甘蔗R2R3-MYB类似基因Sc2RMyb1的克隆及表达特性分析

摘要

R2R3-MYB是MYB转录因子基因家族的主要成员,已被证明在次生代谢和非生物逆境胁迫应答中起重要作用.为获得甘蔗(Saccharum complex) R2R3-MYB转录因子基因序列信息及其在不同非生物因子胁迫下的表达情况,本研究通过对甘蔗EST数据的分析,运用电子克隆获得一个甘蔗R2R3-MYB类似基因序列,进而采用PCR方法从甘蔗中克隆了该基因的基因组DNA和cDNA序列,基因命名为Sc2RMyb1(GenBank序列号:JQ823165).Sc 2RMyb1的基因组DNA全长1 807 bp,由3个外显子和2个内含子构成,编码区长度为1 284 bp,编码427个氨基酸.构建含有Sc2RMyb1基因的原核表达载体并转入大肠杆菌(Escherichia coli),经IPTG诱导产生的重组蛋白相对分子量约为52 kD.在NaC1胁迫的LB培养基上,重组菌生长明显优于对照菌.实时荧光定量PCR分析表明,甘蔗中Sc2RMyb1基因的表达受H2O2和NaC1抑制而下调,推测该基因作为负调节因子参与了NaC1胁迫应答相关基因的调控过程.本研究结果为后续该类转录因子基因在甘蔗抗逆相关机理研究和抗逆育种中的应用提供了基础资料.%R2R3-MYB is a principal member of MYB transcription factor superfamily, which has been showed to play an important role in secondary metabolism and abiotic stress responses. In order to obtain a full-length sequence of R2R3-MYB transcription factor gene and its expression profile under different abiotic stresses, a full-length cDNA sequence of sugarcane (Saccharum complex) R2R3-MYB-like gene named as SdRMybl (GenBank Accession No. JQ823165) was cloned by combined sugarcane EST databases, electronic doing technology and PCR amplification. Genomic DNA sequence of SdRMybl was 1 807 bp in length, including 3 exons and 2 introns, and the complete coding sequence was 1 248 bp encoding 427 amino acids. The recombinant protein with an estimated molecular mass of 52 kD was produced in positive prokaryotic strain induced by IPTG. The recombinant Escherichia coli cells exhibited better growth than that of the negative strain in liquid LB medium with addition of NaCl. When 25rRNA gene was used as the internal control, the expression profiles of SdRMybl gene responsive to different stresses were detected by Real-time qPCR in sugarcane tissue culture seedling. The transcript levels of Sc2RMybl gene in sugarcane seedling reduced under H2O2 and NaCl stress, respectively. It was suggested that the SdRMybl gene, as a negative adjustment factor, might involve in the response to salt stress. The results of this study provide basic information for the further research of the Sc2RMybl gene in sugarcane stress tolerance mechanisms and resistance breeding.