摘要
【目的】克隆枣多聚半乳糖醛酸酶(Polygalacturonase,PG)基因,分析ZjPG基因表达模式并构建过表达载体,为探究ZjPG基因调控枣裂果的分子机制及培育抗裂枣品种提供理论参考。【方法】以壶瓶枣为试验材料,克隆ZjPG基因(ID:LOC107426373),采用ExPASy、SOPMA、SWISS-MODEL等在线工具对ZjPG蛋白进行生物信息学分析;利用MEGA 7.0构建基于PG氨基酸序列的系统发育进化树并分析ZjPG基因启动子顺式作用元件;通过荧光定量PCR检测枣不同组织(幼叶、成熟叶、花、幼果和膨大期、白熟期、脆熟期及完熟期果实)与不同浓度茉莉酸甲酯(MeJA,0.2和0.4 mol/L)和脱落酸(ABA,0.2 mol/L)处理下枣果实及脆熟期正常果实和开裂果实中ZjPG基因的相对表达量,并构建ZjPG基因过表达载体。【结果】壶瓶枣中ZjPG基因的编码区(CDS)序列全长为1353 bp,与NCBI网站ZjPG基因CDS序列相似性为99.70%,氨基酸序列相似性为99.33%,说明成功克隆ZjPG基因。ZjPG蛋白分子量为49145.28 Da,编码450个氨基酸残基,理论等电点为6.27,无跨膜结构域,为稳定亲水性蛋白,含有Pgu1保守序列。其二级结构由12.67%的α-螺旋,32.22%的延伸链,8.00%的β-折叠及47.11%的无规则卷曲组成。系统发育进化树结果显示,ZjPG与拟南芥AtPG和葡萄VvPG的亲缘关系较近,ZjPG与其他9个物种的PG蛋白基序具有高保守性。ZjPG基因的启动子中包含多个激素响应顺式作用元件。实时荧光定量PCR检测结果显示,ABA和MeJA均可诱导ZjPG基因表达;裂果中ZjPG基因相对表达量显著高于正常果(P<0.05);ZjPG基因在壶瓶枣的叶、花和果实中均有表达,在完熟期果实中的相对表达量最高。成功构建pCAMBIA-1300-ZjPG表达载体。【结论】成功克隆壶瓶枣ZjPG基因CDS序列并成功构建该基因的过表达载体,不同物种之间的PG蛋白序列具有高度保守性,ZjPG基因的表达受到ABA和MeJA信号调控,在裂果中高表达,ZjPG基因表达具有组织特异性和时空特异性。
【Objective】This study aimed to clone the polygalacturonase(PG)gene from Chinese jujube(Ziziphus ju juba Mill.),analyze the expression pattern of ZjPG gene and construct an overexpression vector,providing a theoretical reference for exploring the molecular mechanism of ZjPG gene in regulating fruit cracking and breeding crack-resistant jujube varieties.【Method】Using Z.jujuba Mill.cv.Huping as experimental material,the ZjPG gene(ID:LOC107426373)was cloned.The ZjPG protein was analyzed bioinformatically by online tools such as ExPASy,SOPMA and SWISSMODEL.A phylogenetic tree based on PG amino acid sequence was constructed by using MEGA 7.0 software,and the cis-acting elements of the ZjPG promoter were analyzed.The relative expression levels of the ZjPG gene were detected by quantitative real-time PCR(qRT-PCR)in different tissues of Z.jujuba Mill.cv.Huping(young leaves,mature leaves,flowers,fruits at expansion period,white mature period,crisp ripening period and full-ripening periods)as well as nor mal and cracked fruits at the crisp ripening period,which had been all treated with different concentrations of methyl jas monate(MeJA,0.2 and 0.4 mol/L)and abscisic acid(ABA,0.2 mol/L).The ZjPG gene overexpression vector was con structed.【Result】The total length of coding region(CDS)sequence of ZjPG gene in Z.jujuba Mill.cv.Huping was 1353 bp,with 99.70%CDS sequence similarity and 99.33%amino acid sequence similarity to the ZjPG gene in NCBI database,in dicating the successful cloning of the ZjPG gene.The molecular weight of ZjPG protein was 49145.28 Da,encoding 450 amino acids residues,with a theoretical isoelectric point of 6.27.It was a stable hydrophilic protein without transmem brane structural domains,containing a conserved sequence of Pgu1.Its secondary structure consisted of 12.67%α-helix,32.22%extended strand,8.00%β-sheet and 47.11%random coil.Phylogenetic evolutionary tree showed that ZjPG was closely related to AtPG from Arabidopsis thaliana and VvPG from Vitis vinifera.ZjPG and PG protein motifs of other nine species were highly conserved.The ZjPG gene promoter contained multiple hormone-responsive cis-acting elements.The qRT-PCR analysis showed that ABA and MeJA could induce the expression of ZjPG gene.The relative expression level of the ZjPG gene was significantly higher in cracked fruits than in normal fruits(P<0.05).The ZjPG gene was expressed in leaves,flowers and fruits of Z.jujuba Mill.cv.Huping,with the highest relative expression level in full-ripening period fruits.The pCAMBIA-1300-ZjPG expression vector was successfully constructed.【Conclution】The CDS sequence of the ZjPG gene from Z.jujuba Mill.cv.Huping is successfully cloned and its overexpression vector is successfully constructed.The PG protein sequences are highly conserved among different species.The expression of the ZjPG gene is regulated by ABA and MeJA signals,and highly expressed in cracked fruits.The ZjPG gene expression shows tissue and spatiotemporal specificity.
作者
仇力平
晁瑞强
梁晋军
温鹏飞
张鹏飞
QIU Li-ping;CHAO Rui-qiang;LIANG Jin-jun;WEN Peng-fei;ZHANG Peng-fei(College of Horticulture,Shanxi Agricultural University,Jinzhong,Shanxi 030801,China;Shanxi Shilou JujubeScience and Technology Courtyard,Lüliang,Shanxi 032500,China)
出处
《南方农业学报》
CAS
CSCD
北大核心
2024年第6期1682-1691,共10页
Journal of Southern Agriculture
基金
山西重大专项计划“揭榜挂帅”项目(202201140601027)
山西重点研发计划项目(201903D221072)
山西果树产业体系岗位专家项目(2023CYJSTX07-13)。
关键词
壶瓶枣
ZjPG基因
裂果
表达特征
载体构建
Ziziphus jujuba Mill.cv.Huping
ZjPG gene
fruit cracking
expression feature
vector construction
