【作者】 吴燕；

【导师】 陶俊； 孙静；

【作者基本信息】 扬州大学 ， 观赏园艺学， 2020， 硕士

【摘要】 芍药(Paeonia lactiflora Pall.)花型美观、花色丰富,具有很高的观赏价值,是近年来新兴的高档切花。但是芍药单个品种花期短暂,群体品种花期集中,不仅影响芍药的观赏期,也导致芍药切花采切时用工紧张。通过设施栽培可以调控芍药花期,但是生产成本高。近年来,许多开花调控的分子机制研究,为利用分子技术调控植物花期提供了技术支撑。本研究以芍药品种‘大富贵’为试材,采集芍药处于花芽分化临界期的潜在花芽、花芽形态建成后的花芽和开花盛期的花朵进行转录组测序,根据转录组分析结果从中筛选芍药花期调控相关基因;然后,分离和克隆开花相关基因,并构建其表达载体进行遗传转化,旨在为探究芍药开花调控的分子机制、明确调控芍药开花的关键基因提供理论依据。主要研究结果如下:(1)使用Illumina HiSeq平台对芍药‘大富贵’样品进行RNA-Seq 比较转录组分析。构建了 9个cDNA文库(3次生物学重复),共获得92.53 Gb数据,81788个Unigenes,Unigenes的序列长度主要在200-3000 nt之间。对筛选到的Unigenes进行七大功能数据库(KEGG、GO、NR、NT、SwissProt、Pfam 和 KOG)注释,同时注释到的 Unigenes为19,281个(23.57%),注释到任一数据库的Unigenes为56,599个(69.20%)。比对发现芍药‘大富贵’Unigenes与NR数据库中Vitis vinifera的相似度最高(21.41%)。KOG 数据库注释到‘Cell cycle control,cell division,chromosome part’类别的 Unigenes数量为670个。筛选组间差异基因,DEGs的GO功能分类显示T1(花芽分化临界期)、T2(花芽形态建成后)和T3(盛花期)这三个时期注释到GO功能的DEGs数量较前一时期有所增加,组间差异基因在GO功能分类中注释到最多的两个二级类别都相同。对DEGs进行KEGG Pathway分类,获得显著富集的代谢通路为植物昼夜节律和异黄酮生物合成。其中植物昼夜节律通路与花期调控相关,从中筛选出芍药开花调控相关的DEGs 19种,随机对9个基因(TCP21,CHE、CO、PRR5、PAP1,MYB75、HY5、ELF3、GI、FT、CRY1)进行了 qRT-PCR分析,三个时期样品的表达量变化与转录组测序结果一致。(2)分离和克隆了芍药成花相关基因PlFT。从芍药‘大富贵’叶片提取了总RNA,反转录合成cDNA第一链,根据转录组数据中差异基因FT(基因 ID CL8783.Contig2_All)的全长序列克隆得到PlFT基因并对FT基因进行了生物信息学分析。PlFT基因的全长为592 bp,具有完整的开放阅读框522 bp,共编码173个氨基酸,获得基因登录号为MT249229。成功构建了芍药pCAMBIA1301-PlFT超表达载体。测序及菌液PCR验证后,采用冻融法将重组质粒转化农杆菌EHA105,获得用于遗传转化的阳性转化因子。(3)遗传转化突变体拟南芥ft-10(功能回补实验)。通过农杆菌转化法将pCAMBIA1301-plFT导入突变体拟南芥ft-10,获得的种子经过Hyg抗性筛选,最终得到35个转PlFT基因株系,并对部分植株进行了 GUS组织染色鉴定。通过qRT-PCR对10个转基因ft-10拟南芥株系中PlFT的表达量进行分析,结果表明PlFT在转基因突变体拟南芥ft-10中均能成功表达,且表达量显著高于对照组。对转PlFT基因突变体拟南芥ft-10和对照野生型拟南芥Col-0、突变体拟南芥ft-10的开花时间和莲座叶片数进行观察和表型统计分析。转PlFT基因拟南芥各植株均先于对照组拟南芥开花,初开时转基因拟南芥株系的莲座叶片数最少。结合表型和qRT-PCR结果分析表明芍药PlFT具有成花促进作用。更多还原

【Abstract】 Chinese herbaceous peony(Paeonia lactiflora Pall.)is a new high-grade cut flower in recent years.It has beautiful flowers,rich colors and high ornamental value.However,the florescence of single species of Paeonia lactiflora is short,and the florescence of group species is concentrated,which not only affects the ornamental period of Paeonia lactiflora,but also leads to the labor shortage when cutting Paeonia lactiflora flowers.The florescence of Paeonia lactiflora can be controlled by protected cultivation,but the production cost is high.In recent years,many studies on the molecular mechanism of flowering regulation have provided technical support for the use of molecular technology to regulate the florescence of plants.In this study,we used Paeonia lactiflora variety ’Dafugui’ as the test material and collected the potential flower buds that are in the critical period of flower bud differentiation,the flower buds after the completion of flower bud morphology and the flowers in the full-bloom stage of Paeonia lactiflora to sequence the transcriptome According to the transcriptome analysis results,we screened the genes related to the regulation of Paeonia lactiflora flowering;then,we isolated and cloned the gene related to flowering and constructed the expression vector of it for genetic transformation.The aim is to provide theoretical basis for exploring the molecular mechanism of flowering regulation of Paeonia lactiflora and clarifying the key genes regulating Paeonia lactiflora flowering The main results are as follows(1)Using Illumina HiSeq platform to analyze RNA-Seq comparative transcriptome of the Paeonia lactiflora ’Dafugui’ samples.9 cDNA libraries(3 biological repeats)were constructed.92.53 Gb data and 81788 Unigenes were obtained.The sequence length of Unigenes is mainly between 200-3000 nt.Seven functional databases(KEGG,GO,NR,NT,SwissProt,Pfam and KOG)were annotated to the selected Unigenes.19,281(23.57%)Unigenes were annotated together,and 56,599(69.20%)Unigenes were annotated to any database.It was found that the similarity between the Unigenes and Vitis vinifera in NR database was the highest(21.41%).The number of Unigenes annotated by the KOG database to the category of ’Cell cycle control,cell division,chromosome part’ is 670.We screened the intergroup differential expression genes(DEGs).The GO function classification of DEGs showed that the number of DEGs annotated to GO function in T1(critical stage of flower bud differentiation),T2(after flower bud morphogenesis)and T3(full-bloom stage)increased compared with the previous period,and the two most annotated secondary categories are the same in the GO function classification.KEGG pathway analysis were carried out for DEGs and the significant enrichment metabolic pathways are circadian rhythm pathway and isoflavone biosynthesis pathway.Among them,plant circadian rhythm pathway is related to flowering regulation.We selected 19 DEGs related to flowering regulation of Paeonia lactiflora from plant circadian rhythm pathway,and randomly analyzed 9 genes(TCP21,CHE、CO、PRR5、PAP1,MYB75、HY5、ELF3、GI、FT、CRY1)by qRT-PCR.The expression of the samples in the three periods was consistent with the result of transcriptome sequencing(2)PlFT related to Paeonia lactiflora flowering was isolated and cloned.The total RNA was extracted from the leaves of Paeonia lactiflora ’Dafugui’ and the first strand of cDNA was synthesized by reverse transcription.According to the full-length sequence of DEGs FT(gene ID CL8783.Contig2_All)in transcriptome data,PlFT gene was cloned and analyzed by bioinformatics.The total length of PlFT gene is 592 bp,which has a complete open reading frame 522 bp and encodes 173 amino acids.The gene login number is MT249229.The pCAMBIA1301-PlFT over-expression vector was successfully constructed After sequencing and bacterial solution PCR verification,the recombinant plasmid was transformed into Agrobacterium EHA105 by freeze-thaw method,and the positive transformation factors were obtained.(3)The mutant ft-10 Arabidopsis was genetically transformed(functional replenishment experiment).Through Agrobacterium transformation,pCAMBIA1301-PlFT was introduced into mutant Arabidopsis ft-10,and the seeds were screened for hyg resistance.Finally,35 PlFT transgenic lines were obtained,and some of them were identified by GUS tissue staining.The transcription level of PlFT in 10 transgenic ft-10 Arabidopsis lines was analyzed by qRT-PCR.The results showed that PlFT could be expressed successfully in transgenic Arabidopsis lines,and the expression was significantly higher than that in the control group.The phenotypes such as flowering time and leaf number of transgenic mutant Arabidopsis ft-10 of PlFT gene,wild type Arabidopsis Col-0 and mutant Arabidopsis ft-10 were observed and analyzed.All lines of transgenic Arabidopsis with PIFT gene flowered earlier than those of the control group,and the transgenic Arabidopsis lines had the least number of rosette leaves in first blooming.The results of phenotypic and qRT-PCR analysis showed that PlFT could promote flower formation.更多还原