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根据苦荞花期转录组数据,采用RT-PCR技术和PCR技术克隆得到一条新的b HLH类蛋白基因FtbHLH3(登录号:KU296217),并分析了逆境胁迫下该基因在芽期苦荞胚轴和子叶中的表达量。结果显示,Ftb HLH3基因cDNA全长1 273 bp,包含一个957 bp的开放阅读框,编码蛋白含318个氨基酸。蛋白结构域分析表明,FtbHLH3编码蛋白N-端和C-端分别含有一个bHLH家族典型的结构域。系统进化树分析表明,FtbHLH3与其它植物参与抗逆的bHLH蛋白聚为一簇。荧光定量PCR分析表明,UV-B处理苦荞后,胚轴中FtbHLH3相对表达量在6 h内缓慢增加至1.08,12 h后显著上升至48 h的2.96;而子叶中FtbHLH3相对表达量在3 h后就显著上升,至24 h达到最大值6.64后趋于稳定。4℃冷处理苦荞后,胚轴和子叶中FtbHLH3相对表达量均随时间持续上升,并在48 h后达到最大值,分别为3.22和10.27。可见,本研究克隆的Ftb HLH3基因可能参与了苦荞对UV-B和寒冷等非生物胁迫的应答反应。
According to the data of transcriptome of flowering stage of buckwheat, a new b HLH protein gene FtbHLH3 (accession number: KU296217) was cloned by RT-PCR and PCR techniques. Under stress stress, The amount of expression in cotyledons. The results showed that the full length cDNA of Ftb HLH3 gene was 1 273 bp in length and contained a 957 bp open reading frame encoding a protein of 318 amino acids. Protein domain analysis showed that FtbHLH3 encoded protein N-terminal and C-terminal respectively contain a bHLH family of typical domains. Phylogenetic tree analysis showed that FtbHLH3 clustered with bHLH proteins involved in stress resistance in other plants. Fluorescence quantitative PCR analysis showed that the relative expression of FtbHLH3 in hypocotyls increased slowly to 1.08 at 6 h after UV-B treatment, and increased to 2.96 at 48 h after UV-B treatment. The relative expression of FtbHLH3 in cotyledons was 3 h after a significant increase, reaching a maximum of 24 h after 24 h to stabilize. The relative expression of FtbHLH3 in hypocotyls and cotyledons at 4 ℃ cold treatment increased with time and reached the maximum at 48 h, which were 3.22 and 10.27, respectively. It can be seen that the Ftb HLH3 gene cloned in this study may be involved in the response of tartary buckwheat to abiotic stress such as UV-B and cold.