CN110093328A - 一种与水芹木质素合成相关的cad基因序列及其应用 - Google Patents

一种与水芹木质素合成相关的cad基因序列及其应用 Download PDF

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CN110093328A
CN110093328A CN201810100011.5A CN201810100011A CN110093328A CN 110093328 A CN110093328 A CN 110093328A CN 201810100011 A CN201810100011 A CN 201810100011A CN 110093328 A CN110093328 A CN 110093328A
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chinese celery
gene
ojcad
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celery
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熊爱生
仇亮
王枫
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Nanjing Agricultural University
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Abstract

CAD基因是参与木质素合成的关键基因。本发明从水芹中克隆了一个新的CAD基因OjCAD。该基因开放阅读框长为1074bp，编码357个氨基酸。OjCAD编码的蛋白属于疏水蛋白，具有典型的CAD1保守结构域，属于MDR家族。与同属伞形科的胡萝卜CAD进化关系最近，具有高度保守性。荧光定量表达分析表明OjCAD基因在‘八卦洲水芹’中的表达量比‘南选八卦洲紫水芹’中高，在叶片中的表达量显著高于叶柄。本发明利用聚合酶扩增技术从水芹中克隆获得水芹OjCAD基因，该水芹OjCAD基因对水芹植株中木质素的合成有影响。

Description

一种与水芹木质素合成相关的CAD基因序列及其应用
技术领域
本发明属于植物基因工程领域，涉及植物的一个CAD基因及其应用。本发明从水芹品种‘八卦洲水芹’中获得一个与木质素合成相关的基因OjCAD，该基因能够应用于水芹的木质素合成途径研究和应用。
背景技术
水芹[Oenanthe javanica(Blume)DC.]为伞形科水芹属多年生草本植物，是重要的水生蔬菜。水芹既可食用又可入药。除富含人体所必需的营养元素外，水芹还富含抗维生素、膳食纤维、芹菜素、坏血酸、黄酮类化合物等多种成分，可以降压安神、增强人体对外在环境的免疫能力、预防心脑血管等疾病，具有很高的药用价值和保健功能。
木质素是植物中肉桂醇脱氢聚合产生的酚类高分子聚合物，为沉积在高等植物特定细胞壁中第二丰富的天然化合物，是植物细胞壁的重要组成成分(Boudet A M et al.，The News Phytologist，1995，129(2)：203-236.)。木质素是膳食纤维的重要组成部分，能改善人体健康状况，降低一些疾病的风险(Lattimer J M et al.，Nutrients，2010，2(12)：1266-1289.)。但在蔬菜中，木质素含量增加后，植株会变得木质化，很大程度上会影响口感。作为以幼嫩叶和茎为主要食用部位的水芹，木质素含量对其品质有较大的影响。
肉桂醇脱氢酶(cinnamyl alcohol dehydrogenase，CAD)作用于木质素单体合成的最后一步，参与木质素特异合成途径下游酶类的调控。木质素合成过程中，CAD以NADPH为辅酶，将3种肉桂醛还原成肉桂醇，是重要的限速酶。抑制或促进CAD酶的活性，能影响植株中木质素的含量，是调控木质素合成的理想控制点(Tobias C M et al.，Planta，2005，220：678-688)。
发明内容
本发明提供了一种水芹OjCAD基因制备方法和用途。所获得的OjCAD基因有利于深入了解水芹木质素合成途径和OjCAD响应机制。
附图说明
图1.水芹OjCAD氨基酸序列保守域预测
图2.水芹与其他物种CAD蛋白的系统进化树
图3.水芹OjCAD蛋白氨基酸序列的疏水性和亲水性分析
图4.水芹OjCAD蛋白的三级结构
图5.水芹OjCAD基因在‘八卦洲水芹’和‘南选八卦洲紫水芹’的叶片和叶柄中的表达量分析
具体实施方式
1.水芹总RNA的提取及cDNA的合成：将水芹样品利用RNA Simple Total RNA Kit总RNA提取试剂盒提取总RNA(北京Tiangen公司)，并按照Prime Script RT reagent Kit试剂盒说明书将提取的水芹总RNA反转录成cDNA。
2.水芹OjCAD基因的克隆：根据本课题组的水芹转录组数据(Jiang Q et al.，Molecular Genetics & Genomics，2015，290(2)：671-683.)，检索得到水芹OjCAD基因序列，并设计一对克隆引物，正向引物序列：5’-ATGACGCAAACAACTCCCAA-3’，反向引物序列：5’-CTATGGTTTTCCAGCAATGT-3’。以水芹cDNA为模板进行PCR扩增。扩增体系总体积10.0μL，包括0.30ng·μL-1cDNA模板0.5μL、2×TaKaRa Ex Taq Mix(产品编号RR001A)5.0μL、ddH2O3.5μL、0.10μmol·L-1正向引物和反向引物各0.5μL。扩增程序为：94℃预变性5min；94℃变性30s，54℃退火30s，72℃延伸80s，共34个循环；最后72℃延伸10min。将反应所得产物用12g·L-1琼脂糖凝胶电泳分离并回收。回收产物连接到pMD19-T载体后转化到大肠杆菌DH5α，菌液检测后送至南京金斯瑞生物科技有限公司测序鉴定。
3.序列分析：通过DNAMAN 6.0软件确定基因的开放阅读框(ORF)序列，翻译成相应的氨基酸序列后进行氨基酸序列的亲水性和疏水性分析。利用NCBI数据库中的Blast-p进行同源比对和保守域预测，采用MEGA5软件绘制系统进化树。不同物种CAD蛋白的氨基酸组成及理化性质分析均在ExPASy(http://www.expasy.org)上完成。利用SWISS-MODEL(http://swiss-model.expasy.org)进行空间建模分析。
4.实时定量PCR反应：根据OjCAD基因测序结果，利用Primer Premier 6软件设计一对荧光定量PCR引物，正向引物序列：5’-CAGGCACAATCACTCCTTACG-3’，反向引物序列：5’-TCACACTTCAAACACGACGC-3’。以水芹actin基因作为荧光定量数据处理的内参基因(Jiang Qet al.，Plos One，2014，9(3)：e92262.)，正向：5’-CTGGATTCTGGTGATGGT-3’，反向：5’-GCAGTTGTTGTGAAGGAG-3’。荧光定量PCR试验操作步骤按照大连TaKaRa公司的SYBR PremixEx Taq试剂盒的操作说明在iQTM5 Real-Time PCR System上进行，数据处理采用iQTM5Software完成。CAD基因在水芹不同品种和组织中的相对表达量的计算采用2-ΔΔC T方法( et al.，Nucleic Acids Res，2001，29(14)：2994-3005)。
5.实验结果：1).对本发明克隆获得的水芹OjCAD基因编码的氨基酸序列进行保守域预测，发现水芹CAD蛋白具有完整的CAD1结构域以及2个Zn结合位点，属于MDR家族(图1)。2).将本发明克隆获得的水芹OjCAD基因编码的氨基酸序列与其他物种的CAD蛋白序列进行对比，发现水芹的CAD蛋白与同属伞形科胡萝卜CAD蛋白进化关系最近(图2)。3).对本发明克隆获得的水芹OjCAD基因编码的氨基酸序列进行亲疏水性分析，结果显示，水芹OjCAD氨基酸位于第189位的异亮氨酸疏水性最强，位于第221的赖氨酸和223位的丙氨酸亲水性最强。从总体来看，水芹OjCAD属于疏水性蛋白(图3)。4).以PDB ID：1yqd为模型对水芹OjCAD蛋白序列进行同源建模，结果显示，水芹OjCAD蛋白结构主要由7个α-螺旋和17个β-折叠组成(图4)。5).荧光定量PCR结果显示，OjCAD基因在紫色和非紫色水芹叶和叶柄中的表达量有明显的差异。OjCAD基因在‘八卦洲水芹’中的相对表达量是‘南选八卦洲紫水芹’的两倍。在叶中的表达量相对较高，且差异显著，而在叶柄中的表达量极小，无显著差异(图5)。

Claims (5)

1.一种水芹OjCAD基因的获得。
2.权利要求1所述的OjCAD基因编码的CAD蛋白的氨基酸序列。
3.一种制备权利要求1所述的源于OjCAD基因的方法，包括以下步骤：
1)基于水芹转录组测序信息，进行检索分析，获得OjCAD的基因序列；
2)设计引物，正向：5’-ATGACGCAAACAACTCCCAA-3’，反向5’-CTATGGTTTTCCAGCAATGT-3’，通过PCR方法从‘八卦洲水芹’中克隆获得OjCAD基因。
4.权利要求1所述的水芹OjCAD基因功能研究：采用荧光定量PCR方法，完成水芹OjCAD基因在‘八卦洲水芹’和‘南选八卦洲紫水芹’的叶片和叶柄中的表达量分析。
5.权利要求1所述的水芹OjCAD基因应用。
CN201810100011.5A 2018-01-29 2018-01-29 一种与水芹木质素合成相关的cad基因序列及其应用 Pending CN110093328A (zh)

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CN115232827A (zh) * 2022-06-24 2022-10-25 扬州大学 一种与水芹β-石竹烯合成相关的OjTPS1基因序列及其应用

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CN115232827A (zh) * 2022-06-24 2022-10-25 扬州大学 一种与水芹β-石竹烯合成相关的OjTPS1基因序列及其应用
CN115232827B (zh) * 2022-06-24 2023-10-24 扬州大学 一种与水芹β-石竹烯合成相关的OjTPS1基因序列及其应用

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Application publication date: 20190806