【作者】 王晶；

【导师】 邬荣领；

【作者基本信息】 北京林业大学 ， 计算生物学与生物信息学， 2019， 博士

【摘要】 植物嫁接技术具有十分悠久的历史,因操作简便、繁育快并能改良接穗性状等优势,被广泛应用于农林业与园艺业植物繁育与生产等方面。一直以来,研究者都在试图探索嫁接植物砧木与接穗相互作用的机制。随着生物分子技术的快速发展,众多研究逐渐聚焦于探索嫁接植物砧木与接穗之间遗传物质的传递问题。本研究以藏川杨(Populus szechuanica var.tibetica)和毛白杨(Populus tonmetosa Carr.)为实验材料,通过搭建无菌组培体系进行微型异种嫁接实验,并采用转录组测序技术获取基因表达数据,解析藏川杨与毛白杨组织间mRNA的交流情况。主要研究结果如下:1.本研究选用微型嫁接方法降低外界环境对实验结果的影响,因此首先搭建藏川杨与毛白杨无菌组培体系。我们通过设计植物激素浓度梯度来获取藏川杨组织培养各阶段最适培养基配方,成功构建藏川杨组培体系;另外对现有的毛白杨组培材料进行愈伤组织再分化诱导去除年龄效应的影响;最后对两种杨树进行正反嫁接实验,并以自体嫁接作为对照。结果发现藏川杨与毛白杨嫁接亲和性较好,且以藏川杨做砧木嫁接毛白杨的成活率更高。另外,我们通过细胞荧光示踪剂确定了砧木与接穗维管组织重连的时间在嫁接后5～6 d。2.根据维管组织重建时间,分别在嫁接后5 d,6 d,8 d,12 d,18 d,26 d,36 d与48 d采集嫁接材料茎段部位,提取RNA并进行转录组测序。获取原始数据后使用Trinity对对照组样本的clean reads进行组装,去除冗余序列后进行二次拼接,最终获得藏川杨转录本83,222条,毛白杨转录本106,402条。使用RSEM进行基因表达水平进行定量验证组装的准确性,获取的表达量数据在校正批次效应后显示样本重复性好,组装结果与定量结果均具有较高的可靠性。3.将藏川杨与毛白杨转录组序列进行比对筛选两种杨树各自的特异性序列,通过异源组织与特异性序列进行比对定量,获得能够进行双向转运的转录本,包含转运至毛白杨体内的273个,至藏川杨体内的1678个。经过功能富集显示这些能够产生跨越嫁接接合部位转录本的基因与植物生长发育、代谢相关以及相应外界刺激并调节自身基因表达调控等生物过程相关。4.使用ImpulseDE2工具对藏川杨与毛白杨之间交换的基因进行时序性差异表达分析,分别获得在不同受体组织中的差异表达基因,并对这些基因所发挥的功能与命运走向进行了预测。5.首次尝试使用构建基因调控网络的方法解析嫁接机制,将高维微分方程与进化博弈论理论相结合,对藏川杨与毛白杨产生交换的基因进行互作调控关系分析,寻找到关键的枢纽基因,为后续进一步挖掘与验证杨树嫁接过程中起重要作用的影响因子提供了参考方向。更多还原

【Abstract】 Plant grafting,an ancient asexual propogation techniques,has been widely used in agriculture and horticulture for its advantages in easy operation,rapid breeding and trait improvement.Researchers have been working on the mechanism of interaction between rootstocks and scions.In recent years,rapid development of molecular technology made them focus on genetic materials exchange across the graft union.In this study,tissue culture materials of P.szechuanica var.tibetica and P.tomentosa were grafted,and expression data were obtained by transcriptome sequencing to analyze mRNA exchange between rootstocks and scions.The main research contents and results are as follows:1.Micrografting method was chosed to reduce the influence of external environment,and tissue culture materials were established.I designed different concentration gradient of hormones and successfully established tissue culture materials of P.szechuanica var.tibetica.Age effects of P.tomentosa materials existed in our lab were eliminated by induction of callus redifferentiation.Then P.szechuanica var.tibetica was micrografted with P.tomentosa and vice versa,and autograft of this two poplars were used as control.I found the two poplars had good graft compatibility,and taking P.szechuanica var.tibetica as rootstock was the best option.The time of vascular reconstruction between scion and rootstock was 5-6 d after grafting which was detected by 6-CFDA.2.Stems of scions and rootstocks were collected at 5 d,6 d,8 d,12 d,18 d,26 d,36 d and 48 d after grafting,and RNA of each sample was extracted for transcriptome sequencing.Trinity was applied to assemble clean reads of control group for P.szechuanica var.tibetica and P.tomentosa,respectively.After asscmbling,unigenes were sort out and reassembled by GICL.Finally,83,222 contigs for P.szechuanica var.tibetica and 106,402 for P.tomentosa were obtained.RSEM was used for quantitative analysis of gene expression,and correlation detection after eliminating batch effect indicated reliability and accuracy of the results.3.Transcriptome of the two poplars were blasted to obtain specific sequences for each poplar,and then I detected expression of P.szechuanica var.tibetica specific sequences in P.tomentosa samples which had grafted with P.szechuanica var.tibetica and vice versa to identify transcripts exchange between the two poplars.273 transcripts were found move from P.szechuanica var.tibetica to P.tomentosa,and 1678 transcripts move from P.tomentosa to P.szechuanica var.tibetica.Results of functional annotations and classification demonstrated these exchanged genes participated in metabolism,response to stimulus and regulation of biological process.4.ImpulseDE2 was applied to analyze temporal differential expression of exchanged genes between P.szechuanica var.tibetica and P.tomentosa in each recipients,and the possible function and fate of these genes were also predicted.5.Regulatory network of the exchanged genes were constructed by combining high-dimensional differential equations with evolutionary game theory,and some pivotal genes were identified,which give theoretical direction for subsequent studies in validating factors playing important roles in rootstock and scion interaction.更多还原