CN113584210B - Method for screening cinnabar plants based on NtPIF1 gene expression level - Google Patents
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Abstract
The invention relates to a method for screening a cinnabar plant based on the expression level of an NtPIF1 gene, wherein the gene is the NtPIF1 gene; SEQ ID NO.1; the method comprises the following steps: single plant listing number, leaf sampling, RNA extraction, cDNA synthesis, gene expression level detection and bagging self-copulation seed collection. The invention discloses a method for screening a cinnabar plant by utilizing the NtPIF1 gene expression level for the first time, and tobacco leaves are directly sampled and detected without any treatment, and the screened plant leaves show remarkable cinnabar characteristics after baking.
Description
Technical Field
The invention belongs to the technical field of genetic breeding of tobacco, and particularly relates to the technical fields of screening genes, sequences and methods of cinnabar tobacco plants.
Background
Cultivated tobacco (Nicotiana tabacum) is a heterotetraploid derived from two ancestor species Nicotiana villosa (N.tomotosisomici) and Nicotiana woods (N.sylvestris) by interspecific cross doubling, but its leaf major alkaloids are quite different from the two ancestors. In most green and senescent leaves of cultivated tobacco, nicotine is the major alkaloid and accounts for about 90-95% of the total alkaloids. The villous tobacco accumulates the demethylated nicotine in the green and senescent leaves; while woody tobacco accumulates mainly nicotine in green leaves, most of the nicotine is converted to nornicotine during leaf senescence. Burley tobacco is a type of common tobacco, the major alkaloid of which is "non-transformant" is nicotine, but about 20% of individuals in each generation population undergo unknown mutation to become "transformant", and the major alkaloid in "transformant" is nornicotine. Similar mutation can occur in flue-cured tobacco single plants, the tobacco leaves mainly accumulate the nornicotine, the surfaces of the tobacco leaves after the mutant plants are cured show red spots, foreign scholars are called "Cherry-red", and China is called "Cinnabaris cigarette". The occurrence probability of the cinnabar is extremely low, and researches show that 3 cinnabar strains are found in 538 single plants, and the probability is about 0.56%. The cinnabar cigarette has unique 'sticky rice fragrance', and is a popular flavoring raw material when smokers in Yunnan region smoke the water-absorbing chimney. Transcriptome analysis is carried out on the cinnabar resource tobacco leaves and the conventional tobacco leaves, and the difference of the expression level of the NtPIF1 gene between the cinnabar resource tobacco leaves and the conventional tobacco leaves is extremely remarkable, so that the cinnabar resource tobacco leaves and the conventional tobacco leaves can be used as marker genes for screening cinnabar plants.
The cinnabar tobacco leaves are used as special raw materials, and have wide application prospect. However, currently, the production of the cinnabar tobacco mainly adopts a purchasing link to manually select field mutation tobacco leaves with the cinnabar characteristics, so that the yield of the tobacco leaves is unstable, and the quality of the tobacco leaves has a plurality of defects. Screening out the Cinnabaris smoke resource capable of being inherited stably is a precondition for large-scale development of Cinnabaris smoke.
Disclosure of Invention
The cinnabar tobacco is difficult to distinguish from the conventional flue-cured tobacco in the growth stage by appearance, and the cinnabar tobacco plants cannot be screened out and harvested, so that cinnabar-free tobacco seeds are used for agricultural production. The invention aims to solve the defects, and aims to provide a screening gene, a sequence and a method for a cinnabar plant.
The invention is realized by adopting the following technical scheme.
The Cinnabaris tobacco plant screening gene is NtPIF1 gene; the sequence is SEQ ID NO.1.
The method for screening the cinnabar plants based on the genes comprises the following steps: single plant listing number, leaf sampling, RNA extraction, cDNA synthesis, gene expression level detection and bagging self-copulation seed collection.
The method comprises the following steps:
step (1) single plant listing number: all the individual plants of the tobacco to be screened are hung, and the unique numbers are marked;
step (2) sampling the leaves: about 0.1g of tender leaf tissue of the tobacco plant is taken and put into liquid nitrogen for preservation;
step (3) RNA extraction and cDNA synthesis: extracting total RNA of leaf tissues, and reversely transcribing the total RNA into cDNA;
and (4) detecting the gene expression level: designing a specific qPCR primer according to the NtPIF1 gene sequence, and carrying out qPCR analysis by taking the leaf cDNA as a template, wherein the NtPIF1 gene expression level reaches the standard, namely the cinnabar plant;
and (5) finding out plants with corresponding numbers according to qPCR results, bagging and selfing, and obtaining stable cinnabar genetic resources.
The step (4) standard of the invention is as follows: reaching less than 1/100 of the Reference gene action, i.e. Target/Reference <1 x 10 -2 。
The specific qPCR primer in the step (4) is NtPIF1qF1: CGGAGGTGGCAGCGGTGATGC, ntPIF1qR1: ACGTTCGAAGGAGGAGGAGTC;
the NtACTIN gene is used as an internal reference, and is:
NtACTINq_F_1:CTGAGGTCCTTTTCCAACCA、
NtACTINq_R_1:TACCCGGGAACATGGTAGAG。
the standard of the stable cinnabar genetic resource obtained in the step (5) is 8 offspring plants, the NtPIF1 gene expression level of the leaf is lower than 1/100 of the Reference gene action, namely the Target/Reference is less than 1 x 10 -2 The method comprises the steps of carrying out a first treatment on the surface of the All individual baked leaves exhibited significant cinnabar characteristics, indicating successful screening.
The gene is applied to a method for screening cinnabar plants in an expression level.
The application of the method in the screening of the cinnabar plants is disclosed.
The application of the method provided by the invention is that the specific qPCR primer and the NtACTIN internal reference are applied to the screening of cinnabar plants.
SEQ ID NO.1
>NtPIF1
atgaatcattcagttcctgagtttgatatggatgatgactacactattcctacgtcttctggtcttacca
gacctaaaaagtctgcaatggcggaagaggatatcatggaactattgtggcataatggacaagtggttat
gcagagccaaaatcaaagatctctgaagaaatctcacattagcaacggcggtggcggaggtggcagcggt
gatgcgcttattccctccgaacaagctgtcagtagagagatccgacatgtagaggaaactactacaccac
agcaactgtttatgcaggaggacgagatggcctcatggcttcactacccactcgatgactcctcctcctt
cgaacgtgatctttacgccgatctcctttattccacaccgagcgcaaccgttacaaccgctgcgccgccg
cgagaaatccgtacgcccccggtggagatccgtccacctccgccgcatccatcccctgcaccgccgattg
cagtggctccacgaccgcctatacctcctcctgcaagacgtcccggcactgaaagctcacatcggttcca
gaacttcggacacttctcgcgattgcctagtcgaacaaggtcagaacttggtccgtcaaattcgagcaag
tcacctagagaatcaacggttgtggactcaaacgaaactccaatttcagggcctgaatctagggtttcac
aggtagcggataatgtagtaccggttcccggcggaaatggagcatgtggggctgtaaatgtcaacggaac
tgcgactgcgtcaacggcaattagggaaccggcgacaacatgtgagctttcggtgacgtcatctcccggc
tcaggaaacagtataaacgccagcgctgaaccaccgctgtcggaaacggcggcgttggcgacaccgacgg
ctgcggcatcgaatgatcggaaacgcaaaggaatagaaacggacgacggggatggtcagaatgaggacgc
tgaatttgggtctggtgatacaaagaagcatgcacgtggttcaacgtctacaaaacgatctcgtgctgca
gaggtccataatctttcggaaaggagacgtcgagacagaataaatgagaagatgagggctctgcaggaac
tgataccacgctgtaacaagacagacaaagcttcaatgctggatgaggcaattgagtatttgaaatcact
gcaattgcaagtgcagatgatgtccatgggatgcggcatggtcccgatgatgtatcctggaatgcagcaa
tacatgccagctatgggaatgggcatggtgggaatgggtatggagattggcatgaacaggccaatggttc
catatccacctctattaccaggtgcagcgatgcagaatgcagctgcagcagcacaaatgggtcctagatt
tcctatggcaccgtttcatttgccaccagttccagtaccagatccttccaggatgcaagcctcaagtcag
caagatccaatgctaaatccacttgtagcacgtaatcccaaccagccaagacttccgaattttaatgatc
catatcaacagcattttggtctccaccaggcacaagtgcaattaccgcagaatcaggcagtagaacagca
aggttacaataaacccggcagcagcaaagaagttggaaatccagggaatcctcaatctggttga
The invention has the beneficial effects that the invention firstly discloses screening the cinnabar plants by utilizing the NtPIF1 gene expression level, and the tobacco leaves are directly sampled and detected without any treatment, and the screened plants show remarkable cinnabar characteristics after baking.
The invention is further explained below with reference to the drawings and the detailed description.
Drawings
FIG. 1 is a transcriptome analysis chart.
FIG. 2LG2.1367 gene expression level validation panels.
FIG. 3 shows a graph of the verification of the expression level of NtPIF1 gene.
FIG. 4 shows a graph of plant leaf NtPIF1 gene expression level.
FIG. 5 is a leaf phenotype plot of the screened plants after baking.
FIG. 6 shows a graph of the detection of gene expression levels of leaf NtPIF1 in progeny plants.
Detailed Description
The invention is further described below with reference to examples and figures, but is not limited in any way, and any alterations or substitutions based on the teachings of the invention are within the scope of the invention.
The invention relates to a screening gene, a sequence and a method of a cinnabar plant; the method comprises the following specific steps of single plant listing numbering, leaf sampling, RNA extraction, cDNA synthesis, gene expression level detection, bagging selfing seed collection:
(1) Single plant listing number: all the individual plants of the tobacco to be screened are hung, and the unique numbers are marked.
(2) Blade sampling: about 0.1g of tender leaf tissue of the tobacco plant is taken and put into liquid nitrogen for preservation.
(3) RNA extraction and cDNA synthesis: leaf tissue total RNA was extracted and reverse transcribed into cDNA.
(4) Gene expression level detection: design of specificity according to NtPIF1 Gene sequenceqPCR primer, leaf cDNA is used as template to perform qPCR analysis, and NtPIF1 gene expression level is lower than 1/100 of that of internal Reference gene action (Target/Reference <1 x 10) -2 ) Namely cinnabar plants.
(5) And finding out plants with corresponding numbers according to qPCR results, bagging and self-copulation, thus obtaining stable cinnabar genetic resources.
The nucleotide sequence of the NtPIF1 gene is shown as SEQ ID NO.1.
The expression level of the leaf of the NtPIF1 gene is used as an index in the application of the cinnabar plant screening.
Example 1
Taking middle leaves of cinnabar resources and control (with 3 biological repetition) in tobacco germplasm resources, sequencing transcriptome by utilizing an Illumina Hiseq platform, filtering off-machine Data to obtain Clean Data, and comparing the Clean Data with a reference genome to obtain Mapped Data. And performing differential expression analysis among sample groups by using DESeq2 software to obtain a differential expression gene set between the cinnabar and a control. Screening conditions for differential Gene were |log 2 Fold Change|>=1, and FDR<0.05. There were obtained 435 differentially expressed genes, 330 genes whose expression level was increased and 105 genes whose expression level was decreased in Cinnabaris smoke (FIG. 1). Several candidate genes with elevated expression levels in cinnabar smoke were randomly selected and the transcriptome results were verified using qPCR. In the transcriptome analysis, the expression level of the cinnabar LG2.1367 gene is obviously reduced, but the qPCR detection result shows that the expression level of the LG2.1367 gene in the cinnabar and the control is not obviously different (figure 2). In the transcriptome analysis, the expression level of the NtPIF1 gene of the cinnabar is obviously reduced, and the qPCR detection result is consistent with the transcriptome result (figure 3), which shows that the NtPIF1 gene can be used as a candidate target gene for screening the cinnabar.
Example 2
Selecting tobacco plants which normally grow in the field and are not infected by viruses, taking about 0.1g of tender leaf tissues, and immediately placing the tender leaf tissues in liquid nitrogen for storage. Total RNA from tobacco leaves was extracted and reverse transcribed to obtain a first strand of cDNA (PrimeScriptTM RT reagent Kit with gDNA Eraser, takara). The target gene was subjected to fluorescent real-time quantitative PCR (qRT-PCR) analysis (LightCycler 480, roche) using SYBR Green method (SYBR Green l Master Mix, roche), and the NtACTIN gene was used as an internal reference, and the quantitative primers were as follows:
NtPIF1qF1：CGGAGGTGGCAGCGGTGATGC
NtPIF1qR1：ACGTTCGAAGGAGGAGGAGTC
NtACTINq_F_1:CTGAGGTCCTTTTCCAACCA
NtACTINq_R_1:TACCCGGGAACATGGTAGAG
as shown in FIG. 4, the NtPIF1 gene expression level of plant_9 was lower than 1/100 of that of the Reference gene, actin (Target/Reference <1×10) -2 ) Namely, the plant is cinnabar. The single plant is baked after being put on a card, and the baked leaf presents obvious cinnabar characteristics (figure 5), and sensory evaluation shows that the cinnabar smoke has obvious aroma characteristics.
Example 3
After harvesting the plant_9 seeds, sowing, randomly selecting 10 plants on a floating plate, taking about 0.1g of tender leaf tissues, and immediately placing the tender leaf tissues in liquid nitrogen for standby. Total RNA from tobacco leaves was extracted and reverse transcribed to obtain a first strand of cDNA (PrimeScriptTM RT reagent Kit with gDNA Eraser, takara). The target gene was subjected to fluorescent real-time quantitative PCR (qRT-PCR) analysis (LightCycler 480, roche) using SYBR Green method (SYBR Green l Master Mix, roche), and the NtACTIN gene was used as an internal reference, and the quantitative primers were as follows:
NtPIF1qF1：CGGAGGTGGCAGCGGTGATGC
NtPIF1qR1：ACGTTCGAAGGAGGAGGAGTC
NtACTINq_F_1:CTGAGGTCCTTTTCCAACCA
NtACTINq_R_1:TACCCGGGAACATGGTAGAG
as shown in FIG. 6, the NtPIF1 gene expression level of 8 progeny plant (CR 60-1-8) leaves of plant_9 was lower than 1/100 of the Reference gene action (Target/Reference < 1.times.10) -2 ) All the individual plants show remarkable cinnabar characteristics after baking, which indicates that the screened individual plants can be inherited stably.
The foregoing is only a few specific embodiments of the present invention (the embodiments are not intended to be exhaustive, and the scope of the invention is defined by the following claims and other technical gist of the present invention), and the details or common sense of the present invention are not described in any detail herein. It should be noted that the above embodiments do not limit the present invention in any way, and it is within the scope of the present invention for those skilled in the art to obtain the technical solution by equivalent substitution or equivalent transformation. The protection scope of the present application shall be subject to the content of the claims, and the description of the specific embodiments and the like in the specification can be used for explaining the content of the claims.
<110> tobacco agricultural science institute of Yunnan province
<120> a method for screening cinnabar plants based on NtPIF1 gene expression level
<160> 5
<210> 1
<211> 1674
<212> DNA
<213> artificial sequence
<400> 1
atgaatcattcagttcctgagtttgatatggatgatgactacactattcctacgtcttctggtcttacca
gacctaaaaagtctgcaatggcggaagaggatatcatggaactattgtggcataatggacaagtggttat
gcagagccaaaatcaaagatctctgaagaaatctcacattagcaacggcggtggcggaggtggcagcggt
gatgcgcttattccctccgaacaagctgtcagtagagagatccgacatgtagaggaaactactacaccac
agcaactgtttatgcaggaggacgagatggcctcatggcttcactacccactcgatgactcctcctcctt
cgaacgtgatctttacgccgatctcctttattccacaccgagcgcaaccgttacaaccgctgcgccgccg
cgagaaatccgtacgcccccggtggagatccgtccacctccgccgcatccatcccctgcaccgccgattg
cagtggctccacgaccgcctatacctcctcctgcaagacgtcccggcactgaaagctcacatcggttcca
gaacttcggacacttctcgcgattgcctagtcgaacaaggtcagaacttggtccgtcaaattcgagcaag
tcacctagagaatcaacggttgtggactcaaacgaaactccaatttcagggcctgaatctagggtttcac
aggtagcggataatgtagtaccggttcccggcggaaatggagcatgtggggctgtaaatgtcaacggaac
tgcgactgcgtcaacggcaattagggaaccggcgacaacatgtgagctttcggtgacgtcatctcccggc
tcaggaaacagtataaacgccagcgctgaaccaccgctgtcggaaacggcggcgttggcgacaccgacgg
ctgcggcatcgaatgatcggaaacgcaaaggaatagaaacggacgacggggatggtcagaatgaggacgc
tgaatttgggtctggtgatacaaagaagcatgcacgtggttcaacgtctacaaaacgatctcgtgctgca
gaggtccataatctttcggaaaggagacgtcgagacagaataaatgagaagatgagggctctgcaggaac
tgataccacgctgtaacaagacagacaaagcttcaatgctggatgaggcaattgagtatttgaaatcact
gcaattgcaagtgcagatgatgtccatgggatgcggcatggtcccgatgatgtatcctggaatgcagcaa
tacatgccagctatgggaatgggcatggtgggaatgggtatggagattggcatgaacaggccaatggttc
catatccacctctattaccaggtgcagcgatgcagaatgcagctgcagcagcacaaatgggtcctagatt
tcctatggcaccgtttcatttgccaccagttccagtaccagatccttccaggatgcaagcctcaagtcag
caagatccaatgctaaatccacttgtagcacgtaatcccaaccagccaagacttccgaattttaatgatc
catatcaacagcattttggtctccaccaggcacaagtgcaattaccgcagaatcaggcagtagaacagca
aggttacaataaacccggcagcagcaaagaagttggaaatccagggaatcctcaatctggttga
<210> 2
<211> 21
<212> DNA
<213> artificial sequence
<400> 2
CGGAGGTGGCAGCGGTGATGC
<210> 3
<211> 20
<212> DNA
<213> artificial sequence
<400> 3
ACGTTCGAAGGAGGAGGAGTC
<210> 4
<211> 20
<212> DNA
<213> artificial sequence
<400> 4
CTGAGGTCCTTTTCCAACCA
<210> 5
<211> 20
<212> DNA
<213> artificial sequence
<400> 5
TACCCGGGAACATGGTAGAG
Claims (2)
1. The method for screening the cinnabar plants based on the NtPIF1 gene is characterized by comprising the following steps of: the method comprises the following steps of single plant listing numbering, leaf sampling, RNA extraction, cDNA synthesis, gene expression level detection and bagging self-copulation seed collection, wherein the steps are as follows:
step (1) single plant listing number: all the individual plants of the tobacco to be screened are hung, and the unique numbers are marked;
step (2) sampling the leaves: 0.1g of tender leaf tissue of tobacco plant is taken and put in liquid nitrogen for preservation;
step (3) RNA extraction and cDNA synthesis: extracting total RNA of leaf tissues, and reversely transcribing the total RNA into cDNA;
and (4) detecting the gene expression level: designing a specific qPCR primer according to the NtPIF1 gene sequence, and carrying out qPCR analysis by taking the leaf cDNA as a template, wherein the NtPIF1 gene expression level reaches the standard, namely the cinnabar plant;
finding out plants with corresponding numbers according to qPCR results, bagging selfing seeds, and obtaining stable cinnabar genetic resources, wherein the standard of the step (4) is as follows: reaching less than 1/100 of the Reference gene action, i.e. Target/Reference <1 x 10 -2 The NtPIF1 gene sequence is SEQ ID NO.1, the standard of the stable cinnabar genetic resource obtained in the step (5) is 8 offspring plants, the expression level of the NtPIF1 gene of the leaf is lower than 1/100 of that of the internal Reference gene action, namely, the Target/Reference is less than 1 x 10 -2 The method comprises the steps of carrying out a first treatment on the surface of the All individual baked leaves exhibited significant cinnabar characteristics, indicating successful screening.
2. The method of claim 1, wherein the specific qPCR primer of step (4) is NtPIF1qF1: CGGAGGTGGCAGCGGTGATGC, ntPIF1qR1: ACGTTCGAAGGAGGAGGAGTC;
the NtACTIN gene is used as an internal reference, and is:
NtACTINq_F_1:CTGAGGTCCTTTTCCAACCA、
NtACTINq_R_1:TACCCGGGAACATGGTAGAG。
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004087923A1 (en) * | 2003-03-31 | 2004-10-14 | Korea Research Institute Of Bioscience And Biotechnology | A NEW STRESS-RESISTANCE TRANSCRIPTION FACTOR (CaPIF1 AND NbPIF1), ITS GENE AND A STRESS-RESISTANCE PLANT TRANSFECTED BY THE GENE |
| CN103555748A (en) * | 2013-10-30 | 2014-02-05 | 贵州省烟草科学研究院 | Carrier composition for reverse regulation of tobacco NtPSY gene function |
| CN110839367A (en) * | 2019-11-20 | 2020-02-28 | 云南省烟草农业科学研究院 | Method for screening cinnabar tobacco seeds based on nicotine quantitative conversion rate and gene expression |
| CN111690662A (en) * | 2020-06-03 | 2020-09-22 | 吉林大学 | Application of soybean bHLH transcription factor GmPIF1 gene in promotion of isoflavone synthesis |
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2021
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004087923A1 (en) * | 2003-03-31 | 2004-10-14 | Korea Research Institute Of Bioscience And Biotechnology | A NEW STRESS-RESISTANCE TRANSCRIPTION FACTOR (CaPIF1 AND NbPIF1), ITS GENE AND A STRESS-RESISTANCE PLANT TRANSFECTED BY THE GENE |
| CN103555748A (en) * | 2013-10-30 | 2014-02-05 | 贵州省烟草科学研究院 | Carrier composition for reverse regulation of tobacco NtPSY gene function |
| CN110839367A (en) * | 2019-11-20 | 2020-02-28 | 云南省烟草农业科学研究院 | Method for screening cinnabar tobacco seeds based on nicotine quantitative conversion rate and gene expression |
| CN111690662A (en) * | 2020-06-03 | 2020-09-22 | 吉林大学 | Application of soybean bHLH transcription factor GmPIF1 gene in promotion of isoflavone synthesis |
Non-Patent Citations (2)
| Title |
|---|
| PIF transcription factors link a neighbor threat cue to accelerated reproduction in Arabidopsis;Vinicius Costa Galvāo等;《Nature communications》;第10卷(第1期);文献号：4005 * |
| PREDICTED: Nicotiana tabacum transcription factor PIF1-like (LOC107828013), mRNA;GenBank;《GenBank》;XM_016655263 * |
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