海滨雀稗自交结实突变体及野生型幼穗组织的转录组分析
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摘要
二倍体海滨雀稗Adalayd作为自交不亲和的暖季型草坪草,在环境保护及修复上发挥着重要的作用,但由于自身自交不亲和性减缓了其大面积推广。至今海滨雀稗自交不亲和的机制尚且未知并且基因数据库资源十分匮乏。为了研究海滨雀稗自交不亲和的机制,以二倍体海滨雀稗自交亲和体细胞突变体SP2008-3和自交不亲和野生型Adalayd为材料,采用Illumina Hiseq 2000高通量测序技术进行转录组测序。获得2个材料幼穗组织的表达谱,共获得68175654个Raw reads片段,测序结果de novo拼接获得117619个单基因簇(Unigene),其中50%的Unigene被注释。比较两个基因表达谱,发现1303个差异表达基因并对这些差异基因进行了基因本体论(gene ontology, GO)和京都基因与基因组百科全书(kyoto encyclopedia of genes and genomes, KEGG)分类,筛选出了22个与植物自交不亲和反应相关的基因,其中14个钙离子信号通路(钙调素、钙依赖蛋白激酶、类钙调素互作蛋白激酶)、3个F-box和5个硫氧还蛋白(thioredoxin)基因在体细胞突变体SP2008-3和野生型Adalayd之间存在差异表达,测序结果获得了很多与海滨雀稗自交不亲和相关的遗传资源信息。本研究首次将转录组学研究应用于海滨雀稗自交不亲和研究,为海滨雀稗自交不亲和进一步研究提供了宝贵且有价值的基因信息基础。
The warm-season turfgrass seashore paspalum(Paspalum vaginatum) is rapidly gaining recognition as having a potentially important contribution in a range of turf and environment conservation applications. However, self-incompatibility(SI) creates issues for seed production in seashore paspalum, consequently limiting the feasibility of large scale planting. The mechanism of SI response in P. vaginatum is still largely unknown. In addition, the lack of genomic and transcriptomic information makes it highly challenging to clarify the mechanism of SI in seashore paspalum. In order to further understand the mechanism of SI in seashore paspalum, we created a self-compatible(SC) mutant(SP2008-3) and compared the first transcriptome of SI seashore paspalum(variety Adalayd) and the SC mutant using Illumina sequencing technology. Transcriptome analysis using RNA-sequencing was performed to profile gene expression patterns in Adalayd and SP2008-3(the SC mutant). A total of 68.17 million raw reads were obtained, and de novo assembly produced 117619 Unigenes. All assembled Unigenes were annotated by querying against public databases. 58810 Unigenes(50%) were found to be homologous to genes in the NCBI non-redundant protein(Nr) database. In further orthologous analyses, comparison of the transcriptomes found 1303 Unigenes that showed significant differences in transcript abundance between Adalayd and SP2008-3. These Unigenes were functionally annotated within the Gene ontology(GO), Kyoto encyclopedia of genes and genomes(KEGG) pathways. A large number of notable genes potentially involved in SI responses showed differential expression. We conclude these genes may encode critical regulators of SI responses. Examples of these genes include 14 CaM(Calmodulin, calcium-dependent protein kinases and CBL-interacting protein kinases), 3 F-box and 5 THL(Thioredoxin). Our data represent a genetic resource for the discovery of genes related to SI in seashore paspalum. To our knowledge, this is the first study of the transcriptome of seashore paspalum focusing on SI. This resource will be very useful for future studies on the mechanisms of SI in seashore paspalum.
The warm-season turfgrass seashore paspalum(Paspalum vaginatum) is rapidly gaining recognition as having a potentially important contribution in a range of turf and environment conservation applications. However, self-incompatibility(SI) creates issues for seed production in seashore paspalum, consequently limiting the feasibility of large scale planting. The mechanism of SI response in P. vaginatum is still largely unknown. In addition, the lack of genomic and transcriptomic information makes it highly challenging to clarify the mechanism of SI in seashore paspalum. In order to further understand the mechanism of SI in seashore paspalum, we created a self-compatible(SC) mutant(SP2008-3) and compared the first transcriptome of SI seashore paspalum(variety Adalayd) and the SC mutant using Illumina sequencing technology. Transcriptome analysis using RNA-sequencing was performed to profile gene expression patterns in Adalayd and SP2008-3(the SC mutant). A total of 68.17 million raw reads were obtained, and de novo assembly produced 117619 Unigenes. All assembled Unigenes were annotated by querying against public databases. 58810 Unigenes(50%) were found to be homologous to genes in the NCBI non-redundant protein(Nr) database. In further orthologous analyses, comparison of the transcriptomes found 1303 Unigenes that showed significant differences in transcript abundance between Adalayd and SP2008-3. These Unigenes were functionally annotated within the Gene ontology(GO), Kyoto encyclopedia of genes and genomes(KEGG) pathways. A large number of notable genes potentially involved in SI responses showed differential expression. We conclude these genes may encode critical regulators of SI responses. Examples of these genes include 14 CaM(Calmodulin, calcium-dependent protein kinases and CBL-interacting protein kinases), 3 F-box and 5 THL(Thioredoxin). Our data represent a genetic resource for the discovery of genes related to SI in seashore paspalum. To our knowledge, this is the first study of the transcriptome of seashore paspalum focusing on SI. This resource will be very useful for future studies on the mechanisms of SI in seashore paspalum.
引文
[1] Baumann U,Juttner J,Bian X,et al.Self-incompatibility in the grasses.Annals of Botany,2000,85(Supple 1):203-209.
[2] Ran Z W,Li B Y,Yang D Z,et al.Advance in gene mapping of self-incompatibility in Poaceae plants.Chinese Agricultural Science Bulletin,2014,30(3):32-37.冉志伟,李保叶,杨定照,等.禾本科植物自交不亲和基因定位进展.中国农业通报,2014,30(3):32-37.
[3] Richter J.Mutations affecting self-incompatibility in Phalaris-coerulescens Desf (Poaceae).Heredity,1992,68(2):495-503.
[4] Li X,Paech N,Nield J,et al.Self-incompatibility in the grasses:Evolutionary relationship of the S gene from Phalaris coerulescens to homologous sequences in other grasses.Plant Molecular Biology,1997,34(2):223-232.
[5] Van Daele I,Van Bockstaele E,Martens C,et al.Identification of transcribed derived fragments involved in self-incompatibility in perennial ryegrass (Lolium perenne L.) using cDNA-Aflp.Euphytica,2008,163(1):67-80.
[6] Klaas M,Yang B,Bosch M,et al.Progress towards elucidating the mechanisms of self-incompatibility in the grasses:Further insights from studies in Lolium.Annals of Botany,2011,108(4):677-685.
[7] Yue G D,Gao Q,Luo L H,et al.Application of high-throughput sequencing in plant and animal research.Scientia Sinica Vitae,2012,42(2):107-124.岳桂东,高强,罗龙海,等.高通量测序技术在动植物研究领域中的应用.中国科学:生命科学,2012,42(2):107-124.
[8] Zhang W,Wei X,Meng H L,et al.Transcriptomic comparison of the self-pollinated and cross-pollinated flowers of Erigeron breviscapus to analyze candidate self-incompatibility associated genes.BMC Plant Biology,2015,15(1):248.
[9] Duncan R R,Carrow R.Seashore paspalum:The environmental turfgrass.Hoboken:John Wiley & Sons,inc.,1999:4.
[10] Huang B,Duncan R R,Carrow R N.Drought-resistance mechanisms of seven warm-season turfgrasses under surface soil drying:II.root aspects.Crop Science,1997,37(6):1858-1863.
[11] Lu S Y,Guo Z F.Physiological responses of turfgrass to abiotic stresses.Acta Prataculturae Sinica,2003,12(4):7-13.卢少云,郭振飞.草坪草逆境生理研究进展.草业学报,2003,12(4):7-13.
[12] Lee G,Carrow R N,Duncan R R.Growth and water relation responses to salinity stress in halophytic seashore paspalum ecotypes.Scientia Horticulturae,2005,104(2):221-236.
[13] Chen J B,Chu X Q,Li S,et al.Effect of saline water irrigation on growth of 7 genera and 11 species of warm season turfgrasses and their salinity tolerance difference.Pratacultural Science,2012,29(8):1185-1192.陈静波,褚晓晴,李珊,等.盐水灌溉对7属11种暖季型草坪草生长的影响及抗盐性差异.草业科学,2012,29(8):1185-1192.
[14] Cardona C A,Duncan R R,Lindstrom O.Low temperature tolerance assessment in Paspalum.Crop Science,1997,37(4):1283-1291.
[15] Xie X M,Lu X L.Good properties and values for utilization of seashore paspalum germplasm resource.Journal of South China Agricultural University,2004,25(Supple 2):64-67.解新明,卢小良.海雀稗种质资源的优良特性及其利用价值.华南农业大学学报,2004,25(增刊2):64-67.
[16] Liu G D.Tropical forage plant resources in China.Beijing:China Agricultural University Press,1999.刘国道.中国热带饲用植物资源.北京:中国农业大学出版社,1999.
[17] Xu M,Lu T G.Research progress of plant mutagenesis technology.Current Biotechnology,2011,1(2):90-97.徐明,路铁刚.植物诱变技术的研究进展.生物技术进展,2011,1(2):90-97.
[18] Zhong X X,Liu Z W,Chang P P,et al.Acquirement of self-compatible somatic mutants induced by colchicine in Paspalum vaginatum.Acta Prataculturae Sinica,2013,22(6):205-212.钟小仙,刘智微,常盼盼,等.秋水仙素诱导获得自交结实的海滨雀稗体细胞突变体.草业学报,2013,22(6):205-212.
[19] Jing Z B,Wei L,Yu J,et al.Transcription sequencing and its application prospective on discovering the gene resources of forage.Pratacultural Science,2011,28(7):1364-1369.井赵斌,魏琳,俞靓,等.转录组测序及其在牧草基因资源发掘中的应用前景.草业科学,2011,28(7):1364-1369.
[20] Niu J H,Lu X M,Tang J H,et al.Self-incompatibility in Poaceae and its advancement in molecular biological research.Molecular Plant Breeding,2006,4(2):269-274.牛俊海,鲁晓民,汤继华,等.禾本科植物自交不亲和性及其分子生物学研究进展.分子植物育种,2006,4(2):269-274.
[21] Jia X P,Ye X Q,Liang L J,et al.Transcriptome characteristics of Paspalum vaginatum analyzed with illumina sequencing technology.Acta Prataculturae Sinica,2014,23(6):242-252.贾新平,叶晓青,梁丽建,等.基于高通量测序的海滨雀稗转录组学研究.草业学报,2014,23(6):242-252.
[22] Zhang J C,Meng Y H,Song Y H,et al.Research developments of Ca～(2+)-CaM signal system and its regulation in plant.Journal of Chongqing Normal University (Natural Science Edition),2005,22(4):49-52.张君诚,孟玉环,宋育红,等.植物Ca～(2+)-CaM信号系统及其调控研究进展.重庆师范大学学报(自然科学版),2005,22(4):49-52.
[23] Straatman K R,Dove S K,Holdaway-Clarke T,et al.Calcium signalling in pollen of Papaver rhoeas undergoing the self-incompatibility (Si) response.Sexual Plant Reproduction,2001,14(1/2):105-110.
[24] Snowman B N,Geitmann A,Clarke S R,et al.Signalling and the cytoskeleton of pollen tubes of Papaver rhoeas.Annals of Botany,2000,85(Supple A):49-57.
[25] Staiger C,Franklin-Tong V.The actin cytoskeleton is a target of the self-incompatibility response in Papaver rhoeas.Journal of Experimental Botany,2003,54:103-113.
[26] Yang H Y.The role of calcium in the fertilization process in flowering plants.Acta Botannica Sinica,1999,41(10):1027-1035.杨弘远.钙在有花植物受精过程中的作用.植物学报,1999,41(10):1027-1035.
[27] Franklin-Tong V E,Ride J P,Read N D,et al.The self-incompatibility response in Papaver rhoeas is mediated by cytosolic free calcium.The Plant Journal,1993,4(1):163-177.
[28] Jordan N D,Ride J P,Rudd J J,et al.Inhibition of self-incompatible pollen in Papaver rhoeas involves a complex series of cellular events.Annals of Botany,2000,85(Supple A):197-202.
[29] Wehling P,Hackauf B,Wricke G.Identification of S-Locus linked pcr fragments in rye (Secale cereale L.) by denaturing gradient gel electrophoresis.The Plant Journal,1994,5(6):891-893.
[30] Kao T H,Tsukamoto T.The molecular and genetic bases of S-RNase-based self-incompatibility.Plant Cell,2004,16(16):72-83.
[2] Ran Z W,Li B Y,Yang D Z,et al.Advance in gene mapping of self-incompatibility in Poaceae plants.Chinese Agricultural Science Bulletin,2014,30(3):32-37.冉志伟,李保叶,杨定照,等.禾本科植物自交不亲和基因定位进展.中国农业通报,2014,30(3):32-37.
[3] Richter J.Mutations affecting self-incompatibility in Phalaris-coerulescens Desf (Poaceae).Heredity,1992,68(2):495-503.
[4] Li X,Paech N,Nield J,et al.Self-incompatibility in the grasses:Evolutionary relationship of the S gene from Phalaris coerulescens to homologous sequences in other grasses.Plant Molecular Biology,1997,34(2):223-232.
[5] Van Daele I,Van Bockstaele E,Martens C,et al.Identification of transcribed derived fragments involved in self-incompatibility in perennial ryegrass (Lolium perenne L.) using cDNA-Aflp.Euphytica,2008,163(1):67-80.
[6] Klaas M,Yang B,Bosch M,et al.Progress towards elucidating the mechanisms of self-incompatibility in the grasses:Further insights from studies in Lolium.Annals of Botany,2011,108(4):677-685.
[7] Yue G D,Gao Q,Luo L H,et al.Application of high-throughput sequencing in plant and animal research.Scientia Sinica Vitae,2012,42(2):107-124.岳桂东,高强,罗龙海,等.高通量测序技术在动植物研究领域中的应用.中国科学:生命科学,2012,42(2):107-124.
[8] Zhang W,Wei X,Meng H L,et al.Transcriptomic comparison of the self-pollinated and cross-pollinated flowers of Erigeron breviscapus to analyze candidate self-incompatibility associated genes.BMC Plant Biology,2015,15(1):248.
[9] Duncan R R,Carrow R.Seashore paspalum:The environmental turfgrass.Hoboken:John Wiley & Sons,inc.,1999:4.
[10] Huang B,Duncan R R,Carrow R N.Drought-resistance mechanisms of seven warm-season turfgrasses under surface soil drying:II.root aspects.Crop Science,1997,37(6):1858-1863.
[11] Lu S Y,Guo Z F.Physiological responses of turfgrass to abiotic stresses.Acta Prataculturae Sinica,2003,12(4):7-13.卢少云,郭振飞.草坪草逆境生理研究进展.草业学报,2003,12(4):7-13.
[12] Lee G,Carrow R N,Duncan R R.Growth and water relation responses to salinity stress in halophytic seashore paspalum ecotypes.Scientia Horticulturae,2005,104(2):221-236.
[13] Chen J B,Chu X Q,Li S,et al.Effect of saline water irrigation on growth of 7 genera and 11 species of warm season turfgrasses and their salinity tolerance difference.Pratacultural Science,2012,29(8):1185-1192.陈静波,褚晓晴,李珊,等.盐水灌溉对7属11种暖季型草坪草生长的影响及抗盐性差异.草业科学,2012,29(8):1185-1192.
[14] Cardona C A,Duncan R R,Lindstrom O.Low temperature tolerance assessment in Paspalum.Crop Science,1997,37(4):1283-1291.
[15] Xie X M,Lu X L.Good properties and values for utilization of seashore paspalum germplasm resource.Journal of South China Agricultural University,2004,25(Supple 2):64-67.解新明,卢小良.海雀稗种质资源的优良特性及其利用价值.华南农业大学学报,2004,25(增刊2):64-67.
[16] Liu G D.Tropical forage plant resources in China.Beijing:China Agricultural University Press,1999.刘国道.中国热带饲用植物资源.北京:中国农业大学出版社,1999.
[17] Xu M,Lu T G.Research progress of plant mutagenesis technology.Current Biotechnology,2011,1(2):90-97.徐明,路铁刚.植物诱变技术的研究进展.生物技术进展,2011,1(2):90-97.
[18] Zhong X X,Liu Z W,Chang P P,et al.Acquirement of self-compatible somatic mutants induced by colchicine in Paspalum vaginatum.Acta Prataculturae Sinica,2013,22(6):205-212.钟小仙,刘智微,常盼盼,等.秋水仙素诱导获得自交结实的海滨雀稗体细胞突变体.草业学报,2013,22(6):205-212.
[19] Jing Z B,Wei L,Yu J,et al.Transcription sequencing and its application prospective on discovering the gene resources of forage.Pratacultural Science,2011,28(7):1364-1369.井赵斌,魏琳,俞靓,等.转录组测序及其在牧草基因资源发掘中的应用前景.草业科学,2011,28(7):1364-1369.
[20] Niu J H,Lu X M,Tang J H,et al.Self-incompatibility in Poaceae and its advancement in molecular biological research.Molecular Plant Breeding,2006,4(2):269-274.牛俊海,鲁晓民,汤继华,等.禾本科植物自交不亲和性及其分子生物学研究进展.分子植物育种,2006,4(2):269-274.
[21] Jia X P,Ye X Q,Liang L J,et al.Transcriptome characteristics of Paspalum vaginatum analyzed with illumina sequencing technology.Acta Prataculturae Sinica,2014,23(6):242-252.贾新平,叶晓青,梁丽建,等.基于高通量测序的海滨雀稗转录组学研究.草业学报,2014,23(6):242-252.
[22] Zhang J C,Meng Y H,Song Y H,et al.Research developments of Ca～(2+)-CaM signal system and its regulation in plant.Journal of Chongqing Normal University (Natural Science Edition),2005,22(4):49-52.张君诚,孟玉环,宋育红,等.植物Ca～(2+)-CaM信号系统及其调控研究进展.重庆师范大学学报(自然科学版),2005,22(4):49-52.
[23] Straatman K R,Dove S K,Holdaway-Clarke T,et al.Calcium signalling in pollen of Papaver rhoeas undergoing the self-incompatibility (Si) response.Sexual Plant Reproduction,2001,14(1/2):105-110.
[24] Snowman B N,Geitmann A,Clarke S R,et al.Signalling and the cytoskeleton of pollen tubes of Papaver rhoeas.Annals of Botany,2000,85(Supple A):49-57.
[25] Staiger C,Franklin-Tong V.The actin cytoskeleton is a target of the self-incompatibility response in Papaver rhoeas.Journal of Experimental Botany,2003,54:103-113.
[26] Yang H Y.The role of calcium in the fertilization process in flowering plants.Acta Botannica Sinica,1999,41(10):1027-1035.杨弘远.钙在有花植物受精过程中的作用.植物学报,1999,41(10):1027-1035.
[27] Franklin-Tong V E,Ride J P,Read N D,et al.The self-incompatibility response in Papaver rhoeas is mediated by cytosolic free calcium.The Plant Journal,1993,4(1):163-177.
[28] Jordan N D,Ride J P,Rudd J J,et al.Inhibition of self-incompatible pollen in Papaver rhoeas involves a complex series of cellular events.Annals of Botany,2000,85(Supple A):197-202.
[29] Wehling P,Hackauf B,Wricke G.Identification of S-Locus linked pcr fragments in rye (Secale cereale L.) by denaturing gradient gel electrophoresis.The Plant Journal,1994,5(6):891-893.
[30] Kao T H,Tsukamoto T.The molecular and genetic bases of S-RNase-based self-incompatibility.Plant Cell,2004,16(16):72-83.