关键词: 甜菜夜蛾')">甜菜夜蛾, 原代细胞')">原代细胞, 离体培养')">离体培养, 转录组测序')">转录组测序, 细胞周期')">细胞周期

Abstract:

 【Aim】 The aim of this study is to analyze the whole process of establishing cell lines from Spodoptera exigua pupal ovarian cells, and to explore the changes of gene expression at the transcriptional level from in vivo to in vitro culture, so as to provide a theoretical basis for the establishment of insect in vitro culture models. 【Methods】 In this study, the transcriptomes of various stages of S. exigua pupal ovarian cells during ex vivo culture were sequenced by using second-generation transcriptome sequencing technology Illumina Hiseq, and the differentially expressed genes and their related signaling pathways were analyzed. The expression of some cell cycle-related genes (cycd and cdk4), regulatory genes (cdc20, apc1, skp2 and mad1), and proliferation-related molecular markers (mcm4 and pcna) in different stages of S. exigua pupal ovarian cells during ex vivo culture were verified by real-time PCR. 【Results】 The process of ex vivo culture of S. exigua pupal ovarian cells includes five stages: anatomical obtaining of ex vivo ovarian tissues, primary cells isolated after adherent culture of ovarian tissues, transformed cells with re-proliferation ability, first generation cells capable of continuous passage, and cell lines capable of continuous passage for more than 15 generations. A total of 46 796 unigene sequences were obtained from the five stages of S. exigua pupal ovarian cells during ex vivo culture by RNA sequencing, with good sequence length integrity of the assembly. The splicing length distribution of unigene sequences was reasonable, and the Q30 of the sample base was above 94%. Based on the KEGG database, 1 473 unigenes were annotated to be involved in 20 signaling pathways closely related to cellular processes, and 92 unigenes in the cell cycle signaling pathway. Cluster analysis showed that the gene expression patterns of ovarian cells in a rapidly developing state in vivo were very close to that of cell lines which were also in a proliferative state. In addition, 619 differentially expressed genes were screened at the critical transformation stage of primary cells from transient stagnant growth to clustered cells. The expression of cdk4 significantly decreased ex vivo, and the expression level of cycd significantly increased after the critical stage of cell transformation. The expression levels of Cdc20, apc1, skp2, and mad1 were significantly higher in ovarian tissues and cell lines than in primary cells, key transformed cells, and first passage cells. From primary cells to passage, the expression level of cycd was significantly increased by 8.7-fold, which was significantly higher than those of mcm4 and pcna. 【Conclusion】 The sequence quality of five stages of S. exigua pupal ovarian cells during ex vivo culture by transcriptome sequencing met with the basic requirements for transcriptome data analysis. The differentially expressed genes in S. exigua pupal ovarian cells during ex vivo culture were obtained. The reverse cell proliferation of primary cells may be related to the expression of cell cycle regulatory genes such as cdk4, cycd, skp2 and mad1. In addition, cycd can be used as a marker for the ability of primary cells to pass through.

Key words: Spodoptera exigua, primary cell; ex vivo culture, transcriptome sequencing, cell cycle