Phyllostachys Edulis LTR Transposon-Cloning And Transposition Activity Identification Of PHRE6 And Construction Of Transposition Monitoring System

Long terminal repeats(LTR)Retrotransposons are a type of mobile DNA sequence that is commonly found in eukaryotic genomes and are named after having long terminal repeats at both ends.Most LTR retrotransposons are capable of sensing changes in the external environment,with transcriptional activation and transposition activation properties.In this study,a complete LTR retrotransposon was cloned from the genome of Phyllostachys edulis pubescens and named PHRE6(Phyllostachys edulis retrotransposons 6).The structure and properties of the transposon were analyzed and identified,and the bamboo under stress was observed.The activity of the transposon was identified by the changes in RNA level and DNA level of the LTR transposon.1.Using the published Phyllostachys pubescens genomic database,the LTR-STRUC software was used to search for the complete structural LTR transposon in the genome.Based on domain integrity and LTR homology,an LTR transposon PHRE6 was selected.The transposon has a full length of 5620 bp,the nucleotide sequence coding region contains an open reading frame of 48221 bp,GAG(Retrotransposon gag protein,GAG)and intact Pol(Polymerase,Pol)conserved domains required for transposition,and has PBS.(Prime bingding site,PBS),PPT(Polypurine tract,PPT)and LTR sequences at both ends;According to INT(Integrase,INT),RT(Reverse transcriptases,RT),RH(Ribonuclease H,RH),PR in Pol coding region The sequence of(Pepsin-like aspartate proteases,PR)can be used to judge PHRE6 belongs to the Ty1/Copia superfamily in the LTR retrotransposons;the homology of the LTR sequences at both ends is 99.08%,and the insertion time is 346.2 thousand years;The LTR sequence contains a cis-regulatory element and a light-responsive element for the glycometabolism reaction and endosperm expression.2.In order to further confirm the transcription activity and tissue specificity of PHRE6 transposon,three domains were detected by fluorescence quantitative PCR in three different tissues of Moso bamboo root,shoot and leaf,and DNA methylation inhibitors and four stress treatments.Changes in transcription levels in seedlings of Moso bamboo(including irradiation,high temperature,low temperature,and high salt treatment)revealed that the expression level of PHRE6 in leaves was significantly higher than that in roots and shoots;treatment with DNA methylation inhibitors After treatment with high temperature(42°C),low temperature(4°C)and high salt stress,the expression level increased significantly.The expression level after three gradients of irradiation was relative to the untreated wild type.Down-regulation,this result can indicate that PHRE6 is a transcription retroactive retrotransposon and may participate in the process of response to Moso bamboo stress.3.To further confirm the transposition activity of PHRE6 transposons,the SYBR Green fluorescence quantitative PCR was used to detect the change of PHRE6 copy number in the Phyllostachys edulis genome.The study found that high-temperature(42 °C),low temperature(4 °C),three gradients of high salt,100 ?mol / L,150 ?mol / L DNA methylation inhibitor treatment increased the copy number.The analysis of these results indicates that the transposition activity of the PHRE6 transposon can be activated under conditions of DNA methylation inhibition and under certain stress.4.This study constructed a yeast detection system to study the transposition mechanism of the LTR retrotransposons(PHRE2),which is more intuitive than previous methods of detecting insertion polymorphisms,copy number,etc.This method constructs a vector carrying the LTR of the histidine reporter gene with a reversed intron and a needle,transforms the two recombinant vectors into yeast,and screens and screens the defective media.The transposon transposition was performed,and genomic high-throughput sequencing was performed to analyze the new insertion site of the transposon in the yeast genome.In summary,this study cloned a complete LTR retrotransposon PHRE6 from the genome of Phyllostachys edulis,analyzed and identified the structure and characteristics of the transposon,and observed the RNA level of the LTR transposon under the stress.The activity of the transposon was identified by changes in DNA levels and the results indicated that PHRE6 is a potential activating activity LTR retrotransposon.At the same time,a more accurate and direct yeast monitoring system was constructed to identify the transposition activity of LTR retrotransposons.