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低温处理是苹果花药培养诱导胚状体形成的关键步骤,花药中的小孢子在经过一定时间的低温诱导后才能获得胚性潜能。本文通过转录组测序的方法对低温处理前和低温处理30 d的苹果花药进行研究,分析低温诱导条件下花药中的差异表达基因。结果表明:转录组测序共得到10.90 Gb的Clean Data。基因表达分析结果显示,共有4 105个基因发生差异表达,包括表达上调基因1 849个,表达下调基因2 256个。注释到GO、COG、KEGG、Swiss-Prot和nr数据库的差异表达基因分别有3 325个、1 504个、733个、2 993个和3 758个。差异表达基因主要富集在与糖类代谢和激素信号转导有关的过程中,其中在淀粉和蔗糖代谢、植物激素信号转导这两个代谢通路中富集的差异表达基因最多。筛选出的差异表达基因中控制蔗糖合成、细胞分裂素、脱落酸和油菜素内酯信号转导的相关基因表达量上调,控制淀粉合成、生长素信号转导的相关基因表达量下调。差异表达基因的荧光定量PCR结果显示测序结果和实际结果变化趋势完全一致。由此可见,苹果花药经低温诱导后,影响蔗糖、淀粉生物合成和生长素、细胞分裂素、脱落酸、赤霉素和油菜素内酯信号转导相关基因的表达变化是影响小孢子获得胚性潜能的关键。
Cryopreservation is a key step in the induction of embryoid body formation by apple anther culture. The microspore in anther can obtain the embryogenic potential after a certain period of low temperature induction. In this paper, the anther of apple anther treated with low temperature and hypothermia for 30 days was studied by transcriptome sequencing method, and the differentially expressed genes in anthers under low temperature induction were analyzed. The results showed that a total of 10.90 Gb of Clean Data was obtained by transcriptome sequencing. Gene expression analysis showed that a total of 4 105 genes were differentially expressed, including 1 849 up-regulated genes and 2 256 down-regulated genes. There were 3 325, 1 504, 733, 2 993 and 3 758 differentially expressed genes in GO, COG, KEGG, Swiss-Prot and nr databases, respectively. The differentially expressed genes were mainly involved in the process of carbohydrate metabolism and hormone signal transduction. Among them, the most abundant differentially expressed genes were found in the two metabolic pathways of starch and sucrose metabolism and plant hormone signal transduction. Among the differentially expressed genes screened, the expression levels of related genes that control sucrose synthesis, cytokinin, abscisic acid and brassinosteroid signal transduction were up-regulated, and the expression of related genes for starch synthesis and auxin signaling was down-regulated. Fluorescent quantitative PCR results of differentially expressed genes showed that the sequencing results were in good agreement with the actual results. Thus, the change of expression of sucrose, starch biosynthesis and auxin, cytokinin, abscisic acid, gibberellin and brassinosteroid signal transduction related genes in apple anthers after induced by low temperature is the effect of microspore-derived embryos The key to sexual potential.