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马尾松PmAOX基因克隆与不同逆境胁迫表达分析
孙晓波, 陈佩珍, 吴晓刚, 吴帆, 季孔庶
南京林业大学学报（自然科学版） ›› 2020, Vol. 44 ›› Issue (4) : 70-78.
PDF(3429 KB)
PDF(3429 KB)
马尾松PmAOX基因克隆与不同逆境胁迫表达分析
The cloning and expression analysis of PmAOX gene from Pinus massoniana under different stress
揭示马尾松（Pinus massoniana）抗氰呼吸途径的交替氧化酶（alternative oxidase，AOX）基因的功能。
提取15年生马尾松不同组织RNA，运用RT-PCR及RACE技术克隆基因全长。以基因组DNA为模板,利用染色体步移法（chromosome walking，CK）克隆目的基因的启动子区域。通过qRT-PCR技术分析目的基因在不同组织及逆境胁迫下的表达。
克隆获得基因全长为1 610 bp，开放阅读框（open reading finder, ORF）1 221 bp, 编码406个氨基酸，推测氨基酸含有与拟南芥AtAOX基因相同的双铁羧酸结构LET、 NERMHL、LEEA和RADE_H，将其命名为PmAOX。实时荧光定量PCR显示，PmAOX在马尾松花中表达量最高，根中最低；脱落酸（abscisic acid，ABA）（100 μmol/L）胁迫后表达量逐渐下降，持续胁迫至24 h时，表达量增加至最大；经高温（42 ℃）、H2O2、NaCl（200 mol/L）、CO2（0.08% ~ 0.10%）、低温（4 ℃）及干旱（10% PEG6000）处理后，表达量均呈现先升高后降低的趋势；高温42 ℃ 处理至3 h时表达量最高；H2O2处理4 h时，表达量最高； NaCl胁迫至6 h时，表达量最高；CO2、低温及干旱处理至12 h时，表达量最高。克隆得到PmAOX 起始密码子上游1 081bp的启动子区域，经PlantCARE分析显示PmAOX启动子区域具有CAAT-box 及TATA-box 的基本顺式作用元件和低温、干旱等多个胁迫诱导元件，同时还包括脱落酸、茉莉酸甲酯、赤霉素、水杨酸在内的激素调控元件和光及昼夜节律响应元件。
PmAOX 可能与马尾松抗逆性相关，并受激素的诱导和调控。
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.
马尾松 / PmAOX / 基因克隆 / 逆境胁迫 / 荧光定量 / 启动子
Pinus massoniana / PmAOX / gene cloning / adversity stress / fluorescent quantification / promoter
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