CN106997429B - A kind of prediction technique of forest long segment non-coding RNA target gene - Google Patents
A kind of prediction technique of forest long segment non-coding RNA target gene Download PDFInfo
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Abstract
The present invention relates to a kind of prediction techniques of forest long segment non-coding RNA target gene, belong to molecular genetic techniques field.1) prediction technique of forest long segment non-coding RNA target gene provided by the invention is the following steps are included: obtain forest lncRNA sequence；2) target gene of the lncRNA after preliminary screening is obtained using blast prediction technique；3) target gene of the lncRNA after postsearch screening is obtained using RNAplex prediction technique；4) tissue-specific expression pattern of the target gene of the lncRNA in the step 3) and the lncRNA after postsearch screening is detected, calculates the expression correlation of the two, determines the interaction relationship between lncRNA and its target gene.Prediction technique of the present invention can efficiently, steadily detect the effect section of lncRNA and its target gene, and can significantly improve the accuracy of forest lncRNA microRNA target prediction.
Description
Technical field
The present invention relates to molecular genetic technique field more particularly to a kind of forest long segment non-coding RNA target genes
Prediction technique.
Background technique
Long segment non-coding RNA (lncRNA) is that a kind of length is greater than 200 nucleotide, no encoding histone ability or coding
The extremely low rna transcription sheet of ability.Studies have shown that lncRNA can be in transcription, post-transcriptional level and the horizontal shadow of epigenetics
The expression of gene is rung, and then influences the physiology and biochemical process of plant, for example, the regulation etc. of flowering of plant time.In plant
It was found that lncRNA mainly by way of sequence is complementary, is combined with its target gene, to promote or the table of suppressor
It reaches.For example, arabidopsis lncRNA HID 1 (HIDDEN TREASURE 1) by with 3 (PHYTOCHROME- of PIF
INTERACTING FACTOR 3) promoter region base pair complementarity combines, and then inhibits the expression of PIF3.Due to perennial
Forest genome heterozygosity is high, and DNA sequence polymorphism is abundant, utilizes bioinformatic analysis means prediction lncRNA and its target more
The sequence complementary segment of gene, while detecting the expression correlation of lncRNA and its target gene.
In the prior art, the prediction of plant lncRNA target gene, only takes into account the principle of sequence complementation, and prediction technique
Threshold parameter is lower, and causing the target gene predicted, there are false positives.The prior art lacks a kind of higher plant of accuracy
The prediction technique of lncRNA target gene.
Summary of the invention
The purpose of the present invention is to provide a kind of prediction techniques of forest long segment non-coding RNA target gene.The present invention mentions
The prediction technique of confession can efficiently, steadily detect the effect section of lncRNA and its target gene, and can significantly improve woods
The accuracy of the wooden lncRNA microRNA target prediction.
The present invention provides a kind of prediction techniques of forest long segment non-coding RNA target gene, comprising the following steps:
1) forest lncRNA sequence is provided；
2) the lncRNA sequence and the gene with encoding histone function are subjected to sequence ratio using blast prediction technique
It is right, parameter setting are as follows: E-value < 1E-10, the target gene of the lncRNA after obtaining preliminary screening；
3) it is screened, is joined again using target gene of the RNAplex prediction technique to the lncRNA that the step 2) obtains
Number setting are as follows: E-value < -60, the target gene of the lncRNA after obtaining postsearch screening；
4) to the tissue specific expression mould of the target gene of the lncRNA after postsearch screening in lncRNA and the step 3)
Formula is detected, and is calculated the expression correlation of the two, is determined the interaction relationship between lncRNA and its target gene；
The calculating formula is as follows:
The x represents expression quantity of the lncRNA in tissue detected,
The y represents expression quantity of the target gene in tissue detected,
R represents the relative coefficient of lncRNA Yu its expression of target gene amount；
Threshold value r represents the evaluation index to the two expression correlation；R shows x and y expression phase between -0.75~0.75
Closing property is lower；R>0.75 or r<-0.75 show that x is related to the expression poling of y, determine that the target gene is that forest long segment is non-
The target gene of coding RNA.
Preferably, the gene with encoding histone function includes: that the forest corresponds to all tools in species genome
There is the gene of protein coding.
Preferably, the detection method of the step 4) tissue-specific expression pattern is real-time quantitative fluorescence PCR method.
Preferably, the amplification condition of the real-time quantitative fluorescence PCR method are as follows: 95 DEG C of initial denaturation, 30s；PCR reaction 40
A circulation: 95 DEG C, 3s, 60 DEG C, 30s；Solubility curve is drawn in 60 DEG C~95 DEG C sections.
The present invention provides a kind of prediction techniques of forest long segment non-coding RNA target gene.The present invention passes through two kinds
Bioinformatics Prediction method combines, and efficiently, steadily detects the effect section of lncRNA Yu its target gene, and can show
Write the accuracy for improving forest lncRNA microRNA target prediction.Test result shows that the application method can successfully realize that forest is long
The prediction of segment non-coding RNA target gene.
Detailed description of the invention
Fig. 1 is the complementary segment of lncRNA lnc-CK and Pto-CKX6 that the embodiment of the present invention 1 provides；
Fig. 2 is the phase during the lncRNA lnc-CK and Pto-CKX6 that the embodiment of the present invention 1 provides is organized at 8, Chinese white poplar
To the correlation results figure of expression quantity and expression.
Specific embodiment
The present invention provides a kind of prediction techniques of forest long segment non-coding RNA target gene, comprising the following steps:
1) forest lncRNA sequence is provided；
2) the lncRNA sequence and the gene with encoding histone function are subjected to sequence ratio using blast prediction technique
It is right, parameter setting are as follows: E-value < 1E-10, the target gene of the lncRNA after obtaining preliminary screening；
3) it is screened, is joined again using target gene of the RNAplex prediction technique to the lncRNA that the step 2) obtains
Number setting are as follows: E-value < -60, the target gene of the lncRNA after obtaining postsearch screening；
4) to the tissue specific expression mould of the target gene of the lncRNA after postsearch screening in lncRNA and the step 3)
Formula is detected, and is calculated the expression correlation of the two, is determined the interaction relationship between lncRNA and its target gene；
The calculating formula is as follows:
The x represents expression quantity of the lncRNA in tissue detected,
The y represents expression quantity of the target gene in tissue detected,
R represents the relative coefficient of lncRNA Yu its expression of target gene amount；
Threshold value r represents the evaluation index to the two expression correlation；R shows x and y expression phase between -0.75~0.75
Closing property is lower；R>0.75 or r<-0.75 show that x is related to the expression poling of y, determine that the target gene is that forest long segment is non-
The target gene of coding RNA.
After obtaining forest lncRNA sequence, by the lncRNA sequence and there is encoding histone using blast prediction technique
The gene of function carries out sequence alignment, parameter setting are as follows: E-value < 1E-10, the target base of the lncRNA after obtaining preliminary screening
Cause.
In the present invention, the gene with encoding histone function include: the forest correspond in species genome own
Gene with protein coding.In the present invention, the lncRNA sequence and the gene complementation with encoding histone function are long
Degree is preferably 70% or more of lncRNA sequence length.
The present invention is limited to lncRNA sequence to lncRNA sequence and the gene complementation length with protein coding function
70%.
After the target gene of lncRNA after obtaining preliminary screening, the present invention is using RNAplex prediction technique to the step
2) target gene of the lncRNA obtained is screened again, parameter setting are as follows: E-value < -60, after obtaining postsearch screening
The target gene of lncRNA；
After the target gene of lncRNA after obtaining postsearch screening, the present invention is to postsearch screening in lncRNA and the step 3)
The tissue-specific expression pattern of the target gene of lncRNA afterwards is detected, and the expression correlation of the two is calculated, and is determined
Interaction relationship between lncRNA and its target gene；
The calculating formula is as follows:
The x represents expression quantity of the lncRNA in tissue detected,
The y represents expression quantity of the target gene in tissue detected,
R represents the relative coefficient of lncRNA Yu its expression of target gene amount；
Threshold value r represents the evaluation index to the two expression correlation；R shows x and y expression phase between -0.75~0.75
Closing property is lower；R>0.75 or r<-0.75 show that x is related to the expression poling of y, determine that the target gene is that forest long segment is non-
The target gene of coding RNA.
In the present invention, the detection method of the step 4) tissue-specific expression pattern is real-time quantitative fluorescence PCR side
Method.
In the present invention, the amplification condition of the real-time quantitative fluorescence PCR method are as follows: 95 DEG C of initial denaturation, 30s；PCR reaction
40 circulations: 95 DEG C, 3s, 60 DEG C, 30s；Solubility curve is drawn in 60 DEG C~95 DEG C sections.
Below with reference to embodiment, to a kind of prediction technique of forest long segment non-coding RNA target gene provided by the invention
It is described in detail, but they cannot be interpreted as to the restriction to the application protection scope.
Embodiment 1
Using the prediction technique of forest long segment non-coding RNA target gene of the invention, to Chinese white poplar lncRNA lnc-CK
Target gene predicted.
1, the acquisition of lnc-CK sequence.The blade for collecting Populus tomentosa Clones " LM50 ", using CTAB method, to Chinese white poplar
Blade total serum IgE extracts, quality evaluation Hou Song company sequencing analysis, obtains the sequence number of leaves of Populus Tomentosa expression lncRNA
According to.And then obtain the sequence of Chinese white poplar lncRNA lnc-CK, SEQ ID NO.1.
2, joint utilizes the prediction technique of blast and RNAplex, to the target gene of lncRNA obtained in the step 1
Carry out tentative prediction.First with the method for blast, by lnc-CK and the sequence in leaves of Populus Tomentosa tissue cDNA library
(E-value < 1E-10) is compared in column, and the method for recycling RNAplex is further screened (parameter setting are as follows: E-
Value < -60), as a result, it has been found that the 1593-1774 base of the 1-182 base of lncRNA lnc-CK and Pto-CKX6
There are the base pair complementarity area of 182bp, the complementary segment figure of lncRNA lnc-CK and Pto-CKX6 are as shown in Figure 1.Preliminary sieve
Choosing obtains Pto-CKX6 as the target gene (blast:E-value=5E-055 of its prediction；RNAplex:E-value=-76.4),
Wherein, Pto-CKX6 sequence is SEQ ID NO.2.
3, for lncRNA lnc-CK and the special RT-qPCR primer of Pto-CKX6 implementation sequence, with poplar Actin base
The expression quantity of cause is internal reference, is detected to its relative expression quantity.The base sequence of LncRNA lnc-CK primer combination is SEQ
ID NO.3 and SEQ ID NO.4；The base sequence of Pto-CKX6 primer pair is SEQ ID NO.5 and SEQ ID NO.6；Actin
The base sequence of primer combination is SEQ ID NO.7 and SEQ ID NO.8 (see Table 1 for details).
1 primer sequence of table
To eight of annual Populus tomentosa Clones " LM50 " tissues or organ, (i.e. root, mature xylem, prematurity are wooden
Portion, bast, forming layer, tender leaf, old leaf and stem apex) RNA extract, reverse transcription be cDNA carry out RT-qPCR experiment.
The condition of RT-qPCR amplification are as follows: 95 DEG C of 30s of initial denaturation；PCR reacts 40 circulations, including 95 DEG C of 3s, 60 DEG C of 30s；Finally in 60
Draw solubility curves in DEG C of -95 DEG C sections.
Through the above steps, detection lncRNA lnc-CK and Pto-CKX6 is in this eight tissues and the opposite table in organ
Up to amount (using the expression quantity of Actin gene as internal reference), phase of the lncRNA lnc-CK and Pto-CKX6 in 8, Chinese white poplar tissues
To the correlation results figure of expression quantity and expression as shown in Fig. 2 and table 2.According to relative coefficient calculation formulaThe expression correlation coefficient that lncRNA lnc-CK and Pto-CKX6 is calculated is r=
0.92。
Table 2 lnc-CK and Pto-CKX6 expression quantity correlation
By the above implementation steps, it can be deduced that, Pto-CKX6 is the latent effect target gene of lncRNA lnc-CK.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
SEQUENCE LISTING
<110>Beijing Forestry University
<120>a kind of prediction technique of forest long segment non-coding RNA target gene
<130> 2016
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Claims (4)
1. a kind of prediction technique of forest long segment non-coding RNA target gene, comprising the following steps:
1) forest lncRNA sequence is provided；
2) the lncRNA sequence and the gene with encoding histone function are subjected to sequence alignment using blast prediction technique,
Parameter setting are as follows: E-value < 1E-10, the target gene of the lncRNA after obtaining preliminary screening；
3) it is screened again using target gene of the RNAplex prediction technique to the lncRNA that the step 2) obtains, parameter is set
It is set to: < -60 E-value, the target gene of the lncRNA after obtaining postsearch screening；
4) to the tissue-specific expression pattern of the target gene of the lncRNA after postsearch screening in lncRNA and the step 3) into
Row detection, calculates the expression correlation of the two, determines the interaction relationship between lncRNA and its target gene；
The calculating formula is as follows:
The x represents expression quantity of the lncRNA in tissue detected,
The y represents expression quantity of the target gene in tissue detected,
R represents the relative coefficient of lncRNA Yu its expression of target gene amount；
Relative coefficient r represents the evaluation index to the two expression correlation；R shows x and y expression between [- 0.75,0.75]
Correlation is lower；R > 0.75 or r < -0.75 shows that x is related to the expression poling of y, determines that the target gene is forest lengthy motion picture
The target gene of section non-coding RNA.
2. prediction technique according to claim 1, which is characterized in that the gene with encoding histone function includes:
The forest corresponds to all genes with protein coding in species genome.
3. prediction technique according to claim 1, which is characterized in that the inspection of the step 4) tissue-specific expression pattern
Survey method is real-time quantitative fluorescence PCR method.
4. prediction technique according to claim 3, which is characterized in that the amplification item of the real-time quantitative fluorescence PCR method
Part are as follows: 95 DEG C of initial denaturation, 30s；PCR reacts 40 and recycles: 95 DEG C, 3s, 60 DEG C, 30s；It is drawn in [60 DEG C, 95 DEG C] section molten
Solution curve.
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| CN111863127B (en) * | 2020-07-17 | 2023-06-16 | 北京林业大学 | Method for constructing genetic regulation network of plant transcription factor to target gene |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102799796A (en) * | 2011-05-24 | 2012-11-28 | 上海聚类生物科技有限公司 | Method for association analysis of long noncoding ribonucleic acid (LncRNA) and messenger ribonucleic acid (mRNA) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102799796A (en) * | 2011-05-24 | 2012-11-28 | 上海聚类生物科技有限公司 | Method for association analysis of long noncoding ribonucleic acid (LncRNA) and messenger ribonucleic acid (mRNA) |
Non-Patent Citations (3)
| Title |
|---|
| "Association Studies in Populus tomentosa Reveal the Genetic Interactions of Pto-MIR156c and Its Targets in Wood Formation";Mingyang Quan etc.;《ORIGINAL RESEARCH》;20160831;第7卷;论文第1-10页 * |
| "Exploring the Secrets of Long Noncoding RNAs";Mingyang Quan etc.;《International Journal of Molecular Sciences》;20150310;论文第5467-5488页 * |
| "lncRNA的研究进展";邢晓蕊等;《科技风》;20160930(第17期);论文第207-208页 * |
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