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The cloning and expression analysis of PmAOX gene from Pinus massoniana under different stress
SUN Xiaobo, CHEN Peizhen, WU Xiaogang, WU Fan, JI Kongshu
JOURNAL OF NANJING FORESTRY UNIVERSITY ›› 2020, Vol. 44 ›› Issue (4) : 70-78.
PDF(3429 KB)
PDF(3429 KB)
The cloning and expression analysis of PmAOX gene from Pinus massoniana under different stress
The objective was to understand the function of the cyanide-resistant alternate oxidase (AOX) gene on the cyanide-resistant respiration pathway in Pinus massoniana.
RNA was extracted from 15-year-old P. massoniana, and the full length gene was cloned using RT-PCR and RACE. Using genomic DNA as a template, the PmAOX promoter region was cloned using chromosome walking (CK). The expression of target genes in different tissues under different kinds of stress was analyzed using qRT-PCR.
A full-length 1 610 bp gene was cloned to obtain a full-length open reading frame (ORF) of 1 221 bp encoding 406 amino acids. It was speculated that the amino acid sequence included the same ferrous carboxylic acid structure LET as the ArabidopsisAtAOX gene, NERMHL, LEEA, and RADE_H, and it was named PmAOX. The real-time fluorescence quantitative analysis showed that PmAOX expression was the highest in P. massoniana flowers, while it was the lowest in roots. ABA expression (100 μmol/L) decreased under ABA (100 μmol/L) stress and peaked 24 h after ABA (100 μmol/L) stress. After high temperature (42 °C), H2O2, NaCl (200 mmol/L), CO2 (0.08% to 0.1%), low temperature (4 °C), and drought (10% PEG6000) stress, PmAOX expression increased before subsequently decreasing. The highest expression level was as follows: high temperature of 42 °C treatment at 3 h; H2O2 treatment at 4 h; NaCl stress at 6 h; CO2 , low temperature, and drought treatment at 12 h. A 1 081 bp promoter region upstream of the PmAOX start codon was cloned. Based on the PlantCARE analysis, we found that the PmAOX promoter region has basic cis-acting elements, including a CAAT-box, TATA-box, and multiple stress-inducing elements, such as low temperature and drought. It also includes hormonal regulatory elements, including abscisic acid (ABA), methyl jasmonate (MeJA), gibberellin (GA) and salicylic acid (SA), as well as light and circadian rhythm response elements.
PmAOX may be related to the resistance of P. massoniana, and it is induced and regulated by hormones.
Pinus massoniana / PmAOX / gene cloning / adversity stress / fluorescent quantification / promoter
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