目的 通过鉴定曼地亚红豆杉Taxus×media中病程相关基因非表达子1（non-expressor of pathogenesis-related genes 1，NPR1）家族基因，分析并比较其蛋白结构以及基因表达等差异，为利用NPR1基因提高红豆杉紫杉醇含量奠定理论基础。方法 利用本地化blast对曼地亚红豆杉NPR1基因进行鉴定，通过CD-search、MEGA X、DNAMAN以及MEME在线网站等生物信息学技术进行保守结构域、进化树构建、多序列比对以及基序分析，利用blast比对转录组数据，预测所获得的NPR1基因在不同组织以及激素和低温处理后的表达模式。结果 在曼地亚红豆杉中共鉴定出3个NPR1家族基因，命名为TmNPR1～TmNPR3。研究发现，3个NPR1家族成员均含有BTB_POZ保守结构域，并分成2个亚家族。TmNPR1/2与AtNPR3/4聚在一起，TmNPR3与AtNPR5/6亲缘关系较近。TmNPR1/2与AtNPR3/4相似，均含有EAR基序（VDLNETP）。表达模式分析发现3个NPR1基因在不同组织中的表达有所差异，TmNPR1/2在针叶组织中的表达水平最高，TmNPR1响应低温胁迫，且在茉莉酸甲酯和冠菌素激素处理曼地亚红豆杉细胞系中表达量最高。结论 曼地亚红豆杉NPR1家族的3个成员在序列和结构上存在保守性，但也在红豆杉根和针叶的发育以及在低温等不同胁迫和茉莉酸甲酯信号途径中扮演了不同的角色，推测具有不同的功能，为揭示TmNPR1s基因功能和利用分子生物学技术提高红豆杉紫杉醇产量提供理论依据。

Objective To provide a theoretical basis for increasing taxol content of Hongdoushan (Taxus wallichiana var. chinensis) by non-expressor of pathogenesis-related genes 1 (NPR1) gene through identifying the family gene of NPR1 in T.×media and analyzing and comparing its differences in protein structure and gene expression. Methods Localized-blast was used to identify the NPR1 gene. Bioinformatics techniques, such as CD-search, MEGA X, DNAMAN, and MEME online websites were used to construct the conservative domain, phylogenetic tree construction, multiple sequence alignment, and motif analysis. The transcriptome data were compared by blast to predict the expression patterns of the obtained NPR1 gene in different tissues and after hormone and low-temperature treatments. Results A total of three NPR1 genes were identified in T.×media, which were named TmNPR1-3. It was found that all the three NPR1 family members contained BTB_POZ conservative domains and were divided into two subfamilies. Moreover, TmNPR1/2 and AtNPR3/4 were clustered together, and TmNPR3 and AtNPR5/6 were closely related. TmNPR1/2 was similar to AtNPR3/4 in which both of them contained an EAR motif (VDLNETP). The expression pattern analysis showed that the expression of the three NPR1 genes was different in different tissues. It is worth noting that the expression level of TmNPR1/2 was the highest in needles, and TmNPR1 was the highest in methyl jasmonate (MeJA) and coronatine-treated T.×media cell lines in response to low temperature stress. Conclusion The three members of the NPR1 family in T.×media were conserved in sequence and structure, and they may play different roles in the development of roots and needles as well as in hormonal signals and abiotic stresses, such as MeJA signaling pathways and low temperature, which was speculated to have different functions. This study provides a theoretical basis for revealing the function of TmNPR1s genes and improving the taxol content in T. wallichiana var. chinensis by molecular biology technology.

国家自然科学基金资助项目（32371907）