CN103866007B - Method for screening real-time fluorescence quantification PCR internal reference molecules of syntrichia caninervis in desert - Google Patents

Method for screening real-time fluorescence quantification PCR internal reference molecules of syntrichia caninervis in desert Download PDF

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CN103866007B
CN103866007B CN201410057384.0A CN201410057384A CN103866007B CN 103866007 B CN103866007 B CN 103866007B CN 201410057384 A CN201410057384 A CN 201410057384A CN 103866007 B CN103866007 B CN 103866007B
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sequence
real
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time fluorescence
tooth rib
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CN103866007A (en
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张道远
李小双
李海燕
杨红兰
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Xinjiang Institute of Ecology and Geography of CAS
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Xinjiang Institute of Ecology and Geography of CAS
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Abstract

The invention relates to a method for screening real-time fluorescence quantification PCR internal reference molecules of syntrichia caninervis in a desert. The method comprises the following steps: selecting 15 internal reference candidate genes by utilizing a syntrichia caninervis transcriptome database, designing an internal reference gene specific primer of real-time fluorescence quantification PCR by taking 15 internal reference genes as templates; carrying out a fluorescence quantification PCR experiment by selecting syntrichia caninervis gametophytes stressed by 10 non-living things and free of stress (in contrast) as experimental materials; carrying out fluorescence quantification data analysis by using geNorm, NormFinder and Refinder software, so as to screen out the best fit and the most stable internal reference molecules CDPK and alpha-TUB2 of the syntrichia caninervis for developing fluorescence quantification research under various abiotic stresses. By adopting the method disclosed by the invention, the errors of the syntrichia caninervis transcriptome material under different abiotic stresses in ribonucleic acid (RNA) quality, yield, reverse transcription efficiency and the like can be avoided, the real-time fluorescence quantification detection data can be better corrected and standardized, and the accuracy and the reliability of the gene quantification research are improved.

Description

The screening method of desert tooth rib red moss real-time fluorescence quantitative PCR reference molecules
Technical field
The present invention relates to a kind of screening method being applicable to the reference molecules of real-time fluorescence quantitative PCR research under the various abiotic stress of the desert red moss of mosses tooth rib.
Background technology
Arid, the abiotic stress such as high salt are the principal elements of limiting plant growth growth and crop yield, cause crop failure to reach more than 50% every year.Along with global warming, the aggravation of Greenhouse effect and desertification, Arid Problem will become increasingly conspicuous, and arid also will be more serious on the impact of agriculture production.At present, utilize drought stress genes involved to improve the drought-resistant ability of plant, become the molecular biological study hotspot of plant stress-resistance and the important research direction of plant stress-resistance genetically engineered.
The drought-resistant genetic resources that plants in arid region accumulates in long-term evolution process is the driving source of carrying out germplasm innovation.It is special, natural that arid biogeographic zone harsh physical environment breeds, rare adversity plant resource.The red moss of desert extremely drought-enduring mosses tooth rib is Typical Representative.The red moss of tooth rib (Syntrichia caninervis Mitt.) is subordinate to Cong Xian section, red Rhodobryum, for typical poikilohydric plant, the sociales forming mosses skinning, in grey black under seasoning state, desert surface is distributed in like one deck " shell ", be formed with the liver moss biological breadcrust of important ecological significance, not only there is stronger anti-adversity ability, and play an important role in the maintenance stability of desert ecosystem and the restoration and reconstruction of damaged ecosystem.In addition, once run into precipitation, namely in 30s, rapidly by rehydration process, " recoverys ", in fresh and alive green, and starts to perform photosynthesis, the machine-processed study hotspot having become domestic and international related science of molecular regulation of the physiological property of its uniqueness and drought resistance and thermotolerance to the red moss of tooth rib.Meanwhile, long-term environmental stress makes this kind have fabulous drought resisting, and the ability of the multiple abiotic stress of the opposing such as high temperature resistance, must contain abundant adversity gene resource in its body.At present, from the red moss of extreme drought resisting mosses tooth rib, excavate high-quality adversity gene resource launch, particularly the foundation of tooth rib red moss transcript profile database, especially for the gene studies of this kind provides good platform.At present, clone from the red moss of tooth rib, the work analyzing the family genes such as ALDH, HSP70, DREB launches.And the degeneration-resistant mechanism of the red moss of tooth rib to be understood and excavate abundant adversity gene resource, a very crucial method be exactly to be separated these genes and rapidly, the expression of detection by quantitative anti-drought gene under various abiotic stress exactly.Traditional blot hybridization, the means such as biochip technology and RT-PCR are on real-time, detection by quantitative anti-drought gene expression amount, or existing defects, or expensive, waste time and energy.Along with the development of the every technology of molecular biology, real-time fluorescence quantitative PCR has become the main method that current quantitative gene expression detects, this method is not only highly sensitive, sample consumption is few, low abundance expression of target gene amount can be detected, quantitative linearity is wide, but the reference gene that the accuracy that real-time fluorescence quantitative PCR detects depends on screening applicable to a great extent corrects and stdn data, thus avoids the difference that different specimens may exist on the Yield and quality and reverse transcription rate of RNA.Therefore, the selection of suitable stable reference gene is then the basis of the degeneration-resistant mechanism of the red moss of tooth rib and adversity gene research.
The stability study of reference gene was confirmed in the past, the constant expression of what is called of any one reference gene is all the relative constancy under the plant or the effect of particular experiment factor of certain type, in the plant of other types or different experiments process stability inferior be then likely change.If in other species of reference blindly a stable internal reference or or several reference gene, the fine difference of genetic expression may be made on the one hand to be difficult to find, mistake or even contrary conclusion may to be obtained on the other hand.In recent years, the stability problem of reference gene is more and more disputed in recent years and is paid close attention to.Along with people are to the more and more attention of the capable problem of internal reference stable gene, at present, developed based on Excel program various software, for evaluating stability and the variation coefficient of reference gene, wherein applying the most general is GeNorm and NormFinder program.
In sum, the red moss of desert extremely drought-enduring mosses tooth rib is the degeneration-resistant mechanism of research desert mosses and the good material excavating excellent adversity gene.Studying and screen the most stable reference gene in the red moss of tooth rib is the prerequisite of carrying out this kind of gene quantification research (fluorescent quantitative PCR experiment).In recent years, molecular level work as the red moss of tooth rib of extreme drought resisting mosses launches successively, some adversity genes have been cloned from the red moss of tooth rib, as acetaldehyde dehydrogenase gene ALDH21(NCBI accession number: GQ245973), particularly in recent years this seminar to the red moss transcript profile of tooth rib, the acquisition in succession of the high-throughput sequencing results such as protein groups, accelerates the molecular studies work of the red moss of tooth rib especially.Characteristic and the function of a lot of adversity gene are urgently studied, but have no report about the sequence data of the reference gene of this kind, and the research of most suitable reference molecules and screening operation are blank especially.Therefore, the present invention is just intended to find that a kind of to carry out real-time fluorescence quantitative PCR research the red moss of Abiotic stress conditions lower tooth rib the most stable, the screening method of most suitable reference molecules, for carrying out adversity gene characteristic on a large scale and functional study lays the foundation later.
Summary of the invention
The object of the invention is to, the screening method of a kind of desert tooth rib red moss real-time fluorescence quantitative PCR reference molecules is provided, when the method utilizes real time fluorescence quantifying PCR method to study bryophyte anti contravariance related gene function, lack applicable, reliably, express constant reference molecules, propose and be a kind ofly applicable to the desert red moss of mosses tooth rib and under Abiotic stress conditions, carry out the most stable of real-time fluorescence quantitative PCR research, the screening method of most suitable reference molecules; Utilize the red moss transcript profile of tooth rib to check order the database obtained, have chosen 15 tooth rib red moss candidate reference genes, and based on these gene orders, design the reference gene special primer of real-time fluorescence quantitative PCR.Choosing by 10 kinds of abiotic stress and the red moss of tooth rib of not coercing (contrast) is that experiment material carries out fluorescent quantitative PCR experiment checking, three large internal reference stability assessment softwares are used to comprise geNorm, NormFinder and Refinder carries out the analysis of fluorescent quantitation data, thus filter out the red moss of tooth rib and carry out the most applicable of fluorescent quantitation research under various abiotic stress, the most stable reference molecules.Use the reference molecules of the method for the invention screening, can avoid at the red moss sample of multiple abiotic stress lower tooth rib in RNA output, the difference that quality and reverse transcription efficiency may exist, the data that can better detect real-time fluorescence quantitative PCR correct and stdn, improve the accuracy to this kind of gene quantification research and reliability.
The screening method of a kind of desert mosses tooth rib of the present invention red moss real-time fluorescence quantitative PCR reference molecules, follows these steps to carry out:
A, utilize tooth rib red moss transcript profile sequencing data, select 15 tooth rib red moss internal reference candidate genes, and based on select 15 red moss sequences of tooth rib, devise the reference gene primer of 15 pairs of real-time fluorescence quantitative PCRs, wherein the nucleotide sequence of 15 reference genes and 15 pairs of real-time fluorescence quantitative PCR primer sequences of design are:
> sequence 1 tooth rib red moss SyntrichiacaninervisActin (ACT) nucleotide sequence
ATGGCTGATGCTGAGGATGTCCAGCCTTTGGTGTGCGACAATGGATCGGGGATGGTTAAGGCCGGATTCGCCGGAG
ATGACGCCCCGCGTGCCGTATTTCCCAGCATTGTTGGGCGCCCGAGACACACCGGTGTGATGGTGGGCATGGGACA
GAAAGACGCGTATGTGGGCGACGAGGCGCAGTCCAAGAGGGGTATCCTGACGCTGAAGTACCCGATCGAGCACGG
CGTGGTCACCAACTGGGACGACATGGAGAAGATCTGGCACCACACCTTCTACAACGAGCTGCGTGTGGCCCCCGA
GGAGCACCCGGTGCTGCTCACGGAGGCGCCTCTCAATCCCAAGGCCAACAGGGAGAAGATGACGCAGATCATGTT
CGAGACCTTCAACGTGCCGGCCATGTACGTGGCCATCCAGGCGGTGCTGTCGCTGTACGCCAGTGGGCGAACCACC
GGAATTGTGCTGGATAGTGGAGACGGTGTGACTCACACAGTGCCCATCTATGAGGGGTACGCCTTGCCTCACGCCA
TTCTGCGGCTG
GACTTGGCCGGTCGCGACTTGACGGACGCGCTGATGAAGATTCTGACGGAGCGTGGTTACTCGTTCACCACCACGG
CCGAGCGCGAGATCGTGCGTGACATGAAGGAGAAGCTGGCGTACGTGGCAATCGACTTTGAGCAGGAGCTCGACA
CAGCGCGCTCATCGTCCTCGCTGGAGAAGAGCTACGAGCTGCCGGACGGGCAGGTGATCACGATCGGCGCGGAGC
GGTTCAGGTGCCCGGAGGTGCTGTTCAACCCGTCGCTGATCGGGATGGAAGCTGCGGGCATTCACGAGACCACTTA
CAACTCGATCATGAAGTGTGACGTCGATATCCGTAAAGACCTGTACGGAAACATCGTGTTGTCTGGAGGATCGACCA
TGTTCCCGGGCATCGCCGACCGTATGAGCAAGGAGATCACTGCTCTTGCGCCCAGCAGCATGAAGATCAAGGTGGT
TGCGCCGCCGGAGAGGAAGTACAGTGTCTGGATCGGAGGATCCATCTTGGCATCTCTCAGCACCTTCCAGCAGATG
TGGATTGCG
AAGAGCGAGTACGATGAGTCCGGTCCATCGATTGTGCACAGGAAGTGCTTT
Real-time fluorescence quantitative PCR primer sequence: forward primer: TCGTGTTGTCTGGAGGATCG
Reverse primer: TACTCGCTCTTCGCAATCCA
> sequence 2 tooth rib red moss Syntrichiacaninervis actin associated protein (ARP) nucleotide sequence
GTGAGCCATGAGTTGAAACTAATAAATTGGAAAGAAAAACAAGCTCAGAAAATGAGCGTACATCGTTGAGACGG
TCAGACGAACCATTATCTACGGACATGCGTATTGCGTTGTTCAGGAATGTTCTTTGCGTGGAACTCGGTGTCGGTA
ACTGGAAGATGTTGACGCAGTTGCTGCTGTTACCTTGCTGACCAAGTTTAGTCCAACATGGCATTGTACCAAAAA
GCTTGCGCCTGGAGGGTGTTGGAGTCCCTGTACTTTCGGCCGCTGCCCACCATGACCATACCGTCCAACCAGTCCT
CGTACTGCACCCATGAGTCGCGTCCGAAGTCCAAGACGGCGAGAACCGCGCCACCTTTCCACATGATATTCATTG
GAGCCATTCGATTTTGAAGAACCTCCACAGTGTCCACAGCTTCGTCCACAGGTATGGCGTGCAGAACCCTCTCCTC
AACAGCATCAACCAGGCCTTTCGTTAACCCAACGCCGCCAACCAGCTGAATACTCGCGAACAGCTTCTTCTGAAG
ATCCACGCGACCTAACGACAGGATACTTTTCACAATAGCTTGTGCAAGGCCACTTGAAGCTTCTTCCACCTCTTTC
TTCCCTTTATCTTCAAAGTCGTACGGATCGGAATCATTGTTAATAGAGTTTCCATTAACACTTCCGTTGGTTCCTCC
TGTTTCATTTGTCTCAATACGTCGGACAGCTTCAGAAAATGAATCTTCCGCAGTATCTTCATAATCTACGTGATAC
CAGGGTCGAGGAAGAATCGAATACTCTTCCAGTGCAAGTAGCGAAGGGTAAAATAGTCCCATGGGTGGAATGTT
GATGGAAGATAATACGACTGTATATACTCGAGTTGGCTCGCCTGCCATGCGGCACTTGATATCAACCGTCGTGTGT
TGCTCACCTTCCAGAAGCACGCAGTGGGTCTCCTTCAGTTTCTCAAGAGTCAAAAAGTCCAACGGATCACGCAAA
GGATCAGTATCAGCCAGGGGCCATGTCTTCTTTCGTTGTTGGACCCATAACAAACACCTAGTGATGTCATCTCCAC
CATACGGCAGCAGAAAACGTGTTGATGGAATAGCAACCCCTTCCTCAACACACATGCCTGAGATAACTTGTGCAC
CCATGTTTACGATACATCCAGATGAAAAGCCATTCCCAAATGTAGCCGCAACGCATTCCTGGA
Real-time fluorescence quantitative PCR primer sequence: forward primer: GCAAGTAGCGAAGGGTAAA
Reverse primer: TGCCGTATGGTGGAGATG
> sequence 3 tooth rib red moss Syntrichiacaninervis transcriptional elongation factor (SPT) nucleotide sequence
TTGAAGGTCTTTCTGATATAATCTTCCTTCTTTAAGCGAGTCTTTTCCTGCTCGTTGAGTAAGAAGGTGATGTCGGAT
TTAAAACGCTTGTCCCGGTGCTGCGTATCATCCCTAGACTCTTCCCGCATATCGCTTTCCTTGCATGTCAAATCGACC
AAGTACTTATCCTTTTTCACCTCTTTCACTCGGCAAGTCACTGAAGTGCCTTCTGAAAATTTATCTTTAAGTTCAACA
TCGCCGTCATCTGAAACATCTTCCTTCTGAATGAAACCCAGTAATCCACATTCCAATACACACATGAGCTGATGTTCC
TGCACTCTTCTTATTGTTGCTTGTACAATTCCACCTTCAAAGAGTGTCTCTTTGCTCTCCCCGGTCATGAGATAGAAA
CACTCTTCTTGAGACGGCTCCGAGTACGACCTCCGCCATTCTTGAAATCCTCGCTGCAGCTCAGATTTGATCAACTC
CAGTGTCTTCAATCCCCGGCCTTCAACGTGTGCAGATTGGGCATACTCCGCAATGTCTACAGAATCTAAAGCGTTGG
GATTTTTAATCACACGATCTACTGCCATACTTGGCACATCATCGTCCACTTCTTCGTTATTCCGCATGCCTTCCTTCTC
GATCGCAGCTTCAGCTAGGTTCTTTGCCATCTGATACAACTCAGGGTGAATACGGGTGTTGTCTAAAAGCTCTGTAG
GACGTAAATCTGAAGGTGTTTCTCCAATTTCACGCACTCGGATGAAACCAGCGGAATTTGTGAAGACTTTCTCATTC
ACAGCCCGTATGGTCGAGTAAAGACTATCTCGAGTCAATACACGCTTTACCACTTGAAGATTTGTCCGAAACTCACA
AGCCTTCAGAGGGCCAAGACCCGCAACGAACTGAAGGGTCTCAAACAACCAGTCGTGGATAATAGCTAGA
Real-time fluorescence quantitative PCR primer sequence: forward primer: ACTTGGCACATCATCGTCC
Reverse primer: ATTCCGCTGGTTTCATCC
> sequence 4 tooth rib red moss Syntrichiacaninervis calcium dependent protein kinases (CDPK) nucleotide sequence
TGAGATGTTTAAACACTACCTCAATTACATGCTGAAATCCAATACATTTGACGTAGAAGCATCAACAGTGTTTGGC
ACCTAATCATTTCCTCTCGGGCGCCTCTAGCGATGGCACTGAAAATGATACACGGGGACTAAAGGCCATGGCCCT
AGAGCAAGTAACATGACAAACTTTACATATTCAAACTCCCCATGGTAAGGAGAGAGTGCTACTCGCCTCTGTAGA
AGTGACTGGTTCGCACAGCCATGTTGAGGTGCCTACTTCCTGCTGAACTCGGTGTGAACCCTATCCTCCGATCCCC
AACGCGTCGGTCAGATTCAACTGCTGGCTGTTGCGTAGCGATGAGCGACCAGCACCGCCCTTGTTGCCCTTGCGC
ATCATGTTGACGAACTCGGTGTAATCAATCTGGCCATCGTTGTTCTGGTCGACCTCCCTGAGCAGCTCCTCCACGG
ATACGTCTCCCATGTTGAATTCGGCCAACGCATGTTGCAGCTCATCGACCGTCAGATAGCCGCTGTTGTCCTTGTC
TAGGTACGAGAAGGCGGCGAACATGTTCTCATCGCGTTCGACCTTGTTCAGGTGGAGAGTGGCGGCCAGAAACTC
GCCATAATCGATTGCCCCACTGCCGTCGACGTCAGCGGCGTCCATGAGCTGCCGAACGTCTTCCTCTTTGAGATTG
GAACCCACTTTCTTCAGACCCGCCTTCAGCTCCTCGTAGCTGATGGACCCTGAGTTGTCCGTGTCCATCATCTTGA
ACATCTCCTTCAGACCTGCGATCTCCTCCTCCGACAAGGACTCCGCGATCACCCGGATGGCGATCTTCTTCAGCTT
GTTCATTGCGGAGAAGTGCTTGAGCCGGGATTGCACTGCGGAATCGATGGGTCTGTCGGGCGCTACTCCGTCCTC
GCAGATCCAAGGATGTGCCAACACTTGATGGGCTTTCAACCTCTTCGAAGCCTGGGGCGTGAGCATTTTGCGGAT
CAAATCCTTGGCGCTATCAGAAATCTTAGGCCATGGATCTGATGCAAAATCAAGCTCTGATTTCAAAACCTGCTCA
AAGATGCCCTGCTCCGTCTCTGCCCAGAATGGAGGGACTCCGCTGAGAAGGATGTAAAGTATCACACCCGCACTC
CACACATCCGCTTCCGGGCCATAGTTCTTCCTCAACACTTCAGGTGCCACATAGTAAGGGCTTCCAACCACATCTC
TGAAAACTTCTCCAGGTTTAAAGAACACTGATAGCCCAAAATCAGTCGTTTTCAGAGCCGCATCCTCCTTCGTACT
CGAAAACAGGAAGTTCTCCGGCTTCAAATCTCTGTGCATCACGCCAAGAGAATGGCAGGTCTCAACCACGCCCAC
GATAGTACGAGTGAGCACTGCCGCTTTGGCTTCCGAGTACTGTCCTCGTTGAATGATGCGGTCAAACAGCTCACC
GCCTGCACAAAGCTCCATCACCAGATGCACTGAAGCAGGATCCTCATACGCCCCCTTGATCATGACAATGTTCGG
ATGTCCTGAAAGGTGATGCATGATCTGCAGTTCACGGCGAACGTCATCCACGTCTTCCTTGGAGATAAGCTTCCGC
TTGGCAATAGACTTGCATGCGTACTCCCTGTTGGTGGCCTTCTCAATGCACAGGTAGGTGGTGCCGAATTGACCTT
GCCCCAGCTTCTTTCCCAGAACATACAGGTCTCTGAGGTTCTCTGTCTTCCGCTGCAGCACAGAGTGAGTCAGACT
CTGGCCAGGGACGGGCTTCATCTCTCTCCCTTTCTTCATGGCTTCGATCACTTCTTTGAATTGCTGAGTTTCGGCCG
GTTCCACAGCAGGGATGTGAGCTGGGCGTGGCGGTTTCCGTTGAGTCTCCGGGGACTCCGCCTCTGCCCTGCTCCC
CCCTCTGGACTCTGCCTTGGATGGAGTGGGTGATACCTCACTAGGGGATCTGGAGCTACGCCCTCCCAGTGCGATC
ACACTGGTGAAGCCCTGAAAGTACCCAGCCGCTCCTACACAACTGTTGCCCATTCAGACACAATCTGTAACATCC
CTCCACGGGAATCTATTTATCCCGTTCCCCTAAACTTGTCTCTTAAAGCAAAGGCCTAAACATACACTACAACGAA
CTTGATGCCGTTTTTGTCACCATTTGGCCACGTACCTTCGCACAATCATCTGAAGTAAGAACAGGCTGAGGAGAA
AGACCGCCGTCTAGGGTTTCAAGGCTGAGAAGCGAACCCTGAAATGAGTGTAAGGTGGCCGAAACTGACTTCAA
AGAGAGAACTGGCGGCCAGGTTTCCTCAGTTATTCTTTCCAGTCTCAAACCTCGAGCTCCGGCTGCGAAATAATCT
GGCAGCGAAAGTGGCAGCAAAAACAAGCAGCTCTCGCACTATTTTCC
Real-time fluorescence quantitative PCR primer sequence: forward primer: AACACTTCAGGTGCCACATAG
Reverse primer: GCATCATTCAACGAGGACAG
> sequence 5 tooth rib red moss Syntrichiacaninervis α tubulin (α-TUB1) nucleotide sequence
GTGGATGTGACGGAGTTCCAGACGAACTTGGTGCCGTACCCCCGTATCCACTTCATGCTTTCGTCTTACGCGCCGG
TGATCTCGGCCGAGAAGGCGTACCATGAGCAGCTGTCCGTCGCGGAGATCACCAACTCTGCGTTCGAGCCGTCGT
CTATGATGGCGAAGTGCGACCCCCGTCACGGGAAGTACATGGCGTGCTGCCTGATGTACAGAGGAGACGTTGTGC
CGAAGGACGTGAACGCGGCAGTGGCAACGATCAAGACGAAGAGGACGATCCAGTTCGTGGACTGGTGCCCGACC
GGGTTCAAGTGCGGAATCAACTACCAGCCCCCGACGGTGGTGCCCGGAGGCGACCTGGCGAAGGTGCAGCGGGC
GGTGTGCATGATCAGCAACAACACGGCGGTGGCGGAGGTGTTCTCCCGGATCGACCACAAGTTCGATCTCATGTA
CGCGAAGCGCGCGTTCGTGCACTGGTACGTGGGCGAGGGCATGGAAGAGGGTGAGTTTTCGGAGGCCCGTGAGG
ACTTGGCTGCGCTCGAGAAGGACTACGAAGAAGTGGGCGCGGAGTCGACGGACGGCGCCAACGACGACGATGAC
TAC real-time fluorescence quantitative PCR primer sequence: forward primer: CGTCGTCTATGATGGCGAAGT
Reverse primer: TCTTGATCGTTGCCACTGCC
> sequence 6 tooth rib red moss Syntrichiacaninervis α tubulin 2 (α-TUB2) nucleotide sequence
ATGAGAGAGATCATCAGTATCCACATCGGCCAGGCCGGTATCCAGGTCGGCAACGCCTGCTGGGAGCTCTACTGC
CTCGAGCACGGCATCCAGCCCGATGGCCGCATGCCTAGTGACGCGTCTACAGGAGGTGGAGACGATGCGTTCAAC
ACCTTCTTCAGCGAGACCGGGGCGGGGAAGCACGTGCCGCGGGCTGTGTTTGTGGATCTGGAGCCGACCGTGATC
GATGAGGTGCGCACGGGGACGTACCGGCAGCTGTTTCACCCGGAGCAGCTTATTTCCGGGAAGGAGGACGCCGCT
AACAACTTCGCTCGCGGTCATTACACCGTGGGAAAGGAGATTGTGGACTTGTGCTTGGACCGGGTGAGGAAGCTG
GCAGACAACTGCACGGGCCTGCAGGGATTTTTGGTATTCAATGCTGTGGGAGGAGGCACGGGATCGGGACTCGGT
TCGCTGTTGCTGGAGAGGCTCTCAGTAGACTACGGAAAGAAGTCCAAGTTGGGATTCACCATCTACCCGTCTCCTC
AGGTGTCCACCGCAGTTGTGGAGCCGTACAACAGTGTGCTGTCGACTCACAGTCTGTTGGAGCACACCGATGTGG
CGGTGTTGCTGGACAATGAGGCCATTTACGACATCTGCCGGCGCTCGTTGGACATCGAGCGGCCGACGTACACGA
ACCTGAACAGGCTGATCTCGCAGATCATTTCGAGTCTGACGACGAGCCTGCGGTTTGACGGCGCGCTGAACGTGG
ACATCACTGAGTTCCAGACCAACTTGGTGCCG
Real-time fluorescence quantitative PCR primer sequence: forward primer: CGGTCATTACACCGTGGGAA
Reverse primer: CCTCTCCAGCAACAGCGAA
> sequence 7Syntrichia caninervis beta tubulin (β-TUB) nucleotide sequence
ATGAGAGAGATCCTCCACATCCAGGGCGGGCAGTGCGGGAACCAAATCGGTTCCAAGTTCTGGGAGGTGGTGTG
CGAGGAGCACGGGATTGACCCCACCGGCTCGTACCAGGGGGTGTCTGACCTGCAGCTCGAGCGCATCAATGTGTA
CTTTGATGAGGCCAGCGGCGGGCGCTACGTGCCGCGCGCGGTGCTCATGGATCTGGAGCCCGGCACCATGGACAG
CGTCCGCACTGGCCCCTACGGCCAGATCTTCCGCCCCGACAACTTCGTCTTCGGACAGACCGGCGCTGGTAACAA
CTGGGCCAAGGGCCACTACACCGAGGGCGCGGAGCTCATCGACTCAGTGCTGGACGTCGTGCGCAAGGAGGTTG
AGAGCTGCGACTGCCTGCAAGGCTTCCAGTTCTGCCATTCTTTGGGAGGAGGAACGGGATCTGGAATGGGAACGC
TTTTGATATCTAAGATCCGCGAGGAGTACCCCGATAGAATGATGCTTACGTTTTCCGTGTTCCCCTCTCCGAAGGT
GTCCGACACCGTGGTGGAGCCTTACAATGCTACTCTCTCAGTGCATCAGCTGGTGGAGAATGCCGACGAGTGTAT
GGTGTTGGATAACGAGGCGCTGTACGACATCTGCTTTAGGACTTTGAAGCTGATCACCCCCTCATTTGGAGACTTG
AACCATTTGATTTCTGCGACGATGAGTGGTATCACCTGTTGCCTGCGTTTCCCCGGACAGCTGAACTCTGACTTGC
GGAAGCTGGCGGTGAACCTCATCCCGTTCCCCCGGTTGCACTTCTTCATGATCGGTTTCGCGCCTTTGACCTCCCG
TGGCTCACAGCAGTACCGGTCGCTCACCGTGCCGGAGCTGACGCAGCAGATGTGGGACTCAAAGAACATGATGTG
CGCGGCGGATCCGCGTCACGGGCGCTACTTGACGGCTTCCGCGGTGTTCCGCGGCAAGGTGTCCACCAAGGAGGT
CGACGAGCAAATGATCAACGTGCAGAACAAGAACTCATCCTATTTCGTTGAGTGGATCCCGAACAACGTGAAGTC
GTCTGTCTGCGACATCCCCCCGACGGGTCTGAAGATGTCGTCGACGTTCATCGGGAACTCGACGTCCATCCAGGA
AATGTTCCGGCGCGTGTCGGAGCAGTTCACGGCCATGTTCAGGAGGAAGGCGTTTCTGCATTGGTACACGGGCGA
GGGCATGGACGAGATGGAGTTCACAGAGGCGGAGAGCAACATGAACGATCTGGTGTCGGAGTACCAGCAGTACC
AGGACGCGAGCGCCGAAGAGGAAGGCGAGTACGAGGACGACCTTGAGGAGGCC
Real-time fluorescence quantitative PCR primer sequence: forward primer: CTTTGACCTCCCGTGGCTC
Reverse primer: GTTCGGGATCCACTCAACGA
> sequence 8 tooth rib red moss Syntrichia caninervis ubiquitin protein ligase 1 (UBR1) nucleotide sequence
GCGCTTGTGAACGAGCTCATCACCAAGTTGTCCAGCCGGAGCACCAAGGTACATCGGGATGCTGCCAAGACCATA
AGGTGCCTAGCGAAGATAAACAAAGAGAATCGAATTCTTATTGCGGAGCAGGGAGGCATACCTTTCTTGATCAAC
CTTCTCCGATCGCCTGATGAGGAGACGCAGGAGCACGCCATCACCGCGTTGATGAACCTGTCCTTGCATCCAAAC
AACAGAGGGCTCATCATGAGAGCAGGGGCCATAGACGGCATCGTTCACGTAGTGAAGCATGGCGAGAGCACGGA
CGCGCGCGAGAACGCAGCAGCAGCAATCCAGTGCCTCTCGTATGACAACGAGAACAAGATCTCCATCGGCAACA
CGGGTGCGATCCCCGCGCTCGTTCAGCTGCTGCGCACCGGGACTCGCCAGGGCAAGAAGGACGCGGCCCACGCG
CTCTGCAACCTCGTCAGCTACCTGGACGGCAACAAGAAGCGCGCGGTGGACGCCGGCCTCACACCCCTCCTCATG
GCGGTTCTGCGCGAC
Real-time fluorescence quantitative PCR primer sequence: forward primer: GCAGGGAGGCATACCTTTCTT
Reverse primer: ATGAGCCCTCTGTTGTTTGGA
> sequence 9 tooth rib red moss Syntrichia caninervis ubiquitin protein ligase 2 (UBR2) nucleotide sequence
ATGGGGTTAGGGTATGGGCCAGTGGTGGCATTGTACACGGCGTTGAGTGTGGGTGGGATAGCATGGAAGCTCAAC
TCAGCGTGGTGGACTATGACCTACGGCAAGGGTCAAGGAGGTGGTGGGCAGATGGATCCCTCCGAAGCGGCGCG
CACGCTGCAGGCCATGCTGCAATCATCCGTCGTGGTGGCGCTCCTCGTCAACCTTCTCGTCAACTTTTTCCTTCTCG
TCACCCTCTCAATGAAGACTGTGTTCTTCGGTCAGTTGTCTCTGGTTGAGACTCAGAAGGTGGTGGAGCGCCTCAT
CAATTATGTATTATTCAAGGGCCTGTTCTTGACATGGGTCGTGAGGCCAGAAATGATGCAGATAGCTGTGTGGTTG
GCGTGGTTCTCCGTCTTGGGCTTTCTGAAGATGTTCCAAGGTTTGGCAAGAGATCGTTTGGAGCGCTTAAATGCAT
CTCCTTCTGCGACAATGTTGGCTCACATAAGAGTTTATACGGTTCTCTTGTTCGTTCTTCTGTTCGATTTGCTCTGG
ATGCAGCTGTGTCTGCTTTTATTCAAAGACACTGGAACCAGTACCTTCTTGCTACTTTTGTTTGAGCCTCTCAGTAT
AGCATTTGACACTCTTCAGGCAGTAATTGTACACGGTATGCAGCTATTGGACACTTGGCAGCGGCAAAACCTTGA
TATTTCTGCACACGATATAAGTTTGCAGCCTTCTGAGAGGTCTGCTGCAGGTGCAGCTTGGGAGTGGCGTGGAACT
GTTGTAAGAAATTGCAGCTTTGTGATGGATATTGTGAGCTTGTTACTGGCTTTGGGCCATTGCGTACACATCTGGT
GGTTACGTGGGCTTGCTTTTCAAGTGGTGGATGCCATTCTTTTCCTCAACTTGCGCGCGCTATTGAGTGCTATTTCA
AAAAGAATAAAAGGTTTCATGCGTCTTCGGGCAGCCATGACAACTCTGCAGGGTGCGCTTCCCGATGCAACGCAG
GATGAACTCCTTGGATATGAAGATGATTGTGCCATTTGCAAGGAGCCCATGGCTAGGGCAAAACGGCTTCCTTGT
GCTCATCTCTTCCATCTCGCGTGCTTGAGATCCTGGCTAGACCAAGGATTAGCAGACACATACTCTTGCCCGACCT
GCCGTAGACCCCTCTTCATGGGCGGGTTCCGGACTGCAACAAACAATTTGCAGCGCCTGGTAGATGTGCCCACTG
CATCCCCCGGCACAAATGAAACTCAGGCACAGGTTGCACAACGACAAGACTTAGCTGCAGCGCCGTCTCCACCTC
TCACCACCTTGACTCCCGCTCCAGCACCG
Real-time fluorescence quantitative PCR primer sequence: forward primer: AGCTTGTTACTGGCTTTGGG
Reverse primer: GGAGTTCATCCTGCGTTGC
> sequence 10 tooth rib red moss Syntrichia caninervis glyceraldehyde-3-phosphate dehydrogenase 1 (GAPDH1) nucleotide sequence
TCCGGGATCAAGAACACCAATGCCGTCACCTTCGGCAAGCGCACTGATGACCTCGTCAGCGTTGTTGCTGCCCAG
TCTGCTGTGTCTGCTAGCTCCGGAGCCACGAGGGCCGTGACCGAGGCCAAGATCAGGGTTGCCATTAACGGTTTT
GGGCGTATTGGACGCAACTTCGTCCGCTGCTGGCATGGAAGGAAGGACTCGCCTCTCGAGGTCGTCGTGATCAAC
GACACCGGAGGCGTGAAGCAGGCTTCTCACCTGTTGAAGTACGACTCCATGCTCGGCACCTTCAATGCCGACGTG
AAGGTCGACGGTGATGGCATCAGCGTTGATGGCAAGACCATCAAGGTGGTGTCCGACAGGAACCCGCTCAACTTG
CCATGGGGGGATATGGACATCGACCTTGTCATTGAGGGGACCGGAGTGTTCGTCGACGAGGCTGGTGCCGGCAAG
CACTTGCAGGCGGGAGCGAAGAAGGTGCTCATCACCGCCCCAGGAAAGGGTGCCATCCCCACGTACGTGATCGG
AGTAAACGAGCAGGATTACACCCACGCGGACAAGATCATCAGCAACGCGTCGTGCACCACCAACTGCCTGGCCC
CGTTCGTGAAGGTGTTGGACGAGAAGTTCGGCATCATCAAGGGTACCATGACCACCACCCACTCATACACTGGTG
ACCAGAGGCTGCTGGACGCTTCCCACCGCGACTTGAGGAGAGCCCGTGCCGCGGCCCTGAACATCGTGCCCACCT
CCACGGGAGCCGCCAAGGCCGTGGCGCTGGTGCTGCCGAGGCTGAAGGGCAAGATCAACGGAATCGCCCTGCGT
GTGCCCACCCCCAACGTGTCCGTGGTGGACTTGGTGGTGCAGGTGGAGAAGAAGACGTTCGCGGAGGAGGTGAA
CCAGGCGTTCAGGGATGCGTGCGAGACCCCTCAGCTGAAGGGCATTTTGGCAGTGTGCGACGAGCCTTTGGTGTC
GGTGGACTTCAGGTGCACTGACGTGTCGTCCACGGTGGACTCGTCCCTGACCATGGTGATGGGCGACGACATGGT
GAAGGTTGTGGCATGGTACGACAACGAGTGGGGGTACTCGCAGCGGGTGGTGGACCTGGCTAACTTGGTAGCGG
AGAAGTGGCAG
Real-time fluorescence quantitative PCR primer sequence: forward primer: GCGTTGTTGCTGCCCAGTC
Reverse primer: AGGCGAGTCCTTCCTTCCAT
> sequence 11 tooth rib red moss Syntrichia caninervis glyceraldehyde-3-phosphate dehydrogenase 2 (GAPDH2) nucleotide sequence
ATCGGAAGACTCGTCCTCCGTGTGATTCTTGCCAGGGACGATGTGGAAGTTGTTGCCGTCAACGACCCCTTCATTG
ACACCAAGTACATGGCATACCTGTTGAAGTACGATTCCACCCACGGGCTTCTCAAGGCTGATGTGCACGCTGTTG
ATGACAAGACTCTGCAAGTTGCTTCTCAAACCATCAAGGTCTTCGGATGCAGGGATCCCGCGGATATCCCATGGG
GTGAAGCCGGTGCTGATTACGTGGTGGAGTCCACCGGTGTTTTCACCGCCGTTGCCGGCGCCTCGGCCCATTTCAA
GGGAGGAGCCAAGAAGGTTGTTATTTCCGCACCATCGGGAGATGCCCCCATGTTCGTCATGGGTGTGAACCAGAA
CGCTTACAAGGATGACATGAACGTCGTCTCCAATGCCAGCTGCACCACCAACTGCCTTGCTCCCCTAGCTAAGGT
GATCCAGGACGAGTTCGGCATTCTCGAGGGTCTGATGACCACCGTTCACGCCACTACCGCCACCCAGAAGACCGT
CGATGGCCCATCAGGGAAGGACTGGCGTGGAGGACGTGGTGCTGGCCAGAACATCATCCCCAGCTCCACCGGCG
CTGCCAAGGCTGTGGGCAAGGTGTTGCCTGAGCTGAACGGCAAGTTGACCGGCATGGCGTTCCGTGTGCCCACCC
CGAACGTTTCAGTGGTGGACTTGACTGTGCGCCTGGAGAAGGGTGGGTCCTACGAGGACGTCAAGGCAGCCATCA
AGGCCGCCTCTGAAGGACCGATGCATGGCATCTTGGGCTACACCGAAGACGACGTGGTGTCGAACGACTTCATCG
GTGACGCCAGGTCCAGCATCTTCGATGCTAAGGCAGGAATCGCTTTGAACGACAAGTTCGTCAAGTTGGTGTCGT
GGTATGACAACGAGTGGGGCTACAGCAACCGTGTGGTGGACCTG
Real-time fluorescence quantitative PCR primer sequence: forward primer: GGGCTTCTCAAGGCTGATGT
Reverse primer: TCCACCACGTAATCAGCACC
> sequence 12 tooth rib red moss Syntrichia caninervis SAND albumen (SAND) nucleotide sequence
TCGGAAGTGGAAGCTGATAATACAGCGAAACTCTCAAACCTGGACATCAGTAGCAGCACGCAGGAGGACGGTTCA
AAGCTTGAAACTCATGATGCAGAAGATGGTCCAAAAGCCTTGGACATGGGAGATGGAGAAGGTACTGCGCTCGCT
GCTTCTACAACTACTGATGTTGTGCCGGAGAGTACTGTGGATGGAGATACATTGTCTGAAGATGGCATCCATGAGAG
CCCTGTGAGTCCAAGCAGTAGCCGCGAAGGAACCAGTGGCTACTTAGCAGGCAGTGGCAGCAGTACCGCGAGTGT
CTATAGCGCAAGTGGAGCAGATGAAAGTGCCGATCGAATTCTGAATCAGGATTCTATGCCACAAGCGACGAGGCGG
TCTTGGGTCCCTGGAAAACGCCATCCAGATGAAGATGACACGTCTCTCGCTTGGCGAAAGCACAAGAAGCATTTCT
TTATTCTCAGCAATGCAGGAAAACCA
Real-time fluorescence quantitative PCR primer sequence: forward primer: AAAGCCTTGGACATGGGAGA
Reverse primer: CGTCGCTTGTGGCATAGAA
> sequence 13 tooth rib red moss Syntrichia caninervis H3 (HIS3) nucleotide sequence
CTAAAGACGGGAGAACGAAGACCCTTTTCTCCTCCTTCGGCTTCACCTCCCCATCTTTCTCTCTCGCTCACTCTCGA
CATCGTCATCTCTCTCGTATTCTACCTACACCGCCGTTTCATTTCCACAAGTGTCACTATGGCGCGTACGAAGCAG
ACGGCTCGTAAGTCCACCGGAGGAAAGGCTCCGAGGAAGCAGCTCGCCACGAAGGCTGCGAGGAAGTCGGCCCC
CACCACCGGAGGAGTGAAGAAGCCCCATCGCTACAGGCCGGGAACCGTCGCTCTTCGTGAGATCCGCAAGTACC
AGAAGAGTACCGAGTTGCTGATCCGCAAGCTGCCCTTCCAGAGGCTGGTTCGTGAGATCGCCCAGGACTTCAAGA
CCGATCTGCGTTTCCAGAGCCACGCCGTGCTGGCGCTGCAGGAGGCGGCGGAGGCGTACCTGGTGGGTCTGTTCG
AGGACACCAACTTGTGTGCCATCCACGCGAAGAGGGTGACCATCATGCCGAAGGACATCCAGCTGGCCCGCAGA
ATCAGGGGAGAGAGGGCTTAGGCCGGCTCGCCCGGAGTCTGTATGAAAAAACCCAAAAAAATCTAAAAAAAAGT
TTGGGGTTCTTTTGAACCCACCAAAGGGGCGTGTACTCCGTGTATGTGGCATGATGTTACTTGTATGTTACTTGTA
TGTTTGTGCGGAGTGATTGCTGAAGCAGCCGAG
Real-time fluorescence quantitative PCR primer sequence: forward primer: AGGAGTGAAGAAGCCCCATC
Reverse primer: CGAACAGACCCACCAGGTAC
> sequence 14 tooth rib red moss Syntrichia caninervis F-box protein (F-BOX) nucleotide sequence
ATTCGTCTTTTTGTTTGGTTGCGTAAGTGTTTAGGTTGACACAATCCTCGTTTCGCTTCTAGGTTTTGTGTTTTGGCG
TTTAAATCACTGAGGTTTCCCTGAAGACCTTTGATGCCATAGAGATGACGCTTTCCTTGTCCTGCTTACAGCTTGAG
CTGGTGTGACAGTATCCGAATTTGCCCTAATTGGAGGGGCATGTGAAGCTCACGATCTGGGTCCGCTTACAAGCTCA
CAGCTTTAGTCAGTATTGACAACATGGGATGATCGTCTTGCAATCTCGAAGAAGCGATGGAGCAAGACGGATTGGA
TTCATGGAGCAGGCTACCGGAAGATGTCGTGCTGAGAGTGCTCACGCGCCTGCCAATTCCGCATCTTTTCCGCGCG
CGCACGGTCTGCAAGCAATGGAATGCGCTCACTTCGACCCCGGAGTTTCTGGCCATGTGCAGCGCGCGTCACGCGC
CCTACTTCCCGGTGGTGATCTCGCGCCGCTTCTACATGGGCGACCTGGGGATCTGCTTCCTGGGGAACGACTCGCA
GGGCGGCAACGACCTCTTCCGCGTCTTCTTCGGCTACGACCATGAGGCGCAAGCGTGGCAGAAGCTGCCCCCGTTC
GACTTCCTCCCGCACGAGGCGCGCATCCCGGTGGCCTCCGCCGGCGGCCTCATCTGCTTCCGCGGCACCTCCAGGC
TCTTCCTGTGCAACCCCGTCGCCCGGAGCTTCCTGGAGCTCCCCGCCATCACGTACAAGTGG
CCGCCGTCCGTCTCCGTGCACATCCTCGTGGAGAGCAGCGCCTTCAAAGTCATCATCGTGGGGAAGATCCGCCACA
ACTTCGTCACCGACTCCTTCCGCAGCATCGCCATCTACGAGTCCGTCACCAAGGCCTGGCGCGTCGTGGACGCGCC
GCATCCCGCGAAGGTATTCTCCTACGGTCCCACTGCAGCCGTCTGCGGCAACTCCATCTACTGTGAGGCGATTTGCC
ATTGCGGCCAGCTGGGTGTGATGGCCTACGACGTCCAGTCCGAGACGTGGAACCCGGTGTTGCATGAGATTCCGAG
TGATGAACGCGGCGAGTATCAGCTGACGCAAGTTGTG
Real-time fluorescence quantitative PCR primer sequence: forward primer: CGCCTTCAAAGTCATCATCG
Reverse primer: GGCAAATCGCCTCACAGTAG
> sequence 15 tooth rib red moss Syntrichia caninervis18s ribosome-RNA(rRNA) (18S) nucleotide sequence
TTACTATGGTGGTGACGGGTGACGGAGAATTAGGGTTCGATTCCGGAGAGGGAGCCTGAGAAACGGCTACCACATC
CAAGGAAGGCAGCAGGCGCGCAAATTACCCAATCCTGACACGGGGAGGTAGTGACAATAAATAACAATACCGGGC
TCTTTGAGTCTGGTAATTGGAATGAGTACAATCTAAATCCCTTAACGAGGATCCATTGGAGGGCAAGTCTGGTGCCA
GCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTTAAGTTGTTGCAGTTAAAAAGCTCGTAGTTGGACTTTGGGT
TGGGTCGGCCGGTCCGCCTCTGGTGTGCACCGGTCGTCTCGTCCCTTCTGCCGGCGATACGCTCCTGGTCTTAACTG
GCCGGGTCGTGCCTCCGGCGCTGTTACTTTGAAGAAATTAGAGTGCTCAAAGCAAGCCTACGCTCTGGATACATTA
GCATGGGATAACATCATAGGATTTCGGTCCA
Real-time fluorescence quantitative PCR primer sequence: forward primer: GGAGAGGGAGCCTGAGA
Reverse primer: CACCAGACTTGCCCTCCAA;
B, choose arid respectively, high salt, cold, heat, xeothermic mixing, dormin, oxidation, physical abuse, heavy metal, ultraviolet coerces totally 10 kinds of abiotic stress and without Stress treatment, and 11 tooth rib red moss gametophyte sample is experiment material altogether, carries out the experiment of quantitative fluorescent PCR template;
The internal reference estimation of stability software geNorm that c, the data importing two obtained by real-time fluorescence quantitative PCR are the most frequently used, a NormFinder and internal reference stable checking calculation software Refinder carries out internal reference stability and the analysis of internal reference number, filters out optimum reference molecules and reference molecules combination.
Reference gene described in step a is 18s ribosome-RNA(rRNA), Actin muscle, α tubulin 1, α tubulin 2, beta tubulin, glyceraldehyde-3-phosphate dehydrogenase 1, glyceraldehyde-3-phosphate dehydrogenase 2, ubiquitin protein ligase 1, ubiquitin protein ligase 2, calcium dependent protein kinases, F-box protein, transcriptional elongation factor, actin associated protein, SAND albumen, H3.
In step b, 11 tooth rib red moss gametophyte is the leaf tissue of the removal rhizoid after various Stress treatment after complete rehydration 24h altogether.
The condition of abiotic stress process in step b is: use 20% polyethylene glycol 6000 respectively, 250mM sodium-chlor, 100 μMs of dormins, 0.5mM copper sulfate, 50mM hydrogen peroxide, 0.5w/m 2uv-radiation simulating drought, high salt, dormin, heavy metal, oxidation and ultraviolet are coerced; Red for tooth rib moss is placed in respectively temperature 4 DEG C of refrigerators and temperature 42 DEG C of illumination boxs simulation low temperature and high temperature stress; Coerce with the 42 DEG C of dry-hot simulation mixing of 10%PEG+ temperature, it is realize by cutting off blade that physical abuse is coerced; All process are all draw materials after coercing 6h, and the material obtained is placed in liquid nitrogen immediately, and are placed in rapidly temperature-80 DEG C of refrigerator storage.
In step b, real-time fluorescence quantitative PCR template is 1 μ g RNA and reverses 5 times of diluents of the cDNA obtained, and fluorescent quantitation program is: the first step is denaturation: temperature 95 DEG C-30 seconds; Second step is PCR step of reaction, temperature 95 DEG C-5 seconds, temperature 58 DEG C-60 DEG C, 30 seconds, and fluorescent signal is collected in 40 circulations; 3rd step is solubility curve analysis, temperature 65 DEG C-95 DEG C, and temperature 65 DEG C progressively rose to 95 DEG C at 30 seconds, collects first order fluorescence signal for every 0.5 DEG C.
The screening method of desert tooth rib of the present invention red moss real-time fluorescence quantitative PCR reference molecules, the sequences Design of 15 genes described in the method is applicable to the PCR primer of fluorescent quantitation experiment, search for the specificity of primer with Primer-BLAST and local blastn simultaneously, and the solubility curve generated with real-time fluorescence quantitative PCR and PCR primer electrophoresis judge the specificity of primer, and do typical curve to judge the amplification efficiency etc. of this reference gene.Each reference gene fluorescent quantitation primer information is in table 1:
The screening method of desert tooth rib of the present invention red moss real-time fluorescence quantitative PCR reference molecules, the feature of the method is: for the blank of the reference sequences poor information of reference gene and the screening operation of the most stable, most suitable reference molecules in the research of natural drought-relief rare desert mosses tooth rib red moss gene quantification, have selected 12 conventional classical reference molecules genes and 3 new reference molecules genes are candidate's reference gene, and design the primer of special real-time fluorescence quantitative PCR based on these sequences.The expression stability of these 15 genes under 10 kinds of abiotic stress is checked with realtime fluorescent quantitative PCR experiment.Then use three large internal reference analysis software real-time fluorescence quantitative PCR data, shown that reference gene the most stable under the red moss of tooth rib is under abiotic stress and the combination of optimum reference gene are CDPK and α-tub2.Use the reference molecules of the present invention's screening, the red moss material of the tooth rib under different abiotic stress can be avoided in RNA quality, the error that may exist on output and reverse transcription efficiency etc., the data that real time fluorescent quantitative detects can better be corrected and stdn, improve accuracy and the reliability of research.
Accompanying drawing explanation
Fig. 1 is the solubility curve figure of the present invention's 15 candidate's reference molecules.
Fig. 2 is the electrophorogram of 15 reference gene real time fluorescent quantitative products of the present invention.
Fig. 3 is the Cq value figure that under 10 abiotic stress, the real-time fluorescence quantitative PCR of different reference gene obtains, and wherein ■ is Cq mean value, and I is maximum value and minimum value.
Fig. 4 is the optimum reference molecules ordering chart of geNorm software analysis, wherein ← and be least stable internal reference, → be the most stable internal reference.
Fig. 5 is the optimum internal reference number of geNorm software analysis and most suitable reference molecules combined result figure.
Fig. 6 is the reference molecules stability ordering chart of NormFinde software analysis.Wherein ← be least stable internal reference, → be the most stable internal reference.
Fig. 7 is the integrated ordered figure of reference molecules stability of Refinder software analysis.Wherein ← be least stable internal reference, → be the most stable internal reference.
Embodiment
In the following example, experimental implementation is carried out routinely, and the method as described in " fine works molecular biology experiment guide " (F.M Ao Sibai, R.E. James Kingston, J.G Sai Deman edits, Ma Xuejun, and Su Yuelong translates, Beijing: Science Press, 2004) is carried out;
Selecting of reference molecules:
Studying and screen the most stable reference molecules and most suitable internal reference number (internal reference combination optimum in other words) in specified plant is the prerequisite of carrying out fluorescent quantitative PCR experiment.At present, the internal reference being used as gene expression in plants analysis in document mainly contains 18SrRNA, the classical housekeeping gene of these former bibliographical informations such as Actin, GAPDH.More and more report proof now, traditional classical housekeeping gene is so stable not as the imagination, therefore the much trial work to new reference gene launches successively, traditional classical reference gene is all better than under the experiment condition that the stability of reference gene as new in the CDPK that Libault etc. all reports, F-BOX etc. is tested soybean.Therefore, classical reference gene conventional in the main plant Arabidopsis thaliana in mode of the present invention and pattern liver moss small liwan moss is (as ACT, GAPDH, UBR) be reference, also select severally proves that in Arabidopis thaliana and soybean stability is better than the new reference gene of traditional internal reference (as CDPK simultaneously, F-BOX, SAND), from existing tooth rib red moss transcript profile database, select the homologous gene alternatively reference genes of reference gene in the red moss of tooth rib such as 15 above-mentioned Arabidopis thalianas, design each reference gene real time fluorescent quantitative special primer.With the red moss gametophyte of the tooth rib adopted back from ancient capital Xi'an for experiment material, after complete rehydration 24h is carried out to the tooth rib red moss gametophyte material of the drying of adopting back, carry out various Stress treatment, collect the material after Stress treatment 6h, extract RNA, reversion cDNA, carries out the specificity that each primer of internal reference is verified in fluorescent quantitation experiment;
The design of real time fluorescent quantitative internal reference primer:
Based on 15 reference gene sequences, the gene specific primer of design 15 pairs of real-time fluorescence quantitative PCRs, by the similarity analysis tools BLAST search specificity of primer in tooth rib red moss transcript profile storehouse, and verify the specificity of each primer further with the solubility curve of real-time fluorescence quantitative PCR and product electrophoresis; Fig. 1 is the solubility curve figure of 15 genes, as seen from the figure, the solubility curve of each reference gene primer is unimodal, without primer dimer and non-specific amplification, the RT-PCT product electrophorogram of each reference gene also confirms that 15 reference gene amplifications are single specific band, and stripe size is consistent with expection.Above-mentionedly all prove, the primer of 15 reference genes is all special, can be used for the experiment of ensuing fluorescent quantitation;
The collection of plant stress process material:
Choose the tooth rib red moss gametophyte experiment material of rehydration 24h, use 20% polyethylene glycol 6000 respectively, 250mM sodium-chlor, 100 μMs of dormins, 0.5mM copper sulfate, 50mM hydrogen peroxide, 0.5w/m 2uv-radiation carrys out simulating drought, high salt, dormin, heavy metal, and oxidation and ultraviolet are coerced; Red for tooth rib moss is placed in respectively temperature 4 DEG C of refrigerators and temperature 42 DEG C of illumination boxs to simulate low temperature and high temperature stress; Carry out dry-hot simulation mixing by 10% polyethylene glycol 6000+temperature 42 DEG C to coerce; It is by cutting off blade to realize that physical abuse is coerced; All process are all draw materials after coercing 6h, and the material obtained will be placed in liquid nitrogen immediately, and are placed in rapidly temperature-80 DEG C of refrigerator storage;
The extraction of RNA and reverse transcription:
Collect the RNAiso reagent reagent of the RNA extraction TAKARA of each sample, all sample standard deviations 1 μ g RNA carries out reverse transcription;
Realtime fluorescent quantitative PCR experiment:
With 5 times of diluents of the cDNA under each stress conditions of above-mentioned reversion for template, carry out real-time fluorescence quantitative PCR reaction with each fluorescent quantitation special primer;
GeNorm, NormFinder and ReFinder software carries out the real-time fluorescence quantitative PCR data analysis of candidate's reference molecules:
GeNorm software be according to the ratio between two of a certain reference gene and other reference gene expression levels after logarithmic transformation, calculate the mean value M(M value of its average difference as genetic expression stability with 1.5 for stagnation point, be greater than 1.5 to think and be not suitable for doing internal reference, M value is more little more stable), then the expression stability of all internal references is sorted, and the suitableeest number (stagnation point of V is generally defaulted as 0.15) that pairing variance analysis (pairing variation coefficient Vn/Vn+1) judges required reference gene is carried out to normalization factor, the selection result through geNorm screening method obtains: two the most stable reference molecules are calcium dependent protein kinases (CDPK) and α tubulin 2(α-tub2) (Fig. 4), that least stable is 18s ribosome-RNA(rRNA) (18S), the stability sequence of each reference molecules is as follows: calcium dependent protein kinases (CDPK)/α tubulin 2(α-tub2) >SAND albumen (SAND) > Actin muscle (ACT) > glyceraldehyde-3-phosphate dehydrogenase 2(GAPDH2) > beta tubulin (β-tub) > α tubulin 1(α-tub1) > ubiquitin protein ligase 2(UBR2) > H3 (HIS3) > glyceraldehyde-3-phosphate dehydrogenase 1(GAPDH1) > actin associated protein (ARP) >F-box protein (F-BOX) > transcriptional elongation factor (SPT) > ubiquitin protein ligase 1(UBR1) >18s ribosome-RNA(rRNA) (18S), and V2/3=0.139<0.15, needed for illustrating, the suitable number of reference gene is 2, express the most stable calcium dependent protein kinases (CDPK) and α tubulin 2(α-tub2 relatively respectively) (Fig. 5),
NormFinder determines internal reference stability according to the variation of reference gene between group and in group.Pass judgment on the stability (generally with 0.5 for threshold value) of reference gene with stability factor (stability value), this stability factor is less, shows that this reference gene is more stable.Predict the outcome consistent with geNorm, after NormFinder analyzes, the most stable reference gene remains α-tub2, is secondly CDPK, and that least stable is 18S(Fig. 6).The stability of the specific order for: alpha tubulin 2 (alpha tub2) & gt;Calcium dependent protein kinase (CDPK) & gt;Beta tubulin (beta tub) & gt;Glyceraldehyde 2-3 - phosphate dehydrogenase (GAPDH2) & gt;SAND protein (SAND) & gt;Actin (ACT) & gt;Ubiquitin protein ligase (UBR2 2) & gt;Alpha microtubule protein 1 (alpha tub1) & gt;Histone 3 (HIS3) & gt;Glyceraldehyde 1-3 - phosphate dehydrogenase (GAPDH1) & gt;Extension factor (SPT) & gt;Actin related protein (ARP) & gt;F - BOX protein (F - BOX) & gt;Ubiquitin protein ligase (1) UBR1 & gt;18 s ribosomal RNA (18 s);
Refinder software is the online software (http://www.leonxie.com/referencegene.php) of a comprehensive analyzing evaluation internal reference stability, it incorporates above geNorm, the algorithm of the different softwares such as NormFinder, comprehensively analyzes the stability of internal reference and sorts.According to the sequence that each algorithm obtains, Refinder calculates the suitable weight (appropriate weight) of each gene, and the geometric mean then by calculating each weight determines the eventual stabilities sequence of each gene.After Refinder comprehensive sequencing results are as follows: alpha microtubule protein 2 (alpha tub2) & gt;Calcium dependent protein kinase (CDPK) & gt;Beta tubulin (beta tub) & gt;Glyceraldehyde 2-3 - phosphate dehydrogenase (GAPDH2) & gt;SAND protein (SAND) & gt;Actin (ACT) & gt;Actin related protein (ARP) & gt;Alpha microtubule protein 1 (alpha tub1) & gt;Ubiquitin protein ligase (UBR2 2) & gt;Glyceraldehyde 1-3 - phosphate dehydrogenase (GAPDH1) & gt;Histone 3 (HIS3) & gt;F - BOX protein (F - BOX) & gt;Extension factor (SPT) & gt;Ubiquitin protein ligase (1) UBR1 & gt;18 18 s ribosomal RNA (s) and the comprehensive stability of sorting result is similar with geNorm and NormFinder.
Below by experimental technique, concrete screening process of the present invention is described:
Experimental implementation process:
The acquisition of candidate's reference gene:
Based on tooth rib red moss transcript profile database, select 15 internal reference candidate genes, the tooth rib red moss gametophyte transcript profile that all candidate's reference gene sequences obtain from the order-checking of this testing laboratory.
The compare of analysis of 15 reference gene sequences: submit to the BLASTX in NCBI to compare each gene order, each reference gene sequence in the red moss of tooth rib and the homogenic sequence identity of pattern mosses small liwan moss are all more than 58%, and major part is all more than 90%.With the sequence identity of model plant Arabidopis thaliana more than 28%, major part is also more than 90%.Comparison result is in table 2
Real-time fluorescence quantitative PCR design of primers and checking:
Based on these gene orders, with Primer Premier5.0 software design fluorescent quantitation special primer, by the specificity of Primer-BLAST software search primer, and the solubility curve generated with real-time fluorescence quantitative PCR and PCR primer electrophoresis judge the specificity of primer, and do typical curve to judge the amplification efficiency etc. of this reference gene.With the red moss material of the tooth rib of complete rehydration 24h, draw materials after various Stress treatment 6h (the red moss gametophyte of tooth rib removes rhizoid), extract RNA, reverse transcription becomes cDNA, and the PCR primer being applicable to fluorescent quantitation experiment with the sequences Design of above-mentioned 15 genes carries out real time fluorescent quantitative experiment.The making of typical curve is all after gradient dilution, do quantitative fluorescent PCR for template obtain with the reverse cDNA that obtains of the RNA of unscared tooth rib red moss 1 μ g, comprises 10 7, 10 6, 10 5, 10 4, 10 3.10 2six template gradients, each reference gene fluorescent quantitation primer and sequence information are see table 1:
The clip size of internal reference real time fluorescent quantitative as each in table 1 is all at about 100-300bp, and Tm value is all very close, the amplification curve of each reference gene, and typical curve is all fine, and the amplification efficiency (E) as 18SrRNA gene reaches 99.1, relation conefficient (R 2) equal 1, show that the amplification efficiency of this internal reference of 18S is very high, the linear relationship of typical curve is fine.The quantitative fluorescent PCR typical curve of each reference gene, each reference gene specific band all nothing but shown in solubility curve analysis (Fig. 1) and product electrophorogram (Fig. 2), specificity is fine.The internal reference small segment primer etc. that above data all demonstrate design all meets the requirements, and can continue the fluorescent quantitation experiment in downstream.
Realtime fluorescent quantitative PCR experiment:
With 10 kinds of abiotic stress (arid, high salt, cold, heat, xeothermic mixing, dormin, heavy metal, ultraviolet, oxidative stress and physical abuse) under the red moss of tooth rib be experiment material, carry RNA, RNA quality inspection (comprise agarose gel electrophoresis and uv-spectrophotometric detect) and reverse transcription, the PrimeScript Reverse Transcription box of what reverse transcription adopted is TAKARA.Concrete reaction system and program as follows:
Reverse transcription reaction:
5×PrimeScript Buffer:4μl
PrimerScript ThermoScript II mixed solution: 1 μ l
Oligo dT and random six mers primer (50 μMs): 1 μ l
Total serum IgE: 1 μ g
Remove the water of RNA enzyme: up to20 μ l
Reverse transcription reaction condition:
Temperature 37 DEG C, time 30min; Temperature 85 DEG C, time 5s.
With cDNA under each stress conditions of fluorescent quantitation primer amplification of each reference gene:
The instrument of fluorescent quantitation is Bio-rad CFX96., and that real-time fluorescence quantitative PCR adopts is the SYBRPremix Ex Taq of TAKARA tM, reaction system is as follows:
SYBR Premix Ex TaqⅡ(2ⅹ):2μl
PCR forward primer (10 μMs): 0.4 μ l
PCR reverse primer (10 μMs): 0.4 μ l
CDNA solution: 2 μ l
Water: 12.2 μ l
Cumulative volume: 20 μ l
Response procedures is:
The first step: denaturation;
Cycle number 1, temperature 95 DEG C, 30 seconds time;
Second step: PCR reacts;
Cycle number 40, sex change: temperature 95 DEG C, 5 seconds time; Annealing: temperature 58-60 DEG C, 30 seconds time
3rd step: solubility curve makes;
Temperature 65 DEG C-95 DEG C (30s progressively rises to 95 DEG C), collect first order fluorescence signal for every 0.5 DEG C, the Cq mean value of all reference genes that real-time fluorescence quantitative PCR obtains is in table 3 and Fig. 3:
Table 3
As shown in table 3 Fig. 3,15 reference genes differ greatly totally at 10 kinds of abiotic stress and the relative expression quantity under contrast 11 kinds of experiment conditions.In all internal references, 18S gene expression abundance the highest (Cq value is less than normal), ARP and UBR1 gene expression abundance very low (Cq value is bigger than normal).The Cq of other internal references, all between 20-28, meets the requirement of the most suitable Cq value of internal reference in quantitative examination scope.
GeNorm software carries out the real-time fluorescence quantitative PCR data analysis of candidate's reference molecules:
Data analysis adopts Excel2003 and geNorm program to carry out, from http://medgen.ugent.be/jvdesomp/genorm/ website, download geNorm program, with geNorm software carry out internal reference stability and internal reference number analyze time, its step and standard as follows:
GeNorm should be consistent in all samples to the ratio that the principle that reference gene carries out estimation of stability foundation is two desirable reference gene expression levels, no matter at which kind of experiment condition or cell type.The method is not subject to the impact of gene expression abundance difference and extreme ratio between gene, and the increase of ratio degree of variation means the reduction of one or full gene expression stability in the two.; By calculating the evaluation index of average variation degree M as gene stability of the Logarithm conversion value of ratio between two between a certain gene and other all genes: the cycle threshold (Cq) first obtained by quantitative fluorescent PCR is converted to relative quantification data, method finds the gene (expression amount is the highest) of minimum Cq value, minimum Cq value is deducted by the Cq value of every other gene, obtain △ Cq, its value>=0, each gene is 2 relative to the relative expression quantity of most high expression level gene -△ Cq.Then make Excel form, column heading is sample title, and rower is entitled as Gene Name, will calculate 2 of each each gene of sample -△ Cqnumerical value List of input.Under this file is saved in geNorm installation folder, close all Excel programs.Start geNorm program, automatic analysis button is clicked according to operation instructions, program provides M value broken line graph automatically, the figure illustrates the average expression stability numerical value of each reference gene of each step in the reference gene process progressively removing least stably express, M value is less, genetic expression is more stable, and this figure sorts to each house-keeping gene stability.Again click automatic analysis button, to provide pairing analysis of variance Vn/n+1 result figure, the figure illustrates the number needing to select reference gene, suggestion V<0.15 is for selecting threshold value, as V<0.15, show only to select 2 reference genes to make standardized index.Utilize this program investigator can screen multiple house-keeping genes of particular studies, select more than 2 house-keeping genes as internal reference, contribute to corrective system deviation to obtain more reliable result, this is for the accurate quantitative analysis of gene, and the gene expression research of especially tiny differential expression has extremely important value.
In the present invention, first geNorm program achieves by calculation expression stability mean value (M) sequence that 15 house-keeping genes express stability, the most stable internal reference of result display is CDPK and α-TUB2, that least stable is 18S gene (Fig. 4), the stability sequence of each reference molecules is as follows: calcium dependent protein kinases (CDPK)/α tubulin 2(α-tub2) >SAND albumen (SAND) > Actin muscle (ACT) > glyceraldehyde-3-phosphate dehydrogenase 2(GAPDH2) > beta tubulin (β-tub) > α tubulin 1(α-tub1) > ubiquitin protein ligase 2(UBR2) > H3 (HIS3) > glyceraldehyde-3-phosphate dehydrogenase 1(GAPDH1) > actin associated protein (ARP) >F-box protein (F-BOX) > transcriptional elongation factor (SPT) > ubiquitin protein ligase 1(UBR1) >18s ribosome-RNA(rRNA) (18S), then geNorm calculates again pairing variation coefficient V, result obtains V2/3=0.139, <0.15(Fig. 5), therefore, for the quantitative examination of the red moss gametophyte of tooth rib under abiotic stress experiment condition, only need jointly to correct with two internal references just can obtain quantitative result accurately with stdn.These two internal references are the most stable calcium dependent protein kinases (CDPK) and α tubulin 2(α-tub2 respectively);
NormFinder software carries out the real-time fluorescence quantitative PCR data analysis of candidate's reference molecules:
NormFinder determines internal reference stability according to the variation of reference gene between group and in group, and pass judgment on the stability of reference gene with stability factor, this stability factor is less, shows that this reference gene is more stable.The data-switching of NormFinder is identical with geNorm, namely the cycle threshold Cq first quantitative fluorescent PCR obtained) be converted to relative quantification data, method finds the gene (expression amount is the highest) of minimum Cq value, minimum Cq value is deducted by the Cq value of every other gene, obtain △ Cq, its value>=0, each gene is 2 relative to the relative expression quantity of most high expression level gene -△ Cq.Then make Excel form, with geNorm algorithm unlike, the rower that NormFinder software data imports is entitled as sample ID, and column heading is Gene Name, will calculate 2 of each each gene of sample -△ Cqnumerical value List of input.Relative quantification data importing NormFinder software is carried out internal reference stability analysis.
In the present invention, the internal reference stability result that NormFinder analyzes is shown in Fig. 6, predict the outcome consistent with geNorm, after NormFinder analyzes, the most stable reference gene remains α tubulin 2(α-tub2), next is calcium dependent protein kinases (CDPK), and that least stable is 18s ribosome-RNA(rRNA) (18S) (Fig. 6).The stability of the specific order for: alpha tubulin 2 (alpha tub2) & gt;Calcium dependent protein kinase (CDPK) & gt;Beta tubulin (beta tub) & gt;Glyceraldehyde 2-3 - phosphate dehydrogenase (GAPDH2) & gt;SAND protein (SAND) & gt;Actin (ACT) & gt;Ubiquitin protein ligase (UBR2 2) & gt;Alpha microtubule protein 1 (alpha tub1) & gt;Histone 3 (HIS3) & gt;Glyceraldehyde 1-3 - phosphate dehydrogenase (GAPDH1) & gt;Extension factor (SPT) & gt;Actin related protein (ARP) & gt;F - BOX protein (F - BOX) & gt;Ubiquitin protein ligase (1) UBR1 & gt;18 s ribosomal RNA (18 s);
Refinder software carries out the real-time fluorescence quantitative PCR data analysis of candidate's reference molecules:
Refinder software is the online software (http://www.leonxie.com/referencegene.php) of a comprehensive analyzing evaluation internal reference stability, it incorporates above geNorm, the algorithm of the different softwares such as NormFinder, comprehensively analyzes the stability of internal reference and sorts.According to the sequence that each algorithm obtains, Refinder calculates the suitable weight (appropriate weight) of each gene, and the geometric mean then by calculating each weight determines the eventual stabilities sequence of each gene.After Refinder comprehensive sequencing results are as follows: alpha microtubule protein 2 (alpha tub2) & gt;Calcium dependent protein kinase (CDPK) & gt;Beta tubulin (beta tub) & gt;Glyceraldehyde 2-3 - phosphate dehydrogenase (GAPDH2) & gt;SAND protein (SAND) & gt;Actin related protein (ARP) & gt;Alpha microtubule protein 1 (alpha tub1) & gt;Ubiquitin protein ligase (UBR2 2) & gt;Glyceraldehyde 1-3 - phosphate dehydrogenase (GAPDH1) & gt;Histone 3 (HIS3) & gt;F - BOX protein (F - BOX) & gt;Extension factor (SPT) & gt;Ubiquitin protein ligase (1) UBR1 & gt;18 s ribosomal RNA (18) s (figure 7).The comprehensive stability ranking results that Refinder provides is very similar to geNorm with NormFinder.And the most stable two internal references are also α tubulin 2(α-tub2) and calcium dependent protein kinases (CDPK), that least stable is 18s ribosome-RNA(rRNA) (18S);
Comprehensive improvement is sorted in table 4 to the stability of three fatware:
(table 4)
Show from table 4 result: although the algorithm of three softwares is different, but the internal reference stability obtained sequence is very consistent, also illustrate that the reliability of this experimental result, i.e. calcium dependent protein kinases (CDPK) and α tubulin 2(α-tub2) be the most stable two, next is beta tubulin (β-tub), Actin muscle (ACT) and glyceraldehyde-3-phosphate dehydrogenase 2(GAPDH2), and least stable be 18s ribosome-RNA(rRNA) (18S) always, ubiquitin protein ligase 1(UBR1), transcriptional elongation factor (SPT), actin associated protein (ARP) and F-box protein (F-BOX), the stability of other gene is placed in the middle.
Denomination of invention: the screening method of desert tooth rib red moss real-time fluorescence quantitative PCR reference molecules
Applicant: Xinjiang Inst. of Ecology and Geography, Chinese Academy of Sciences
The nucleotide sequence of 15 reference genes and 15 of design pairs of real-time fluorescence quantitative PCR primer sequences are:
The red moss of > sequence 1 tooth rib syntrichiacaninervisactin (ACT) nucleotide sequence
ATGGCTGATGCTGAGGATGTCCAGCCTTTGGTGTGCGACAATGGATCGGGGATGGTTAAGGCCGGATTCGCCGGAGATGACGCCCCGCGTGCCGTATTTCCCAGCATTGTTGGGCGCCCGAGACACACCGGTGTGATGGTGGGCATGGGACAGAAAGACGCGTATGTGGGCGACGAGGCGCAGTCCAAGAGGGGTATCCTGACGCTGAAGTACCCGATCGAGCACGGCGTGGTCACCAACTGGGACGACATGGAGAAGATCTGGCACCACACCTTCTACAACGAGCTGCGTGTGGCCCCCGAGGAGCACCCGGTGCTGCTCACGGAGGCGCCTCTCAATCCCAAGGCCAACAGGGAGAAGATGACGCAGATCATGTTCGAGACCTTCAACGTGCCGGCCATGTACGTGGCCATCCAGGCGGTGCTGTCGCTGTACGCCAGTGGGCGAACCACCGGAATTGTGCTGGATAGTGGAGACGGTGTGACTCACACAGTGCCCATCTATGAGGGGTACGCCTTGCCTCACGCCATTCTGCGGCTG
GACTTGGCCGGTCGCGACTTGACGGACGCGCTGATGAAGATTCTGACGGAGCGTGGTTACTCGTTCACCACCACGGCCGAGCGCGAGATCGTGCGTGACATGAAGGAGAAGCTGGCGTACGTGGCAATCGACTTTGAGCAGGAGCTCGACACAGCGCGCTCATCGTCCTCGCTGGAGAAGAGCTACGAGCTGCCGGACGGGCAGGTGATCACGATCGGCGCGGAGCGGTTCAGGTGCCCGGAGGTGCTGTTCAACCCGTCGCTGATCGGGATGGAAGCTGCGGGCATTCACGAGACCACTTACAACTCGATCATGAAGTGTGACGTCGATATCCGTAAAGACCTGTACGGAAACATCGTGTTGTCTGGAGGATCGACCATGTTCCCGGGCATCGCCGACCGTATGAGCAAGGAGATCACTGCTCTTGCGCCCAGCAGCATGAAGATCAAGGTGGTTGCGCCGCCGGAGAGGAAGTACAGTGTCTGGATCGGAGGATCCATCTTGGCATCTCTCAGCACCTTCCAGCAGATGTGGATTGCG
AAGAGCGAGTACGATGAGTCCGGTCCATCGATTGTGCACAGGAAGTGCTTT
Real-time fluorescence quantitative PCR primer sequence: forward primer: TCGTGTTGTCTGGAGGATCG
Reverse primer: TACTCGCTCTTCGCAATCCA
 
The red moss of > sequence 2 tooth rib syntrichiacaninervisactin associated protein (ARP) nucleotide sequence
GTGAGCCATGAGTTGAAACTAATAAATTGGAAAGAAAAACAAGCTCAGAAAATGAGCGTACATCGTTGAGACGGTCAGACGAACCATTATCTACGGACATGCGTATTGCGTTGTTCAGGAATGTTCTTTGCGTGGAACTCGGTGTCGGTAACTGGAAGATGTTGACGCAGTTGCTGCTGTTACCTTGCTGACCAAGTTTAGTCCAACATGGCATTGTACCAAAAAGCTTGCGCCTGGAGGGTGTTGGAGTCCCTGTACTTTCGGCCGCTGCCCACCATGACCATACCGTCCAACCAGTCCTCGTACTGCACCCATGAGTCGCGTCCGAAGTCCAAGACGGCGAGAACCGCGCCACCTTTCCACATGATATTCATTGGAGCCATTCGATTTTGAAGAACCTCCACAGTGTCCACAGCTTCGTCCACAGGTATGGCGTGCAGAACCCTCTCCTCAACAGCATCAACCAGGCCTTTCGTTAACCCAACGCCGCCAACCAGCTGAATACTCGCGAACAGCTTCTTCTGAAGATCCACGCGACCTAACGACAGGATACTTTTCACAATAGCTTGTGCAAGGCCACTTGAAGCTTCTTCCACCTCTTTCTTCCCTTTATCTTCAAAGTCGTACGGATCGGAATCATTGTTAATAGAGTTTCCATTAACACTTCCGTTGGTTCCTCCTGTTTCATTTGTCTCAATACGTCGGACAGCTTCAGAAAATGAATCTTCCGCAGTATCTTCATAATCTACGTGATACCAGGGTCGAGGAAGAATCGAATACTCTTCCAGTGCAAGTAGCGAAGGGTAAAATAGTCCCATGGGTGGAATGTTGATGGAAGATAATACGACTGTATATACTCGAGTTGGCTCGCCTGCCATGCGGCACTTGATATCAACCGTCGTGTGTTGCTCACCTTCCAGAAGCACGCAGTGGGTCTCCTTCAGTTTCTCAAGAGTCAAAAAGTCCAACGGATCACGCAAAGGATCAGTATCAGCCAGGGGCCATGTCTTCTTTCGTTGTTGGACCCATAACAAACACCTAGTGATGTCATCTCCACCATACGGCAGCAGAAAACGTGTTGATGGAATAGCAACCCCTTCCTCAACACACATGCCTGAGATAACTTGTGCACCCATGTTTACGATACATCCAGATGAAAAGCCATTCCCAAATGTAGCCGCAACGCATTCCTGGA
Real-time fluorescence quantitative PCR primer sequence: forward primer: GCAAGTAGCGAAGGGTAAA
Reverse primer: TGCCGTATGGTGGAGATG
 
The red moss of > sequence 3 tooth rib syntrichiacaninervistranscriptional elongation factor (SPT) nucleotide sequence
TTGAAGGTCTTTCTGATATAATCTTCCTTCTTTAAGCGAGTCTTTTCCTGCTCGTTGAGTAAGAAGGTGATGTCGGATTTAAAACGCTTGTCCCGGTGCTGCGTATCATCCCTAGACTCTTCCCGCATATCGCTTTCCTTGCATGTCAAATCGACCAAGTACTTATCCTTTTTCACCTCTTTCACTCGGCAAGTCACTGAAGTGCCTTCTGAAAATTTATCTTTAAGTTCAACATCGCCGTCATCTGAAACATCTTCCTTCTGAATGAAACCCAGTAATCCACATTCCAATACACACATGAGCTGATGTTCCTGCACTCTTCTTATTGTTGCTTGTACAATTCCACCTTCAAAGAGTGTCTCTTTGCTCTCCCCGGTCATGAGATAGAAACACTCTTCTTGAGACGGCTCCGAGTACGACCTCCGCCATTCTTGAAATCCTCGCTGCAGCTCAGATTTGATCAACTCCAGTGTCTTCAATCCCCGGCCTTCAACGTGTGCAGATTGGGCATACTCCGCAATGTCTACAGAATCTAAAGCGTTGGGATTTTTAATCACACGATCTACTGCCATACTTGGCACATCATCGTCCACTTCTTCGTTATTCCGCATGCCTTCCTTCTCGATCGCAGCTTCAGCTAGGTTCTTTGCCATCTGATACAACTCAGGGTGAATACGGGTGTTGTCTAAAAGCTCTGTAGGACGTAAATCTGAAGGTGTTTCTCCAATTTCACGCACTCGGATGAAACCAGCGGAATTTGTGAAGACTTTCTCATTCACAGCCCGTATGGTCGAGTAAAGACTATCTCGAGTCAATACACGCTTTACCACTTGAAGATTTGTCCGAAACTCACAAGCCTTCAGAGGGCCAAGACCCGCAACGAACTGAAGGGTCTCAAACAACCAGTCGTGGATAATAGCTAGA
Real-time fluorescence quantitative PCR primer sequence: forward primer: ACTTGGCACATCATCGTCC
Reverse primer: ATTCCGCTGGTTTCATCC
 
The red moss of > sequence 4 tooth rib syntrichiacaninerviscalcium dependent protein kinases (CDPK) nucleotide sequence
TGAGATGTTTAAACACTACCTCAATTACATGCTGAAATCCAATACATTTGACGTAGAAGCATCAACAGTGTTTGGCACCTAATCATTTCCTCTCGGGCGCCTCTAGCGATGGCACTGAAAATGATACACGGGGACTAAAGGCCATGGCCCTAGAGCAAGTAACATGACAAACTTTACATATTCAAACTCCCCATGGTAAGGAGAGAGTGCTACTCGCCTCTGTAGAAGTGACTGGTTCGCACAGCCATGTTGAGGTGCCTACTTCCTGCTGAACTCGGTGTGAACCCTATCCTCCGATCCCCAACGCGTCGGTCAGATTCAACTGCTGGCTGTTGCGTAGCGATGAGCGACCAGCACCGCCCTTGTTGCCCTTGCGCATCATGTTGACGAACTCGGTGTAATCAATCTGGCCATCGTTGTTCTGGTCGACCTCCCTGAGCAGCTCCTCCACGGATACGTCTCCCATGTTGAATTCGGCCAACGCATGTTGCAGCTCATCGACCGTCAGATAGCCGCTGTTGTCCTTGTCTAGGTACGAGAAGGCGGCGAACATGTTCTCATCGCGTTCGACCTTGTTCAGGTGGAGAGTGGCGGCCAGAAACTCGCCATAATCGATTGCCCCACTGCCGTCGACGTCAGCGGCGTCCATGAGCTGCCGAACGTCTTCCTCTTTGAGATTGGAACCCACTTTCTTCAGACCCGCCTTCAGCTCCTCGTAGCTGATGGACCCTGAGTTGTCCGTGTCCATCATCTTGAACATCTCCTTCAGACCTGCGATCTCCTCCTCCGACAAGGACTCCGCGATCACCCGGATGGCGATCTTCTTCAGCTTGTTCATTGCGGAGAAGTGCTTGAGCCGGGATTGCACTGCGGAATCGATGGGTCTGTCGGGCGCTACTCCGTCCTCGCAGATCCAAGGATGTGCCAACACTTGATGGGCTTTCAACCTCTTCGAAGCCTGGGGCGTGAGCATTTTGCGGATCAAATCCTTGGCGCTATCAGAAATCTTAGGCCATGGATCTGATGCAAAATCAAGCTCTGATTTCAAAACCTGCTCAAAGATGCCCTGCTCCGTCTCTGCCCAGAATGGAGGGACTCCGCTGAGAAGGATGTAAAGTATCACACCCGCACTCCACACATCCGCTTCCGGGCCATAGTTCTTCCTCAACACTTCAGGTGCCACATAGTAAGGGCTTCCAACCACATCTCTGAAAACTTCTCCAGGTTTAAAGAACACTGATAGCCCAAAATCAGTCGTTTTCAGAGCCGCATCCTCCTTCGTACTCGAAAACAGGAAGTTCTCCGGCTTCAAATCTCTGTGCATCACGCCAAGAGAATGGCAGGTCTCAACCACGCCCACGATAGTACGAGTGAGCACTGCCGCTTTGGCTTCCGAGTACTGTCCTCGTTGAATGATGCGGTCAAACAGCTCACCGCCTGCACAAAGCTCCATCACCAGATGCACTGAAGCAGGATCCTCATACGCCCCCTTGATCATGACAATGTTCGGATGTCCTGAAAGGTGATGCATGATCTGCAGTTCACGGCGAACGTCATCCACGTCTTCCTTGGAGATAAGCTTCCGCTTGGCAATAGACTTGCATGCGTACTCCCTGTTGGTGGCCTTCTCAATGCACAGGTAGGTGGTGCCGAATTGACCTTGCCCCAGCTTCTTTCCCAGAACATACAGGTCTCTGAGGTTCTCTGTCTTCCGCTGCAGCACAGAGTGAGTCAGACTCTGGCCAGGGACGGGCTTCATCTCTCTCCCTTTCTTCATGGCTTCGATCACTTCTTTGAATTGCTGAGTTTCGGCCGGTTCCACAGCAGGGATGTGAGCTGGGCGTGGCGGTTTCCGTTGAGTCTCCGGGGACTCCGCCTCTGCCCTGCTCCCCCCTCTGGACTCTGCCTTGGATGGAGTGGGTGATACCTCACTAGGGGATCTGGAGCTACGCCCTCCCAGTGCGATCACACTGGTGAAGCCCTGAAAGTACCCAGCCGCTCCTACACAACTGTTGCCCATTCAGACACAATCTGTAACATCCCTCCACGGGAATCTATTTATCCCGTTCCCCTAAACTTGTCTCTTAAAGCAAAGGCCTAAACATACACTACAACGAACTTGATGCCGTTTTTGTCACCATTTGGCCACGTACCTTCGCACAATCATCTGAAGTAAGAACAGGCTGAGGAGAAAGACCGCCGTCTAGGGTTTCAAGGCTGAGAAGCGAACCCTGAAATGAGTGTAAGGTGGCCGAAACTGACTTCAAAGAGAGAACTGGCGGCCAGGTTTCCTCAGTTATTCTTTCCAGTCTCAAACCTCGAGCTCCGGCTGCGAAATAATCTGGCAGCGAAAGTGGCAGCAAAAACAAGCAGCTCTCGCACTATTTTCC
Real-time fluorescence quantitative PCR primer sequence: forward primer: AACACTTCAGGTGCCACATAG
Reverse primer: GCATCATTCAACGAGGACAG
 
The red moss of > sequence 5 tooth rib syntrichiacaninervisα tubulin (α-TUB1) nucleotide sequence
GTGGATGTGACGGAGTTCCAGACGAACTTGGTGCCGTACCCCCGTATCCACTTCATGCTTTCGTCTTACGCGCCGGTGATCTCGGCCGAGAAGGCGTACCATGAGCAGCTGTCCGTCGCGGAGATCACCAACTCTGCGTTCGAGCCGTCGTCTATGATGGCGAAGTGCGACCCCCGTCACGGGAAGTACATGGCGTGCTGCCTGATGTACAGAGGAGACGTTGTGCCGAAGGACGTGAACGCGGCAGTGGCAACGATCAAGACGAAGAGGACGATCCAGTTCGTGGACTGGTGCCCGACCGGGTTCAAGTGCGGAATCAACTACCAGCCCCCGACGGTGGTGCCCGGAGGCGACCTGGCGAAGGTGCAGCGGGCGGTGTGCATGATCAGCAACAACACGGCGGTGGCGGAGGTGTTCTCCCGGATCGACCACAAGTTCGATCTCATGTACGCGAAGCGCGCGTTCGTGCACTGGTACGTGGGCGAGGGCATGGAAGAGGGTGAGTTTTCGGAGGCCCGTGAGGACTTGGCTGCGCTCGAGAAGGACTACGAAGAAGTGGGCGCGGAGTCGACGGACGGCGCCAACGACGACGATGACTACReal-time fluorescent quantitative PCR primers sequence: forward primer: CGTCGTCTATGATGGCGAAGT
Reverse primer: TCTTGATCGTTGCCACTGCC
 
The red moss of > sequence 6 tooth rib syntrichiacaninervisα tubulin 2 (α-TUB2) nucleotide sequence
ATGAGAGAGATCATCAGTATCCACATCGGCCAGGCCGGTATCCAGGTCGGCAACGCCTGCTGGGAGCTCTACTGCCTCGAGCACGGCATCCAGCCCGATGGCCGCATGCCTAGTGACGCGTCTACAGGAGGTGGAGACGATGCGTTCAACACCTTCTTCAGCGAGACCGGGGCGGGGAAGCACGTGCCGCGGGCTGTGTTTGTGGATCTGGAGCCGACCGTGATCGATGAGGTGCGCACGGGGACGTACCGGCAGCTGTTTCACCCGGAGCAGCTTATTTCCGGGAAGGAGGACGCCGCTAACAACTTCGCTCGCGGTCATTACACCGTGGGAAAGGAGATTGTGGACTTGTGCTTGGACCGGGTGAGGAAGCTGGCAGACAACTGCACGGGCCTGCAGGGATTTTTGGTATTCAATGCTGTGGGAGGAGGCACGGGATCGGGACTCGGTTCGCTGTTGCTGGAGAGGCTCTCAGTAGACTACGGAAAGAAGTCCAAGTTGGGATTCACCATCTACCCGTCTCCTCAGGTGTCCACCGCAGTTGTGGAGCCGTACAACAGTGTGCTGTCGACTCACAGTCTGTTGGAGCACACCGATGTGGCGGTGTTGCTGGACAATGAGGCCATTTACGACATCTGCCGGCGCTCGTTGGACATCGAGCGGCCGACGTACACGAACCTGAACAGGCTGATCTCGCAGATCATTTCGAGTCTGACGACGAGCCTGCGGTTTGACGGCGCGCTGAACGTGGACATCACTGAGTTCCAGACCAACTTGGTGCCG
Real-time fluorescence quantitative PCR primer sequence: forward primer: CGGTCATTACACCGTGGGAA
Reverse primer: CCTCTCCAGCAACAGCGAA
 
> sequence 7 syntrichia caninervisbeta tubulin (β-TUB) nucleotide sequence
ATGAGAGAGATCCTCCACATCCAGGGCGGGCAGTGCGGGAACCAAATCGGTTCCAAGTTCTGGGAGGTGGTGTGCGAGGAGCACGGGATTGACCCCACCGGCTCGTACCAGGGGGTGTCTGACCTGCAGCTCGAGCGCATCAATGTGTACTTTGATGAGGCCAGCGGCGGGCGCTACGTGCCGCGCGCGGTGCTCATGGATCTGGAGCCCGGCACCATGGACAGCGTCCGCACTGGCCCCTACGGCCAGATCTTCCGCCCCGACAACTTCGTCTTCGGACAGACCGGCGCTGGTAACAACTGGGCCAAGGGCCACTACACCGAGGGCGCGGAGCTCATCGACTCAGTGCTGGACGTCGTGCGCAAGGAGGTTGAGAGCTGCGACTGCCTGCAAGGCTTCCAGTTCTGCCATTCTTTGGGAGGAGGAACGGGATCTGGAATGGGAACGCTTTTGATATCTAAGATCCGCGAGGAGTACCCCGATAGAATGATGCTTACGTTTTCCGTGTTCCCCTCTCCGAAGGTGTCCGACACCGTGGTGGAGCCTTACAATGCTACTCTCTCAGTGCATCAGCTGGTGGAGAATGCCGACGAGTGTATGGTGTTGGATAACGAGGCGCTGTACGACATCTGCTTTAGGACTTTGAAGCTGATCACCCCCTCATTTGGAGACTTGAACCATTTGATTTCTGCGACGATGAGTGGTATCACCTGTTGCCTGCGTTTCCCCGGACAGCTGAACTCTGACTTGCGGAAGCTGGCGGTGAACCTCATCCCGTTCCCCCGGTTGCACTTCTTCATGATCGGTTTCGCGCCTTTGACCTCCCGTGGCTCACAGCAGTACCGGTCGCTCACCGTGCCGGAGCTGACGCAGCAGATGTGGGACTCAAAGAACATGATGTGCGCGGCGGATCCGCGTCACGGGCGCTACTTGACGGCTTCCGCGGTGTTCCGCGGCAAGGTGTCCACCAAGGAGGTCGACGAGCAAATGATCAACGTGCAGAACAAGAACTCATCCTATTTCGTTGAGTGGATCCCGAACAACGTGAAGTCGTCTGTCTGCGACATCCCCCCGACGGGTCTGAAGATGTCGTCGACGTTCATCGGGAACTCGACGTCCATCCAGGAAATGTTCCGGCGCGTGTCGGAGCAGTTCACGGCCATGTTCAGGAGGAAGGCGTTTCTGCATTGGTACACGGGCGAGGGCATGGACGAGATGGAGTTCACAGAGGCGGAGAGCAACATGAACGATCTGGTGTCGGAGTACCAGCAGTACCAGGACGCGAGCGCCGAAGAGGAAGGCGAGTACGAGGACGACCTTGAGGAGGCC
Real-time fluorescence quantitative PCR primer sequence: forward primer: CTTTGACCTCCCGTGGCTC
Reverse primer: GTTCGGGATCCACTCAACGA
 
The red moss of > sequence 8 tooth rib syntrichia caninervisubiquitin protein ligase 1 (UBR1) nucleotide sequence
GCGCTTGTGAACGAGCTCATCACCAAGTTGTCCAGCCGGAGCACCAAGGTACATCGGGATGCTGCCAAGACCATAAGGTGCCTAGCGAAGATAAACAAAGAGAATCGAATTCTTATTGCGGAGCAGGGAGGCATACCTTTCTTGATCAACCTTCTCCGATCGCCTGATGAGGAGACGCAGGAGCACGCCATCACCGCGTTGATGAACCTGTCCTTGCATCCAAACAACAGAGGGCTCATCATGAGAGCAGGGGCCATAGACGGCATCGTTCACGTAGTGAAGCATGGCGAGAGCACGGACGCGCGCGAGAACGCAGCAGCAGCAATCCAGTGCCTCTCGTATGACAACGAGAACAAGATCTCCATCGGCAACACGGGTGCGATCCCCGCGCTCGTTCAGCTGCTGCGCACCGGGACTCGCCAGGGCAAGAAGGACGCGGCCCACGCGCTCTGCAACCTCGTCAGCTACCTGGACGGCAACAAGAAGCGCGCGGTGGACGCCGGCCTCACACCCCTCCTCATGGCGGTTCTGCGCGAC
Real-time fluorescence quantitative PCR primer sequence: forward primer: GCAGGGAGGCATACCTTTCTT
Reverse primer: ATGAGCCCTCTGTTGTTTGGA
 
The red moss of > sequence 9 tooth rib syntrichia caninervisubiquitin protein ligase 2 (UBR2) nucleotide sequence
ATGGGGTTAGGGTATGGGCCAGTGGTGGCATTGTACACGGCGTTGAGTGTGGGTGGGATAGCATGGAAGCTCAACTCAGCGTGGTGGACTATGACCTACGGCAAGGGTCAAGGAGGTGGTGGGCAGATGGATCCCTCCGAAGCGGCGCGCACGCTGCAGGCCATGCTGCAATCATCCGTCGTGGTGGCGCTCCTCGTCAACCTTCTCGTCAACTTTTTCCTTCTCGTCACCCTCTCAATGAAGACTGTGTTCTTCGGTCAGTTGTCTCTGGTTGAGACTCAGAAGGTGGTGGAGCGCCTCATCAATTATGTATTATTCAAGGGCCTGTTCTTGACATGGGTCGTGAGGCCAGAAATGATGCAGATAGCTGTGTGGTTGGCGTGGTTCTCCGTCTTGGGCTTTCTGAAGATGTTCCAAGGTTTGGCAAGAGATCGTTTGGAGCGCTTAAATGCATCTCCTTCTGCGACAATGTTGGCTCACATAAGAGTTTATACGGTTCTCTTGTTCGTTCTTCTGTTCGATTTGCTCTGGATGCAGCTGTGTCTGCTTTTATTCAAAGACACTGGAACCAGTACCTTCTTGCTACTTTTGTTTGAGCCTCTCAGTATAGCATTTGACACTCTTCAGGCAGTAATTGTACACGGTATGCAGCTATTGGACACTTGGCAGCGGCAAAACCTTGATATTTCTGCACACGATATAAGTTTGCAGCCTTCTGAGAGGTCTGCTGCAGGTGCAGCTTGGGAGTGGCGTGGAACTGTTGTAAGAAATTGCAGCTTTGTGATGGATATTGTGAGCTTGTTACTGGCTTTGGGCCATTGCGTACACATCTGGTGGTTACGTGGGCTTGCTTTTCAAGTGGTGGATGCCATTCTTTTCCTCAACTTGCGCGCGCTATTGAGTGCTATTTCAAAAAGAATAAAAGGTTTCATGCGTCTTCGGGCAGCCATGACAACTCTGCAGGGTGCGCTTCCCGATGCAACGCAGGATGAACTCCTTGGATATGAAGATGATTGTGCCATTTGCAAGGAGCCCATGGCTAGGGCAAAACGGCTTCCTTGTGCTCATCTCTTCCATCTCGCGTGCTTGAGATCCTGGCTAGACCAAGGATTAGCAGACACATACTCTTGCCCGACCTGCCGTAGACCCCTCTTCATGGGCGGGTTCCGGACTGCAACAAACAATTTGCAGCGCCTGGTAGATGTGCCCACTGCATCCCCCGGCACAAATGAAACTCAGGCACAGGTTGCACAACGACAAGACTTAGCTGCAGCGCCGTCTCCACCTCTCACCACCTTGACTCCCGCTCCAGCACCG
Real-time fluorescence quantitative PCR primer sequence: forward primer: AGCTTGTTACTGGCTTTGGG
Reverse primer: GGAGTTCATCCTGCGTTGC
 
The red moss of > sequence 10 tooth rib syntrichia caninervisglyceraldehyde-3-phosphate dehydrogenase 1 (GAPDH1) nucleotide sequence
TCCGGGATCAAGAACACCAATGCCGTCACCTTCGGCAAGCGCACTGATGACCTCGTCAGCGTTGTTGCTGCCCAGTCTGCTGTGTCTGCTAGCTCCGGAGCCACGAGGGCCGTGACCGAGGCCAAGATCAGGGTTGCCATTAACGGTTTTGGGCGTATTGGACGCAACTTCGTCCGCTGCTGGCATGGAAGGAAGGACTCGCCTCTCGAGGTCGTCGTGATCAACGACACCGGAGGCGTGAAGCAGGCTTCTCACCTGTTGAAGTACGACTCCATGCTCGGCACCTTCAATGCCGACGTGAAGGTCGACGGTGATGGCATCAGCGTTGATGGCAAGACCATCAAGGTGGTGTCCGACAGGAACCCGCTCAACTTGCCATGGGGGGATATGGACATCGACCTTGTCATTGAGGGGACCGGAGTGTTCGTCGACGAGGCTGGTGCCGGCAAGCACTTGCAGGCGGGAGCGAAGAAGGTGCTCATCACCGCCCCAGGAAAGGGTGCCATCCCCACGTACGTGATCGGAGTAAACGAGCAGGATTACACCCACGCGGACAAGATCATCAGCAACGCGTCGTGCACCACCAACTGCCTGGCCCCGTTCGTGAAGGTGTTGGACGAGAAGTTCGGCATCATCAAGGGTACCATGACCACCACCCACTCATACACTGGTGACCAGAGGCTGCTGGACGCTTCCCACCGCGACTTGAGGAGAGCCCGTGCCGCGGCCCTGAACATCGTGCCCACCTCCACGGGAGCCGCCAAGGCCGTGGCGCTGGTGCTGCCGAGGCTGAAGGGCAAGATCAACGGAATCGCCCTGCGTGTGCCCACCCCCAACGTGTCCGTGGTGGACTTGGTGGTGCAGGTGGAGAAGAAGACGTTCGCGGAGGAGGTGAACCAGGCGTTCAGGGATGCGTGCGAGACCCCTCAGCTGAAGGGCATTTTGGCAGTGTGCGACGAGCCTTTGGTGTCGGTGGACTTCAGGTGCACTGACGTGTCGTCCACGGTGGACTCGTCCCTGACCATGGTGATGGGCGACGACATGGTGAAGGTTGTGGCATGGTACGACAACGAGTGGGGGTACTCGCAGCGGGTGGTGGACCTGGCTAACTTGGTAGCGGAGAAGTGGCAG
Real-time fluorescence quantitative PCR primer sequence: forward primer: GCGTTGTTGCTGCCCAGTC
Reverse primer: AGGCGAGTCCTTCCTTCCAT
 
The red moss of > sequence 11 tooth rib syntrichia caninervisglyceraldehyde-3-phosphate dehydrogenase 2 (GAPDH2) nucleotide sequence
ATCGGAAGACTCGTCCTCCGTGTGATTCTTGCCAGGGACGATGTGGAAGTTGTTGCCGTCAACGACCCCTTCATTGACACCAAGTACATGGCATACCTGTTGAAGTACGATTCCACCCACGGGCTTCTCAAGGCTGATGTGCACGCTGTTGATGACAAGACTCTGCAAGTTGCTTCTCAAACCATCAAGGTCTTCGGATGCAGGGATCCCGCGGATATCCCATGGGGTGAAGCCGGTGCTGATTACGTGGTGGAGTCCACCGGTGTTTTCACCGCCGTTGCCGGCGCCTCGGCCCATTTCAAGGGAGGAGCCAAGAAGGTTGTTATTTCCGCACCATCGGGAGATGCCCCCATGTTCGTCATGGGTGTGAACCAGAACGCTTACAAGGATGACATGAACGTCGTCTCCAATGCCAGCTGCACCACCAACTGCCTTGCTCCCCTAGCTAAGGTGATCCAGGACGAGTTCGGCATTCTCGAGGGTCTGATGACCACCGTTCACGCCACTACCGCCACCCAGAAGACCGTCGATGGCCCATCAGGGAAGGACTGGCGTGGAGGACGTGGTGCTGGCCAGAACATCATCCCCAGCTCCACCGGCGCTGCCAAGGCTGTGGGCAAGGTGTTGCCTGAGCTGAACGGCAAGTTGACCGGCATGGCGTTCCGTGTGCCCACCCCGAACGTTTCAGTGGTGGACTTGACTGTGCGCCTGGAGAAGGGTGGGTCCTACGAGGACGTCAAGGCAGCCATCAAGGCCGCCTCTGAAGGACCGATGCATGGCATCTTGGGCTACACCGAAGACGACGTGGTGTCGAACGACTTCATCGGTGACGCCAGGTCCAGCATCTTCGATGCTAAGGCAGGAATCGCTTTGAACGACAAGTTCGTCAAGTTGGTGTCGTGGTATGACAACGAGTGGGGCTACAGCAACCGTGTGGTGGACCTG
Real-time fluorescence quantitative PCR primer sequence: forward primer: GGGCTTCTCAAGGCTGATGT
Reverse primer: TCCACCACGTAATCAGCACC
 
The red moss of > sequence 12 tooth rib syntrichia caninervissAND albumen (SAND) nucleotide sequence
TCGGAAGTGGAAGCTGATAATACAGCGAAACTCTCAAACCTGGACATCAGTAGCAGCACGCAGGAGGACGGTTCAAAGCTTGAAACTCATGATGCAGAAGATGGTCCAAAAGCCTTGGACATGGGAGATGGAGAAGGTACTGCGCTCGCTGCTTCTACAACTACTGATGTTGTGCCGGAGAGTACTGTGGATGGAGATACATTGTCTGAAGATGGCATCCATGAGAGCCCTGTGAGTCCAAGCAGTAGCCGCGAAGGAACCAGTGGCTACTTAGCAGGCAGTGGCAGCAGTACCGCGAGTGTCTATAGCGCAAGTGGAGCAGATGAAAGTGCCGATCGAATTCTGAATCAGGATTCTATGCCACAAGCGACGAGGCGGTCTTGGGTCCCTGGAAAACGCCATCCAGATGAAGATGACACGTCTCTCGCTTGGCGAAAGCACAAGAAGCATTTCTTTATTCTCAGCAATGCAGGAAAACCA
Real-time fluorescence quantitative PCR primer sequence: forward primer: AAAGCCTTGGACATGGGAGA
Reverse primer: CGTCGCTTGTGGCATAGAA
 
The red moss of > sequence 13 tooth rib syntrichia caninervish3 (HIS3) nucleotide sequence
CTAAAGACGGGAGAACGAAGACCCTTTTCTCCTCCTTCGGCTTCACCTCCCCATCTTTCTCTCTCGCTCACTCTCGACATCGTCATCTCTCTCGTATTCTACCTACACCGCCGTTTCATTTCCACAAGTGTCACTATGGCGCGTACGAAGCAGACGGCTCGTAAGTCCACCGGAGGAAAGGCTCCGAGGAAGCAGCTCGCCACGAAGGCTGCGAGGAAGTCGGCCCCCACCACCGGAGGAGTGAAGAAGCCCCATCGCTACAGGCCGGGAACCGTCGCTCTTCGTGAGATCCGCAAGTACCAGAAGAGTACCGAGTTGCTGATCCGCAAGCTGCCCTTCCAGAGGCTGGTTCGTGAGATCGCCCAGGACTTCAAGACCGATCTGCGTTTCCAGAGCCACGCCGTGCTGGCGCTGCAGGAGGCGGCGGAGGCGTACCTGGTGGGTCTGTTCGAGGACACCAACTTGTGTGCCATCCACGCGAAGAGGGTGACCATCATGCCGAAGGACATCCAGCTGGCCCGCAGAATCAGGGGAGAGAGGGCTTAGGCCGGCTCGCCCGGAGTCTGTATGAAAAAACCCAAAAAAATCTAAAAAAAAGTTTGGGGTTCTTTTGAACCCACCAAAGGGGCGTGTACTCCGTGTATGTGGCATGATGTTACTTGTATGTTACTTGTATGTTTGTGCGGAGTGATTGCTGAAGCAGCCGAG
Real-time fluorescence quantitative PCR primer sequence: forward primer: AGGAGTGAAGAAGCCCCATC
Reverse primer: CGAACAGACCCACCAGGTAC
 
The red moss of > sequence 14 tooth rib syntrichia caninervisf-box protein (F-BOX) nucleotide sequence
ATTCGTCTTTTTGTTTGGTTGCGTAAGTGTTTAGGTTGACACAATCCTCGTTTCGCTTCTAGGTTTTGTGTTTTGGCGTTTAAATCACTGAGGTTTCCCTGAAGACCTTTGATGCCATAGAGATGACGCTTTCCTTGTCCTGCTTACAGCTTGAGCTGGTGTGACAGTATCCGAATTTGCCCTAATTGGAGGGGCATGTGAAGCTCACGATCTGGGTCCGCTTACAAGCTCACAGCTTTAGTCAGTATTGACAACATGGGATGATCGTCTTGCAATCTCGAAGAAGCGATGGAGCAAGACGGATTGGATTCATGGAGCAGGCTACCGGAAGATGTCGTGCTGAGAGTGCTCACGCGCCTGCCAATTCCGCATCTTTTCCGCGCGCGCACGGTCTGCAAGCAATGGAATGCGCTCACTTCGACCCCGGAGTTTCTGGCCATGTGCAGCGCGCGTCACGCGCCCTACTTCCCGGTGGTGATCTCGCGCCGCTTCTACATGGGCGACCTGGGGATCTGCTTCCTGGGGAACGACTCGCAGGGCGGCAACGACCTCTTCCGCGTCTTCTTCGGCTACGACCATGAGGCGCAAGCGTGGCAGAAGCTGCCCCCGTTCGACTTCCTCCCGCACGAGGCGCGCATCCCGGTGGCCTCCGCCGGCGGCCTCATCTGCTTCCGCGGCACCTCCAGGCTCTTCCTGTGCAACCCCGTCGCCCGGAGCTTCCTGGAGCTCCCCGCCATCACGTACAAGTGG
CCGCCGTCCGTCTCCGTGCACATCCTCGTGGAGAGCAGCGCCTTCAAAGTCATCATCGTGGGGAAGATCCGCCACAACTTCGTCACCGACTCCTTCCGCAGCATCGCCATCTACGAGTCCGTCACCAAGGCCTGGCGCGTCGTGGACGCGCCGCATCCCGCGAAGGTATTCTCCTACGGTCCCACTGCAGCCGTCTGCGGCAACTCCATCTACTGTGAGGCGATTTGCCATTGCGGCCAGCTGGGTGTGATGGCCTACGACGTCCAGTCCGAGACGTGGAACCCGGTGTTGCATGAGATTCCGAGTGATGAACGCGGCGAGTATCAGCTGACGCAAGTTGTG
Real-time fluorescence quantitative PCR primer sequence: forward primer: CGCCTTCAAAGTCATCATCG
Reverse primer: GGCAAATCGCCTCACAGTAG
 
The red moss of > sequence 15 tooth rib syntrichia caninervis18s ribosome-RNA(rRNA) (18S) nucleotide sequence
TTACTATGGTGGTGACGGGTGACGGAGAATTAGGGTTCGATTCCGGAGAGGGAGCCTGAGAAACGGCTACCACATCCAAGGAAGGCAGCAGGCGCGCAAATTACCCAATCCTGACACGGGGAGGTAGTGACAATAAATAACAATACCGGGCTCTTTGAGTCTGGTAATTGGAATGAGTACAATCTAAATCCCTTAACGAGGATCCATTGGAGGGCAAGTCTGGTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTTAAGTTGTTGCAGTTAAAAAGCTCGTAGTTGGACTTTGGGTTGGGTCGGCCGGTCCGCCTCTGGTGTGCACCGGTCGTCTCGTCCCTTCTGCCGGCGATACGCTCCTGGTCTTAACTGGCCGGGTCGTGCCTCCGGCGCTGTTACTTTGAAGAAATTAGAGTGCTCAAAGCAAGCCTACGCTCTGGATACATTAGCATGGGATAACATCATAGGATTTCGGTCCA
Real-time fluorescence quantitative PCR primer sequence: forward primer: GGAGAGGGAGCCTGAGA
Reverse primer: CACCAGACTTGCCCTCCAA.

Claims (5)

1. a screening method for desert mosses tooth rib red moss real-time fluorescence quantitative PCR reference molecules, is characterized in that following these steps to carry out:
A, utilize tooth rib red moss transcript profile sequencing data, select 15 tooth rib red moss internal reference candidate genes, and based on select 15 red moss sequences of tooth rib, devise the reference gene primer of 15 pairs of real-time fluorescence quantitative PCRs, wherein the nucleotide sequence of 15 reference genes and 15 pairs of real-time fluorescence quantitative PCR primer sequences of design are:
Sequence 1 tooth rib red moss SyntrichiacaninervisActin nucleotide sequence
ATGGCTGATGCTGAGGATGTCCAGCCTTTGGTGTGCGACAATGGATCGGGGATGGTTAAGGCCGGATTCGCCGGAG
ATGACGCCCCGCGTGCCGTATTTCCCAGCATTGTTGGGCGCCCGAGACACACCGGTGTGATGGTGGGCATGGGACA
GAAAGACGCGTATGTGGGCGACGAGGCGCAGTCCAAGAGGGGTATCCTGACGCTGAAGTACCCGATCGAGCACGG
CGTGGTCACCAACTGGGACGACATGGAGAAGATCTGGCACCACACCTTCTACAACGAGCTGCGTGTGGCCCCCGA
GGAGCACCCGGTGCTGCTCACGGAGGCGCCTCTCAATCCCAAGGCCAACAGGGAGAAGATGACGCAGATCATGTT
CGAGACCTTCAACGTGCCGGCCATGTACGTGGCCATCCAGGCGGTGCTGTCGCTGTACGCCAGTGGGCGAACCACC
GGAATTGTGCTGGATAGTGGAGACGGTGTGACTCACACAGTGCCCATCTATGAGGGGTACGCCTTGCCTCACGCCA
TTCTGCGGCTG
GACTTGGCCGGTCGCGACTTGACGGACGCGCTGATGAAGATTCTGACGGAGCGTGGTTACTCGTTCACCACCACGG
CCGAGCGCGAGATCGTGCGTGACATGAAGGAGAAGCTGGCGTACGTGGCAATCGACTTTGAGCAGGAGCTCGACA
CAGCGCGCTCATCGTCCTCGCTGGAGAAGAGCTACGAGCTGCCGGACGGGCAGGTGATCACGATCGGCGCGGAGC
GGTTCAGGTGCCCGGAGGTGCTGTTCAACCCGTCGCTGATCGGGATGGAAGCTGCGGGCATTCACGAGACCACTTA
CAACTCGATCATGAAGTGTGACGTCGATATCCGTAAAGACCTGTACGGAAACATCGTGTTGTCTGGAGGATCGACCA
TGTTCCCGGGCATCGCCGACCGTATGAGCAAGGAGATCACTGCTCTTGCGCCCAGCAGCATGAAGATCAAGGTGGT
TGCGCCGCCGGAGAGGAAGTACAGTGTCTGGATCGGAGGATCCATCTTGGCATCTCTCAGCACCTTCCAGCAGATG
TGGATTGCG
AAGAGCGAGTACGATGAGTCCGGTCCATCGATTGTGCACAGGAAGTGCTTT
Real-time fluorescence quantitative PCR primer sequence: forward primer: TCGTGTTGTCTGGAGGATCG
Reverse primer: TACTCGCTCTTCGCAATCCA
Sequence 2 tooth rib red moss Syntrichiacaninervis actin associated protein nucleotide sequence
GTGAGCCATGAGTTGAAACTAATAAATTGGAAAGAAAAACAAGCTCAGAAAATGAGCGTACATCGTTGAGACGG
TCAGACGAACCATTATCTACGGACATGCGTATTGCGTTGTTCAGGAATGTTCTTTGCGTGGAACTCGGTGTCGGTA
ACTGGAAGATGTTGACGCAGTTGCTGCTGTTACCTTGCTGACCAAGTTTAGTCCAACATGGCATTGTACCAAAAA
GCTTGCGCCTGGAGGGTGTTGGAGTCCCTGTACTTTCGGCCGCTGCCCACCATGACCATACCGTCCAACCAGTCCT
CGTACTGCACCCATGAGTCGCGTCCGAAGTCCAAGACGGCGAGAACCGCGCCACCTTTCCACATGATATTCATTG
GAGCCATTCGATTTTGAAGAACCTCCACAGTGTCCACAGCTTCGTCCACAGGTATGGCGTGCAGAACCCTCTCCTC
AACAGCATCAACCAGGCCTTTCGTTAACCCAACGCCGCCAACCAGCTGAATACTCGCGAACAGCTTCTTCTGAAG
ATCCACGCGACCTAACGACAGGATACTTTTCACAATAGCTTGTGCAAGGCCACTTGAAGCTTCTTCCACCTCTTTC
TTCCCTTTATCTTCAAAGTCGTACGGATCGGAATCATTGTTAATAGAGTTTCCATTAACACTTCCGTTGGTTCCTCC
TGTTTCATTTGTCTCAATACGTCGGACAGCTTCAGAAAATGAATCTTCCGCAGTATCTTCATAATCTACGTGATAC
CAGGGTCGAGGAAGAATCGAATACTCTTCCAGTGCAAGTAGCGAAGGGTAAAATAGTCCCATGGGTGGAATGTT
GATGGAAGATAATACGACTGTATATACTCGAGTTGGCTCGCCTGCCATGCGGCACTTGATATCAACCGTCGTGTGT
TGCTCACCTTCCAGAAGCACGCAGTGGGTCTCCTTCAGTTTCTCAAGAGTCAAAAAGTCCAACGGATCACGCAAA
GGATCAGTATCAGCCAGGGGCCATGTCTTCTTTCGTTGTTGGACCCATAACAAACACCTAGTGATGTCATCTCCAC
CATACGGCAGCAGAAAACGTGTTGATGGAATAGCAACCCCTTCCTCAACACACATGCCTGAGATAACTTGTGCAC
CCATGTTTACGATACATCCAGATGAAAAGCCATTCCCAAATGTAGCCGCAACGCATTCCTGGA
Real-time fluorescence quantitative PCR primer sequence: forward primer: GCAAGTAGCGAAGGGTAAA
Reverse primer: TGCCGTATGGTGGAGATG
Sequence 3 tooth rib red moss Syntrichiacaninervis transcriptional elongation factor nucleotide sequence
TTGAAGGTCTTTCTGATATAATCTTCCTTCTTTAAGCGAGTCTTTTCCTGCTCGTTGAGTAAGAAGGTGATGTCGGAT
TTAAAACGCTTGTCCCGGTGCTGCGTATCATCCCTAGACTCTTCCCGCATATCGCTTTCCTTGCATGTCAAATCGACC
AAGTACTTATCCTTTTTCACCTCTTTCACTCGGCAAGTCACTGAAGTGCCTTCTGAAAATTTATCTTTAAGTTCAACA
TCGCCGTCATCTGAAACATCTTCCTTCTGAATGAAACCCAGTAATCCACATTCCAATACACACATGAGCTGATGTTCC
TGCACTCTTCTTATTGTTGCTTGTACAATTCCACCTTCAAAGAGTGTCTCTTTGCTCTCCCCGGTCATGAGATAGAAA
CACTCTTCTTGAGACGGCTCCGAGTACGACCTCCGCCATTCTTGAAATCCTCGCTGCAGCTCAGATTTGATCAACTC
CAGTGTCTTCAATCCCCGGCCTTCAACGTGTGCAGATTGGGCATACTCCGCAATGTCTACAGAATCTAAAGCGTTGG
GATTTTTAATCACACGATCTACTGCCATACTTGGCACATCATCGTCCACTTCTTCGTTATTCCGCATGCCTTCCTTCTC
GATCGCAGCTTCAGCTAGGTTCTTTGCCATCTGATACAACTCAGGGTGAATACGGGTGTTGTCTAAAAGCTCTGTAG
GACGTAAATCTGAAGGTGTTTCTCCAATTTCACGCACTCGGATGAAACCAGCGGAATTTGTGAAGACTTTCTCATTC
ACAGCCCGTATGGTCGAGTAAAGACTATCTCGAGTCAATACACGCTTTACCACTTGAAGATTTGTCCGAAACTCACA
AGCCTTCAGAGGGCCAAGACCCGCAACGAACTGAAGGGTCTCAAACAACCAGTCGTGGATAATAGCTAGA
Real-time fluorescence quantitative PCR primer sequence: forward primer: ACTTGGCACATCATCGTCC
Reverse primer: ATTCCGCTGGTTTCATCC
Sequence 4 tooth rib red moss Syntrichiacaninervis calcium dependent protein kinases nucleotide sequence
TGAGATGTTTAAACACTACCTCAATTACATGCTGAAATCCAATACATTTGACGTAGAAGCATCAACAGTGTTTGGC
ACCTAATCATTTCCTCTCGGGCGCCTCTAGCGATGGCACTGAAAATGATACACGGGGACTAAAGGCCATGGCCCT
AGAGCAAGTAACATGACAAACTTTACATATTCAAACTCCCCATGGTAAGGAGAGAGTGCTACTCGCCTCTGTAGA
AGTGACTGGTTCGCACAGCCATGTTGAGGTGCCTACTTCCTGCTGAACTCGGTGTGAACCCTATCCTCCGATCCCC
AACGCGTCGGTCAGATTCAACTGCTGGCTGTTGCGTAGCGATGAGCGACCAGCACCGCCCTTGTTGCCCTTGCGC
ATCATGTTGACGAACTCGGTGTAATCAATCTGGCCATCGTTGTTCTGGTCGACCTCCCTGAGCAGCTCCTCCACGG
ATACGTCTCCCATGTTGAATTCGGCCAACGCATGTTGCAGCTCATCGACCGTCAGATAGCCGCTGTTGTCCTTGTC
TAGGTACGAGAAGGCGGCGAACATGTTCTCATCGCGTTCGACCTTGTTCAGGTGGAGAGTGGCGGCCAGAAACTC
GCCATAATCGATTGCCCCACTGCCGTCGACGTCAGCGGCGTCCATGAGCTGCCGAACGTCTTCCTCTTTGAGATTG
GAACCCACTTTCTTCAGACCCGCCTTCAGCTCCTCGTAGCTGATGGACCCTGAGTTGTCCGTGTCCATCATCTTGA
ACATCTCCTTCAGACCTGCGATCTCCTCCTCCGACAAGGACTCCGCGATCACCCGGATGGCGATCTTCTTCAGCTT
GTTCATTGCGGAGAAGTGCTTGAGCCGGGATTGCACTGCGGAATCGATGGGTCTGTCGGGCGCTACTCCGTCCTC
GCAGATCCAAGGATGTGCCAACACTTGATGGGCTTTCAACCTCTTCGAAGCCTGGGGCGTGAGCATTTTGCGGAT
CAAATCCTTGGCGCTATCAGAAATCTTAGGCCATGGATCTGATGCAAAATCAAGCTCTGATTTCAAAACCTGCTCA
AAGATGCCCTGCTCCGTCTCTGCCCAGAATGGAGGGACTCCGCTGAGAAGGATGTAAAGTATCACACCCGCACTC
CACACATCCGCTTCCGGGCCATAGTTCTTCCTCAACACTTCAGGTGCCACATAGTAAGGGCTTCCAACCACATCTC
TGAAAACTTCTCCAGGTTTAAAGAACACTGATAGCCCAAAATCAGTCGTTTTCAGAGCCGCATCCTCCTTCGTACT
CGAAAACAGGAAGTTCTCCGGCTTCAAATCTCTGTGCATCACGCCAAGAGAATGGCAGGTCTCAACCACGCCCAC
GATAGTACGAGTGAGCACTGCCGCTTTGGCTTCCGAGTACTGTCCTCGTTGAATGATGCGGTCAAACAGCTCACC
GCCTGCACAAAGCTCCATCACCAGATGCACTGAAGCAGGATCCTCATACGCCCCCTTGATCATGACAATGTTCGG
ATGTCCTGAAAGGTGATGCATGATCTGCAGTTCACGGCGAACGTCATCCACGTCTTCCTTGGAGATAAGCTTCCGC
TTGGCAATAGACTTGCATGCGTACTCCCTGTTGGTGGCCTTCTCAATGCACAGGTAGGTGGTGCCGAATTGACCTT
GCCCCAGCTTCTTTCCCAGAACATACAGGTCTCTGAGGTTCTCTGTCTTCCGCTGCAGCACAGAGTGAGTCAGACT
CTGGCCAGGGACGGGCTTCATCTCTCTCCCTTTCTTCATGGCTTCGATCACTTCTTTGAATTGCTGAGTTTCGGCCG
GTTCCACAGCAGGGATGTGAGCTGGGCGTGGCGGTTTCCGTTGAGTCTCCGGGGACTCCGCCTCTGCCCTGCTCCC
CCCTCTGGACTCTGCCTTGGATGGAGTGGGTGATACCTCACTAGGGGATCTGGAGCTACGCCCTCCCAGTGCGATC
ACACTGGTGAAGCCCTGAAAGTACCCAGCCGCTCCTACACAACTGTTGCCCATTCAGACACAATCTGTAACATCC
CTCCACGGGAATCTATTTATCCCGTTCCCCTAAACTTGTCTCTTAAAGCAAAGGCCTAAACATACACTACAACGAA
CTTGATGCCGTTTTTGTCACCATTTGGCCACGTACCTTCGCACAATCATCTGAAGTAAGAACAGGCTGAGGAGAA
AGACCGCCGTCTAGGGTTTCAAGGCTGAGAAGCGAACCCTGAAATGAGTGTAAGGTGGCCGAAACTGACTTCAA
AGAGAGAACTGGCGGCCAGGTTTCCTCAGTTATTCTTTCCAGTCTCAAACCTCGAGCTCCGGCTGCGAAATAATCT
GGCAGCGAAAGTGGCAGCAAAAACAAGCAGCTCTCGCACTATTTTCC
Real-time fluorescence quantitative PCR primer sequence: forward primer: AACACTTCAGGTGCCACATAG
Reverse primer: GCATCATTCAACGAGGACAG
Sequence 5 tooth rib red moss Syntrichiacaninervis α tubulin nucleotide sequence
GTGGATGTGACGGAGTTCCAGACGAACTTGGTGCCGTACCCCCGTATCCACTTCATGCTTTCGTCTTACGCGCCGG
TGATCTCGGCCGAGAAGGCGTACCATGAGCAGCTGTCCGTCGCGGAGATCACCAACTCTGCGTTCGAGCCGTCGT
CTATGATGGCGAAGTGCGACCCCCGTCACGGGAAGTACATGGCGTGCTGCCTGATGTACAGAGGAGACGTTGTGC
CGAAGGACGTGAACGCGGCAGTGGCAACGATCAAGACGAAGAGGACGATCCAGTTCGTGGACTGGTGCCCGACC
GGGTTCAAGTGCGGAATCAACTACCAGCCCCCGACGGTGGTGCCCGGAGGCGACCTGGCGAAGGTGCAGCGGGC
GGTGTGCATGATCAGCAACAACACGGCGGTGGCGGAGGTGTTCTCCCGGATCGACCACAAGTTCGATCTCATGTA
CGCGAAGCGCGCGTTCGTGCACTGGTACGTGGGCGAGGGCATGGAAGAGGGTGAGTTTTCGGAGGCCCGTGAGG
ACTTGGCTGCGCTCGAGAAGGACTACGAAGAAGTGGGCGCGGAGTCGACGGACGGCGCCAACGACGACGATGAC
TAC real-time fluorescence quantitative PCR primer sequence: forward primer: CGTCGTCTATGATGGCGAAGT
Reverse primer: TCTTGATCGTTGCCACTGCC
Sequence 6 tooth rib red moss Syntrichiacaninervis α tubulin 2 nucleotide sequence
ATGAGAGAGATCATCAGTATCCACATCGGCCAGGCCGGTATCCAGGTCGGCAACGCCTGCTGGGAGCTCTACTGC
CTCGAGCACGGCATCCAGCCCGATGGCCGCATGCCTAGTGACGCGTCTACAGGAGGTGGAGACGATGCGTTCAAC
ACCTTCTTCAGCGAGACCGGGGCGGGGAAGCACGTGCCGCGGGCTGTGTTTGTGGATCTGGAGCCGACCGTGATC
GATGAGGTGCGCACGGGGACGTACCGGCAGCTGTTTCACCCGGAGCAGCTTATTTCCGGGAAGGAGGACGCCGCT
AACAACTTCGCTCGCGGTCATTACACCGTGGGAAAGGAGATTGTGGACTTGTGCTTGGACCGGGTGAGGAAGCTG
GCAGACAACTGCACGGGCCTGCAGGGATTTTTGGTATTCAATGCTGTGGGAGGAGGCACGGGATCGGGACTCGGT
TCGCTGTTGCTGGAGAGGCTCTCAGTAGACTACGGAAAGAAGTCCAAGTTGGGATTCACCATCTACCCGTCTCCTC
AGGTGTCCACCGCAGTTGTGGAGCCGTACAACAGTGTGCTGTCGACTCACAGTCTGTTGGAGCACACCGATGTGG
CGGTGTTGCTGGACAATGAGGCCATTTACGACATCTGCCGGCGCTCGTTGGACATCGAGCGGCCGACGTACACGA
ACCTGAACAGGCTGATCTCGCAGATCATTTCGAGTCTGACGACGAGCCTGCGGTTTGACGGCGCGCTGAACGTGG
ACATCACTGAGTTCCAGACCAACTTGGTGCCG
Real-time fluorescence quantitative PCR primer sequence: forward primer: CGGTCATTACACCGTGGGAA
Reverse primer: CCTCTCCAGCAACAGCGAA
Sequence 7Syntrichia caninervis beta tubulin nucleotide sequence
ATGAGAGAGATCCTCCACATCCAGGGCGGGCAGTGCGGGAACCAAATCGGTTCCAAGTTCTGGGAGGTGGTGTG
CGAGGAGCACGGGATTGACCCCACCGGCTCGTACCAGGGGGTGTCTGACCTGCAGCTCGAGCGCATCAATGTGTA
CTTTGATGAGGCCAGCGGCGGGCGCTACGTGCCGCGCGCGGTGCTCATGGATCTGGAGCCCGGCACCATGGACAG
CGTCCGCACTGGCCCCTACGGCCAGATCTTCCGCCCCGACAACTTCGTCTTCGGACAGACCGGCGCTGGTAACAA
CTGGGCCAAGGGCCACTACACCGAGGGCGCGGAGCTCATCGACTCAGTGCTGGACGTCGTGCGCAAGGAGGTTG
AGAGCTGCGACTGCCTGCAAGGCTTCCAGTTCTGCCATTCTTTGGGAGGAGGAACGGGATCTGGAATGGGAACGC
TTTTGATATCTAAGATCCGCGAGGAGTACCCCGATAGAATGATGCTTACGTTTTCCGTGTTCCCCTCTCCGAAGGT
GTCCGACACCGTGGTGGAGCCTTACAATGCTACTCTCTCAGTGCATCAGCTGGTGGAGAATGCCGACGAGTGTAT
GGTGTTGGATAACGAGGCGCTGTACGACATCTGCTTTAGGACTTTGAAGCTGATCACCCCCTCATTTGGAGACTTG
AACCATTTGATTTCTGCGACGATGAGTGGTATCACCTGTTGCCTGCGTTTCCCCGGACAGCTGAACTCTGACTTGC
GGAAGCTGGCGGTGAACCTCATCCCGTTCCCCCGGTTGCACTTCTTCATGATCGGTTTCGCGCCTTTGACCTCCCG
TGGCTCACAGCAGTACCGGTCGCTCACCGTGCCGGAGCTGACGCAGCAGATGTGGGACTCAAAGAACATGATGTG
CGCGGCGGATCCGCGTCACGGGCGCTACTTGACGGCTTCCGCGGTGTTCCGCGGCAAGGTGTCCACCAAGGAGGT
CGACGAGCAAATGATCAACGTGCAGAACAAGAACTCATCCTATTTCGTTGAGTGGATCCCGAACAACGTGAAGTC
GTCTGTCTGCGACATCCCCCCGACGGGTCTGAAGATGTCGTCGACGTTCATCGGGAACTCGACGTCCATCCAGGA
AATGTTCCGGCGCGTGTCGGAGCAGTTCACGGCCATGTTCAGGAGGAAGGCGTTTCTGCATTGGTACACGGGCGA
GGGCATGGACGAGATGGAGTTCACAGAGGCGGAGAGCAACATGAACGATCTGGTGTCGGAGTACCAGCAGTACC
AGGACGCGAGCGCCGAAGAGGAAGGCGAGTACGAGGACGACCTTGAGGAGGCC
Real-time fluorescence quantitative PCR primer sequence: forward primer: CTTTGACCTCCCGTGGCTC
Reverse primer: GTTCGGGATCCACTCAACGA
Sequence 8 tooth rib red moss Syntrichia caninervis ubiquitin protein ligase 1 nucleotide sequence
GCGCTTGTGAACGAGCTCATCACCAAGTTGTCCAGCCGGAGCACCAAGGTACATCGGGATGCTGCCAAGACCATA
AGGTGCCTAGCGAAGATAAACAAAGAGAATCGAATTCTTATTGCGGAGCAGGGAGGCATACCTTTCTTGATCAAC
CTTCTCCGATCGCCTGATGAGGAGACGCAGGAGCACGCCATCACCGCGTTGATGAACCTGTCCTTGCATCCAAAC
AACAGAGGGCTCATCATGAGAGCAGGGGCCATAGACGGCATCGTTCACGTAGTGAAGCATGGCGAGAGCACGGA
CGCGCGCGAGAACGCAGCAGCAGCAATCCAGTGCCTCTCGTATGACAACGAGAACAAGATCTCCATCGGCAACA
CGGGTGCGATCCCCGCGCTCGTTCAGCTGCTGCGCACCGGGACTCGCCAGGGCAAGAAGGACGCGGCCCACGCG
CTCTGCAACCTCGTCAGCTACCTGGACGGCAACAAGAAGCGCGCGGTGGACGCCGGCCTCACACCCCTCCTCATG
GCGGTTCTGCGCGAC
Real-time fluorescence quantitative PCR primer sequence: forward primer: GCAGGGAGGCATACCTTTCTT
Reverse primer: ATGAGCCCTCTGTTGTTTGGA
Sequence 9 tooth rib red moss Syntrichia caninervis ubiquitin protein ligase 2 nucleotide sequence
ATGGGGTTAGGGTATGGGCCAGTGGTGGCATTGTACACGGCGTTGAGTGTGGGTGGGATAGCATGGAAGCTCAAC
TCAGCGTGGTGGACTATGACCTACGGCAAGGGTCAAGGAGGTGGTGGGCAGATGGATCCCTCCGAAGCGGCGCG
CACGCTGCAGGCCATGCTGCAATCATCCGTCGTGGTGGCGCTCCTCGTCAACCTTCTCGTCAACTTTTTCCTTCTCG
TCACCCTCTCAATGAAGACTGTGTTCTTCGGTCAGTTGTCTCTGGTTGAGACTCAGAAGGTGGTGGAGCGCCTCAT
CAATTATGTATTATTCAAGGGCCTGTTCTTGACATGGGTCGTGAGGCCAGAAATGATGCAGATAGCTGTGTGGTTG
GCGTGGTTCTCCGTCTTGGGCTTTCTGAAGATGTTCCAAGGTTTGGCAAGAGATCGTTTGGAGCGCTTAAATGCAT
CTCCTTCTGCGACAATGTTGGCTCACATAAGAGTTTATACGGTTCTCTTGTTCGTTCTTCTGTTCGATTTGCTCTGG
ATGCAGCTGTGTCTGCTTTTATTCAAAGACACTGGAACCAGTACCTTCTTGCTACTTTTGTTTGAGCCTCTCAGTAT
AGCATTTGACACTCTTCAGGCAGTAATTGTACACGGTATGCAGCTATTGGACACTTGGCAGCGGCAAAACCTTGA
TATTTCTGCACACGATATAAGTTTGCAGCCTTCTGAGAGGTCTGCTGCAGGTGCAGCTTGGGAGTGGCGTGGAACT
GTTGTAAGAAATTGCAGCTTTGTGATGGATATTGTGAGCTTGTTACTGGCTTTGGGCCATTGCGTACACATCTGGT
GGTTACGTGGGCTTGCTTTTCAAGTGGTGGATGCCATTCTTTTCCTCAACTTGCGCGCGCTATTGAGTGCTATTTCA
AAAAGAATAAAAGGTTTCATGCGTCTTCGGGCAGCCATGACAACTCTGCAGGGTGCGCTTCCCGATGCAACGCAG
GATGAACTCCTTGGATATGAAGATGATTGTGCCATTTGCAAGGAGCCCATGGCTAGGGCAAAACGGCTTCCTTGT
GCTCATCTCTTCCATCTCGCGTGCTTGAGATCCTGGCTAGACCAAGGATTAGCAGACACATACTCTTGCCCGACCT
GCCGTAGACCCCTCTTCATGGGCGGGTTCCGGACTGCAACAAACAATTTGCAGCGCCTGGTAGATGTGCCCACTG
CATCCCCCGGCACAAATGAAACTCAGGCACAGGTTGCACAACGACAAGACTTAGCTGCAGCGCCGTCTCCACCTC
TCACCACCTTGACTCCCGCTCCAGCACCG
Real-time fluorescence quantitative PCR primer sequence: forward primer: AGCTTGTTACTGGCTTTGGG
Reverse primer: GGAGTTCATCCTGCGTTGC
Sequence 10 tooth rib red moss Syntrichia caninervis glyceraldehyde-3-phosphate dehydrogenase 1 nucleotide sequence
TCCGGGATCAAGAACACCAATGCCGTCACCTTCGGCAAGCGCACTGATGACCTCGTCAGCGTTGTTGCTGCCCAG
TCTGCTGTGTCTGCTAGCTCCGGAGCCACGAGGGCCGTGACCGAGGCCAAGATCAGGGTTGCCATTAACGGTTTT
GGGCGTATTGGACGCAACTTCGTCCGCTGCTGGCATGGAAGGAAGGACTCGCCTCTCGAGGTCGTCGTGATCAAC
GACACCGGAGGCGTGAAGCAGGCTTCTCACCTGTTGAAGTACGACTCCATGCTCGGCACCTTCAATGCCGACGTG
AAGGTCGACGGTGATGGCATCAGCGTTGATGGCAAGACCATCAAGGTGGTGTCCGACAGGAACCCGCTCAACTTG
CCATGGGGGGATATGGACATCGACCTTGTCATTGAGGGGACCGGAGTGTTCGTCGACGAGGCTGGTGCCGGCAAG
CACTTGCAGGCGGGAGCGAAGAAGGTGCTCATCACCGCCCCAGGAAAGGGTGCCATCCCCACGTACGTGATCGG
AGTAAACGAGCAGGATTACACCCACGCGGACAAGATCATCAGCAACGCGTCGTGCACCACCAACTGCCTGGCCC
CGTTCGTGAAGGTGTTGGACGAGAAGTTCGGCATCATCAAGGGTACCATGACCACCACCCACTCATACACTGGTG
ACCAGAGGCTGCTGGACGCTTCCCACCGCGACTTGAGGAGAGCCCGTGCCGCGGCCCTGAACATCGTGCCCACCT
CCACGGGAGCCGCCAAGGCCGTGGCGCTGGTGCTGCCGAGGCTGAAGGGCAAGATCAACGGAATCGCCCTGCGT
GTGCCCACCCCCAACGTGTCCGTGGTGGACTTGGTGGTGCAGGTGGAGAAGAAGACGTTCGCGGAGGAGGTGAA
CCAGGCGTTCAGGGATGCGTGCGAGACCCCTCAGCTGAAGGGCATTTTGGCAGTGTGCGACGAGCCTTTGGTGTC
GGTGGACTTCAGGTGCACTGACGTGTCGTCCACGGTGGACTCGTCCCTGACCATGGTGATGGGCGACGACATGGT
GAAGGTTGTGGCATGGTACGACAACGAGTGGGGGTACTCGCAGCGGGTGGTGGACCTGGCTAACTTGGTAGCGG
AGAAGTGGCAG
Real-time fluorescence quantitative PCR primer sequence: forward primer: GCGTTGTTGCTGCCCAGTC
Reverse primer: AGGCGAGTCCTTCCTTCCAT
Sequence 11 tooth rib red moss Syntrichia caninervis glyceraldehyde-3-phosphate dehydrogenase 2 nucleotide sequence
ATCGGAAGACTCGTCCTCCGTGTGATTCTTGCCAGGGACGATGTGGAAGTTGTTGCCGTCAACGACCCCTTCATTG
ACACCAAGTACATGGCATACCTGTTGAAGTACGATTCCACCCACGGGCTTCTCAAGGCTGATGTGCACGCTGTTG
ATGACAAGACTCTGCAAGTTGCTTCTCAAACCATCAAGGTCTTCGGATGCAGGGATCCCGCGGATATCCCATGGG
GTGAAGCCGGTGCTGATTACGTGGTGGAGTCCACCGGTGTTTTCACCGCCGTTGCCGGCGCCTCGGCCCATTTCAA
GGGAGGAGCCAAGAAGGTTGTTATTTCCGCACCATCGGGAGATGCCCCCATGTTCGTCATGGGTGTGAACCAGAA
CGCTTACAAGGATGACATGAACGTCGTCTCCAATGCCAGCTGCACCACCAACTGCCTTGCTCCCCTAGCTAAGGT
GATCCAGGACGAGTTCGGCATTCTCGAGGGTCTGATGACCACCGTTCACGCCACTACCGCCACCCAGAAGACCGT
CGATGGCCCATCAGGGAAGGACTGGCGTGGAGGACGTGGTGCTGGCCAGAACATCATCCCCAGCTCCACCGGCG
CTGCCAAGGCTGTGGGCAAGGTGTTGCCTGAGCTGAACGGCAAGTTGACCGGCATGGCGTTCCGTGTGCCCACCC
CGAACGTTTCAGTGGTGGACTTGACTGTGCGCCTGGAGAAGGGTGGGTCCTACGAGGACGTCAAGGCAGCCATCA
AGGCCGCCTCTGAAGGACCGATGCATGGCATCTTGGGCTACACCGAAGACGACGTGGTGTCGAACGACTTCATCG
GTGACGCCAGGTCCAGCATCTTCGATGCTAAGGCAGGAATCGCTTTGAACGACAAGTTCGTCAAGTTGGTGTCGT
GGTATGACAACGAGTGGGGCTACAGCAACCGTGTGGTGGACCTG
Real-time fluorescence quantitative PCR primer sequence: forward primer: GGGCTTCTCAAGGCTGATGT
Reverse primer: TCCACCACGTAATCAGCACC
Sequence 12 tooth rib red moss Syntrichia caninervis SAND protein nucleotide sequence
TCGGAAGTGGAAGCTGATAATACAGCGAAACTCTCAAACCTGGACATCAGTAGCAGCACGCAGGAGGACGGTTCA
AAGCTTGAAACTCATGATGCAGAAGATGGTCCAAAAGCCTTGGACATGGGAGATGGAGAAGGTACTGCGCTCGCT
GCTTCTACAACTACTGATGTTGTGCCGGAGAGTACTGTGGATGGAGATACATTGTCTGAAGATGGCATCCATGAGAG
CCCTGTGAGTCCAAGCAGTAGCCGCGAAGGAACCAGTGGCTACTTAGCAGGCAGTGGCAGCAGTACCGCGAGTGT
CTATAGCGCAAGTGGAGCAGATGAAAGTGCCGATCGAATTCTGAATCAGGATTCTATGCCACAAGCGACGAGGCGG
TCTTGGGTCCCTGGAAAACGCCATCCAGATGAAGATGACACGTCTCTCGCTTGGCGAAAGCACAAGAAGCATTTCT
TTATTCTCAGCAATGCAGGAAAACCA
Real-time fluorescence quantitative PCR primer sequence: forward primer: AAAGCCTTGGACATGGGAGA
Reverse primer: CGTCGCTTGTGGCATAGAA
Sequence 13 tooth rib red moss Syntrichia caninervis H3 nucleotide sequence
CTAAAGACGGGAGAACGAAGACCCTTTTCTCCTCCTTCGGCTTCACCTCCCCATCTTTCTCTCTCGCTCACTCTCGA
CATCGTCATCTCTCTCGTATTCTACCTACACCGCCGTTTCATTTCCACAAGTGTCACTATGGCGCGTACGAAGCAG
ACGGCTCGTAAGTCCACCGGAGGAAAGGCTCCGAGGAAGCAGCTCGCCACGAAGGCTGCGAGGAAGTCGGCCCC
CACCACCGGAGGAGTGAAGAAGCCCCATCGCTACAGGCCGGGAACCGTCGCTCTTCGTGAGATCCGCAAGTACC
AGAAGAGTACCGAGTTGCTGATCCGCAAGCTGCCCTTCCAGAGGCTGGTTCGTGAGATCGCCCAGGACTTCAAGA
CCGATCTGCGTTTCCAGAGCCACGCCGTGCTGGCGCTGCAGGAGGCGGCGGAGGCGTACCTGGTGGGTCTGTTCG
AGGACACCAACTTGTGTGCCATCCACGCGAAGAGGGTGACCATCATGCCGAAGGACATCCAGCTGGCCCGCAGA
ATCAGGGGAGAGAGGGCTTAGGCCGGCTCGCCCGGAGTCTGTATGAAAAAACCCAAAAAAATCTAAAAAAAAGT
TTGGGGTTCTTTTGAACCCACCAAAGGGGCGTGTACTCCGTGTATGTGGCATGATGTTACTTGTATGTTACTTGTA
TGTTTGTGCGGAGTGATTGCTGAAGCAGCCGAG
Real-time fluorescence quantitative PCR primer sequence: forward primer: AGGAGTGAAGAAGCCCCATC
Reverse primer: CGAACAGACCCACCAGGTAC
Sequence 14 tooth rib red moss Syntrichia caninervis F-box protein nucleotide sequence
ATTCGTCTTTTTGTTTGGTTGCGTAAGTGTTTAGGTTGACACAATCCTCGTTTCGCTTCTAGGTTTTGTGTTTTGGCG
TTTAAATCACTGAGGTTTCCCTGAAGACCTTTGATGCCATAGAGATGACGCTTTCCTTGTCCTGCTTACAGCTTGAG
CTGGTGTGACAGTATCCGAATTTGCCCTAATTGGAGGGGCATGTGAAGCTCACGATCTGGGTCCGCTTACAAGCTCA
CAGCTTTAGTCAGTATTGACAACATGGGATGATCGTCTTGCAATCTCGAAGAAGCGATGGAGCAAGACGGATTGGA
TTCATGGAGCAGGCTACCGGAAGATGTCGTGCTGAGAGTGCTCACGCGCCTGCCAATTCCGCATCTTTTCCGCGCG
CGCACGGTCTGCAAGCAATGGAATGCGCTCACTTCGACCCCGGAGTTTCTGGCCATGTGCAGCGCGCGTCACGCGC
CCTACTTCCCGGTGGTGATCTCGCGCCGCTTCTACATGGGCGACCTGGGGATCTGCTTCCTGGGGAACGACTCGCA
GGGCGGCAACGACCTCTTCCGCGTCTTCTTCGGCTACGACCATGAGGCGCAAGCGTGGCAGAAGCTGCCCCCGTTC
GACTTCCTCCCGCACGAGGCGCGCATCCCGGTGGCCTCCGCCGGCGGCCTCATCTGCTTCCGCGGCACCTCCAGGC
TCTTCCTGTGCAACCCCGTCGCCCGGAGCTTCCTGGAGCTCCCCGCCATCACGTACAAGTGG
CCGCCGTCCGTCTCCGTGCACATCCTCGTGGAGAGCAGCGCCTTCAAAGTCATCATCGTGGGGAAGATCCGCCACA
ACTTCGTCACCGACTCCTTCCGCAGCATCGCCATCTACGAGTCCGTCACCAAGGCCTGGCGCGTCGTGGACGCGCC
GCATCCCGCGAAGGTATTCTCCTACGGTCCCACTGCAGCCGTCTGCGGCAACTCCATCTACTGTGAGGCGATTTGCC
ATTGCGGCCAGCTGGGTGTGATGGCCTACGACGTCCAGTCCGAGACGTGGAACCCGGTGTTGCATGAGATTCCGAG
TGATGAACGCGGCGAGTATCAGCTGACGCAAGTTGTG
Real-time fluorescence quantitative PCR primer sequence: forward primer: CGCCTTCAAAGTCATCATCG
Reverse primer: GGCAAATCGCCTCACAGTAG
Sequence 15 tooth rib red moss Syntrichia caninervis18s ribosome-RNA(rRNA) nucleotide sequence
TTACTATGGTGGTGACGGGTGACGGAGAATTAGGGTTCGATTCCGGAGAGGGAGCCTGAGAAACGGCTACCACATC
CAAGGAAGGCAGCAGGCGCGCAAATTACCCAATCCTGACACGGGGAGGTAGTGACAATAAATAACAATACCGGGC
TCTTTGAGTCTGGTAATTGGAATGAGTACAATCTAAATCCCTTAACGAGGATCCATTGGAGGGCAAGTCTGGTGCCA
GCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTTAAGTTGTTGCAGTTAAAAAGCTCGTAGTTGGACTTTGGGT
TGGGTCGGCCGGTCCGCCTCTGGTGTGCACCGGTCGTCTCGTCCCTTCTGCCGGCGATACGCTCCTGGTCTTAACTG
GCCGGGTCGTGCCTCCGGCGCTGTTACTTTGAAGAAATTAGAGTGCTCAAAGCAAGCCTACGCTCTGGATACATTA
GCATGGGATAACATCATAGGATTTCGGTCCA
Real-time fluorescence quantitative PCR primer sequence: forward primer: GGAGAGGGAGCCTGAGA
Reverse primer: CACCAGACTTGCCCTCCAA;
B, choose arid respectively, high salt, cold, heat, xeothermic mixing, dormin, oxidation, physical abuse, heavy metal, ultraviolet coerces totally 10 kinds of abiotic stress and without Stress treatment, and 11 tooth rib red moss gametophyte sample is experiment material altogether, carries out the experiment of quantitative fluorescent PCR template;
The internal reference estimation of stability software geNorm that c, the data importing two obtained by real-time fluorescence quantitative PCR are the most frequently used, a NormFinder and internal reference stable checking calculation software Refinder carries out internal reference stability and the analysis of internal reference number, filters out optimum reference molecules and reference molecules combination.
2. method according to claim 1, is characterized in that the reference gene described in step a is 18s ribosome-RNA(rRNA), Actin muscle, α tubulin 1, α tubulin 2, beta tubulin, glyceraldehyde-3-phosphate dehydrogenase 1, glyceraldehyde-3-phosphate dehydrogenase 2, ubiquitin protein ligase 1, ubiquitin protein ligase 2, calcium dependent protein kinases, F-box protein, transcriptional elongation factor, actin associated protein, SAND albumen, H3.
3. method according to claim 1, is characterized in that in step b, 11 tooth rib red moss gametophyte is the leaf tissue of the removal rhizoid after various Stress treatment after complete rehydration 24h altogether.
4. method according to claim 1, is characterized in that the condition of abiotic stress process in step b is: use 20% polyethylene glycol 6000 respectively, 250mM sodium-chlor, 100 μMs of dormins, 0.5mM copper sulfate, 50mM hydrogen peroxide, 0.5w/m 2uv-radiation simulating drought, high salt, dormin, heavy metal, oxidation and ultraviolet are coerced; Red for tooth rib moss is placed in respectively temperature 4 DEG C of refrigerators and temperature 42 DEG C of illumination boxs simulation low temperature and high temperature stress; Coerce with the 42 DEG C of dry-hot simulation mixing of 10%PEG+ temperature, it is realize by cutting off blade that physical abuse is coerced; All process are all draw materials after coercing 6h, and the material obtained is placed in liquid nitrogen immediately, and are placed in rapidly temperature-80 DEG C of refrigerator storage.
5. method according to claim 1, it is characterized in that real-time fluorescence quantitative PCR template in step b is 1 μ gRNA and reverses 5 times of diluents of the cDNA obtained, and fluorescent quantitation program is: the first step is denaturation: temperature 95 DEG C-30 seconds; Second step is PCR step of reaction, temperature 95 DEG C-5 seconds, temperature 58 DEG C-60 DEG C, 30 seconds, and fluorescent signal is collected in 40 circulations; 3rd step is solubility curve analysis, temperature 65 DEG C-95 DEG C, and temperature 65 DEG C progressively rose to 95 DEG C at 30 seconds, collects first order fluorescence signal for every 0.5 DEG C.
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