白花泡桐纤维素合成酶PfCesA2及PfCesA8基因克隆及初步表达分析

为了获得白花泡桐纤维素生物合成途径关键酶基因-纤维素合成酶家族基因,本研究采用PCR技术和RACE技术,利用Oligo、Primer5等软件进行引物设计,从白花泡桐中克隆出纤维素合成酶基因两条,NCBI分析后,分别命名为PfCesA2(NCBI登录号:MK340935)及PfCesA8(NCBI登录号:MK340937)。PfCesA2基因的cDNA全长4164 bp,其开放阅读框(ORF)长度为3273 bp,能够编码含有1090个氨基酸的蛋白质,此蛋白质的相对分子质量为123431.95,等电点为6.70。PfCesA8基因的cDNA全长为3341 bp,其开放阅读框(ORF)长度为2955 bp,能够编码含有984个氨基酸的蛋白质,此蛋白质的相对分子质量为110241.71,等电点为6.20。二级结构分析表明二者都以α-螺旋和无规则卷曲为主。结构域和跨膜区分析表明二者在N端都含有锌指结构域,在C端都含有6个跨膜区,这些结构域能够表现纤维素合成酶的特征。系统进化树分析表明在所选取的物种当中,白花泡桐与芝麻、紫花风铃的亲缘关系较近。组织特异性分析结果表明,二者在茎中的表达量最高,根部次之,叶部最少;且在茎中,木质部的表达量高于韧皮部。说明二者可能主要参与次生壁的建成。本研究所克隆的两个基因属于白花泡桐纤维素合成酶家族基因,这在丰富该领域研究的同时,也能够为利用基因工程手段对泡桐木进行遗传改良提供重要的基因资源。

Cloning and Preliminary Expression Analysis of Cellulose Synthase PfCesA2 and PfCesA8 from Paulownia fortunei

In order to obtain the cellulose synthase family genes which are the key enzyme genes of cellulose biosynthesis pathway from Paulownia fortunei.Using PCR and RACE techniques and primers designed with softwares like Oligo and Primer5,two cellulose synthase genes were cloned from Paulownia fortunei.Two enzyme genes,after NCBI analysis,were named PfCesA2(NCBI accession number:MK340935)and PfCesA8(NCBI accession number:MK340937).The cDNA of PfCesA2 gene is 4164 bp in length and its open reading frame(ORF)length is 3273 bp,capable of encoding a protein containing 1090 amino acids.The relative molecular mass of this protein is 123431.95 and the isoelectric point is 6.70.The full-length cDNA of the PfCesA8 gene is 3341 bp,and its open reading frame(ORF)is 2955 bp in length,capable of encoding a protein of 984 amino acids with a relative molecular mass of 110241.71 and an isoelectric point of 6.20.Secondary structure analysis showed that both proteins were dominated byα-helix and random coil.Meanwhile domain and transmembrane region analysis indicated that both of them contain a zinc finger domain at the N-terminus and six transmembrane regions at the C-terminus,which characterize cellulose synthase.Phylogenetic tree analysis showed that among the selected species,Paulownia fortunei is closely related to Sesamum indicum and Handroanthus impetiginosus.The results of tissue specific analysis showed that the expression of PfCesA2 and PfCesA8 were the highest in the stem,followed by the root,and the leaf was the least.The expression in the xylem and stem were higher than that of the phloem indicating that PfCesA2 and PfCesA8 may mainly participate in the construction of the secondary wall.The two genes cloned from Paulownia fortunei in this study belong to the family of cellulose synthase gene.While enriching the research in this field,they can provide important genetic resources for genetic improvement of paulownia wood by genetic engineering.