CN117660493A - Gene for regulating and controlling morchella flavor and screening method thereof - Google Patents

Abstract

A gene for regulating and controlling the flavor of Morchella and a screening method thereof are provided, wherein the nucleotide sequence of the gene for regulating and controlling the flavor of Morchella is shown as SEQ ID No. 1. The invention screens the related genes of the Morchella flavor through different culture temperatures, and can be used for developing the Morchella flavor enhancer to exert the Morchella flavor enhancing effect.

Description

Gene for regulating and controlling morchella flavor and screening method thereof

Technical Field

The invention belongs to the technical fields of molecular biology and edible fungi, and relates to a gene for regulating and controlling Morchella flavor and a screening method thereof.

Background

Morchella spp is a rare edible and medicinal fungus, is crispy and delicious, has special flavor, and is rich in proteins, vitamins and the like, polysaccharides and a plurality of bioactive components. Morchella artificial cultivation has a history of centuries, but still faces the problems of difficult quality assurance, unstable yield and the like. In recent years, china gradually forms a morchella field ecological cultivation mode, and standardized cultivation of morchella is started. The environmental temperature can not be accurately regulated during field cultivation, and the temperature stress has a remarkable influence on the growth of Morchella.

Morchella is a low temperature mushroom, and the temperature is one of the key factors for the growth and development of Morchella. The temperature required by Morchella is lower in the growth process, the temperature difference is larger, and the mycelium differentiation is conveniently stimulated. Studies have shown that low temperature stress causes oxidative damage to edible fungi, and in the low temperature stress process, the activities of superoxide dismutase (superoxide dismutase, SOD) are increased and decreased in the early and later stages, the activities of Catalase (CAT) and ascorbate peroxidase (aseorbate peroxidase, APX) are increased as a whole, and the content of glutathione (reduced glutathione, GSH) is increased; heat stress causes plants to produce antioxidants, accumulate and regulate compatible solutes, induce mitogen-activated protein kinase (MAPK) and calcium-dependent protein kinase (CDPK) cascades, and the like. These controls are achieved through complex interactions between members of different gene families, such as involving cell signaling pathways, and the like. In the prior art, the influence of temperature and the like on the growth of edible fungi is generally studied by a transcriptome method.

Transcriptome refers to the complete transcripts and transcript numbers formed at a certain developmental stage or under a certain physiological condition in a cell, consisting mainly of ribosomal RNA (80% -90%, rRNA), transfer RNA (5% -15%, tRNA), messenger RNA (2% -4%, mRNA) and non-coding RNA (1%, ncRNA) within or between genes. The histology technique has high sensitivity and wide application range, and is favored by researchers. The application of the histology technology in the edible fungi research is a great breakthrough in the edible fungi research, so that the edible fungi research is changed from artificial cultivation to basic research. Among the different tissue types of edible fungi, these transcriptome components have respective unique biological functions. Transcriptomics research on different types of edible fungi can help people to better reveal the action mode and the regulation and control process of functional genes, so that edible fungus breeding selection and cultivation practice are improved.

Disclosure of Invention

The invention aims to provide a gene for regulating and controlling Morchella flavor and a screening method thereof.

To achieve the above and other related objects, the present invention provides the following technical solutions: a gene for regulating and controlling the flavor of Morchella is provided, and the nucleotide sequence of the gene for regulating and controlling the flavor of Morchella is shown as SEQ ID No. 1.

To achieve the above and other related objects, the present invention provides the following technical solutions: a screening method of a gene for regulating and controlling Morchella flavor comprises the following steps:

step 1: taking Morchella pieces with uniform diameter after activation, transferring the Morchella pieces to a culture medium center, and culturing at different temperatures for the same time;

step 2: calculating the growth speed of the hypha according to the diameter of the colony marked by the extension degree of the hypha to the flat plate;

step 3: observing microscopic morphology of hyphae using an inverted microscope;

step 4: collecting Morchella mycelium at different temperatures, extracting RNA, and performing transcriptome sequencing;

step 5: comparing and analyzing the sequencing sequence with a designated Morchella reference genome, and counting the expression level and annotation information of genes to obtain differential expression genes of mycelia at different culture temperatures;

step 6: constructing a hierarchical clustering tree of the differential expression genes by utilizing weighted gene co-expression network analysis, and exploring the association relation between different gene modules and mycelium culture temperature in the clustering tree;

step 7: and constructing a collinear network diagram based on the interrelation among genes in each module, and excavating Morchella flavor related genes involved in temperature regulation.

Due to the application of the technical scheme, compared with the prior art, the invention has the advantages that:

morchella is taken as a precious edible fungus resource, is fragrant, crisp and delicious, has delicious taste, has flavor, is rich in nutrition, is rich in protein, vitamins and the like, polysaccharides and a plurality of bioactive components, and is increasingly favored by consumers. The flavor is determined by the balance and interaction of the various different tasting free amino acids. The amino acid is one of the characteristic flavor compounds of Morchella esculenta, and Morchella esculenta mycelium is rich in amino acid. Morchella is a low temperature mushroom, and the temperature is one of the key factors for the growth and development of Morchella. At present, less reports are reported on Morchella flavor control by the culture temperature. The invention screens the related genes of the Morchella flavor through different culture temperatures, and can be used for developing the Morchella flavor enhancer to exert the Morchella flavor enhancing effect.

Drawings

FIG. 1 shows a comparison of hypha growth rates of test strains at different temperatures.

FIG. 2 shows the microscopic hyphae morphology of the test strain at different temperatures. H1, H5, H6, H8, H18, M4, M15, M24, MZ-14, MZ-20, MZ-22, T6 and T7 are sequentially arranged from top to bottom; from left to right, 5 ℃,10 ℃, 15 ℃, 20 ℃, 25 ℃, 30 ℃.

FIG. 3 shows a graph of a gene coliform network of negative regulation of Morchella mycelium by culture temperature.

Detailed Description

Further advantages and effects of the present invention will become apparent to those skilled in the art from the disclosure of the present invention, which is described by the following specific examples.

Please refer to fig. 1-3. It should be understood that the structures, proportions, sizes, etc. shown in the drawings are for illustration purposes only and should not be construed as limiting the invention to the extent that it can be practiced, since modifications, changes in the proportions, or otherwise, used in the practice of the invention, are not intended to be critical to the essential characteristics of the invention, but are intended to fall within the spirit and scope of the invention. Also, the terms such as "upper," "lower," "left," "right," "middle," and "a" and the like recited in the present specification are merely for descriptive purposes and are not intended to limit the scope of the invention, but are intended to provide relative positional changes or modifications without materially altering the technical context in which the invention may be practiced.

Example 1: gene for regulating and controlling morchella flavor and screening method thereof

1. Screening the optimal temperature range for the growth of Morchella mycelium

(1) Six sister (m.sexteleata), seven sister (m.septimeleata) and ladder (m.importuna) were selected for a total of 13 strains of Morchella strains tested, and each strain was transferred from the tube to PDA medium on an ultra clean bench with a sterile inoculating needle for activation.

(2) Taking Morchella esculenta blocks with the diameter of 5mm×5mm, placing in the center of PDA culture medium with the diameter of 90mm, and culturing in dark constant temperature incubator at 5 deg.C, 10 deg.C, 15 deg.C, 20 deg.C, 25 deg.C and 30 deg.C respectively.

(3) The extent of mycelium extension to the medium at each culture temperature was recorded for 24h and 48h, for calculating the mycelium growth rate in each treatment group, i.e. mycelium growth rate = (48 h measured diameter-24 h measured diameter)/d, 5 replicates per treatment.

(4) Six temperature-treated groups of medium plates were placed under an inverted microscope (zeiss LSM 880) and the branching of the hyphae was observed at 40 x magnification, 5 replicates per treatment group.

(5) And (3) performing significant difference analysis on the data by adopting SPSS (version 26.0) software, and determining that the optimal culture temperature for hypha growth of 13 tested Morchella strains is 15-20 ℃ according to the growth speed and hypha bifurcation condition.

2. Screening temperature-regulated flavor genes in Morchella mycelium

(1) Taking the test strain H6 Hesister (M.sextelata) as a representative Morchella strain, respectively culturing at 5 deg.C, 10 deg.C, 15 deg.C, 20 deg.C, 25 deg.C and 30 deg.C for 5 days;

(2) Mycelium in the culture medium of each temperature treatment group was collected by a sterile spatula, placed in a sterile bag, and dried in an oven, 3 replicates per treatment group.

(3) The dried mycelium was placed in liquid nitrogen and ground to a powder, 100mg of the powder was placed in a 1.5mL centrifuge tube without RNase. 1mL Trizol was added, and after shaking vigorously for 30 seconds, the mixture was allowed to stand on ice for 3 minutes to allow the mycelium cells to lyse sufficiently.

(4) 200. Mu.L of chloroform was added, the tube cap was closed, and after shaking vigorously for 15 seconds, the mixture was allowed to stand on ice for 5 minutes, and the mixture was centrifuged at 12000rpm at 4℃for 15 minutes to separate phases.

(5) Transfer 500. Mu.L of the upper aqueous phase into a new centrifuge tube, add 500. Mu.L of isopropanol, mix well, stand on ice for 10min, centrifuge at 10000rpm, centrifuge at 4℃for 10min to precipitate RNA.

(6) 1mL of 75% ethanol was added and rinsed 3 times, and the RNA was washed by centrifugation at 7000rpm at 4℃for 5min and the supernatant was removed.

(7) The RNA pellet was dried in an ultra clean bench at room temperature, and then 50. Mu.L of DEPC water was added for sufficient dissolution to obtain mycelium RNA for each treatment group.

(8) The concentration and purity of the extracted RNA are detected by using a Nanodrop2000, the integrity of the RNA is detected by agarose gel electrophoresis, the RIN value is measured by Agilent5300, and the RNA is stored at-80 ℃.

(9) mRNA was isolated from total RNA of each treatment group using Oligo dT-bearing magnetic beads. Fragmentation buffer is added to randomly break mRNA into small fragments of about 300 bp.

(10) The mRNA is used as a template to reversely synthesize cDNA under the action of reverse transcriptase. Then, an End pair Mix was added to make the cohesive End of the double-stranded cDNA blunt, and an A base was added to the 3' -End to join the Y-adaptor. The product after ligation to the adapter was enriched using Illumina NovaSeq Reagent Kit (Illumina, san Diego, CA, USA) and quantitated using 2% agarose to recover the target band for Qubit 4.0 to give the final library, which was then sequenced on the Illumina NovaSeq6000 platform.

(11) And performing data quality control on the original sequence by utilizing Trimmemic (version 0.36) software, including removing reads without inserted fragments or with the N content exceeding 10%, and removing sequences with quality values smaller than 10 or lengths smaller than 20bp, so as to obtain optimized high-quality data clean data.

(12) Comparing the clear data after quality control with Morchella reference genome (GCF_020137385.1_ASM2013738v1), and counting the expression level and function annotation information of the genes.

(13) Mycelium cultured at 20 ℃ is used as a control group, and the differential expression genes of the mycelium between the culture temperatures are obtained.

(14) Constructing a hierarchical clustering tree of the differential expression genes by utilizing weighted gene co-expression network analysis (WGCNA), searching a synergistically expressed gene module according to different branches of the clustering tree, and exploring the association relation between each gene module and mycelium culture temperature.

(15) And constructing a collinear network diagram based on the interrelation among genes in each module, displaying the regulation and control relation of the genes in the modules, and excavating Morchella flavor related genes involved in temperature regulation and control.

3. Analysis of results

(1) Test strain

TABLE 1 sources of test strains

(2) Effect of different temperature treatments on mycelium growth Rate

TABLE 2 hypha growth rates of test strains at different temperatures

Note that: data are expressed as mean ± variance (n=10); different lower case letters indicate that the difference between different temperatures of the same strain is significant (P < 0.5)

FIG. 1 is a bar graph of growth rates of different Morchella strains at the same temperature, and it can be seen from the graph that the growth rate difference of each strain is obvious below 20 ℃, and the growth rate difference of each strain becomes smaller when the temperature is above 20 ℃. At 20℃the growth rate of 9 strains reached 14.00mm/d.

(3) Influence of different temperature treatments on microcosmic morphology of Morchella mycelium

Overall, morchella mycelium tip bifurcation increased with increasing temperature (fig. 2). When the temperature is low, the mycelium is loose and straight, and as the temperature is increased, the mycelium is bent and kinked, and the mycelium density is increased. Taking H8 as an example, hypha is straight at 5 ℃, front branches are shorter, and the number of branches is small; the branches at the front end of the mycelium are prolonged at 10 ℃ and the number of branches is small; the same is true at 15 ℃. When the temperature is low, hyphae are supposed to maintain vitality, and concentrated growth reduces branches. The number of branches of hyphae is increased at 20 ℃, and the density of the hyphae is increased; the number of the mycelium branches is the greatest at 25 ℃, the mycelium is the thickest, the temperature is higher at the moment, the mycelium branches rapidly, and the adaptability of the mycelium to the environment is improved. The hypha bending phenomenon is obvious at 30 ℃.

(4) Differential expression genes of mycelium in different temperature treatment groups

Taking 20 ℃ group as a control group, wherein the total number of 10 ℃ groups is 2334, and the up-regulated expression is 669 and the time-regulated expression is 1665; 15 ℃ group total 1950, with up-regulated expression 752, time-regulated expression 1198; a total of 1655 in 25 ℃ group, 1129 of which are up-regulated and 526 of which are time-regulated; the 30℃group had a total of 1861, of which 1299 were up-regulated and 562 were time-regulated.

TABLE 3 differential expression genes for different temperature treatment groups

(5) WGCNA analysis

739 genes (r= -0.896, p < 0.001) were obtained in total by WGCNA, which were significantly inversely related to the culture temperature. A collinear network diagram is constructed for these negative regulatory genes, as shown in FIG. 3. The collinear network diagram consists of 122 nodes and 4,710 links, all of which are divided into three main modules, namely module I, module II and module III. The three modules contained 70, 38 and 14 genes, respectively (table 4). By gene function annotation, the gene H2S33_ 001094 (flavo protein WrbA) related to Morchella flavor is found in module III, and the nucleotide sequence of the gene H2S33_ 001094 is shown as SEQ ID No. 1. This result indicates that the flavor of Morchella decreases with increasing mycelium culture temperature.

TABLE 4 details of Gene function of each Module

4. Morchella is taken as a precious edible fungus resource, is fragrant, crisp and delicious, has delicious taste, has flavor, is rich in nutrition, is rich in protein, vitamins and the like, polysaccharides and a plurality of bioactive components, and is increasingly favored by consumers. The flavor is determined by the balance and interaction of the various different tasting free amino acids. The amino acid is one of the characteristic flavor compounds of Morchella esculenta, and Morchella esculenta mycelium is rich in amino acid. Morchella is a low temperature mushroom, and the temperature is one of the key factors for the growth and development of Morchella. At present, less reports are reported on Morchella flavor control by the culture temperature. The embodiment screens the related genes of the morchella flavor through different culture temperatures, and can be used for developing the morchella flavor enhancer to exert the function of improving the flavor of the morchella.

SEQ ID No.1：

Atggctccccgcgtcgctatcattatctactccatgtacgggcacattactcagcttgctgaggccgagaaggctggtatcgaggctgctggtggttctgctaccatcctccagtgagttatgctcaattcctactattgaagcattgcttgcaagctgcttcaaaagtcgattgatctcctaatacttacaatcttttctttcttcagggttcctgagactcttcctgaggaggttcttggaaagatgcacgctgcccccaagcctaactaccccatcattaccgccccggaactgaagaactacgatgccttcctcttcggtatccccactcgttacggaaacttccccggccagtggaaggccttcatcgacaccactggtggcctctggggtgagggtgctctctccggaaagtacgccggtctctttatctctaccggcacccagggcggtggacaggagattactgccttaaacgccatgtccaccctcgcccaccacggaataatctacgttcccttcggatacaagcacgctttccccatccttgcctccaacgaggaggtccgcggtggatccccatggggtgctggtacctttgctgtaagttttctctttgtttgtgaggtttaagttttccccttcagattactgattgcgtctgttacagggtgccgatggttcccgccagccctcagcgaaggagctcgagattgctcagatccatggcaagtccttctacgagactgtcgctcgtgtcaacttcgcctga。

The foregoing description of the preferred embodiment of the invention is not intended to be limiting in any way, but rather, it is intended to cover all modifications or variations of the invention which fall within the spirit and scope of the invention.

Claims (2)

1. A gene for regulating and controlling morchella flavor, which is characterized in that: the nucleotide sequence of the gene for regulating and controlling the flavor of Morchella esculenta is shown as SEQ ID No. 1.

2. A screening method of a gene for regulating and controlling Morchella flavor is characterized by comprising the following steps: comprises the following steps:

step 1: taking Morchella pieces with uniform diameter after activation, transferring the Morchella pieces to a culture medium center, and culturing at different temperatures for the same time;

step 2: calculating the growth speed of the hypha according to the diameter of the colony marked by the extension degree of the hypha to the flat plate;

step 3: observing microscopic morphology of hyphae using an inverted microscope;

step 4: collecting Morchella mycelium at different temperatures, extracting RNA, and performing transcriptome sequencing;

step 5: comparing and analyzing the sequencing sequence with a designated Morchella reference genome, and counting the expression level and annotation information of genes to obtain differential expression genes of mycelia at different culture temperatures;

step 6: constructing a hierarchical clustering tree of the differential expression genes by utilizing weighted gene co-expression network analysis, and exploring the association relation between different gene modules and mycelium culture temperature in the clustering tree;

step 7: and constructing a collinear network diagram based on the interrelation among genes in each module, and excavating Morchella flavor related genes involved in temperature regulation.