胡萝卜中油菜素内酯信号途径相关转录因子BZR1的克隆与表达分析
时间:2018-10-13 10:22 来源:毕业论文 作者:毕业论文 点击:次
| 摘要：BZR1转录因子是油菜素内酯最关键的转录因子之一，为研究胡萝卜(Daucus carota L. var. sativa Hoffm.)发育过程中BZR1的表达情况，本实验以胡萝卜品种黑田五寸(Heitianwucun)为试验材料，通过RT-PCR的方法克隆BZR1基因，采用生物学信息学的方法分析其氨基酸组成等理化性质，并进行实时荧光定量PCR从而探究该基因在黑田五寸生长过程中的表达。序列分析表明黑田五寸的BZR1基因全长为945bp，编码315个氨基酸，预测属于疏水性氨基酸。分析不同植物BZR1基因编码的氨基酸，发现各植物BZR1在进化过程中较保守。进化树分析显示同科植物间进化关系最近。实时荧光定量PCR表明，黑田五寸BZR1基因在根、叶柄和叶片中均有表达且存在组织特异性，其中叶片中含量总体最高。不同生长发育阶段中，该基因的相对表达水平规律不同。29038 毕业论文关键词：胡萝卜；转录因子BZR1；油菜素内酯；克隆；实时荧光定量PCR； Cloning and expression of transcription factor BZR1 in brassinosteroids signal transduction of carrot Abstract:BRASSINAZOLE-RESISTANT1（BZR1）is one of key transcription factors of brassinolide, which plays an important role in regulating plants growth. Carrot(Daucus carota L. var. sativa Hoffm.) is an biennial vegetable of the Apiaceae family, which is a great source of carotene and vitamin. In order to investigate the expression of BZR1 in the development of carrot, thus we can get more information about the connection between BZR1 and brassinolide biosynthesis pathway. This study using reverse transcript PCR (RT-PCR) to clone the BZR1 from one carrot cultivar called Heitianwucun based on the carrot transcriptome database. Sequence alignments and homology sequences are carried out using NCBI BLAST webpage. Phylogenetic relationships among sequences are performed with MEGA6.0. Analysis of hydrophobicity and hydrophilicity, theoretical PI, relative molecular mass are displayed by DNAMAN 6.0. The information of amino acid is acquired by BioXM 2.6 and the SMS website program. The expression of BZR1 is demonstrated by means of Real-time quantitative PCR. Sequence analysis shows: the BZR1 from Heitianwucun contains an open reading frame (ORF) with the full length of 948 bp, which encodes 315 amino acids. Theoretical relative molecular mass of protein encoded by BZR1 from Heitianwucun is 33.85kDa, and theoretical pI is 8.89. Itis predicted to be hydrophobic amino acid and belong to the BES1_N superfamily. Based on multiple sequence alignment, the homology of amino acid sequences encoded by BZR1gene from Heitianwucun, V.vinifera and M.domesticareaches86.40%, which is highly conserved.Analysis of amino acid composition and physicochemical properties indicates that BZR1 in different kinds of plants remain conserved through evolution. Phylogenetic treeilluminates that the BZR1 of the same family such as Cucurbitaceae and Rosaceaeare clustered in the same branch. This phenomenon demonstates clearly that BZR1 exists high evolutionary conservation among different species. Real-time quantitative PCR shows that the gene was tissue-specific and highly expressed in leaf in total. Furthermore, the gene expression were significantly different under different growth stages. Key words:Daucus carota L. var. sativa Hoffm.;transcription factor BZR1; brassinolide; gene clone; Real-time quantitative PCR; 目录 摘要1 关键词1 Abstract1 Key words1 引言2 1材料与方法3 1.1植物材料、菌种与质粒3 1.2胡萝卜总RNA的提取与cDNA的合成3 1.3BZR1基因的克隆与序列检测3 1.4序列分析3 1.5实时荧光定量PCR4 2试验结果与分析4 2.1黑田五寸转录因子BZR1基因的克隆4 2.2黑田五寸转录因子BZR1氨基酸序列的结构域分析5 2.3黑田五寸转录因子BZR1氨基酸亲水性/疏水性分析5 2.4黑田五寸转录因子BZR1氨基酸序列与葡萄、苹果比较6 (责任编辑：qin) |