【作者】 安娜；

【导师】 韩明玉； 任小林；

【作者基本信息】 西北农林科技大学 ， 果树学， 2018， 博士

【摘要】 嫁接是苹果生产上广泛应用的繁殖技术。利用矮化砧木（如M9、T337和M26等）嫁接接穗,能够使苹果树体矮化、早花、丰产及果实品质提升。当前,富士作为我国苹果主栽品种,总栽培面积和产量均在75%以上,但其存在成花难、花芽质量差及大小年结果严重等生产问题,严重制约我国苹果产业的优质高效发展。生产实践中,应用矮化砧木对于改善富士成花难等问题具有积极的调控作用,但砧穗间差异表达的mRNAs、miRNAs和lncRNAs如何通过嫁接改变接穗成花性状的调控机理目前尚不清楚,由此形成的分子调控网络也有待确定,因此本研究通过高通量测序对苹果砧穗间差异表达的RNAs进行分析,从中筛选响应嫁接并参与接穗成花调控的关键mRNAs、miRNAs和lncRNAs,为苹果砧穗互作基因调控网络鉴定提供基础数据;以筛选得到的关键候选基因MdAGL24为研究对象,分析其表达模式,筛选其互作蛋白,研究其通过嫁接参与苹果成花调控的分子网络,并鉴定其功能,取得的主要结果如下:（1）以盛花期30 d的‘长富2号’/M9嫁接苗为材料,利用RNA-seq测序获得嫁接口上、下10 cm处韧皮部转录组数据（3个生物学重复）。在6个样本中,共获得约36 G测序数据。结果表明:1)在砧穗间共筛选获得1593个差异表达基因,其中在嫁接口上10 cm处韧皮部中分别有971和622个上调和下调表达。GO和KEGG分析发现,这些差异基因主要涉及光合、代谢、糖酵解、碳固定和果糖代谢等生物学过程。另外,鉴定发现差异基因中有45个为转录因子,且大部分属于MADS-box家族蛋白,暗示MADS-box家族蛋白通过嫁接参与苹果接穗成花调控中发挥重要作用。2)分析发现,生长素原初响应基因Aux/IAA家族成员在嫁接苹果砧穗间显著差异表达,进一步通过生物信息学方法鉴定得到苹果中有54个Aux/IAA基因家族成员,分布在苹果16条染色体上,qRT-PCR结果表明MdIAA22（MD09G1202300）和MdIAA51（MD17G1183500）在嫁接口下10 cm处韧皮部的表达量显著低于嫁接口上方,说明其具有通过嫁接参与接穗性状调控的重要作用。（2）以盛花期30 d的‘长富2号’自根苗顶梢（BS）、M9自根砧根尖（RS）以及‘长富2号’/M9嫁接苗的顶梢（BG）、嫁接口上10 cm处韧皮部（UP）、嫁接口下10 cm处韧皮部（DP）和根尖（RG）为材料,构建了18个sRNA文库（3个生物学重复）。结果表明:1)在BS、RS、BG、UP、DP和RG共6个样本中分别获得11,686,623、11,590,477、11,775,098、11,874,809、11,663,829和11,896,826个clean reads。总计鉴定获得206个已知miRNAs属于42个保守miRNA家族以及976个新miRNAs。2)聚类分析结果表明,所有miRNAs在6个样本中均有6类表达模式显著响应嫁接。在已知miRNAs中,有30个在嫁接苗顶梢中相对于‘长富2号’自根苗顶梢上调表达,43个在嫁接苗根尖中相对于M9自根苗根尖上调表达,115个在嫁接口上韧皮部相对于嫁接口下韧皮部中上调表达;在新miRNAs中,分别有168、268和54个具有同样的表达模式。3)进一步通过生物信息学预测得知这些已知和新miRNAs分别有1051个和7473个靶基因,结合其注释信息及所有miRNA在砧穗间各组织中的表达特点,分析发现mdm-miR156-MdSPLs、mdm-miR159-MdMYB、mdm-miR171-MdGRAS、mdm-miR171-MdAP2等在砧穗间差异表达,说明这些miRNAs及其靶基因可以通过嫁接参与调控苹果接穗成花。（3）以4年生‘长富2号’/M9的幼果（YT）,顶梢（ST）,韧皮部（SP）和根尖（RT）为材料,构建了12个RNA-Seq文库（3个生物学重复）。结果表明:1)分别在ST、SP、YF和RT中获得125,890,873、127,949,159、127,839,931和127,821,622个clean reads。经过滤筛选,共鉴定得到1726个苹果hc-lncRNA（high confidence lncRNA）。2)在YF、RT、ST和SP中分别鉴定到522、551、553和631个hc-lncRNAs,其中分别有85、33、36和45个在这4个组织中唯一表达。有850个hc-lncRNA可预测到靶基因。3)进一步比较砧穗间差异表达的lncRNA,发现在ST和SP中有43和16个相对于RT上调表达,76和25个下调表达,包括TCONS₀0118115、TCONS₀0090580和TCONS₀0043576,其靶基因分别为AP2和AGL24,说明lncRNA也参与并构成了砧穗间苹果成花的分子调控网络。（4）通过前3个组学数据综合分析,筛选确定MdAGL24为研究对象,分析其表达模式,筛选其互作蛋白并鉴定其功能。1)以‘长富2号’短枝顶芽cDNA为模板克隆得到MdAGL24编码基因全长765 bp,编码254个氨基酸;含有高度保守的MADS保守结构与和K-box结构域;定位于细胞核中。组织特异性定量分析表明,MdAGL24在芽中表达量最高;不同激素处理结果发现,MdAGL24主要响应GA₃诱导表达,而对ABA、SA以及MeJA处理微弱响应或不响应。2)通过GUS染色鉴定了MdAGL24启动子的核心活性片段,并构建了MdAGL24-pAbAi载体,利用酵母单杂交技术筛选得到MdAGL24的上游调控因子MdHY5。通过酵母单杂交和双荧光素酶试验证明MdHY5可以靶向调控MdAGL24。3)构建了MdAGL24-pGBKT7载体,利用不同发育时期短枝顶芽的酵母cDNA文库筛选得到49个互作蛋白,测序结果显示互作蛋白中有9个为转录因子;进一步通过BIFC试验证实,MdAGL24与多个直接调控成花相关的蛋白,如MdAGL19、MdAGL42、MdSOC1a/b和MdTIFY6b蛋白存在互作关系。4)在番茄中过量表达MdAGL24基因,发现转基因株系中成花相关基因FT和SPL3等的表达量相对于野生型均显著上调,且转基因株系具有明显早花和早果表型,说明MdAGL24在苹果成花调控中具有重要作用。综上所述,本研究通过鉴定分析苹果砧穗中差异表达的RNAs,构建了由mRNAs、miRNAs和lncRNAs形成的苹果矮化砧木通过嫁接影响接穗成花的分子调控网络,并进一步对通过嫁接参与接穗成花调控的苹果MADS-box基因家族关键成员MdAGL24进行互作蛋白筛选,通过遗传转化番茄对其进行功能验证,为解析MdAGL24基于嫁接调控苹果成花的分子机理提供理论基础。更多还原

【Abstract】 Grafting is widely used in apple industry.Grafting scions on dwarfing rootstocks（such as M9,T337 and M26）is beneficial to dwarf apple tree,accelerate flower,and improve fruit yield as well as quality.At present,‘Fuji’is the main cultivar of apple in China,and its total cultivated area accounts for about 75%.However,‘Fuji’shows difficultly flowering,poor quality of flower bud,and alternate bearing,which seriously restrict the development of higher-quality and higher-benefit in apple industry.The application of dwarfing rootstocks effectively solves the problems on flowering of Fuji.However,the molecular mechanism of dwarfing rootstocks regulating the flowering characters of Fuji scions is not clear.Therefore,we used‘Nagafu No.2’self-rooted seedling,M9 self-rooted seedling and‘Nagafu No.2’/M9grafted seedlings to analyze differentially expressed mRNAs,miRNAs and lncRNAs as well as flowering related RNAs which were caused by grafting in this study;this research will lay the foundation for elucidating the molecular regulation mechanism of dwarfing rootstocks promoting flowering of scion.Moreover,the key flowering gene MdAGL24 was selected as research object.We cloned MdAGL24,and analyzed its promoter and coding sequences,subcellular localization,and expression pattern.The function of MdAGL24 was studied in transgenic tomato.Yeast two-hybird and one-hybird assays were used to screen interacting proteins and transcriptional factors which regulated MdAGL24,and we verified interacting components of MdAGL24 through bimolecular fluorescence complementation（BIFC）and dual luciferase technologies.The main results were obtained as follows:1.Using phloem of the up and down of graft union of‘Nagafu No.2’/M9 at the thirtieth day after full blooming,RNA-seq was completed with three biological repetitions.There were about 36G sequencing data in 6 libraries.（1）there were 1,593 differentially expressed genes on up graft union/down graft union,of which 971 genes were up-regulated and 622 genes were down-regulated.These differentially expressed genes mainly involved in photosynthetic process,metabolic process,secondary metabolism,glycolysis,carbon fixation and fructose metabolism through GO and KEGG analyses.There were 45 transcription factors among them,and most of transcription factors belong to apple MADS family protein,such as MdAGL24,suggesting their important roles in dwarf rootstocks-mediated scion flowering.（2）In addition,lots of auxin Aux/IAA genes were significantly differentially expressed between on up and down graft union,indicating their important functions in flowering control.Therefore,we identified Aux/IAA（IAA）family gene in apple,analyzed their chromosomal location and synteny of between MdIAAs and Arabidopsis IAA genes;Real time fluorescence quantitative PCR（qRT-PCR）analysis showed that the expression levels of MdIAA22（MD09G1202300）和MdIAA51（MD17G1183500）were significantly lower in down graft union than up graft union,indicating that they may play important roles in dwarfing rootstocks inducing scion flowering.2.A total of 18 sRNA libraries with three biological repetitions were constructed using shoot tips of‘Nagafu No.2’（BS）,shoot tips of M9（RS）,and shoot tips（BG）,phloem of the up（UP）and down（DP）of graft union,as well as root tips（RG）of‘Nagafu No.2’/M9.（1）In BS,RS,BG,UP,DP and RG,11,686,623、11,590,477、11,775,098、11,874,809、11,663,829and 11,896,826 clean reads were respectively obtained.206 known miRNAs belonged to 42conserved miRNA families,and 976 new miRNAs were identified.（2）The results of cluster analysis indicated that all known and novel miRNAs had six significant response types to grafting of the six samples.Among the known miRNAs,30 were up-regulated in BG relative to BS,and 43 were up-regulated in RG relative to RS,115 were up-regulated in UP relative to DP;in the novel miRNAs,168,268 and 54 have the same expression pattern,respectively.（3）There were separately 1,051 and 7,473 target genes for known and novel miRNAs,such as MdmiR156-MdSPLs,MdmiR172-MdAP2,MdmiR171-MdAP2,MdmiR159-MdMYB and MdmiR393-MdARFs,respectively.These miRNAs and their target genes were predicted to involve in growth and development,and flowering of scion.3.Twelve RNA-Seq libraries with three biological repetitions were constructed with the young fruit（YT）,shoot tips（ST）,phloem（SP）and root tip（RT）from 4-year-old‘Nagafu No.2/T337/Malus Robusta’.（1）129,626、770,132、222,062、134,814、512 and 132,220,946 clean reads were identified in ST,SP,YF and RT,respectively.After filtering,1,726 hc-lncRNA(high confidence lncRNA were obtained.（2）There were respectively 522、551、553 and 631hc-lncRNAs in YF,RT,SP and ST;and 85、33、36 and 45 target genes of 850 hc-lncRNAs were characterized in above four tissues.（3）Further,the comparison of differentially expressed lncRNAs between rootstock-sicon revealed that 25 down-regulated and 16up-regulated lncRNAs in SP and 76 down-regulated and 43 up-regulated lncRNAs in ST compared with RT,including TCONS₀0118115,TCONS₀0090580 and TCONS₀0043576,with target genes of AP2 and AGL24,respectively.Our results indicated that lncRNA also participates in the molecular regulatory network of apple flowering.4.In order to reveal the function of MdAGL24,we cloned it,analyzed its promoter activity and expression pattern,constructed its transgenic tomato,and screened its interacting proteins and transcription factors.（1）Its length was 765 bp,encoding 254 amino acids;it contained highly conserved MADS and K-box domains.Subcellular localization analysis revealed that it was located in nucleus.qRT-PCR analysis showed that it mainly expressed in buds;moreover,it was obviously induced by GA₃ treatment,while did not respond to ABA,SA and MeJA treatment.（2）The core sequences of its promoter were identified by activity assay.Yeast one hybrid was used to screen upstream regulators of MdAGL24,and we identify that MdHY5 can target MdAGL24 through yeast one hybrid and double luciferase technologies.（3）A yeast two hybrid library was constructed with short shoot buds at different developmental stages;a total of 49 proteins which interact with MdAGL24 were obtained from the yeast two hybrid library,and 9 of them were transcription factors;Through BIFC,MdAGL24 has been found to interact with several proteins that directly regulate flowering,such as MdAGL19,MdAGL42,MdSOC1a/b and MdTIFY6b,indicating that MdAGL24 plays a direct role in flowering control.（4）The expression levels of flowering related genes MdFT and MdSPL3 was higher in overexpressing MdAGL24 tomato than wild type plants,and transgenic plants showed early flowering and early fruit.In summary,the expression patterns of differentially expressed mRNAs,miRNAs and lncRNAs in grafted apples were identified,which constructed the regulation network of dwarfing rootstocks-mediatied scion flowering induction.Moreover,the function of MdAGL24 and its interaction proteins were characterized.The findings in this study laid foundation for analyzing physiological and molecular mechanism of apple dwarf rootstock mediated scion flower formation in future.更多还原