目的 探知浙贝母Fritillaria thunbergii（“浙贝1号”与“浙贝3号”）2个品种鳞茎形成与发育产生差异的分子机制，对“浙贝1号”与“浙贝3号”进行转录组测序，挖掘导致差异形成的关键基因。方法 以“浙贝1号”和“浙贝3号”成熟期鳞茎为材料，采用Illumina HiSeq高通量测序平台进行转录组测序，组装与注释后进行差异基因的富集并筛选与鳞茎发育相关的基因，对获得的候选基因进行功能验证。结果 转录组测序后共获得35 509个Unigene注释信息，其中差异表达基因共295个。对京都基因和基因组百科全书（Kyoto encyclopedia of genes and genomes，KEGG）、基因本体论（gene ontology，GO）、同源蛋白簇（clusters of orthologous groups of proteins，KOG）富集结果进行筛选，获得了132个与鳞茎形成及发育相关的候选基因，涉及淀粉合成、蛋白质合成、植物激素及信号转导。淀粉合成相关基因与鳞茎膨大相关。淀粉与蛋白质的合成可能是新鳞茎形成的物质基础。与赤霉素（gibberellin，GA）、脱落酸（abscisic acid，ABA）、细胞分裂素（cytokinin，CTK）和吲哚乙酸（indole acetic acid，IAA）相关DEGs可能是浙贝3号鳞茎繁殖系数提高的原因。通过浙贝母鳞茎内源激素与质量的相关性分析，发现“浙贝1号”与“浙贝3号”鳞茎中CTK与质量呈极显著正相关。对筛选出的CKX3和CKX9进行生物信息学分析，结果显示2个基因都符合细胞分裂素脱氢酶的基本特征。在本氏烟草上进行瞬时过表达及亚细胞定位实验，结果显示CKX3和CKX9表达量提高且能降低CTK的含量，GFP信号定位在细胞膜上。结论 通过对“浙贝1号”和“浙贝3号”转录组测序，初步揭示了导致繁殖系数及形态特征产生差异的候选基因，并对CKX3和CKX9进行了功能验证，可为浙贝母鳞茎发育研究提供科学依据。

Objective To elucidate the molecular mechanisms underlying the differences in bulb formation and development between the two varieties of Fritillaria thunbergii(“Zhebei No. 1” and “Zhebei No. 3”), transcriptome sequencing was performed on both varieties, with a focus on identifying key genes associated with these differences. Methods The mature bulbs of Zhebei No.1 and Zhebei No.3 were used as materials for transcriptome sequencing by Illumina HiSeq high-throughput sequencing platform. After assembly and annotation, differential genes were enriched and genes related to bulb formation and development were screened, and the functions of the obtained candidate genes were verified. Results A total of 35 509 Unigene annotation messages were obtained after transcriptome sequencing, including 295 differentially expressed genes. The results of KEGG, GO and KOG enrichment were screened, and 132 candidate genes related to bulb formation and development were obtained, including starch synthesis, protein synthesis, plant hormones and signal transduction. Genes related to starch synthesis were associated with bulb enlargement. The synthesis of starch and protein may be the material basis for the formation of new bulbs. DEGs related to GA, ABA, CTK and IAA may be the reason for the increase of reproductive coefficient of Zhebei No.3 bulb. Through the correlation analysis of endogenous hormones and quality in bulb, it was found that CTK in the bulbs of Zhebei No.1 and Zhebei No.3 is extremely significantly positively correlated with quality. The selected CKX3 and CKX9 genes were analyzed by bioinformatics, and the results showed that the both were consistent with the basic characteristics of cytokinin dehydrogenase. Transient overexpression and subcellular localization were carried out on Nicotiana benthamiana, and the results showed that CKX3 and CKX9 expression levels were increased and CTK content was decreased, and GFP signal was localized on the cell membrane.Conclusion By sequencing the transcriptome of Zhebei No.1 and Zhebei No.3, the candidate genes that lead to the differences in reproductive coefficient and morphological characteristics were initially revealed, and the functions of CKX3 and CKX9 were verified, which can provide basis for the study of bulb development of F. thunbergii.

R286.12

浙江省中药材新品种选育重大科技专项（2021C02074-1）;宁波市重点研发计划暨“揭榜挂帅”项目（2023Z120）;宁波市特色农产品质量安全检测与控制重点实验室开放项目（KF202302）