拟南芥CYCD3;1基因植物表达载体的构建 及在烟草中的遗传转化分析

为了分析植物D型细胞周期蛋白在细胞周期中的作用,采用PCR方法从拟南芥花序中克隆出At CYCD3;1基因的全长c DNA序列。该基因的开放读码框为1131 bp,预测编码376个氨基酸,蛋白质的分子量为42701.6 Da,蛋白质的等电点为4.89。构建植物表达载体p ROKII-At CYCD3;1,并利用农杆菌介导的叶盘法将外源基因导入野生型烟草中。通过PCR及q RT-PCR检测显示,At CYCD3;1成功插入烟草基因组并在转录水平表达。表型观察显示转基因株系与野生型株系相比主要表现为花冠宽度变大,花瓣和萼片长度变长,果实变大,茎干弯曲,叶片卷曲,根尖细胞变小。综上,拟南芥At CYCD3;1基因的过量表达影响了植物的生长发育。

Construction of plant expression vector and genetic transformation analysis of Arabidopsis thaliana CYCD3;1 gene in Nicotiana tabacum

In order to analyze the role of plant D-type cyclins in plant development, a cDNA sequence of a homologous gene of CYCD3;1 was cloned from Arabidopsis inflorescences (AtCYCD3;1) using PCR. The open reading frame (ORF) of AtCYCD3;1 was 1131 bp in length, which encoded a protein containing 376 amino acids, while the estimated molecular weight and isoelectric point of the putative protein were 42701.6 Da and 4.89. A plant overexpression vector pROKII-AtCYCD3;1 was constructed and transformed into a wild-type tobacco by Agrobacterium-mediated leaf disc transformation The transgenic tobacco plants were confirmed by PCR, indicating that the AtCYCD3;1 gene was integrated into tobacco genome. The result of qRT-PCR of the T₂ generation plants showed the expression of AtCYCD3;1 in transgenic tobaccos. There were significant differences in phenotype between the transgenic and wild-type plants, such as increased width of the corolla, length of the petal and sepal, and the volume of seed pod. Moreover, transgenic plants showed curved stems, curled leaves, and smaller seeds compared with the wild type. Additionally, the root tip cells of transgenic plants were smaller than those of the wild type. Results suggested that overexpression of AtCYCD3;1 obviously influenced the growth and development of plants.