deficiencies in the prior art, the present invention provides a method for differential expression analysis of tannin synthesis-related genes in Eucalyptus grandis, which has the advantages of accelerating the improvement process of tannin synthesis-related genes in Eucalyptus grandis, thereby increasing its tannin content, and solves the problems in the above-mentioned background technology.

[0006] (II) Technical solution

[0007] In order to achieve the above-mentioned purpose of accelerating the improvement process of tannin synthesis-related genes in Eucalyptus grandis, thereby increasing its tannin content, the present invention provides the following technical solution: a method for differential expression analysis of tannin synthesis-related genes in Eucalyptus grandis, comprising the following steps:

[0008] S1: Based on the preliminary investigation results, Eucalyptus grandis was divided into 4 groups according to the tannin content for sampling, with 4 biological replicates in each group. 200g of leaves were taken from each plant, and the leaves were wrapped in tin foil and placed in a liquid nitrogen box;

[0009] S2: Extracting tannin from Eucalyptus grandis;

[0010] S3: Antibacterial test using tannins in each group;

[0011] S4: qPCR experiments were performed based on the known gene sequences of CHS, CH I, F3H, and LAR families and the published genome of Eucalyptus grandis to verify the transcriptional expression of these gene families;

[0012] S5: Based on the qPCR experimental results of CHS, CHI, F3H, and LAR families, the quantitative expression of tannin synthesis-related genes in Eucalyptus grandis was analyzed to explore their relationship with tannin traits.

[0013] Preferably, S2 further comprises:

[0014] S21: Take dry Eucalyptus grandis 1-2cm 2 The fragmented raw materials are crushed and passed through a 50-mesh sieve, and the coarse powder is collected for later use;

[0015] S22: Weigh a certain amount of coarse powder, add 60% ethanol with a solid-liquid ratio of 1:4 to soak, and add 0.2% biological enzyme cellulase and hemicellulase based on the weight of the coarse powder, and soak for enzymolysis at a pH of 5 and a temperature of 50° C. for 2 hours;

[0016] S23: placing the above enzymatic hydrolysate in a constant temperature water bath at 50° C. for 100 minutes and concentrating;

[0017] S24: passing the obtained concentrated solution through a macroporous adsorption resin column WLD, first eluting with 4 column volumes of anhydrous sodium sulfate, then eluting with 70% ethanol, and collecting the ethanol eluate;

[0018] S25: Recover the reagent, the yellow or brown powdery substance is tannin;

[0019] S26: Obtain the tannin content by UV detection.

[0020] Preferably, S3 further comprises:

[0021] S31: Weigh 33g of nutrient agar, add 1L of distilled water, stir, heat and boil until completely dissolved, divide into conical bottles, and sterilize at 121℃ for 20 minutes. When the nutrient agar cools to 45℃, pour 15ml of nutrient agar into each culture dish on a clean workbench and leave it to solidify. Use a sterilized L-shaped glass rod to evenly spread the bacteria on the surface of the nutrient agar, then invert the culture dish in a constant temperature box at 37℃ and culture for 48 hours;

[0022] S32: On a clean workbench, add the same concentration of bacteria and 5 groups of different concentrations of tannins into the culture solution, place it in a constant temperature box at 37°C, and culture it for 24 hours. Set up a blank control and conduct 3 groups of experiments in parallel. Count the number of bacteria in each drop of culture solution under a microscope and compare the number of bacteria at each concentration with the blank group.

[0023] S33: According to the results of the antibacterial test, the MIC50 of the tannin was calculated using the formula to obtain the inhibition rate of bacteria.

[0024] Preferably, the S33 further comprises:

[0025] MIC 50 =log -1 [X m -i(∑P-0.5)]

[0026] Among them, X m represents the logarithmic value of the maximum dose, i represents the absolute value of the difference between two adjacent logarithmic doses, and ∑P represents the overall inhibition rate.

[0027] Preferably, the S4 further comprises:

[0028] S41: The reagents used in the experiment include polysaccharide and polyphenol plant total RNA extraction kit, PrimeScript TM RT kit, TransStart Top Green qPCR SuperMix, 2×T5 Super PCR Mix, DL2000Plus DNA Marker (Vazyme), and anhydrous ethanol;

[0029] S42: The instruments and equipment used in the experiment include analytical balance, fluorescence quantitative PCR instrument, desktop high-speed centrifuge, gel imager, electrophoresis tank, electrophoresis instrument, visible spectrophotometer, 4℃ and -20℃ refrigerators, and -80℃ ultra-low temperature refrigerator.

[0030] Preferably, S4 further comprises the following experimental steps:

[0031] Nucleic acid extraction: extracting target DNA or RNA from samples;

[0032] Reverse transcription process: converting the extracted RNA into the corresponding cDNA;

[0033] Sample purification: purify the extracted DNA or cDNA to remove contaminants;

[0034] Primer and probe design: Design specific primers and fluorescent probes to specifically bind to the target sequence;

[0035] PCR cycle implementation: Place the reaction system into a thermal cycler and perform PCR cycles to monitor DNA denaturation, primer binding, amplification, and changes in fluorescence signals;

[0036] Data analysis: According to the changes in fluorescence signals, analyze the Ct value and calculate the relative quantity of the target sequence.

[0037] Preferably, the S5 further comprises:

[0038] S51: Investigate the tannin content of Eucalyptus grandis. According to the tannin content, Eucalyptus grandis was divided into 4 groups for sampling, with 4 biological replicates in each group, and the tannin concentration, antibacterial effect and related characteristics were analyzed;

[0039] S52: Quantitative information on tannin synthesis-related genes CHS, CH I, F3H, and LAR in Eucalyptus grandis was obtained through qPCR experiments. Samples were taken from four groups of Eucalyptus grandis materials showing obvious differences in tannin traits, RNA was extracted, and qPCR experiments were performed to obtain the expression of related genes CHS, CH I, F3H, and LAR in different Eucalyptus grandis families;

[0040] S53: Analysis of the phylogenetic relationships, DNA duplication, colinearity, and gene functions of the CHS, CH I, F3H, and LAR gene families in Eucalyptus grandis;

[0041] S54: To explore the relationship between the main components of Eucalyptus grandis tannins and the genes related to tannin synthesis, and to identify the main genes for tannin synthesis and their modes of action.

[0042] (III) Beneficial effects

[0043] Compared with the prior art, the present invention provides a method for differential expression analysis of tannin synthesis-related genes in Eucalyptus grandis, which has the following beneficial effects:

[0044] The present invention uses known genes related to tannin synthesis to analyze the tannin trait data of Eucalyptus grandis and the expression of tannin-related synthesis gene families CHS, CHI, F3H, and LAR, and uses this as a starting point to verify the real-time quantitative expression of these genes. This method can directly reveal the relationship between tannin traits and the expression of its synthesis genes, thereby efficiently and accurately identifying the main effect genes of tannin synthesis in Eucalyptus grandis. This efficient and direct research idea is the innovation and feature of this study. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Schematic diagram of the method of the present invention. DETAILED DESCRIPTION

[0046] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0047] Example 1

[0048] The present invention provides a technical solution: a method for differential expression analysis of tannin synthesis-related genes in Eucalyptus grandis, comprising the following steps:

[0049] S1: Based on the preliminary investigation results, Eucalyptus grandis was divided into 4 groups according to the tannin content for sampling, with 4 biological replicates in each group. 200g of leaves were taken from each plant, and the leaves were wrapped in tin foil and placed in a liquid nitrogen box;

[0050] S2: Extracting tannin from Eucalyptus grandis;

[0051] S3: Antibacterial test using tannins in each group;

[0052] S4: qPCR experiments were performed based on the known gene sequences of CHS, CHI, F3H, and LAR families and the published genome of Eucalyptus grandis to verify the transcriptional expression of these gene families;

[0053] S5: Based on the qPCR experimental results of CHS, CHI, F3H, and LAR families, the quantitative expression of tannin synthesis-related genes in Eucalyptus grandis was analyzed to explore their relationship with tannin traits.

[0054] S2 further includes:

[0055] S21: Take dry Eucalyptus grandis 1-2cm 2 The fragmented raw materials are crushed and passed through a 50-mesh sieve, and the coarse powder is collected for later use;

[0056] S22: Weigh a certain amount of coarse powder, add 60% ethanol with a solid-liquid ratio of 1:4 to soak, and add 0.2% biological enzyme cellulase and hemicellulase based on the weight of the coarse powder, and soak for enzymolysis at a pH of 5 and a temperature of 50° C. for 2 hours;

[0057] S23: placing the above enzymatic hydrolysate in a constant temperature water bath at 50° C. for 100 minutes and concentrating;

[0058] S24: passing the obtained concentrated solution through a macroporous adsorption resin column WLD, first eluting with 4 column volumes of anhydrous sodium sulfate, then eluting with 70% ethanol, and collecting the ethanol eluate;

[0059] S25: Recover the reagent, the yellow or brown powdery substance is tannin;

[0060] S26: Obtain the tannin content by UV detection.

[0061] S3 further includes:

[0062] S31: Weigh 33g of nutrient agar, add 1L of distilled water, stir, heat and boil until completely dissolved, divide into conical bottles, and sterilize at 121℃ for 20 minutes. When the nutrient agar cools to 45℃, pour 15ml of nutrient agar into each culture dish on a clean workbench and leave it to solidify. Use a sterilized L-shaped glass rod to evenly spread the bacteria on the surface of the nutrient agar, then invert the culture dish in a constant temperature box at 37℃ and culture for 48 hours;

[0063] S32: On a clean workbench, add the same concentration of bacteria and 5 groups of different concentrations of tannins into the culture solution, place it in a constant temperature box at 37°C, and culture it for 24 hours. Set up a blank control and conduct 3 groups of experiments in parallel. Count the number of bacteria in each drop of culture solution under a microscope and compare the number of bacteria at each concentration with the blank group.

[0064] S33: According to the results of the antibacterial test, the MIC50 of the tannin was calculated using the formula to obtain the inhibition rate of bacteria.

[0065] S33 further comprises:

[0066] MIC 50 =log -1 [X m -i(∑P-0.5)]

[0067] Among them, X m represents the logarithmic value of the maximum dose, i represents the absolute value of the difference between two adjacent logarithmic doses, and ∑P represents the total inhibition rate. S4 further includes: S41: The reagents used in the experiment include polysaccharide and polyphenol plant total RNA extraction kit, PrimeScript TMRT kit, TransStart Top Green qPCR SuperMix, 2×T5 SuperPCR Mix, DL2000Plus DNA Marker (Vazyme) and anhydrous ethanol; S42: The instruments and equipment used in the experiment include analytical balance, fluorescence quantitative PCR instrument, desktop high-speed centrifuge, gel imager, electrophoresis tank, electrophoresis instrument, visible spectrophotometer, 4℃ and -20℃ refrigerators, and -80℃ ultra-low temperature refrigerators. S4 further includes the following experimental steps: Nucleic acid extraction: extracting target DNA or RNA from the sample; Reverse transcription process: converting the extracted RNA into the corresponding cDNA; Sample purification: purifying the extracted DNA or cDNA to remove contaminants; Primer and probe design: designing specific primers and fluorescent probes for specific binding of target sequences; PCR cycle implementation: placing the reaction system in a thermal cycler and performing PCR cycles to monitor DNA denaturation, primer binding, amplification and changes in fluorescence signals; Data analysis: analyzing Ct values ​​based on changes in fluorescence signals and calculating the relative number of target sequences. S5 further includes: S51: Investigate the tannin content of Eucalyptus grandis, divide Eucalyptus grandis into 4 groups according to the tannin content for sampling, with 4 biological replicates in each group, and analyze the tannin concentration, antibacterial effect and related characteristics; S52: Obtain quantitative information on tannin synthesis-related genes CHS, CHI, F3H, and LAR in Eucalyptus grandis through qPCR experiments, take samples from four groups of Eucalyptus grandis materials showing obvious differences in tannin traits, extract RNA, and conduct qPCR experiments to obtain the expression of related genes CHS, CHI, F3H, and LAR in different Eucalyptus grandis families;

[0068] S53: Analysis of the phylogenetic relationships, DNA duplication, colinearity, and gene functions of the CHS, CH I, F3H, and LAR gene families in Eucalyptus grandis;

[0069] S54: To explore the relationship between the main components of Eucalyptus grandis tannins and the genes related to tannin synthesis, and to identify the main genes for tannin synthesis and their modes of action.

[0070] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0071] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for differential expression analysis of tannin synthesis-related genes in Eucalyptus grandis, characterized in that: The following steps are involved: S1: Based on the preliminary investigation results, Eucalyptus grandis was divided into 4 groups according to the tannin content for sampling, with 4 biological replicates in each group. 200g of leaves were taken from each plant, and the leaves were wrapped in tin foil and placed in a liquid nitrogen box; S2: Extracting tannin from Eucalyptus grandis; S3: Antibacterial test using tannins in each group; S4: qPCR experiments were performed based on the known gene sequences of CHS, CHI, F3H, and LAR families and the published genome of Eucalyptus grandis to verify the transcriptional expression of these gene families; S5: Based on the qPCR experimental results of CHS, CHI, F3H, and LAR families, the quantitative expression of tannin synthesis-related genes in Eucalyptus grandis was analyzed to explore their relationship with tannin traits.

2. The method for differential expression analysis of tannin synthesis-related genes in Eucalyptus grandis according to claim 1, characterized in that: The S2 further comprises: S21: Take dry Eucalyptus grandis 1-2cm 2 The fragmented raw materials are crushed and passed through a 50-mesh sieve, and the coarse powder is collected for later use; S22: Weigh a certain amount of coarse powder, add 60% ethanol with a solid-liquid ratio of 1:4 to soak, and add 0.2% biological enzyme cellulase and hemicellulase based on the weight of the coarse powder, and soak for enzymolysis at a pH of 5 and a temperature of 50° C. for 2 hours; S23: placing the above enzymatic hydrolysate in a constant temperature water bath at 50° C. for 100 minutes and concentrating; S24: passing the obtained concentrated solution through a macroporous adsorption resin column WLD, first eluting with 4 column volumes of anhydrous sodium sulfate, then eluting with 70% ethanol, and collecting the ethanol eluate; S25: Recover the reagent, the yellow or brown powdery substance is tannin; S26: Obtain the tannin content by UV detection.

3. The method for differential expression analysis of tannin synthesis-related genes in Eucalyptus grandis according to claim 1, characterized in that: The S3 further comprises: S31: Weigh 33g of nutrient agar, add 1L of distilled water, stir, heat and boil until completely dissolved, divide into conical bottles, and sterilize at 121℃ for 20 minutes. When the nutrient agar cools to 45℃, pour 15ml of nutrient agar into each culture dish on a clean workbench and leave it to solidify. Use a sterilized L-shaped glass rod to evenly spread the bacteria on the surface of the nutrient agar, then invert the culture dish in a constant temperature box at 37℃ and culture for 48 hours; S32: On a clean workbench, add the same concentration of bacteria and 5 groups of different concentrations of tannins into the culture solution, place it in a constant temperature box at 37°C, and culture it for 24 hours. Set up a blank control and conduct 3 groups of experiments in parallel. Count the number of bacteria in each drop of culture solution under a microscope and compare the number of bacteria at each concentration with the blank group. S33: According to the results of the antibacterial test, the MIC50 of the tannin was calculated using the formula to obtain the inhibition rate of bacteria.

4. The method for differential expression analysis of tannin synthesis-related genes in Eucalyptus grandis according to claim 1, characterized in that: The S33 further comprises: MIC 50 =log -1 [X m -i(∑P-0.5)] Among them, X m represents the logarithmic value of the maximum dose, i represents the absolute value of the difference between two adjacent logarithmic doses, and ∑P represents the overall inhibition rate.

5. The method for differential expression analysis of tannin synthesis-related genes in Eucalyptus grandis according to claim 1, characterized in that: The S4 further comprises: S41: The reagents used in the experiment include polysaccharide and polyphenol plant total RNA extraction kit, PrimeScript TM RT reagent kit, TransStart Top Green qPCR SuperMix, 2×T5 Super PCR Mix, DL2000 Plus DNA Marker (Vazyme), and anhydrous ethanol; S42: The instruments and equipment used in the experiment include analytical balance, fluorescence quantitative PCR instrument, desktop high-speed centrifuge, gel imager, electrophoresis tank, electrophoresis instrument, visible spectrophotometer, 4℃ and -20℃ refrigerators, and -80℃ ultra-low temperature refrigerator.

6. The method for differential expression analysis of tannin synthesis-related genes in Eucalyptus grandis according to claim 1, characterized in that: The S4 further comprises the following experimental steps: Nucleic acid extraction: extracting target DNA or RNA from samples; Reverse transcription process: converting the extracted RNA into the corresponding cDNA; Sample purification: purify the extracted DNA or cDNA to remove contaminants; Primer and probe design: Design specific primers and fluorescent probes to specifically bind to the target sequence; PCR cycle implementation: Place the reaction system into a thermal cycler and perform PCR cycles to monitor DNA denaturation, primer binding, amplification, and changes in fluorescence signals; Data analysis: According to the changes in fluorescence signals, analyze the Ct value and calculate the relative quantity of the target sequence.

7. The method for differential expression analysis of tannin synthesis-related genes in Eucalyptus grandis according to claim 1, characterized in that: The S5 further comprises: S51: Investigate the tannin content of Eucalyptus grandis. According to the tannin content, Eucalyptus grandis was divided into 4 groups for sampling, with 4 biological replicates in each group, and the tannin concentration, antibacterial effect and related characteristics were analyzed; S52: Quantitative information on tannin synthesis-related genes CHS, CHI, F3H, and LAR in Eucalyptus grandis was obtained through qPCR experiments. Samples were taken from four groups of Eucalyptus grandis materials showing obvious differences in tannin traits, RNA was extracted, and qPCR experiments were performed to obtain the expression of related genes CHS, CHI, F3H, and LAR in different Eucalyptus grandis families; S53: Analysis of the phylogenetic relationships, DNA duplication and colinearity, and gene functions of the CHS, CHI, F3H, and LAR gene families in Eucalyptus grandis; S54: To explore the relationship between the main components of Eucalyptus grandis tannins and the genes related to tannin synthesis, and to identify the main genes for tannin synthesis and their modes of action.

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