Biotechnology Bulletin ›› 2022, Vol. 38 ›› Issue (4): 202-216.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0830

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

HAO Qing-qing(), YAO Sheng, LIU Jia-he, CHEN Pei-zhen, ZHANG Meng-yang, JI Kong-shu()   

  1. 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
  • Received:2021-06-30 Online:2022-04-26 Published:2022-05-06
  • Contact: JI Kong-shu E-mail:hqq@njfu.edu.cn;ksji@njfu.edu.cn

Abstract:

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 1 726 bp and open reading frame was 1 209 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 1 492 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.

Key words: Pinus massoniana, NAC, codon bias, subcellular localization, abiotic stress, promoter