橡胶树红根病病原菌木聚糖酶编码基因GpTR1774的克隆与表达分析  

Cloning and Expression Analysis of Xylanase GpTR1774 Gene from Ganoderma pseudoferreum

作　　者：伏雪 涂敏[2] 蔡海滨 张红骥[1] 于德才 曾霞[2] FU Xue;TU Min;CAI Haibin;ZHANG Hongji;YU Decai;ZENG Xia(Plant Protection College,Yunnan Agricultural University,Kunming,Yunnan 650201,China;Rubber Research Institute,Chi-nese Academy of Tropical Agricultural Sciences,Haikou,Hainan 571101,China)

机构地区：[1]云南农业大学植物保护学院,云南昆明650201 [2]中国热带农业科学院橡胶研究所,海南海口571101

出　　处：《热带作物学报》2023年第12期2461-2468,共8页Chinese Journal of Tropical Crops

基　　金：海南省重点研发计划项目(No.ZDYF2021XDNY291);海南省优秀人才团队项目(No.20210203)。

摘　　要：细胞壁降解酶是诸多病原真菌的重要致病因子,其中木聚糖酶作为最重要的半纤维素酶,在丝状真菌降解细胞壁侵染寄主的过程中占有重要作用。本研究以橡胶树红根病病原菌HD3为材料,采用RT-PCR克隆木聚糖酶编码基因GpTR1774的基因序列,对其进行生物信息学分析;用HD3侵染橡胶树热研73397组培苗根部,电镜观测病原菌对根部细胞的侵染和破坏过程,并利用qRT-PCR方法测定不同侵染时间GpTR1774基因的表达量。结果表明:GpTR1774基因cDNA全长为780bp,编码259个氨基酸,其中含量最丰富的氨基酸为丙氨酸(Ala),占15.1%;预测蛋白分子量为28.12 kDa,脂肪系数为81.93,等电点为9.07,属亲水性蛋白,共有15个磷酸化位点,无信号和肽跨膜域,定位于细胞溶质;α-螺旋和无规则卷曲是GpTR1774蛋白二级结构的主要元件,分别占氨基酸序列的38.10%和41.31%。系统进化树分析显示,GpTR1774基因与狭长孢灵芝木聚糖酶基因相似度最高,达87.5%。qRT-PCR显示,木聚糖酶GpTR1774基因表达量整体趋势为先上升后下降,侵染3 d和4 d表达量极显著上升,4 d达到最高水平,约为侵染初始的16倍。本研究结果初步表明,木聚糖酶编码基因GpTR1774很可能参与橡胶树红根病病原菌的致病过程,为橡胶树红根病的致病机理解析和绿色防控提供参考。Cell wall degrading enzymes are important pathogenic factors of many pathogenic fungi,among which xy-lanase,as the most important hemicellulase,plays an important role in the process of host cell wall degrading by fila-mentous fungi to achieve host infection.In this study,RT-PCR was used to clone the coding region of xylanase GpTR1774 gene of Ganoderma pseudoferreum strain HD3 and analyze its bioinformatics.The root of Hevea tissue cul-tured seedlings Reyan 73397 were infected with HD3,meanwhile infection and destruction process of root cells were observed by electron microscopy.The gene expression of GpTR1774 was determined by qRT-PCR.The results showed that the total length of GpTR1774 cDNA was 780 bp,encoding 259 amino acids,among which the most abundant amino acid was alanine(Ala),accounting for 15.1%.The molecular weight of GpTR1774 protein was 28.12 kDa,fat coeffi-cient was 81.93,and the isoelectric point was 9.07.It was a hydrophilic protein with 15 phosphorylation sites,no signal and peptide transmembrane domain,and was located in the cell solute.The main components of the secondary structureα-helix and random curling are the main components of the secondary structure of GpTR1774 protein,accounting for 38.10%and 41.31%of the amino acid sequence,respectively.Phylogenetic analysis showed that GpTR1774 gene had the highest similarity with xylanase gene of Ganoderma boninense,reaching 87.5%.qRT-PCR showed that the overall gene expression trend of xylanase GpTR1774 was firstly increased and then decreased.The expression level of GPTR1774 increased significantly on the 3rd and 4th day after infection,and reached the highest level on the 4th day,about 16 times of the initial level.The results of this study indicate that the xylanase GpTR1774 gene was likely to be involved in the pathogenesis of G.psedoferreum,providing reference for the pathogenesis analysis and green prevention and control of G.psedoferreum.

关 键 词：热研73397 红根病病原菌 GpTR1774基因 侵染过程 QRT-PCR 

分 类 号：S763.7[农业科学—森林保护学]