葫芦藓LEAFY基因和启动子的克隆及表达分析  

Cloning and Expression Analysis of LEAFY Gene and Promoter of Funaria hygrometrica

作　　者：赵冬林 王琦 邓莉兰 Zhao Donglin;Wang Qi;Deng Lilan(College of Landscape Architecture,Southwest Forestry University,Kunming,650224)

机构地区：[1]西南林业大学园林学院,昆明650224

出　　处：《基因组学与应用生物学》2021年第1期357-364,共8页Genomics and Applied Biology

基　　金：云南省县城生物多样性保护规划编制研究项目(09960/309622)资助。

摘　　要：本研究以孢子植葫芦藓为试验材料,采用Tail-PCR与RT-PCR相结合的方法克隆得到葫芦藓LFY基因(FhLFY)的完整片段,该基因DNA全长为2 527 bp,包含4个外显子和3个内含子序列,有1个1 050 bp的完整开放阅读框,编码349个氨基酸。通过Tail-PCR技术克隆得到905 bp的FhLFY基因启动子序列,利用PlantCARE启动子在线预测工具分析表明该序列含有CAT-box、CATT-box等启动子的特定结构,还包含低温响应元件、光响应元件等。通过采用荧光定量PCR方法对葫芦藓不同发育时期与不同组织的LFY基因的表达进行检测,发现LFY基因表达量在葫芦藓孢子体世代的孢子体中最高,推测LFY基因可能与孢子发育有关。在脱水胁迫条件处理下,葫芦藓LFY基因随处理时间的增加其表达量也随之增加,在3.5 h时表达量达到最高,且表达量差异最显著,推测LFY基因的表达受干旱因子的调控。本研究为LFY基因在苔藓植物中的表达模式分析以及苔藓植物在系统演化等方面的研究提供了基础资料,同时也为探索内含子的起源与进化提供了有价值的研究线索。This study uses the Funaria hygrometrica as the test material. A DNA fragment of Funaria hygrometrica was isolated and cloned by RT-PCR and Tail-PCR method. The DNA clone contained a complete sequence of 2 527 bp LFY homologous gene which included 3 intron, 4 exons and a complete open reading frame of 1 050 bp encoding 349 amino acids. A 905 bp fragment of LFY promoter was cloned by Thermal Asymmetric Interlaced PCR(Tail-PCR). Promoter sequence analysis showed that this promoter fragment had specific structure of the promoter, such as CAT-box, CAAT-box, etc, and some other regulatory sequences, such as light adjustment related components and low temperature response element. LFY gene expression in different tissues and at different development stage of Funaria hygrometrica was studied by real time fluorescent quantitative PCR(RT-qPCR) technology, the RT-qPCR analysis showed that the expression level of the LFY reached the highest peak at sporophyte in sporophyte generation, implyng that LFY gene is related to spore development. Under the treatment of dehydration stress, the expression of LFY gene of Funaria hygrometrica increased with the increase of treatment time, and reached the highest expression level at 3.5 h, with the most significant difference in expression level. It was speculated that expression of FhLFY gene was regulated by drought factor. This may provide a basis at the analysis of expression patterns of LFY gene in bryophytes and the systematic evolution of bryophytes, as well as valuable research clues for the exploration of the origin and evolution of introns.

关 键 词：葫芦藓(Funaria hygrometrica) LEAFY基因 启动子 表达分析 

分 类 号：Q943.2[生物学—植物学]