Isolation and identification of genes expressed differentially in rice inflorescence meristem with suppression subtractive hybridization

Abstract

A subtracted cDNA library of rice (Oryza sativa L.) inflorescence meristem (IM) was constructed using the suppression subtractive hybridization (SSH) method. The cDNAs of the rice shoot apical meristem (SAM) were used as "driver" and inflorescence meristem ( I . ) as "tester" in the experiment, respectively. Forty of 250 randomly chosen cDNA clones were identified by differential screening, which were IM-specific or IM-highly expressed. Most of the rice IM cDNAs cloned by SSH appear to represent rare transcripts, 40% of which were derived from truly differentially expressed genes. Of all the forty sequenced cDNA inserts, eleven contain the regions with 60%--90% identity to their homolog in GenBank, eighteen are expected to be new genes, only two correspond to published rice genes.Keywords: rice (Oryza sativa L.) cDNA, suppression subtractive hybridization, shoot apical meristem, inflorescence meristem.The suppression subtractive hybridization (SSH) is a new method developed for identifying differentially expressed genes between two different mRNA populations. Based on suppression PCR and suppression subtractive hybridization, SSH is designed to normalize and to amplify selectively cDNAs from different mRNA species with different abundances. Because selective amplification of target cDNA fragment (suppressing amplification of nontarget cDNA simultaneously) avoids laborious repeated hybridization and physical separation of ss and ds cDNA, SSH makes cloning of differentially expressed genes (including high, middle, low, rare abundance transcripts) as much as possible. The efficiency and reproducibility of SSH contributes its successful application in different studies for the identification of tissue-specific, developmental or induced, differentially expressed genes['41.In this note we report the results of identifying genes differentially expressed in rice inflorescence meristem other than that in shoot apical meristem with SSH technique. The transition from vegetative growth to reproductive development is a critical stage for the production of crops. cDNAs synthesized from inflorescence meristem poly(A+) RNA were subtracted against cDNAs from shoot apical meristem. The subtracted cDNAs were then cloned into pGEM-T vector to construct a subtracted library with 500 clones. 40 clones showing IM-specific or IM-increasing expression pattern have been identified by differentially screening with forward and reverse subtracted cDNA probes. Materials and methods( i ) Rice meristem materials. Rice (Oryza sativa L.) was planted in field. At the stages of vegetative growth and reproductive development initiation, shoot apical meristems and inflorescence meristems were collected after being detected under microscope, respectively.( ii ) mRNA isolation. Total RNA was isolated from plant material by a published method (Sacco de Vries 1998). Poly(A+) mRNA was purified from total RNA by two cycles of oligo(dT)-cellu-lose chromatography ( mRNA purification kit, Pharmacia ).(iii) Suppression subtractive hybridization. cDNA synthesis...

Alternative splicing and expression analysis ofOsFCA(FCAinOryza sativaL.), a gene homologous toFCAinArabidopsis

“…The cDNA from rice calli as driver, and the cDNA from differentiating calli as tester. After two round hybridizations, a primary PCR was applied to selectively amplify the differentially expressed fragments and the secondary PCR was performed with nested primers (Diatchenko et al 1996;Liu et al 2001). The products was ligated to pGEM-T vector (Promega), then transformed into Escheria coli strain JM109 (Sambrook et al 1989).…”

Cloning and Characterization of OsORC2, A New Member of Rice Origin Recognition Complex

“…However, in plants, the only cloned putative homologs of ORC2 are the Arabidopsis ORC2 and the maize ORC2. SSH is accepted as a highly efficient and powerful method for the identification of differentially expressed genes between different tissues or different developmental processes because of the normalization of frequent and rare cDNAs and the subtraction of cDNAs common between two populations (Liu et al 2001). In this paper, we have identified, isolated and characterized a new member of rice ORC use SSH and RACE method.…”

“…The cDNA from rice shoot apical meristem was used as the driver and the cDNA from inflorescence meristem as the tester. After two rounds of hybridizations, a primary PCR was used to amplify selectively the differentially expressed fragments and the secondary PCR was performed with nested primers (Liu et al 2001). The product was ligated to pGEM-T vector (Promega), and then transformed into an Escherichia coli strain JM109.…”

Analysis of highly expressed genes in the late zygotene to pachytene stages of meiotic prophase I in david lily

“…Follow ing PCR amplification, two copies of Hybond N + nylon membrane with the same lattice pattern were prepared for differential screening with two DIG labeled probes: forward subtracted and reverse sub tracted products. Hybridization and washing were car ried out by the conventional protocol [8]. The detec tion procedure was done according to the manufac turer's recommendations (Roche Molecular Biochemicals).…”