CN113584210A - 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 a NtPIF1 gene, wherein the gene is a NtPIF1 gene; the sequence is SEQ ID NO. 1; the method comprises the following steps: numbering the single plant, sampling leaves, extracting RNA, synthesizing cDNA, detecting gene expression level, bagging and self-crossing. The invention discloses screening of cinnabar plants by using the NtPIF1 gene expression level for the first time, and the tobacco leaves are directly sampled and detected without any treatment, and the screened plant-roasted leaves show obvious cinnabar characteristics.
Description
Technical Field
The invention belongs to the technical field of tobacco genetic breeding, and particularly relates to the technical field of screening genes, sequences and methods of cinnabar plants.
Background
Cultivated tobacco (Nicotiana tabacum) is an heterotetraploid derived from two ancestral species, villiform tobacco (n.tominosiformis) and forest tobacco (n.sylvestris), which is doubled by interspecific hybridization, but the major alkaloids of its leaves are quite different from those of the two ancestors. In most of green and aged leaves of cultivated tobacco, nicotine is the main alkaloid and accounts for about 90-95% of the total alkaloid. The villous tobacco accumulates demethylated nicotine in both green and senescent leaves; while forest tobacco accumulates nicotine mainly in green leaves, most of the nicotine is converted into demethylated nicotine during the leaf senescence process. Burley tobacco is a type of common tobacco in which the main alkaloid of the "non-transformants" is nicotine, but about 20% of individuals in each generation population undergo an unknown mutation to become "transformants" in which the main alkaloid is demethylated nicotine. Similar mutation can also occur in single flue-cured tobacco plant, the tobacco leaves mainly accumulate demethylated nicotine, red spots appear on the surface of the mutant cured tobacco leaves, foreign scholars are called as 'Cherry-red', and Chinese is called as 'cinnabar'. The probability of the occurrence of the cinnabar is extremely low, and researches show that 3 cinnabar strains are found in 538 single strains, and the probability is about 0.56 percent. The cinnabar cigarette has unique glutinous rice fragrance, and is a well-received flavoring material for smokers in Yunnan area to smoke hookah. The method comprises the steps of collecting the resource tobacco leaves of the cinnabar smoke and the conventional tobacco leaves, and carrying out transcriptome analysis to find that the expression level difference of the NtPIF1 gene between the two is extremely obvious, so that the gene can be used as a marker gene for screening cinnabar smoke plants.
The cinnabar tobacco leaves are used as a characteristic raw material and have wide application prospect. However, in the existing cinnabar production, field mutant tobacco leaves with cinnabar characteristics are mainly selected in a purchasing link, 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 cinnabar resource capable of stabilizing inheritance is a prerequisite basis for large-scale development of cinnabar.
Disclosure of Invention
As the cinnabar and the conventional flue-cured tobacco are difficult to distinguish by appearance in the growth stage, cinnabar plants cannot be screened out and harvested, and the cinnabar-free seeds are used for agricultural production. The invention aims to solve the problems and defects, and aims to provide a screening gene, a screening sequence and a screening method of a cinnabar plant.
The invention is realized by adopting the following technical scheme.
Screening genes of cinnabar plants, wherein the genes are NtPIF1 genes; the sequence is SEQ ID NO. 1.
The method for screening the cinnabar plants based on the gene comprises the following steps: numbering the single plant, sampling leaves, extracting RNA, synthesizing cDNA, detecting gene expression level, bagging and self-crossing.
The steps of the invention are specifically as follows:
step (1), numbering the single hanging plate: all the tobacco to be screened are hung and marked with unique numbers;
step (2), sampling the leaves: taking about 0.1g of young and tender leaf tissues of tobacco plants, and preserving in liquid nitrogen;
and (3) RNA extraction and cDNA synthesis: extracting total RNA of leaf tissue and carrying out reverse transcription to obtain 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 the plant with the corresponding number according to the qPCR result, bagging and self-mating the plant to obtain the stable genetic resource of the cinnabar.
The step (4) standard of the invention is as follows: reach 1/100 lower than the Reference gene Actin, i.e. Target/Reference <1 x 10-2。
The specific qPCR primer in the step (4) is NtPIF1qF 1: CGGAGGTGGCAGCGGTGATGC, NtPIF1qR 1: ACGTTCGAAGGAGGAGGAGTC, respectively;
the NtAcTN gene is used as an internal reference and comprises the following components:
NtACTINq_F_1:CTGAGGTCCTTTTCCAACCA、
NtACTINq_R_1:TACCCGGGAACATGGTAGAG。
the standard of the genetic resources of the cinnabar obtained in the step (5) of the invention is 8 offspring plants, the expression level of the NtPIF1 gene of the leaf is lower than 1/100 of the internal Reference gene Actin, namely Target/Reference <1 x 10-2(ii) a All the roasted leaves of the single plants show obvious cinnabar characteristics, which indicates that the screening is successful.
The invention relates to application of a gene, in particular to a method for screening cinnabar plants by using the gene in expression level.
The application of the method disclosed by the invention is the application of the method in screening of the cinnabar plants.
The application of the method disclosed by the invention is the application of the specific qPCR primer and the NtActin internal reference in screening of the cinnabar plant.
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 screening method has the beneficial effects that the screening method utilizes the NtPIF1 gene expression level to screen the cinnabar plants for the first time, the tobacco leaves are directly sampled and detected without any treatment, and the screened plant-roasted leaves show obvious cinnabar characteristics.
The invention is further explained below with reference to the drawings and the detailed description.
Drawings
FIG. 1 transcriptome analysis chart.
FIG. 2 is a verification drawing of the expression level of LG2.1367 gene.
FIG. 3 is a verification diagram of the expression level of NtPIF1 gene.
FIG. 4 is a graph showing the expression level of plant leaf NtPIF1 gene.
FIG. 5 is a table diagram of leaves of the selected plants after roasting.
FIG. 6 is a graph showing the expression level of NtPIF1 gene in the leaves of progeny plants.
Detailed Description
The present invention is further illustrated by the following examples and the accompanying drawings, but the present invention is not limited thereto in any way, and any modifications or alterations based on the teaching of the present invention are within the scope of the present invention.
The invention relates to a screening gene, a screening sequence and a screening method of a cinnabar plant; the method comprises the following steps of single plant listing numbering, leaf sampling, RNA extraction and cDNA synthesis, gene expression level detection, bagging self-cross seed collection, and comprises the following specific steps:
(1) the single plant is numbered: all the tobacco to be screened are hung and marked with unique numbers.
(2) Leaf sampling: taking about 0.1g of young leaf tissue of the tobacco plant, and preserving in liquid nitrogen.
(3) RNA extraction and cDNA synthesis: total RNA from leaf tissue was extracted and reverse transcribed into cDNA.
(4) And (3) detecting the gene expression level: specific qPCR primers are designed according to the NtPIF1 gene sequence, qPCR analysis is carried out by taking leaf cDNA as a template, and the expression level of the NtPIF1 gene is lower than 1/100 of internal Reference gene Actin (Target/Reference <1 x 10)-2) Namely the cinnabar plant.
(5) Finding out the plant with the corresponding serial number according to the qPCR result, bagging and self-mating the plant to obtain the stable genetic resource of the cinnabar.
The nucleotide sequence of the NtPIF1 gene is shown in SEQ ID NO. 1.
The NtPIF1 gene and the expression level of the leaf thereof are used as indexes for screening the cinnabar plant.
Example 1
Taking the middle leaf of the cinnabar resource and the control (3 biological repeats) in the tobacco germplasm resource, sequencing the transcriptome by using an Illumina HiSeq platform, filtering off-machine Data to obtain Clean Data, and performing sequence comparison with a reference genome to obtain Mapped Data. Differential expression analysis between sample groups was performed using the DESeq2 software to obtain a differentially expressed gene set between cinnabar smoke and controls. The screening condition of the differential gene is | log2Fold Change|>1, and FDR<0.05. 435 differentially expressed genes were obtained in total, of which 330 genes were increased in the presence of Cinnabaris and 105 genes were decreased in the presence of Cinnabaris (FIG. 1). Randomly selecting a plurality of candidate genes with increased expression level in the cinnabar smoke, and verifying the transcriptome result by utilizing qPCR. In transcriptome analysis, the expression level of the LG2.1367 gene of cinnabar is obviously reducedHowever, the results of the qPCR assay showed no significant difference in the expression level of LG2.1367 gene in the cinnabar and the control (fig. 2). In transcriptome analysis, the expression level of the cinnabar NtPIF1 gene is obviously reduced, and the qPCR detection result is consistent with the transcriptome result (figure 3), which indicates that the NtPIF1 gene can be used as a candidate target gene for screening cinnabar.
Example 2
Selecting tobacco plants which grow normally in the field and are not infected by virus, taking about 0.1g of young leaf tissues, and immediately storing in liquid nitrogen for later use. Total RNA from tobacco leaves was extracted and reverse transcribed to obtain the first strand cDNA (PrimeScriptTM RT reagent Kit with gDNA Eraser, Takara). Fluorescence real-time quantitative PCR (qRT-PCR) analysis (LightCycler 480, Roche) was performed on the target gene by SYBR Green method (SYBR Green Master Mix, Roche), NtACCIN gene was used as 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 expression level of NtPIF1 gene of plant _9 is lower than 1/100 of internal Reference gene Actin (Target/Reference <1 x 10)-2) Namely, the plant is cinnabar. The single plant is baked after being hung with the card, the baked blade presents obvious cinnabar characteristics (figure 5), and sensory evaluation shows that the fragrant characteristic of cinnabar smoke is obvious.
Example 3
After the seeds of the plant _9 are harvested, sowing is carried out, 10 plants are randomly selected on a floating disc, about 0.1g of young and tender leaf tissues are taken, and the young and tender leaf tissues are immediately placed in liquid nitrogen for storage and standby. Total RNA from tobacco leaves was extracted and reverse transcribed to obtain the first strand cDNA (PrimeScriptTM RT reagent Kit with gDNA Eraser, Takara). Fluorescence real-time quantitative PCR (qRT-PCR) analysis (LightCycler 480, Roche) was performed on the target gene by SYBR Green method (SYBR Green Master Mix, Roche), NtACCIN gene was used as 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 expression level of the NtPIF1 gene of the leaves of 8 progeny plants (CR60_1-8) of the plant _9 is lower than that of 1/100 of the internal Reference gene Actin (Target/Reference <1 x 10)-2) All the roasted leaves of the single plant show obvious cinnabar characteristics, which indicates that the screened single plant can be stably inherited.
The above description is only a part of specific embodiments of the present invention (since the formula of the present invention belongs to the numerical range, the embodiments are not exhaustive, and the protection scope of the present invention is subject to the numerical range and other technical point ranges), and the detailed contents or common knowledge known in the schemes are not described too much. It should be noted that the above-mentioned embodiments do not limit the present invention in any way, and all technical solutions obtained by means of equivalent substitution or equivalent transformation for those skilled in the art are within the protection scope of the present invention. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.
<110> research institute of tobacco agricultural science in Yunnan province
<120> 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 (9)
1. The screening gene of the cinnabar plant is characterized in that the gene is NtPIF1 gene; the sequence is SEQ ID NO. 1.
2. The method for screening a cinnabar plant based on the gene of claim 1, wherein the method comprises the following steps: numbering the single plant, sampling leaves, extracting RNA, synthesizing cDNA, detecting gene expression level, bagging and self-crossing.
3. The method according to claim 2, characterized in that the steps are in particular:
step (1), numbering the single hanging plate: all the tobacco to be screened are hung and marked with unique numbers;
step (2), sampling the leaves: taking about 0.1g of young and tender leaf tissues of tobacco plants, and preserving in liquid nitrogen;
and (3) RNA extraction and cDNA synthesis: extracting total RNA of leaf tissue and carrying out reverse transcription to obtain 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 the plant with the corresponding number according to the qPCR result, bagging and self-mating the plant to obtain the stable genetic resource of the cinnabar.
4. The method of claim 3, wherein the step (4) criteria are: reach 1/100 lower than the Reference gene Actin, i.e. Target/Reference <1 x 10-2。
5. The method of claim 3, wherein the specific qPCR primers of step (4) are NtPIF1qF 1: CGGAGGTGGCAGCGGTGATGC, NtPIF1qR 1: ACGTTCGAAGGAGGAGGAGTC, respectively;
the NtAcTN gene is used as an internal reference and comprises the following components:
NtACTINq_F_1:CTGAGGTCCTTTTCCAACCA、
NtACTINq_R_1:TACCCGGGAACATGGTAGAG。
6. the method as claimed in claim 3, wherein the standard of genetic resources of the cinnabar obtained in step (5) is 8 offspring plants, and the expression level of NtPIF1 gene of leaf is lower than 1/100 of internal Reference gene Actin, namely Target/Reference <1 x 10-2(ii) a All the roasted leaves of the single plants show obvious cinnabar characteristics, which indicates that the screening is successful.
7. The use of the gene as claimed in claim 1, wherein the gene is used in a method for screening the plant with the expression level of Cinnabaris.
8. The use of the method according to claim 3, wherein the method is used in screening of a Cibotium japonicum plant.
9. The use of the method as claimed in claim 5, wherein the specific qPCR primer and the NtActin internal reference are used in screening of the cinnabar plant.
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2021
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