苹果紫色酸性磷酸酶相关基因MdPAP10的克隆及功能鉴定

Molecular Cloning and Functional Characterization of Purple Acid Phosphatase Related Gene MdPAP10 of Apple

【目的】克隆苹果紫色酸性磷酸酶相关基因Md PAP10,研究其组织表达模式和低磷响应,并进一步研究Md PAP10在低磷条件下的功能,为深入研究Md PAP10在果树中参与紫色酸性磷酸酶分泌和影响磷吸收的分子机理奠定基础。【方法】本研究以‘嘎啦’苹果(Malus×domestica‘Royal Gala’)为试材,利用同源序列比对和PCR技术,克隆苹果紫色酸性磷酸酶相关基因Md PAP10。通过NCBI分析Md PAP10的蛋白质结构并获得白梨、桃和草莓等10个物种的PAP10氨基酸序列,利用MEGA5.0构建系统进化树。利用q RT-PCR检测Md PAP10在苹果不同组织的表达情况和对低磷胁迫的响应特性。将Md PAP10连接到植物过表达载体p BI121,转化LBA4404农杆菌,用于侵染苹果愈伤组织。通过在抗性培养基上筛选和PCR鉴定,获得Md PAP10转基因愈伤组织。在低磷培养基上培养Md PAP10转基因愈伤组织检测其酸性磷酸酶积累情况以及对低磷胁迫的耐受性和磷含量。最后利用q RT-PCR检测Md PAP10转基因愈伤组织中磷相关基因的表达量。【结果】克隆获得苹果紫色酸性磷酸酶基因Md PAP10(基因序列号:MDP0000272096),开放阅读框为1 332 bp,编码含有443个氨基酸的蛋白。蛋白质结构分析显示,Md PAP10包含一个信号肽和一个磷酸酶结构域。基因结构分析显示,Md PAP10含有5个外显子和4个内含子。进化树分析显示,苹果Md PAP10与白梨Pb PAP10同源性最高,亲缘关系最近。表达分析显示,Md PAP10在根、茎、叶、花、果中均有表达,并且在根中的表达量最高。Md PAP10对低磷条件有明显响应,在根中表达量逐渐升高,在6 h达到最大后逐渐下降;在叶中的表达量始终低于对照组。Md PAP10转基因愈伤组织在低磷条件下能够明显促进酸性磷酸酶的分泌。在低磷条件下培养转基因愈伤组织20 d发现过表达Md PAP10提高了愈伤组织对低磷胁迫的耐受性,并且提高了对磷的吸收。q RT-PCR结果显示,过表达Md PAP10能够明显促进苹果磷相关基因的表达。【结论】Md PAP10能够对低磷胁迫有明显响应,在低磷条件下能够促进磷吸收和酸性磷酸酶的分泌。Md PAP10在响应低磷胁迫过程中发挥着重要的正调控作用。

【Objective】This study is aiming at cloning apple purple acid phosphatase gene MdPAP10 and studying its expression pattern and low phosphorus response process. The mechanism of action of MdPAP10 under low phosphorus condition was further studied. This study laid the foundation for further study of the molecular mechanism of MdPAP10 affecting phosphorus uptake in woody fruit trees. 【Method】MdPAP10 gene was cloned by homology sequence alignment and PCR technique from Malus× domestica ‘Royal Gala＇ apple. The protein structure of MdPAP10 was analyzed by NCBI and the PAP10 amino acid sequences of 10 species such as white pear, peach and strawberry were obtained. The phylogenetic tree was constructed by MEGA5.0. The induced expression and tissue-specific expression profiles of MdPAP10 gene in apple with low phosphorus stress were detected by real-time fluorescent quantitative PCR（q RT-PCR）. MdPAP10 was ligated into the plant overexpression vector p BI121. The resultant construct was transformed into LBA4404 Agrobacterium, for the infection of apple calli. MdPAP10 transgenic calli were obtained by screening on resistant medium and PCR identification. The acid phosphatase accumulation and the tolerance to low phosphorus stress and phosphorus content were detected by culturing MdPAP10 transgenic calli on low phosphorus medium. Finally, q RT-PCR was used to detect the expression of phosphorus-related genes in MdPAP10 transgenic calli.【Result】An apple purple acid phosphatase gene MdPAP10（MDP0000272096） was cloned from Malus × domestica ‘Royal Gala＇. Sequence analysis showed that the open reading frame（ORF） of MdPAP10 is 1 332 bp, which encoded 443 amino acids. Protein structure analysis showed that MdPAP10 contained a signal peptide and a phosphatase domain. Gene structure analysis showed that MdPAP10 contained 5 exons and 4 introns. A phylogenetic tree indicated that the apple MdPAP10 exhibited the highest sequence similarity to Pyrus bretschneideri Pb PAP10. Expression analysis showed that MdPAP10 was expressed in roots, stems, leaves, flowers and fruits, and the expression level in roots was the highest. MdPAP10 had a significant response to low phosphorus conditions, and the expression level in roots increased gradually, reached the maximum at 6 h and then decreased gradually. The expression level in leaves was always lower than that in the control group. MdPAP10 transgenic calli were obtained by Agrobacterium tumefaciens infection and verified by PCR and q RT-PCR. MdPAP10 transgenic calli can significantly advance the secretion of acid phosphatase in low phosphorus conditions. Overexpression of MdPAP10 in transgenic calli under low phosphorus conditions improved the tolerance of calli to low phosphorus stress and increased the uptake of phosphorus. The result of q RT-PCR showed that overexpression of MdPAP10 could significantly promote the expression of apple phosphorus-related genes. 【Conclusion】MdPAP10 can respond significantly to low phosphorus stress and promote phosphorus uptake and acid phosphatase secretion under low phosphorus conditions. MdPAP10 plays an important regulatory role in response to low-phosphorus stress.