【作者】 王伟；

【导师】 赖钟雄； 苏明华；

【作者基本信息】 福建农林大学 ， 花卉与景观园艺， 2018， 博士

【摘要】 金线莲是多年生珍稀草本植物,兼具观赏和药用价值,野生资源稀缺且生长缓慢。本试验以大棚栽培福建金线莲(Anoectochilus roxburghii)为材料,研究了夜间补光的光质、时间和光强对其产量和次生代谢物含量的影响。在此基础上,采用超高效液相色谱(UPLC)和串联质谱(MS/MS)对两个比较组(最佳光质与对照组)进行了代谢组学研究,筛选差异代谢物并进行KEGG代谢通路富集分析;采用Illumina高通量测序技术,建立了金线莲蓝光和对照组的转录组数据库,筛选两个比较组的差异基因并进行功能注释和KEGG代谢通路分析,筛选了苯丙烷及类黄酮代谢途径关键酶并利用qRT-PCR对其进行表达验证;同时,利用高通量测序建立了金线莲小RNA组库,鉴定了大量miRNA,并对差异表达miRNA的靶基因进行了功能注释和KEGG代谢通路分析。本研究从转录组学、小RNA组学和代谢组学等多组学角度解析蓝光诱导金线莲类黄酮化合物积累的分子机理,并为生产上利用补光提高金线莲产量和药用成分含量提供科学依据。主要研究结果如下:1.不同LED光质、补光时间和光强对金线莲产量和次生代谢产物含量的影响①采用红、蓝、黄、绿、白等5种LED光源以及无补光对照进行补光光质对金线莲产量和次生代谢产物含量影响的比较研究。结果表明:蓝光处理下金线莲获得了最大生物量,其具有最高的叶片数、茎粗、鲜重和干重,其鲜重和干重分别比对照高17%和33.3%。并且蓝光显著促进总黄酮合成,其总黄酮含量比对照高24.2%。②进一步选用蓝光进行补光时间比较试验,设置0、4、8、12 h·d-1等4个处理,发现8 h/d蓝光能显著提高金线莲叶片数、茎粗、根长、鲜重、干重等,且显著促进总黄酮积累;③同时,针对蓝光进行光强比较试验,设置0、12、25、50 μmol·m2-·s1-等4个处理,发现25μmol·m2-·s1-能显著提高叶片数、茎粗、鲜重、干重等,且显著促进总黄酮含量。2.金线莲响应蓝光的代谢组学研究根据前面的研究结果,进行金线莲响应蓝光的代谢组学研究,结果表明:①金线莲样本共检测出代谢物543种,并鉴定出其中519种,鉴定比例为95.58%。其中90种是类黄酮化合物。②从两个比较组中筛选出差异代谢物70个,其中蓝光组相比对照组有显著上调代谢物55个,显著下调15个。差异代谢物有16个类黄酮化合物。③差异代谢物的KEGG通路富集分析表明:黄酮和黄酮醇的生物合成(Flavone and flavonolbiosynthesis)这一代谢通路是蓝光诱导的差异代谢物所参与的最主要代谢途径,而芦丁(Rutin)和木犀草苷(Luteoloside)是本试验蓝光造成金线莲总黄酮含量差异的最主要成分。差异代谢物中4种具有高生物活性的类黄酮葡萄糖苷化合物:异奥卡宁-7-葡萄糖苷(Isookanin-7-glucoside)、槲皮素3-O-葡萄糖苷(Quercetin-3-O-glucoside(Isoquercitrin))、木犀草素 7-O-葡萄糖苷(Luteolin 7-O-glucoside)和山奈酚3-O-葡萄糖苷(紫云英苷)(Kaempferol 3-O-glucoside(Astragalin))等在蓝光处理下含量显著提高,这说明蓝光能显著提高金线莲的药用价值。3.金线莲响应蓝光的转录组组学研究针对总黄酮含量显著差异的蓝光和对照组,采用Illumina高通量测序进行金线莲响应蓝光的转录组学研究。结果表明:①测序得到有效数据4.0×108条,数据量达到57.7 G。数据组装共获得63025条 Unigene,Unigene 平均长度为 826 bp,N50 为 1479 bp。通过本试验建立了福建金线莲转录组库,获得了大量高质量金线莲序列信息。②将 Unigene 与 Nr、Swissprot、KOG 和 KEGG 等 NCBI 的 4大数据库进行Blast比对,共有26363个Unigene得到了注释,其注释比例是41.83%。Nr数据库比对发现与金线莲亲缘关系较近的的物种是:油棕、海枣、小果野蕉、芦笋、红掌等。③使用MISA软件对所有Unigene进行搜索共获得SSR标记10592个,且种类丰富,涵盖了一至六碱基重复的几个类型SSR标记,也包括复合型SSR。④本实验共鉴定出1066个转录因子,可归类到55个家族,数量前五的家族是bHLH(75个)、C2H2(73个)、MYB related(61个)、ERF(59个)和FAR1(59个)。⑤两个比较组筛选得到差异基因382个。其中,显著上调基因183个,显著下调基因为199个。对差异基因进行GO功能注释,在生物学过程分类中,其差异表达基因主要注释在代谢过程(24个);在细胞组分分类中,主要注释在细胞(17个);在分子功能分类中,主要注释在催化活性(22个)。⑥差异基因显著富集的前10个代谢通路为:光合天线蛋白、植物-病原体互作、淀粉和蔗糖代谢、自噬调节和戊糖以及葡糖醛酸相互转化、乙醛酸和二羧酸代谢、叶酸生物合成、苯丙素生物合成、氨基糖和核苷酸糖代谢,丙氨酸和天冬氨酸以及谷氨酸代谢。其中,苯丙烷生物合成途径(Phenylpropanoid biosynthesis)与黄酮类物质合成代谢密切相关,类黄酮代谢是苯丙烷代谢的分支途径。这说明苯丙烷合成途径上富集的差异基因——过氧化物酶(POD)在苯丙烷以及类黄酮代谢中扮演了重要角色。⑦对苯丙烷代谢途径上的5个关键基因以及类黄酮代谢途径的13个关键结构基因并进行qRT-PCR表达验证,对转录组数据和qRT-PCR结果进行比较,这18个基因上调或下调表达的趋势基本一致,证明了转录组数据的可靠性。4.金线莲类黄酮代谢结构基因CHS的功能分析从金线莲转录组测序数据挖掘得到CHS全长基因,并经过测序验证。构建过表达载体将其转入本氏烟草,结果发现CHS基因的转入过表达导致本氏烟植株呈现矮壮化,转基因植株株高相比对照降低11.3%、茎粗相比对照增加了 10.4%,开花时间提早,其种子百粒重比对照植株低11%,而其总黄酮含量比对照高出13.5%。这说明CHS基因在植物类黄酮物质代谢过程中发挥了重要作用,其对植物的生长及种子发育也产生着影响。5.金线莲响应蓝光的小RNA组学研究针对总黄酮含量显著差异的蓝光和对照组,采用高通量测序进行金线莲响应蓝光的小RNA组学研究。研究结果表明:①本实验以蓝光组和对照组叶片为材料,构建两个比较组的小RNA组及数字化表达谱,得到了大量小RNA序列信息。共鉴定到430个已知miRNA,预测到449个新miRNA。针对这879个miRNA进行靶基因预测,预测到靶基因15960个,全部靶基因位点33039个。②两个比较组之间筛选得到差异表达miRNA 52个,蓝光组相比对照组显著上调表达27个,显著下调表达25个。③对差异表达miRNA靶基因进行KEGG Pathway富集分析,显著富集的前6个代谢通路是:ABC转运器、莨菪烷类和哌啶以及吡啶生物碱生物合成、酪氨酸代谢、苯丙氨酸代谢、甘油磷脂代谢。其中,苯丙氨酸代谢(Phenylalanine metabolism)与黄酮类化合物密切相关,苯丙氨酸是黄酮类化合物代谢的起始底物,该途径miRNA重点调控的3个靶基因为:咖啡酰辅酶A氧甲基转移酶(CCoAOM)、多胺氧化酶(PAO)、天冬氨酸转氨酶(AST);另外,类黄酮生物合成途径(Flavonoid biosynthesis)富集到的差异miRNA 靶基因也是CCoAOMT;苯丙烷生物合成途径(Phenylpropanoid biosynthesis)富集到的差异表达miRNA靶基因是CCoAOMT和β-葡萄糖苷酶(BGL1)。综上:差异表达 miRNA(novel-m0027-3p,miR9662-y 和 novel-m0076-3p)及其靶基因(CCoAOMT、AST、PAO和BGL1)在本研究中响应蓝光,促进苯丙氨酸的代谢、抑制木质素的合成,最终导致黄酮类物质含量的上升。④为验证本实验金线莲small RNA测序结果的准确性,并探讨其与靶基因的关系,本研究挑选了8个在蓝光处理组和对照组之间差异表达的miRNAs,设计高效率特异扩增引物茎环qRT-PCR验证,结果表明qRT-PCR验证结果与small RNA测序结果基本一致,证明了 small RNA测序结果的可靠性。综上所述,本研究从光照条件三要素(光质、补光时间、光强)进行筛选并建立了促进金线莲生长及类黄酮化合物积累的补光模型;在此基础上,采用代谢物组学、转录组学、小RNA组学等多组学手段,解析了蓝光诱导金线莲类黄酮代谢的分子机理,为生产上利用补光提高金线莲产量和药用成分含量提供科学依据。更多还原

【Abstract】 Anoectochilus is a perennial herbal medicine with distinct ornamental and medicinal value.Its wild resources are scarce and grow slowly.This paper study the light quality,supplemental lighting time and light intensity in the overnight supplementary lighting of the cultivation stage of A.roxburghiiis in order to increase its planting yield and medicinal value.Based on that,UPLC and MS/MS were used to analyze the discrepant metabolites of the two comparative groups(the best light quality and the control group)and the metabolic pathways were analyzed.Then the illumina high-throughput sequencing technology was carried out to establish a transcriptome database of blue light and CK group of A.roxburghiiis.The DGEs of two comparative groups were screened for functional annotation and KEGG metabolic pathway analysis.The key enzymes of flavonoids metabolism pathway were screened and the qRT-PCR expression verification was performed.At the same time,the HiSeq high-throughput sequencing was carried out to establish a small RNA database of blue light and CK groups of A.roxburghiiis.A large number of miRNAs were identified,and functional annotation and KEGG metabolic pathways of the target genes of differentially expressed miRNAs were also analyzed.This study intended to analyze the molecular mechanisms of blue light-induced accumulation of flavonoids in A.roxburghiiis from the perspective of multiple omics such as transcriptomics,sRNAomics,and metabonomics,it also provides the scientific basis for increasing the yield and the content of medicinal components by using the supplementary lighting in the production of A.roxburghiiis.The main conclusions are as follows:1.Influence of light quality,supplemental lighting time,light intensity of LED light source on yield and secondary metabolite content of A.roxburghiiis①Red light(RL),blue light(BL),yellow light(YL),green light(GL)and white light(WL)were applied as supplemental lighting at night to A.roxburghiiis in comparison with darkness(CK).The results showed that the supplementation of BL had a positive effect on A.roxburghii with the biggest leaf numbers,stem diameter,fresh weight and dry weight,the fresh weight and dry weight were 17%and 33.3%higher than CK respectively.The total flavonoids content increased significantly which was 24.2%higher than CK.②BL was furtherly applied to A.roxburghiiis to investigate the effects of different supplemental lighting time(0,4,8,12 h·d-1)on the growth and accumulation of medicinal components.The results showed that 8h/d treatment had a positive effect on the growth of A.roxburghiiis,and the leave numbers,stem diameter,root length,fresh weight and dry weight increased significantly.And the total flavonoids content were significantly higher than CK.③ At the same time,BL was applied to A.roxburghiiis to investigate the effects of different light intensities(0,12,25,50 μmol·m-2·s-1)on the growth and accumulation of medicinal components.We found that 25 μmol·m-2·s-1 treatment had a positive effect on the growth of A.roxburghiiis,and the leave numbers,stem diameter,fresh weight and dry weight increased observably.And the accumulation of total flavonoids were significantly promoted.2.The metabolomics study of A.roxburghiiis in response to blue lightBased on the results of the previous studies,the metabolomic study of A.roxburghiiis in response to blue light was studied.The results are as follows:① There were 543 kinds of metabolites detected in A.roxburghiiis,of which 519 kinds of metabolites have been identified,the identification ratio was 95.58%.90 of them are flavonoids.② There were 70 differential metabolites in total between the blue light and control groups,in which 55 metabolites were significantly up-regulated and 15 were significantly down-regulated.The differential metabolites are mostly flavonoids.There are 16 flavonoids in the differential metabolites.③ The KEGG pathway enrichment analysis of differential metabolites showed that flavone and flavonol biosynthesis is the major metabolic pathway in which blue-induced differential metabolites are involved,while Rutin and Luteoloside are the main components of the difference in the total flavonoid content induced by BL of A.roxburghiiis.The significant increase in the content of four highly active flavonoid glucosides compounds:Isookanin-7-glucoside,Quercetin-3-O-glucoside(Isoquercitrin),Luteolin 7-O-glucoside and Kaempferol 3-O-glucoside(Astragalin)under blue light treatment indicates that blue light can significantly increase the medicinal value of A.roxburghiiis.3.The transcriptomics study of A.roxburghiiis in response to blue lightUsing illumina high-throughput sequencing to study the transcriptomics of blue light and the control group which have significant difference on total flavonoids content,the results are as follows:①By assembly,4*108(57.7 G)clean reads and 63025 unigenes were obtained.The average unigene length was 826 bp,and the N50 was 1479 bp.Through this experiment,we established the transcription library of A.roxburghiiis and obtained a large number of high-quality sequence information.② The Blast comparisons of unigene with Nr,Swissprot,KOG and KEGG databases of NCBI were performed and a total of 26,363 unigenes were annotated,the annotation ratio was 41.83%.The Nr database comparison showed that these species closely related to A.roxburghiiis are Elaeis guineensis,Phoenix dactylifera,Musa acuminata,Asparagus officinalis,Anthurium amnicola and so on.③ All the Unigenes were searched using the MISA software to obtain a total of 10,592 SSR markers of various types,covering several SSR of one to six base repeats and complex SSR.④ A total of 1066 transcription factors were identified and could be classified into 55 families.The top five families were bHLH(75),C2H2(73),MYB related(61),ERF(59)and FAR1(59).⑤There were 382 DEGs in the two comparison groups.Among them,183 unigene were significantly up-regulated while 199 genes were significantly downregulated.GO function annotation was performed on the DEGs.In the biological process classification,the DEGs were mainly annotated in the metabolic process(24).In the cell component classification,the main annotation was in cells(17).In the molecular function classification,the DEGs were mainly annotated in catalytic activity(22).⑥The KEGG metabolism pathway analysis shown that the first 10 metabolic pathways which DEGs significantly enriched were photosynthesis-antenna proteins,plant-pathogen interaction,starch and sucrose metabolism,autophagy regulation,pentose and glucuronate interconversions,glyoxylate and dicarboxylate metabolism,folate biosynthesis,phenylpropanoid biosynthesis,amino sugar and nucleotide sugar metabolism,alanine and aspartate and glutamate metabolism.Among them,the phenylpropanoid biosynthesis pathway is closely related to the anabolism of flavonoids,which flavonoids metabolism is the branch pathway of it.This suggests that peroxidase,the DEGs enriched in the phenylpropanoid biosynthesis pathway,which may play an important role in the metabolism of phenylpropanoids and flavonoids.⑦ The five key genes on the phenylpropanoid metabolic pathway and thirteen key structural genes of flavonoid metabolic pathways were excavated and performed qRT-PCR expression validation.By comparing the transcriptional data with the qRT-PCR results,we found that the up-regulation or down-regulation trend of the expression of these 18 genes is consistent which proves the reliability of the transcriptional data.4.Functional analysis of the structural gene CHS of the flavonoids of A.roxburghiiisThe CHS full-length gene was obtained from the transcriptome sequencing data of A.roxburghiiis and verified.The vector was constructed and transferred into N.benthamiana.It was found that the transgenic overexpression of CHS gene resulted in the plant height of the transgenic plants decreased by 11.3%compared with the control,and the stem diameter increased by 10.4%.Flowering was early and the seed weight was 11%lower than that of the control,and the total flavonoids content was 13.5%higher than the control.This indicates that the CHS gene plays an important role in the metabolism of plant flavonoids,which also has an impact on plant growth and seed development.5.The sRNAomics study of A.roxburghiiis in response to blue lightUsing high-throughput sequencing to study the sRNAomics of blue light and the control group which have significant difference on total flavonoids content,the results are as follows:① In this experiment,we constructed the small RNA group and digital expression profiles of blue light and control groups.A large number of small RNA sequences were obtained.A total of 430 known miRNAs were identified and 449 new miRNAs were predicted.And 15960 target genes and 33039 target sites were predicted from these 879 miRNAs.② 52 differentially expressed miRNAs were screened between the two comparison groups,27 of them were up-regulated while 25 of them were down-regulated.③KEGG Pathway enrichment analysis of differentially expressed miRNA target genes revealed that the first six significantly enriched metabolic pathways were:ABC transporters,tropane,piperidine and pyridine alkaloid biosynthesis,tyrosine metabolism,phenylalanine metabolism,glycerophospholipid metabolism,isoquinoline alkaloid biosynthesis.Among them,phenylalanine metabolism is closely related to flavonoids.Phenylalanine is the starting material of the metabolism of flavonoids.The three target genes regulated by miRNA in this pathway are caffeoyl-CoA-O-methyltransferase,primary-amine oxidase,aspartate aminotransferase.In addition,the differential miRNA target genes enriched in flavonoid biosynthesis pathway are CCoAOMT;the differentially expressed miRNA target genes enriched in the phenylpropanoid biosynthesis pathway are CCoAOMT andβ-glucosidase(BGL1).In summary,differentially expressed miRNAs(novel-m0027-3p,miR9662-y and novel-m0076-3p)and their target genes(CCoAOMT,AST,PAO and BGL1)responded to blue light and promoted the metabolism of phenylalanine and inhibited the synthesis of lignin and eventually led to an increase of flavonoids content.④In order to verify the accuracy of the small RNA sequencing results of this experiment,and to explore its relationship with the target gene,eight miRNAs differentially expressed between the blue-light treatment group and the control group were selected,and high-efficiency specific amplification primers were designed for stem-loop qRT-PCR verification.The results showed that the qRT-PCR results were basically consistent with the results of small RNA sequencing,which proved the reliability of small RNA sequencing results.In summary,this study screened from the three factors(light quality,supplemental lighting time,light intensity)of light conditions and established a light-supplementary model for promoting growth and flavonoids metabolism of A.roxburghiiis,the molecular mechanism of blue light-induced flavonoids metabolism in A.roxburghiiis was explored from the perspective of multiple omics such as transcriptomics,sRNAomics,and metabonomics,which provided a scientific basis for increasing the yield and the content of medicinal components by using the supplementary lighting in the production of A.roxburghiiis.更多还原