新疆野苹果种子MsWRKY33基因的克隆、亚细胞定位及表达分析  

Cloning,Subcellular Localization and Expression Analysis of MsWRKY33 Gene in Malus sieversii Seeds

作　　者：李静[1] 房震 叶春秀 LI Jing;FANG Zhen;YE Chunxiu(College of Forestry and Landscape Architecture,Xinjiang Agricultural University,Urumqi 830000,China)

机构地区：[1]新疆农业大学林学与风景园林学院,乌鲁木齐830000

出　　处：《西北农业学报》2024年第12期2328-2336,共9页Acta Agriculturae Boreali-occidentalis Sinica

基　　金：新疆农业大学高层次人才引进项目(2521GCCRC)。

摘　　要：WRKY转录因子能够通过调节种子休眠和发芽来适当介导生命周期的启动。为探究新疆野苹果种子WRKY33转录因子在低温层积中的作用机理,通过对新疆野苹果[Malus sieversii(Ledeb.)M.Roem.]种子MsWRKY33进行克隆,生物信息学分析、亚细胞定位及不同低温层积时期种子的基因表达量水平检测,初步揭示MsWRKY33基因在新疆野苹果种子低温层积下的功能。采用沙藏法进行低温(4℃)层积处理,常规保存(室温)种子作为对照,以0 d、30 d、60 d、90 d处理的新疆野苹果种子种胚为研究材料,提取种胚RNA,利用同源重组的方法克隆MsWRKY33,测序后进行生物信息学分析。并构建表达载体,进行亚细胞定位分析。最后通过实时荧光定量(qRT-PCR)分析新疆野苹果种子在不同低温层积时期MsWRKY33的表达水平。结果表明,MsWRKY33全长CDS序列为1560 bp,编码520个氨基酸。保守结构域表明,MsWRKY33为GroupⅠ家族WRKY转录因子成员;序列相似性和系统进化树分析结果显示:新疆野苹果MsWRKY33与NP_001281056.1(栽培苹果Malus domestica)、XP_050143309.1(欧洲野苹果Malus sylvestris)序列相似性度极高,亲缘关系最近;亚细胞定位分析显示MsWRKY33定位在细胞核中;不同低温层积时期qRT-PCR表达分析发现,MsWRKY33基因在低温层积60 d时表达水平最高,显著高于未层积时表达量。由此说明,新疆野苹果种子基因MsWRKY33在低温层积过程对解除种子休眠具有调控功能。WRKY transcription factors mediate the initiation of life cycle by regulating seed dormancy and germination.In order to explore the mechanism of WRKY33 transcription factor in low temperature stratification of Malus sieversii(Ledeb.)M.Roem seeds,the function of MsWRKY33 gene in low temperature stratification of Malus sieversii seeds was preliminarily revealed by cloning MsWRKY33 of Malus sieversii seeds,bioinformatics analysis,subcellular localization,and detection of gene expression in seeds at different low temperature stratification stages.In this study,the sand storage method was used for low temperature(4℃)stratification treatment,and the conventional preservation(room temperature)seeds were used as control.The seed embryos of Malus sieversii seeds treated for 0 day,30 days,60 days and 90 days were used as materials to extract RNA.MsWRKY33 was cloned by homologous recombination and sequenced for bioinformatics analysis.The expression vector was constructed,and subcellular localization was analyzed.The expression vector was constructed and subcellular localization was analyzed.Finally,the expression level of MsWRKY33 in Malus sieversii seeds at different low temperature stratification periods was analyzed by real-time fluorescence quantification(qRT-PCR).The results showed that the full-length CDS sequence of MsWRKY33 was 1560 bp,encoding 520 amino acids.The conserved domain indicated that MsWRKY33 was a member of GroupⅠfamily WRKY transcription factor.The sequence similarity between MsWRKY33 and NP_001281056.1(Malus domestica)and XP_050143309.1(Malus sylvestris)was very high,and the genetic relationship was closest.Subcellular localization analysis showed that MsWRKY33 was located in the nucleus.qRT-PCR expression analysis during different low-temperature stratification periods found that the expression level of MsWRKY33 was the highest at 60 days of low-temperature stratification,which was significantly higher than that without stratification.The seed gene MsWRKY33 of Malus sieversii has a regulatory fu

关 键 词：新疆野苹果 种子 MsWRKY33 亚细胞定位 表达分析 

分 类 号：S661.1[农业科学—果树学]