CN112501160A

CN112501160A - Cloning method and application of mulberry anthocyanin-3-O-glucoside-2-O-glucuronic acid transferase gene

Info

Publication number: CN112501160A
Application number: CN202011524163.1A
Authority: CN
Inventors: 曾益春; 黄盖群; 刘刚; 危玲; 代洁; 郑继川; 姚永权; 李永远
Original assignee: Sericultural Research Institute of Sichuan Academy of Agricultural Sciences
Current assignee: Sericultural Research Institute of Sichuan Academy of Agricultural Sciences
Priority date: 2020-12-22
Filing date: 2020-12-22
Publication date: 2021-03-16

Abstract

The invention discloses a method for cloning a mulberry anthocyanin-3-O-glucoside-2-O-glucuronic acid transferase gene and application thereof, wherein the cloning method comprises the following steps: mulberry is used for extracting total RNA of a sample and synthesizing a first strand of cDNA; cloning of mulberry MaUGAT gene. The invention also discloses application of the mulberry anthocyanin-3-O-glucoside-2-O-glucuronic acid transferase gene cloned by the method in a mulberry germplasm cultivation process. The invention compares and identifies 1 anthocyanin-3-O-glucoside-2-O-glucuronic acid transferase gene from a Chuan mulberry genome database, and clones the gene from the mulberry of Guangdong mulberry 10 by designing a specific primer.

Description

Cloning method and application of mulberry anthocyanin-3-O-glucoside-2-O-glucuronic acid transferase gene

Technical Field

The invention belongs to the technical field of biology, and relates to a cloning method and application of a mulberry anthocyanin-3-O-glucoside-2-O-glucuronic acid transferase gene.

Background

Anthocyanins (Anthocynin) are one of important pigments for determining the color of plant flowers, fruits, seeds, etc., and belong to flavone polyphenols. In recent years, a great deal of research finds that the anthocyanin has multiple biological activities of resisting inflammation, resisting tumor, regulating blood fat and the like, and shows that the anthocyanin can play an important role in resisting plant stress and preventing human chronic diseases. The development and utilization of mulberry anthocyanins are now hot due to their high content, but the research on the biosynthesis pathway and mechanism of mulberry anthocyanins is still less. Anthocyanin biosynthesis belongs to a part of flavonoid biosynthesis pathway, the pathway comprises a large number of genes and complex functions, and because expression of different genes has difference, synergy and competition exist, so that a metabolite is not anthocyanin.

Shinya S et al identified an anthocyanin-3-O-glucoside-2-O-glucuronic acid transferase (BpUGAT) gene in the anthocyanin biosynthesis pathway of red daisy (Bellis perennis), quantitative PCR analysis showed that the transcription product of the BpUGAT gene could be specifically detected in red petals, consistent with the temporal and spatial distribution of enzyme activity in plants, functional analysis confirmed that it catalyzed the transfer of glucuronic acid residues from UDP-glucuronic acid to the 2 "-hydroxyl group of the 3-glucosyl moiety of anthocyanin 3-O-6" -O-malonyl glucoside, indicating that UGAT gene is downstream of UDP-glucose-flavonoid-3-O-glucosyl transferase gene (UFGT). Research shows that anthocyanin-3-O-glucoside-2-O-glucuronic acid transferase (UGAT) catalyzes anthocyanin-3-O-glucoside to be converted into anthocyanin 3-O- (2-O-beta-D-glucuronic acid group) -beta-D-glucoside, and finally stable anthocyanin substances are formed. Therefore, the anthocyanin-3-O-glucoside-2-O-glucuronic acid transferase can participate in the regulation of the metabolic process of anthocyanin in mulberry, and the anthocyanin has an important regulation effect in the accumulation process of the anthocyanin. However, the research on the cloning and application of the mulberry anthocyanin-3-O-glucoside-2-O-glucuronic acid transferase (MaUGAT) gene is not reported at present.

Disclosure of Invention

The invention aims to provide a cloning method and application of a mulberry anthocyanin-3-O-glucoside-2-O-glucuronic acid transferase gene.

The technical scheme is as follows:

a method for cloning a mulberry anthocyanin-3-O-glucoside-2-O-glucuronic acid transferase gene comprises the following steps:

step 1, extracting total RNA of mulberry test sample and synthesizing cDNA first strand

Grinding a mulberry sample of Guangdong mulberry 10 into powder by using liquid nitrogen, extracting total RNA of the sample according to a standard extraction step of a kit, detecting the extraction quality of the total RNA by using a Nano Drop2000 micro spectrophotometer, calculating the ratio of OD260/OD280, and indicating that the extraction quality of the total RNA is better when the ratio is about 1.8-2.2. Configuring 20 microliter reaction system according to the first chain cDNA synthesis kit operation process, and synthesizing cDNA 1 st chain by reverse transcription.

Step 2, cloning of mulberry MaUGAT gene

Referring to a gene sequence of red daisy (BpUGAT), uploading the gene sequence to a Chuansang genome database (https:// mours. swu. edu. cn/moursdb /) for sequence alignment, and designing a specific primer according to the gene sequence, wherein the specific primer is shown as SEQ ID: NO 1-SEQ ID: NO 2, and the Morus016781 gene with the highest similarity rate is a mulberry (MaUGAT) gene;

PCR amplification is carried out by taking 10 sample mixed cDNA of Guangdong mulberry as template, wherein the amplification reaction system (25 mu L) is 10 XPCR buffer 2.5 mu L, 25mmol/L MgCl₂mu.L of 2. mu.L, 2. mu.L of 20mmol/L dNTP, 1. mu.L of each of 10. mu. mol/L upstream and downstream primers, 1. mu.L of cDNA template, 0.2. mu.L of 5U/. mu.L DNA polymerase, and the balance deionized water. And immediately centrifuging for 15s by using a PCR tube centrifuge at 5000 r/min to ensure that the reaction system is completely mixed and reduce errors. The reaction condition is that the mixture is pre-denatured for 3min at 98 ℃; at 98 ℃ for 10s, at 59 ℃ for 15s, at 72 ℃ for 20s, for 39 cycles; extending for 5min at 72 ℃, and storing at 4 ℃. And (3) carrying out agarose gel electrophoresis on the amplification product, rapidly cutting off an adhesive tape containing a target strip under an ultraviolet lamp compared with a Marker, and recovering by adopting a column type trace agarose gel recovery kit. And connecting the recovered product with a pMD19-T vector, then transforming an escherichia coli competent cell, sequencing the positive clone, and finally obtaining gene sequence information.

The mulberry anthocyanin-3-O-glucoside-2-O-glucuronic acid transferase gene cloned by the method disclosed by the invention is applied to the mulberry germplasm cultivation process.

Step 1, extracting total RNA of mulberry test sample

1) Mulberry tissue samples were ground to a powder in liquid nitrogen, immediately added to 1ml Trizol and thoroughly mixed for lysis.

2) 200ul of chloroform was added thereto, followed by vigorous shaking and mixing, and centrifugation at 12000rpm at 4 ℃ for 10 minutes.

3) And (3) taking the supernatant, adding the same volume of phenol-formaldehyde, fully oscillating and uniformly mixing, and centrifuging at 12000rpm at 4 ℃ for 10 minutes.

4) The supernatant was taken, added with chloroform of equal volume, sufficiently shaken and mixed, and centrifuged at 12000rpm at 4 ℃ for 10 minutes.

5) The supernatant was taken, added with an equal volume of isopropanol, mixed by inversion and precipitated at-20 ℃ for 1 hour. Centrifuge at 12000rpm for 15 minutes at 4 ℃.

6) The supernatant was discarded, and 0.5ml of 75% ethanol was added thereto at 4 ℃ 8000-.

7) And repeating the previous step.

8) The supernatant was discarded and centrifuged briefly, and the ethanol was blotted off with a pipette and dried in vacuo for 2 min.

9) Adding 20ul RNA-Free water, dissolving at room temperature for 5min, mixing, and centrifuging to obtain RNA solution.

Step 2, reverse transcription

A20. mu.l reaction was set up with 50ng-2ug total RNA according to the reverse transcription kit (R223) instructions.

1) Thawing mulberry RNA on ice; 4 XgDNA wiper Mix, 5 XHiScript | qRT Supermix |, RNase-Free ddH₂O was thawed at room temperature (15-25 ℃ C.), and placed on ice immediately after thawing. Each solution was vortexed, shaken well before use, and briefly centrifuged to collect the residual liquid on the tube wall.

2) A mixed solution was prepared according to the genomic DNA removal system of Table 1, and thoroughly mixed. Briefly centrifuged and incubated at 42 ℃ for 2 min. Then placed on ice.

TABLE 1 gDNA removal reaction System

Composition of matter	Amount of the composition used
		4×gDNA wiper Mix	4μl
Total RNA	The total amount is not more than 2ug
		RNase-Free ddH₂O	Make up to 16. mu.l

3) A mixed solution was prepared according to the reverse transcription reaction system of Table 2.

TABLE 2 reverse transcription reaction System

Composition of matter	Amount of the composition used
		Reaction solution for gDNA removal step	16μl
5×HiScript‖qRT SuperMix‖	4μl

4) Mixing well.

5) Incubate at 50 ℃ for 50 min.

6) The cDNA obtained after incubation for 5min at 95 ℃ was used for the subsequent experiments.

Step 3, quantitative analysis and application of gene

1) A20. mu.l reaction system was prepared according to the following table

2) Reaction conditions

Pre-denaturation at 95 deg.C for 90s, (denaturation at 95 deg.C for 5s, denaturation at 60 deg.C for 15s, denaturation at 72 deg.C for 20s, and 40 cycles), extension at 72 deg.C for 5min, and storage at 4 deg.C.

3) And (3) melting curve analysis: the temperature is 65-95 ℃.

Step 4, data calculation

Experiment according to 2^-△△CtThe method processes the data. Wherein, delta Ct (target gene average Ct-reference average Ct); delta. DELTA. Ct ═

(target gene delta Ct in the sample to be tested-target gene delta Ct in the reference sample).

Δ Ct mean ═ AVERAGE (complex Ct values for all samples);

ct SD ═ STDEV (all samples replicate Ct values)

Error＝SE Ct sample

The invention has the beneficial effects that:

the invention compares and identifies 1 anthocyanin-3-O-glucoside-2-O-glucuronic acid transferase gene (MaUGAT) from a Chuansang (Morus nodobiis) genome database, clones the gene from Morus yunnanensis 10 through designing a specific primer, and further detects the expression modes of the gene in different growth and development stages of different varieties through real-time fluorescence quantitative PCR (polymerase chain reaction), thereby not only enriching related data in Morus and even Moraceae plants, but also providing candidate genes for mulberry genetic improvement, promoting the mulberry quality to be improved, and better serving the mulberry industrialization.

Drawings

FIG. 1 is an electrophoretogram of RNA, in which M: marker, 1: large 10 total RNA of Guangdong mulberry;

FIG. 2 shows the target gene amplification electrophoretogram, wherein M: marker, 1: mulberry MaUGAT gene;

FIG. 3 expression analysis of mulberry MaUGAT gene in different varieties and different maturation stages, wherein S1: the olive period; s2: a color conversion period; s3: and (5) a mature period.

Detailed Description

The technical solution of the present invention will be described in further detail with reference to specific embodiments.

1.1 materials with Primary reagents

The tested variety of the mulberry is Guangdong mulberry 10 (preserved in mulberry germplasm resource nursery of silkworm research institute of academy of agricultural sciences of Sichuan province), and the fruit (mulberry) of Guangdong mulberry 10 is purple black in the mature period. The adopted plant total RNA rapid extraction kit (B518631-0100), AMV first strand cDNA synthesis kit (B532445-0020) and reverse transcription kit (R223) are purchased from biological engineering (Shanghai) GmbH, plasmid vector pMD19-T, DNA polymerase and real-time fluorescent quantitative PCR reagent are purchased from biological engineering (Dalian) GJH, primers are synthesized by biological engineering (Shanghai) GmbH, Escherichia coli competent cells are prepared by laboratory, and other reagents are analytically pure.

1.2 Total RNA extraction and first cDNA Strand Synthesis of samples from Mulberry

1.3 cloning of Mulberry MaUGAT Gene

Obtaining mulberry MaUGAT gene sequence by comparing and identifying from Sichuan mulberry genome database by referring to the gene sequence of red daisy (BpUGAT), and designing specific primers, wherein the primer sequence is as follows:

MaUGAT-F1—ATGCAAGCGATCAAGCCTAG

MaUGAT-R1—CTACACATTGAACTGCAAGTTTTC

PCR amplification is carried out by taking 10 sample mixed cDNA of Guangdong mulberry as template, wherein PCR reaction system (25 mu L) is 10 XPCR buffer 2.5 mu L, 25mmol/L MgCl₂mu.L of 2. mu.L, 2. mu.L of 20mmol/L dNTP, 1. mu.L of each of 10. mu. mol/L upstream and downstream primers, 1. mu.L of cDNA template, 0.2. mu.L of 5U/. mu.L DNA polymerase, and the balance deionized water. And immediately centrifuging for 15s by using a PCR tube centrifuge at 5000 r/min to ensure that the reaction system is completely mixed and reduce errors. The reaction condition is that the mixture is pre-denatured for 3min at 98 ℃; at 98 ℃ for 10s, at 59 ℃ for 15s, at 72 ℃ for 20s, for 39 cycles; extending for 5min at 72 ℃, and storing at 4 ℃. And (3) carrying out agarose gel electrophoresis on the amplification product, rapidly cutting off an adhesive tape containing a target strip under an ultraviolet lamp compared with a Marker, and recovering by adopting a column type trace agarose gel recovery kit. And connecting the recovered product with a pMD19-T vector, then transforming an escherichia coli competent cell, sequencing the positive clone, and finally obtaining gene sequence information.

1.4RNA extraction and electrophoresis

Through detection, the ratio of OD260/OD280 of the total RNA of the extracted sample is 2.19, which indicates that the extracted total RNA has high quality and meets the requirements of subsequent tests, and the gel electrophoresis detection is shown in figure 1:

1.5 cloning of Mulberry MaUGAT Gene

According to the above gene cloning method, the MaUGAT gene band (FIG. 2) was successfully amplified by Applied

Sequencing by a Biosystems 3730XL type sequencer, wherein a target band is 1397bp in length and 383 amino acids are coded, and through DNAman software comparison and analysis, the comparison similarity of a target gene band sequence and a retrieval sequence of a Chuangsang genome database reaches 97.35 percent, which indicates that the mulberry MaUGAT gene is successfully cloned by the method. By usinghttps://www.genes.mit.edu/ GENSCAN.htmlThe encoded protein was analyzed to have a molecular weight of 43.54392kDa and an isoelectric point of 5.50.

Base sequence of mulberry MaUGAT gene:

ATGCAAGCGATCAAGCCTATCCAACATTTCATGAGCATTCTAATGCTTCCATGGTTAGCTCACGGTCACGTATCACCCTACCTAGAGCTAGCCAAGAGGCTGGCGGAGAAAAATTTCCACGTCTACTTCTGCTCAACCCCGGCAAATCTCGTCTCTATCAAAACCAAAATTCCTAACAAATATTCTGATCACGAAAATCACTCTCTTGGCATTATTGAACTTGTGGAGCTTCATCTTCCCGAGCTACCCGACCTTCCTCGCCATTACCACACCACAAATGGCCTCCCACCACATCTCATGCCGACTCTCAAGAAGGCCTTCGACATGTCAAGCCCGTCTTTCGCGAAAATCCTCGATGATCTCGAGCCAGAGTTGCTCATTTACGACTTTCTTCAGCCTTGGGCTCCAACGCTGGCTTCTCAGAGGAACATTCCCGCGGTCGAGTTCTTGAGCTGCAGTGCCAGCATGACTTCGTTTTGTCTTCATTGGCGGAGCAAAGGAGGCGTTGAGTTTCCTTTTCCGACGATTTATTTGAAGGGTTATGAAGTTAGTGGCTTCAACAATCTGCTGGAGTCGTCGGCGAATGATGTCAAGGACAAAGAAAGGGTACGGCGGTGCAGTGAACAATCTTGCAATATTGTTTTGGTGAAGTCTTTTTCTGATGTTGAAGACAAATACATCGATTATCTTTCTGTTCTGTTGGGTAAGAAGATTGTGCCGGTCGGTTCGCTTGTTGATGACGGCAAAGATTCCAACGATTTAGAAGATTATAATAATGTGATAAAATGGCTCGATAGCAAGGAAAAGTCTTCGGTGGTGTTTGTCTCCTTTGGAAGCGAGTATTTTCTGTCCAAAGATGAGATGAGGGAGATAGCTTATGGTTTAGAACTCAGTGGAGTGAGTTCATTTGGGTGGTTAGATTCCCGGTGGGAGAGAAATGAACATTGAAGAAGCCTTACCAAATGGTTTTCTCAAAaGGGTTGAAAGAAAGGGAATGATAATAGAGAAATGGGCACCCCAAAGGGAAGTTCTCAAGAGCAAAAGCATTGGCGGGTTCGTGAGTCATTGCGGGTGGAGCTCTGTAATGGAGAGCATGAAATTTGGGGTTCCTATAATAGCAATGCCAATGCATCTTGACCAGCCTATTAATGCAAGATTGGTGGAGGAGGTTGGTGTAGGTTTCGAGGTCGAGAGAGACGAGAACGGGAGGATTCGGAGTGAGGAATTAGTGAAAGCAGTTCGAAAGGTGGTGTTGGAAAAAAGTGGGGAGAGTGTGAGGAACAAGGCAGTGGAAATGGGGGAGAAAATGAGGAGGAGAGGAGATGAAGAGATGGAAGAAGTGGTGAAGGAGCTCGTGCAACTTTGTGGGAAAGAAAACTTGCAGTTCAATGTGTAG

the mulberry MaUGAT gene codes an amino acid sequence:

MQAIKPIQHFMSILMLPWLAHGHVSPYLELAKRLAEKNFHVYFCSTPANLVSIKTKIPNKYSDHENHSLGIIELVELHLPELPDLPRHYHTTNGLPPHLMPTLKKAFDMSSPSFAKILDDLEPELLIYDFLQPWAPTLASQRNIPAVEFLSCSASMTSFCLHWRSKGGVEFPFPTIYLKGYEVSGFNNLLESSANDVKDKERVRRCSEQSCNIVLVKSFSDVEDKYIDYLSVLLGKKIVPVGSLVDDGKDSNDLEDYNNVIKWLDSKEKSSVVFVSFGSEYFLSKDEMREIAYGLELSGPINARLVEEVGVGFEVERDENGRIRSEELVKAVRKVVLEKSGESVRNKAVEMGEKMRRRGDEEMEEVVKELVQLCGKENLQFNV

1.6 expression analysis of Mulberry MaUGAT Gene in different varieties and different maturation periods

The transcription level detection application of the identified mulberry MaUGAT gene is carried out through real-time fluorescent quantitative PCR, and the test materials are in the same batch. First strand cDNA was synthesized using a reverse transcription kit (R223), and gene copy specific primers for qPCR were designed using software Primer Premier 5. Morus022526 is used as an internal reference gene to verify the transcription level of the MaUGAT gene in three stages of different mulberry varieties. Three independent biological replicates of each gene were performed to ensure reproducibility and reliability.

1.7 specific primer design

Designing a primer according to the sequence of the mulberry MaUGAT gene obtained by cloning, wherein the primer sequence is as follows:

MaUGAT-F2 GTTCGCTTGTTGATGATGGC，

MaUGAT-R2 CGCTTCCAAAGGAGACAAACAC。

RT-PCR was performed using the mulberry cDNA of 3 different stages of two different varieties of fruit color mulberry as a template to perform the expression pattern analysis and application of MaUGAT gene in mulberry (FIG. 3).

1.8 Total RNA extraction from Mulberry samples

7) And repeating the previous step.

1.9 reverse transcription

TABLE 1 gDNA removal reaction System

TABLE 2 reverse transcription reaction System

4) Mixing well.

5) Incubate at 50 ℃ for 50 min.

1.10 quantitative analysis of Gene and its application

1) A20. mu.l reaction system was prepared according to the following table

2) Reaction conditions

3) And (3) melting curve analysis: the temperature is 65-95 ℃.

1.11 data calculation

Δ Ct mean ═ AVERAGE (complex Ct values for all samples);

ct SD ═ STDEV (all samples replicate Ct values)

Error＝SE Ct sample

The results show that the MaUGAT gene (figure 3) is not expressed at the S1 stage of Guangdong morula 10(Da10) mulberry, starts to be expressed at the S2 stage and is obviously up-regulated at the S3 stage; it was not expressed in both S1 and S2 stages of Morus alba (Baiang) and Morus alba, and was expressed in a small amount in S3 stage. The enzyme gene catalyzes anthocyanin-3-O-glucoside to be converted into anthocyanin 3-O- (2-O-beta-D-glucuronyl) -beta-D-glucoside, and finally stable anthocyanin substances are formed. Due to the obvious down-regulation expression of anthocyanin-3-O-glucoside-2-O-glucuronic acid transferase gene (MaUGAT), anthocyanin substances are abnormally metabolized in the development process of mulberry of Hongtiang white mulberry, so that anthocyanin cannot be synthesized and accumulated in mulberry, and finally, anthocyanin is white when the mulberry is mature.

The above description is only a preferred embodiment of the present invention, and the scope of the present invention is not limited thereto, and any simple modifications or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the technical scope of the present invention are within the scope of the present invention.

Sequence listing

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Claims

1. A method for cloning a mulberry anthocyanin-3-O-glucoside-2-O-glucuronic acid transferase gene is characterized by comprising the following steps:

Grinding a mulberry sample of Guangdong mulberry 10 into powder by using liquid nitrogen, extracting total RNA of the sample according to a standard extraction step of a kit, detecting the extraction quality of the total RNA by using a Nano Drop2000 micro spectrophotometer, and calculating the ratio of OD260/OD280, wherein the ratio is 1.8-2.2; preparing a 20 mu L reaction system according to the operation flow of the first chain cDNA synthesis kit, and synthesizing the 1 st chain cDNA by reverse transcription;

step 2, cloning of mulberry MaUGAT gene

Referring to a BpUGAT gene sequence of the daisy red, uploading to a Chuansang genome database https:// mouus.swu.edu.cn/mousdb/performing sequence comparison, and designing a specific primer according to the gene sequence, wherein the gene with the highest similarity rate is Morus016781 gene, namely a mulberry MaUGAT gene, and the primer sequence is shown as SEQ ID: NO 1-SEQ ID: NO 2;

PCR amplification is carried out by taking 10 sample mixed cDNA of Guangdong mulberry as template, wherein 25 mu L of amplification reaction system is 10 XPCR buffer 2.5 mu L, and 25mmol/L MgCl₂mu.L, 2 mu.L of 20mmol/L dNTP, 1 mu.L of each of 10 mu mol/L upstream primer and downstream primer, 1 mu.L of cDNA template, 0.2 mu.L of 5U/mu L DNA polymerase and the balance of deionized water; centrifuging for 15s at 5000 rpm by using a PCR tube centrifuge immediately to ensure that a reaction system is completely mixed and reduce errors; the reaction condition is that the mixture is pre-denatured for 3min at 98 ℃; at 98 ℃ for 10s, at 59 ℃ for 15s, at 72 ℃ for 20s, for 39 cycles; extending for 5min at 72 deg.C, and storing at 4 deg.C; carrying out agarose gel electrophoresis on the amplification product, rapidly cutting off an adhesive tape containing a target strip under an ultraviolet lamp compared with a Marker, and recovering by adopting a column type trace agarose gel recovery kit; and connecting the recovered product with a pMD19-T vector, then transforming an escherichia coli competent cell, sequencing the positive clone, and finally obtaining gene sequence information.

2. The application of mulberry anthocyanin-3-O-glucoside-2-O-glucuronic acid transferase gene cloned by the method in mulberry germplasm cultivation.

3. Use according to claim 2, characterized in that it comprises the following steps:

step 1, extracting total RNA of mulberry test sample

1) Grinding mulberry tissue samples in liquid nitrogen to powder, immediately adding 1ml of Trizol, fully mixing and cracking;

2) adding 200ul chloroform, fully oscillating and mixing uniformly with force, and centrifuging at 12000rpm at 4 ℃ for 10 minutes;

3) taking the supernatant, adding the same volume of phenol-formaldehyde, fully oscillating and uniformly mixing, and centrifuging at 12000rpm at 4 ℃ for 10 minutes;

4) taking the supernatant, adding equal volume of chloroform, fully oscillating and uniformly mixing, and centrifuging at 4 ℃ and 12000rpm for 10 minutes;

5) taking the supernatant, adding isopropanol with the same volume, reversing and uniformly mixing, and precipitating for 1 hour at the temperature of minus 20 ℃; centrifuging at 12000rpm at 4 ℃ for 15 minutes;

6) discarding the supernatant, adding 0.5ml of 75% ethanol, and stirring at 4 ℃ 8000-;

7) repeating the previous step;

8) centrifuging for a short time after discarding the supernatant, sucking dry ethanol by a pipette gun, and vacuum-drying for 2 min;

9) adding 20ul RNA-Free water, dissolving at room temperature for 5min, mixing, and centrifuging to obtain RNA solution;

step 2, reverse transcription

According to the instruction of a reverse transcription kit R223, a 20 mu l reaction system is established for 50ng-2ug of total RNA;

1) thawing mulberry RNA on ice; 4 XgDNA wiper Mix, 5 XHiScript | qRT Supermix |, RNase-Free ddH₂Unfreezing O at the room temperature of 15-25 ℃, and quickly placing on ice after unfreezing; before use, each solution is vortexed, oscillated and uniformly mixed, and liquid remained on the tube wall is collected after short centrifugation;

2) preparing a mixed solution according to a genome DNA removal system of the gDNA removal reaction system, and thoroughly mixing the mixed solution; centrifuging briefly, and incubating at 42 deg.C for 2 min; then placing on ice;

the gDNA removal reaction system is as follows:

3) preparing a mixed solution according to a reverse transcription reaction system, wherein the reverse transcription reaction system specifically comprises the following steps:

usage amount of composition

Reaction solution for gDNA removal step 16. mu.l

5×HiScript‖qRT SuperMix‖ 4μl；

4) Fully and uniformly mixing;

5) incubating for 50min at 50 ℃;

6) incubating at 95 ℃ for 5min to obtain cDNA for subsequent experiments;

step 3, quantitative analysis and application of gene

Designing a primer according to the sequence of the cloned mulberry MaUGAT gene, wherein the sequence of the MaUGAT-F2 primer is shown as SEQ ID NO. 1-SEQ ID NO. 2;

1) 20. mu.l of the reaction system 20. mu.l, specifically:

2) reaction conditions

Pre-denaturation at 95 deg.C for 90s, denaturation at 95 deg.C for 5s, denaturation at 60 deg.C for 15s, denaturation at 72 deg.C for 20s, and 40 cycles, extending at 72 deg.C for 5min, and storing at 4 deg.C;

3) and (3) melting curve analysis: the temperature is 65-95 ℃;

step 4, data calculation

Experiment according to 2^-△△CtThe method processes data; wherein, the delta Ct is the average Ct of the target gene-the average Ct of the internal reference; delta Ct is the target gene delta Ct in the sample to be detected-the target gene delta Ct in the reference sample;

Δ Ct mean is the complex well Ct value for all samples of AVERAGE;

ct SD is the Ct value of all sample replicate wells of STDEV;

Error＝SE Ct sample