Biological Activity Analysis Of Reverse Transcriptase In Gossypium Hirsutum And Gossypium Arboreum

Cotton is one of the most important cash crops in the world.As raw materials,cotton fiber and seeds are widely used in the fields of textile and oil.Cotton is not only a model organism for study heterologous polyploidy of plants,but also a good material for cell elongation and plant genome evolution research.Among them,Gossypium hirsutum(n=2x=26,AD1)has the largest planting area,and Gossypium arboreum(n=x=13,A2),as a descendant of the possible ancestor A0 of Gossypium hirsutum,also has a great research value.According to the newly assembled genomic data,the transposable elements content of Gossypium hirsutum accounted for 64.06%,and the scale in Gossypium arboreum reached 80.06%,the LTR retrotransposons is the most abundant element.However,only a few LTR retrotransposons can encode full-length proteins.LTR retrotransposons classic structure contains five domains,such as IN,RT,GAG,RH,AP.RT domain,which can encode reverse transcriptase,is the core domain among the Class I retrotransposon.This research take reverse transcriptase as the core research object,use Ch IP-seq,Westernblot,biological information analysis and other methods,investigate the reverse transcriptase biological functions in Gossypium hirsutum,Gossypium arboreum and Arabidopsis thaliana,the main results are as follows:(1)Genome-wide analysis of the distribution and enrichment of reverse transcriptase binding sites.We building Ch IP-seq database with the anti-RT specific antibody,it was found that the enrichment ratio in both Gossypium hirsutum and Gossypium arboreum was about five times,widely distributed in the euchromatin region far from the centromere.Data analysis showed that IP signals were concentrated and co-localized with genes,suggesting that reverse transcriptase may widely involve the gene expression.Further analysis showed that promoters and exons accounted for about 75% of the reverse transcriptase binding sites in the two cotton,concentrated downstream of the transcription start site.(2)Co-localization analysis,experimental verification of reverse transcriptase and epigenetic modification.By comparing the enrichment sites of reverse transcriptase and H3K27 ac and H3K4me3 in the two cottons.it was found that there was more than 85%overlap between the modification sites of reverse transcriptase and the two markers.The reverse transcriptase bound motif and KEGG-Orthlogs and IPR enrichment pathway analysis showed that methyltransferases and acetyltransferases were present in the reverse transcriptase binding sites,and transcription factor binding sites such as ZNF and IRF were also present in the binding motifs.Finally,the binding of reverse transcriptase and histone modify enzyme was further confirmed by IP-Western blot.(3)Construction and phenotype analysis of the reverse transcriptase overexpress Arabidopsis thaliana lines.By cloning the full-length sequence of reverse transcriptase and its necessary functional protein genes,connecting the 35 S strong promoter overexpression vector,and transfecting the inflorescence of Arabidopsis thaliana by invasion,the Arabidopsis thaliana overexpressed reverse transcriptase lines were constructed.They were further identified as positive in both DNA and protein levels.Meanwhile,RNA levels of reverse transcriptase and integrase in wild-type Arabidopsis thaliana and overexpressed lines were measured,respectively.It was found that the reverse transcriptase content in overexpressed Arabidopsis thaliana lines was significantly higher than that wild type,both in terms of transcription level and protein level.Compared with the Colombia wild-type Arabidopsis thaliana,the overexpressed lines leaf were bigger.Above all,in the process of plant evolution,reverse transcriptase was lossing the activity of transposable,then combined with the acetyl transferase to regulate plant gene expression widely.It is noteworthy that reverse transcriptase play a key role in regulating leaf size in Arabidopsis thaliana.This study provide a reference for the research in the biological activity of reverse transcriptase in plants.