CN106997429A - A kind of Forecasting Methodology of forest long segment non-coding RNA target gene - Google Patents

Abstract

The present invention relates to a kind of Forecasting Methodology of forest long segment non-coding RNA target gene, belong to molecular genetic techniques field.The Forecasting Methodology for the forest long segment non-coding RNA target gene that the present invention is provided comprises the following steps：1) forest lncRNA sequences are obtained；2) target gene of the lncRNA after preliminary screening is obtained using blast Forecasting Methodologies；3) target gene of the lncRNA after postsearch screening is obtained using RNAplex Forecasting Methodologies；4) to the step 3) in lncRNA and the tissue-specific expression pattern of target gene of the lncRNA after postsearch screening detect, calculate both expression correlations, determine the interaction relationship between lncRNA and its target gene.Forecasting Methodology of the present invention can efficiently, stably detect the effect section of lncRNA and its target gene, and can significantly improve the accuracy of forest lncRNA microRNA target predictions.

Description

A kind of Forecasting Methodology of forest long segment non-coding RNA target gene

Technical field

The present invention relates to molecular genetic technique field, more particularly to a kind of forest long segment non-coding RNA target gene Forecasting Methodology.

Background technology

Long segment non-coding RNA (lncRNA) is that a class length is more than 200 nucleotides, no encoding histone ability or coding The extremely low rna transcription sheet of ability.Research shows 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, regulation and control of flowering of plant time etc..In plant It was found that, lncRNA mainly by the complementary form of sequence, is combined with its target gene, thus promote or suppressor table Reach.For example, arabidopsis lncRNA HID 1 (HIDDEN TREASURE 1) by with (the PHYTOCHROME- of PIF 3 INTERACTING FACTOR 3) promoter region base pair complementarity is combined, and then inhibit PIF3 expression.Due to perennial Forest genome heterozygosity is high, and DNA sequence polymorphism enriches, utilize 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 genes, only takes into account the complementary principle of sequence, and Forecasting Methodology Threshold parameter is relatively low, causes the target gene of prediction to there is false positive.Prior art lacks a kind of higher plant of accuracy The Forecasting Methodology of lncRNA target genes.

The content of the invention

It is an object of the invention to provide a kind of Forecasting Methodology of forest long segment non-coding RNA target gene.The present invention is carried The Forecasting Methodology of confession can efficiently, stably detect the effect section of lncRNA and its target gene, and can significantly improve woods The accuracy of wooden lncRNA microRNA target predictions.

The invention provides a kind of Forecasting Methodology of forest long segment non-coding RNA target gene, comprise the following steps：

1) forest lncRNA sequences are provided；

2) the lncRNA sequences are subjected to sequence ratio with the gene with encoding histone function using blast Forecasting Methodologies Right, parameter is set to：E-value<1E-10, obtains the target gene of the lncRNA after preliminary screening；

3) using RNAplex Forecasting Methodologies to the step 2) obtained lncRNA target gene screens, joins again Number is set to：E-value<- 60, obtain the target gene of the lncRNA after postsearch screening；

4) to lncRNA and the step 3) in lncRNA after postsearch screening target gene tissue specific expression mould Formula is detected, is calculated both expression correlations, is determined the interaction relationship between lncRNA and its target gene；

The calculating is as follows with formula：

The x represents expression quantity of the lncRNA in the tissue detected,

The y represents expression quantity of the target gene in the tissue detected,

R represents lncRNA and its expression of target gene amount relative coefficient；

Threshold value r represents the evaluation index to both expression correlations；R shows x and y expression phases between -0.75~0.75 Closing property is relatively low；r>0.75, or r<- 0.75 shows that x is related to y expression poling, determines that the target gene is non-for forest long segment The target gene of coding RNA.

Preferably, the gene with encoding histone function includes：All tools in forest correspondence species genome There is the gene of protein coding.

Preferably, the step 4) tissue-specific expression pattern detection method be real-time quantitative fluorescence PCR method.

Preferably, the amplification condition of the real-time quantitative fluorescence PCR method is：95 DEG C of pre-degeneration, 30s；PCR reactions 40 Individual circulation：95 DEG C, 3s, 60 DEG C, 30s；In 60 DEG C~95 DEG C interval drafting solubility curves.

The invention provides a kind of Forecasting Methodology of forest long segment non-coding RNA target gene.The present invention is by by two kinds Bioinformatics Prediction method is combined, and efficiently, stably detects lncRNA and the effect section of its target gene, and can show Write the accuracy for improving forest lncRNA microRNA target predictions.Result of the test shows that the application method can successfully realize forest length The prediction of fragment non-coding RNA target gene.

Brief description of the drawings

Fig. 1 is lncRNA lnc-CK and Pto-CKX6 that the embodiment of the present invention 1 is provided complementary segment；

Fig. 2 is phases of the lncRNA lnc-CK and Pto-CKX6 of the offer of the embodiment of the present invention 1 in 8 tissues of Chinese white poplar To expression quantity and the correlation results figure of expression.

Embodiment

The invention provides a kind of Forecasting Methodology of forest long segment non-coding RNA target gene, comprise the following steps：

1) forest lncRNA sequences are provided；

2) the lncRNA sequences are subjected to sequence ratio with the gene with encoding histone function using blast Forecasting Methodologies Right, parameter is set to：E-value<1E-10, obtains the target gene of the lncRNA after preliminary screening；

3) using RNAplex Forecasting Methodologies to the step 2) obtained lncRNA target gene screens, joins again Number is set to：E-value<- 60, obtain the target gene of the lncRNA after postsearch screening；

4) to lncRNA and the step 3) in lncRNA after postsearch screening target gene tissue specific expression mould Formula is detected, is calculated both expression correlations, is determined the interaction relationship between lncRNA and its target gene；

The calculating is as follows with formula：

The x represents expression quantity of the lncRNA in the tissue detected,

The y represents expression quantity of the target gene in the tissue detected,

R represents lncRNA and its expression of target gene amount relative coefficient；

Threshold value r represents the evaluation index to both expression correlations；R shows x and y expression phases between -0.75~0.75 Closing property is relatively low；r>0.75, or r<- 0.75 shows that x is related to y expression poling, determines that the target gene is non-for forest long segment The target gene of coding RNA.

Obtain after forest lncRNA sequences, using blast Forecasting Methodologies by the lncRNA sequences with having encoding histone The gene of function carries out sequence alignment, and parameter is set to：E-value<1E-10, obtains the target base of the lncRNA after preliminary screening Cause.

In the present invention, the gene with encoding histone function includes：Own in forest correspondence species genome Gene with protein coding.In the present invention, the lncRNA sequences and the gene complementation with encoding histone function are long Degree is preferably more than the 70% of lncRNA sequence lengths.

The present invention is defined to lncRNA sequences to lncRNA sequences with the gene complementation length with protein coding function 70%.

After the target gene for obtaining the lncRNA after preliminary screening, the present invention is using RNAplex Forecasting Methodologies to the step 2) lncRNA obtained target gene is screened again, and parameter is set to：E-value<- 60, obtain after postsearch screening LncRNA target gene；

After the target gene for obtaining the lncRNA after postsearch screening, the present invention to lncRNA and the step 3) in postsearch screening The tissue-specific expression pattern of the target gene of lncRNA afterwards is detected, calculates both expression correlations, it is determined that Interaction relationship between lncRNA and its target gene；

The calculating is as follows with formula：

The x represents expression quantity of the lncRNA in the tissue detected,

The y represents expression quantity of the target gene in the tissue detected,

R represents lncRNA and its expression of target gene amount relative coefficient；

Threshold value r represents the evaluation index to both expression correlations；R shows x and y expression phases between -0.75~0.75 Closing property is relatively low；r>0.75, or r<- 0.75 shows that x is related to y expression poling, determines that the target gene is non-for forest long segment The target gene of coding RNA.

In the present invention, the step 4) tissue-specific expression pattern detection method be real-time quantitative fluorescence PCR side Method.

In the present invention, the amplification condition of the real-time quantitative fluorescence PCR method is：95 DEG C of pre-degeneration, 30s；PCR reacts 40 circulations：95 DEG C, 3s, 60 DEG C, 30s；In 60 DEG C~95 DEG C interval drafting solubility curves.

With reference to embodiment, a kind of Forecasting Methodology of the forest long segment non-coding RNA target gene provided the present invention It is described in detail, but they can not be interpreted as to the restriction to the application protection domain.

Embodiment 1

Using the Forecasting Methodology of the forest long segment non-coding RNA target gene of the present invention, to Chinese white poplar lncRNA lnc-CK Target gene be predicted.

1st, the acquisition of lnc-CK sequences.The blade of Populus tomentosa Clones " LM50 " is collected, using CTAB methods, to Chinese white poplar Blade total serum IgE is extracted, quality evaluation Hou Song companies sequencing analysis, obtains the sequence number that leaves of Populus Tomentosa expresses lncRNA According to.And then obtain Chinese white poplar lncRNA lnc-CK sequence, SEQ ID NO.1.

2nd, joint utilizes blast and RNAplex Forecasting Methodology, to the lncRNA obtained in the step 1 target gene Carry out tentative prediction.First with blast method, by lnc-CK with coming from the sequence in leaves of Populus Tomentosa tissue cDNA library (E-value is compared in row<1E-10), recycle RNAplex method carry out further screening (parameter is set to：E- value<- 60) lncRNA lnc-CK 1-182 base and Pto-CKX6 the 1593-1774 base, are as a result found There is 182bp base pair complementarity area, lncRNA lnc-CK and Pto-CKX6 complementary segment figure are as shown in Figure 1.Preliminary sieve Choosing obtains the target gene (blast that Pto-CKX6 is its prediction：E-value=5E-055；RNAplex：E-value=-76.4), Wherein, Pto-CKX6 sequences are SEQ ID NO.2.

3rd, for the special RT-qPCR primers of lncRNA lnc-CK and Pto-CKX6 implementation sequences, with willow Actin bases The expression quantity of cause is internal reference, and its relative expression quantity is detected.The base sequence of LncRNA lnc-CK primers combination is SEQ ID NO.3 and SEQ ID NO.4；The base sequence of Pto-CKX6 primer pairs 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 (referring to table 1).

The primer sequence of table 1

To eight of annual Populus tomentosa Clones " LM50 " tissues or organ, (i.e. root, maturation xylem, prematurity are wooden Portion, bast, forming layer, tender leaf, old leaf and stem apex) RNA extracted, reverse transcription be cDNA carry out RT-qPCR experiments. RT-qPCR amplification condition be：95 DEG C of 30s of pre-degeneration；PCR reacts 40 circulations, including 95 DEG C of 3s, 60 DEG C of 30s；Most after 60 DEG C -95 DEG C interval to draw solubility curves.

By above-mentioned steps, detection lncRNA lnc-CK and Pto-CKX6 is in this eight tissues and the relative table in organ Up to amount (using the expression quantity of Actin genes as internal reference), phases of the lncRNA lnc-CK and Pto-CKX6 in 8 tissues of Chinese white poplar To expression quantity and the correlation results figure of expression as shown in Fig. 2 and table 2.According to relative coefficient calculation formulaThe expression correlation coefficient for calculating lncRNA lnc-CK and Pto-CKX6 is r= 0.92。

The lnc-CK of table 2 and Pto-CKX6 expression quantity correlations

By above implementation steps, it can be deduced that, Pto-CKX6 is lncRNA lnc-CK latent effect target gene.

Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should It is considered as protection scope of the present invention.

SEQUENCE LISTING

<110>Beijing Forestry University

<120>A kind of Forecasting Methodology of forest long segment non-coding RNA target gene

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Claims (4)

1. a kind of Forecasting Methodology of forest long segment non-coding RNA target gene, comprises the following steps：

1) forest lncRNA sequences are provided；

2) the lncRNA sequences are subjected to sequence alignment with the gene with encoding histone function using blast Forecasting Methodologies, Parameter is set to：E-value<1E-10, obtains the target gene of the lncRNA after preliminary screening；

3) using RNAplex Forecasting Methodologies to the step 2) obtained lncRNA target gene screens again, and parameter is set It is set to：E-value<- 60, obtain the target gene of the lncRNA after postsearch screening；

4) to lncRNA and the step 3) in the tissue-specific expression pattern of target gene of lncRNA after postsearch screening enter Row detection, calculates both expression correlations, determines the interaction relationship between lncRNA and its target gene；

The calculating is as follows with formula：

$r=\frac{N\mathrm{\&Sigma;}XY-\mathrm{\&Sigma;}X\mathrm{\&Sigma;}Y}{\sqrt{{\mathrm{N\&Sigma;X}}^2-{\left(\mathrm{\&Sigma;}X\right)}^2}\sqrt{{\mathrm{N\&Sigma;Y}}^2-{\left(\mathrm{\&Sigma;}Y\right)}^2}};$

The x represents expression quantity of the lncRNA in the tissue detected,

The y represents expression quantity of the target gene in the tissue detected,

R represents lncRNA and its expression of target gene amount relative coefficient；

Threshold value r represents the evaluation index to both expression correlations；R shows x and y expression correlations between -0.75~0.75 It is relatively low；r>0.75, or r<- 0.75 shows that x is related to y expression poling, and it is forest long segment non-coding to determine the target gene RNA target gene.

2. Forecasting Methodology according to claim 1, it is characterised in that the gene with encoding histone function includes： All genes with protein coding in forest correspondence species genome.

3. Forecasting Methodology according to claim 1, it is characterised in that the step 4) inspection of tissue-specific expression pattern Survey method is real-time quantitative fluorescence PCR method.

4. Forecasting Methodology according to claim 3, it is characterised in that the amplification bar of the real-time quantitative fluorescence PCR method Part is：95 DEG C of pre-degeneration, 30s；PCR reacts 40 circulations：95 DEG C, 3s, 60 DEG C, 30s；Dissolved in 60 DEG C~95 DEG C interval draw Curve.