杉木紫色酸性磷酸酶基因ClPAP18b的克隆及表达分析

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

【目的】以速生丰产型杉木无性系洋023、洋036、洋6421和拟南芥为材料,研究杉木中紫色酸性磷酸酶(PAPs)的功能作用,以筛选具有磷素高效利用特性的杉木无性系。【方法】采用PCR技术克隆PAP18b基因,分析其序列特征和同源性,并对杉木无性系洋023、洋036、洋6421进行正常供磷(1.0 mmol/L KH2PO4)和低磷胁迫(0.1 mmol/L KH_(2)PO_(4),0.9 mmol/L KCl)砂培盆栽处理0、10、15、30和60天,测定酸性磷酸酶活性及全磷含量,定量分析根和叶中ClPAP18b基因表达量、磷含量及酸性磷酸酶活性的关系,并将ClPAP18b基因过表达至拟南芥,进行该基因的功能验证。【结果】成功克隆获得杉木PAP18b基因CDS序列(1212 bp),命名为ClPAP18b,该基因编码404个氨基酸,亚细胞定位于胞间区,这表明ClPAP18b基因可能发挥调控酸性磷酸酶分泌至胞外的功能。酸性磷酸酶活性测定结果表明,30天磷处理后,正常供磷和低磷处理下,杉木洋036和洋6421无性系根中的酸性磷酸酶活性均高于叶,而根中酸性磷酸酶活性低磷处理下高于正常供磷处理;洋023的根和叶中酸性磷酸酶活性在低磷胁迫诱导下多高于正常供磷条件下根和叶中酸性磷酸酶活性。磷含量分析结果表明,杉木洋023、洋036和洋6421无性系的地上部磷含量高于地下部,不同水平供磷处理后,不同杉木无性系或同一杉木不同组织磷含量存在差异。RT-qPCR结果显示,低磷胁迫诱导杉木ClPAP18b基因表达。低磷条件下,ClPAP18b过表达拟南芥植株长势优于对照组,且过表达植株中的酸性磷酸酶活性叶高于对照组,但花青素积累量低于对照组。此外,相比于正常供磷处理植株,过表达ClPAP18b拟南芥中PHT1;2、PHT1;8和AtPAP26等与磷胁迫相关的基因表达量显著高于对照组,而AtPAP12和AtPAP17基因表达量明显降低。【结论】低磷胁迫诱导杉木ClPAP18b基因表达和酸性磷酸酶活性增强,但不同杉木无性系对磷缺乏的适应性存在明显差异,过表达ClPAP18b基因可促进拟南芥植株耐低磷胁迫,ClPAP18b基因可能在杉木低磷胁迫调节机制中发挥调控作用,可作为改良杉木耐低磷的重要候选基因。

【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 KH_(2)PO_(4))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.RTqPCR 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.