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ISSN 0582-9879 Acta Biochimica et Biophysica Sinica 2004, 36(7): 467–476 CN 31-1300/Q

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Isolation and Partial Characterization of a Novel Pollen-specific cDNA with Multiple Polyadenylation Sites from Wheat

Yong-Feng JIN* and Teng-Fei BIAN

Institute of Biochemistry, Zhejiang University, Hangzhou 310029, China

Abstract A novel pollen-specific full-length cDNA clone PSG076 was isolated using suppression subtractive hybridization and 5'/3' RACE techniques. PSG076 was shown to exhibit multi-site polyadenylation by sequencing the 3' ends of the cDNAs. At least six transcripts with different length were produced from the single gene based on different poly(A) tail attachment sites. However, polyadenylation consensus sequence AAUAAA was not seen at the 3'-untranslated sequence. PSG076 contained a 299 bp 5' untranslated region and an open reading frame of 663 bp encoding a 221 amino acid peptide with pI of 4.31. A blast search revealed that this sequence did not show a significant similarity to any genes deposited in the public database. Southern blot indicated that PSG076 was a single copy gene. Northern blot and RT-PCR analysis indicated that PSG076 transcripts showed specific expression in mature pollen, and weak or undetectable signals in uninucleate microspore, immature seed, stem, young leave, root and ovary. Further analysis of the expression pattern in gametophyte showed that PSG076 transcripts were undetectable in uninucleate, binucleate microspore and pollen at early stage, and were first detectable and increased rapidly at middle and late stages of pollen development with the maximum level in mature pollen and also expressed in germinating pollen in vivo, suggesting that PSG076 might play a role in pollen germination and pollen tube growth in addition to its function in maturation. The evidences gathered in this work indicated that the six different transcripts from the single gene were differentially expressed during pollen development.

Key words wheat (Triticum aestivum L.); pollen-specific cDNA; suppression subtractive hybridization; multiple-site polyadenylation; differential expression

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Received: April 15, 2004 Accepted: May 25, 2004

This work is supported by a grant from the National Natural Science Foundation of China (No. 30370759)

*Corresponding author: Tel, 86-571-86971414; Fax, 86-571-86971501; E-mail, [email protected]