In Silico Cloning Of Functional Genes In Rice And Construction Of A Salt Induced CDNA Library

In silico cloning is recently developed with the development of genome, EST projects and bioinformatics to rapidly know gene DNA or cDNA sequences by assembling homogous genome or EST sequences of target genes with the help of computer, and then to get the full length clone by RT-PCR. In silico cloning was applied first in human according to the abundant human genome or EST information. Lower cost, rapid, simple and clear objective are all advantages of in silico cloning, compared with traditional strategies, such as map-based cloning or transposable element tagging. In silico cloning is also considered to replace RACE(rapid amplification of cDNA ends ) or screening cDNA library for isolating full length gene.Using the wheat cytosolic glucose-6-phosphate dehydrogenase (G6PDH) cDNA as a query probe, homogous rice genome sequences from rice nr library in GenBank was discoverd. Full length rice G6PDH cDNA(OsG6PDH) sequence was attained by assembling the genome sequence and then cloned by RT-PCR. The full length 6-phosphaogluconate dehydrogenase (6PGDH) cDNA sequence was also attained by assembling the ESTs from rice dbEST library in GenBank with the maize cytosolic 6PGDH cDNA as the query probe. Os6PGDH was then isolated by RT-PCR. Both OsG6PDH and Os6PGDH coding proteins are key enzymes in pentose phosphate pathway(PPP) and first cloned in one crop, especially in mode plant. The cloning of OsG6PDH and Os6PGDH must be helpful to deeply researching the subcell distrubtion of PPP, and for prepare in genetic utilization of G6PDH+6PGDH. OsG6PDH is 5 kb genomic length with 15 exons. Seven putative promoters were discovered at the 5' portion and the promoter region with the highest possibility includes TATA-box. OsG6PDH is all expressed in immature spike,embryo, root and leaf, but slightly higher in immature spike and root. The genomic sequence of Os6PGDH is only 3 kb length including only two exons and specially, there is no start code existing in the first exon and the only intron lasts 1,250 bp. The promoter region was discovered in 5' portion of Os6PGDH genome sequence. According to RT-PCR result, Os6PGDH was expressed in all four tissues above and highest in immature spike. Phylogenetic tree analysis of G6PDH and 6PGDH show that both cytosolic and plastid 6PGDH of higher plant may originate from cyanobacterial while the origin of G6PDH geneis ambiguous. The mRNA expression pattern and genomic structure analysis of OsG6PDH and Os6PGDH above may indicate their different origin.A rice C2H2 type zinc finger protein cDNA was isolated by in silico cloning. The cloning strategy is to search rice dbEST library using a 500 bp rice EST sequence, which is originated from cDNA library, as query probe and let the homogous ESTs assemble to one longest EST contig. The clone, OsZFP, is also attained by RT-PCR. The zinc finger region of OsZFP has typical QALGGH motif, which is found only in plant and only one other rice gene with this type is previously isolated.Duing to incomplete rice genome library and smaller contigs, in silico cloning of longer fragment is difficult. It is also difficult for in silico cloning to get the full 5' UTR and 3' UTR unless much more ESTs. But screening cDNA library can solve these problems easily. A salt induced cDNA library of rice salt tolerance cultivar "Jiu Caiqing" was constructed. The original library was titered in 1.08X 107. The 99% recombined ratio and 1.1 kb average insert fragment were determined. The liter of amplified library was 0.8 X 10 10, which attained the saving requirement of library.OsG6PDH, Os6PGDH and OsZFP discribed in this paper were registered in GenBank with their accession numbers AY078072. AF486280 and AY077725 respectively.