CN113249512B - Method for identifying different cytoplasm of kenaf based on mitochondrial gene transcript difference and application thereof - Google Patents

Method for identifying different cytoplasm of kenaf based on mitochondrial gene transcript difference and application thereof Download PDF

Info

Publication number
CN113249512B
CN113249512B CN202110590238.4A CN202110590238A CN113249512B CN 113249512 B CN113249512 B CN 113249512B CN 202110590238 A CN202110590238 A CN 202110590238A CN 113249512 B CN113249512 B CN 113249512B
Authority
CN
China
Prior art keywords
gene
cox3
atp1
atp9
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202110590238.4A
Other languages
Chinese (zh)
Other versions
CN113249512A (en
Inventor
廖小芳
唐兴富
赵艳红
侯文焕
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Guangxi Zhuang Nationality Autonomous Region Academy of Agricultural Sciences
Original Assignee
Guangxi Zhuang Nationality Autonomous Region Academy of Agricultural Sciences
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Guangxi Zhuang Nationality Autonomous Region Academy of Agricultural Sciences filed Critical Guangxi Zhuang Nationality Autonomous Region Academy of Agricultural Sciences
Priority to CN202110590238.4A priority Critical patent/CN113249512B/en
Publication of CN113249512A publication Critical patent/CN113249512A/en
Application granted granted Critical
Publication of CN113249512B publication Critical patent/CN113249512B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Genetics & Genomics (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Botany (AREA)
  • Mycology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention belongs to the field of molecular genetics, relates to identification of different cytoplasm of kenaf plants, and particularly relates to a method and application for identifying different cytoplasm of kenaf based on mitochondrial gene transcript difference. The method comprises the following steps: weighing the kenaf anther to be identified, and extracting RNA; self-ligating the extracted RNA to form an RNA cyclization product; reverse transcribing the cyclized product into cDNA; designing a reverse nest primer of the 5 'end and the 3' end of a known sequence of a target mitochondrial gene, performing two rounds of reverse nest PCR, detecting by gel electrophoresis, and recovering a second round of nest PCR product for sequencing; comparing and analyzing the sequencing result with the CDS conservation region of the target mitochondrial gene to obtain the CDS full length of the target mitochondrial gene and the full length of transcripts from 5 'to 3' ends; analyzing the sequence characteristics of CDS full length and/or transcript full length of mitochondrial gene to identify different types of cytoplasm of kenaf. The invention can also be applied to the comparison research of transcript abundance and transcript ends of target genes in different material treatments or different varieties.

Description

Method for identifying different cytoplasm of kenaf based on mitochondrial gene transcript difference and application thereof
Technical Field
The invention belongs to the field of molecular genetics, relates to identification of different cytoplasm of kenaf plants, and particularly relates to a method and application for identifying different cytoplasm of kenaf based on mitochondrial gene transcript difference.
Background
The full-length acquisition of the target gene and the characteristic analysis of the transcript are the precondition of the research of gene functions and the expression regulation. In recent years, whole genome sequencing and transcriptome sequencing provide good technical methods for the feature analysis and expression analysis regulation research of biological genes, but for most organisms, whole genome sequences are not obtained yet, and the full-length sequences of certain genes are unknown; the cost of expression regulation of a single target gene is too high through the transcriptome mode, and a large amount of redundant information irrelevant to the expression of the target gene exists in transcriptome data. The traditional method obtains the target gene sequence through the RACE kit or TAIR-PCR, and the RACE kit is expensive, high in cost and complicated in steps, so that mRNA is easy to lose or degrade in the operation process; the TAIR-PCR method has the characteristics that only one end is a gene specific primer, and the other end is a random degenerate primer, the success rate of PCR amplified products is low, the specificity is not strong, and the like, so that the subsequent experiment requirements are difficult to meet.
Weston Blot is a traditional method for researching gene transcript expression, but the method has long operation period and complicated steps, and is difficult to accurately detect some transcripts with low abundance expression; in addition, the Weston Blot method also fails to perform recovery sequencing analysis on transcript bands of the detected genes.
Patent CN 201110195025.8 discloses a method and application of reverse transcription technology using nested PCR in combination with gene specific primers to mine low abundance expressed genes and systematically identify splice variants, which is based on reverse transcription PCR of 3' GSP1, using gradient PCR to find the optimal annealing temperature for each pair of primer combinations for each gene, nested PCR sequencing based on GSP and based on sequence analysis to determine if a new splice variant is present.
Based on the existing researches, various methods for amplifying the full length of the target gene exist, and researchers aim at the technical problems to be solved based on the existing methods, and the methods often need to be improved. The application aims at solving the technical problem of distinguishing different cytoplasm of kenaf by mitochondrial gene transcript difference, and improves and futures the prior art method.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for identifying different cytoplasm of kenaf based on mitochondrial gene transcript difference and application thereof.
The technical scheme of the invention is realized as follows:
the method for identifying the different cytoplasm of the kenaf based on the mitochondrial gene transcript difference comprises the following steps:
(1) Weighing the kenaf anther to be identified, and extracting RNA;
(2) Cyclizing the RNA extracted in the step (1), and then reversely transcribing the RNA cyclized product to form cDNA;
(3) Respectively designing two pairs of reverse nested primers at the 5 'and 3' ends of a target mitochondrial gene conservation region, performing two rounds of reverse nested PCR, and performing recovery sequencing on a second round of nested PCR product after gel electrophoresis detection;
(4) Comparing and analyzing the sequencing result with the CDS conservation region of the target mitochondrial gene, and comparing and analyzing the CDS full length of the target mitochondrial gene and the transcript full length of the 5 'to 3' ends of the mitochondrial gene through ORF finder on-line software and homologous sequences;
(5) Analyzing the sequence characteristics of CDS full length and/or 5 'to 3' end transcript full length of the mitochondrial gene obtained in the step (4) to identify different cytoplasm of kenaf.
The mitochondrial gene of interest in step (3) isatp1，atp9Andcox3in reverse nested PCR, the first round of nested PCR is performed by two primers which are far from the boundaries of the 5 'and 3' ends of the conserved region of the target gene, and the second round of nested PCR is performed by two primers which are near to the boundaries of the 5 'and 3' ends of the conserved region of the target gene.
The objective mitochondrial gene isatp1When the primer is used, two primers far from the boundary of the 5 'end and the 3' end of the conserved region of the target gene are respectivelyatp1-AP1 andatp1-the SP1 and two primers which are closer to the boundaries of the 5 'end and the 3' end of the conserved region of the target gene are respectivelyatp1-AP2 and AP2atp1-SP2 whereinatp1-The sequence of the AP1 is 5'-CGATCTCATCCACTTGAAAATTCGT-3',atp1-the sequence of the AP2 is 5'-TTAGTTCTGCAGCTCTGGGAGAGA-3',atp1-the SP1 sequence was set to 5'-GGCTTACAGAAGTACCGAAACAACC-3',atp1-SP2 sequence 5'-GGATTCTGTGATCGAATGCCATTAG-3'.
The objective mitochondrial gene isatp9When the primer is used, two primers far from the boundary of the 5 'end and the 3' end of the conserved region of the target gene are respectivelyatp9-AP1 andatp9-the SP1 and two primers which are closer to the boundaries of the 5 'end and the 3' end of the conserved region of the target gene are respectivelyatp9-AP2 and AP2atp9-SP2 whereinatp9-The sequence of the AP1 is 5'-GCACCCATTAATTTTGCACCTTC-3',atp9-the sequence of the AP2 is 5'-ATTCTCGTCACGCGCTTTATCATTC-3',atp9-the SP1 sequence was set to 5'-ATTTTGGGCTTTGCTCTAACCGAAG-3',atp9-SP2 sequence 5' -GAAGCTATTGCATTGTTTGCCCTAA-3’。
The objective mitochondrial gene iscox3When the primer is used, two primers far from the boundary of the 5 'end and the 3' end of the conserved region of the target gene are respectivelycox3-AP1 andcox3-the SP1 and two primers which are closer to the boundaries of the 5 'end and the 3' end of the conserved region of the target gene are respectivelycox3-AP2 and AP2cox3-SP2 whereincox3-The sequence of the AP1 is 5'-GCCCAAACTGAGAAGTCTTGCAC-3',cox3-the sequence of the AP2 is 5'-CCACTTTGCCTCGGTTGTATGTAA-3',cox3-the SP1 sequence was set to 5'-GGTCATCTGACTAAAGAGCATCACG-3',cox3-SP2 sequence 5'-GCATTTTGTAGACGTGGTTCGG-3'.
The reverse nest PCR reaction system in the step (3) is as follows: in a 20. Mu.L reaction system, 10. Mu.L of 2X Phanta Max Master Mix (Nanjinozan), 0.6. Mu.M AP primer, 0.6. Mu.M SP primer, 50 ng template, add ddH 2 O is added to the total volume of 20 mu L; the amplification procedure was: 95. pre-denaturation at 95deg.C for 3 min, denaturation at 95deg.C for 30 sec, annealing at 58-60deg.C for 30 sec, extension at 72deg.C for 1min, reaction cycle number of 35, extension at 72deg.C for 5 min, and preservation at 4deg.C.
The standard for identifying the different cells of the kenaf in the step (5) is as follows: to be used foratp1In the case of a gene of interest in mitochondria,atp1three specific bands were amplified in the sterile line cytoplasm, only one in the maintainer line cytoplasm, and in F 1 Two specific bands are arranged in cytoplasm; to be used foratp9For the mitochondrial gene of interestatp9When the gene is the mitochondria target gene, a specific band with the size of 400 bp is amplified in a sterile line, and the size of the amplified specific band in a maintainer line and F1 generation is 450 bp; to be used forcox3In the case of a gene of interest in mitochondria,cox35 specific bands were amplified in the cytoplasm of the sterile line, with the weak band between the two major bands, only one specific band in the maintainer line, and two adjacent major bands in the F1 generation.
The method is used for analysis/identificationatp1，atp9Or (b)cox3Use of the full length of the gene CDS.
The method is used for analysis/identificationatp1，atp9Or (b)cox3Transcription of genes in the cytoplasm of kenaf under testApplication.
The method is used for analysis/identificationatp1，atp9Or (b)cox3The use of the gene for the differences in transcript abundance (transcript band brightness) in different cytoplasm of kenaf.
The method is used for analysis/identificationatp1，atp9Or (b)cox3Use of the gene for the terminal differences of the 5 'and 3' transcripts in the different cytoplasm of kenaf.
The method is used for analysis/identificationatp1，atp9Or (b)cox3The application of the gene in detecting whether the cytoplasm of the kenaf is regulated by nuclear-cytoplasmic interaction.
The invention has the following beneficial effects:
1. the invention is based on that the 5 'phosphate group and the 3' hydroxyl of mRNA form a ring structure by self-linking under the action of RNA linking enzyme, cDNA is synthesized under the action of reverse transcriptase, and the cDNA is used as a template to carry out PCR amplification by using an inverse nested PCR method to obtain a target sequence.
2. The invention is used for gel electrophoresisatp1Andcox3nest primer pair of kenaf sterile line UG93A, maintainer line UG93B and F 1 Performing nested PCR amplification on the cDNA product after (UG 93A/UG 93R) cyclization, and performing nested PCR amplification on the cDNA product after the second round of amplification,atp1andcox3the amplified band sizes were different in the sterile line and the maintainer line; and even in the same cytoplasm, the brightness of different bands is different, namely transcripts with different sizes have inconsistent transcript abundance; although the sterile line is consistent with the cytoplasmic type in F1, the difference between F1 transcripts and the sterile line is caused by F1 carrying nuclear recovery genes, further explainingatp1Andcox3is regulated by nuclear-cytoplasmic interactions。Although UG93A, UG B and F1atp9Only one band is used as the first and second round amplification products of (a) but the sterile line isatp9Is smaller than the maintainer line and F1, indicating that in different cytoplasmatp9Is also regulated by nuclear-cytoplasmic interactions. Therefore, the method of the invention can not only identify the transfer of mitochondrial genes in different cytoplasmThe difference of the sizes of the transcripts can also identify whether the abundance of transcripts with different sizes in the same cytoplasm is different or not, and can also identify whether the transcription of the gene is regulated by nuclear-cytoplasmic interaction or not.
3. When the transcripts obtained by sequencing are analyzed, the application finds that,atp1andcox3multiple transcription initiation sites exist at the 5' end of (a), but the frequency of generation of each initiation site is not the same in different cytoplasm. In the case of the UG93A,atp13 transcription initiation sites located at-78 bp, -176 bp and-213 bp upstream of the initiation codon, at frequencies of 87.5% (14/16), 6.25% (1/16) and 6.25% (1/16), respectively; the 5' of UG93B has only one transcription initiation site located at-138 bp upstream of the initiation codon; the 5' -end of F1 had 2 transcription initiation sites located at-158, bp and-213, bp upstream of the initiation codon, at frequencies of 11.11% (2/18) and 88.89% (16/18), respectively.cox3There is only one transcription initiation site at the 5' end of the maintainer line UG93B, located-313 bp upstream of the initiation codon; and in UG93A and F1cox35 different transcription initiation sites were located at the 5' end of each of the sequences located at-164, -313, -442, -814 and-856 bp upstream of the initiation codon, respectively. Although UG93A and F1cox3But the frequency of the start sites is not the same for each transcript in different nuclear contexts: in UG93A, the transcript frequencies at positions-164 and-856 upstream of the start codon were not 30.43% (7/23) and 56.52% (13/23), respectively, whereas the transcript frequencies at positions-313, -442 and-814 upstream of the start codon were 4.34% (1/23); the transcript frequencies at positions-164, -814 and-856 upstream of the start codon in F1 were 4.3% (1/23); the transcription frequencies at positions-313 and-442 upstream of the start codon were 30.43% (7/23) and 56.52% (13/23), respectively. In different cytoplasmatp9Is identical at the 5 'transcript end, but differs at the 3' transcription termination site. In sterile linesatp9The 3' transcript end of (2) was located +271bp downstream of the stop codon, while the transcript end in maintainer line and F1 was located +397 bp downstream of the stop codon. Therefore, the method of the invention can not only identify the size and the transcription of mitochondrial genes in different cytoplasm by monoclonal sequencingThe difference of transcript abundance can also identify the difference of the 5 'end and the 3' end transcript ends of different mitochondrial genes in different cytoplasm; and identifying whether transcription of mitochondrial genes is regulated by nuclear-nuclear interactions.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of RNA circularized nested PCR, wherein the green part is the core conservation region of the gene CDS, the red part is the primer combination AP1/SP1 initial amplification region, the yellow part is the primer combination AP2/SP2 initial amplification region, the blue part is the unknown sequences at the 5 'end and the 3' end of the gene CDS sequence, and the white part is the non-coding regions at the 5 'end and the 3' end.
FIG. 2 is a view of kenafatp1，atp9Andcox3and detecting the reverse nest PCR amplification result, wherein M is 100 bp Marker.
FIG. 3 is a schematic view ofatp1Reverse nested PCR amplification result sequence alignment, wherein the green background sequence isatp1Is shown in the dark blue background as CDS conserved sequence of (2)atp1 reverse nest PCR amplified 5 'end and 3' end CDS sequence, light blue background isatp15 'and 3' UTR sequences, respectively, the reverse nested primer sequences are shown as red boxes, and the reverse nested primer sequences are shown as yellow boxesatp1The start codon and the stop codon of (2), the blue boxes are respectivelyatp1Variable region of the 5' UTR sequence of (C).
FIG. 4 is a diagram ofatp1The transcript ends were amplified in different cytoplasm of kenaf, wherein the right hand score of the picture indicates the proportion of each transcription start site present.
FIG. 5 is a schematic view of a displayatp9Reverse nested PCR amplification result sequence alignment, wherein the green background sequence isatp9Is shown in the dark blue background as CDS conserved sequence of (2)atp9Reverse nested PCRAmplified 5 'and 3' CDS sequences, light blue backgroundatp9The 5 'and 3' UTR sequences, the red boxes are reverse nested primer sequences, respectively, and the red boxes and yellow boxes are respectivelyatp1Initiation codon and termination codon of (c).
FIG. 6 is a diagram of different cytoplasmsatp9Transcript end characterization.
FIG. 7 is a diagram ofcox3Reverse nested PCR amplification result sequence alignment, wherein the green background sequence iscox3Is shown in the dark blue background as CDS conserved sequence of (2)atp9CDS sequences at the 5 'end and the 3' end amplified by reverse nested PCR, and the light blue background iscox3The 5 'and 3' UTR sequences, the red boxes are reverse nested primer sequences, respectively, and the red boxes and yellow boxes are respectivelyatp1Initiation codon and termination codon of (c).
FIG. 8 is a diagram ofcox3Transcript ends amplify ratios in different cytoplasm of kenaf.
Detailed Description
The invention is carried out by adopting the following method:
extraction of RNA
(1) The anther, which is 0.2. 0.2 g fresh or frozen and preserved by liquid nitrogen, is immediately placed in a precooled mortar, and sufficient liquid nitrogen is added to quickly grind the anther into powder, and the powder is quickly transferred into a 2mL RNase-free centrifuge tube.
(2) To the centrifuge tube, 1mL of CTAB lysate (0.1M Tris-HCl pH8.0, 0.025M EDTA-Na) preheated at 65℃was added 2 pH8.0, 2M NaCl, 2% CTAB (w/v), vortex shaking 30-60 s,65 ℃, water bath 10min.
(3) An equal volume of chloroform was added: isoamyl alcohol (25:24:1), thoroughly mixed, centrifuged at 12,000 rpm at 4℃for 10min.
(4) Taking supernatant, adding saturated guanidine isothiocyanate (5M) into the supernatant according to a certain proportion (supernatant: guanidine isothiocyanate=1.7:1), adding 1/2 volume of absolute ethyl alcohol, and mixing the mixture uniformly in a reverse manner.
(5) The supernatant was aspirated and the mixture was transferred to an RNA purification column.
(6) The liquid in the collection tube was discarded at 12000 rpm for 30 s at 4℃and this step was repeated until the mixture was completely transferred.
(7) 700. Mu.L of 75% ethanol was added at 4℃and 12000 rpm for 30 s, the liquid in the collection tube was discarded, and the procedure was repeated 1 time.
(8) The column was thrown empty at 4℃at 12000 rpm for 2 min.
(9) The column was placed in a new 1.5 ml RNase-free EP tube, and 30. Mu.l RNase-free ddH was added to the center of the column 2 0 water and standing on ice for 2 min.
(10) Centrifuging at 12000 rpm at 4 ℃ for 1min, discarding the centrifugal column, and collecting the liquid from the centrifugal tube to obtain the purified RNA.
(11) The purity and integrity of RNA were checked by using a Nanodrop nucleic acid concentration detector and a gel electrophoresis apparatus, and stored at-80℃for further use.
Cyclization of RNA
(1) To 200. Mu.L of RNase free PCR tubes, were added: 1. Mu.g total RNA, 1. Mu.L 10 Reaction Buffer, 20U T4 RNA Ligase and 1. Mu.L 1 mg/ml BSA, followed by RNase-free ddH 2 0 up to 10 μl (all reagents were purchased from us Thermo Scientific).
(2) Incubate 4 h at 37℃with constant temperature (this step was performed on a PCR instrument).
(3) The enzyme was inactivated by reaction at 70℃for 10min.
Reverse transcription of RNA cyclization products
(1) To the circularized RNA mixture, 2.5. Mu.L of 5 XgDNA wipe Mix (Nanjinovirzan) was added, and the mixture was blown and mixed with a pipette at 42℃for 2 minutes.
(2) The following reagents are respectively added into the mixed solution in the last step: 2.5 Mu L10 xRT Mix,2.5 mu L HiScriptIII Enzyme Mix,1.25 mu L Oligo (dT) 20 And 6.25. Mu.L RNase-free ddH 2 O, the total volume of the reaction solution was 25. Mu.L (the above reagents were all purchased from Nanjinozan Biotech Co., ltd.).
(3) The cDNA synthesis reaction was performed according to the following reaction conditions: 25 ℃ for 5 min;50 ℃;45 min;85 ℃,5 sec.
(4) The cDNA product was transferred to a 1.5. 1.5 mL sterile centrifuge tube and stored at-20℃until use.
(IV) reverse nest PCR (Reverse Nesting PCR) Gene full Length
At the position ofatp1，atp9Andcox3respectively designing two reverse primers with unequal distances from the boundary of the reverse primers at the 5 'end and the 3' end of the gene conserved region (wherein the 5 'end primers are represented by AP1 and AP2, the 3' end primers are represented by SP1 and SP 2), carrying out PCR amplification reaction by using cDNA which is reversely transcribed after RNA cyclization as a template in the first round of PCR reaction and combining two primers far from the boundary of the 5 'end and the 3' end of the target gene conserved region into a primer pair (AP 1/SP 1); the PCR products of the first round of PCR reaction are taken as templates, two primers which are closer to the boundary of the 5 'end and the 3' end of a target gene conservation region are combined into a primer pair (AP 2/SP 2), the PCR amplification reaction is carried out, and the annealing temperature of the primers is designed to be 58-60 ℃ in order to ensure the specificity of the PCR amplification products; the PCR reaction system is as follows: in a 20. Mu.L reaction system, 10. Mu.L of 2X Phanta Max Master Mix (Nanjinozan), 0.6. Mu.M AP primer, 0.6. Mu.M SP primer, 50 ng template, add ddH 2 O is added to the total volume of 20 mu L; the PCR amplification procedure was: 95. pre-denaturation at 95deg.C for 3 min, denaturation at 95deg.C for 30 sec, annealing at 58-60deg.C for 30 sec, extension at 72deg.C for 1min, reaction cycle number of 35, extension at 72deg.C for 5 min, and preservation at 4deg.C. The first round and the second round of PCR products were spotted in two adjacent gel wells of 1.5% agarose, respectively, and electrophoresis was performed simultaneously, if the difference in the sizes of the first round and the second round of amplified PCR products was exactly identical to the difference in the sizes of the first round and the second round of nested primers, the fragment was indicated as the specific amplified target fragment (FIG. 1). All specific amplified product fragments of the second round were recovered by a gel recovery kit (nanking praziram), linked to T-vector, and positive monoclonal was sent to the wuhan Jin Kairui bioengineering company for sequencing, at least 15 clones per recovered product.
TABLE 1 reverse nested PCR primers
Figure DEST_PATH_IMAGE002
。
The technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without any inventive effort, are intended to be within the scope of the invention.
Examples
In the embodiment, the kenaf sterile line UG93A and the maintainer lines UG93B and F 1 (UG 93A/UG 93R) as an implementation object, and obtaining mitochondrial genes by RNA cyclization and reverse nest-PCR methodatp1，atp9Andcox3and 5 'and 3' non-coding sequences. In the second round of nested PCR products,atp1three specific bands were amplified in UG93A, comprising a main band of 450 bp and two weak bands of 550 bp and 350 bp, respectively, with only one specific band of 450 bp in UG93B and F 1 (UG 93A/UG 93R) there are two bands, the main band of 450 bp and the weak band of 350 bp (FIG. 2A).atp9In kenaf UG93A, UG B and F 1 All of which amplify a specific band, the amplified products in UG93A have the size of 400 bp, UG93B and F 1 The amplified specific band size was 450 bp (FIG. 2B).cox3In UG93A and F 1 Five specific bands were amplified in the same order as 1300 bp,1200 bp,850 bp,750 bp and 600 bp, respectively. Wherein the bands 1300 bp and 600 bp in UG93A are the main bands of the nested PCR amplification product, and F 1 The size of the middle nested PCR amplified main band product is 850 bp and 750 bp; only one specific band of size 700 bp is present in UG 93B. Experimental results indicate that although F 1 Is identical to UG93A due to F 1 The action of the nuclear restorer gene results incox3The transcript expression was significantly different from UG93A, indicatingcox3Is regulated by nuclear restorer genes.
Recovering and sequencing the second round of nested PCR product through the target geneatp1，atp9Andcox3mitochondrial genes were obtained by CDS conserved region alignment analysis and ORF finder (https:// www.ncbi.nlm.nih.gov/ordered /) online softwareatp1，atp9Andcox3CDS full length sequence of (c), and full length 5 'and 3' transcripts. Reverse nested PCR results showed that UG93A, UG93B and F1atp1The total length of CDS of 1524 bp, in different cytoplasm, atp1 only 1 transcription termination site at the 3' end of (a), i.e. 49 bp after the termination codon. Whileatp1A plurality of transcription initiation sites exist at the 5' end of UG93A,atp13 transcription initiation sites located at-78 bp, -176 bp, and-213 bp upstream of the initiation codon, respectively; the 5' of UG93B had only one transcription start site located 138 bp upstream of the start codon; the 5' -end of F1 has 2 transcription initiation sites located at-158, bp and-213, bp upstream of the initiation codon, respectively. In addition, the transcription initiation sites of sterile and fertile cytoplasm were upstream of-11 bp and-12 bp and between-92 bp and-114 bp with 2 bp base deletions and 8 base mutation sites, respectively (FIG. 3). In UG93A and F1atp1Multiple transcription initiation sites exist at the 5' end of (a), but each transcription initiation site occurs at a different frequency. From monoclonal sequencing, it was found that UG93Aatp1The 5' transcript ends of (a) were found most frequently at translation initiation site-176, accounting for 87.5% (14/16) of the entire clonality sequencing, and the transcript frequencies at translation initiation sites-78 and 213 were each lowest, accounting for 6.25% (1/16) of the number of clones. Whereas the transcription frequency at translation initiation site-213 in F1 was highest, accounting for 87.5% (14/16) of the number of clones (FIG. 4).
Example 1 of the embodiment
Reverse nested PCR results showed that UG93A, UG93B and F1atp9The total length of CDS of 264 bp and only one transcript in each of the different cytoplasm (fig. 5, 6). Of UG93A, UG93B and F1,atp9the 5 'transcripts of (2) are identical at their ends and all located-71 bp upstream of the start codon, while the 3' transcription termination sites in the different cytoplasm are different. The 3' transcript ends of UG93A were located +271bp downstream of the stop codon, while the transcript ends in UG93B and F1 were located +397 bp downstream of the stop codon (fig. 5). Although UG93A and hybrid F1 had the same cytoplasmic background, their 3' -terminal transcripts were not identical, indicating mitochondriaatp9Is also regulated by nuclear-nuclear interactions.
Example 2 of the embodiment
Reverse nested PCR results showed that UG93A, UG93B and F1cox3The CDS total length of (2) is 843bp; in UG93Bcox3Only one 5' end of (C) located at-313 bp upstream of the start codon; and in UG93A and F1cox35 different transcription initiation sites located at-164, -313, -442, -814 and-856 bp upstream of the initiation codon, respectively; while in three cytoplasmcox3Only one at each end of the 3' transcript downstream of the stop codon 314 bp. In addition, the transcription initiation sites of sterile cytoplasm and fertile cytoplasm were upstream-274 and bp had a single base mutation site (FIG. 7). Monoclonal sequencing results showed that even in UG93A and F1cox3The number of transcripts was consistent but the frequency of amplification was not the same for each sequence site in different nuclear contexts. In UG93A, the transcript frequencies at positions-164 and-856 upstream of the start codon were not 30.43% (7/23) and 56.52% (13/23), respectively, whereas the transcript frequencies at positions-313, -442 and-814 upstream of the start codon were 4.34% (1/23); the transcript frequencies at positions-164, -814 and-856 upstream of the start codon in F1 were 4.3% (1/23); the transcription frequencies at positions-313 and-442 upstream of the start codon were 30.43% (7/23) and 56.52% (13/23), respectively (FIG. 8). It has been shown that, in a different nuclear context,cox3is also regulated by nuclear-cytoplasmic interactions.
In summary, the CDS conservation sequence of the target gene can be used for amplifying the CDS full length of the target gene by adopting an RNA self-ligation and reverse nested PCR method, and simultaneously, the full length of transcripts at the 5 'end and the 3' end of the target gene can be obtained, and compared with the traditional RACE kit for amplifying the full length of the 5 'end and the 3' end of the target gene, the method has the advantages of low cost and higher cost performance; compared with the method for obtaining the target gene two terminal sequences by using a TAIL-PCR method, the method has stronger specificity and higher efficiency. The invention can synchronously amplify whether the target gene has a plurality of transcripts or different transcript abundance, and is more sensitive and faster than the traditional Weston Blot method for analyzing the gene transcription. In addition, the invention can also be applied to the comparison research of transcript abundance and transcript tail ends of target genes under different condition treatments or different varieties, and has important significance for the subsequent research of the shearing mode of target gene RNA and the research of transcription regulation mechanism.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.

Claims (6)

1. The method for identifying whether the kenaf variety is regulated by nuclear-cytoplasmic interaction based on mitochondrial gene transcript difference is characterized by comprising the following steps:
(1) Weighing the kenaf anther to be identified, and extracting RNA;
(2) Cyclizing the RNA extracted in the step (1), and then carrying out reverse transcription on the RNA cyclized product to form cDNA;
(3) Respectively designing two pairs of reverse nested primers at the 5 'and 3' ends of a target mitochondrial gene conserved region, carrying out two rounds of reverse nested PCR, carrying out a first round of PCR reaction by taking cDNA which is reversely transcribed after RNA cyclization as a template, combining two primers far from the boundaries of the 5 'and 3' ends of the target mitochondrial gene conserved region into a primer pair AP1/SP1, and carrying out PCR amplification reaction; the PCR products of the first round of PCR reaction are used as templates, two primers which are closer to the boundary of the 5 'end and the 3' end of a target gene conservation region are combined into a primer pair AP2/SP2, PCR amplification reaction is carried out, and after gel electrophoresis detection, the second round of nested PCR products are recovered and sequenced; the objective mitochondrial gene isatp1，atp9Andcox3；
(4) Comparing the sequencing result with the CDS conservation region of the target mitochondrial gene, and comparing the CDS full length of the target mitochondrial gene and the transcript full length of the 5 'to 3' ends of the mitochondrial gene through ORF finder on-line software and homologous sequences;
(5) Analyzing the sequence characteristics of CDS full length and/or 5 'to 3' end transcript full length of the mitochondrial gene obtained in the step (4), and identifying different cytoplasm of kenaf; the standard for identifying the different cytoplasm of the kenaf in the step (5) is as follows: to be used foratp1For mitochondrial purposesIn the case of the gene,atp1three specific bands were amplified in the sterile line cytoplasm, only one in the maintainer line cytoplasm, and in F 1 Two specific bands are arranged in cytoplasm; to be used foratp9When the gene is the mitochondria target gene, a specific band with the size of 400 bp is amplified in a sterile line, and the size of the amplified specific band in a maintainer line and F1 generation is 450 bp; to be used forcox3In the case of a gene of interest in mitochondria,cox35 specific bands were amplified in the cytoplasm of the sterile line, with the weak band between the two major bands, only one specific band in the maintainer line, and two adjacent major bands in the F1 generation.
2. The method of claim 1, wherein the mitochondrial gene of interest isatp1When the primer is used, two primers far from the boundary of the 5 'end and the 3' end of the conserved region of the target gene are respectivelyatp1-AP1 andatp1-the SP1 and two primers which are closer to the boundaries of the 5 'end and the 3' end of the conserved region of the target gene are respectivelyatp1-AP2 and AP2atp1-SP2 whereinatp1-The Ap1 sequence was 5'-CGATCTCATCCACTTGAAAATTCGT-3' and,atp1-the sequence of the AP2 is 5'-TTAGTTCTGCAGCTCTGGGAGAGA-3',atp1-the SP1 sequence was set to 5'-GGCTTACAGAAGTACCGAAACAACC-3',atp1-SP2 sequence 5'-GGATTCTGTGATCGAATGCCATTAG-3'.
3. The method of claim 1, wherein the mitochondrial gene of interest isatp9When the primer is used, two primers far from the boundary of the 5 'end and the 3' end of the conserved region of the target gene are respectivelyatp9-AP1 andatp9-the SP1 and two primers which are closer to the boundaries of the 5 'end and the 3' end of the conserved region of the target gene are respectivelyatp9-AP2 and AP2atp9-SP2 whereinatp9-The sequence of the AP1 is 5'-GCACCCATTAATTTTGCACCTTC-3',atp9-the sequence of the AP2 is 5'-ATTCTCGTCACGCGCTTTATCATTC-3',atp9-the SP1 sequence was set to 5'-ATTTTGGGCTTTGCTCTAACCGAAG-3',atp9-SP2 sequence 5'-GAAGCTATTGCATTGTTTGCCCTAA-3'.
4. The method according to claim 1, characterized in thatWherein the mitochondrial gene of interest iscox3When the primer is used, two primers far from the boundary of the 5 'end and the 3' end of the conserved region of the target gene are respectivelycox3-AP1 andcox3-the SP1 and two primers which are closer to the boundaries of the 5 'end and the 3' end of the conserved region of the target gene are respectivelycox3-AP2 and AP2cox3-SP2 whereincox3-The sequence of the AP1 is 5'-GCCCAAACTGAGAAGTCTTGCAC-3',cox3-the sequence of the AP2 is 5'-CCACTTTGCCTCGGTTGTATGTAA-3',cox3-the SP1 sequence was set to 5'-GGTCATCTGACTAAAGAGCATCACG-3',cox3-SP2 sequence 5'-GCATTTTGTAGACGTGGTTCGG-3'.
5. The method according to any one of claims 2 to 4, wherein the reverse nested PCR reaction system in step (3) is as follows: in a 20. Mu.L reaction system containing 10. Mu.L of 2X Phanta Max Master Mix, 0.6. Mu.M AP primer, 0.6. Mu.M SP primer, 50 ng template, add ddH 2 O is added to the total volume of 20 mu L; the amplification procedure was: 95. pre-denaturing at 95deg.C for 3 min, denaturing at 95deg.C for 30 sec, annealing at 58-60deg.C for 30 sec, extending at 72deg.C for 1min, setting the number of reaction cycles to 35, extending at 72deg.C for 5 min, and preserving at 4deg.C; the first and second rounds of PCR products were each spotted in two adjacent gel wells of 1.5% agarose for gel electrophoresis analysis.
6. The method of claim 5 in analysis/identificationatp1，atp9Or (b)cox3The application of the gene in the nuclear-cytoplasmic interaction regulation of the kenaf variety to be detected.
CN202110590238.4A 2021-05-28 2021-05-28 Method for identifying different cytoplasm of kenaf based on mitochondrial gene transcript difference and application thereof Active CN113249512B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110590238.4A CN113249512B (en) 2021-05-28 2021-05-28 Method for identifying different cytoplasm of kenaf based on mitochondrial gene transcript difference and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110590238.4A CN113249512B (en) 2021-05-28 2021-05-28 Method for identifying different cytoplasm of kenaf based on mitochondrial gene transcript difference and application thereof

Publications (2)

Publication Number Publication Date
CN113249512A CN113249512A (en) 2021-08-13
CN113249512B true CN113249512B (en) 2023-04-28

Family

ID=77185226

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110590238.4A Active CN113249512B (en) 2021-05-28 2021-05-28 Method for identifying different cytoplasm of kenaf based on mitochondrial gene transcript difference and application thereof

Country Status (1)

Country Link
CN (1) CN113249512B (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107475399A (en) * 2017-09-06 2017-12-15 西北农林科技大学 A kind of cytoplasmic authentication method of K-type wheat cytoplasmic male sterilty
CN109796527A (en) * 2019-03-07 2019-05-24 广西大学 A kind of bluish dogbane mitochondrial protein COX3 antigen polypeptide and the methods and applications for preparing polyclonal antibody

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2716374B1 (en) * 1994-02-18 1996-04-19 Sederma Sa New cosmetic or dermopharmaceutical compositions containing plant extracts.
CN102260670B (en) * 2010-07-12 2013-04-03 中国人民解放军军事医学科学院放射与辐射医学研究所 Method for excavating low-abundance expression gene and system identification splicing variants by using reverse transcription technique combining nested PCR (Polymerase Chain Reaction) with gene-specific primers (GSP), and application thereof
CN103667495B (en) * 2013-12-19 2015-11-11 广西大学 A kind of molecular assay method of bluish dogbane male sterile cytoplasm
CN112877468B (en) * 2021-04-21 2022-08-05 广西壮族自治区农业科学院 CMS molecular tag based on kenaf mitochondrial gene non-coding region, primer pair and application thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107475399A (en) * 2017-09-06 2017-12-15 西北农林科技大学 A kind of cytoplasmic authentication method of K-type wheat cytoplasmic male sterilty
CN109796527A (en) * 2019-03-07 2019-05-24 广西大学 A kind of bluish dogbane mitochondrial protein COX3 antigen polypeptide and the methods and applications for preparing polyclonal antibody

Also Published As

Publication number Publication date
CN113249512A (en) 2021-08-13

Similar Documents

Publication Publication Date Title
CN110567951A (en) Apple stem groove virus visual detection system based on CRISPR-Cas12a technology and detection method thereof
CN104962643B (en) Stablize reference gene, its screening technique and the application of expression in white backed planthopper different tissues position
CN107385117B (en) Detection method for composite virus disease (SPVD) pathogen of sweet potato seed
CN113249512B (en) Method for identifying different cytoplasm of kenaf based on mitochondrial gene transcript difference and application thereof
CN113604598A (en) Molecular marker primer and method for identifying common camellia oleifera and small camellia oleifera
CN115852028B (en) Triple PCR primer group, kit and detection method for detecting rice blast fungus non-toxic genes
CN112342300A (en) Fluorescence quantitative detection reference gene of 7 tissues of red crayfish and screening method and application thereof
CN107779521B (en) DNA bar code and application thereof in identifying muscadine grapes
CN114622041A (en) Primer and TaqMan probe for detecting canine torque teno virus and application thereof
CN110964837B (en) Primer group and detection kit for detecting horse, donkey, horse mule and donkey mule-derived components
CN107541521A (en) A kind of authentication method of radix coniti coreani DNA bar code and radix coniti coreani based on big data
CN109456967B (en) Specific nucleotide, labeled primer and identification method of physalis macrocarpa
CN113430276A (en) Method for identifying sheep wool and goat wool based on CO I gene
CN112899389B (en) Identifying primer and molecular identifying method for dalbergia odorifera
CN115341048B (en) Molecular marker related to presence or absence of phlorizin in malus plants and application thereof
CN109468398B (en) Characteristic sequence, primer and method for identifying polygonatum longstem and polygonatum cyrtonema
CN108277294A (en) Specific primer and probe for detecting maize dna and real-time fluorescence quantitative PCR kit
CN116497138B (en) Detection method and kit for identifying and detecting mycoplasma bovis and mycoplasma caprae
CN113481220A (en) Blood coding gene CYP1A1 related to dairy cow ketosis and PCR detection kit thereof
Meena et al. Isolation of genomic dna from sheep and goat Milk
CN105385687B (en) The cloning process of tobacco 26S RNA reference genes and its application
CN117965809A (en) RT-PCR detection primer and detection method for citrus delta split virus 1
JP2006254710A (en) Primer set for strawberry variety identification and method for strawberry variety identification using the same
CN110656194A (en) Callicarpa nudiflora DNA bar code standard detection sequence and application thereof
CN116497131A (en) Rana chensinensis specific sequence, primer and method for identifying rana chensinensis or rana chensinensis oil by using same

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant