CN109456980B - Bletilla striata high-temperature internal reference genes Bs18S rRNA and BUBI - Google Patents

Abstract

The invention belongs to the technical field of bletilla striata genomes, and particularly relates to two high-temperature reference genes stably expressed under a high-temperature conditionBs18S rRNAAndBsUBI. The above-mentionedBs18S rRNAGene, containing 1792 bp; the above-mentionedBsUBIGene, 422 bp. In the application, based on the comparison between the normal growth temperature and the high-temperature stress condition, the real-time fluorescence quantitative PCR analysis is carried out on the expression stability of part of candidate reference genes, and the comprehensive analysis result shows that:Bs18S rRNAandBsUBIthe two genes have the most stable expression conditions in different time and different tissues under normal growth temperature and high temperature stress, so that a certain application foundation can be laid for the research of gene expression stability and reliability under the high temperature stress condition and the screening and cultivation of high temperature resistant rhizoma bletillae germplasm resources.

Description

Bletilla striata high-temperature internal reference genes Bs18S rRNA and BUBI

Technical Field

The invention belongs to the technical field of bletilla striata genomes, and particularly relates to two high-temperature reference genes stably expressed under a high-temperature conditionBs18S rRNAAndBsUBI。

background

Rhizoma Bletillae (Bletilla striata (Thunb.) Reihb. f.), and rhizoma Bletillae, rhizoma Alpiniae Officinarum, herba Violae, herba Glechomae, radix Melandrii Szechuanensis, radix seu herba Thalictri Elaeagni, radix Callicarpae Formosanae, rhizoma Helminthostachydis Zeylanicae, belonging to the family Orchidaceae and genus rhizoma Bletillae, is the basic plant of rhizoma Bletillae, and has effects of invigorating lung, relieving swelling, stopping bleeding, promoting granulation, etc. The method is mainly distributed in the shady and humid areas or hillside grass clusters under forests with the elevation of 100-3200 m in south China and south China. Bletilla striata is warm in nature, cool in the shade and relatively humid in the shade, and the growth of bletilla striata is obviously influenced when the temperature is higher than 36 ℃. Therefore, heat-resistant resources and gene mining are one of the important targets of bletilla striata breeding work.

The reference gene is basically a gene that is constantly expressed under a certain condition, and is mainly used as a reference for detecting the expression level of the target gene under a specific condition. Many studies have shown that the expression level of the reference gene varies under different conditions in different species, and the reference gene which can be stably expressed under any condition is almost nonexistent. Therefore, the selection of proper reference genes under different conditions is one of the keys of the real-time fluorescent quantitative PCR research. Therefore, when studying the expression of a target gene under a certain specific condition in a certain species, an internal reference gene capable of being stably expressed under such a condition should be selected.

In terms of the gene research of the bletilla striata, due to the thermolabile characteristic of the bletilla striata, when heat-resistant resources are screened, the internal reference gene which can be stably expressed under high-temperature conditions is screened and determined, and the gene research method has very important application significance for related gene analysis and genetic breeding research work.

Disclosure of Invention

The application aims to provide two reference genes which can be stably expressed in different tissues and different growth stages under the conditions of normal growth temperature and high temperature stressBs18S rRNAAndBsUBIthereby laying a foundation for researching related functional genes of the bletilla striata and screening high-temperature resistant bletilla striata resources.

The technical solution adopted in the present application is detailed as follows.

Rhizoma bletillae high-temperature reference geneBs18S rRNAAndBsUBIthe two genes are relatively stable and consistent in expression under the conditions of normal growth and high-temperature stress of the rhizoma bletillaeBs18S rRNAThe gene contains 1792bp, and the base sequence is shown in SEQ ID NO. 6; the above-mentionedBsUBIThe gene contains 422bp, and the base sequence is shown in SEQ ID NO. 7.

Used for obtaining rhizoma bletillae high-temperature reference gene by PCR amplificationBs18S rRNAAndBsUBIthe degenerate primers of (1) are as follows:

Bs18S rRNA-F：5’- TGGTTGATCCTGCCAGTAGT-3’，

Bs18S rRNA-R：5’- GTTCACCTACGGAAACCTTG-3’ ，

BsUBI-F：5’- ATGCAGATCTTCGTGAARACCCT-3’ ，

BsUBI-R：5’- CAGTAGTGGCGRTCGAAGTGGT-3’。

utilizing the degenerate primer to obtain rhizoma bletillae high-temperature reference genesBs18S rRNAAndBsUBIthe PCR amplification method of (1), comprising the steps of:

(1) extracting total RNA and performing reverse transcription to obtain cDNA as an amplification template;

(2) the degenerate primers were used for PCR amplification, and during PCR amplification, 20. mu.L of the reference design of the amplification system was as follows:

10×PCR Buffer，2.0 μL；

dNTP（2.5 mmol/L），1.6 μL；

primers F and R (both 10 mmol/L), each 0.5. mu.L;

cDNA template, 0.8. mu.L;

rTaqDNA polymerase (5U/. mu.L), 0.2. mu.L;

ddH₂O，14.4 μL；

the PCR amplification procedure was: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 40 s, annealing at 58 ℃ for 40 s, and extension at 72 ℃ for 80 s, for 35 cycles; finally, extension is carried out for 10 min at 72 ℃.

The rhizoma bletillae high-temperature reference geneBs18S rRNAAndBsUBIuse in bletilla striata, as reference gene, in particular for the functional gene ribulose-1, 5-bisphosphate carboxylase/oxygenase large subunit gene ((R))rbcL) The fluorescent quantitative detection analysis of (1).

A fluorescent quantitative detection method for ribulose-1, 5-diphosphocarboxylase/oxygenase large subunit gene is disclosed, which is characterized by thatBs18S rRNAAnd/orBsUBIThe method is used as an internal reference gene and specifically comprises the following steps:

(1) design of fluorescent quantitative primer as follows

Bs18S rRNA-F：5’-TTTATGAAAGACGAACCACTGC-3’，

Bs18S rRNA-R：5’-TCGGCATCGTTTATGGTTG-3’；

BsUBI-F：5’-CGCCGATTACAACATCCAGAA-3’，

BsUBI-R：5’-TTCTTGGGCTTGGTGTATGTC-3’；

qBsrbcl-F：5’-CCAAAACTTTCCAAGGTCCG-3’，

qBsrbcl-R：5’- TCCACCCCGTAGACATTCATA-3’；

(2) Extracting total RNA and performing reverse transcription to obtain cDNA as an amplification template, wherein when fluorescent quantitative PCR detection is performed, a 20.0 mu L reaction system is designed as follows:

2×SYBR Premix Ex Taq Ⅱ，10.0 μL；

primers F and R, each 0.8. mu.L;

cDNA template, 2.0 μ L;

ddH₂O，6.4 μL；

the reaction conditions are as follows: pre-denaturation at 95 ℃ for 30 s, denaturation at 95 ℃ for 15 s, annealing at 58 ℃ for 15 s, and extension at 72 ℃ for 15 s, for 40 cycles.

In the prior art, partial functional genes of bletilla striata genome are studied, but no genes of 3-glyceraldehyde phosphate dehydrogenase are found: (GAPDH) 18S ribosomal RNA genes（18S rRNA) The transcription elongation factor 1A gene (EF1α) Alpha tubulin gene (a)TUA) Beta tubulin gene (a)TUB) Ubiquitin elongase Gene: (UBI) NAC domain protein gene (NAC)NAC) Detailed study of these 7 genes.

In the application, based on the comparison between the normal growth temperature and the high-temperature stress condition, the expression stability of the 7 candidate reference genes is subjected to real-time fluorescent quantitative PCR analysis, and the comprehensive analysis result shows that:Bs18S rRNAandBsUBIthe two genes have the most stable expression conditions in different time and different tissues under normal growth temperature and high temperature stress, so that a certain application foundation can be laid for the research of gene expression stability and reliability under the high temperature stress condition and the screening and cultivation of high temperature resistant rhizoma bletillae germplasm resources.

Drawings

FIG. 1 is an electrophoretogram of total RNA of extracted rhizoma Bletillae;

FIG. 2 is a schematic view ofBs18S rRNAFor the internal referencerbcLRelative expression level of the gene;

FIG. 3 is a schematic view ofBsUBIFor the internal referencerbcLRelative expression level of gene.

Detailed Description

The technical solution of the present application is further explained with reference to the following embodiments, and before the detailed description, the following embodiments briefly describe the background of some biological materials, experimental reagents, experimental devices, etc. involved in the following embodiments.

Biological material:

bletilla striata, a common medicinal, ornamental orchidaceous plant, the experimental material used in the following examples was taken from the orchid engineering research center of Zhengzhou academy of education, intelligent sunlight greenhouse;

the synthesis and sequencing work of related primer sequences is completed by the Shanghai Jun biotechnology company;

experimental reagent:

ampicillin, IPTG, X-Gal, DH5 alpha competent cells, pGEM-T easy vector, PrimeScript RT reagent Kit with gDNA Eraser Kit, SYBR Premix Ex Taq^TM

RevertAId First Strand cDNA Synthesis Kit, purchased from TaKaRA;

agarose gel recovery kit, Omega 6837-01 plant total RNA extraction kit, and the like, purchased from Tiangen reagent company;

experimental equipment:

microspectrophotometer (for measuring RNA concentration), quewell Q5000, usa;

mastercycler ep realplex of fluorescent quantitative PCR instrument²Product of Eppendorf company, Germany;

a gene amplification PCR apparatus T3000 Thermocycler, product of Germany Biometra;

phytoclimatic chamber, USA PERCIVAL E-41HO 2.

Example 1

Based on the assumption that the inventor carries out actual screening verification work, the present embodiment briefly introduces the relevant screening work process and the relevant screening results as follows.

High temperature stress treatment and cDNA template preparation

In the present application, the high temperature stress pretreatment is specifically performed by: selecting two-year-old rhizoma Bletillae seedling with the same growth period in a greenhouse, pre-culturing in a plant artificial climate box for 7d (culture conditions: light-dark ratio of 12 h/12 h, temperature of 26 deg.C, relative humidity of 70%, and illumination of 100 μmol. m)^-2·s^-1) High temperature treatment at 40 ℃ for different time, 10 strains each are treated, and three biological repetitions are set.

Respectively taking roots (respectively treated for 0h and 8 h), stems (respectively treated for 0h and 8 h) and leaves (respectively treated for 0h, 1h, 2h, 4h, 8h, 12h, 24h and 48 h) of common bletilla pseudobulb seedlings subjected to high-temperature stress treatment for different time as samples (the treatment for 0h is equivalent to normal growth and is taken as a control), randomly taking three common bletilla pseudobulb sample sources to be uniformly mixed, and fully grinding the samples in liquid nitrogen; the material without high temperature treatment was used as a control.

Extracting total RNA of different samples by adopting an Omega 6837-01 plant total RNA extraction kit according to the kit instruction; subjecting the total RNA to agarose gel electrophoresis to detect its integrity, and measuring its concentration and OD with Quawell Q5000 microspectrophotometer₂₆₀/₂₈₀The value is obtained.

The analysis of the results of the electrophoretic detection (FIG. 1) shows that: the electrophoresis has three bands, 28S and 18S bands are clear, and the brightness of the 28S band is about 2 times that of the 18S band; the Quawell Q5000 assay results show that: OD₂₆₀/₂₈₀The total RNA concentration is 1.8-2.1, and the total RNA concentration is 200-400 ng/muL. These results indicate that the integrity of the total RNA extracted is good, the extraction results of different samples are stable and consistent, and the total RNA can be used for preparing cDNA by subsequent reverse transcription.

Further, referring to the instruction book, synthesizing a First cDNA chain by using a RevertAID First Strand cDNA Synthesis Kit, and cloning bletilla striata internal reference genes by using the First cDNA chain as a template of a common PCR reaction; the first cDNA chain reverse transcribed by using the PrimeScript RT reagent Kit with gDNA Eraser Kit is used as a template of real-time fluorescence quantitative PCR reaction of rhizoma bletillae reference genes.

In the process of preparing cDNA used for real-time fluorescence quantitative PCR, each sample cDNA is diluted into a reaction system with the concentration of 100 ng/20 mu L by Easy Dilution and is directly applied, or is stored at the temperature of minus 20 ℃ for later use.

(II) cloning of candidate high-temperature reference genes

In view of the previous research work of the inventors, based on the existing reference gene of Orchidaceae, the inventors considered that glyceraldehyde-3-phosphate dehydrogenase gene(s) ((GAPDH) 18S ribosomal RNA Gene: (18S rRNA) The transcription elongation factor 1A gene (EF1α) Alpha tubulin gene (a)TUA) Beta tubulin gene (a)TUB) Ubiquitin elongase Gene: (UBI) NAC domain protein gene (NAC)NAC) These 7 genes may be used as reference genes, so the inventors designed degenerate primer sequences of cloned common bletilla pseudobulb reference genes as follows:

。

using cDNA prepared in the step (I) as a template (using sample cDNA which is not processed at high temperature as the template), and using the primer sequence to carry out common PCR amplification, wherein the reference design of a 20-mu-L amplification system is as follows:

10×PCR Buffer，2.0 μL；

dNTP（2.5 mmol/L），1.6 μL；

primers F and R (both 10 mmol/L), each 0.5. mu.L;

cDNA template, 0.8. mu.L;

rTaqDNA polymerase (5U/. mu.L), 0.2. mu.L;

ddH₂O，14.4 μL；

the PCR amplification procedure was: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 40 s, annealing at 58 ℃ for 40 s, and extension at 72 ℃ for 80 s, for 35 cycles; finally, extension is carried out for 10 min at 72 ℃.

(III) sequence identification and analysis

And (3) carrying out 1% agarose gel electrophoresis detection on the PCR amplification product obtained in the step (II), cutting off the fragment with the correct position, carrying out gel recovery (by using an agarose gel recovery kit), detecting the correct position of the recovered target fragment by gel electrophoresis, connecting the target fragment to a pGEM-T easy cloning vector, transferring the cloned vector into DH5 alpha competent cells, and carrying out blue-white screening and colony PCR identification on the obtained product to send the positive bacterial liquid to Shanghai Ying Jun biotechnologies company for sequencing.

Specific glyceraldehyde-3-phosphate dehydrogenase gene (a)GAPDH) The transcription elongation factor 1A gene (EF1α) Alpha tubulin gene (a)TUA) Beta tubulin gene (a)TUB) NAC domain protein gene (NAC)NAC) 18S ribosomal RNA Gene: (18S rRNA) Ubiquitin elongase Gene: (UBI) The base sequences are respectively shown as SEQ ID NO. 1-7, and specifically as follows:

BsGAPDH(shown in SEQ ID NO.1, 875 bp):

AAAATCGGAATCAATGGATTCGGAAGGATCGGTCGGCTTGTGGCCAGGGTTGCGCTTTTGAGTGAGGATGTGGAGCTGGTCGCTGTCAACGATCCGTTCATTACCACCGAATACATGACATTCATGTTCAAGTATGACACTGTGCACGGTCAGTGGAAACATCATGAGATAACTGTGAAGGACTCCAAGACCCTTCTCTTTGGAGAGAAACCTGTTGCAGTCTTTGGCGTCAGGAACCCTGAGGAGATCCCATGGGGTGAGGCTGGTGCTGACTATGTTGTGGAGTCCACTGGAGTCTTTACGGACAAGGACAAGGCTGCCGCTCACCTGAAGGGTGGTGCCAAAAAGGTTATCATCTCTGCACCTAGCAAAGATGCTCCAATGTTTGTTGTTGGTGTTAATGAGCATGAGTACAAGGCTGATATTGATATTGTTTCTAATGCTAGCTGCACAACCAATTGTTTGGCTCCCCTTGCAAAGGTCATTCATGACAGGTTTGGCATTATTGAGGGCTTGATGACTACTGTTCACTCAATCACCGCCACTCAGAAGACTGTTGATGGTCCATCAAGCAAGGACTGGAGGGGCGGAAGAGCTGCTAGTTTCAATATCATTCCTAGCAGCACTGGAGCTGCTAAGGCTGTTGGGAAAGTGCTCCCTGCTCTGAATGGGAAATTGACTGGTATGTCCTTCCGTGTACCGACTGTTGATGTTTCAGTTGTTGATCTTACTGTGAGAATTGAGAAGAAGGCCTCTTATGATGAAATCAAGGCTGCTATAAAGGAAGAATCGGAGGGAAATCTCAAAGGCATCATTGGCTACACTGACGAGGACCTTGTGTCCACTGACTTTTTGGGTGACAGCAGGTCAAGCAT

BsEF1α(shown in SEQ ID NO.2, 620 bp):

ACATCAACATTGTGGTCATTGGTCATGTCGACTCTGGGAAATCGACCACCACTGGTCATCTCATATACAAGCTTGGCGGTATTGATAAACGTGTGATCGAGAGGTTTGAGAAGGAAGCTGCTGAGATGAACAAAAGATCATTCAAGTACGCATGGGTGCTCGACAAGCTCAAAGCTGAGCGTGAGCGCGGTATTACCATTGATATCGCTCTGTGGAAATTCGAGACCACGAAATACTATTGCACTGTCATTGATGCCCCCGGACATCGCGATTTTATCAAGAACATGATCACCGGAACCTCTCAGGCAGATTGTGCCGTCCTTATTATTGATTCCACGACTGGAGGCTTTGAAGCTGGTATCTCCAAAGATGGCCAGACTCGTGAACATGCCCTCCTTGCTTTTACTCTCGGTGTTAAACAGATGATTTGTTGCTGCAACAAGATGGATGCAACTACACCCAAGTACTCAAAAGCAAGGTATGATGAGATTGTGAAGGAGGTTTCCTCCTACCTCAAGAAGGTTGGTTACAATCCTGATAAGATCCCATTTGTACCAATCTCCGGATTCGAAGGTGACAACATGATTGACAGATCAACCAACCTTGACTGGTACAAAGGC

BsTUA：(shown in SEQ ID NO.3, 1205 bp)

GGTATCCAGGTCGGAAACGCATGCTGGGAGCTTTACTGCCTTGAGCATGGTATCCAGCCTGATGGCCAGATGCCAAGCGACAAAACAGTGGGTGGAGGTGATGATGCTTTCAACACCTTCTTCAGCGAGACAGGAGCGGGGAAACATGTTCCGCGAGCCGTGTTCGTAGATCTGGAGCCAACTGTTATTGATGAGGTGAGGACCGGAGGCTACCGCCAGCTCTTCCACCCCGAGCAGCTCATCAGTGGCAAGGAAGATGCAGCCAACAACTTTGCTCGTGGCCATTATACAATTGGCAAAGAGATTGTTGATCTATGCCTTGACCGCATCAGGAAGCTTGCTGATAACTGCACCGGCCTTCAAGGGTTCCTTGTCTTCAATGCTGTAGGTGGAGGCACTGGCTCAGGACTTGGTTCTCTTCTTCTTGAGAGGCTCTCTGTTGACTATGGAAGGAAGTCGAAGCTTGGGTTTACTGTATATCCCTCGCCACAGGTGTCCACCTCTGTGGTCGAGCCTTACAACAGCGTCCTCTCCACACACTCTTTGCTCGAACACACTGATGTGGCTGTTCTTCTGGATAATGAAGCCATATATGACATCTGCAGGCGTTCCCTTGACATTGAACGCCCCACCTACACCAACTTGAACCGCTTGGTGTCGCAGGTGATTTCATCCCTTACCGCCTCACTAAGGTTTGATGGAGCCTTGAATGTGGATGTAACTGAGTTCCAAACCAACTTAGTTCCATACCCGAGAATCCACTTCATGCTCTCATCCTATGCTCCTGTAATCTCAGCCGAAAAAGCCTATCATGAGCAACTCTCCGTTGCCGAAATAACCAACAGCACCTTCGAACCATCCTCCATGATGGCCAAATGCGATCCCCGCCATGGCAAATACATGGCTTGCTGCTTGATGTATCGCGGCGATGTCGTGCCCAAGGATGTCAATGCCGCCGTCGCCACCATCAAGACCAAACGCACGATCCAGTTTGTCGACTGGTGTCCGACCGGCTTCAAATGCGGGATCAACTACCAGCCGCCGACCGTTGTGCCTGGTGGTGATCTTGCCAAGGTGCAGAGGGCTGTGTGCATGATCTCGAACTCGACTAGCGTTGCGGAGGTGTTCTCTCGAATTGATCTTAAGTTTGATCTGATGTATTCGAAGAGGGCGTTCGTGCACTGGTACGTCGGTGAGGGTATGGA

BsTUB(shown in SEQ ID NO.4, 1055 bp):

ATGAGAGAGATCCTTCACATCCAGGGAGGGCAATGTGGCAACCAGATCGGTGCGAAGTTCTGGGAGGTGATTTGTGATGAGCACGGGATCGATCACACCGGTAAGTACAGTGGAGACTCCGAACTTCAACTCGAGCGGATCAACGTCTATTACAATGAGGCGAGCGGAGGGAGGTATGTTCCGAGGGCTGTGCTTATGGATCTGGAGCCGGGGACGATGGATTCGGTGAGATCGGGTCCGTTCGGGCAGATCTTTCGTCCGGATAATTTTGTGTTCGGGCAATCGGGTGCGGGGAATAATTGGGCAAAGGGGCACTATACAGAGGGTGCGGAGCTCATTGATTCTGTGTTGGATGTGGTGAGGAAGGAGGCGGAGAACTGCGATTGCTTGCAAGGATTCCAAGTATGCCATTCCTTGGGTGGAGGAACAGGATCTGGCATGGGCACCCTTCTGATTTCAAAGATCAGAGAGGAGTATCCTGACCGCATGATGCTGACATTCTCTGTTTTCCCATCACCAAAGGTCTCCGATACTGTTGTGGAGCCATACAATGCTACTCTCTCAGTTCACCAGCTTGTTGAAAATGCTGACGAGTGCATGGTCCTCGACAATGAGGCGCTTTATGATATTTGTTTCCGCACTCTCAAGCTTGCTACTCCTACATTTGGTGATCTCAATCACCTCATCTCTGCCACCATGAGTGGTGTCACTTGCTGCCTGCGTTTCCCTGGACAGCTGAATTCTGATCTCCGGAAGCTTGCCGTCAACCTAATCCCCTTCCCTCGCCTCCACTTCTTCATGGTCGGCTTTGCTCCCCTAACATCTCGGGGGTCGCAGCAATACCGCGCCCTTACTGTCCCTGAACTGACTCAGCAGATGTGGGATTCCAAGAACATGATGTGTGCAGCTGATCCACGCCATGGCCGTTACCTCACCGCCTCTGCCATGTTTCGCGGGAAGATGAGCACTAAAGAGGTGGACGAGCAAATGATTAACGTTCAGAACAAGAACTCATCCTACTTTGTTGAGTGGATCCCCAACAACGTCAAGTCCAG

BsNAC：(shown in SEQ ID NO.5, 357 bp)

TTCAGTTTCCACCCGACCGATGAGGAGCTCATCCTCCATTACCTCTGCAAGCGGGCAGCATCCGCTCCTTGTCCAGTCGCGATCATCGCCGATATCAATATATACAAGTTCAATCCATGGGATCTGCCTGCAAAAGCTATGTTTGGGGAGAGAGAATGGTATTTCTTCACCCCGCGAGACCGCAAGTACCCAAACGGAGTGCGGCCTAACAGGGCAGCAGGGTCGGGTTACTGGAAGGCAACCGGCACCGACAAACCGATAACGGCGGCCCGAAGGAATGAGAACATTGGCGTTAAGAAAGCTCTGGTGTTCTACAAGGGAAAGCCTCCCAGGGGCATAAGACCAACTGGATCATGC

Bs18S rRNA: (shown in SEQ ID NO.6, 1792 bp)

TGGTTGATCCTGCCAGTAGTCATATGCTTGTCTCAAAGATTAAGCCATGCATGTGTAAGTATGAACTATTTCAGACTGTGAAACTGCGAATGGCTCATTAAATCAGTTATAGTTTGTTTGATGGTACGTGCTACTCGGATAACCGTAGTAATTCTAGAGCTAATACGTGCACCAAACCCCCGACTTCTGGAAGGGATGCATTTATTAGGTAAAAGGTCAATGCGGGCTCTTGCCCGTTGCCCTGATGATTCATGATAACTCGTCGGATCGCACGGCCCTTGTGCCGGCGACGCATCATTCGAATATCTGCCCTATCAACTTTCGATGGTAGGATAGGGGCCTACCATGGTGGTGACGGGTGACGGAGAATTAGGGTTCGATTCCGGAGAGGGAGCCTGAGAGACGGCTACCACATCCAAGGAAGGCAGCAGGCGCGCAAATTACCCAATCCTGACACGGGGAGGTAGTGACAATAAATAACAATACCGGGCTCTTCGAGTCTGGTAATTGGAATGAGTACAATCTAAACCCCTTAACGAGGATCCATTGGAGGGCAAGTCTGGTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTTAAGTTGTTGCAGTTAAAAAGCTCGTAGTTGGACTTTGGGTTGGGTCGGTCGGTCCGCCTCACGGTGTGCACCGCCCGTCTCGTCCCTTATGCCGGCGATGCGTTCCTGGCCTTAATTGGCCGGGTCGTGCCTCCGGCGCCGTTACTTTGAAGAAATTAGAGTGCTCAAAGCAAGCCCACGCTCTGGATACATTAGCATGGGATAACACCACAGGATTTCGGTCCTATTGTGTTGGCCTTTGGGATCGGAGTAATGATTAAGAGGGACGGTCGGGGGCATTCGTATTTCATAGTCAGAGGTGAAATTCTTGGATTTATGAAAGACGAACCACTGCGAAAGCATTTGCCAAGGATGTTTTCATTAATCAAGAACGAAAGTTGGGGGCTCGAGGACGATCAGATACCGTCCTAGTCTCAACCATAAACGATGCCGACCAGGGATCGGCGGATGTTGCTTTTAGGACTCCGCCGGCACCTTGTGAGAAATCAAAGTTTTTGGGTTCCGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACCAGGAGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTTACCAGGTCCAGACATAGTAAGGATTGACAGACTGAGAGCTCTTTCTTGATTCTATGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGCGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTCAGCCTGCTAACTAGCTATGCGGAGTTCAACCTGCGCGGCCAGCTTCTTAGAGGGACTATGGCCGTTTAGGCCACGGAAGTTTGAGGCAATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGCACGCGCGCTACACTGATGTATTCAACGAGTATATAGCCTTGGCCGACAGGCCCGGGTAATCTTTAGAAAATTTCATCGTGATGGGGATAGATCATTGCAATTGTTGGTCTTCAACGAGGAATTCCTAGTAAGCGCGAGTCATCAGCTCGCGTTGACTACGTCCCTGCCCTTTGTACACACCGCCCGTCGCTCCTACCGATTGAATGGTCCGGTGAAGTGTTCGGATCGCTGTGACGCGGGCGGTTCGCCGCCCGTGACTCTGCGAGAAGTCCATTGAACCTTATCATTTAGAGGAAGGAGAAGTCGTAACAAGGTTTCCGTAGGTGAAC

BsUBI：(shown in SEQ ID NO.7, 422 bp)

ATGCAGATCTTCGTGAAAACCCTAACGGGGAAGACGATCACCCTCGAGGTGGAGTCGTCCGACACGATCGACAACGTTAAAGCAAAGATTCAGGACAAGGAGGGCATCCCACCAGATCGACAGCGCCTAATCTTTGCCGGGAAGCAGCTTGAGGACGGCCGCACCCTCGCCGATTACAACATCCAGAAGGAATCTACGCTTCACCTCGTTCTCCGCCTCCGCGGGGGTGCCAAAAAGCGCAAGAAGAAGACATACACCAAGCCCAAGAAGATCAAGCACAAGAAGAAGAAGGTTAAGCTCGCCGTGCTGCAGTTCTACAAAGTCGGCGACGCCGGAAAAGTGGCGCGCCTGCGCAAGGAGTGCCCCAACGCTGAGTGTGGAGCGGGGACATTCATGGCGAACCACTTCGACCGCCACTACTG。

Example 2

Based on the sequencing work of example 1, the inventors have detected the expression of 7 candidate reference genes by using a real-time fluorescence quantification technique, and the specific experimental conditions are briefly described as follows.

(I) designing primers for real-time fluorescent quantitative PCR and fluorescent quantitative PCR detection

Based on the results of sequencing 7 candidate reference genes in example 1, PCR primers for real-time fluorescence quantification were redesigned as follows:

。

SYBR Premix Ex Taq was used^TM

And (3) carrying out real-time fluorescent quantitative PCR reaction detection on the expression condition of each gene after different high-temperature stress treatment time, wherein the reaction system is designed to be 20.0 mu L, and the specific steps are as follows:

2×SYBR Premix Ex Taq

，10.0 μL；

primers F and R, each 0.8. mu.L;

cDNA template, 2.0 μ L;

ddH₂O，6.4 μL；

the reaction conditions are as follows: pre-denaturation at 95 ℃ for 30 s, denaturation at 95 ℃ for 15 s, annealing at 58 ℃ for 15 s, and extension at 72 ℃ for 15 s, for 40 cycles.

The reaction is carried out in an Eppendorf Mastercycler ep realplex²The fluorescent quantitative PCR instrument is used for carrying out the reaction, each sample is carried out for 3 times, and negative control is carried out. After the PCR reaction is finished, the amplification condition of each gene is checked through automatic analysis of an instrument, and a corresponding Ct value is derived.

(II) expression stability of candidate reference gene in leaf

Based on the fluorescence quantitative PCR detection result in the step (I), the Ct average values of 7 candidate reference genes in the leaves processed at high temperature for different time are counted and summarized, and the results are shown in the following table:

。

based on the above results, the expression stability of 7 candidate reference genes was analyzed by using analysis software GeNorm, Bestkeeper and NormFinder, respectively, and the results are shown in the following table:

。

as can be seen from the above table, GeNorm analysis indicates that the 2 most stably expressed reference genes in bletilla striata leaves at different times of high temperature stress areBs18S rRNAAndBsUBI(ii) a The results of the NormFinder analysis suggest that the most stably expressed reference gene isBsTUBSecondly isBsGAPDH(ii) a The Bestkeeper analysis result shows that the most stably expressed reference gene isBs18S rRNASecondly isBsEF1α。

(III) analysis of expression stability in different tissues

Based on the fluorescence quantitative PCR detection result in the step (I), the Ct average values of 7 candidate reference genes in different tissues (roots, stems and leaves) processed at high temperature for different time are counted and summarized, and the results are shown in the following table:

。

based on the above results, the expression stability of 7 candidate reference genes was analyzed by using analysis software GeNorm, Bestkeeper and NormFinder, respectively, and the results are shown in the following table:

。

as can be seen from the above table, GeNorm analysis showed that the 2 most stably expressed reference genes in different tissues of bletilla under high temperature stress areBsEF1αAndBsUBI(ii) a The results of the NormFinder analysis showed that the most stably expressed reference gene was BsABI, followed by BsABIBs18S rRNA(ii) a The Bestkeeper analysis result shows that the most stably expressed reference gene isBs18S rRNASecondly isBsGAPDH。

(IV) comprehensive analysis of expression stability of reference genes

And (4) carrying out comprehensive analysis by using analysis software RefFinder based on the analysis results of the step (II) and the step (III) to obtain a comprehensive index ranking, wherein the smaller the index is, the more stable the expression of the reference gene is. The specific results are shown in the following table:

。

as can be seen from the above table, under different high temperature stress time, the 2 most stable internal reference genes expressed in the bletilla striata leaves areBs18S rRNAAndBsTUB(ii) a Under the condition of high-temperature stress, 2 reference genes which are most stably expressed in different tissues of bletilla areBsUBIAndBs18S rRNA(ii) a And on the basis of comprehensive consideration, can selectBs18S rRNAAndBsUBIthe gene expression analysis of the 2 reference genes is optimal for gene expression analysis in different tissue samples at different processing times under normal or high-temperature stress of the bletilla striata, and the 2 reference genes are further used for screening and researching heat-resistant gene resources of the bletilla striata.

Example 3

Determined finally in example 2Bs18S rRNAAndBsUBIas an internal reference gene, the inventor uses a specific functional gene ribulose-1, 5-diphosphocarboxylase/oxygenase large subunit gene (rbcL) For example, the expression of the functional gene in bletilla striata is subjected to real-time fluorescence quantitative detection and analysis, and related experimental processes are briefly introduced as follows.

(1) Design of primers

Detection of genesrbcLThen, the primers were designed as follows:

qBsrbcl-F: 5’-CCAAAACTTTCCAAGGTCCG-3’，

qBsrbcl-R: 5’- TCCACCCCGTAGACATTCATA-3’；

the PCR amplification length is 155 bp;

aiming at internal reference geneBs18S rRNAAndBsUBIprimer sequences reference example 2;

(2) preparation of cDNA and real-time fluorescent quantitative PCR detection

Taking samples of different parts processed at normal temperature and high temperature for different time as materials, extracting total RNA, performing reverse transcription to obtain cDNA as a template, and referring to example 1 in a real-time fluorescence quantitative PCR reaction system and a reaction program.

PCR results were in terms of relative expression amount rel. Exp = 2^-ΔΔCtAnd (4) calculating.

To be provided withBs18S rRNAAs reference gene pairrbcLThe results of the gene detection are shown in FIG. 2, toBsUBIAs reference gene pairrbcLThe results of the gene detection are shown in FIG. 3. Analysis can see that:

regardless of which of the reference genes is selected,rbcLthe expression and the change trend of the genes are similar, namely:rbcLthe gene is mainly expressed in the leaf, but the expression level of the gene is in a trend of gradually decreasing along with the prolonging of the high-temperature treatment time;rbcLthe gene is absent in the rootThe expression is increased in tubers along with the prolonged high-temperature treatment time. It can be seen thatBs18S rRNAAndBsUBIcan be used as an internal reference gene to be applied to the screening research of rhizoma bletillae heat-resistant gene resources.

SEQUENCE LISTING

<110> Zheng State college of education

<120> Bletillae hyperthermia reference genes Bs18S rRNA and BsbUBI

<130> none

<160> 7

<170> PatentIn version 3.5

<210> 1

<211> 875

<212> DNA

<213> Bletilla striata

<400> 1

aaaatcggaa tcaatggatt cggaaggatc ggtcggcttg tggccagggt tgcgcttttg 60

agtgaggatg tggagctggt cgctgtcaac gatccgttca ttaccaccga atacatgaca 120

ttcatgttca agtatgacac tgtgcacggt cagtggaaac atcatgagat aactgtgaag 180

gactccaaga cccttctctt tggagagaaa cctgttgcag tctttggcgt caggaaccct 240

gaggagatcc catggggtga ggctggtgct gactatgttg tggagtccac tggagtcttt 300

acggacaagg acaaggctgc cgctcacctg aagggtggtg ccaaaaaggt tatcatctct 360

gcacctagca aagatgctcc aatgtttgtt gttggtgtta atgagcatga gtacaaggct 420

gatattgata ttgtttctaa tgctagctgc acaaccaatt gtttggctcc ccttgcaaag 480

gtcattcatg acaggtttgg cattattgag ggcttgatga ctactgttca ctcaatcacc 540

gccactcaga agactgttga tggtccatca agcaaggact ggaggggcgg aagagctgct 600

agtttcaata tcattcctag cagcactgga gctgctaagg ctgttgggaa agtgctccct 660

gctctgaatg ggaaattgac tggtatgtcc ttccgtgtac cgactgttga tgtttcagtt 720

gttgatctta ctgtgagaat tgagaagaag gcctcttatg atgaaatcaa ggctgctata 780

aaggaagaat cggagggaaa tctcaaaggc atcattggct acactgacga ggaccttgtg 840

tccactgact ttttgggtga cagcaggtca agcat 875

<210> 2

<211> 620

<212> DNA

<213> Bletilla striata

<400> 2

acatcaacat tgtggtcatt ggtcatgtcg actctgggaa atcgaccacc actggtcatc 60

tcatatacaa gcttggcggt attgataaac gtgtgatcga gaggtttgag aaggaagctg 120

ctgagatgaa caaaagatca ttcaagtacg catgggtgct cgacaagctc aaagctgagc 180

gtgagcgcgg tattaccatt gatatcgctc tgtggaaatt cgagaccacg aaatactatt 240

gcactgtcat tgatgccccc ggacatcgcg attttatcaa gaacatgatc accggaacct 300

ctcaggcaga ttgtgccgtc cttattattg attccacgac tggaggcttt gaagctggta 360

tctccaaaga tggccagact cgtgaacatg ccctccttgc ttttactctc ggtgttaaac 420

agatgatttg ttgctgcaac aagatggatg caactacacc caagtactca aaagcaaggt 480

atgatgagat tgtgaaggag gtttcctcct acctcaagaa ggttggttac aatcctgata 540

agatcccatt tgtaccaatc tccggattcg aaggtgacaa catgattgac agatcaacca 600

accttgactg gtacaaaggc 620

<210> 3

<211> 1205

<212> DNA

<213> Bletilla striata

<400> 3

ggtatccagg tcggaaacgc atgctgggag ctttactgcc ttgagcatgg tatccagcct 60

gatggccaga tgccaagcga caaaacagtg ggtggaggtg atgatgcttt caacaccttc 120

ttcagcgaga caggagcggg gaaacatgtt ccgcgagccg tgttcgtaga tctggagcca 180

actgttattg atgaggtgag gaccggaggc taccgccagc tcttccaccc cgagcagctc 240

atcagtggca aggaagatgc agccaacaac tttgctcgtg gccattatac aattggcaaa 300

gagattgttg atctatgcct tgaccgcatc aggaagcttg ctgataactg caccggcctt 360

caagggttcc ttgtcttcaa tgctgtaggt ggaggcactg gctcaggact tggttctctt 420

cttcttgaga ggctctctgt tgactatgga aggaagtcga agcttgggtt tactgtatat 480

ccctcgccac aggtgtccac ctctgtggtc gagccttaca acagcgtcct ctccacacac 540

tctttgctcg aacacactga tgtggctgtt cttctggata atgaagccat atatgacatc 600

tgcaggcgtt cccttgacat tgaacgcccc acctacacca acttgaaccg cttggtgtcg 660

caggtgattt catcccttac cgcctcacta aggtttgatg gagccttgaa tgtggatgta 720

actgagttcc aaaccaactt agttccatac ccgagaatcc acttcatgct ctcatcctat 780

gctcctgtaa tctcagccga aaaagcctat catgagcaac tctccgttgc cgaaataacc 840

aacagcacct tcgaaccatc ctccatgatg gccaaatgcg atccccgcca tggcaaatac 900

atggcttgct gcttgatgta tcgcggcgat gtcgtgccca aggatgtcaa tgccgccgtc 960

gccaccatca agaccaaacg cacgatccag tttgtcgact ggtgtccgac cggcttcaaa 1020

tgcgggatca actaccagcc gccgaccgtt gtgcctggtg gtgatcttgc caaggtgcag 1080

agggctgtgt gcatgatctc gaactcgact agcgttgcgg aggtgttctc tcgaattgat 1140

cttaagtttg atctgatgta ttcgaagagg gcgttcgtgc actggtacgt cggtgagggt 1200

atgga 1205

<210> 4

<211> 1055

<212> DNA

<213> Bletilla striata

<400> 4

atgagagaga tccttcacat ccagggaggg caatgtggca accagatcgg tgcgaagttc 60

tgggaggtga tttgtgatga gcacgggatc gatcacaccg gtaagtacag tggagactcc 120

gaacttcaac tcgagcggat caacgtctat tacaatgagg cgagcggagg gaggtatgtt 180

ccgagggctg tgcttatgga tctggagccg gggacgatgg attcggtgag atcgggtccg 240

ttcgggcaga tctttcgtcc ggataatttt gtgttcgggc aatcgggtgc ggggaataat 300

tgggcaaagg ggcactatac agagggtgcg gagctcattg attctgtgtt ggatgtggtg 360

aggaaggagg cggagaactg cgattgcttg caaggattcc aagtatgcca ttccttgggt 420

ggaggaacag gatctggcat gggcaccctt ctgatttcaa agatcagaga ggagtatcct 480

gaccgcatga tgctgacatt ctctgttttc ccatcaccaa aggtctccga tactgttgtg 540

gagccataca atgctactct ctcagttcac cagcttgttg aaaatgctga cgagtgcatg 600

gtcctcgaca atgaggcgct ttatgatatt tgtttccgca ctctcaagct tgctactcct 660

acatttggtg atctcaatca cctcatctct gccaccatga gtggtgtcac ttgctgcctg 720

cgtttccctg gacagctgaa ttctgatctc cggaagcttg ccgtcaacct aatccccttc 780

cctcgcctcc acttcttcat ggtcggcttt gctcccctaa catctcgggg gtcgcagcaa 840

taccgcgccc ttactgtccc tgaactgact cagcagatgt gggattccaa gaacatgatg 900

tgtgcagctg atccacgcca tggccgttac ctcaccgcct ctgccatgtt tcgcgggaag 960

atgagcacta aagaggtgga cgagcaaatg attaacgttc agaacaagaa ctcatcctac 1020

tttgttgagt ggatccccaa caacgtcaag tccag 1055

<210> 5

<211> 357

<212> DNA

<213> Bletilla striata

<400> 5

ttcagtttcc acccgaccga tgaggagctc atcctccatt acctctgcaa gcgggcagca 60

tccgctcctt gtccagtcgc gatcatcgcc gatatcaata tatacaagtt caatccatgg 120

gatctgcctg caaaagctat gtttggggag agagaatggt atttcttcac cccgcgagac 180

cgcaagtacc caaacggagt gcggcctaac agggcagcag ggtcgggtta ctggaaggca 240

accggcaccg acaaaccgat aacggcggcc cgaaggaatg agaacattgg cgttaagaaa 300

gctctggtgt tctacaaggg aaagcctccc aggggcataa gaccaactgg atcatgc 357

<210> 6

<211> 1792

<212> DNA

<213> Bletilla striata

<400> 6

tggttgatcc tgccagtagt catatgcttg tctcaaagat taagccatgc atgtgtaagt 60

atgaactatt tcagactgtg aaactgcgaa tggctcatta aatcagttat agtttgtttg 120

atggtacgtg ctactcggat aaccgtagta attctagagc taatacgtgc accaaacccc 180

cgacttctgg aagggatgca tttattaggt aaaaggtcaa tgcgggctct tgcccgttgc 240

cctgatgatt catgataact cgtcggatcg cacggccctt gtgccggcga cgcatcattc 300

gaatatctgc cctatcaact ttcgatggta ggataggggc ctaccatggt ggtgacgggt 360

gacggagaat tagggttcga ttccggagag ggagcctgag agacggctac cacatccaag 420

gaaggcagca ggcgcgcaaa ttacccaatc ctgacacggg gaggtagtga caataaataa 480

caataccggg ctcttcgagt ctggtaattg gaatgagtac aatctaaacc ccttaacgag 540

gatccattgg agggcaagtc tggtgccagc agccgcggta attccagctc caatagcgta 600

tatttaagtt gttgcagtta aaaagctcgt agttggactt tgggttgggt cggtcggtcc 660

gcctcacggt gtgcaccgcc cgtctcgtcc cttatgccgg cgatgcgttc ctggccttaa 720

ttggccgggt cgtgcctccg gcgccgttac tttgaagaaa ttagagtgct caaagcaagc 780

ccacgctctg gatacattag catgggataa caccacagga tttcggtcct attgtgttgg 840

cctttgggat cggagtaatg attaagaggg acggtcgggg gcattcgtat ttcatagtca 900

gaggtgaaat tcttggattt atgaaagacg aaccactgcg aaagcatttg ccaaggatgt 960

tttcattaat caagaacgaa agttgggggc tcgaggacga tcagataccg tcctagtctc 1020

aaccataaac gatgccgacc agggatcggc ggatgttgct tttaggactc cgccggcacc 1080

ttgtgagaaa tcaaagtttt tgggttccgg ggggagtatg gtcgcaaggc tgaaacttaa 1140

aggaattgac ggaagggcac caccaggagt ggagcctgcg gcttaatttg actcaacacg 1200

gggaaactta ccaggtccag acatagtaag gattgacaga ctgagagctc tttcttgatt 1260

ctatgggtgg tggtgcatgg ccgttcttag ttggtggagc gatttgtctg gttaattccg 1320

ttaacgaacg agacctcagc ctgctaacta gctatgcgga gttcaacctg cgcggccagc 1380

ttcttagagg gactatggcc gtttaggcca cggaagtttg aggcaataac aggtctgtga 1440

tgcccttaga tgttctgggc cgcacgcgcg ctacactgat gtattcaacg agtatatagc 1500

cttggccgac aggcccgggt aatctttaga aaatttcatc gtgatgggga tagatcattg 1560

caattgttgg tcttcaacga ggaattccta gtaagcgcga gtcatcagct cgcgttgact 1620

acgtccctgc cctttgtaca caccgcccgt cgctcctacc gattgaatgg tccggtgaag 1680

tgttcggatc gctgtgacgc gggcggttcg ccgcccgtga ctctgcgaga agtccattga 1740

accttatcat ttagaggaag gagaagtcgt aacaaggttt ccgtaggtga ac 1792

<210> 7

<211> 422

<212> DNA

<213> Bletilla striata

<400> 7

atgcagatct tcgtgaaaac cctaacgggg aagacgatca ccctcgaggt ggagtcgtcc 60

gacacgatcg acaacgttaa agcaaagatt caggacaagg agggcatccc accagatcga 120

cagcgcctaa tctttgccgg gaagcagctt gaggacggcc gcaccctcgc cgattacaac 180

atccagaagg aatctacgct tcacctcgtt ctccgcctcc gcgggggtgc caaaaagcgc 240

aagaagaaga catacaccaa gcccaagaag atcaagcaca agaagaagaa ggttaagctc 300

gccgtgctgc agttctacaa agtcggcgac gccggaaaag tggcgcgcct gcgcaaggag 360

tgccccaacg ctgagtgtgg agcggggaca ttcatggcga accacttcga ccgccactac 420

tg 422

Claims (6)

1. Rhizoma bletillae high-temperature reference geneBs18S rRNAAndBsUBIcharacterized in thatBs18S rRNAThe gene has a base sequence shown in SEQ ID NO. 6; the above-mentionedBsUBIThe gene and the base sequence are shown in SEQ ID NO. 7.

2. Used for obtaining rhizoma bletillae high-temperature reference gene by PCR amplificationBs18S rRNAAndBsUBIthe primer of (2), wherein the specific primer design is as follows:

Bs18S rRNA-F：5’- TGGTTGATCCTGCCAGTAGT-3’，

Bs18S rRNA-R：5’- GTTCACCTACGGAAACCTTG-3’，

BsUBI-F：5’- ATGCAGATCTTCGTGAARACCCT-3’，

BsUBI-R：5’- CAGTAGTGGCGRTCGAAGTGGT-3’。

3. by using the apparatus of claim 2The primer obtains the rhizoma bletillae high-temperature reference geneBs18S rRNAAndBsUBIthe PCR amplification method of (1), characterized by comprising the steps of:

(1) extracting total RNA and performing reverse transcription to obtain cDNA as an amplification template;

(2) PCR amplification was performed using primers.

4. Rhizoma bletillae high-temperature reference geneBs18S rRNAAndBsUBIthe application in rhizoma bletillae is characterized in that the rhizoma bletillae is used as an internal reference gene;

the above-mentionedBs18S rRNAThe gene has a base sequence shown in SEQ ID NO. 6; the above-mentionedBsUBIThe gene and the base sequence are shown in SEQ ID NO. 7.

5. The rhizoma bletillae hyperthermia reference gene of claim 4Bs18S rRNAAndBsUBIuse in bletilla striata for the functional gene ribulose-1, 5-bisphosphate carboxylase/oxygenase large subunit generbcLThe fluorescent quantitative detection analysis of (1).

6. A method for detecting ribulose-1, 5-diphosphocarboxylase/oxygenase large subunit gene by fluorescence quantitative detection is characterized in that the detection is carried out byBs18S rRNAAnd/orBsUBIThe method is used as an internal reference gene and specifically comprises the following steps:

(1) primers for fluorescent quantitative detection were designed as follows:

Bs18S rRNA-F：5’-TTTATGAAAGACGAACCACTGC-3’，

Bs18S rRNA-R：5’-TCGGCATCGTTTATGGTTG-3’；

BsUBI-F：5’-CGCCGATTACAACATCCAGAA-3’，

BsUBI-R：5’-TTCTTGGGCTTGGTGTATGTC-3’；

qBsrbcl-F：5’-CCAAAACTTTCCAAGGTCCG-3’，

qBsrbcl-R：5’- TCCACCCCGTAGACATTCATA-3’；

(2) extracting rhizoma bletillae total RNA and performing reverse transcription to obtain cDNA serving as an amplification template, and performing fluorescent quantitative PCR detection.

Images