학술논문
橡胶树甾醇14α-去甲基化酶基因（HbCYP51）的克隆及机械损伤和外源激素对其表达的影响 / Cloning of Sterol 14α-demethylase Gene (HbCYP51) and Effects of Mechanical Damage and Exogenous Phytohormones on Its mRNA Expression in Hevea brasiliensis
Document Type
Academic Journal
Source
农业生物技术学报 / Journal of Agricultural Biotechnology. (12):1525-1535
Subject
Language
Chinese
ISSN
1674-7968
Abstract
甾醇14α-去甲基化酶(sterol 14α-demethylase, CYP51)是细胞色素P450家族的重要成员之一。基于从巴西橡胶(Hevea brasiliensis)胶乳转录组文库分离出的目的基因，利用实时荧光定量PCR方法研究橡胶树在不同机械损伤(割次)中和外施乙烯利和茉莉酸后，其胶乳中HbCYP51 mRNA表达差异，探讨不同刺激方式和水平对HbCYP51基因表达的影响。BLAST结果表明，本研究克隆了1个与细胞色素P450相关的基因，该基因与毛果杨(Populus trichocarpa)(90%)和苹果(Malus domestica)(88%)中已报道的CYP51同源性最高，命名为HbCYP51(GenBank登录号：KM203677)，含有3个外显子和2个内含子，其cDNA全长2305 bp，包括1461 bp的开放阅读框(ORF)，推测编码1个含486个氨基酸的蛋白。定量RT-PCR分析表明，胶乳HbCYP51基因是诱导型表达，并被割胶、乙烯利和茉莉酸调控表达，其中受24 h茉莉酸诱导上调表达最显著(P＜0.01)，首次证明了割胶、乙烯利和茉莉酸可激活HbCYP51的表达。相关性分析表明，割胶刀次与HbCYP51基因的表达量和胶乳产量均呈极显著正相关(P＜0.01)，而胶乳产量与HbCYP51基因的表达量无显著相关。研究结果初步说明，HbCYP51可能是巴西橡胶的重要防御因子，直接或间接参与对割胶和乙烯利的防御反应。
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.
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.