大白菜BrMLP328的克隆、表达及功能验证

Cloning,Expression and Functional Identification of BrMLP328 Gene in Brassica rapa subsp.pekinensis

【目的】克隆大白菜BrMLP328基因,对其表达模式进行分析,并验证对花期调控的功能,为进一步探究该基因在大白菜成花调控过程的作用机理奠定基础。【方法】运用RT-PCR克隆BrMLP328,并进行生物信息学分析;利用RT-qPCR测定该基因的相对表达量;构建过表达载体并通过蘸花法转化野生型拟南芥,比较T_(2)代植株与野生型的开花时间差异。【结果】BrMLP328的CDS全长为456 bp,编码151个氨基酸,蛋白相对分子质量为17493.82 Da,定位于细胞核。BrMLP328在大白菜茎中的表达量最高,根中次之,花蕾中最低;茎尖生长点中的表达量表现为春化后升高,之后在花芽分化阶段迅速下降,并维持在很低的水平。过表达BrMLP328的拟南芥开花时间比野生型延迟了1.46-3.09 d。【结论】从大白菜中克隆得到BrMLP328基因,其表达量在不同组织及不同成花阶段有所不同,该基因能够延迟开花。

【Objective】The BrMLP328 gene of Chinese cabbage(Brassica rapa subsp.pekinensis)was cloned,its expression pattern was analysed,and its function in regulating flowering was verified.This may lay the basis for further investigation into the mechanism of the role of this gene in regulating flower formation in Chinese cabbage.【Method】BrMLP328 was cloned by RT-PCR and analysed by bioinformatics.RT-qPCR was used to determine the relative expression of the BrMLP328.An overexpression vector was constructed and transformed into wildtype Arabidopsis thaliana by flower-dipping method,and the difference in flowering time between the T_(2) generation plants and the wild type was compared.【Result】The coding sequence(CDS)of BrMLP328 has a total length of 456 bp,encoding 151 amino acids.The relative molecular weight of the protein was 17493.82 Da and was localized in the nucleus.The expression of BrMLP328 was the highest in the stems of Chinese cabbage,followed by roots and lowest in flower buds.The expression in the growing point of the stem tip showed an elevated level after vernalization,after which it declined rapidly at the stage of flower bud differentiation and was maintained at a very low level.Compared with the wild type,the flowering time of the BrMLP328 of T_(2) generation of transgenic plants delayed by 1.46-3.09 d.【Conclusion】BrMLP328 was cloned from Chinese cabbage.The expression of BrMLP328 was different in different tissues and different flowering stages,and the gene could delay flowering.