光周期诱导马铃薯淀粉积累响应基因的识别及功能研究

发布时间：2019-02-24 13:47

【摘要】：同一物种不同基因型对长日照或短日照响应不同,淀粉积累起始时间、积累量也会存在明显差异；调控植物对光周期的响应及淀粉积累起始时间,无疑是增加淀粉积累不可或缺的选择之一；然而,目前关于植物如何在基因表达水平响应光周期却知之甚少。本实验以不同光周期处理马铃薯叶片为材料,通过转录组测序,识别淀粉积累响应光周期编码基因,实验结果如下：(1)长、短日照和对照处理样品测序共识别27,022个转录本,转录本序列平均长度为1640bp-其中,光照和黑暗下共表达基因数量分别为22,033和19,935个：KEGG富集分析显示,121个代谢途径参与了本研究设置的长、短日照处理。(2)GO功能注释和分化表达基因分析(DEG),筛选了上游关键响应基因1,985个；其中处理特异DNA结合(TSDB)蛋白基因208个,部分短日照处理TSDBs基因进行qPCR定量分析,结果表明,表达量倍数变化与其RPKM值基本一致。(3)不同光周期下,17个淀粉生物合成酶编码基因呈现显著差异表达；其中,4个基因光照下表达显著高于黑暗,1个在黑暗下显著高于光照；生物信息学分析显示,转录因子AP2等直接参与了17个基因的转录起始。(4)克隆了EPIDERMAL PATTERNING FACTOR 2 (EPF2), YABBY-like transcription factor GRAMINIFOLIA (GRAM)和cup-shaped protein (CUC3) 3个基因全长cDNA,构建了实现体内过量和抑制表达的目的融合基因6个。(5)筛选获得epf2+和epf2-阳性转化株系,其气孔印记观察结果显示,epf2+和epf2-阳性转化株系气孔呈现复杂的打开和关闭模式,体内过量和抑制EPF2表达与光周期间存在相互作用,调控气孔打开和关闭。(6)淀粉含量测定结果表明,不同光周期下epf2+与对照叶片淀粉积累趋势一致；长日照光周期显著减少epf2-株系淀粉积累,短和中日照下epf2-株系与对照株系淀粉积累模式相反,即黑夜淀粉积累显著高于日照。
[Abstract]:Different genotypes of the same species had different responses to long or short days, and there were significant differences in the initial time and amount of starch accumulation. Regulating the response of plants to photoperiod and the starting time of starch accumulation is undoubtedly one of the indispensable options for increasing starch accumulation. However, little is known about how plants respond to photoperiod at the level of gene expression. In this experiment, potato leaves treated with different photoperiod were used as materials, and 27022 transcripts were identified by transcriptome sequencing. The results were as follows: (1) 27022 transcripts were identified by long, short sunlight and control samples sequencing. The average length of the transcribed sequence was 1640bp-among which, the number of co-expressed genes under light and dark was 22033 and 19935 respectively. KEGG enrichment analysis showed that 121 metabolic pathways were involved in the length of this study. (2) GO functional annotation and differentiation expression gene analysis (DEG), screened 1985 upstream key response genes; 208 genes of (TSDB) protein were treated with specific DNA, and the quantitative qPCR analysis of TSDBs gene was carried out by partial short-day irradiation. The results showed that the change of expression multiple was basically the same as its RPKM value. (3) under different photoperiod, the expression of TSDBs gene was similar to its RPKM value. 17 starch biosynthesis coding genes showed significant differential expression. Among them, four genes were significantly higher in light than in dark, and one in dark was significantly higher than that in light. Bioinformatics analysis showed that transcription factor AP2 was directly involved in the transcription initiation of 17 genes. (4) three full-length cDNA, genes, EPIDERMAL PATTERNING FACTOR 2 (EPF2), YABBY-like transcription factor GRAMINIFOLIA (GRAM) and cup-shaped protein (CUC3), were cloned. Six fusion genes were constructed to achieve excessive and inhibitory expression in vivo. (5) epf2 and epf2- positive transformed lines were screened, and stomatal imprinting was observed. The stomata of epf2 and epf2- positive transformation lines showed complex opening and closing modes, and there was interaction between in vivo overdose and inhibition of EPF2 expression and photoperiod, which regulated stomatal opening and closing. (6) starch content determination showed that there was no significant difference in stomatal opening and closing. The trend of starch accumulation in epf2 leaves was the same as that in control leaves under different photoperiod. The starch accumulation of epf2- strain decreased significantly under long sunlight, and the starch accumulation pattern of epf2- strain was opposite to that of control line under short and middle sunshine, that is, the starch accumulation of dark night was significantly higher than that of sunshine.
【学位授予单位】：宁夏大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S532

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