摘要
在从白菜‘矮脚黄’自交系Bcajh97-01中克隆获得多聚半乳糖醛酸酶基因BcPG17及其启动子序列的基础上,采用qRT-PCR、原位杂交和启动子融合表达载体瞬时转化技术,分析了该基因的表达特征。BcPG17的DNA全长为2105 bp,包含9个外显子和8个内含子。ORF序列长度为1344 bp,编码447个氨基酸残基。预测的编码蛋白的相对分子量为48.61kD,理论等电点为8.39,含有1个跨膜结构域,N末端具有信号肽序列,具有被分泌到细胞膜外起作用的特征;其氨基酸序列具有PG蛋白的4个典型结构域,与十字花科芸薹属的其他物种亲缘关系较近。通过qRT-PCR分析发现,BcPG17在开花期花茎中的表达水平最高,且在花粉发育的四分体时期和单核小孢子时期有表达。原位杂交试验结果也显示,BcPG17在开花期花茎的所有组织中均存在强烈的表达信号。通过对克隆获得的1442 bp的BcPG17启动子序列分析,发现该序列含有与分生组织表达有关的顺式作用元件1个、与植物激素响应相关的作用基序和元件多个,以及花药特异基序4个和绒毡层降解延迟蛋白(TDR)结合位点2个;能够启动GUS信号在开花期花茎的第2节间和节处强烈表达,在花发育中期的花药中也存在明显的GUS信号。上述结果表明,BcPG17可能通过与植物激素代谢相关蛋白相互作用调控花茎的伸长,受到绒毡层降解相关蛋白的调控参与花粉的发育。
In the present study,a PG gene BcPG17 and its promoter were cloned from Brassica campestris L.ssp.chinensis Makino’Aijiaohuang’inbred-line Bcajh97-01.Gene expression and promoter activity were explored by q RT-PCR,in situ hybridization and transient transformation of promoter-GUS fusion.The genomic DNA fragment of BcPG17 was 2015 bp in length,containing nine exons and eight introns.The ORF of BcPG17 was 1344 bp in length,encoding 447 amino acids.The predicted molecular weight and the theoretical p I of BcPG17 were 48.61 kD and 8.39,respectively.The protein contained a transmembrane domain with a signal peptide sequence at the N-terminus,indicating that the protein may be associated with the cell membrane.Analysis of the amino acid sequence indicated the presence of four typical domains of PG protein,closely relating to other species of Brassica.The highest expression level of BcPG17 was found in the inflorescence stem during flowering,then to a less level in the tetrad stage and uninucleate microspore stage of pollen development.The results of in situ hybridization showed that BcPG17 had a strong signal in all tissues of inflorescence stems during flowering.The 1442 bp promoter contained at least one cis-acting element related to meristem expression,multiple motifs or elements related to plant hormone response,and four anther-specific motifs,as well as two tapetum-degenerationretardation(TDR)binding sites.Moreover,transient expression of the promoter-GUS showed strong signal in the second internode and node of the inflorescence stem during flowering,and there was also a significant GUS signal in the anthers in the middle development stage of flower.The above results illustrated that the BcPG17 may regulate the elongation of the inflorescence stem by interacting with other proteins,presumably related to plant hormone metabolism,and it may be involved in the development of the pollen wall by the regulation of TDR protein.
作者
吕美玲
曹家树
LÜMeiling;CAO Jiashu(College of Horticulture,Fujian Agriculture and Forestry University,Fuzhou 350002,China;Laboratory of Cell&Molecular Biology,Institute of Vegetable Science,Zhejiang University,Hangzhou 310058,China)
出处
《园艺学报》
CAS
CSCD
北大核心
2021年第3期505-517,共13页
Acta Horticulturae Sinica
基金
福建省自然科学基金项目(2017J01431)。
关键词
白菜
BcPG17
多聚半乳糖醛酸酶
花茎
表达分析
Brassica campestris
BcPG17
polygalacturonase
inflorescence stem
expression analysis
