ABSTRACT
With the development of structural and functional genomics, nowadays specific plant genome and transcriptome sequences can be cloned much easier and faster. Next step is to identify the functions of different genes and regulating elements to unravel the genetic mechanisms behind plant growth and development. Expression and its regulation are the language and dynamic property of genetic material, so expression and regulation analysis of target genes and sequences in plant cell is the basis for function study. Besides stable genetic transformation, plant transient expression system gains broad application in recent years, and its combination with other new technologies as gene shuffling, VIGS and RNAi plays a more and more important role in plant functional genomics.
Subject(s)
Gene Expression Profiling , Genome, Plant , Genetics , Genomics , Methods , Immunity, Innate , Genetics , Plant Diseases , Genetics , Plants , Genetics , Plants, Genetically Modified , Genetics , RNA InterferenceABSTRACT
A pair of degenerate primers were designed based on NBS (nucleotide binding site, NBS) domain of resistance(R) gene and used to perform PCR with cDNA from the translocation line 6VS/6AL of Triticum aestivum-Haynaldia villosa. A clone (N7) characterized with NBS was obtained by sequencing analysis. Two specific primers were designed from the N7 sequence and used to screen a genomic TAC (transformation-competent artificial chromosome, TAC) library of 6VS/6AL consisting of ca. 2 x 10(6) clones. The library was stored as clone pools in twenty-two 96-well plates, each well containing approximately 1000 TAC clones. TAC plasmids were prepared from all the 2112 pools. Using a pooled PCR screening procedure, a positive TAC clone having a 40 kb insert was obtained. The positive clone was confirmed by Southern hybridization with the NBS fragment as a probe. The results indicate that the pooled PCR method is effective for screening of genomic libraries having large number of clones.