葡萄CHS基因家族鉴定与表达分析  

Identification and expression analysis of CHS gene family in grape

作　　者：成永娟 张明月 曹雪璟 毛娟 陈佰鸿 CHENG Yongjuan;ZHANG Mingyue;CAO Xuejing;MAO JUAN;CHEN Baihong(College of Horticulture,Gansu Agricultural University,Lanzhou 730000,Gansu,China)

机构地区：[1]甘肃农业大学园艺学院,兰州730000

出　　处：《果树学报》2023年第5期861-874,共14页Journal of Fruit Science

基　　金：甘肃农业大学国家级大学生创新创业训练计划(202010733039);甘肃农业大学伏羲杰出人才培育计划(Gaufx-03J02)。

摘　　要：【目的】明确葡萄查尔酮合成酶(chalconesynthase,CHS)基因家族响应外源激素和非生物胁迫的表达模式。【方法】利用生物信息学方法对葡萄CHS基因家族成员进行理化性质、系统进化、共线性、顺式作用元件等分析。采用实时荧光定量PCR(quantitativereal-timePCR,qRT-PCR)检测VvCHS基因在外源激素和非生物胁迫下的表达情况,将筛选出的VvCHS3基因构建植物表达载体并进行烟草瞬时转化以确定其表达位置。【结果】在葡萄基因组中共鉴定出7个VvCHS基因成员,分布于5条不同的染色体上,蛋白质二级结构以α-螺旋和不规则卷曲为主。系统进化和基因对选择压表明,CHS基因家族基因可分为三大亚族,葡萄CHS基因家族与其他物种之间主要进行纯化选择。共线性分析表明,VvCHS3与VvCHS4、VvCHS3与VvCHS5、VvCHS4与VvCHS5之间存在共线性关系。顺式作用元件预测结果表明,大部分VvCHS基因成员可能同时对多种逆境胁迫有响应。qRT-PCR分析发现,VvCHS基因具有明显的组织特异性,在根中表达水平最高,茎中表达水平最低。在5mmol·L-1水杨酸(salicylicacid,SA)处理下,VvCHS3基因在茎中的表达水平显著高于对照,为对照的44.3倍,在0.1mmol·L-1茉莉酸甲酯(methyljasmonate,MeJA)处理下,VvCHS3基因在叶中显著上调表达,为对照的36.1倍。在400mmol·L-1NaCl处理下,VvCHS5基因在根中相对表达量高于对照,为对照的30.6倍。在4℃和10%聚乙二醇(polyethyleneglycol,PEG)处理下,VvCHS4基因在根中显著上调表达。成功克隆得到VvCHS3基因,经农杆菌介导瞬时转化烟草下表皮细胞,荧光倒置显微镜检测表明VvCHS3基因定位于细胞核和细胞膜,与预测结果相符。【结论】葡萄CHS基因家族参与SA和MeJA等外源激素的调控,同时响应低温、干旱和高盐胁迫。【Objective】This paper aimed to clarify the expression pattern of the grape chalcone syn-thase(CHS)gene family in response to exogenous hormones and abiotic stresses.【Methods】The CHS gene family members were identified from grape genome using Blast and HMMER.The CHS gene fam-ily was analyzed using bioinformatics,including physicochemical property,gene structure,subcellular location,phylogenetic relationship,and cis-acting element distribution.In vitro propagated plantlets of Vitis vinifera L.‘Pinot Noir’under different treatments were used as the experiment materials.The ex-perimental materials were cultured in a growth chamber under the conditions of 25℃,16 h light/22℃,8 h dark.After 30 d of culture,these plantlets were transferred to GS liquid media with 0.2 mmol·L-1 ABA(abscisic acid),50 mg·L-1 GA3(gibberellin),0.05 mg·mL-1 NAA(naphthylacetic acid),5 mmol·L-1(salicylic acid)SA,or 0.1 mmol·L-1 MeJA(methyl jasmonate).Some plantlets were subject-ed to the treatments of low temperature(4℃),400 mmol·L-1 NaCl(sodium chloride)or 10%PEG(polyethylene glycol).Meanwhile,the same volume of sterile water and test-tube plantlets with normal growth were used as control.Each treatment had three replicates.qRT-PCR was carried out to detect the expression levels of VvCHS genes in different tissues under different hormone treatments and abiotic stresses.The genes with significant relative expression were selected for cloning and subcellular local-ization verification.【Results】The isoelectric point of grape CHS gene family members ranged from 5.61 to 8.86,and the instability indexes were between 36.64 and 43.50,five of them were stable pro-teins,and VvCHS1,VvCHS2,VvCHS3,VvCHS4 were acidic proteins.Phylogenetic tree was con-structed from CHS protein sequences of Vitis vinifera,Arabidopsis thaliana,Citrus sinensis and Sola-num lycopersicum.The 26 CHS proteins from the four species were divided into three subgroups ac-cording to genetic distance.Both CitCHS and SlCHS proteins were clustered in GroupⅠ,Gro

关 键 词：葡萄 查尔酮合酶 生物信息学 QRT-PCR 基因克隆 

分 类 号：S663.1[农业科学—果树学]