Cloning And Functional Analysis Of Rice And Maize Tissue Specific Expressed Genes

Two subtracted libraries of rice differentiating callus and maize hybrid were established by suppression subtractive hybridization (SSH) in order to clone the rice differentiating callus-specific expressed genes, and maize hybrid specific expressed genes. 420 clones were obtained and the sequence comparison showed that the majority of ESTs from rice SSH library were belonged to the regulation gene which play important roles in plant growth and development, including rice RNase L inhibitor, FCA protein, SWI/SNF transcriptional factors and so on. Most of ESTs from maize SSH library involved in photosynthesis and metabolism of carbon, and others involved in the processing of mRNA, turnover of proteins and cell structure proteins. Dot blots were carried out using DIG-labeled rice cDNA as probe to screen the rice and maize SSH library in order to find out the differential expressed genes between the rice F1 and its parents. 22 of 223 (9.9%) rice ESTs showed differential expression between SHANYOU 63 and its parents, and 33 of 223 (14.8%) rice ESTs showed differential expression between Nippobare/9311 and its parents. Among 190 ESTs from maize SSH library, 18 ESTs were differentially expressed between SHANYOU 63 and its parents (the ratio is 9.5%), and 25 ESTs were differentially expressed between Nippobare/9311 and its parents (the ratio is 13.2%). The result indicated that there exist more differential-expressed genes in the indica/indica hybrid compared with the indica /japonica hybrid. RLI is a critical element of control of mRNA turnover rate. A novel cDNA fragment of rice RNase L inhibitor (RLI) was isolated by screening the subtractive library, which had a higher expression level in differentiating callus than in callus. The full-length cDNA of rice-RLI was obtained by the method of rapid amplification of cDNA ends, which contained a 1812 bp open reading frame encoding a 604 amino acid polypeptide. After sequence alignment, it was found that rice-RLI was highly homologous to human-RLI and mu-RLI. Homologous analysis showed that rice-RLI contained the conserved motifs (two repeated P-loops, two ATP-binding boxes and an iron-sulfur binding motif). The fluorescence quantitative PCR analysis showed that it was a constitutive expressed gene but up-regulated in abiotic stress (low temperature and NaCl treatment) and down-regulated under the treatments of NAA and IAA. FCA plays a key role in contolling followering-time of Arabidopsis. The homologous rFCA was cloned from rice by screening the SSH library and RACE. Four alternative splicing of rFCA were cloned from the leaves of rice, and designated as rFCA-1, rFCA-2, rFCA-3 and rFCA-4 respectively. rFCA-1 was homologous to FCA-gamma of Arabidopsis and contained conserved domains (two RNA-binding domains and a WW-domain). rFCA-2 was 102 bp shorter than rFCA-1 which caused the WW-domain deletion. The proteins encoded by rFCA-3 and rFCA-4 were 101 amino acids shorter than rFCA-1 at the N-terminal which is a glycine-rich region. The fluorescence quantitative PCR analysis showed that the mRNA of rFCA-1 is most abundant in the four alternative splicing of rice FCA, and its expression level is much higher in differentiating callus than in callus; The expression of rFCA-1 is steadly in the leaves of all rice varieties, but up-regulated in young spike of primary branch-differentiating stage of ZHENSHAN 97B and down-regulated in young spike of pistil and stamen-differentiating stage of ZHENSHAN 97B.