Functional annotation and expression analysis of novel sequences associated with aposporous development.

OCHOGAVIA A; SEIJO G; GONZÁLEZ AM; LASPINA N; CAMPOS CARNEIRO V; PESSINO S

Brasilia, Brasil

Congreso; XX International Congress on Sexual Plant Reproduction, Brasilia, Brasil; 2008

Plant Reproduction International Society

Recently, a group of 65 cDNA fragments corresponding to genes differentially expressed in sexual and aposporous P. notatum were identified. One-third of the sequences showed no homology to the gene databases, which prevented their functional annotation. These novel sequences probably correspond to genes that remain uncharacterised in plants, or display low levels of conservation. This work was aimed at a progressive investigation of the possible functional role for each one of these novel candidates. Characterization started with the selection of 5 clones which originated from differential display experiments (N2, N13, N11, N17, N22), based on its particular expression patterns and/or their weak homology to interesting candidates in the data banks. Total RNA was extracted from immature inflorescences of an obligate aposporous P. notatum genotype (Q4117). A cDNA amplification library was constructed using the Marathon cDNA Amplification kit (BD Biosciences, Clontech). Two upper and lower nested primers were designed for each one of the sequences, in order to perform 5’ and 3’ RACE (Rapid Amplification of Complementary DNA Ends) experiments. Flanking regions of clones N13 and N17 were successfully amplified after two rounds of selective nested amplification, cloned in PGemTeasy (Promega) and double-strand sequenced. Initial alignment of partial RACE sequences was done with Clustal W2 from the EBI-EMBL website (http://www.ebi.ac.uk/Tools/clustalw2/) and the consensus percentage and full sequence was obtained with Consensus program (http://structure.bu.edu/cgi-bin/consensus/consensus.cgi). At first, full sequences were BLAST-compared with the general data bases at the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi), and with specific data bases of rice and green plants at the Gramene website (http://www.gramene.org/multi/blastview). Sequences were translated into the six possible frames (http://us.expasy.org/tool/dna.html) and analysed using some Prosite and ProDom tools (http://ca.expasy.org/tools/scanprosite/) to determine the presence of conserved domains, which were further characterized using the Pfam server (http://pfam.sanger.ac.uk/). Dendrograms representing genetic distances were constructed with MegAlign (DNAstar). Consensus sequence for N13 presented homology to AJ877257, an Amborella trichopoda mRNA for a putative crabs claw transcription factor (CRC gene), a specialized member of the YABBY family, whose expression is restricted to carpels and nectaries. Homology was detected in approximately 50% of the sequence (including the 5´and 3´terminal ends), while the central section was highly variable, as is usual in other members of the crabs claw family. Meanwhile, clone N17 displayed homology to Os10g21160, a retrotransposon protein-like protein. In situ hybridization experiments were carried out in order to analyze more conclusively if differential expression between the appropriate tissues can be directly associated with the phenotype. Clone 13 hybridized strongly in ovaries, showing differential signals between apomictic and sexual plants. Results reported here allowed characterization of the structure, possible function and detailed pattern of expression of two novel genes involved in aposporous development in Paspalum notatum.