Journal
MOLECULES
Volume 23, Issue 3, Pages -Publisher
MDPI
DOI: 10.3390/molecules23030608
Keywords
3 '-RACE; high-throughput sequencing; alternative splicing; alternative polyadenylation; Populus
Funding
- National Natural Science Foundation of China [31570639]
- Nanjing Forestry University
- Priority Academic Program Development of Nanjing Forestry University
- Poplar Germplasm Nursery Project (Jiangsu Provincial Platform for Conservation and Utilizations for Agricultural Germplasm)
Ask authors/readers for more resources
Detection of complex splice sites (SSs) and polyadenylation sites (PASs) of eukaryotic genes is essential for the elucidation of gene regulatory mechanisms. Transcriptome-wide studies using high-throughput sequencing (HTS) have revealed prevalent alternative splicing (AS) and alternative polyadenylation (APA) in plants. However, small-scale and high-depth HTS aimed at detecting genes or gene families are very few and limited. We explored a convenient and flexible method for profiling SSs and PASs, which combines rapid amplification of 3'-cDNA ends (3'-RACE) and HTS. Fourteen NAC (NAM, ATAF1/2, CUC2) transcription factor genes of Populus trichocarpa were analyzed by 3'-RACE-seq. Based on experimental reproducibility, boundary sequence analysis and reverse transcription PCR (RT-PCR) verification, only canonical SSs were considered to be authentic. Based on stringent criteria, candidate PASs without any internal priming features were chosen as authentic PASs and assumed to be PAS-rich markers. Thirty-four novel canonical SSs, six intronic/internal exons and thirty 3'-UTR PAS-rich markers were revealed by 3'-RACE-seq. Using 3'-RACE and real-time PCR, we confirmed that three APA transcripts ending in/around PAS-rich markers were differentially regulated in response to plant hormones. Our results indicate that 3'-RACE-seq is a robust and cost-effective method to discover SSs and label active regions subjected to APA for genes or gene families. The method is suitable for small-scale AS and APA research in the initial stage.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available