Cloning of Sterol 14α-demethylase Gene (HbCYP51) and Effects of Mechanical Damage and Exogenous Phytohormones on Its mRNA Expression in Hevea brasiliensis
Abstract：Sterol 14α-demethylase (CYP51), an important member of the cytochrome P450 family, is the key enzyme in sterol biosynthesis pathway. The objective of the study was to clone CYP51 gene and illustrate its function in latex production and drainage, and in defense of rubber tree against both tapping and ethephon stimulation. The target gene, encoding sterol 14α-demethylase, was separated from the rubber latex transcriptome library of elite Hevea brasiliensis material CATAS 7-33-97 and the mRNA expression difference, which stimulated by tapping and ethephon or jasmonic acid, was identified by Real-time quantitative PCR (qRT-PCR). BLAST analysis indicated that the cloned gene from Hevea brasiliensis was related to cytochrome P450 and named as HbCYP51 (GenBank No. KM203677), which had high homologies with the sterol 14α-demethylase reported in Populus trichocarpa (90%) and Malus domestica (88%). DNA sequence analysis showed that the size of the HbCYP51 cDNA was 2 305 bp, which included an open reading frame(ORF) of 1 461 bp, and encoded a polypeptide of 486 amino acids residues. The genome sequence length of HbCYP51 gene was 5 271 bp, consisting of 3 exons and 2 introns, and the first intron presented in 5'-UTR of the gene. The phylogenetic analysis suggested that HbCYP51 and CYP51 of Solanaceae (Nicotiana tabacum, Solanum chacoense, Solanum lycopersicum and Petunia×hybrida) were in the same branch group, indicating that the HbCYP51 had closer genetic relationship with other CYP51 of Solanaceae than that of Geum rivale, Fragaria vesca and Arabidopsis thaliana. qRT-PCR analysis showed that HbCYP51 was induced in the latex. HbCYP51 was regulated by tapping, ethephon and jasmonic acid, and significantly up-regulated by mechanical damage at the 7th tapping and ethephon at 48 h, and extremely significantly up-regulated by jasmonic acid at 24 h. The result confirmed that tapping, ethephon and jasmonic acid activated the expression of HbCYP51. Correlation analysis revealed that times of tapping had significantly positive correlation with expression of HbCYP51 gene (P＜0.05), and with latex yield (P＜0.01), but there was no significant correlation between latex yield and expression of HbCYP51 gene. In conclusion, the HbCYP51 gene might play an important role in the defense of rubber tree, and is directly or indirectly involved in the defense response against tapping and ethephon.
