摘要：

光周期是影响植物抽薹开花最重要的环境因子之一,光周期信号通过植物的光敏色素蛋白调控植物的抽薹开花,其中PHYB介导的信号网络对植物抽薹开花有重要的抑制作用。前期研究发现,大白菜耐抽薹材料06-247与易抽薹材料He102的PHYB基因启动子存在大片段插入/缺失差异,为了进一步研究启动子突变对PHYB及其下游途径关键基因的影响,以耐抽臺材料06-247与极易抽臺材料He102为试验材料,采用生物信息法分析了大白菜基因组中光敏色素基因的冗余特点,发现大白菜基因组包含6个光敏色素基因,其中PHYA有2个拷贝,PHYB、PHYC、PHYD和PHYE都仅有1个拷贝。进一步通过氨基酸序列比对筛选出了PHYB的特异序列、设计了抗原决定簇并制备了抗大白菜PHYB的特异抗体,采用荧光定量RT-PCR技术和Western Blot技术研究了06-247与He102中PHYB的相对含量,同时比较了PHYB下游光周期通路关键调控基因CCA1、FLC、CO和FT的动态变化。结果表明:启动子突变造成PHYB在06-247中表达水平显著升高并进而造成PHYB蛋白水平显著升高,同时下游调控基因CCA1、FLC、CO和FT均在06-247中高水平表达,这对06-247的耐抽薹特性有重要影响。

关键词: 大白菜, PHYB, 光周期, 抽薹, 蛋白质印迹

Abstract:

Photoperiod is one of important environmental factors affecting plant bolting and flowering which are regulated by plant phytochrome proteins.The signal network mediated by PHYB has an important inhibitory effect on plant bolting and flowering.Previous studies revealed that there were large segment insertion/deletion differences between the PHYB gene promoter of the Chinese cabbage late-bolting line 06-247 and the easy-bolting line He102.In order to further investigate the impact of promoter mutation on PHYB and the key genes of its downstream pathways,this study was conducted.Based on bioinformatics methods,the redundancy characteristics of phytochrome genes in genome Chinese cabbage were firstly analyzed.It was found that the Chinese cabbage genome contained six phytochrome genes,of which PHYA had two copies,and PHYB,PHYC,PHYD,and PHYE all had only one copy.Then amino acid sequence alignment was used for screening of the specific sequence of PHYB.Antigenic determination clusters were designed based on the specific sequence and the antibodies against PHYB was prepared.The fluorescence quantitative RT-PCR technology and Western Blot technology were used to study the relative content of PHYB in 06-247 and He102.At the same time,the dynamic changes of key regulatory genes such as CCA1,FLC,CO and FT which in the downstream of PHYB pathway were also compared.The results showed that the promoter mutation caused significant differences both in level of mRNA in 06-247 and then significantly increased the protein level of PHYB.At the same time,the downstream regulatory genes such as CCA1,FLC,CO and FT were highly expressed in 06-247,which had an important impact on bolting resistance of 06-247.

Key words: Chinese cabbage, Phytochrome B, Photoperiod, Bolting, Western Blot