CN108070673B - Fluorescent quantitative reference gene for leaf tissues of different tree parts of two kinds of tree pears and primer and application thereof - Google Patents
Fluorescent quantitative reference gene for leaf tissues of different tree parts of two kinds of tree pears and primer and application thereof Download PDFInfo
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Abstract
The invention discloses fluorescent quantitative reference genes of leaf tissues of different tree body parts of two kinds of tree-shaped pears, primers and application thereof. According to the invention, expression levels of 13 candidate reference genes in pear leaf tissue samples of different tree body parts of two tree forms are analyzed by qRT-PCR, expression stability values of the candidate reference genes are evaluated by using geNorm software, and ARM and Actin2 are determined to be the best reference gene combination; different internal reference genes are used for respectively carrying out relative expression standardization analysis on the APX, and the reliability of the internal reference gene combination ARM and Actin2 serving as standardization factors is high.
Description
Technical Field
The invention belongs to the field of plant molecular biology, and particularly relates to fluorescent quantitative reference genes of leaf tissues of different tree parts of two tree pears, primers and application thereof.
Background
The pear is one of the most economic fruit trees in the world and China. Different tree structures influence the distribution and penetration of photosynthetically active radiation in the canopy, and thus have a regulating effect on fruit quality. The tree body of the traditional pear tree is tall and big, and the interior of a canopy is easy to close; in recent years, the trellis tree shape is widely popularized in China, the tree structure is opened, the illumination is uniformly received, and the commodity performance of fruits is favorably improved. The research on the expression level change of related genes at each part of different pear trees is helpful to analyze the molecular mechanism of the tree structure regulation and control library source relationship.
The real-time quantitative PCR (qRT-PCR) technology has been widely used for gene expression level analysis due to its advantages of rapidness, sensitivity, accuracy, high repeatability, wide practicability, etc. When using the relative quantification method, in order to eliminate the deviation of initial template amount, RNA quality and the like of different samples, the experimental data needs to be corrected by using the reference gene with stable expression. However, the expression level of any one gene is not kept constant under all experimental conditions. Even for the same species, when the experimental conditions are changed, the stability of the reference gene is also changed, and it cannot be absolutely considered that the reference gene stably expressed under a certain experimental condition can be applied to all other experimental conditions, otherwise the experimental data is likely to generate deviation and even wrong conclusion. Therefore, before analyzing gene expression, an internal reference gene stably expressed under the specific condition should be screened, which is very important for obtaining accurate qRT-PCR experimental results. In addition, selection of multiple reference gene combinations allows for more accurate correction and normalization of relative quantitative data relative to a single reference gene.
Disclosure of Invention
The invention aims to develop a double-internal reference gene and a specific primer thereof, which can be applied to gene expression analysis in different tree forms (trellis tree forms and evacuation layering forms) and leaf tissues of all tree body parts (external parts, middle parts and internal parts) aiming at the defect that the accuracy of the traditional single internal reference gene is not high, and provide reliable guarantee for accurate quantification of the gene under the experimental condition.
The expression levels of pear leaf tissue samples of 13 candidate reference genes at different tree body parts (outer part, middle part and inner part) of two tree forms (trellis tree form and sparse layering form) are analyzed by using qRT-PCR; evaluating the expression stability value of the candidate reference gene by using geNorm software, and determining ARM and Actin2 as the optimal reference gene combination; and respectively carrying out relative expression standardization analysis on the APX by using different internal reference genes, and verifying that the gene combination has reliability as a standardization factor.
The first purpose of the invention is to provide fluorescent quantitative reference genes of leaf tissues of different tree body parts of two tree-shaped pears. The fluorescent quantitative reference genes of the leaf tissues of different tree body parts of the two tree-shaped pears are reference genes Actin2 and ARM.
Preferably, the nucleotide sequence of the reference gene Actin2 is shown in SEQ ID NO. 1; the nucleotide sequence of the internal reference gene ARM is shown in SEQ ID NO. 4.
The second purpose of the invention is to provide specific primers of the fluorescent quantitative reference genes of the leaf tissues of different tree body parts of the two tree-shaped pears.
Preferably, the specific primers of the fluorescent quantitative reference genes of the leaf tissues of different tree body parts of the two tree pears are respectively shown as SEQ ID No.2 and SEQ ID No.3, and the forward primer and the reverse primer of the specific primer of the reference gene Actin2 are respectively shown as SEQ ID No.2 and SEQ ID No. 3; the forward primer and the reverse primer of the specific primer of the internal reference gene ARM are respectively shown as SEQ ID NO.5 and SEQ ID NO. 6.
The third purpose of the invention is to provide the application of the internal reference gene or the specific primer in the preparation of a fluorescence quantitative kit.
Preferably, the kit further comprises conventional PCR reagents.
The reference gene Actin2 and the reference gene ARM are used as the fluorescent quantitative reference genes of leaf tissues of different tree body parts of two tree-shaped pears.
The specific primers are applied as fluorescent quantitative specific primers of leaf tissues of different tree body parts of two tree-shaped pears.
Preferably, the application is fluorescent quantitative PCR, the fluorescent quantitative PCR system is 10 mu L, the system comprises 5 mu L of 2 xSYBRGreen PCR Master Mix, 0.5 mu L of each of 10 mu mol/L forward primer and reverse primer, 0.5 mu L of cDNA template, and ddH2O to 10 μ L; the fluorescent quantitative PCR reaction program was 50 ℃ for 5min, followed by 40 cycles of 95 ℃ for 10min, 94 ℃ for 15sec, and 60 ℃ for 1 min.
The two tree forms are a shed frame tree form and an evacuation layered form, and different tree body parts are the outside of the tree body which is 1m away from the central trunk, the middle of the tree body which is 0.5-1.0m away from the central trunk and the inside of the tree body which is 0-0.5m away from the central trunk.
The expression conditions of candidate reference genes of different tree body parts of two tree-shaped pears are comprehensively analyzed; screening out reference gene combinations Actin2 and ARM for gene expression analysis of different tree pears in leaf tissues with different spatial distributions, and verifying that the gene combinations are strong in reliability as standardization factors.
The invention has the advantages that:
(1) the method screens out reference gene combinations Actin2 and ARM suitable for gene expression analysis in leaf tissues of different trees and different spatial parts for the first time.
(2) And (6) reliability. The influence of two factors of different tree shapes and different tree body parts on the gene expression stability is considered at the same time, the expression level Ct values of the 13 candidate reference genes are subjected to stability evaluation through the geNorm software, and further verification shows that the combination of the Actin2 and the ARM reference genes has reliability when being applied to target gene standardization. The Actin2 and ARM of the invention are stably expressed in pear leaf tissues of different tree parts of two trees, are the optimal dual-reference gene combination for fluorescence quantitative analysis of the different tree parts of the two trees, and the application of the gene combination is obviously superior to the standard effect of a common single-reference gene. The ARM is used as a new candidate internal reference gene screened by the inventor, and is not used for standardized analysis of other purposes at present.
(3) The invention has wide applicability. The method can be used for reference genes in gene expression analysis of pear leaf tissue samples of different tree forms and different tree body parts, and provides a stable comparative standard for gene expression analysis of pear leaf tissues in spatial distribution.
Drawings
FIG. 1 is a boxplot of Ct value distributions of 13 candidate reference genes in samples of different tree body parts of two kinds of tree pears, wherein upper and lower boundaries of a box, straight lines in the box, end points of a target and circles respectively represent 25/75 quartile points, median values, maximum/small values and abnormal values.
FIG. 2 is a graph of the expression stability of 13 candidate reference genes in leaf tissues of different treelike parts of two kinds of pear trees ranked by the geNorm software.
FIG. 3 is a graph of the number of reference genes determined by geonorm to be optimal for accurate quantitative analysis, and the pairwise differences between the nth normalization factor and the n +1 normalization factor are analyzed, with asterisks indicating the most appropriate number of reference genes.
FIG. 4 is a diagram showing the analysis of the expression amount change of the APX gene IN the leaf tissues of different tree parts of two kinds of treelises by using different reference genes as normalization factors, wherein DP represents a trellis tree shape, SP represents a sparse stratification shape, and the rear sides-EX, -CE and-IN represent leaf tissue samples of the outer part (1 m away from the central trunk), the middle part (0.5-1.0 m away from the central trunk) and the inner part (0-0.5 m away from the central trunk) of the tree shape, for example, DP-EX represents a leaf tissue sample of a pear at the outer part of the trellis tree shape, and the like.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1: screening an optimal dual-reference gene combination of Actin2 and ARM for fluorescence quantitative analysis of different tree body parts of two kinds of tree pears
1. Sampling experimental materials: taking adult leaf tissues of 'Yuanhuang' pears of two tree forms (a trellis tree form and an evacuation layering form) as experimental materials, and respectively adopting leaf tissues of the outer part (1 m away from a central stem), the middle part (0.5-1.0 m away from the central stem) and the inner part (0-0.5 m away from the central stem) of the tree body in 6 th of 2016. Selecting 3 trees from each tree-shaped pear tree as 3 biological repeats, quickly freezing a leaf tissue sample in liquid nitrogen after being separated from the body, and then storing at-80 ℃.
2. Extracting total RNA of leaf materials: total RNA from leaf tissue samples was extracted using the RNAprep Pure Plant Kit (Tiangen) RNA extraction Kit according to the instructions.
3. Total RNA integrity test: electrophoresis on 1.2% agarose gel, 1 XTAE buffer, 100V, 20 min; monitoring quality and concentration; using a NanoPhotometerTMThe OD260/280 value and the concentration are detected by a spectrophotometer of a spectrophotometer (IMPLEN), and the value is between 1.9 and 2.1, and the sample is qualified.
Synthesis of the first Strand of cDNA: according to RevertAIdTMFirst Strand cDNA Synthesis Kit (Fermentas) instructions reverse the total RNA extracted in step 2 (test-eligible samples) to cDNA for use as a qRT-PCR template.
1) Prepare reaction mixture on ice: mu.g of total RNA, 1. mu.l of Oligo (dT) primer, and 12. mu.l of DEPC water were added in this order.
2) Bathing at 65 deg.C for 5min, and cooling on ice for 2 min.
3) Slightly centrifuging; the centrifuge tube was placed on ice and 4. mu.l of 5 × Reaction buffer, 1. mu.l of Ribonuclose inhibitor Ribonuclease inhibitor, 2. mu.l of 10mM dNTPmix and 1. mu.l of reverse Aid were added in this orderTMM-MuLV reverse transcriptase (200U/. mu.l).
4) Mixing and centrifuging.
5) Bathing at 42 deg.C for 60min, bathing at 70 deg.C for 5min, cooling on ice, and storing at-20 deg.C.
5. The cDNA synthesized by reverse transcription is taken as a template, Actin2, ARM, EF1a, GAPC, Histone H3, MYB10, SAND, SKD1, SR34A, TIP41-like, TUB7, UBQ5 and YLS8 are selected as candidate internal reference genes, and primers are designed to carry out qRT-PCR reaction. The above-mentioned 13 candidate reference genes are based onThe transcriptome analysis result obtained by the laboratory of the inventor and other reported pear reference gene sequence information are screened and determined, and corresponding amplification primers are designed aiming at the candidate genes. The primer sequences and amplification characteristics of the candidate reference genes are shown in Table 1, the fluorescent quantitative PCR system is 10 muL, and comprises 5 muL of 2 XSYBR Green PCR Master Mix, 0.5 muL of each of 10 mumol/L forward primer and reverse primer, 0.5 muL cDNA template, and ddH2O to 10 μ L; the fluorescent quantitative PCR reaction program is 5min at 50 ℃; then, 40 cycles of 95 ℃ for 10min, 94 ℃ for 15sec, and 60 ℃ for 1min were performed. And (3) judging that the used primer is a target fragment specifically amplified by observing that a dissolution curve generated after qRT-PCR reaction is a single peak.
TABLE 1 candidate reference Gene names, primer sequences and amplification product information
6. Reading the cycle number, namely Ct value, which is passed when the fluorescence signal in each reaction tube reaches a set threshold value. As shown in FIG. 1, the transcription levels of different reference genes show different expression change amplitudes, and the Ct values of 13 candidate reference genes range from 15.93 to 26.86; GAPC and Histone H3 showed relatively high transcription levels with Ct averages of 18.90 and 16.75, respectively; MYB10 mean expression level was lowest, Ct mean 26.21.
7. The stability of 13 internal reference genes is calculated and sequenced by using a geonorm program, and as shown in FIG. 2, the stability of the internal reference genes is sequenced from strong to weak as Actin2/ARM > SR34A > SAND > GAPC > YLS8> MYB10> UBQ5> TIP41-like > EF1a > SKD1> TUB7> Histone H3.
8. After determining the expression stability of the candidate reference genes, geNorm can be analyzed by calculating the normalization factor pair difference (V)n/n+1) The value is used to determine the most appropriate number of reference genes if Vn/n+1A value less than 015, it is not necessary to introduce the (n + 1) th reference gene. The result is shown in FIG. 3, where the first column in the histogram to the left has a height of less than 0.15, indicated by an asterisk, and corresponds to a horizontal axis V2/V3I.e., n is 2. Therefore, the minimum number of the stably expressed reference gene combinations in the leaf tissues of different tree parts of the two tree forms is 2, namely the optimal reference gene combinations are Actin2 and ARM. Wherein the forward primer of the specific primer of the reference gene Actin2 is 5'-CTCCCAGGGCTGTGTTTCCTA-3' (shown as SEQ ID NO. 2), the reverse primer is 5'-CTCCATGTCATCCCAGTTGCT-3' (shown as SEQ ID NO. 3), and the nucleotide sequence of the amplified product of the reference gene Actin2 amplified by the primer pair is shown as SEQ ID NO. 1; the forward primer of the specific primer of the internal reference gene ARM is 5-CAAGGGCATTCTTTCGG-3 '(shown as SEQ ID NO. 5), the reverse primer is 5-CAGGGACATCATTAGGAACAT-3' (shown as SEQ ID NO. 6), and the nucleotide sequence of the amplified product of the internal reference gene ARM amplified by the primer pair is shown as SEQ ID NO. 4.
Example 2: different reference genes respectively calculate the change of the APX gene expression level of the pear
The APX gene was normalized using the optimal combination of internal reference genes (Actin2 and ARM), 2 unstable internal reference genes (Histone H3 and TIP41-like), and 2 common internal reference genes (UBQ10 and TUB β), respectively, as normalization factors (FIG. 4). The result shows that the gene quantification is most accurate when 2 most stable internal reference genes of Actin2, ARM and the combination thereof are selected. In the case of quantitative analysis using unstably expressed reference genes and 2 commonly used reference genes, the expression level of APX gene was greatly deviated in some samples.
Sequence listing
<110> research institute for fruit tree tea of academy of agricultural sciences of Hubei province
<120> two fluorescent quantitative reference genes of leaf tissues of different tree body parts of pear trees as well as primers and application thereof
<160> 6
<170> SIPOSequenceListing 1.0
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<211> 173
<212> DNA
<213> Pear (Pyrus spp.)
<400> 1
ctcccagggc tgtgtttcct agtattgttg gtcgcccacg acacacaggt gtcatggttg 60
gtatgggtca gaaggatgcc tatgtaggtg atgaagcaca gtcgaaaaga ggtatcctta 120
ccttgaagta tcccattgag cacggtatag tgagcaactg ggatgacatg gag 173
<210> 2
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 2
ctcccagggc tgtgtttcct a 21
<210> 3
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<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 3
ctccatgtca tcccagttgc t 21
<210> 4
<211> 101
<212> DNA
<213> Pear (Pyrus spp.)
<400> 4
caagggcatt ctttcggagc aactggtttc tgtgaaagaa gaaagcatga gaatattgaa 60
ggacttcatc accagacaca atgttcctaa tgatgtccct g 101
<210> 5
<211> 17
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 5
caagggcatt ctttcgg 17
<210> 6
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 6
cagggacatc attaggaaca t 21
Claims (4)
1. Internal reference geneActin2AndARMthe combination of the two kinds of the tree-shaped pears is used as the fluorescent quantitative reference gene of the leaf tissues of different tree body parts of the two kinds of tree-shaped pears, and is characterized in that the reference geneActin2The nucleotide sequence is shown as SEQ ID NO. 1; the reference geneARMThe nucleotide sequence of the tree is shown as SEQ ID number 4, and the two trees are trellis trees and sparse layered trees.
2. Internal reference geneActin2Specific primers and reference genes of (3)ARMThe application of the specific primer combination as the specific primer of the fluorescent quantitative internal reference gene of the leaf tissues of different tree body parts of two tree-shaped pears is characterized in that the internal reference geneActin2The forward primer and the reverse primer of the specific primer of (1) are respectively shown as SEQ ID NO.2 and SEQ ID NO. 3; the reference geneARMThe forward primer and the reverse primer of the specific primer are respectively shown as SEQ ID NO.5 and SEQ ID NO.6, and the two tree forms are a trellis tree form and an evacuation layering form.
3. The use of claim 2, wherein the use is of a fluorescent quantitative PCR system of 10. mu.L comprising 5. mu.L of 2 XSSYBR Green PCR Master Mix, 0.5. mu.L each of 10. mu. mol/L forward and reverse primers, 0.5. mu.L cDNA template, plus ddH2O to 10 μ L; the fluorescent quantitative PCR reaction program was 50 ℃ for 5min, followed by 40 cycles of 95 ℃ for 10min, 94 ℃ for 15sec, and 60 ℃ for 1 min.
4. The use according to claim 2, wherein said different trunk parts are an outer part of the trunk outside of 1m from the central trunk, a middle part of the trunk between 0.5 and 1.0m from the central trunk and an inner part of the trunk between 0 and 0.5m from the central trunk.
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