Abstract
With the successful completion of genomic sequencing for Brassica napus, identification of novel genes, determination of functions performed by genes, and exploring the molecular mechanisms underlying important agronomic traits were challenged. Mutagenesis-based functional genomics techniques including chemical, physical, and insertional mutagenesis have been used successfully in the functional characterization of genes. However, these techniques had their disadvantages and inherent limitations for allopolyploid Brassica napus, which contained a large number of homologous and redundant genes. Long intron-spliced hairpin RNA (ihpRNA) constructs which contained inverted repeats of the target gene separated by an intron, had been shown to be very effective in triggering RNAi in plants. In the present study, the genome-wide long ihpRNA library of B. napus was constructed with the rolling circle amplification (RCA)-mediated technology. Using the phytoene desaturase (PDS) gene as a target control, it was shown that the RCA-mediated long ihpRNA construct was significantly effective in triggering gene silence in B. napus. Subsequently, the resultant long ihpRNA library was transformed into B. napus to produce corresponding RNAi mutants. Among the obtained transgenic ihpRNA population of B. napus, five ihpRNA lines with observable mutant phenotypes were acquired including alterations in the floral model and the stamen development. The target genes could be quickly identified using specific primers. These results showed that the RCA-mediated ihpRNA construction method was effective for the genome-wide long ihpRNA library of B. napus, therefore providing a platform for study of functional genomics in allopolyploid B. napus.
Highlights
The complete sequencing of the genome of Brassica napus [1] has facilitated in the identification of novel genes and their corresponding functions, as well as in the exploration of the molecular mechanisms underlying important agronomic traits
Sci. 2020, 21, 7243 identified using specific primers, and five intron-spliced hairpin RNA (ihpRNA) lines with observable mutant phenotypes were acquired including alterations in the floral model and the stamen development. These results showed that the rolling circle amplification (RCA)-mediated ihpRNA construction method was effective for the genome-wide long ihpRNA library of B. napus, providing a platform for functional studies of genes on a genome-wide scale in B. napus
The improved intermediate cloning vector Pkan-BsaI was highly efficient for cDNA cloning, containing the ccdB lethal gene and facilitating in the insertion of double strand cDNAs (Step 1; Figure 1)
Summary
The complete sequencing of the genome of Brassica napus [1] has facilitated in the identification of novel genes and their corresponding functions, as well as in the exploration of the molecular mechanisms underlying important agronomic traits. Mutagenesis-based functional genomics techniques, including chemical (e.g., sodium azide or ethyl methanesulphonate), physical (e.g., c-radiation), and insertional mutagenesis (e.g., transposons or T-DNAs), have been successfully used in the functional characterization of genes [2,3,4,5,6,7,8]. These techniques have disadvantages and inherent limitations. To obtain mutants covering genome-wide genes, a large and sufficient mutant population should be produced. It is highly difficult to obtain mutants of essential genes because functional defects in these genes are often lethal
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
