【作者】 李越；

【导师】 郭庆勋；

【作者基本信息】 吉林大学 ， 农业推广（专业学位）， 2017， 硕士

【摘要】 辣椒(Capsicum ssp.)是深受人们青睐的蔬菜和调味品,果实具有独特的辛辣味,其成分是辣椒素类物质;同时,辣椒素类物质又是重要的次生代谢物,具有抗癌、镇痛和减肥等医疗和保健作用。目前栽培辣椒(Capsicum annuum)果实辣椒素含量很低,通过怎样途径促进辣椒素合成和积累,为辣椒素提取提供优质原料成为重要研究课题。辣椒基因组测序已经完成,辣椒素途径已经明确。Pun1是辣椒素合成途径中关键基因,它决定果实辣味的有无和辣椒素含量的多少,因此,研究该基因的表达调控,为解析辣椒素生物合成调控的分子机理,为辣椒种质资源遗传改良提供重要理论依据。为此,本论文对AP2/ERF家族转录因子与辣椒素生物合成关键基因Pun1的关系进行了探讨,主要研究结果如下:1.ERF/JERF转录因子的克隆和生物信息学分析在辣椒中克隆出ERF家族转录因子名为ERF和JERF的两个基因,Gen Bank登录号分别为KF060657,KF169943。ERF基因全长950bp,编码264个氨基酸,在序列533-706位置编码一个含有58个氨基酸的AP2结构域。JERF基因全长1219bp,编码145个氨基酸,在序列736-912位置编码一个含有58个氨基酸的AP2结构域。运用生物信息学的软件工具分析预测ERF和JERF序列所包含的基因信息。利用Infusion技术,将ERF和JERF基因序列连接于载体p GAD424上,得到重组载体p GAD424-ERF和p GAD424-JERF。2.不同辣椒品种的不同发育时期胎座中基因表达采用RT-QPCR技术,分析ERF,JERF和Pun1基因在3个辣椒品种果实不同发育时期的表达量,结果表明这3个品种内的3个基因的表达量变化趋势基本一致,表明ERF和JERF与Pun1基因的表达有关系。3.Pun1基因的启动子的克隆与生物信息学分析设计引物克隆出Pun1基因的启动子序列,全长1535bp,其含有G-BOX,TAAT-BOX等相关顺式作用元件。利用Infusion技术,构建Pun1基因启动子与p Lac Zi的重组载体p Lac Zi-Pun1Pro。4.ERF/JERF转录因子与Pun1相互作用的验证利用酵母单杂交技术先后将AD目标载体p Lac Zi-Pun1Pro,BD报告载体p GAD424-ERF/JERF转入酵母菌株YM4271,以实验室保存的FTG-cry2为阳性对照,分别用LEU单缺,LEU/URA双缺板筛选培养,液体显色反应结果显示,ERF转录因子与Pun1之间的相互作用较弱,JERF转录因子与Pun1的相互作用较强。更多还原

【Abstract】 Capsicum(Capsicum ssp.)is a popular choice of vegetables and spices,with a distinctive pungent flavor,which is a substance called Capsicum.At the same time,the capsaicin substance is an important secondary metabolite,which has the role of anti-cancer,analgesic and weight loss.The current cultivation of pepper(Capsicum annuum),capsaicin content of it is low,so how to promote the synthesis of capsaicin and accumulation,by for the extraction of capsaicin to provide quality raw materials become an important research subject.The capsaicin genome has been sequenced and the capsaicin pathway has been clear.Pun1 is key genes in the capsaicin synthesis pathway,it decided to spicy fruit whether and how many levels of capsaicin,therefore,to study the gene expression regulation and to analyze molecular mechanism of capsaicin biosynthesis regulation,provide important theoretical basis for pepper germplasm genetic improvement.In this paper,the relationship between the AP2 / ERF family transcription factor and the key gene Pun1 of capsaicin biosynthesis is discussed,and the main results are as follows:1.Cloning and bioinformatics analysis of ERF/JERF transcription factorCloning two ERF transcription factors gene ERF and JERF in pepper,Genbank login number are KF060657 and KF169943.The full-length of ERF gene is 950 bp, encoding 264 amino acids,has one AP2 domain which containing 58 amino acids in the sequence of 533-706 position;the full-length of JERF gene is1219 bp,encoding145 amino acids,has one AP2 domain which containing 58 amino acid in the sequence of 736-912 position.Using Infusion technology,the ERF and JERF sequences were connected to the vector p GAD424,and the recombinant vectors are p GAD424-ERF and p GAD424-JERF.2.Gene expressions of different Pepper Cultivars at different developmental stages in placentaUsing real-time quantitative PCR technology to analysis the expression of ERF,JERF gene and Pun1 gene in different pepper cultivars at different developmental stages in placenta.The results showed that the expression change trends of these three genes in the three varieties of peppers were basically the same,which indicated that ERF and JERF could promote the expression of Pun1.3.Cloning and bioinformatics analysis of Pun1 promoterPrimers were designed to clone the promoter sequence of Pun1 gene,the full-length of promoter sequence of Pun1 gene is 1535 bp,which contains G-BOX,TAAT-BOX and other related boxes.Infusion technology was used to construct recombinant vector of Pun1 gene promoter and p Lac Zi which is p Lac Zi-Pun1 Pro.4.Verification of interaction between ERF,JERF transcription factors and Pun1By using yeast one hybrid technology,transform AD target vector p Lac Zi-Pun1 Pro,BD reporter vector p GAD424-ERF/JERF into yeast strain YM4271,use FTG-cry2 as positive control group which is preserved in laboratory,use with LEU single missing,LEU/URA double missing boards to screen cultures.The results show that the interaction between transcription factor ERF and Pun1 is weak,JERF transcription factor and Pun1 is strong.更多还原