A Preliminary Study On The Alternative Cleavage And Polyadenylation Of NAC Transcription Factor Genes In Populus

Alternative splicing(AS)and alternative polyadenylation(APA)events widely exist in eukaryotic gene transcription,and have a significant effect on gene expression regulation in eukaryotes.This study combined the traditional 3' rapid amplification technology(3'-RACE)with high-throughput sequencing technologies,and set up a method for the identification of gene splicing sites and polyadenylation sites at the same time,termed 3'-RACE-seq.After artificial data statistical analysis,theoretical derivation and experimental verification,we presented a set of strict data filter criterions.The method was used for studying 14 poplar NAC transcription factor genes,and revealed the complex alternative splicing and alternative polyadenylation events.On the basis of 3'-RACE-seq results,the functions of the genes and their alternative isoforms were studied preliminarily.The main results were as follows:(1)According to the annotation information of Populus genome database Phytozome P.trichocarpa v3.0,we choose 14 poplar NAC transcription factor genes as the target genes.The 3'-RACE-seq methods was established to achieve depth sequencing of the target genes.Experiments were designed to explore the causes of false positive splicing sites and set the filter criterions.A total of 72 splicing sites,including 34 new-found splicing sites,were annotationed to 14 comospore NAC transcription factor genes in polar,and revealed the complex alternative splicing events.Further,experiments were performed to verify the splice sites identified by the 3'-RACE-seq method.(2)3'-RACE-seq could be used for the identification of polyadenylation sites(PAS).Experiments were designed to explore the causes of false positive polyadenylation sites and set the filter criterions.A total of 30 polyadenylation sites(PAS)/ polyadenylation sites clusters(PAC)were identified in the 3'-UTR regions of primary gene model from 10 poplar NAC genes,and revealed tandem 3'-UTR APA events.Moreover,2 sites(PAS/PAC)in exon and 4 sites(PAS/PAC)in intron were found in PtNAC052,PtATAF1.2 and PtNAC035.For the three alternative polyadenylation isoforms of PtNAC052,experiments were performed to verify the polyadenylation sites revealed by 3'-RACE-seq.(3)On the basis of 3'-RACE-seq results,the full-length cDNA of the primary gene models and the altenative isoforms of six poplar NAC genes were cloned.Analysis of the sequences revealed that the subcellular localization and function might be different between the primary gene models and the altenative isoforms.By using poplar protoplast transient expression system,the subcellular localization of the coding proteins were observed,and localization of the isoforms,PtNAC053 c and PtATAF1.1b,showed different from their primary models.(4)From the 6 groups(the primary gene model and the altenative isoforms)of poplar NAC genes cloned before,PtATAF1.2 and its altenative isoform were selected for the further study.Using the method of quantitative real-time PCR analysis,the expression levels of PtATAF1.2 gene and its altenative isoform under different processing conditions were analysed.The results showed that,the expression of PtATAF1.2a and its isoform PtATAF1.2b were induced under the treatment of ABA or Flg22.It may associated with the response of abiotic stress and biological stress.Moreover,the drought assays and the pathogen infection assays of transgenic Arabidopsis thaliana plants showed: compared to the wild type(Col-0)control,transgenic plants overexpressing PtATAF1.2b showed a little more resistance to drought or Botrytis cinerea infection;while the phenotypes of transgenic plants overexpressing PtATAF1.2a were less obvious.This study presented a method combined traditional experimental method and high-throughput sequencing method,termed 3'-RACE-seq.Using theoretical analysis combined with experimental method,the false positive problems were explored,and these filter criterions were put forward for the splicing sites? and altenative polyadenylation sites? annotation of the 14 NAC genes and their alternative isoforms in P.trichocarpa.Based on cloning and analysis of the six poplar NAC genes and their variable,the difference in function between gene and its alternative isoforms were revealed,provided the basis for further studies on poplar NAC gene alternative splicing and alternative polyadenylation events in the future.