Construction of an SSH Library of Pinellia ternata Under Heat Stress, and Expression Analysis of Four Transcripts

Abstract

Suppression subtractive hybridization (SSH) was used to clone transcripts upregulated during heat stress in leaves of Pinellia ternata. The complementary structures of deoxyribonucleic acid (cDNAs) of heat-stressed plants were used as the “tester,” and cDNAs of unstressed plants were used as the “driver.” Sequencing the whole SSH library yielded 303 expressed sequence tags (ESTs). Among these, 61 showed insignificant homology to any previously identified genes. Forty four of the remaining 242 ESTs represented singletons. Real-time quantitative reverse transcriptase polymerase chain reaction analysis of the expression patterns of four transcripts showed that three of these were upregulated after heat shock, and one was downregulated. The sequences showed high homology to small heat shock protein (sHSP), heat shock protein 90 (HSP90), heat shock protein 70 (HSP70), and stearoyl-ACP-protein desaturase (SAD), respectively. Rapid amplification of cDNA ends (RACE) was used to obtain full-length cDNAs of PtSAD and PtsHSP, and characterization and phylogenetic analyses were carried out according to their sequences.