蜜环菌与天麻共生分子机制的转录组分析
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摘要
目的以蜜环菌和昭通乌天麻营养繁殖茎为实验材料,通过转录组测序比较分析,初步揭示蜜环菌天麻共生的分子机制。方法采用Trizol Reagent(Invitrogen)提取营养繁殖茎样品中的RNA,建立测序文库并进行测序。结果蜜环菌与天麻共生时有38 838条序列得到注释,在FDR(False Discovery Rate)<0.05和log2|FC|>1筛选条件下,共有23 333条基因发生显著差异表达,其中1 595条基因呈上调表达,21 738条呈下调表达。基于转录组数据分析结果,与胞外酶纤维素酶、木聚糖酶、漆酶以及多聚半乳糖醛酸酶相关的Unigene分别有21、6、39和6个,集中差异表达基因分别有9、3、23和4个,除1个纤维素酶基因上调表达外,其他均呈下调表达;与抗氧化酶超氧化物歧化酶、过氧化物酶和过氧化氢酶有关的Unigene基因分别有13、7和17个,其中差异表达的基因分别有6、7和7个,都呈下调表达。此外,q RT-PCR分析验证了这些基因呈下调表达。结论蜜环菌侵染天麻与其共生的过程中,蜜环菌的生命活动减弱,同时未受到天麻胁迫作用。这为进一步研究天麻蜜环菌共生分子机制提供了大量有价值的基因资源。
Objective In this study, Armillaria mellea and Zhaotong Gastrodia elata vegetative stems were used as the experimental material to reveal the symbiosis molecular mechanism of A. mellea infecting G. elata through comparative transcriptome sequencing analysis. Methods Trizor reason(invitrogen) was used to extract RNA from vegetative propagation stem samples, and the sequencing library was established and sequenced. Results The results showed that 38 838 sequences were annotated when G. elata symbiosis with A. mellea. Under false discovery rate(FDR) < 0.05 and log2 value(fold change) > 1 screening conditions, there were significant differences in expression levels among the 23 333 genes, in which 1 595 genes up-regulated and 21 738 down-regulated expression. Based on the transcriptome analysis, unigenes associated with the extracellular enzyme gene cellulase, xylanase, laccase, and polygalacturonase genes were 21, 6, 39, and 6 genes, respectively, in which the number of corresponding differentially expressed genes were 9, 3, 23, and 4, respectively. The expression of all these unigenes were down-regulated except for one cellulase genes. The number of unigenes related to anti-oxidant enzymes superoxide dismutase, peroxidase, and catalase were 13, 7, and 17, respectively. The differentially expressed genes respectively were 6, 7, and 7, all of which were down-regulated. In addition, these genes were selectively confirmed by real-time PCR. Conclusion The life activites of A. mellea which infect and symbiosis with G. elata declined, meanwhile G. elata did not produce biological stress for A. mellea. This study provides a lot of valuable genetic resources for further studying the symbiosis molecular mechanism of A. mellea and G. elata.
Objective In this study, Armillaria mellea and Zhaotong Gastrodia elata vegetative stems were used as the experimental material to reveal the symbiosis molecular mechanism of A. mellea infecting G. elata through comparative transcriptome sequencing analysis. Methods Trizor reason(invitrogen) was used to extract RNA from vegetative propagation stem samples, and the sequencing library was established and sequenced. Results The results showed that 38 838 sequences were annotated when G. elata symbiosis with A. mellea. Under false discovery rate(FDR) < 0.05 and log2 value(fold change) > 1 screening conditions, there were significant differences in expression levels among the 23 333 genes, in which 1 595 genes up-regulated and 21 738 down-regulated expression. Based on the transcriptome analysis, unigenes associated with the extracellular enzyme gene cellulase, xylanase, laccase, and polygalacturonase genes were 21, 6, 39, and 6 genes, respectively, in which the number of corresponding differentially expressed genes were 9, 3, 23, and 4, respectively. The expression of all these unigenes were down-regulated except for one cellulase genes. The number of unigenes related to anti-oxidant enzymes superoxide dismutase, peroxidase, and catalase were 13, 7, and 17, respectively. The differentially expressed genes respectively were 6, 7, and 7, all of which were down-regulated. In addition, these genes were selectively confirmed by real-time PCR. Conclusion The life activites of A. mellea which infect and symbiosis with G. elata declined, meanwhile G. elata did not produce biological stress for A. mellea. This study provides a lot of valuable genetic resources for further studying the symbiosis molecular mechanism of A. mellea and G. elata.
引文
[1]谢果珍,申爱荣,谭著明,等.天麻共生菌研究进展[J].湖南中医杂志,2015,31(4):206-208.
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[3]Kusano S.Gastrodia elata and its symbiotic association with Armillaria mellea[J].J Coll Agric Amp Univ Tokyo,1911,4(1):1-66.
[4]郭顺星,徐锦堂.天麻消化紫箕小菇及蜜环菌过程中细胞超微结构变化的研究[J].真菌学报,1990,9(3):218-225.
[5]徐锦堂,冉砚珠,郭顺星.天麻生活史的研究[J].中国医学科学院学报,1989,11(4):237-241.
[6]徐锦堂,牟春.天麻原球茎生长发育与紫箕小菇及蜜环菌的关系[J].植物学报,1990,32(1):26-31.
[7]邹宁,柏新富,吕剑涛,等.组织培养中天麻与蜜环菌共生机理的研究[J].安徽农业科学,2010,38(34):19312-19313.
[8]Guterman I,Shalit M,Menda N,et al.Rose scent:Genomics approach to discovering novel floral fragrancerelated genes[J].Plant Cell,2002,14(10):2325-2338.
[9]李福后,王伟霞.5株蜜环菌产几种胞外酶活性比较[J].淮海工学院学报:自然科学版,2006,15(3):58-61.
[10]王玉万,徐文玉.构菌栽培过程中对木质纤维素的降解和几种多糖分解酶活性的变化[J].微生物学通报,1989,16(3):137-140.
[11]包燚,狄永国,操璟璟,等.四株昭通乌天麻共生蜜环菌的生理生化特征及其分子鉴定[J].中国微生态学杂志,2017,29(7):761-770.
[12]杨舒贻,陈晓阳,惠文凯,等.逆境胁迫下植物抗氧化酶系统响应研究进展[J].福建农林大学学报:自然科学版,2016,45(5):481-487.
[13]徐锦堂,范黎.天麻种子/原球茎和营养繁殖茎被菌根真菌定殖后的细胞分化[J].植物学报,2001,43(10):1003-1010.
[14]赵香娜,胡亚平,张鹏,等.蜜环菌的特性及其对天麻生长的影响[J].中国果菜,2016,36(6):57-59.
[2]兰进,徐锦堂,李京淑.蜜环菌和天麻共生营养关系的放射性自显影研究[J].真菌学报,1994,13(3):219-222.
[3]Kusano S.Gastrodia elata and its symbiotic association with Armillaria mellea[J].J Coll Agric Amp Univ Tokyo,1911,4(1):1-66.
[4]郭顺星,徐锦堂.天麻消化紫箕小菇及蜜环菌过程中细胞超微结构变化的研究[J].真菌学报,1990,9(3):218-225.
[5]徐锦堂,冉砚珠,郭顺星.天麻生活史的研究[J].中国医学科学院学报,1989,11(4):237-241.
[6]徐锦堂,牟春.天麻原球茎生长发育与紫箕小菇及蜜环菌的关系[J].植物学报,1990,32(1):26-31.
[7]邹宁,柏新富,吕剑涛,等.组织培养中天麻与蜜环菌共生机理的研究[J].安徽农业科学,2010,38(34):19312-19313.
[8]Guterman I,Shalit M,Menda N,et al.Rose scent:Genomics approach to discovering novel floral fragrancerelated genes[J].Plant Cell,2002,14(10):2325-2338.
[9]李福后,王伟霞.5株蜜环菌产几种胞外酶活性比较[J].淮海工学院学报:自然科学版,2006,15(3):58-61.
[10]王玉万,徐文玉.构菌栽培过程中对木质纤维素的降解和几种多糖分解酶活性的变化[J].微生物学通报,1989,16(3):137-140.
[11]包燚,狄永国,操璟璟,等.四株昭通乌天麻共生蜜环菌的生理生化特征及其分子鉴定[J].中国微生态学杂志,2017,29(7):761-770.
[12]杨舒贻,陈晓阳,惠文凯,等.逆境胁迫下植物抗氧化酶系统响应研究进展[J].福建农林大学学报:自然科学版,2016,45(5):481-487.
[13]徐锦堂,范黎.天麻种子/原球茎和营养繁殖茎被菌根真菌定殖后的细胞分化[J].植物学报,2001,43(10):1003-1010.
[14]赵香娜,胡亚平,张鹏,等.蜜环菌的特性及其对天麻生长的影响[J].中国果菜,2016,36(6):57-59.