Analysis Of Expressed Sequence Tags (ESTs) From Chimonanthus Praecox Link Flower And Isolation And Function Analysis Of Cplectin Gene

Calycanthaceae are a family of 10 species in three genera, Calycanthus L. with three species, Chimonanthus Lindley with six, and Idiospermum Blake with a single species. Wintersweet (Chimonanthus praecox Link) is an important species originating from China, where it is cultivated with a long history owing to its important characteristics, such as blooming in deep winter, having strong fragrance and stress resistance et al.. However, the genic expression situations of wintersweet are not yet reported. In this dissertation, we report the construction of the cDNA library from wintersweet flower, sequence and functional analysis of the expressed sequence tags (ESTs), cloning and functional identification of a lectin gene (Cplectin )from the library. The main results are as follows:1. Construction of wintersweet flower cDNA libraryA cDNA library was constructed with SMART cDNA Library Construction Kit using the RNA from the wintersweet flowers of sprout period, flower-bud period, display-petal period, initiating bloom period, bloom period and wither period. The primary library had a high titer of 1.4×10⁶, in which 96% clones were recombinant and the length of insert cDNAs was over 0.5 kb. The amplified library had a titer of 10¹⁰. The positive signals of PI, AP3 and LTP were detected by PCR in the amplified library, although their expression abundances were different. The LTP gene from the amplified library had no intron, which showed no genomic DNA in the library. These meant that a high quality cDNA library of wintersweet flower was obtained.2. Sequencing and assembling of expressed sequence tags (ESTs)...