Differentially Expression of Tua1, a Tubulin-encoding Gene, during Flowering of Tea Plant Camellia sinensis (L.) O. Kuntze Using cDNA Amplified Fragment Length Polymorphism Technique

Abstract The complementary DNA (cDNA) amplified fragment length polymorphism technique was used to isolate transcript-derived fragments corresponding to genes involved in the flowering of tea plant. Comparative sequence analysis of an approximately 300 bp differential fragment amplified by primer combi-nation E11M11 revealed 80%−84 % similarity to the corresponding part of an α-tubulin gene of other species. The complete cDNA sequence of this α-tubulin was cloned by the rapid amplification of cDNA ends technique; its full length is 1537 bp and contains an open reading frame of 450 amino acid residues with two N-glycosylation sites and four protein kinase C phosphorylation sites. The deduced amino acid sequences did show significant homology to the α-tubulin from other plants that has been reported to be a pollen-specific protein and could be correlated with plant cytoplasm-nucleus-interacted male sterility. We named this complete cDNA Tua1. The nucleotide and amino acid sequence data of Tua1 have been recorded in the GenBank sequence database with the accession No. DQ340766. This Tua1 gene was cloned into the pET-32a expression system and expressed in Escherichia coli BL21trxB(DE3). The molecular weight of expressed protein was deduced to be approximately 49 kDa. Western blot analysis was used to identify the temporal expression of Tua1 in tea plant. Further studies of the effect of Tua1 protein on pollen tube growth indicated the Tua1 solution obviously promoted the growth of tea pollen tube