Laser microdissection and fluorescence in situ hybridization reveal the tissue-specific gene expression in the ovules of P. tabulaeformis Carr

Abstract

Ovules are important carriers for seed plant reproduction, and ovules of gymnosperms are composed mainly of female gametophyte (FG) and adjacent diploid tissue (ADT). To investigate tissue-specific genes in the ovules of Pinus tabulaeformis Carr., we used laser microdissection (LMD) to separate FGs and ADTs, and performed linear amplification to construct cDNA libraries, obtaining a total of 156 expressed sequence tags (EST). Furthermore, some differentially expressed genes between FG and ADT of P. tabulaeformis ovule were screened by the analysis of EST. In addition, the expression levels of key genes in fertile line (FL) and sterile line (SL) ovules during development were verified by RT-qPCR, and we found that both PtRPL7a and PtDHN4 were more highly expressed in FL in each period (at least 1.7 times that of SL). Finally, fluorescence in situ hybridization (FISH) was used to reveal the temporal and spatial expression patterns of PtRPL7a and PtDHN4 in the ovules of P. tabuliformis during ovule development between FL and SL. Our results indicate that the expression levels and the locations of PtRPL7a and PtDHN4 show significant differences in different tissues during ovule development between FL and SL. This study further elucidates the molecular mechanism of the ovule abortion of P. tabulaeformis and provides a theoretical basis for the germplasm optimization of gymnosperms.