【作者】 栗燕；

【导师】 杨秋生；

【作者基本信息】 河南农业大学 ， 森林培育， 2017， 博士

【摘要】 牡丹(Paeonia suffruticosa)是众人皆知的观赏花卉,却不适宜盆栽种植,大大阻碍了其工厂化育苗的步伐。本研究以凤丹(Paeonia ostii)和芍药(Paeonia broteri)为砧木嫁接的‘洛阳红’牡丹(Paeonia suffruticosa‘Luo Yanghong’)为试材,研究了容器栽培条件下盆栽牡丹各生长发育阶段的形态特征指标、可溶性糖组分的变化、蔗糖在植株体内的转运路径、蔗糖转运蛋白基因(PsSUT2)的克隆及表达以及与蔗糖转运和代谢相关基因的挖掘等内容,为拓宽牡丹栽培范围、实现分子育种和遗传改良提供理论和技术方面支持。主要研究结果如下:1.形态特征指标年生长周期中,两种砧木嫁接的牡丹株高、冠幅变化趋势相似,差异不显著,而芍药砧‘洛阳红’新枝长、成花数量、花蕾的高度和直径等均优于凤丹砧牡丹,差异显著,芍药砧牡丹较凤丹砧牡丹更适宜用作容器栽培。2.可溶性糖组分的变化年生长周期中,蔗糖在凤丹砧牡丹叶片中的积累量明显高于芍药砧牡丹叶片(夏季休眠期除外),与凤丹砧‘洛阳红’成花数量少,库强不足有一定关系;叶柄是蔗糖运输的主要通道,有时也可以暂时贮存蔗糖;花后生长期是鳞芽积累蔗糖及生理代谢高峰期,因此花后增施磷钾肥有利于牡丹的花芽发育;冬季休眠期两根砧根和老茎是贮存蔗糖的主要器官,凤丹砧老茎表现出比芍药砧老茎更强的贮存蔗糖能力,芍药砧老茎中蔗糖则更多地回流到根中贮存,而整个生长期芍药砧根中蔗糖水平持续下降,为牡丹的生长源源不断地提供碳源物质;而在秋后生长期,当凤丹砧‘洛阳红’叶、叶柄和芽代谢活动基本停止的时候,芍药砧‘洛阳红’叶、叶柄和芽依然保持较高的代谢状态,秋后生长期的延长更有利于芍药砧叶片利用光合作用合成蔗糖流向根中贮存。3.蔗糖在植株体内的转运途径CF(Carboxyfluorescein,羧基荧光素)荧光示踪结果表明:生长期和即将休眠期,叶片主脉CF荧光被限制在韧皮部细胞内,并未向周围薄壁细胞扩散,说明主脉中SE/CC与PP处于共质体隔离状态;而叶柄、新茎、老茎、根中尽管CF荧光信号强弱不同,生长期和即将休眠期均可以观察到CF荧光向周围薄壁细胞扩散的情况,说明在‘洛阳红’牡丹的运输韧皮部(叶柄、新茎和老茎)中及贮藏器官(根)中,共质体是广泛联系的,两根砧牡丹间荧光示踪结果未见明显差异。超微结构观察进一步显示两根砧牡丹的主脉、叶柄、花瓣、生长期的根、即将休眠嫁接部位、即将休眠的凤丹根砧中胞间连丝数量较少,并观察到筛管、伴胞细胞壁的内突生长、壁旁体和囊泡的出现,薄壁细胞胞间隙较大、内部含有囊泡结构等特征,意味着这些部位中同化物的转运可能以质外体途径为主,以共质体途径为辅,而即将休眠期芍药砧根中蔗糖转运采取共质体途径。4.蔗糖转运蛋白基因(PsSUT2)的克隆及表达以‘洛阳红’牡丹叶片为材料,利用RT-PCR和RACE方法从叶片中克隆了蔗糖转运蛋白基因,将此基因命名为PsSUT2,GenBank登录号为KC542396。该基因c DNA全长2 347 bp,编码601个氨基酸,相对分子量为64.6 KD,等电点为6.14,编码的氨基酸序列与葡萄同源性最高,为75.27%。该蛋白有12个跨膜螺旋,属于MFS蛋白家族。采用实时荧光定量RT-q PCR法,分析了PsSUT2基因在凤丹砧和芍药砧‘洛阳红’不同生育时期不同器官中的表达情况,结果表明PsSUT2在两根砧牡丹的不同器官中表达趋势相似,且主要在秋后生长期的叶、叶柄、鳞芽、新茎、老茎、根和盛花期的花瓣中表达,为该时期蔗糖在植株体营养器官和生殖器官中的转运做出积极贡献。5.蔗糖转运及代谢相关基因的挖掘通过新一代测序技术(RNA-seq)对小风铃期和盛花期‘洛阳红’花瓣转录组数据进行分析,共有59 616条unigenes被组装,有33 919条unigenes得到功能注释,筛选得到差异表达基因共有4 224个,其中有1 886个上调表达,2 338个下调表达。通过KEGG分析,共有698条差异基因归为118条代谢途径,其中碳代谢、淀粉与蔗糖代谢是主要的代谢途径,该途径中表达量明显发生变化的基因在牡丹开花过程中发挥着重要作用。测序结果显示牡丹蔗糖转运家族中PsSUT1基因上调表达,还发现了与蔗糖转运有关的另一个新家族SWEET成员,其中8个基因差异表达(3个上调表达,5个下调表达);参与蔗糖代谢的关键酶基因8个差异表达,其中转化酶基因4个(2个上调表达,2个下调表达),蔗糖合成酶基因4个均上调表达,而蔗糖磷酸合成酶基因和PsSUT2未见差异表达。这17个差异表达的基因在‘洛阳红’牡丹开花过程的蔗糖转运和代谢中起到关键作用。更多还原

【Abstract】 The tree peony(Paeonia suffruticosa)is a worldwide well-known ornamental flower,however,it is couldn’t be live for long in pot cultivation.Which is an obstracle in the tree peony industial seedlings.To broaden the cultivation scope of tree peony,the morphological indexes of potted peony,the rule of soluble sugar content in different development stages,the sucrose transporting pathway,some key genes such as sucrose transporter genes(Ps SUT2)were studied with Paeonia suffruticosa ‘Luo Yanghong’ as the scions,and Paeonia ostii and Paeonia broteri as stocks,separately.The paper provides theory and technology support in molecular breeding and genetic improvement of the tree peony.The main results were as follows:1.The morphological indexesThere were no significant differences in the indexes such as the plant height,the crown wide of the two kinds of tree peony in the annual growth period.,however,In Paeonia suffruticosa ‘Luo Yanghong’ grafted in Paeonia broteri,the annual branch length,the bud number,bud height,bud diameter were all bigger than Paeonia suffruticosa ‘Luo Yanghong’ grafted in Paeonia osti.,and the differences were significant.The Paeonia broteri is a better material as stock of potted peony.2.The soluble sugar contentThe leaf sucrose content of Paeonia suffruticosa ‘Luo Yanghong’ grafted in Paeonia ostii was higher than the one grafted in Paeonia broteri in the annual growth period(exclusion of summer dormancy stage).These results indicated of small bud number and the weaker sink strength in the peony in Paeonia ostii.And also,it was root provided carbon source material for bud development in the tree peony.Leaf stipe was a main channel for sucrose transportation,and sometimes it could be used to store the sucrose temporarily.The post-anthesis stage is crucial for the sucrose accumulation and metabolism in bud,so increase of P,K fertilizer is beneficial to the bud development.Root and stem were the main organs for sucrose storage in the winter dormancy stage.Stem showed more capable on the sucrose storage in the peony grafted in Paeonia ostii,while root more capable in the peony grafted in Paeonia broteri.When the tree peony was germinant,the sucrose content continuously decreased in the grafted Paeonia broteri root,we speculate that it provides carbohydrate for bud developement.In the growth stage after autumn,Paeonia suffruticosa ‘Luo Yanghong’ grafted in Paeonia broteri was still vigorous in the leaves,stipes and buds compared with the one grafted in Paeonia ostii.The prolong of the growth stage after autumn help the leaves of the peony grafted in Paeonia broteri compound more sucrose transporting to the root,and it is the reason why peony grafted in Paeonia broteri breeds more buds.3.The sucrose transporting pathwayThe results showed that the CF was confined in the phloem cells,and the SE/CC was isolated to the PP in the main vein of the leaf in the growth stage and before the dormancy stage.while the CF was diffused in the stipe,the current stem,the perennial stem and the root in spite of the different signal intensity.It was considered that the symplasmic unloading pathway of the sucroseexist in the transport phloem cells including the stipe,the current stem,the perennial stem and the root.There was no significant difference in the Paeonia suffruticosa ‘Luo Yanghong’ grafted together with the two kinds of stock.According to the ultrastructure of phloem and its surrounding parenchyma cells in the main vein of the leaf,the stipe,the petal,the root in the growth stage,the grafted location before the winter dormancy stage,the grafted Paeonia ostii root before the winter dormancy stage,the SE/CC complex and their surrounding PP cells have seldom plasmodesmata,and at the same time,there were paramural body,vesicles,cell wall ingrowth of SE and CC,the intercellular space was bigger between the PP cells and the vesicles in them.As a result,the apoplasmic unloading pathways exist mainly between them.The symplasmic unloading in the grafted Paeonia broteri root may prompt the sucrose backflow to the root.4.The cloning and expression analysis of sucrose transporter gene Ps SUT2Sucrose transporter genes was cloned from the leaves of Paeonia suffruticosa ‘Luo Yanghong’ by RACE technique,named Ps SUT2,accession number was KC542396 in the Gen Bank.The full length c DNA of Ps SUT2 was 2 347 bp,encoding a protein of 601 amino acids.Its molecular mass is 64.6 KD,and p I is 6.14.The identities of putative amino acid sequence of Ps SUT2 compared with that of SUT gene from Vitis vinifera is 75.27%.Ps SUT2 protein contains twelve transmembrane helical structures,which belongs to the major facilitator superfamily.Expression of Ps SUT2 gene in different organs were performed with RT-q PCR at the different developmental stage of Paeonia suffruticosa ‘Luo Yanghong’ grafted in the two kinds of stocks.The main results showed that the profile of Ps SUT2 gene was similar in the two kinds of stock,and Ps SUT2 expressed mainly in the leaf,the stipe,the bud,the current stem,the perennial stem,petal and the root in the autumn growth stage.5.Mining of key genes involving in sucrose transporting and metabolizing pathway in Paeonia suffruticosa ‘Luo Yanghong’ by RNA-seq.The transcriptome of Paeonia suffruticosa ‘Luo Yanghong’ petals in small bell stage and flowering stage were studied by RNA-seq.De novo assemblies yielded 59 616 unigenes and 33919 unigenes were annotated.There were 4 324 differentially expressed genes in flowering,the number of up-regulated genes and down-regulated genes were 1 886 and 2 338 respectively.KEGG pathway enrichment analysis of differentially expressed genes showed that there were 118 pathways enriched,the carbon metabolism and starch and the sucrose metabolism were the important pathways.The genes which significantly differentially expressed in these pathways may playe an important role in the regulation of Paeonia suffruticosa ‘Luo Yanghong’ response to flower.Ps SUT1 gene was up-regulated in the SUT family,and 8 new members(SWEET)for sucrose transporting in the Paeonia suffruticosa ‘Luo Yanghong’ were founded(3 up-regulated and 5 down-regulated).8 sucrose metabolism enzyme genes differentially expressed,Inv genes 4(2 up-regulated and 2 down-regulated),SS genes 4(all up-regulated),while SPS genes and Ps SUT2 were not differentially expressed.We speculate that these 17 differentially expressed genes may play an important part in the sucrose transportation and metabolism during the flowering of Paeonia suffruticosa ‘Luo Yanghong’.更多还原