Abstract
The cis-prenyltransferase gene LLA66 (Lilium longiflorum anther-66), the first prenyltransferase to be identified in the tapetum and microspores, was selected from a suppression subtractive cDNA library during microspore development in the anther of L. longiflorum. The LLA66 cDNA encodes a polypeptide of 308 amino acids with a calculated molecular mass of 35.7 kDa. Thermal asymmetric interlaced-PCR was employed to obtain the 5'-regulatory region of LLA66. Sequence alignment revealed that the LLA66 protein shares 30-41% identity with cis-prenyltransferases of various broad-spectrum species and is phylogenetically distinct from other monocot cis-prenyltransferases. Based on critical regulatory domains in cis-prenyltransferase, LLA66 was concluded to catalyze the production of long-chain polyprenyl products. RNA blot analysis indicated that the LLA66 gene is anther specific and differentially expressed during microspore development in the anther. In situ hybridization with the digoxigenin-labeled antisense riboprobe of LLA66 showed strong signals at the tapetal layer of the anther wall. The LLA66 mRNA was also coordinately detected in the microspores. Furthermore, gibberellin inhibitor analysis indicated that the LLA66 gene is endogenously induced by gibberellin, but its induction is independent of ethylene regulation. Reverse transcription-PCR analysis indicated that gene expression of LLA66 both in the microspore and in the anther wall increased to the maximum level, at which stage the tapetum became highly active and secretory. The enzyme activity of prenyltransferases in various stages of microspore development correlated with tapetal growth and disintegration. LLA66 was introduced into Saccharomyces cerevisiae, and the His-tagged LLA66 protein was affinity purified using Ni(2+)-nitrilotriacetic acid-agarose. The involvement of cis-prenyltransferase in the anther in the synthesis of dolichols and polyprenols is discussed.
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