关键词: 生菜, LsPHYB, 可变剪接体, 高温胁迫, 响应

Abstract: 【Objective】Phytochrome B (PHYB) is a receptor for both light and temperature. The biological functions of alternative splice variants of LsPHYB in lettuce in response to high environmental temperatures were investigated through cloning them and analyzing their expression patterns in order to provide a theoretical basis for breeding heat-tolerant lettuce.【Method】The cDNA sequences of LsPHYB were searched from the genomic database of lettuce by bioinformatics method. The cloned LsPHYB1, LsPHYB2 and LsPHYB3 were subjected to multi-sequence alignment, alternative splice variants form analysis and phylogenetic analysis. Protein properties, such as molecular weight, theoretical isoelectric point, hydrophilicity and hydrophobicity, were predicted by online software. Secondary structure, tertiary structure and conserved domains were analyzed by bioinformatics software. Three alternative splice variants were characterized for expression after high temperature treatment assayed by RT-PCR. 【Result】There were three alternative splice variants of LsPHYB obtained by cloning, namely LsPHYB1, LsPHYB2 and LsPHYB3, with their CDS lengths of 3 509, 3 877 and 2 690 bp, which encoded 1 094, 960 and 853 amino acids, respectively. Alternative splice forms of LsPHYB1 were alternative 3′ splice site and skipped exon. Alternative splice forms of LsPHYB2 were alternative polyA and retain intron. An alternative splice form of LsPHYB3 was skipped exon. Conserved structural domain analysis showed that the N-terminal of PHYB2 lacked the PAS and PHY domains. The N-terminal of PHYB3 lacked the PAS and PHY domains, and its C-terminal lacked the HisKA domain. Phylogenetic analysis showed that three alternative splice variants were clustered into a clade. qRT-PCR analysis showed that the expression of LsPHYB3 was highest at the first day of high temperature treatment; LsPHYB2 had higher expression than LsPHYB1 and LsPHYB3 at days 5-9 of high temperature treatment; and at day 11 of high temperature treatment, the expression of LsPHYB1 was higher than that of LsPHYB2 and LsPHYB3. The three alternative splice variants peaked at different times during the 11 days of high temperature treatment. 【Conclusion】There are three alternative transcript variants of LsPHYB, named LsPHYB1, LsPHYB2 and LsPHYB3. LsPHYB1 encodes PHYB1 with intact functional domains; LsPHYB2 encodes a truncated PHYB2 without PAS and PHY domains; LsPHYB3 encodes a truncated PHYB3 without PAS, PHY and HisKA domains. LsPHYB3 expression was highest in the early stage of high temperature treatment, LsPHYB2 in the middle stage, and LsPHYB1 in the late stage, suggesting that the three alternative splice variants are functionally differentiated in response to high temperature stress.

Key words: lettuce, LsPHYB, alternative splice variants, heat stress, response