基于转录组测序分析象草木质素合成的研究

doi:10.11686/cyxb2018303

摘要/Abstract

摘要： 采用高通量测序技术Illumlna HiSeq 2000对高木质素的象草品系eg7和低木质素的象草品系eg87(对照)茎组织进行转录组比较测序。测序获得了169630902个序列读取片段(reads),包含13788439920 nt碱基信息。对reads进行序列组装,获得87641个单基因簇(unigene),平均长度580 nt。从长度分布、GC含量等方面对unigene进行评估,数据显示测序质量好,可信度高。将获得的unigene与Nr、Nt、Swiss-Prot、COG、GO和KEGG数据库进行序列同源性比较和功能分析,62557个unigene与其他生物的已知基因具有不同程度的同源性,象草与高粱序列同源性最高。共鉴定出33323个差异表达基因,其中上调基因9704个(29.12%),下调基因23619个(70.88%);GO分析显示39968个unigene归为54个功能类别,大量unigene与细胞进程、代谢过程、催化活性等相关;KEGG pathway分析富集得到127条代谢通路,包括光合作用、betalain生物合成、苯丙烷类代谢、苯丙氨酸代谢等,苯丙烷类代谢途径差异基因富集程度高、差异基因数目最多,达285条,该途径中64条木质素单体合成酶基因表达上调,79条ClassⅢ型植物过氧化物酶基因表达下调、22条上调。挑选9个差异基因进行qRT-PCR验证,9个基因的表达趋势与高通量测序结果一致。为象草的分子生物学研究提供了宝贵的基因组数据,对于了解象草茎生物合成与木质素调控基因挖掘和多用途定向育种具有指导意义。

关键词: 象草, 高通量测序, 转录组, 木质素

Abstract: Elephant grass (Cenchrus purpureus) is an excellent forage crop and a promising lignocelluloses energy crop; its stem characteristics are very important for forage and feedstock quality. In order to understand the gene expression profile of elephant grass stem in the absence of a reference genome, we performed de novo transcriptome sequencing and compared the transcriptome of stems with a high lignin content plant (HLCP) eg7 and a low lignin content plant (LLCP) eg87 in elephant grass using Illumina HiSeq 2000 platform. A total of 169630902 high qualities reads were obtained, which were then assembled into 87641 unigenes with an average length of 580 nt. All of the 87641 unigenes were compared against the non-redundant protein database (Nr), non-redundant nucleotide database (Nt), swisssprot protein sequence database (Swiss-Prot), gene ontology (GO), cluster of orthologous groups (COG) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases for functional annotation; a total of 62557 unigenes were annotated. Most homologous sequences were found between elephant grass with sorghum. 33323 unigenes showed significant differences in expression between eg7 and eg87 (control), 9704 (29.1%) genes were up-regulated and 23619 (70.9%) were down-regulated. GO and KEGG pathway analysis revealed that the genes related to photosynthesis-antenna proteins, photosynthesis, betalain biosynthesis, phenylpropanoid biosynthesis are genes of interest. 9 monolignol biosynthesis-related genes and 8 ClassⅢ peroxidase genes were selected as candidate genes. qRT-PCR analysis showed that the expression of 9 selected unigenes were consistent with the transcriptome data. Our study presented comprehensive transcriptomic data and gene function analysis of C. purpureus stem, providing a valuable resource for future studies of plant breeding in the genus Pennisetum and comparative genome analysis for C₄ grasses.

Key words: Cenchrus purpureus, high-throughput sequencing, transcriptome, lignin

吴娟子, 钱晨, 刘智微, 潘玉梅, 钟小仙. 基于转录组测序分析象草木质素合成的研究[J]. 草业学报, 2019, 28(1): 150-161.

WU Juan-zi, QIAN Chen, LIU Zhi-wei, PAN Yu-mei, ZHONG Xiao-xian. De novo transcriptomic analysis for lignin synthesis in Cenchrus purpureus using RNA-seq[J]. Acta Prataculturae Sinica, 2019, 28(1): 150-161.

参考文献

[1] Río J C D, Prinsen P, Rencoret J, et al. Structural characterization of the lignin in the cortex and pith of elephant grass (Pennisetum purpureum) stems. Journal of Agricultural and Food Chemistry, 2012, 60(14): 3619-3634.
[2] Nyambati E M, Sollenberger L E, Kunkle W E.Feed intake and lactation performance of dairy cows offered napiergrass supplemented with legume hay. Livestock Production Science, 2003, 83(2): 179-189.
[3] Strezov V, Evans T J, Hayman C.Thermal conversion of elephant grass (Pennisetum purpureum Schum) to bio-gas, bio-oil and charcoal. Bioresource Technology, 2008, 99: 8394-8399.
[4] Liu X H, Shen Y X, Lou L Q, et al. Copper tolerance of the biomass crops elephant grass (Pennisetum purpureum Schum), vetiver grass (Vetiveria zizanioides) and the upland reed (Phragmites australis) in soil culture. Biotechnology Advances, 2009, 27(5): 633-640.
[5] Somerville C, Youngs H, Taylor C, et al. Feedstocks for lignocellulosic biofuels. Science, 2010, 329(5993): 790-792.
[6] Bhandari A P, Sukanya D H, Ramesh C R.Application of isozyme data in fingerprinting napiergrass (Pennisetum purpureum Schum) for germplasm management. Genetic Resources and Crop Evolution, 2006, 53(2): 253-264.
[7] Jakob K, Zhou F, Paterson A H.Genetic improvement of C₄ grasses as cellulosic biofuel feedstocks. In vitro Cellular and Developmental Biology Plant, 2009, 45(3): 291-305.
[8] Grabherr M G, Haas B J, Yassour M, et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nature Biotechnology, 2011, 29(7): 644-652.
[9] Liu W X, Zhang Z S, Chen S Y, et al. Global transcriptome profiling analysis reveals insight into saliva-responsive genes in alfalfa. Plant Cell Reports, 2016, 35(3): 561-571.
[10] Li X D, Wang X L, Chen X, et al. Transcriptome profiling analysis of phosphate-solubilizing mechanism of the white clover rhizosphere strain RW8. Acta Prataculturae Sinica, 2017, 26(8): 168-179.
李小冬, 王小利, 陈锡, 等. 转录组解析白三叶根际溶磷菌株RW8的解磷机制. 草业学报, 2017, 26(8): 168-179.
[11] Leng N, Liu X W, Zhang N, et al. Differential gene analysis of Poa pratensis in response to drought stress. Acta Prataculturae Sinica, 2017, 26(12): 128-137.
冷暖, 刘晓巍, 张娜, 等. 草地早熟禾干旱胁迫转录组差异性分析. 草业学报, 2017, 26(12): 128-137.
[12] Zhao J B, Hou X Y, Wu Z N, et al. Transcriptome analysis of Leymus chinensis under different mowing intensities. Acta Prataculturae Sinica, 2018, 27(2): 105-116.
赵劲博, 侯向阳, 武自念, 等. 不同刈割强度下羊草转录组研究. 草业学报, 2018, 27(2): 105-116.
[13] Zhang J L, Wu J Z, Qian C, et al. Study on biomass yield and lignocellulosic ethanol production potential of elephant grass. Jiangsu Agricultural Sciences, 2016, 44(8): 503-505.
张建丽, 吴娟子, 钱晨, 等. 不同品系象草的生物产量及木质纤维素乙醇生产潜力研究. 江苏农业科学, 2016, 44(8): 503-505.
[14] Soest P J V. The use of detergents in the analysis of fibrous feeds: II. a rapid method for the determination of fiber and lignin. Journal of the Association of Official Agriculture Chemists, 1963, 46: 829-835.
[15] Vogel K P, Pedersen J F, Masterson S D, et al. Evaluation of a filter bag system for NDF, ADF, and IVDMD forage analysis. Crop Science, 1999, 39: 276-279.
[16] Reis-Filho J S. Next-generation sequencing. Breast Cancer Research, 2009, 11(Supply 3): S12.
[17] Altschul S F, Madden T L, Schaffer A A, et al. Gapped BLAST and PSIBLAST: A new generation of protein database search programs. Nucleic Acids Research, 1997, 25(17): 3389-3402.
[18] Ashburner M, Ball C A, Blake J A, et al. Gene ontology: Tool for the unification of biology. Nature Genetics, 2000, 25: 25-29.
[19] Apweiler R, Bairoch A, Wu C H, et al. UniProt: The universal protein knowledgebase. Nucleic Acids Research, 2004, 32(database issue): 115-119.
[20] Kanehisa M, Goto S, Kawashima S, et al. The KEGG resource for deciphering the genome. Nucleic Acids Research, 2004, 32(database issue): 277-280.
[21] Mortazavi A, Williams B A, McCue K, et al. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nature Methods, 2008, 5(7): 621-628.
[22] Himmel M E, Ding S Y, Johnson D K, et al. Biomass recalcitrance: Engineering plants and enzymes for biofuels production. Science, 2007, 315(5813): 804-807.
[23] Simmons B A, Loque D, Ralph J.Advances in modifying lignin for enhanced biofuel production. Current Opinion in Plant Biology, 2010, 13(3): 313-320.
[24] Bonawitz N D, Chapple C.The genetics of lignin biosynthesis: Connecting genotype to phenotype. Annual Review of Genetics, 2010, 44: 337-363.
[25] Fornale S, Capellades M, Encina A, et al. Altered lignin biosynthesis improves cellulosic bioethanol production in transgenic maize plants down-regulated for cinnamyl alcohol dehydrogenase. Molecular Plant, 2011, 5(4): 817-830.
[26] Li X, Ximenes E, Kim Y, et al. Lignin monomer composition affects Arabidopsis cell-wall degradability after liquid hot water pretreatment. Biotechnol Biofuels, 2010, 3: 27-33.
[27] Pilate G, Guiney E, Holt K, et al. Field and pulping performances of transgenic trees with altered lignification. Nature Biotechnology, 2002, 20(6): 607-612.
[28] Tu Y, Rochfort S, Liu Z, et al. Functional analyses of caffeic acid O-methyltransferase and cinnamoyl-CoA-reductase genes from perennial ryegrass (Lolium perenne). Plant Cell, 2010, 22(10): 3357-3373.
[29] Wang Z Y, Li R Y, Xu J L, et al. Sodium hydroxide pretreatment of genetically modified switchgrass for improved enzymatic release of sugars. Bioresource Technology, 2012, 110(3): 364-370.
[30] Francoz E, Ranocha P, Nguyen-Kim H, et al. Roles of cell wall peroxidases in plant development. Phytochemistry, 2015, 112: 15-21.
[31] Meng Y Y, Fan S L, Song M Z, et al. Advance in research on Class Ⅲ peroxidases and its function in plants. Acta Botanica Boreali-Occidentalia Sinica, 2011, 31(9): 1908-1916.
孟艳艳, 范术丽, 宋美珍, 等. Class Ⅲ过氧化物酶在植物中的作用及其研究进展. 西北植物学报, 2011, 31(9): 1908-1916.
[32] Schopfer P, Plachy C, Frahry G.Release of reactive oxygen intermediates (superoxide radicals, hydrogen peroxide, and hydroxyl radicals) and peroxidase in germinating radish seeds controlled by light, gibberellin, and abscisic acid. Plant Physiology, 2001, 125(4): 1591-1602.
[33] Cosio C, Vuillemin L, De Meyer M, et al. An anionic class III peroxidase from zucchini may regulate hypocotyl elongation through its auxin oxidase activity. Planta, 2009, 229: 823-836.
[34] De O M, Caparro’s-Ruiz D, Vignols F, et al. Characterisation of maize peroxidases having differential patterns of mRNA accumulation in relation to lignifying tissues. Gene, 2003, 309: 23-33.
[35] Mansouri I E, Mercado J A, Santiago-Dómenech N, et al. Biochemistry and metabolism biochemical and phenotypical characterization of transgenic tomato plants over expressing a basic peroxidase. Physiologia Plantarum, 1999, 106(4): 355-362.
[36] Blee K A, Choi J W, O’Connell A P, et al. A lignin-specific peroxidase in tobacco whose antisense suppression leads to vascular tissue modification. Phytochemistry, 2003, 64(1): 163-176.
[37] Gabaldón C, Lópezserrano M, Pedreño M A, et al. Cloning and molecular characterization of the basic peroxidase isoenzyme from Zinnia elegans, an enzyme involved in lignin biosynthesis. Plant Physiology, 2005, 139(3): 1138-1154.
[38] Gutiérrez J, López Núñez-Flores M J, Gómez-Ros L V, et al. Hormonal regulation of the basic peroxidase isoenzyme from Zinnia elegans. Planta, 2009, 230(4): 767-778.
[39] Núñez-Flores M J, Gutiérrez J, Gómez-Ros L V, et al. Down regulation of the basic peroxidase isoenzyme from Zinnia elegans by gibberellic acid. Journal of Integrative Plant Biology, 2010, 52(2): 244-251.
[40] Herrero J, Esteban-Carrasco A, Zapata J M, et al. Looking for Arabidopsis thaliana peroxidases involved in lignin biosynthesis. Plant Physiology and Biochemistry, 2013, 67(3): 77-86.