目的 克隆西伯利亚白刺Nitraria sibirica维生素C合成过程中L-半乳糖-1-磷酸磷酸酶（L-galactose-1-phosphate phosphatase，GPP）编码基因。方法 利用生物信息学方法对西伯利亚白刺三代转录组数据进行分析，共鉴定出2个GPP基因，并从西伯利亚白刺中成功克隆得到2条NsGPP基因序列，进一步利用生物信息学方法对其理化性质、蛋白结构、保守序列以及系统进化等进行分析，并结合RNA-seq数据分析其在盐胁迫下的表达模式，利用qRT-PCR实验检测其在不同组织中的表达水平。结果 从西伯利亚白刺中成功鉴定克隆出2个NsGPP基因，2个NsGPP基因所编码蛋白均含有保守的FBPase/IMPase/glpX-like（FIG）结构域，且与其他植物GPP蛋白具有较高的相似性。利用多个物种的GPP蛋白序列构建系统进化树，结果显示西伯利亚白刺与灌木或半木质化草本植物亲缘关系更近。Motif分析发现NsGPP蛋白与拟南芥等GPP蛋白较为相似，但也存在差异，并且2个NsGPP蛋白之间也存在差异。此外，NsGPP1与NsGPP2的蛋白三维结构之间也存在微小差别。RNA-seq分析表明，不同浓度NaCl处理下，NsGPP1的表达逐步升高的趋势，而NsGPP2的表达量则较低，且在盐胁迫后其表达水平逐渐降低。qRT-PCR实验结果显示，白刺GPP基因的表达存在组织表达特异性，且在同一组织中NsGPP1的表达水平显著高于NsGPP2。结论 从西伯利亚白刺中成功克隆到2个NsGPP基因，其编码蛋白均含有保守的FIG结构域，且与拟南芥等草本植物的GPP蛋白更为相似。NsGPP基因在不同组织中表达水平具有显著差异，其中NsGPP1可能参与西伯利亚白刺维生素C生物合成并参与白刺抵抗盐胁迫。为进一步解析白刺维生素C生物合成以及耐盐分子机制提供了理论依据。

Objective The aim of this study is to clone the gene encoding the L-galactose-1-phosphate phosphatase (GPP), an important rate-limiting step in vitamin C biosynthesis in Nitraia sibirica. Methods Basing on the third-generation transcriptome data of N. sibirica in NCBI public database, GPP genes were identified and cloned. With the bioinformatics methods, the structure, chemical properties, conserved sequenceand phylogentic evolution were analyzed. Moreover, the expression pattern of NsGPP genes under salt stress was analyzed via RNA-seq data, and the expression levels in different tissues were analyzed by qRT-PCR. Results The results showed that two proteins encoded by the NsGPP1/2 genes were similar to the other plant GPP proteins, which have typical conserved FBPase/IMPase/glpX-like(FIG) domain. Moreover, from an evolutionary perspective, NsGPPs may be more closely related to GPPs of shrubs or robust herbs. At the same time, with motif analysis, a few differences between NsGPP and GPP of herbaceous were found. In addition, the three-dimensional structures of NsGPP1 and NsGPP2 were also slightly different. With the transcriptome data, the expression level of NsGPP1 was high and gradually increased under different concentrations of NaCl treatment, while the expression level of NsGPP2 was low and gradually decreased. According to the experimental data of qRT-PCR, NsGPP1/2 were specifically expressed in different tissues in N. sibirica. Moreover, the expression level of NsGPP1 was significantly higher than that of NsGPP2. Conclusion In this study, two NsGPP genes were identified and cloned from N. sibirica, the structures of these two proteins were relatively conservative and closely related to GPP of herbaceous plants. Furthermore, they were expressed in fruits, stems and leaves, and had obvious tissue specificity. Among them, NsGPP1 may be involved in vitamin C biosynthesis and salt tolerance in N. sibirica. This study is helpful to further study of the molecular mechanism of vitamin C biosynthesis and salt tolerance in N. sibirica.

R286.12

牧草与特色作物生物学教育部重点实验室开放课题资助（FECBOF2021010）