【作者】 刘佳；

【导师】 孟庆瑞；

【作者基本信息】 河北农业大学 ， 风景园林（专业学位）， 2020， 硕士

【摘要】 菊花(Chrysanthemum morifolium)兼具观赏、食用、药用和医疗保健等多种价值,是我国传统名花和传统中草药植物,也是当前世界四大切花之一。菊花是典型的短日照开花植物,主要受光周期途径调控开花。隐花色素(Cryptochrome,CRY)是植物体三大光受体之一,它能够感受外界环境中的蓝光和近紫外光信号,在植物生长发育的各个阶段具有重要的调控作用,尤其在植物感受光信号,参与光周期调控植物开花进程中发挥着重要作用。隐花色素的功能在长日模式植物拟南芥(Arabidopsis thaliana)中研究的比较清楚,在短日模式植物水稻(Oryza sativa)中也被证明具有调控开花的作用。菊属植物甘菊(Chrysanthemum lavandulifolium)中已有研究表明CRY基因能够响应光周期信号,并通过与节律钟基因互作调控COL等基因表达,调控开花。但是CRY基因如何响应光信号,如何参与复杂的分子调控网络,并精准控制菊花开花仍需深入研究。本研究从早花菊花‘粉旋球’(C92)、晚花菊花‘粉莲’(C30)中分离到隐花色素基因CRY1和CRY2,并对其进行生物信息学分析、表达特性分析以及对其在植物体内的生物学功能等进行了分析和鉴定。主要的研究结果如下:1.栽培菊花‘粉莲’和‘粉旋球’中CRY1、CRY2基因的克隆与分析(1)利用巢式PCR,从栽培菊花‘粉莲’和‘粉旋球’的叶片中分离得到4个CRY基因,分别命名为:C30-CmCRY1、C92-CmCRY1、C30-CmCRY2和C92-CmCRY2。CRY1和CRY2基因在两个品种间的保守性较高:菊花C30-CmCRY1和C92-CmCRY1基因序列全长均为2108 bp,开放阅读框(Open Reading Frame,ORF)长度为2025 bp,编码674个氨基酸;C30-CmCRY1编码的蛋白相对分子量为76799.29 Da,理论等电点值PI=5.21;C92-CmCRY1基因编码蛋白相对分子量为76767.23 Da,理论等电点值PI=5.21。C30-CmCRY2和C92-CmCRY2基因长度为1839 bp,编码612个氨基酸;C30-CmCRY2编码的蛋白相对分子量为69692.18 Da,理论等电点值PI=5.44;C92-CmCRY2编码的蛋白相对分子量为69790.33 Da,理论等电点值PI=5.95。(2)构建不同物种基于CRY蛋白的系统进化树,CRY1和CRY2蛋白分为两大分支。C30-CmCRY1 和 C92-CmCRY1 蛋白与黄花蒿(Artemisia annua)、菊蒿(Chrysanthemum boreale)等菊科植物CRY1蛋白亲缘关系最近,聚在同一分支上;C30-CmCRY2和C92-CmCRY2蛋白亦与向日葵(Helianthusannuus)、黄花蒿等其它菊科植物的CRY2蛋白聚在一支。2.菊花CmCRY1和CmCRY2基因的表达模式分析(1)对 C30～CmCRY1、C92-CmCRY1、C30-CmCRY2和C92-CmCRY2 在菊花不同器官中的表达情况进行检测,结果显示,CmCRY1和CmCRY2在菊花根、茎、叶中均有表达,属于组成型表达的基因,但其在叶片中的表达显著高于根和茎,表明其主要在叶片发挥功能,这与植物主要通过叶片响应光信号的结论一致。(2)对单个短日照光周期(24 h)内C30-CmCRY1,C92-CmCRY1,C30-CmCRY2和C92-CmCR Y2的变化规律进行了分析,发现4个基因均表现出一定的周期性变化,CmCRY2在两个品种间表达变化规律差异较大,与CRY2相比,CRY1基因在两个品种中均表现出更强的节律性。(3)检测 C30-CmCRY1,C92-CmCRY1,C30-CmCRY2和C92-CmCRY2在连续短日照处理不同时期(1,4,7,10,13,16和19 d)叶片中的表达情况,结果显示两个品种叶片均可以对短日照做出响应,同一品种内不同CRY表达模式相似,C30-CmCRY1和C30-CmCRY2的表达模式基本一致,出现两个表达高峰,分别在短日照处理的第4 d和13 d;C92-CmCRY1和C92-CmCRY2表达模式相似,在处理的第16 d时表达量达到最高。3.菊花CmCRY1和CmCRY2基因的功能研究分别构建了‘粉莲’(C30)、‘粉旋球’(C92)的CmCRY1和CmCRY2基因的过表达载体:pMD-C85-C30-CmCRY1、pMD-C85-C92-CmCRY1、pMD-C85-C30-CmCRY2和pMD-C85-C92-CmCRY2,利用农杆菌介导法转化拟南芥,通过潮霉素抗性筛选和RT-PCR鉴定转基因植株阳性苗,并利用RT-PCR对抗性植株进行了验证。对转基因植株表型分析显示:CmCRY1和CmCRY2拟南芥转基因植株均在不同程度上提前开花,早花品种‘粉旋球’来源的基因作用更强,说明CmCRY1和CmCRY2可能是引起‘粉莲’和‘粉旋球’花期差异的基因。更多还原

【Abstract】 Chrysanthemum morifolium,one of the traditionally famous flowers in China and one of the four best-known cut flowers worldwide,was an important cash crop used for ornamental purposes and also in tea and medicine.C.morifolium is a typical short-day flowering plant,and its flowering is mainly regulated by the photoperiod pathway.Cryptochrome(CRY)is one of the three photoreceptors in plants,it can sense blue and near-ultraviolet light signals in the external environment.In different stage of plant growth,especially in photoperiod flowering pathway,CRYs plays important regulatory role.The functions of CRYs were well studied in the long-day model plant Arabidopsis thaliana,and the similar regulation roles were also proved in the short-day model plant rice.In Chrysanthemum lavandulifolium,another plant of the genus of chrysanthemum,CRY has proved regulated flowering time by interactive with circadian clock genes and regulated the expression of COL gene.However,in Chrysanthemum,how CRYs responds to photoperiod,and how they participated into the complex molecular regulatory network of flowering time regulation,is still not very clear.In this study,four CRY genes in early-flowering variety ’Fen Xuanqiu’(C92)and late-flowering variety ’Fen Lian’(C30),were cloned and their expression patern and function were invested.The main results are as follows:1.Cloning and analysis of CRY1 and CRY2 genes in chrysanthemum variety ’Fen Lian’ and Ten Xuanqiu’(1)Four CRY genes were isolated from leaves of’Fenlian’ and ’Fenxuanqiu’ using nest-PCR,and named them as C30-CmCRYl,C92-CmCRY1,C30-CmCRY2 and C92-CmCRY2 respectively.CRY1 and CRY2 genes are highly conservative between the two varieties:C30-CmCRY1 and C92-CmCRY1 are 2108 bp in length,and the lengths of their open reading frame(ORF)are 2025 bp,encoding 674 Amino acids.The relative molecular weight of the protein encoded by C30-CmCRY1 is 76799.29 Da,and the theoretical isoelectric point is 5.21;the relative molecular weight of the protein encoded by C92-CmCRY1 gene is 76767.23 Da,and the theoretical isoelectric point is 5.21.C30-CmCRY2 and C92-CmCRY2 genes are 1839 bp in length and encode 612 amino acids;the relative molecular weight of the protein encoded by C30-CmCRY2 is 69692.18 Da;the theoretical isoelectric point is 5.44,and the relative molecular weight of the protein encoded by C92-CmCRY2 is 69790.33 Da;Theoretical isoelectric point is 5.95.(2)Phylogenetic tree of different species based on CRY protein was constructed,and CRY1 and CRY2 proteins from different plants are perfectly divided into two branches.In CYR1 branch C30-CmCRY1 and C92-CmCRY1 colsely clustered together with other CRY1 proteins from compositeae plants such as Artemisia annua,Chrysanthemum boreale,and then together with other plants;In CRY2 branch C30-CmCRY2 and C92-CmCRY2 aslo firstly had a convergence with other Compositae plants such as Helianthus annuus and Artemisia annua,and then groupded together with CRY2 from other plants.2.Analysis of Expression Patterns of CmCRY1 and CmCRY2 Genes in Chrysanthemum morifolium(1)The expression of C30-CmCRY1,C92-CmCRY1,C30-CmCRY2 and C92-CmCRY2 in different organs of chrysanthemum was detected,and the results showed that CmCRY1 and CmCRY2 are expressed in Chrysanthemum roots,stems and leaves,which indicated that they are constitutive expression genes.The expression level of CmCRY1 and CmCRY2 in leaves were significantly higher than that in roots and stems,indicated they may mainly functions in leaves.This is consistent with the conclusion that leaf is the main light receptor in plants.(2)The expression of C30-CmCRY1,C92-CmCRY1,C30-CmCRY2 and C92-CmCRY2 at different stage within a single short-day light cycle(24 h)was examined,and all of the four CRY genes showed certain periodic changes,the expression of CmCRY2 differs greatly between the two varieties,compared with CmCRY2,the CmCRY1 gene shows stronger rhythm in both varieties.(3)To detect the expression of C30-CmCRY1,C92-CmCRY1,C30-CmCRY2 and C92-CmCRY2 in the leaves of different periods(1,4,7,10,13,16 and 19 d)of continuous short day treatment,the results showed that the expression patterns of CRYs were very similar in the same variety.Two expression peaks in the expression curve of C30-CmCRY1 and C30-CmCRY2 and appears at 4 d and 13 d under short day respectively.Expression peaks of C92-CmCRY1 and C92-CmCRY2 appear at 16th d of short day treatment.3.Function studies of CmCRY1 and CmCRY2 genes in Chrysanthemum morifoliumOver expression vectors of CmCRY1 and CmCRY2 from early-flowering Chrysanthemum variety’Fenlian’(C30)and late-flowering Chrysanthemum variety ’Fenxuanqiu’(C92)were successfully constructed respectively and named as pMD-C85-C30-CmCRY1,pMD-C85-C92-CmCRY1,pMD-C85-C30-CmCRY2 and pMD-C85-C92-CmCRY2.The CYRs were transformed into Arabidopsis thaliana by Agrobacterium-mediated method.The transgenic plants were selected by Hygromycin B,and CmCRYs were proved expression at transcription level by RT-PCR method.The phenotype analysis of the transgenic plants showed that all the CmCRYs of Chrysanthemum can promote flowering in Arabidopsis.However,the transgenic plants of CRYs isolated from early-flowering Chrysanthemum variety ’Fenlian’(C30)showed much ealer flowering than that with CRYs from late-flowering Chrysanthemum indicated that CRYs are one of the reasons lead to different flowering time between’Fenlian’ and ’Fenxuanqiu’.更多还原