大白菜BrCYP83B1基因的克隆及表达分析  

Cloning and Expression Analysis of BrCYP83B1 Gene in Chinese Cabbage

作　　者：王玉书[1] 赵琳琳 赵爽 胡琦 白慧霞 王欢[2] 曹业萍 范震宇[1] WANG Yu-shu;ZHAO Lin-lin;ZHAO Shuang;HU Qi;BAI Hui-xia;WANG Huan;CAO Ye-ping;FAN Zhen-yu(College of Life Sciences,Agriculture and Forestry,Qiqihar University,Heilongjiang Provincial Key Laboratory of Resistance Gene Engineering and Preservation of Biodiversity in Cold Areas,Qiqihar 161006;College of Chemistry and Chemical Engineering,Qiqihar University,Qiqihar 161006)

机构地区：[1]齐齐哈尔大学生命科学与农林学院黑龙江省抗性基因工程与寒地生物多样性保护重点实验室,齐齐哈尔161006 [2]齐齐哈尔大学化学与化学工程学院,齐齐哈尔161006

出　　处：《生物技术通报》2024年第6期152-160,共9页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31401908);黑龙江省自然科学基金项目(C2016056);黑龙江省省属高等学校基本科研业务费科研项目(145209513,145209319,135509702);黑龙江省普通高等学校青年创新人才培养计划(UNPYSCT-2017155);黑龙江省高等教育教学改革研究与实践项目(SJGY20220406)。

摘　　要：【目的】细胞色素P450家族是十字花科植物硫苷合成重要的酶系,其中CYP83亚家族主要参与核心结构的合成,旨在探究大白菜(Brassica rapa ssp.pekinensis)CYP83B1基因的功能。【方法】利用RT-PCR技术克隆BrCYP83B1基因,通过生物信息学软件分析其编码蛋白理化性质、同源性及启动子顺式作用元件,利用RT-qPCR技术分析BrCYP83B1的表达模式,并构建其植物超表达载体。【结果】BrCYP83B1 cDNA序列全长为1500 bp,编码499个氨基酸,编码蛋白属于细胞色素P450超家族,主要定位于细胞质,二级结构主要由α-螺旋和无规则卷曲构成,与甘蓝型油菜、青花菜的CYP83B1蛋白具有较高的同源性。启动子分析表明,该基因启动子区域包含水杨酸、脱落酸及茉莉酸甲酯等激素响应的顺式作用元件,说明BrCYP83B1基因表达可能受激素调控。RT-qPCR分析结果表明,BrCYP83B1基因在大白菜的根、茎、叶、花和果中均有表达,且以叶中的表达量最高;茉莉酸甲酯够显著促进该基因的表达,而水杨酸处理对其表达具有一定的抑制作用,脱落酸处理下基因先上调后又下调。【结论】BrCYP83B1可能参与大白菜对激素的响应调控。【Objective】The cytochrome P450 family is an important enzyme system involved in glucosinolate synthesis in cruciferous plants,where in the CYP83 subfamily plays a predominant role in core structure formation,this work aims to investigate the Chinese cabbage(Brassica rapa ssp.Pekinensis)CYP83B1 gene functional role.【Method】RT-PCR was used to clone the CYP83B1 gene.Bioinformatics analysis software was utilized to predict the encoded protein's physicochemical properties,homology of the encoded protein,and promoter cis-acting elements.The expression pattern of BrCYP83B1 was analyzed by RT-qPCR,and a plant overexpression vector was constructed for further experimentation.【Result】The cDNA length of BrCYP83B1 was 1500 bp,encoding a total of 499 amino acids.The protein belonged to cytochrome P450 superfamily and predominantly located in the cytoplasm.Its secondary structure primarily comprised ofα-helixes and irregular coil.Homologous comparison illustrated that BrCYP83B1 had close relationship with Brassica napus L and Brassica oleracea L.var.italica.The BrCYP83B1 promoter contained cis-acting elements that were involved in the response to salicylic acid(SA),abscisic acid(ABA),and methyl jasmonate(MeJA),suggesting that the expression of BrCYP83B1 gene may be regulated by hormones.The expression of BrCYP83B1 was detected in various plant organs,including the roots,stems,leaves,flowers and fruits from RT-qPCR results.Notably,the highest expression was observed in the leaves.Moreover,BrCYP83B1 significantly presented induction upon treatment with MeJA,while its expression was repressed by SA.Additionally,ABA treatment initially up-regulated and subsequently down-regulated the gene.【Conclusion】BrCYP83B1 may be involved in the response regulation of Chinese cabbage to hormones.

关 键 词：大白菜 BrCYP83B1 基因克隆 植物激素 表达分析 超表达载体 

分 类 号：Q943.2[生物学—植物学] S634.1[农业科学—蔬菜学]