马尾松NAC转录因子基因PmNAC8的克隆及表达分析  

Cloning and Expression Analysis of NAC Transcription Factor PmNAC8 in Pinus massoniana

作　　者：镐青青 姚圣 刘佳禾 陈佩珍[1] 张梦洋 季孔庶[1] HAO Qing-qing;YAO Sheng;LIU Jia-he;CHEN Pei-zhen;ZHANG Meng-yang;JI Kong-shu(Nanjing Forestry University,Co-Innovation Center for Sustainable Forestry in Southern China,Key Laboratory of Forest Genetics&Biotechnology of the Ministry of Education,Nanjing 210037)

机构地区：[1]南京林业大学南方现代林业协同创新中心林木遗传与生物技术教育部重点实验室,南京210037

出　　处：《生物技术通报》2022年第4期202-216,共15页Biotechnology Bulletin

基　　金：国家重点研发计划(2017YFD0600304);江苏高校优势学科建设工程资助项目(PAPD)。

摘　　要：NAC(NAM,ATAF1/2,CUC2)家族基因参与调控植物生长发育、响应非生物胁迫及激素信号转导。研究马尾松中NAC转录因子在非生物胁迫中的应答,为阐明基因功能提供理论依据。以马尾松(Pinus massoniana)为试验材料,通过RTPCR及RACE技术克隆一个NAC家族基因,并对其进行生物信息学分析。运用qRT-PCR检测其组织表达特性及对逆境胁迫的响应情况;以基因组DNA为模板,运用染色体步移(genome walking)技术克隆该基因上游启动子序列;通过农杆菌注射本氏烟草(Nicotiana benthamiana),观察其亚细胞定位。该基因全长1726 bp,开放阅读框为1209 bp,编码402个氨基酸,将其命名为PmNAC8,GenBank登录号为MZ291447;亚细胞定位结果显示,该基因定位于细胞核;其密码子使用偏性较弱,偏好使用A/T结尾的密码子,且烟草与酵母更适合作为PmNAC8的异源表达受体;qRT-PCR显示PmNAC8在根部表达量最高,且受机械损伤及植物激素GA、ABA的诱导;克隆获得PmNAC8上游1492 bp的启动子区域,含有多个胁迫诱导元件、赤霉素调控元件和光响应元件。PmNAC8参与多种逆境胁迫,可能在赤霉素信号途径中发挥重要作用。NAC(NAM,ATAF1/2,CUC2)family of genes are involved in regulation of plant growth and development,abiotic stress response and hormone signal transduction.Studying the responses of NAC transcription factors in Pinus massoniana to abiotic stress would provide theoretical basis for elucidating gene function.A NAC family’s gene from P.massoniana was cloned by RT-PCR and RACE techniques,and then analyzed by bioinformatics.And the expression patterns in different tissues and its responses to stresses were detected by qRT-PCR.Using genomic DNA as a template,the upstream promoter region of the gene was cloned by the genome walking techniques.By agrobacterium injection of Nicotiana benthamiana,the subcellular localization was observed.The full length of the gene was 1726 bp and open reading frame was 1209 bp,encoding 402 amino acids,and named as PmNAC8.GenBank accession number is MZ291447.Subcellular localization showed that the gene was located in the nucleus and the codon usage bias was low,it preferred to use the codon ending in A/T,and tobacco and Accharomyces cerevisiae are more suitable as heterologous expression receptor materials for PmNAC8.The qRT-PCR demonstrated that PmNAC8 was expressed in all tissues of P.massoniana,and the highest expression was found in the root,and was induced by mechanical damage and plant hormones GA and ABA.The 1492 bp promoter region upstream of PmNAC8 contained multiple stress components,gibberellin control components and light response components.These results suggest that PmNAC8 is a transcription factor involved in various stresses and may play an important role in gibberellin signaling.

关 键 词：马尾松 NAC 密码子偏性 亚细胞定位 非生物胁迫 启动子 

分 类 号：S791.248[农业科学—林木遗传育种] Q943.2[农业科学—林学]