CN114196772B - Real-time quantitative PCR reference gene under different tissues and stress treatment conditions of tilia miqueliana as well as screening method and application thereof - Google Patents

Abstract

The invention discloses a real-time quantitative PCR internal reference gene under different tissues and stress treatment conditions of tilia miqueliana and a screening method and application thereof, belonging to the technical field of plant genetic engineering. The 15 internal reference genes are derived from full-length transcriptome sequences of different tissues of tilia miqueliana, 15 pairs of specific real-time quantitative PCR primers are designed according to sequence information, real-time quantitative PCR amplification is carried out on plant materials subjected to different tissues and stress treatment of tilia miqueliana, ct values of the internal reference genes are obtained, stability of candidate internal reference genes is ordered from strong to weak, and finally comprehensive evaluation is carried out on the ordering results, and the most stable internal reference genes and combinations under different tissues and stress treatment conditions are obtained through screening. The reference gene and the primer thereof obtained by screening have the advantages of strong specificity and wide applicability, and fill the blank of the research on the reference gene of Tilia miqueliana.

Description

Real-time quantitative PCR reference gene under different tissues and stress treatment conditions of tilia miqueliana as well as screening method and application thereof

Technical Field

The invention belongs to the technical field of plant genetic engineering, and relates to a real-time quantitative PCR internal reference gene under different tissues and stress treatment conditions of tilia miqueliana, and a screening method and application thereof.

Background

Tilia miqueliana is an important tree species in China in the countryside and is also an excellent ornamental tree species in gardens and urban street trees. Meanwhile, tilia miqueliana is a multipurpose economic tree species for collecting honey sources, fragrant flowers, materials, fibers, medicines and the like. With the gradual development of cultivation and popularization work, tilia miqueliana cultivation initial scale is developed nationwide. However, the research field of genetic breeding and functional genes of plants of the genus is still blank. In Tilia miqueliana, only a key enzyme HMGR coding gene in a medicinal component terpene synthesis pathway is separated and identified, and an actin gene is adopted as an internal reference gene in gene expression analysis. However, it was found in the study that the stably expressed reference genes in different species and tissues are not identical, and that the reference genes are not identical even under different physiological conditions of the same species. The selection of reference genes in plants is not absolutely versatile. Therefore, in analyzing the expression pattern of the target gene, an appropriate reference gene should be selected according to the change of experimental conditions. At present, no report is found on the screening of Tilia miqueliana internal reference genes.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, provides a real-time quantitative PCR internal reference gene under different tissues and stress treatment conditions of Tilia miqueliana, and a screening method and application thereof, and performs screening of the internal reference gene under different tissues and different stress treatment conditions of Tilia miqueliana, thereby providing reliable guarantee for functional gene separation and expression analysis of Tilia miqueliana and laying a foundation for revealing molecular mechanism aspects of biological characteristics of Tilia miqueliana.

In order to solve the technical problems, the invention provides real-time quantitative PCR internal reference genes under different tissues and stress treatment conditions of tilia miqueliana, which comprise 15 internal reference genes, wherein the internal reference genes are respectively: 18S ribosomal RNA (18 srRNA), actin (ACT 1), clathrin (AP 47), aquaporin (AQP), elongation factor (EF 1 alpha), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone (HIS), protein phosphatase (PP 2 alpha), ribosomal protein S13 (RPS 13), S-adenosylmethionine decarboxylase (SAMDC), SKI interacting protein (SKIP), tubulin TUA, tubulin TUB, ubiquitin conjugating enzyme (UBC), polyubiquitin gene (UBQ 10), the nucleotide sequences of the reference genes are shown in SEQ ID NO. 1-SEQ ID NO.15, respectively.

The invention also provides a primer for amplifying the internal reference gene of the real-time quantitative PCR under the conditions of different tissues and stress treatment of the tilia miqueliana by using real-time fluorescent PCR, wherein the primer sequence for amplifying 18srRNA is SEQ ID NO.16/SEQ ID NO.17, the primer sequence for amplifying actin is SEQ ID NO.18/SEQ ID NO.19, the primer sequence for amplifying clathrin is SEQ ID NO.20/SEQ ID NO.21, the primer sequence for amplifying aquaporin is SEQ ID NO.22/SEQ ID NO.23, the primer sequence for amplifying an extension factor is SEQ ID NO.24/SEQ ID NO.25, the primer sequence for amplifying glyceraldehyde-3-phosphate dehydrogenase is SEQ ID NO.26/SEQ ID NO.27, the primer sequence for amplifying a protein phosphatase is SEQ ID NO.28/SEQ ID NO.29, the primer sequence for amplifying a protein phosphose is SEQ ID NO.30/SEQ ID NO.31, the primer sequence for amplifying a tubulin S13 is SEQ ID NO.20/SEQ ID NO.21, the primer sequence for amplifying a microtubule NO.34 is SEQ ID NO.34, and the primer sequence for amplifying a tubulin is SEQ ID NO.34/SEQ ID NO. 34.

The invention also provides a screening method of real-time quantitative PCR internal reference genes under different tissues and stress treatment conditions of Tilia miqueliana, which comprises the following steps:

(1) Constructing a full-length cDNA library by adopting equal RNA mixed samples of different tissues (6 tissues of roots, stems, leaves, bracts, flowers and fruits) of tilia miqueliana, and sequencing transcriptome; the obtained full-length transcriptome sequencing data of Tilia miqueliana are utilized, the corresponding 15 internal reference gene sequences in Arabidopsis are taken as references, and the 15 internal reference gene sequences of Tilia miqueliana are obtained by adopting a local blast; designing a full-length primer according to the internal reference gene sequence, amplifying the sequence by taking tilia miqueliana cDNA as a template, and sequencing and verifying an amplified product;

(2) Designing a real-time fluorescence quantitative PCR primer by taking the verified sequence as a template and adopting oligo7 software, wherein the length of a required primer amplified fragment is 100-250 bp, then verifying the primer amplified specificity by adopting PCR amplification combined with agarose gel electrophoresis, carrying out real-time fluorescence quantitative PCR amplification on the specifically amplified primer by taking Tilia miqueliana cDNA as a template and carrying out dissolution curve analysis to obtain the primer with the 15 reference genes and the real-time fluorescence quantitative PCR dissolution curve showing single-peak amplification;

(3) Respectively collecting different tissues of Tilia miqueliana and different tissue samples after stress treatment, quick freezing with liquid nitrogen, and storing in a refrigerator at-70 ℃ to-80 ℃;

(4) And respectively extracting total RNA of the samples, and obtaining complementary cDNA samples through reverse transcription reaction. Carrying out real-time fluorescent quantitative PCR detection by taking cDNA obtained after reverse transcription as a template, and recording a Ct value;

(5) Analyzing the Ct value data obtained by real-time fluorescence quantitative PCR, analyzing the internal reference stability and the internal reference number under different tissues and stress conditions, screening out the optimal internal reference gene and the combination of the internal reference genes, and finally comprehensively sequencing the stability of the internal reference genes;

(6) Under different experimental conditions, 2 is adopted ^-ΔΔCt And respectively selecting the most stable reference genes and the least stable reference genes by the method to correct the expression of the target genes for verification.

Preferably, in the step (2), primer amplification specificity detection in Tilia miqueliana real-time quantitative PCR is performed by adopting a dissolution curve method generated by common PCR product electrophoresis and real-time fluorescent quantitative PCR reaction, wherein single bands are detected by electrophoresis, the fragment size is 100-250 bp, and the real-time fluorescent quantitative PCR dissolution curve is unimodal, so that the specificity of fluorescent quantitative PCR primer amplification is proved, the primer amplification efficiency is calculated by adopting LinRegPCR software, and the amplification efficiency is ensured to be more than 1.80.

Preferably, in the step (3), the different tissue samples are taken from 9 tissues including tender roots, tender stems, mature leaves, bracts, buds, blooming fruits, immature seeds and mature seeds of 15-year-old Tilia miqueliana Maxim mature plants, stress treatment comprises 7 treatments including low temperature, high temperature, flooding, drought and salt stress, tissue materials of Tilia miqueliana Maxim tissue culture seedlings are collected at 0, 1, 3, 6, 12, 24 and 48 hours after the treatments, wherein only leaf tissues are collected in the high temperature and low temperature stress treatment, and two tissues of the leaf and root are collected in the flooding, drought and salt stress treatment.

Preferably, the stress treatment conditions are: the low temperature stress temperature is 0-8 ℃, the high temperature stress temperature is 36-42 ℃, the flooding stress is that the water surface is 2cm higher than the surface of the culture medium, the salt stress is simulated by adding 3 permillage NaCl into Hoagland's water culture nutrient solution, the pH value of the water culture nutrient solution is=5.5, the drought is simulated by adding 15% PEG-6000 into Hoagland's water culture nutrient solution, and the pH value of the water culture nutrient solution is=5.5.

Preferably, the amount of the RNA template used in the reverse transcription reaction of the test material under the conditions of each tissue and treatment in the step (4) is 0.5 mug, the cDNA stock solution obtained after reverse transcription is diluted 5 times, 1 mug is taken for real-time fluorescence quantitative PCR amplification, the real-time fluorescence quantitative PCR reaction system is 10 mug, and the concentration of the upstream primer and the downstream primer is 0.5 mug.

Preferably, the real-time fluorescent quantitative PCR reaction procedure in step (4) is: the first step is pre-denaturation at 94 ℃ for 30s; the second PCR reaction is carried out for 15s at 95 ℃ for 10s and 60 ℃ for 40 cycles; and step three, gradually heating the solvent curve at 60-95 ℃.

Preferably, geNorm, normFinder, bestkeeper and DeltaCt 4 algorithms are adopted in the step (5), and the stability of reference genes is analyzed and sequenced; and comprehensively ranking the candidate reference gene stability ranking results obtained by the 4 algorithms by using the refFinder to obtain comprehensive ranking results.

Preferably, in the step (6), the target gene expression amount analysis verification is performed on the application reference gene by using a 2-delta Ct method, delta ct=ct (target gene) -Ct (reference gene), delta ct=delta Ct (treatment) -delta Ct (control), 2 ^-ΔΔCt Relative expression amount of target gene.

The invention also provides application of the real-time quantitative PCR internal reference genes under different tissues and stress treatment conditions of tilia miqueliana in calculating the relative expression quantity of target genes, wherein 3 genes with the top two and the last two of the stability ranks under the selected experimental conditions are named as PP2 alpha, SAMCD and 18srRNA (flooding stress leaves), TUB, TUA and UBC (flooding stress roots), EF1 alpha, GAPDH and UBC (drought stress leaves), PP2 alpha, HIS and RPS13 (drought stress roots), TUB, GAPDH and UBC (salt stress leaves) and TUB, RPS13 and GAPDH (salt stress roots), the expression quantity of the target genes MYB in different stress samples is calculated respectively, and then the availability of the stable expression internal reference genes is obtained through screening under different experimental conditions is verified.

The invention has the beneficial effects that:

(1) The invention fills the blank of screening the stable internal reference genes of tilia miqueliana, selects 15 candidate internal reference genes based on the full-length transcriptome data of 6 tissues of tilia miqueliana root, stem, leaf, bract, flower and fruit, and designs specific real-time fluorescence quantitative PCR primers.

(2) The invention adopts 4 internal reference gene stability analysis algorithms to analyze the expression stability of 15 candidate genes, adopts a refFinder algorithm to comprehensively sort the results obtained by the 4 algorithms, and obtains stable expression genes in different tissues, namely PP2 alpha and SKIP; the stable expression genes under high temperature stress are RPS13 and SAMCD; stable expression genes under low temperature stress are SAMCD and EF1 alpha; stable expression genes in the flooding stress leaves are PP2 alpha and SAMCD, and stable expression genes in the roots are TUA and TUB; stable expression genes in drought stress leaves are EF1 alpha and GAPDH, and stable expression genes in roots are PP2 alpha and HIS; the stable expression genes in salt stress leaves are TUB and GAPDH, and the stable expression genes in roots are TUB and RPS13. The reference genes which are most stable under all conditions at the same time are ACT1 and TUB.

Drawings

FIG. 1 shows a real-time quantitative PCR primer melting curve of Tilia miqueliana genes;

FIG. 2 is a box diagram of Ct values of a real-time fluorescence quantitative PCR reference gene;

FIG. 3 average (M) of the expression stability of 15 candidate genes under different tissues and conditions of Tilia miqueliana based on GeNorm algorithm;

FIG. 4 analysis of comparative variation (Vn/Vn+1) of 15 candidate internal genes under different tissue and processing conditions based on GerNorm algorithm;

FIG. 5 relative expression levels of MYB target genes under different stress treatments.

Detailed Description

The invention is further described below with reference to the drawings and examples. The following examples are only for more clearly illustrating the technical aspects of the present invention, and are not intended to limit the scope of the present invention.

Example 1

The embodiment is the extraction and verification of the 15 tilia miqueliana candidate internal reference gene sequences.

6 tissue RNAs of Tilia miqueliana, stems, leaves, bracts, flowers and fruits are respectively extracted, and after equal amount mixing, a full-length cDNA library is constructed, and full-length transcriptome sequencing data of Tilia miqueliana is obtained through transcriptome sequencing and data analysis. According to the 18srRNA, ACT1, AP47, AQP, EF1 alpha, GAPDH, HIS, PP alpha, RPS13, SAMDC, SKIP, TUA, TUB, UBC and UBQ10 gene sequences in the TAIR Arabidopsis database, the sequence numbers are shown in Table 1, and the homologous sequences in the full-length transcriptome sequence of Tilia miqueliana are obtained by adopting local Blast. The CDS region of the 15 homologous sequences of tilia miqueliana is analyzed by adopting bioXM software sequence, and primers are designed at two ends of the CDS region by adopting oligo7 software. And amplifying CDS regions of 15 homologous sequences by adopting PCR reaction, and carrying out multiple sequence comparison on the CDS regions obtained by sequencing and homologous sequences obtained by blast in a transcriptome by using DNAman software, wherein the result is consistent, and the results prove that the homologous gene sequences of tilia miqueliana obtained by blast are true and reliable. The 15 candidate internal reference gene sequences of Tilia miqueliana are respectively: the nucleotide sequence of 18S ribosomal RNA (18 srRNA) is SEQ ID NO.1, the nucleotide sequence of actin (ACT 1) is SEQ ID NO.2, clathrin (AP 47) SEQ ID NO.3, aquaporin (AQP) SEQ ID NO.4, elongation factor (EF 1 alpha) SEQ ID NO.5, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) SEQ ID NO.6, histone (HIS) SEQ ID NO.7, protein phosphatase (PP 2 alpha) SEQ ID NO.8, ribosomal protein S13 (RPS 13) SEQ ID NO.9, S-adenosylmethionine decarboxylase (SAMDC) SEQ ID NO.10, SKI interacting protein (SKIP) SEQ ID NO.11, tubulin (TUA) SEQ ID NO.12, tubulin (TUB) SEQ ID NO.13, ubiquitin conjugating enzyme (UBC) SEQ ID NO.14, polyubiquitin gene (Q10) SEQ ID NO.15.

TABLE 1.15 candidate reference genes

Numbering device	Gene abbreviations	Gene	Arabidopsis thaliana homologous sequences
				1	18srRNA	18s ribosomal RNA	AT3G41768
2	ACT1	Actin protein	AT5G09810
				3	AP47	Clathrin protein	AT5G46630
4	AQP	Aquaporin	AT2G37170
				5	EF1α	Elongation factor	AT5G60390
6	GAPDH	Glyceraldehyde-3-phosphate dehydrogenase	AT2G24270
				7	HIS	Histones (His)	AT4G40040
8	PP2α	Protein phosphatase	AT3G25800
				9	RPS13	Ribosomal protein S13	AT4G00100
10	SAMDC	S-adenosylmethionine decarboxylase	AT3G02470
				11	SKIP	SKI interaction protein	AT1G45020
12	TUA	Tubulin	AT5G19780
				13	TUB	Tubulin	AT1G75780
14	UBC	Ubiquitin conjugating enzyme	AT1G64230
				15	UBQ10	Ubiquitin family genes	AT3G52590

Example 2

This example provides fluorescent quantitative PCR primers for amplifying the 15 candidate reference genes of example.

Primer design was performed using oligo7 software based on the 15 candidate reference gene sequences in example 1. The design principle follows the following principle: (1) the length of the amplified product is 100 bp-250 bp; (2) primer length is between 15-22 bp; (3) a dissolution temperature (Tm) between 57-60 ℃; (3) a GC content of between 40-60%; (4) without hairpin structure and primer dimer.

After the primer design was completed, the primers were initially selected by PCR amplification, and the total of the PCR reaction system was 10. Mu.l, wherein 1. Mu.l of cDNA template was used, and the upstream and downstream primers were 0.5mM each. The PCR reaction procedure was: (1) Pre-denaturation: 94 ℃ for 5min; (2) denaturation: 94-10 s, (3) annealing: 56-10 s, (4) extension: 72-10 s for 35 cycles total; (5) final extension: 72-10 min. The 5 mu LPCR product was detected by 1.5% agarose gel electrophoresis, and a single amplified product was selected and the PCR product with a band size consistent with the expected size was sequenced, and the corresponding primer was retained when the sequencing result was consistent with the corresponding sequence in example 1.

After the comparison results are consistent, the corresponding primers are selected for real-time fluorescence quantitative PCR amplification, the reaction system is 10 μl, and the concentration of the upstream primer and the downstream primer is 0.5 μM. The reaction procedure is: the first step is pre-denaturation for 94-30 s; the second PCR reaction is carried out for 95-10 s, 60-15 s and 40 cycles; and step three, gradually heating the solvent curve at 60-95 ℃. The LinRegPCR software is adopted to calculate the amplification efficiency of the primer (see figure 1) with the real-time fluorescence quantitative PCR dissolution curve in a single peak, and the amplification efficiency is ensured to be more than 1.8.

The primer sequence for amplifying the 18srRNA is SEQ ID NO.16/SEQ ID NO.17, and the amplification efficiency is 1.88; the primer sequence for amplifying ACT1 is SEQ ID NO.18/SEQ ID NO.19, and the amplification efficiency is 1.87; the primer sequence for amplifying the AP47 is SEQ ID NO.20/SEQ ID NO.21, and the amplification efficiency is 1.79; the primer sequence for amplifying the AQP is SEQ ID NO.22/SEQ ID NO.23, and the amplification efficiency is 1.87; the primer sequence for amplifying EF1 alpha is SEQ ID NO.24/SEQ ID NO.25, and the amplification efficiency is 1.88; the primer sequence for amplifying GAPDH is SEQ ID NO.26/SEQ ID NO.27, and the amplification efficiency is 1.90; the primer sequence for amplifying the HIS is SEQ ID NO.28/SEQ ID NO.29, and the amplification efficiency is 1.90; the primer sequence for amplifying the PP2 alpha is SEQ ID NO.30/SEQ ID NO.31, and the amplification efficiency is 1.92; the primer sequence for amplifying RPS13 is SEQ ID NO.32/SEQ ID NO.33, and the amplification efficiency is 1.88; the primer sequence for amplifying the SAMDC is SEQ ID NO.34/SEQ ID NO.35, and the amplification efficiency is 1.90; the primer sequence for amplifying SKIP is SEQ ID NO.36/SEQ ID NO.37, and the amplification efficiency is 1.84; the primer sequence for amplifying TUA is SEQ ID NO.38/SEQ ID NO.39, and the amplification efficiency is 1.89; the primer sequence for amplifying TUB is SEQ ID NO.40/SEQ ID NO.41, and the amplification efficiency is 1.87; the primer sequence for amplifying UBC is SEQ ID NO.42/SEQ ID NO.43, and the amplification efficiency is 1.90; the primer sequence for amplifying UBQ10 is SEQ ID NO.44/SEQ ID NO.45, and the amplification efficiency is 1.90; .

Example 3

The embodiment provides a material and a processing method for screening candidate internal reference genes of tilia miqueliana.

Different tissue samples: the tender root, tender stem, mature leaf, bract, bud, flower (full bloom), fruit, immature seed and mature seed of Tilia miqueliana mature plant of 15 years old have 9 tissues, and 3 biological repeats of each tissue. Selecting Tilia miqueliana tissue culture seedlings with consistent growth, and carrying out different stress treatments: (1) Placing the high-temperature stress plants in an illumination incubator, wherein the photoperiod is set to be 16h illumination/8 h darkness, and the temperature is set to be 42 ℃; (2) Placing the low-temperature stress plants in an illumination incubator, wherein the photoperiod is set to be 16h illumination/8 h darkness, and the temperature is set to be 4 ℃; (3) Flooding stress potted plants are subjected to flooding treatment, and the water surface is about 2cm higher than the surface of the substrate; (4) Planting the tissue culture seedling of Tilia miqueliana under drought stress in Hoagland's water culture nutrient solution (pH=5.5), and adding 15% PEG-6000 into the nutrient solution; (5) Salt stress Tilia miqueliana tissue culture seedlings are planted in Hoagland's water culture nutrient solution (pH=5.5), and 3 permillage NaCl is additionally added into the nutrient solution. In each stress treatment, tissue materials of tissue culture seedlings of Tilia miqueliana are collected at treatments 0, 1, 3, 6, 12, 24 and 48 hours respectively, wherein only leaf tissues are collected in the high-temperature and low-temperature stress treatment, and 2 tissues of the leaf and root are collected in the flooding, drought and salt stress treatments respectively. 3 biological replicates were set at each time point, and after sample collection, the samples were snap frozen with liquid nitrogen and stored in a-80 ℃ freezer.

Example 4

The embodiment provides a real-time fluorescent quantitative PCR reaction experimental method for screening Tilia miqueliana candidate reference genes.

The RNA of each sample in example 3 is extracted by using a polysaccharide polyphenol plant RNA extraction kit (No: 0416-50GK, hua-Viea, beijing), the total RNA integrity of the RNA sample is detected by 1.2% agarose gel electrophoresis, the purity and concentration of the RNA are detected by using a ultramicro ultraviolet spectrophotometer (Colibri LB 915), the integrity is well reserved, the concentration is more than 50 ng/. Mu.l, and the OD260/280 value is about 2.0. By Goldenstar ^TM cDNA synthesis was performed in RT6 cDNA Synthesis Kit (TSK 301S, nanjing qing family Biotech Co., ltd.). A total of 20. Mu.l of reverse transcription system, wherein RNA-0.5. Mu.g, oligo dT and random primer-0.5 mM, other components are referred to the specification; the reaction procedure: 25-10 min, 50-15 min and 85-5 min. The reverse transcription product is diluted 5 times and then stored at-20 ℃ for standby.

Using the cDNA obtained in the above reaction as a template, 15 pairs of fluorescent quantitative primers obtained in example 2 were selected, and a kit of ChamQ Universal SYBR qPCR Master Mix (Q711-02, nanjinouzan Biotechnology Co., ltd.) was usedIn StepOne ^TM Real-Time fluorescent quantitative PCR amplification was performed on Real-Time PCR System, with 3 replicates per sample. The PCR reaction was 10. Mu.l, with a concentration of the upstream and downstream primers of 0.5. Mu.M and a cDNA template of 1. Mu.l. The reaction procedure is: the first step is pre-denaturation for 94-30 s; the second PCR reaction is carried out for 95-10 s, 60-15 s and 40 cycles; and step three, gradually heating the solvent curve at 60-95 ℃ and collecting fluorescent signals every 0.5 ℃. The Ct average value of all reference genes obtained by real-time fluorescence quantitative PCR is shown in FIG. 2.

As shown in FIG. 2, the Ct values of all 15 candidate genes range from 13 to 31, wherein the expression level of 18srRNA is highest, and the Ct average value is 12.68; TUA expression was the lowest and Ct mean was 30.81. Meanwhile, SKIP is the most stably expressed gene, and the standard deviation (1.75) of Ct values is the smallest in all samples; UBC is the least stably expressed gene, with the greatest standard deviation (4.42) of Ct values in all samples.

Example 5

The embodiment provides a method for carrying out real-time fluorescence quantitative candidate internal reference gene stability sequencing analysis on tilia miqueliana by adopting a GeNorm algorithm.

The GeNorm software download address is https:// gene.cmgg.be/, and the steps and standards are as follows:

GeNorm first calculates the average value (M) of the expression stability of candidate reference genes, and the smaller the M value is, the better the stability of the reference genes is, and the calculated results are shown in Table 2 and FIG. 3. When the M value of the candidate reference gene is lower than 1.5, the candidate reference gene can be used as an alternative to the reference gene. In tissue samples, the two most stable genes are HIS and PP2 a, the most unstable is UBQ, where the M values of UBQ, APQ, RPS, AP47, TUA, SKIP and TUB genes are greater than 1.5, and are not suitable as alternative reference genes. Under stress treatment conditions, wherein in high temperature stress, the most stable two genes are SAMCD and TUA, the most unstable is UBC, and the M values of all candidate reference genes are less than 1.5; in low temperature stress, the two most stable genes are EF1 alpha and SAMCD, the least stable gene is 18srRNA, and the M value of all candidate internal reference genes is less than 1.5; in the flooding stress leaf, the most stable two genes are PP2 alpha and SKIP, the least stable is 18srRNA, and the M values of all candidate internal reference genes are less than 1.5; in drought stress leaves, the two most stable genes are ACT1 and EF1 alpha, and the most unstable is UBC, wherein the M value of the genes is more than 1.5, and the genes are not suitable to be used as alternative reference genes; in the salt stress leaf, the most stable two genes are ACT1 and SAMCD, the most unstable is UBC, and the M value of all candidate internal reference genes is less than 1.5; in flooding stress roots, the most stable two genes are ACT1 and EF1 alpha, the most unstable is SKIP, and the M values of all candidate internal reference genes are less than 1.5; in drought stress roots, the two most stable genes are EF1 alpha and HIS, the most unstable is UBC, and the M values of all candidate internal reference genes are less than 1.5; in the salt stress root slice, the most stable two genes are ACT1 and EF1 alpha, the most unstable is UBC, and the M values of all candidate internal reference genes are less than 1.5; of all samples, the most stable two genes were ACT1 and EF 1. Alpha. And the least stable was UBQ, with M values of UBQ, UBC, AP, 18srRNA and AQP genes greater than 1.5, which are not suitable as alternative reference genes.

Secondly, obtaining a variation value V of the pairwise comparison by using GeNorm software through a standardization factor pairing difference analysis, and finally selecting the proper quantity of the reference genes. When the Vn/n+1 value is smaller than the threshold value of 0.2, the most suitable gene reference number is n, and the first n genes of the reference gene expression stability average value (M) are selected as the reference genes. The GeNorm program can be used for screening any number of reference genes under any condition, more than two reference genes are selected instead of the conventional single reference gene, which is favorable for correcting systematic deviation, obtaining more reliable accurate quantitative results of genes and has extremely important significance for biological research of fine expression differences. The research result shows that V6/7 (0.193) is less than 0.2 in the analysis of Tilia miqueliana tissue material, so that 6 internal reference genes are required to be corrected and standardized together to obtain accurate quantification; in 8 groups of materials of high temperature stress, low temperature stress, flooding stress blade, drought stress blade, salt stress blade, flooding stress root, drought stress root and salt stress root, the variation value V is less than 0.2, so that the accurate expression quantity can be obtained only by calibrating 1 reference gene; V5/V6 (0.194) is less than 0.2 when the total material is comprehensively analyzed, so that 6 reference genes are required to be corrected and standardized together to obtain accurate quantification.

TABLE 2 ranking of stability of 15 candidate internal reference genes under different Material and stress treatments based on different algorithms

The following is noted: genes with a gene M value greater than 1.5 marked by boxes in the GeNorm algorithm sequencing result are not suitable as alternative reference genes under the condition.

Example 6

The embodiment provides a method for carrying out the stability sequencing analysis of the Tilia miqueliana real-time fluorescence quantitative candidate internal reference genes by adopting a NormFinder algorithm.

The NormFinder software download address in the invention is https:// www.moma.dk/NormFinder-software/, and the steps and standards are as follows:

normFinder is an algorithm based on analysis of variance, which evaluates the stability of an internal reference by calculating a Stable Value (SV) of gene expression, the smaller the value, the more the stability is; whereas the worse the stability. The stability analysis results of the reference gene are shown in Table 2. After NormFinder analysis, in the tissue sample, the most stable gene was SKIP and the least stable gene was UBQ; under high temperature stress, the most stable gene is RPS13, and the least stable gene is UBC; under low temperature stress, the most stable gene is SAMCD and the least stable gene is SAMCD; in leaves under flooding stress, the most stable gene is AP47, and the least stable gene is 18srRNA; in drought stress leaves, the most stable gene is GAPDH and the least stable gene is UBC; in salt stress leaves, the most stable gene is HIS and the least stable gene is UBC; in roots under flooding stress, the most stable gene is TUB and the least stable gene is SKIP; in drought stressed roots, the most stable gene is PP2 alpha, and the least stable gene is UBC; in salt stressed roots, the most stable gene is TUB and the least stable gene is UBC; comprehensive analysis results of all materials show that the most stable gene is TUB and the least stable gene is UBQ. Comparing NormFinder to GeNrom results shows that there is some difference in the most stable genes obtained by the two algorithms, but the least stable genes are identical.

Example 7

The embodiment provides a method for carrying out the stability sequencing analysis of the Tilia miqueliana real-time fluorescence quantitative candidate internal reference genes by adopting a BestKeeper algorithm.

The download address of the BestKeeper software is https:// www.gene-identification.de/BestKeeper.html, and the steps and standards are as follows:

the bestdeeper program selects the gene that is most stably expressed by comparing the standard deviation and the coefficient of variation of Ct values, and the smaller the standard deviation and the coefficient of variation, the better the stability, and conversely the worse the stability. The stability analysis results of the reference gene are shown in Table 2. After BestKeeper analysis, in the tissue sample, the most stable gene is UBC and the least stable gene is UBQ; under high temperature stress, the most stable gene is ACT1, and the least stable gene is UBC; under low temperature stress, the most stable gene is RPS13, and the least stable gene is 18srRNA; in leaves under flooding stress, the most stable gene is SAMCD and the least stable gene is 18srRNA; in drought stress leaves, the most stable gene is RPS13, and the least stable gene is 18srRNA; in salt stress leaves, the most stable gene is EF1 alpha and the least stable gene is UBC; in roots under flooding stress, the most stable gene is SKIP, and the least stable gene is GAPDH; in drought stressed roots, the most stable gene is UBQ and the least stable gene is SAMCD; in salt stressed roots, the most stable gene is UBC and the least stable gene is EF1 a; comprehensive analysis results of all materials show that the most stable gene is SKIP and the least stable gene is UBQ. As compared with the results of GeNrom and NormFinder, the most stable genes were slightly different, but the least stable genes were substantially identical.

Example 8

The embodiment provides a method for carrying out the stability sequencing analysis of the Tilia miqueliana real-time fluorescence quantitative candidate internal reference genes by adopting a Delta Ct algorithm.

In the invention, delta Ct algorithm refers to: : silver N, best S, jiang J, thein SL.selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR.BMC Molecular Biology,2006,7:33. The steps and standards are as follows:

the Delta Ct algorithm determines the stability of a reference gene by comparing the relative expression levels of paired genes in the same sample. The Standard Deviation (SD) of the reference gene was used as the standard for sequencing, the smaller the standard deviation, the better the stability, and conversely the worse the stability. The stability analysis results of the reference gene are shown in Table 2. After Delta Ct analysis, in the tissue sample, the most stable gene is SKIP and the least stable gene is UBQ; under high temperature stress, the most stable gene is RPS13, and the least stable gene is UBC; under low temperature stress, the most stable gene is SAMCD and the least stable gene is 18srRNA; in leaves under flooding stress, the most stable gene is UBQ and the least stable gene is 18 srrrna; in drought stress leaves, the most stable gene is GAPDH and the least stable gene is UBC; among the salt stress leaves, the most stable gene is TUB and the least stable gene is AP47; in roots under flooding stress, the most stable gene is TUB and the least stable gene is SKIP; in drought stressed roots, the most stable gene is PP2 alpha, and the least stable gene is UBC; in salt stressed roots, the most stable gene is TUB and the least stable gene is UBC; comprehensive analysis results of all materials show that the most stable gene is ACT1 and the least stable gene is UBQ. As compared with GeNrom, normFinder and Bestkeeper results, the most stable genes have a certain difference, but the least stable genes are basically consistent.

Example 9

The embodiment provides comprehensive sequencing of the stability results of the candidate internal reference genes of Tilia miqueliana by adopting a RefFinder algorithm and adopting real-time fluorescence quantification of Tilia miqueliana obtained by different algorithms.

The RefFinder software download address in the invention is https:// wwleonxie.com/preferencene.php, and the steps and standards are as follows:

the refFinder program calculates the geometric mean of the stability sequences of the 15 candidate reference genes by four algorithms, namely GeNorm, normfinder, bestKeeper and Delta CT, to obtain the final comprehensive ranking. The stability analysis results of the reference gene are shown in Table 2. After refFinder analysis, in the tissue sample, the most stable gene was PP 2. Alpha. And the least stable gene was UBQ; under high temperature stress, the most stable gene is RPS13, and the least stable gene is UBC; under low temperature stress, the most stable gene is SAMCD and the least stable gene is 18srRNA; in leaves under flooding stress, the most stable gene is PP2 alpha and the least stable gene is 18 srrrna; among drought stress leaves, the most stable gene is GA PDH, and the least stable gene is UBC; in salt stress leaves, the most stable gene is EF1 alpha and the least stable gene is UBC; in roots under flooding stress, the most stable gene is TUB and the least stable gene is UBC; in drought stressed roots, the most stable gene is PP2 alpha, and the least stable gene is RPS13; in salt stressed roots, the most stable gene is TUB and the least stable gene is GAPDH; comprehensive analysis results of all materials show that the most stable gene is ACT1 and the least stable gene is UBQ.

Example 10

This example demonstrates the effect of the candidate reference gene application provided by the present invention. MYB gene is selected as target gene, and the nucleotide sequence is SEQ ID NO.46. The gene is homologous to the AT4G38620 nucleotide sequence in the TAIR Arabidopsis database. MYB transcription factors are widely existing transcription factor families in plants, are involved in various secondary metabolic reaction processes such as flavonoid metabolic pathways, biosynthesis of thioglucoside, biosynthesis of anthocyanin and formation of plant secondary cell walls, and play an important role in cell cycle regulation, secondary metabolism, stress response and other vital activities. Research shows that MYB transcription factor can regulate and control the expression of stress resistance related gene indirectly or directly to make plant resist drought, low temperature, high salt, illumination and other bad environment. Therefore, the two reference genes with the best stability and the 1 reference gene with the worse stability are respectively selected in the embodiment, the expression quantity of the tilia miqueliana MYB gene in leaves and roots under the treatment of flooding stress, drought stress and salt stress is verified by utilizing a real-time quantitative PCR reaction, and the application effects are compared. The method comprises the following specific steps:

(1) Test material and method of treatment

The experimental materials are selected from Tilia miqueliana tissue culture seedlings, and after the tissue culture seedlings are subjected to seedling hardening and transplanting for 30 days, the following stress treatment is carried out. Wherein, the flooding stress adopts a basin covering method, and the water surface is kept to be 2cm higher than the surface of the culture medium during the treatment period; the drought stress is simulated by adding PEG-6000 under the condition of water culture, and 15% PEG-6000 is added after the test material is transferred into Hoagland's water culture nutrient solution (pH=5.5); the salt stress is simulated by adding NaCl under the water culture condition, and 3 permillage NaCl is added after the test material is transferred into Hoagland's water culture nutrient solution (pH=5.5).

After treatment, 7 time points of Tilia miqueliana tissue culture seedlings are respectively collected at 0, 1, 3, 6, 12, 24 and 48 hours, and 2 tissue materials of leaves and roots are respectively collected. 3 biological replicates were set at each time point, and after sample collection, the samples were snap frozen with liquid nitrogen and stored in a-80 ℃ freezer.

(2) RNA extraction and cDNA Synthesis

Extracting RNA of each sample by using a polysaccharide polyphenol plant RNA extraction kit (No: 0416-50GK, hua-Viea, beijing), detecting the total RNA integrity of the RNA sample by 1.2% agarose gel electrophoresis, detecting the purity and concentration of the RNA by using a superfine ultraviolet spectrophotometer (Colibri LB 915), and preserving the RNA sample with good integrity, concentration of more than 50 ng/mul and OD260/280 value of about 2.0.

By Goldenstar ^TM cDNA synthesis was performed in RT6 cDNA Synthesis Kit (TSK 301S, nanjing qing family Biotech Co., ltd.). A total of 20. Mu.l of reverse transcription system, wherein RNA-0.5. Mu.g, oligo dT and random primer-0.5 mM, other components are referred to the specification;the reaction procedure: 25-10 min, 50-15 min and 85-5 min. The reverse transcription product is diluted 5 times and then stored at-20 ℃ for standby.

(3) MYB target gene real-time quantitative PCR primer design and specificity verification

According to the nucleotide sequence SEQ ID NO.46 of the Tilia miqueliana MYB gene, primer design is carried out by adopting oligo7 software. The design principle follows the following principle: (1) the length of the amplified product is 100 bp-250 bp; (2) primer length is between 15-22 bp; (3) a dissolution temperature (Tm) between 57-60 ℃; (3) a GC content of between 40-60%; (4) without hairpin structure and primer dimer.

After the primer design was completed, the primers were initially selected by PCR amplification, and the total of the PCR reaction system was 10. Mu.l, wherein 1. Mu.l of cDNA template was used, and the upstream and downstream primers were 0.5mM each. The PCR reaction procedure was: (1) Pre-denaturation: 94 ℃ for 5min; (2) denaturation: 94-10 s, (3) annealing: 56-10 s, (4) extension: 72-10 s for 35 cycles total; (5) final extension: 72-10 min. And detecting the 5 mu LPCR product by 1.5% agarose gel electrophoresis, selecting a single amplified product, sequencing the PCR product with the band size meeting the expectations, and retaining the primer when the sequencing result is consistent with the target gene sequence.

After the comparison results are consistent, the corresponding primers (SEQ ID NO.47 and SEQ ID NO. 48) are selected for real-time fluorescence quantitative PCR amplification, the reaction system is 10 mu l, and the concentration of the upstream primer and the downstream primer is 0.5 mu M. The reaction procedure is: the first step is pre-denaturation for 94-30 s; the second PCR reaction is carried out for 95-10 s, 60-15 s and 40 cycles; and step three, gradually heating the solvent curve at 60-95 ℃. The amplification efficiency of the primers with a single peak of the real-time fluorescent quantitative PCR dissolution curve (see FIG. 1) was calculated to be 1.921 using LinRegPCR software.

(4) Real-time quantitative PCR reaction

The 2 most stable genes and the one most unstable gene under each stress condition obtained by the analysis in example 9 were selected (see table 3). And (3) taking the cDNA obtained in the step (2) as a template, and simultaneously carrying out real-time fluorescence quantitative PCR amplification on the selected and verified internal reference gene and target gene to obtain a corresponding Ct value. During the test, using the ChamQ Universal SYBR qPCR Master Mix (Q711-02, nanjinouzan Biotechnology Co., ltd.) kit, at StepOne ^TM Real-Time fluorescent quantitative PCR amplification was performed on Real-Time PCR System, with 3 replicates per sample. The PCR reaction was 10. Mu.l, with a concentration of the upstream and downstream primers of 0.5. Mu.M and a cDNA template of 1. Mu.l. The reaction procedure is: the first step is pre-denaturation for 94-30 s; the second PCR reaction is carried out for 95-10 s, 60-15 s and 40 cycles; and step three, gradually heating the solvent curve at 60-95 ℃ and collecting fluorescent signals every 0.5 ℃.

TABLE 3 selection of reference genes under different stress conditions

(1) Data processing and analysis

The Ct value is inversely proportional to the expression level of the gene, and the larger the Ct value is, the lower the expression level of the gene is, whereas the smaller the Ct value is, the higher the expression level of the gene is; the method for calculating the relative expression quantity of the genes adopts a 2-delta Ct method and comprises the following specific steps: delta ct=ct (target gene) -Ct (reference gene), delta ct=delta Ct (treatment) -delta Ct (control), 2 ^-ΔΔCt Relative expression amount.

(2) Test results and analysis

As shown in FIG. 5, the relative expression levels of the reference gene MYB under flooding stress treatment conditions are shown in FIGS. 5a and 5 b. In leaf tissues, when PP2 alpha and SAMCD with better stability are used as reference genes, the relative expression quantity of the target gene MYB is more consistent, namely the expression quantity is obviously reduced after treatment; whereas 18srRNA with poor stability was used as an internal reference gene, the magnitude of MYB gene down-regulation was overestimated to some extent (see FIG. 5 a). In root tissues, when TUB and TUA with better stability are used as internal references, the relative expression quantity of target genes is more consistent, MYB genes are up-regulated for expression, peak values are respectively reached at 1h/12h and 3h/12h, and then the expression quantity is gradually reduced; whereas UBC with poor stability is used as an internal reference gene, the up-regulation of MYB gene was severely underestimated (see FIG. 5 b).

Under drought stress conditions, the relative expression levels of the reference gene MYB are shown in fig. 5c and 5 d. In leaf tissues, when EF1 alpha and GAPDH with better stability are adopted as reference genes, the relative expression quantity of a target gene MYB is more consistent, namely the expression quantity is obviously reduced after treatment; and UBC with poor stability is adopted as an internal reference gene, and the reduction amplitude of MYB genes is overestimated to a certain extent (see FIG. 5C). In root tissues, when PP2 alpha and HIS with better stability are used as internal references, the relative expression quantity of target genes is more consistent, and the MYB expression quantity is obviously reduced when the treatment is carried out for 1 h; and RPS13 with poor stability is adopted as an internal reference gene, and the relative expression quantity of MYB genes shows abnormal up-regulation trend after treatment (see figure 5D).

Under salt stress conditions, the relative expression levels of the reference gene MYB are shown in fig. 5e and 5 f. In leaf tissues, when TUB and GAPDH with better stability are adopted as reference genes, the relative expression quantity of the target gene MYB is more consistent, namely the relative expression quantity is obviously reduced after treatment; whereas UBC with poor stability was used as an internal reference gene, MYB genes exhibited abnormal upregulation at 3h and 48h post-treatment (see FIG. 5E). In root tissues, when TUB and RPS13 with better stability are adopted as internal references, the relative expression quantity of target genes is more consistent, namely the expression quantity of MYB genes shows an ascending trend, and peaks appear when the treatment is carried out for 6h and 48 h; and GAPDH with poor stability is adopted as an internal reference gene, the relative expression quantity of MYB genes is abnormally increased, and the maximum value is reached 24h after treatment (see figure 5F).

The foregoing is merely a preferred embodiment of the present invention, and it should be noted that modifications and variations could be made by those skilled in the art without departing from the technical principles of the present invention, and such modifications and variations should also be regarded as being within the scope of the invention.

Sequence listing

<110> institute of plant Material of national academy of sciences of China, jiangsu province

<120> real-time quantitative PCR reference gene under different tissues and stress treatment conditions of Tilia miqueliana, and screening method and application thereof

<160> 48

<170> SIPOSequenceListing 1.0

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<212> DNA

<213> Tilia miqueliana (Tilia miqueliana Maxim)

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aaggaatgct acctggttga tcctgccagt agtcatatgc ttgtctcaaa gattaagcca 60

tgcatgtgta agtatgaact aattcagact gtgaaactgc gaatggctca ttaaatcagt 120

tatagtttgt ttgatggtat ttgctactcg gataaccgta gtaattctag agctaatacg 180

tgcaacaaac cccgacttct ggaagggatg catttattag ataaaaggtc gacgcgggct 240

tcgcccgttg ctctgatgat tcatgataac tcgacggatc gcacggcctt tgtgccggcg 300

acgcatcatt caaatttctg ccctatcaac tttcgatggt aggatagtgg cctactatgg 360

tggtgacggg tgacggagaa ttagggttcg attccggaga gggagcctga gaaacggcta 420

ccacatccaa ggaaggcagc aggcgcgcaa attacccaat cctgacacgg ggaggtagtg 480

acaataaata acaataccgg gctctatgag tctggtaatt ggaatgagta caatctaaat 540

cccttaacga ggatccattg gagggcaagt ctggtgccag cagccgcggt aattccagct 600

ccaatagcgt atatttaagt tgttgcagtt aaaaagctcg tagttggacc tagggttggg 660

tcggccggtc cgcctcacgg tgtgcaccgg tcggctcgtc cctactgccg gcgatgcgct 720

cctggcctta actggccggg tcgttcctcc ggcgctgtta ctttgaagaa attagagtgc 780

tcaaagcagg cctacgcttg tatacattag catgggataa catcatagga tttcggtcct 840

attctgttgg ccttcgggat cggagtaatg attaacaggg acagtcgggg gcattcgtat 900

ttcatagtca gaggtgaaat tcttggattt atgaaagacg aacaactgcg aaagcatttg 960

ccaaggatgt tttcattaat caagaacgaa agttgggggc tcgaagacga tcagataccg 1020

tcctagtctc aaccataaac gatgccgacc agggatcggc ggatgttgct tttaggactc 1080

cgccggcacc ttatgagaaa tcaaagtctt tgggttccgg ggggagtatg gtcgcaaggc 1140

tgaaacttaa aggaattgac ggaagggcac caccaggagt ggagcctgcg gcttaatttg 1200

actcaacacg gggaaactta ccaggtccag acatagtaag gattgacaga ctgagagctc 1260

tttcttgatt ctatgggtgg tggtgcatgg ccgttcttag ttggtggagc gatttgtctg 1320

gttaattccg ttaacgaacg agacctcagc ctgctaacta gctacgcgga ggtgatcctc 1380

cgtggctagc ttcttagagg gactatggcc gcttaggcca aggaagtttg aggcaataac 1440

aggtctgtga tgcccttaga tgttctgggc cgcacgcgcg ctacactgat gtattcaacg 1500

agtctatagc cttggccgac aggcccgggt aatctttgaa atttcatcgt gatggggata 1560

gatcattgca attgttggtc ttcaacgagg aattcctagt aagcgcgagt catcagctcg 1620

cgttgactac gtccctgccc tttgtacaca ccgcccgtcg ctcctaccga ttgaatggtc 1680

cggtgaagtg ttcggatcgc ggcgacgtgg gcggttcgct gcccgcgacg tcgcgagaag 1740

tccactgaac cttatcattt agaggaagga gaagtcgtaa caaggtttcc gtaggtgaac 1800

ctgcggaagg atcattgtcg aaacctgcct agcagaacga cccgcgaacg tgttaactaa 1860

acaccgaggg gagcgaggat gccttcgtgc cccgagcccc cctcagagcc tgggcgtgcg 1920

cgaccgtggc ttcactttgc cttatgggcg ggtgagacgc gcggtcacgt cgcccgtggc 1980

caaacaacga acccccggcg tgaattgcgc caaggaacca tataacgaaa aaagagcacg 2040

ttggcagttg ccgcccgttc gcggtgtgcg cggaccgcct cgtgactcct ttcttgtgaa 2100

aatttaaaac gactctcggc aacggatatc tcggctctcg catcgatgaa gaacgtagcg 2160

aaatgcgata cttggtgtga attgcagaat ccgtgaacca tcgagtcttt gaacgcaagt 2220

tgcgcccaag ccattaggcc gagggcacgc ctgcctgggt gtcacgcatc gtcgccccct 2280

ccacccttta gcc 2293

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<212> DNA

<213> Tilia miqueliana (Tilia miqueliana Maxim)

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tttgtagtat tttaaaagat ggctgatgct gaggatattc aaccccttgt ctgtgacaat 60

ggaactggaa tggtgaaggc tggttttgca ggtgatgatg ctcccagggc agtttttccc 120

agtattgttg gtcgaccccg acacactggt gtcatggttg ggatgggtca gaaggatgcc 180

tatgtaggag atgaggcaca atctaaaaga ggtatcctta ctttgaaata tcccattgag 240

catggtattg tcagtaactg ggatgatatg gaaaagatct ggcatcatac attctacaat 300

gagctccgtg ttgctcctga ggagcaccct gtacttctca cagaggcacc tctcaacccc 360

aaggctaaca gagaaaagat gacccagatc atgtttgaga ccttcaatgt acctgccatg 420

tatgttgcca tccaggctgt tctctctttg tatgccagtg gtcgtacaac gggtattgta 480

ctggattctg gtgatggtgt ttctcacact gtgccaatct atgaagggta tgctcttcca 540

catgccatcc tccgcctaga ccttgctggt cgtgatctca ctgatgcttt gatgaagatt 600

cttactgaga gaggttacat gttcaccacc actgctgaac gggaaattgt ccgtgacatg 660

aaggagaagc ttgcttatgt tgccctggac tacgagcagg aactggagac tgccaagagc 720

agctcttctg ttgagaagaa ctatgagtta cctgatggac aagtcattac tatcggagct 780

gagagattcc gttgtccaga agtcctcttc cagccatctc tcattgggat ggaagctgct 840

ggaatccatg aaactaccta caactctatc atgaagtgtg atgtggatat caggaaggat 900

ctttatggta acattgtgct tagtggtggt tcaaccatgt tccctggtat tgcagaccgt 960

atgagcaagg agatcactgc ccttgctcca agcagcatga agattaaggt tgttgcacca 1020

ccagagagaa agtacagtgt ctggattgga ggatctatct tggcatcact cagcaccttc 1080

cagcagatgt ggatttccaa gggcgagtat gatgagtctg gtccatccat tgtccacagg 1140

aagtgcttct aagtttta 1158

<210> 3

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<212> DNA

<213> Tilia miqueliana (Tilia miqueliana Maxim)

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tggatcgaat atgccggtgg ccgcttctgc catctacttc ttgaatctcc gcggtgatgt 60

cctcatcaat cgtctctacc gcgacgatgt cgggggaaac atggtggatg cctttcgaat 120

gcacattatg caaacgaaag aactcggtac atgtccggtg cgacagattg gaggttgctc 180

tttcttttac atgagaatca gcaatgtata catagtaatt gttgtcagca gcaatgctaa 240

tgtagcttgt gctttcaagt ttgttgttga ggctgttgct ttgttcaaat cttattttgg 300

tggtgctttt gatgaagatg ccattcgtaa taatttcgtt ctgatttatg aattgttgga 360

tgaaatcatg gactttggct acccacaaaa tctttctcct gaaattttaa agctttacat 420

tactcaggaa ggcgtgcggt ccccattttc atccaaggcc ttagataagc ctgttccaaa 480

tgcaacttta caagttacag gtgctgttgg ctggcgaaga gagggacttg tttataaaaa 540

gaatgaggtg tttctggaca tcgtggaaag tgtaaaccta cttatgtctt caaaaggcag 600

tgttctgcgt tgtgatgtta ctggaaaggt tcttatgaag tgcttcctct ctggaatgcc 660

tgatctgaag ttgggcttga atgataagat tgggcttgag aaagaatcac aactaaaatc 720

ccgtcctact aaaagtggca agactattga gctggacgat gtcactttcc atcagtgtgt 780

gaatcttaca agattcaact ctgaaaagac tgttagtttt gtgccaccag atggtgaatt 840

tgaattgatg aagtaccgta ttaccgaggg tgttaatctt cctttccggg tattgccaac 900

aatcaaggaa cttggtcgaa cacgaatgga agtaaatgtt aaggttaaaa gtgtctttgg 960

agcaaaaatg tttgctcttg gagttgtcat caaaattcct gtcccaaaac aaacagcgaa 1020

aacaagtttc caagtcacat caggtcgagc aaagtataat gctgctattg attgcttggt 1080

ttggaagata agaaaattcc ctggacaaac agagccaacc ttgagtgcag aagttgagtt 1140

aatttctaca atggcagaaa agaagtcttg gactaagcca ccaattcaga tggaatttca 1200

ggttccaatg tttactgcct ctggtttacg tgtccgattt cttaaggtgt gggagaagag 1260

tgggtataac acagttgagt gggttcgtta tattaccaag gctggatcat atgagatcag 1320

gtgctagaaa catgggattg g 1341

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<213> Tilia miqueliana (Tilia miqueliana Maxim)

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tctaaagcac taagaggcca tggtaaagaa cattgaggtt ggtggtgagt tccaagccaa 60

ggactaccat gatcctccac cagctccgtt ggttgacgcc gaggagttaa cacagtggtc 120

cttttacagg gctataatag ctgagttcat tgccacgctg ttgtttttgt acattactgt 180

gttgacagtg atcggttaca agagccagac tgctaagggc gatgatgact gtggtggtgt 240

tggcattctt ggcattgctt gggcttttgg tggcatgatc tttatccttg tttactgcac 300

tgctggtatc tcaggaggac acatcaaccc agcagtgacc tttgggcttt tcctggctag 360

gaaggtgtcc ttggtccgag ccatattgta catagctgct caatgtttgg gagccatctg 420

tggatgtggg ttggtgaagg cattccaaaa gtcttactac aatcagtatg gaggaggagc 480

caacagcctc tctgaaggat acagcactgg aaccggtttg gcagctgaaa ttatcggcac 540

ctttgttctt gtctacactg ttttctctgc aactgatccc aagaggaatg caagagactc 600

ccatgtccct gtcttggcac cacttcccat tggatttgct gtgttcatgg ttcacttggc 660

cactattccg atcactggca ccggtatcaa ccctgctcgt agttttggag ctgctgttat 720

ctacaaccag aacaagccat gggatgacca gtggattttc tgggttggac ctttcattgg 780

tgctgccatt gctgcaatct atcaccagta tatcctgagg gcaggtgctg ctaaagctct 840

gggatcctac aggagcagct ctgtcatata aaaaaattaa gcaa 884

<210> 5

<211> 1380

<212> DNA

<213> Tilia miqueliana (Tilia miqueliana Maxim)

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taagttaata ctgcatcatg ggtaaggaga aggttcacat taacattgtg gttattggcc 60

atgtcgactc tggaaagtcg accaccactg gtcacttgat ctacaagctt ggaggtattg 120

acaagcgtgt gattgagagg ttcgagaagg aggctgctga gatgaacaag agatcattca 180

agtatgcttg ggtgcttgac aagttgaagg ctgagcgtga gcgtggtatt accattgata 240

ttgccttgtg gaagtttgag accaccaaat actactgcac tgtcattgat gcccctggac 300

atcgtgactt tatcaagaac atgattaccg gtacctctca ggctgactgt gctgtcctta 360

tcattgactc caccactgga ggttttgaag ctggtatttc caaggatgga cagacccgtg 420

agcatgctct ccttgccttc actcttggtg tgaagcaaat gatttgctgc tgcaacaaga 480

tggatgccac aactcccaaa tactcaaagg caaggtatga tgaaatcgtg aaggaagttt 540

cttcctacct gaagaaggtt ggttacaacc ctgacaagat tccattcgtt cccatctctg 600

gttttgaggg tgacaacatg attgagaggt ccaccaacct tgactggtac aagggtccaa 660

ccctccttga ggcacttgac cagatcaatg agcctaagag acccactgac aagccactcc 720

gtctcccact tcaggatgtc tacaagattg gaggtattgg aacagtccca gtgggtcgtg 780

ttgaaactgg tgtcctcaag cctggtatgg ttgttacctt tggtccctct ggattgtcca 840

ctgaagttaa gtccgttgag atgcatcatg aggctcttca ggaggctctt cctggtgaca 900

atgttggctt caatgttaag aatgttgctg tcaaggatct caagcgagga tttgtcgcct 960

caaactccaa ggatgatcct gccaagggag cagccaactt cacttcccaa gtcatcatca 1020

tgaaccatcc aggtcagatt ggaaatggtt atgctcctgt ccttgattgc cacacttccc 1080

acattgctgt caagtttgca gagatcttga ccaagattga caggcgttct ggtaaggagc 1140

ttgagaagga gcccaagttc ttgaagaacg gtgatgctgg ttttgttaag atgattccca 1200

ccaagcccat ggttgtggaa actttctctg aataccctcc acttggacgt tttgccgtga 1260

gggacatgag gcagacagtt gctgttggtg tcatcaagag tgtggataaa aaggacccaa 1320

ctggagccaa ggtgaccaag tctgctgcca agaagggtgg caagtgaacc gtgcaggttt 1380

<210> 6

<211> 1044

<212> DNA

<213> Tilia miqueliana (Tilia miqueliana Maxim)

<400> 6

ttttccgtta tggggaagat caagatcgga atcaacggat ttggaaggat cgggcgtttg 60

gttgcgagag ttgctcttca aagcgaagat gttgagctcg ttgctgttaa tgatcctttc 120

atcactactg attacatgac ctacatgttt aagtacgaca gtgttcacgg tcaatggaag 180

caccatgagc ttaaggtgaa ggactcgaag acccttctct ttggggaaaa acctgtcacc 240

gtttttggcg tcagaaaccc tgaggaaatc ccatgggctg agactggagc tgattatgtt 300

gtcgaatcta ccggtgtttt cactgacaag gacaaggctg ctgctcactt gaagggtggt 360

gcaaagaagg tgatcatttc tgctcccagt aaggatgccc ccatgtttgt tgtgggtgtc 420

aatgagaagg aatacaagcc tgaccttaac attgtctcca atgctagctg cactaccaac 480

tgccttgctc cattggctaa ggtcatccac gacaaatttg gcattgttga gggtcttatg 540

accactgtcc attcgattac tgctacccaa aagactgttg atggcccatc aatgaaggac 600

tggagaggtg gtagagctgc ttccttcaat atcattccca gcagtactgg agctgccaag 660

gctgttggca aagtgttgcc atcattgaat ggcaagttga ctggaatggc tttccgtgtt 720

cccactgttg atgtctctgt ggtcgacctc acggtaagac ttgagaagaa ggcttcttat 780

gaagatatta aggctgctat caagaaggaa tctgaaacca acttgaaggg aattcttggt 840

tatgtagacg aagatttggt ctcaactgac tttattggag acaaaaggtc aagcattttt 900

gatgccaagg ctggaattgc tttgaatgac aactttgtta agcttgtcgc gtggtatgac 960

aacgagtggg gttacagttc ccgtgtgatt gacttgatcc ggcacatgga ttctgttgct 1020

aagtgagtga ttttggttta tcta 1044

<210> 7

<211> 433

<212> DNA

<213> Tilia miqueliana (Tilia miqueliana Maxim)

<400> 7

aaatttaaga gatggctcgt accaagcaaa ctgctcgtaa gtcaactgga ggaaaggctc 60

ctaggaagca actcgctaca aaggctgcac gtaagtctgc cccaaccacc ggtggtgtga 120

agaagcccca ccgttaccgt cctggaactg tcgccctccg tgaaattcgt aagtaccaga 180

agagtactga gcttcttatc aggaaattgc ccttccagag gcttgtccgt gaaattgccc 240

aggacttcaa gactgatttg cgtttccaga gccatgctgt cctggctctc caggaagctg 300

cagaggcata ccttgtgggt cttttcgaag ataccaactt gtgcgccatc cacgccaagc 360

gtgtaacaat catgcccaag gatatccagc tggctcgtag gatcaggggt gagcgtgctt 420

aagtgtcaag tgt 433

<210> 8

<211> 1795

<212> DNA

<213> Tilia miqueliana (Tilia miqueliana Maxim)

<400> 8

aagatcttgt gagttaacat ggcaatgatt gatgagcctt tgtatccaat tgcggtgttg 60

atcgatgagc tcaagaatga ggatatccag cttcgtttga actcgatccg tagactctct 120

acgattgcac gtgcacttgg ggaagaaagg acacgaaagg aattgattcc gtttctgagt 180

gagaacaatg atgacgatga tgaagttctg cttgccatgg cagaagaatt gggagttttt 240

attccttatg ttggaggtgt ggaatatgct aatgtattgt tacctccatt ggagactctt 300

tgcactgttg aggaaacttg tgtgagggat aaggctgtcg agtcgttgtg tagaattggt 360

gcacagatga gggagcagga cttggttgag tattttattc cactagtgaa gagattggct 420

tctggtgagt ggtttacagc tcgcgtttcg tcatgtggat tgtttcatat tgcataccca 480

agtgcctctg agacattaaa aactgaacta agagcgatat acagtcagct ttgccaagat 540

gacatgccca tggttaggag atctgctgcg acaaatcttg gaaaatttgc tgctactgta 600

gaagcacctc atttgaaggt ggacatcatg tctatgtttg atgatttgac tcaagatgat 660

caagattctg ttcgcttatt agctgttgag ggttgtgcag ctcttggaaa gttgttggag 720

ccccaagatt gtgtggcaca tattctccct gtcatagtta atttctcaca ggataagtct 780

tggcgcgtcc gttacatggt tgcaaatcaa ttatatgagc tgtgtgaagc tgttggacct 840

gagcaaacta ggtcggacct ggtacctgca tatgttcgcc tacttcgtga taatgaagct 900

gaagtacgga tagctgctgc tggaaaagta actaagtttt gccgaattct gaatccagaa 960

ctatcaattc aacatattct tccttgtgtc aaggaactat caacagattc atcccagcat 1020

gttcgttctg ctttggcttc agttataatg ggaatggctc ccgttttagg gaaggatgcg 1080

accatagagc aattgcttcc catatttctt tctcttttga aagatgaatt tccagatgtc 1140

agactgaata tcattagcaa gcttgatcaa gtcaaccagg tgattggaat tgatctactg 1200

tcccagtctc tattgccagc aattgtggag cttgcagagg ataggcactg gagggttcgg 1260

cttgcaataa tagaatacat acctttattg gcaagtcagt tgggtgttgg ctttttcgat 1320

gacaaacttg gtgcgctatg catgcaatgg ttgaaagata aggtttattc cattcgtgat 1380

gctgctgcta ataatgtgaa gcgcctggca gaagaatttg gaccagattg ggcaatgcag 1440

catatagttc cacaggtttt ggacatgatt aacaatcctc attatctgta tcggatgacc 1500

attctacatg cagtttctct acttgcccct gttatgggct cagatattac ttgttctaaa 1560

cttctccctg tggttattaa tgcttcaaaa gatagggtac ccaacatcaa gtttaatgtg 1620

gccaaggtgc tgcagtcact tattcctatt gttgatcaat ctgtggtgga gaagacaatc 1680

cgtccatgct tagttgaatt gagtgaggat cccgacgtcg atgtcaggtt tttcgccaat 1740

caagccctag agtcaagtga tcaggtcatg atgtctagct agattgtgac attag 1795

<210> 9

<211> 480

<212> DNA

<213> Tilia miqueliana (Tilia miqueliana Maxim)

<400> 9

actctgccac catgggtcgt atgcacagcc gaggtaaggg tatttccgca tctgctcttc 60

cctacaagcg aactcctcct agttggttga agatctcttc tcaagatgtt gatgagaata 120

tttgcaagtt tgcaaagaag ggtttgaccc catctcaaat tggagtcatt ctccgtgatt 180

ctcatgggat tgctcaggtg aagagtgtta ctggcagcaa gattttgcgg atactgaaag 240

cacatggtct tgctcccgaa attcctgagg atttgtacca cctgattaag aaagctgttg 300

ccatcagaaa gcatcttgag aggaacagga aagacaagga ttccaagttt aggttgatct 360

tggttgagag cagaattcac cgacttgccc gctattacaa gacgacaaag aagctgccac 420

ctgtgtggaa atatgagtct accaccgcca gcacccttgt agcttagagc tggcacattt 480

<210> 10

<211> 1098

<212> DNA

<213> Tilia miqueliana (Tilia miqueliana Maxim)

<400> 10

tggttgacat agcaatggca gtatctgcca ttggatttga gggttatgag aagaggcttg 60

aaatttcctt ttttgagcct ggcctctttg ttgatcctga aggaaagggt cttcaatcat 120

tgtcaaaagc ccagttggat gagattctag gtccagctga gtgcaccata gtttcttcac 180

tgtcaaacaa gcatgtggac tcctatgtcc tttctgagtc tagtctcttt gtctatcctt 240

acaagatcat catcaaaacc tgtgggacaa caaagttgct tcttgcaatc ccacccatct 300

tgaagttggc tggcagtctt tctcttgcca taaaatctgt cagatatact cgtgggagtt 360

ttatcttccc tggtgctcag ccatatcctc accgtaactt ctctgaagaa gttgctatcc 420

ttgatagcta ttttgggaag cttggcgctg gcagcaaggc ttattttatg ggtggcttgg 480

ataaacagaa atggcatgtg tactctgctt ctgctgaatc agttatctca aatggtccta 540

tttacactgt ggagatgtgc ttaactggtt tggacaggga aatggcttca gttttctaca 600

aagatcaatc aggctcggca gctgtgatga ccattcattc tggcataagg aagatccttc 660

cagattctga gatatgtgat tttgattttg atccctgtgg ttattcaatg aatgcaatcg 720

aaggggatgc gatttctact atccatgtta caccagaaga tgggtttagt tatgcaagct 780

ttgaagctgt gggctatgat ctgaaagatt tgaatctgaa gcagttggta gaaagggtgt 840

tggtttgctt caaaccgaat gacttttctg tagcggtgca cgttgatgtt gcgggacagt 900

cacttgagca caactgtctg ctggatgtga aaggatactg ttgcggagag aggagcaatg 960

aagagcttgg aattggtggt tcaatcatgt accagaaatt caacagcact ggtagctgtg 1020

gatctcctcg atcaactctg aaatgctgct ggaaagagga agaagaggag gaggaagagt 1080

agttaaataa atggttta 1098

<210> 11

<211> 945

<212> DNA

<213> Tilia miqueliana (Tilia miqueliana Maxim)

<400> 11

ccaaacgaat catgaccact gaacccgaaa gccgtaattg gctggagctt cccttggacg 60

tgacggcctc gattctttct cggctaggtg caattgagat cttgaacagc gcccaaaatg 120

tctgctctct gtggcgaaag atctgcaaag acccattaat gtggagatcc attgatatgc 180

acaatttggg tgaggtgtgg gacatggact atgatcccga gaagatgtgc gtccacgctg 240

tcgatcgcag ctgcggccac ttgattgaca tcaatctcga atattttggt agcgatgacc 300

ttctaattca tatcgctgag aggtctagtc atcttaaacg tcttcgactt gtatcgtgct 360

ataatatttc agatgaagga ttaagtgaag cagctttaaa acttcatttt ctagaagaac 420

ttgaaatttc atactgcttc attttgaaaa atgccctgga aactattggg cgttgttgcc 480

ctcacttgaa atcattcaaa ttcaatgttc agggatgcag acgcttccac ttagagtctg 540

acgatgaggc actagctatt gcacaaacta tgcccgaatt acgccacctc caactttttg 600

ggaacaagct gacaaatgtt ggcttgcaag ccattctcga cggttgtcct caccttgaat 660

cacttgactt gcggcagtgt ttcaatgtta gtttgggacg gaacttggag aaaagatgtg 720

ttgaacgcat aaaaaatttg cgacgtccaa atgattcaac tcatgattat gagtttaatt 780

cagaagttca tgatacttgg tcatcagatg aagattaccc attgggaatt tctgacattg 840

acttaatgtc tgatgattat gatgaatatt ttgagttctc aggggccagt gactttgatt 900

atgatgatga atacttgctt tttgactaat ggtttgttca tgaag 945

<210> 12

<211> 1386

<212> DNA

<213> Tilia miqueliana (Tilia miqueliana Maxim)

<400> 12

agaaaaagga gagaaatgag ggagattata agcatccata tagggcaagc tgggattcag 60

gttgggaact catgctggga gctctattgt cttgaacatg aaatccaacc tgatggaatg 120

atgcccagtg acacctctgt gggtgtggca catgatgctt tcaacacttt cttcagcgaa 180

acaggttcag gaaagcatgt tcctagagct atctttgttg atctggaacc cactgttatc 240

gatgaagtta ggactggcac ttaccgccaa cttttccacc ctgagcagct tatttctggc 300

aaagaagatg ctgccaataa ctttgccaga ggccactata caattgggaa ggaaattgtc 360

gatctctgcc ttgatcgagt taggaagtta gctgataact gcacaggttt gcaagggttt 420

ctggtgttta atgctgttgg cggtggaact ggttctggtc tgggatcttt gttgttagag 480

cgcttgtctg tagattatgg aaagaagtca aagcttgggt tcaccatcta tccttcaccg 540

caggtctcaa ctgcagttgt ggagccttac aacagcgttc tgtccactca ttcccttctt 600

gagcacacag atgtggctgt gctcttggac aatgaagcaa tttatgacat atgtcgcaga 660

tccctcgata ttgagaggcc aacttacacc aatttgaacc gattgatatc tcaaatcata 720

tcatccttga caacttcttt aaggtttgat ggagccatta atgtggacat tacagagttc 780

caaaccaatc ttgttccata tcctcgtatc catttcatgc tctcttcata tgcacctgtt 840

atctcagctg agaaagcata ccatgagcaa ttatctattc ctgagatcac aaatgctgtg 900

tttgagccct caagcatgat ggcaaagtgt gatcctaggc atgggaagta catggcatgc 960

tgcctgatgt atcgaggaga tgttgtcccc aaggatgtta atgctgctgt ttccacggtg 1020

aaaacaaaga ggactgtgca gtttgttgac tggtgcccaa ctggctttaa atgcggtatt 1080

aactatcagc ccccaactgt tgtacctggt ggagatcttg ctaaggtgca gagagctgtt 1140

tgcatgataa gcaataatac agcagtgact gaggtgtttg cacgcattga ccacaagttt 1200

gatctcatgt actcgaagag agctttcgtg cattggtatg ttggtgaggg catggaagaa 1260

ggtgaattct cagaagcccg tgaggatctg gctgctcttg agaaagatta tgaagaagtt 1320

ggtgctgaag gtgtagatga tgaagaggaa ggtgaaggtg aagactattg atgtacttat 1380

gcaaaa 1386

<210> 13

<211> 1380

<212> DNA

<213> Tilia miqueliana (Tilia miqueliana Maxim)

<400> 13

tcattttaaa caacatgaga gaaatccttc acattcaagg aggccaatgc gggaaccaaa 60

tcgggtccaa gttttgggaa gtgatctgcg acgagcacgg cgtggatcct atcggaaggt 120

acaatggaga tggaccgtct gatctccaac tggagcggat caatgtgtat tacaatgagg 180

cttctggtgg aaggtacgtt cctcgtgcgg ttctcatgga tcttgaaccc ggtactatgg 240

atagtatcag atccggtccc ttcggccaga tatttaggcc tgataatttt gtgtttggac 300

aatccggtgc cgggaacaac tgggccaaag gtcattacac tgaaggagcc gaattgattg 360

atgctgttct tgatgttgtt cgtaaagagg ctgagaattg tgattgcttg caaggcttcc 420

aggtatgcca ttcacttgga ggaggcacag gctctggcat gggaactctc ctgatatcaa 480

aaatcagaga ggaatatcca gacaggatga tgatgacgtt ctctgttttc ccttcaccta 540

aagtctctga cacagttgtg gagccttata atgccaccct ctctgtgcac cagttggtag 600

agaatgctga cgaatgcatg gttcttgaca atgaggcact ttatgatatt tgcttccgga 660

ctctaaagct cactactcca agctttggag atcttaacca cttgatttct gcaactatga 720

gtggtgtaac atgctgcctg aggttccctg gacagctgaa ctctgacctt cggaagttgg 780

ctgtgaacct gatcccgttc ccacgtcttc acttctttat ggttgggttt gcaccactca 840

catcccatgg ttcccagcag tacatttcgc tcacagtccc ggagctgact cagcaaatgt 900

gggatgccaa aaacatgatg tgtgctgctg accctcgcca tggtcgctac ctgacagcct 960

cagctatgtt ccgaggcaag atgagcacca aagaggttga tgaacaaatt attaatgttc 1020

agaacaagaa ttcatcatac tttgtggagt ggattcccaa taatgtgaag tccagtgttt 1080

gtgatattcc accaaagggt ctgaggatgt catctacttt tgttgggaat tcgacatcaa 1140

tccaggagat gtttaggagg gtaagtgagc aattcacagc aatgttccgt cgcaaggcct 1200

ttttgcattg gtatactgga gaggggatgg atgagatgga gttcacggaa gcagagagca 1260

acatgaatga cttggtggca gagtatcagc aataccagga tgccactgtg gaggatgagg 1320

gtgagtacga ggaggaggag ggtctggagg agaactatga gacctaaagg tgtgggcaat 1380

<210> 14

<211> 470

<212> DNA

<213> Tilia miqueliana (Tilia miqueliana Maxim)

<400> 14

ctcggactct gtcgatggct tcgaagcgga tcttaaagga gctcaaggat ctccagaaag 60

atcctcctac ttcatgcagc gctggtcctg tagctgaaga catgttccat tggcaagcca 120

caataatggg tccccctgat agtccttatt ctggaggtgt ctttctagta acaattcatt 180

ttcctcctga ttatcctttt aaaccaccta aggttgcatt tagaaccaaa gtttttcacc 240

caaatatcaa cagcaatggt agtatatgcc ttgacatact aaaggagcag tggagccctg 300

ccctgacaat atcaaaggtg ttgctttcta tctgctcact cttgacggac ccaaaccctg 360

atgatcccct ggtgccagaa attgctcata tgtacaagac agacaaggct aagtatgaat 420

caactgctag gagctggacc cagaagtatg ccatgggcta gtctaataat 470

<210> 15

<211> 948

<212> DNA

<213> Tilia miqueliana (Tilia miqueliana Maxim)

<400> 15

aattgctcgt agaagatgca gatctttgtg aaaaccctta ccggcaaaac cataaccctc 60

gaggtggaga gctcagacac tatcgataac gttaaggcca agatccaaga caaggaaggc 120

atccctcctg atcaacagcg cctgatcttc gccgggaagc aactcgagga cggccgtacc 180

ctcgctgact ataacatcca gaaggaatcg actctccacc tcgtcctccg gctccgtggc 240

ggcatgcaga tcttcgtcaa aaccctaacc ggaaagacca tcactcttga agtcgaaagc 300

tcggatacga ttgacaatgt caaggccaag atccaagaca aggaaggcat tcctcctgat 360

caacagcgcc tgatcttcgc cgggaagcaa cttgaagacg gccgtactct cgctgactat 420

aacatccaga aggaatcgac tctccacctt gtcctccgtc tccgtggcgg catgcagatc 480

ttcgtcaaaa ccctaaccgg caagaccatc actcttgaag tcgaaagctc tgacacgatc 540

gacaatgtta aggccaagat ccaagacaag gaagggatcc ctccagatca gcaaaggttg 600

attttcgccg ggaagcagct ggaggatggc aggactctgg cagattacaa catccagaaa 660

gagtctactc ttcatcttgt gctgaggctt cgtggaggga tgcagatctt cgtgaagacg 720

ctgactggga agacgattac gcttgaggtg gagagctctg atacaataga taatgtgaag 780

gcgaaaattc aagataagga agggattcca cctgatcagc agaggctcat tttcgctggg 840

aagcaattgg aggacgggcg tactcttgca gattacaaca tccagaagga gtctaccctt 900

caccttgtcc taaggcttcg tggtggtgcc aagtaattag agcttttc 948

<210> 16

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

accgatagcg aacaagtacc g 21

<210> 17

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

tcccgacaat ttcaagcact 20

<210> 18

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 18

caaggctaac agagaaaaga 20

<210> 19

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 19

actggataac agagaaaaga 20

<210> 20

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 20

aacagagcca accttgagtg c 21

<210> 21

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 21

acgagaggct gtaaacattg ga 22

<210> 22

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 22

gagttcattg ccacgctgtt g 21

<210> 23

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 23

gccaagaatg ccaacaccac 20

<210> 24

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 24

gccacacttc ccavattgct 20

<210> 25

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 25

accagcatca ccgttcttca a 21

<210> 26

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 26

ggtgtcaatg agaaggaata c 21

<210> 27

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 27

cagtaatcga atggacagtg g 21

<210> 28

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 28

gtaagtctgc cccaaccacc 20

<210> 29

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 29

caatttcacg gacaagcctc 20

<210> 30

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 30

ccaatcctta ttaccagcca tcg 23

<210> 31

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 31

ccaacaccta actgacttgc c 21

<210> 32

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 32

tgaggatttg taccacctga t 21

<210> 33

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 33

ccttgtcttt cctgttcctc tc 22

<210> 34

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 34

ctcagccata tcctcaccgt a 21

<210> 35

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 35

tgccatttct gtttatccaa gc 22

<210> 36

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 36

gaattacgcc acctccaact 20

<210> 37

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 37

tttctccaag ttccgtccca a 21

<210> 38

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 38

cagccagatc ttcacgagct t 21

<210> 39

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 39

gttctcgcgc attgaccata 20

<210> 40

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 40

tgaaccactt gatctctgcg acta 24

<210> 41

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 41

cagcttgcgg aggtctgagt 20

<210> 42

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 42

acaaatttcc cggtacgttt 20

<210> 43

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 43

tggatcacaa agcaatgac 19

<210> 44

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 44

atttggttct gcgccttaga gga 23

<210> 45

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 45

cagccagagt acgaccgtcc 20

<210> 46

<211> 803

<212> DNA

<213> Tilia miqueliana (Tilia miqueliana Maxim)

<400> 46

tttcccacaa cttgtaaaat tttctactac tcaaatggga aggtctcctt gctgtgagaa 60

agctcacaca aacaaaggtg catggaccaa agaagaagac gatcgcctca tcgcttacat 120

ccgagcccat ggtgaaggtt gctggcgctc actccctaaa gctgctggcc ttctccgttg 180

tggcaaaagt tgtagacttc gatggatcaa ctacttaaga cctgacctta aacgtggcaa 240

tttcactgaa gaagaagatg agctcattat caagctgcat agccttcttg gtaacaagtg 300

gtctcttata gctgggagat taccaggaag aacagataat gagataaaga attactggaa 360

cacacatata agaaggaagc tattgaacag aggtattgat cctgcaactc acaggccact 420

caatgaggct gctcaggatg taacaacaat atctttcggt ggtgctaaag aagagaaaga 480

taagattaat aatcctagtg gatttgtcaa caaagatgag aaaaaaatcc cagttcaaga 540

aaggtgtcca gacttgaatt tggacctcag aattagccct ccttaccatc aaagccagtc 600

gccagagcca ttgaagactg gggggaggat tctttgtttt atttgcagtt tgggagtgaa 660

aaatagcaaa gactgcactt gcagcatcgg tggtactgct ggtagcagca gtagcaacag 720

caacaatggt tatgatttct taggcttgaa aactggcttc ttagattata gaggtttgga 780

aatgaaataa gagggaattt aga 803

<210> 47

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 47

caacaatatc tttcggtggt gct 23

<210> 48

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 48

ccccagtctt caatggctct 20

Claims (3)

1. The real-time quantitative PCR internal reference gene of Tilia miqueliana under different tissues and stress treatment conditions is characterized by comprising the following components: the stable expression genes in the flooding stress leaves are PP2 alpha and SAMCD, and the nucleotide sequences of the genes are respectively shown as SEQ ID NO.8 and SEQ ID NO. 10; the stable expression genes in the flooding stress roots are TUA and TUB, and the nucleotide sequences of the genes are respectively shown as SEQ ID NO.12 and SEQ ID NO. 13; the stable expression genes in drought stress leaves are EF1 alpha and GAPDH, and the nucleotide sequences of the genes are respectively shown as SEQ ID NO.5 and SEQ ID NO. 6; the stable expression genes in drought stress roots are PP2 alpha and HIS, and the nucleotide sequences of the genes are shown as SEQ ID NO.8 and SEQ ID NO.7 respectively; the stable expression genes in the salt stress leaf are TUB and GAPDH, and the nucleotide sequences of the genes are respectively shown as SEQ ID NO.13 and SEQ ID NO. 6; the stable expression genes in the salt stress root are TUB and RPS13, and the nucleotide sequences of the genes are respectively shown as SEQ ID NO.13 and SEQ ID NO. 9.

2. The real-time quantitative PCR reference gene under different tissues and stress treatment conditions of Tilia miqueliana according to claim 1, wherein the primer sequence for amplifying PP2 alpha is SEQ ID NO. 30-SEQ ID NO.31, the primer sequence for amplifying SAMCD is SEQ ID NO. 34-SEQ ID NO.35, the primer sequence for amplifying TUA is SEQ ID NO. 38-SEQ ID NO.39, the primer sequence for amplifying TUB is SEQ ID NO. 40-SEQ ID NO.41, the primer sequence for amplifying EF1 alpha is SEQ ID NO.24-SEQ ID NO.25, the primer sequence for amplifying GAPDH is SEQ ID NO.26-SEQ ID NO.27, and the primer sequence for amplifying HIS is SEQ ID NO. 28-SEQ ID NO.29, and the primer sequence for amplifying RPS13 is SEQ ID NO. 32-SEQ ID NO.33.

3. The application of the Tilia miqueliana in calculating the relative expression of target gene by real-time quantitative PCR reference genes under stress treatment conditions of different tissues of Tilia miqueliana as claimed in claim 1.