【作者】 王欢；

【导师】 祝朋芳；

【作者基本信息】 沈阳农业大学 ， 风景园林学， 2020， 硕士

【摘要】 羽衣甘蓝(Brassica oleracea var.acephala)属十字花科芸薹属,是甘蓝种的变种。作为观叶植物,其叶色鲜艳美丽,叶形皱缩特殊,且因具有较强的耐寒性,在晚秋、冬季及早春应用广泛,具有较强的观赏价值和经济价值。花青素(anthocyanin)是水溶性类黄酮色素,存在于液泡中使植物呈现粉、红、紫等颜色。本研究以羽衣甘蓝为主要试材,利用定量PCR和半定量RT-PCR对叶色相关基因BoDFR、BoMYB2进行表达量分析,同时将过表达BoDFR基因载体转化羽衣甘蓝和拟南芥,沉默表达BoDFR基因载体转化羽衣甘蓝,以期探明控制羽衣甘蓝花青素生物合成的关键基因及其功能。并开发设计了功能标记,为羽衣甘蓝分子标记辅助育种体系的建立提供参考。本研究的主要结果如下:1.选取五种红色、三种白色心叶羽衣甘蓝,红色、绿色心叶白菜,红色、绿色心叶油菜,红色、绿色心叶甘蓝,红色、绿色心叶菜薹,红色、绿色心叶花椰菜,对BoDFR和BoMYB2基因的表达量进行分析,我们发现:在羽衣甘蓝、油菜、白菜中,红色心叶材料中BoDFR的表达量明显高于白/绿色心叶材料中BoDFR的表达量,而BoMYB2表达量在红色心叶和白/绿色心叶材料中无明显规律性差异,推测BoDFR可能是控制羽衣甘蓝、油菜、白菜红色心叶表型的关键基因;而在菜薹、花椰菜、甘蓝中,红色心叶材料中BoDFR和BoMYB2的表达量均高于白/绿色心叶材料中BoDFR和BoMYB2的表达量,推测BoDFR是控制菜薹、花椰菜、甘蓝红色叶表型的关键基因,BoMYB2是主要的调节因子。2.将过表达BoDFR基因载体经农杆菌介导的子叶转化法转入白色羽衣甘蓝‘1631’和‘D10’,共得到27株卡那抗性植株,PCR鉴定18株为阳性;将过表达BoDFR基因载体经农杆菌介导的花序浸染法转入野生型拟南芥,共得到T2代转基因拟南芥9株,PCR鉴定均为阳性,花青素含量较野生型明显提高。3.将沉默表达BoDFR基因载体经TRV介导的VIGS(virus-induced gene silencing)技术转入羽衣甘蓝?0835?、?千宝菜?、?红钻油菜?叶片中。结果显示,沉默BoDFR基因后,沉默组叶片红色明显变淡,叶片中能检测到TRV病毒分子;在?0835?、?红钻油菜?、?千宝菜?中,BoDFR基因表达量明显下降,分别为对照组的11.7%、22.3%、26.0%,花青素含量分别是对照组的27.3%、11.5%、20.3%。4.根据红色、白色心叶羽衣甘蓝BoDFR基因序列差异,设计了两对显性标记DFR1(红色序列特有)、DFR2(白色序列特有)和一组共显性标记DFR3,并在八种红色、四种白色心叶羽衣甘蓝中进行验证,结果发现:显性引物DFR1可以区分八种红色心叶和三种白色心叶羽衣甘蓝,鉴定率91.7%;显性引物DFR2可以区分八种红色心叶和四种白色心叶羽衣甘蓝,鉴定率100%;共显性引物DFR3可以区分八种红色心叶和四种白色心叶羽衣甘蓝,鉴定率100%。更多还原

【Abstract】 Kale(Brassica oleracea var.Acephala)belongs to the Brassicaceae Brassica genus and is a variant of the cabbage species.As a leaf-viewing plant,its leaf color is bright and beautiful,its leaf shape is shrinking,and because of its strong cold resistance,it is widely used in late autumn,winter and early spring,and has strong ornamental and economic value.Anthocyanin is a water-soluble flavonoid pigment,which exists in vacuoles to make plants show rich colors such as pink,red,and purple.In this study,kale was used as the main sample.The quantitative analysis of leaf color-related genes Bo DFR and Bo MYB2 was performed by quantitative PCR and semi-quantitative RT-PCR.At the same time,the overexpression Bo DFR gene vector was transformed into kale and Arabidopsis,and the silent expression Bo DFR gene vector was transformed into kale,with a view to identifying the key genes and functions of controlling the anthocyanin biosynthesis of kale.A functional marker was developed and designed to provide a reference for the establishment of a molecular marker-assisted breeding system for kale.The main results of this study are as follows:1.Five kinds of red and three white heart leaf kale,red and green heart leaf chinese cabbage,red and green heart leaf rape,red and green heart leaf cabbage,red and green heart leaf bolt,red and green heart leaf cauliflower were selected.Analyzing the expression levels of Bo DFR and Bo MYB2 genes,we found that in kale,rape,and chinese cabbage,the expression of Bo DFR in red heart leaf material was significantly higher than that of white / green heart leaf material,while Bo MYB2 There is no significant difference in expression between red heart leaf and white / green heart leaf materials.It is speculated that Bo DFR may be a key gene controlling the red heart leaf phenotype of kale,rape,and chinese cabbage;And in red cabbage,bolt,and cauliflower,red heart,the expression levels of Bo DFR and Bo MYB2 in leaf material were higher than those of white / green heart leaf material.It is speculated that Bo DFR is a key gene that controls the red leaf phenotype of cabbage,bolt,and cauliflower and Bo MYB2 is the main regulator.2.The over-expressing Bo DFR gene vector was transformed into white kale "1631" and "D10" by Agrobacterium-mediated cotyledon transformation.A total of 27 kana resistant plants were obtained,and 18 were positive by PCR;over-expression The Bo DFR gene vector was transferred into wild-type Arabidopsis thaliana by Agrobacterium-mediated inflorescence staining,and a total of 9 T2 generation Arabidopsis thaliana were obtained.The PCR identification was positive,and the anthocyanin content was significantly increased compared with the wild-type.3.Transfer the silently expressed Bo DFR gene vector into a ’ 0835’ 、’Qianbaocai’and ?Red Diamond Rape?,through TRV-mediated gene silencing(VIGS)technology.The results showed that after the Bo DFR gene was silenced,the red color of the leaves of the silent group became significantly lighter,and TRV virus molecules could be detected in the leaves.The decrease was 11.7%,22.3%,and 26.0% in the control group,and the anthocyanin content was 27.3%,11.5%,and 20.3% in the control group,respectively.4.Based on the Bo DFR gene sequence differences between red and white heart kale,two pairs of dominant markers DFRp and DFRw were designed,and a set of co-dominant markers DFRpw was verified in eight red and four white heart kale The results showed that the dominant primer DFRp can distinguish eight red heart leaves and three white heart kale,with a discrimination rate of 91.7%;the dominant primer DFRw can distinguish eight red heart leaves and four white heart leaf kale with a discrimination rate of 100%;the co-dominant primer DFRpw can distinguish eight kinds of red heart leaves and four kinds of white heart kale with a discrimination rate of 100%.更多还原