利用同源克隆的方法对11个立枯丝核菌标准融合群菌株的GPD基因进行了分离。分析发现不同融合群立枯丝核菌GPD基因在外显子数目、编码蛋白长度及内含子剪切方式等方面存在差异，如AG22ⅢB、AG22Ⅳ、AG8三个融合群的GPD基因的外显子个数为10个，其他融合群均为11个；AG8融合群的GPD基因的编码蛋白长度为267个氨基酸，其余融合群的GPD基因的编码蛋白长度约为340个氨基酸。AG22ⅢB、AG22Ⅳ、AG8三个融合群的GPD基因在98、98、272氨基酸位点的内含子发生了缺失。基于蛋白序列的进化分析表明不同融合群之间GPD基因编码蛋白存在明显差异，可以有效地将不同融合群菌株区分开来；同时，不同物种之间GPD基因在进化上仍具有一定保守性，立枯丝核菌自身的GPD基因蛋白序列归为一类且与担子菌门同源性最高。这些结果表明进行保守基因序列差异分析是进行立枯丝核菌分类分群研究的新思路。
王艳丽,任衍春,张震,王教瑜,毛雪琴,姜华,邱海萍,柴荣耀,杜新法,孙国昌*
. 立枯丝核菌不同融合群菌株GPD基因的克隆及序列特征分析[J]. 中国水稻科学, 2013
, 27(6)
: 639
-646
.
DOI: 10.3969/j.issn.1001-7216.2013.06.011
Full length cDNA sequences of glyceraldehyde3phosphate dehydrogenase (GPD) gene from 11 anastomosis groups (AGs) of Rhizoctonia solani were isolated by PCRbased strategies. The open reading frames (ORFs) of the GPD genes(from ATG to TGA) showed some differences in exon numbers, splicing modes and lengths of coding sequences. GPD genes from AG22ⅢB, AG22Ⅳand AG8 have 10 exons, while those from the other AGs have 11 exons; GPD gene from AG8 encodes a protein of 267 amino acids, those from the other AGs encode proteins with about 340 amino acids. Meanwhile, intron deletions were found in the GPD genes from AG22ⅢB, AG22Ⅳ and AG8 at amino acid 98, 98 and 272, respectively. Phylogenetic analysis show that the GPD genes can effectively distinguish the different AGs. Further, the GPD genes from different AGs can differentiate all the AGs of R. solani from other fungi, implying the GPD gene is a useful alternative to the 5.8S rDNAITS gene for determination of phylogenetic relationships among different AGs and between Rhizoctonia and other fungi.
\[1\]刘力, 葛起新. 华东地区立枯丝核菌融合群鉴定. 浙江农业大学学报, 1987, 13(3): 227233.
\[2\]Anderson N A. The genetics and pathology of Rhizoctonia solani. Ann Rev Phytopathol, 1982(20): 329347.
\[3\]张天晓, 张志光. 立枯丝核菌 Rhizoctonia solani Kühn 的研究. 湖南师范大学：自然科学学报, 1986, 9(1): 7682.
\[4\]Carling D E, Kuninaga S, Brainard K A. Hyphal anastomosis reactions, rDNAinternal transcribed spacer sequences, and virulence levels among subsets of Rhizoctonia solani anastomosis group2 (AG2) and AGBI. Phytopathology, 2002, 92: 4350.
\[5\]李华荣. 丝核菌的菌丝融合群及其遗传多样性研究的新进展. 菌物系统, 1999, 18(2): 100107.
\[6\]Redkar R J, Herzog R W, Singh N K. Transcriptional activation of the Aspergillus nidulans gpdA promoter by osmotic signals. Appl Environ Microbiol, 1998, 64: 22292231.
\[7\]Jeong M J, Park S C, Kwon H B, et al. Isolation and characterization of the gene encoding glyceraldehyde3phosphate dehydrogenase. Biochem Biophysic Res Commun,2000, 278: 192196.
\[8\]Schaeffer H J, Forstheoefel N R, Cushman J C. Identification of enchancer and silencer regions involved in saltresponsive expression of Crassulacean acid metabolism (CAM) genes in the facultative halophyte Mesembryanthemum crystallinum. Plant Mol Biol, 1995, 28: 205218.
\[9\]Vernon D M, Ostrem J A, Bohnert H J. Stress perception and response in a facultative halophyte: The regulation of salinity induced genes in Mesembryanthemum crystallinum. Plant Cell Environ, 1993, 16: 437444.
\[10\]曾娟， 王源超， 申贵， 等. 大豆疫霉甘油醛3磷酸脱氢酶基因在病原物植物互作中诱导表达及其抗氧化作用在酵母遗传互补系统中的功能验证. 科学通报, 2006, 51(10): 11751181.
\[11\]肖川, 刘晓斐, 詹树萱, 等. 水稻甘油醛3磷酸脱氢酶cDNA结构分析和分子进化. 自然科学进展, 1998, 8(4): 411419.
\[12\]宋时英, 郭剑, 李军, 等. 甘油醛3磷酸脱氢酶结构的保守性. 生物物理学报, 1998, 14(3): 401406.
\[13\]Ogoshi A. Ecology and pathogenicity of anastomosis and intraspecific groups of Rhizoctonia solani Kühn. Ann Rev Phytopathol, 1987, 25: 125143.
\[14\]Ogoshi A. Studies on the taxonomy of the genus Rhizoctonia. Annu Phytopathol Soc Japan, 1984, 50: 307309.
\[15\]Parmeter J R, Sherwood R T, Platt W D, et al. Anastomosis grouping among isolates of Thanatephorus cucumeris. Phytopathology,1969, 59(1): 12701278.
\[16\]方中达. 常用培养基的配制. 植病研究方法. 北京: 农业出版社, 1982: 8387.
\[17\]朱旭芬. 基因工程实验指导. 北京: 高等教育出版社, 2006: 1825.
\[18\]Rosewich U L, Pettway R, McDonald B, et al. High levels of gene flow and heterozygote excess characterize Rhizoctonia solani AG1 IA (Thanatephorus cucumeris) from texas. Fungal Genet Biol,1999, 28(3): 148159.
\[19\]Duncan S, Barton J E, Obrien P A. Analysis of variation in isolates of Rhizoctoniasolani by random amplified polymorphic DNA assay. Mycolog Res,1993, 97: 10751082.
\[20\]Ceresini P C, Shew H D, Vilgalys R J, et al. Genetic structure of populations of Rhizoctonia solani AG3 on potato in eastern North Carolina. Mycologia, 2002, 94(3): 450460.
\[21\]PadashtDehkaei F, Ceresini P, Zala M, et al. Population genetic evidence that basidiospores play an important role in the disease cycle of riceinfecting populations of Rhizoctonia solani AG1 IA in Iran. Plant Pathol, 2013, 62: 4958.
\[22\]Sharon M, Kuninaga S, Hyakumachi M, et al. The advancing identification and classification of Rhizoctonia spp. using molecular and biotechnological methods compared with the classical anastomosis grouping. Mycoscience, 2006, 47(6): 299316.
\[23\]Kuninaga S, Natsuaki T, Takeuchi T,et al. Sequence variation of the rDNA ITS regions within and between anastomosis groups in Rhizoctonia solani. Cur Genet, 1997, 32(3): 237243.
\[24\]陈涛，张震，柴荣耀，等．浙江省水稻纹枯病菌的遗传分化与致病力研究．中国水稻科学, 2010, 24(1): 6772
\[25\]Takeda K, Kang Y, Yazawa K, et al. Phylogenetic studies of Nocardia species based on gyrB gene analyses. J Med Microbiol, 2010, 59: 165171.
\[26\]Kwok A Y. and Chow A W. Phylogenetic study of Staphylococcus and Macrococcus species based on partial hsp60 gene sequences. Int J Syst Evol Microbiol, 2003, 53(1): 8792.
\[27\]梁日深，周爱国， 陈金涛，等. 基于RAG2 基因序列分析仿石鲈科鱼类及其相关种类系统进化关系.水产学报, 2013, 37(5): 651660.
\[28\]Zheng A, Lin R, Zhang D, et al. The evolution and pathogenic mechanisms of the rice sheath blight pathogen. Nat Commun, 2013, 4: 1424.
