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编码区序列，并进行生物信息学分析，利用clustalW1.83软件构建系统进化树，在CtPAL4基因序列2端引入酶切位点Bgl II和BstE II，构建含有35 S启动子的植物超表达载体pCAMBIA3301-CtPAL4，并在红花悬浮细胞中进行超表达。结果 红花基因组中注释到6个PAL基因，分析显示5个基因具有典型的苯丙氨酸解氨酶的功能结构域及活性位点，并进行启动子和进化分析。CtPAL4基因编码707个氨基酸，相对分子质量为76 820，具有典型的苯丙氨酸解氨酶（PAL）基因的功能结构域及活性位点，编码蛋白的三维结构与PAL的X射线蛋白质晶体结构类似。系统进化分析表明，CtPAL4基因编码的蛋白与拟南芥的亲缘关系最近。GUS染色结果证实，成功建立红花悬浮细胞遗传转化体系，并初步实现了CtPAL4基因在红花悬浮细胞中的过表达。结论 成功克隆1条红花PAL4编码区基因CtPAL4，并构建了植物表达载体pCAMBIA3301-CtPAL4，初步在红花悬浮细胞中实现了超表达，为研究CtPAL4基因在红花黄酮代谢途径的功能奠定基础。

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.

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国家自然科学基金资助项目（31771868）；国家自然科学基金资助项目（31501366）；吉林省科技厅项目（20190201172JC）；吉林省科技厅项目（20190201175JC）；吉林农业大学大学生创新创业训练计划项目