Cloning and expression analysis of Cunninghamia lanceolata purple acid phosphatase gene ClPAP18b

Objectives The fast-growing and high-yielding Chinese fir clones Yang 023, Yang 036, Yang 6421 and Arabidopsis thaliana were used to study the function of purple acid phosphatases (PAPs) and to screen the clones with high P-efficiency utilization.

Methods The PAP18b gene was cloned and its sequence characteristics and homology were analyzed. The Yang 023, Yang 036, and Yang 6421 were cultivated with normal phosphorus (1.0 mmol/L KH₂PO₄) and low phosphorus (0.1 mmol/L KH₂PO₄, 0.9 mmol/L KCl) under the sand culture treatment for 0, 10, 15, 30 and 60 days, respectively. The acid phosphatase activity and total phosphorus content were determined, and the relationship between the expression of ClPAP18b gene, the phosphorus content and acid phosphatase activity in roots and leaves was quantitatively analyzed. The ClPAP18b gene was overexpress in Arabidopsis thaliana for gene function verification.

Results The CDS sequence (1212 bp) was cloned and named ClPAP18b, which encoded 404 amino acids. The subcellular location was in the intercellular region, indicating that the ClPAP18b gene may play a function of regulating the secretion of acid phosphatase to extracellular. The acid phosphatase activity measurement showed that after 30 days of phosphorus treatment, under normal phosphorus supply and low phosphorus treatment, the acid phosphatase activity in the roots of Yang 036 and Yang 6421 clones was higher than that in the leaves, while the acid phosphatase activity in roots under low phosphorus treatment was higher than that in normal phosphorus supply. The acid phosphatase activity in the roots and leaves of Yang 023 under low phosphorus stress was lower than that under normal phosphorus supply. The analysis of phosphorus content showed that the above-ground phosphorus content of Yang 023, Yang 036 and Yang 6421 was higher than that of the underground ones. After different levels of phosphorus treatment, the phosphorus content of different Chinese fir clones or different tissues was different. RT-qPCR results showed that low phosphorus stress induced the expression of Chinese fir ClPAP18b gene. The growth of ClPAP18b overexpression Arabidopsis thaliana plants was better than that of the control group, and the acid phosphatase activity in the leaves of overexpression plants was higher than that in the control group, but the accumulation of anthocyanins was lower than that in the control group. In addition, compared with the normal phosphorus treatment plants, the expression levels of PHT1;2, PHT1;8 and AtPAP26 genes in Arabidopsis thaliana overexpressing ClPAP18b were significantly increased, while the expression of AtPAP12 and AtPAP17 genes was significantly decreased.

Conclusions Low P stress induced increased expression of ClPAP18b gene and acid phosphatase activity in Chinese fir, but the adaptability of different Chinese fir clones to phosphorus deficiency was significantly different. Overexpression of ClPAP18b gene can promote Arabidopsis thaliana plants tolerant to phosphorus stress. ClPAP18b gene may play a regulatory role in the regulation mechanism of Chinese fir phosphorus stress, and can be used as an important candidate gene to improve the low phosphorus tolerance of Chinese fir.