红花PAL家族全基因组分析及其在悬浮细胞中的表达

目的 完成红花Carthamus tinctorius PAL基因家族的生物信息学分析,克隆1条红花PAL4基因编码区序列,通过农杆菌介导的方法,实现CtPAL4在红花悬浮细胞中的过表达.方法 在红花基因组测序基础上,对红花PAL家族进行全基因组分析,利用荧光定量PCR (RT-PCR)方法克隆1条红花PAL4编码区序...

Genome analysis of PAL gene family from Carthami Flos and its expression in suspension cells

Objective The bioinformatics analysis of the PAL gene family from Carthami Flos were performed, and cloned a sequence encoding the PAL4 gene of Carthami Flos. The overexpression of CtPAL4 in Carthami Flos suspension cells was analyzed, laying a foundation for the study of the function of CtPAL4 gene in Carthami Flos flavonoids metabolism pathway. Methods On the basis of Carthami Flos genome sequencing, the PAL family of Carthami Flos was analyzed. Coding region of PAL4 was cloned by RT-PCR and bioinformatics analysis was performed. The phylogenetic tree was constructed by clustalW 1.83 software, Bgl II and BstE II restriction sites were introduced to construct over-expression vector pcambia3301-CtPAL4 containing 35 S promoter. It was overexpressed in suspension cells of Carthami Flos through agrobacterium mediated method. Results Six PAL genes were annotated in Carthami Flos genome sequencing. The analysis showed that five genes of PAL gene family had typical functional domain and active site of phenylalanine ammonia lyase, and promoter and evolution analysis were carried out. The CtPAL4 gene encoded 707 amino acids with a molecular weight of 76 820. The CtPAL4 gene had typical functional domain and active site of phenylalanine ammonia-lyase (PAL) gene. The three-dimensional structure of the coding protein was similar to the crystal structure of the X-ray protein in PAL family. Phylogenetic analysis showed that the protein encoded by CtPAL4 was the closest relationship of Arabidopsis thaliana. GUS characterization results confirmed that the genetic transformation system of Carthami Flos suspension cells was established successfully, and CtPAL4 gene was expressed preliminarily in Carthami Flos suspension cells. Conclusion Coding region gene of Carthami Flos PAL4 was cloned and a plant expression vector pCAMBIA3301-CtPAL4 was constructed successfully, which was initially overexpressed in Carthami Flos suspension cells.