红皮西洋梨果皮红色消退候选基因的筛选分析

发布时间：2018-07-11 11:14

本文选题：红巴梨 + 褪色　；参考：《西北农林科技大学》2017年硕士论文

【摘要】：颜色是果皮重要的外观品质,红皮西洋梨由于其优良品质深受消费者喜爱,但成熟果实果皮红色消退问题严重影响了其商品价值。花青苷是红色果皮形成的物质基础,要确定红皮西洋梨果皮褪色的分子机理,必须弄清花青苷合成与消退的关键基因。为此本研究以果实发育后期不褪色的‘红星’(Pyrus communis L.)和果实发育后期严重褪色的‘红巴梨’(Pyrus communis L.)为试验材料,测定果实不同发育时期花青苷含量,分析果皮中花青苷的合成速率和降解速率的变化;根据花青苷含量变化模式,选择花后35、75天的果皮材料进行转录组测序,结合荧光定量表达分析的结果,确定‘红巴梨’果皮红色消退的候选基因。研究结果如下:1.花后55天,两者花青苷含量达到最高;果实发育后期,‘红巴梨’花青苷含量显著下降,‘红星’下降但不显著;整个发育时期,‘红巴梨’花青苷含量始终低于‘红星’,与果皮表面颜色变化相一致。2.花青苷高峰期之前(花后35-55天),‘红巴梨’花青苷合成速率降解速率,花青苷累积含量增加,且保持较高水平;花青苷高峰期之后(花后55-75天),花青苷合成速率显著降低,降解速率明显上升,最终合成速率降解速率,花青苷由积累模式转变消耗模式,果皮花青苷含量明显减少。因此‘红巴梨’果皮红色消退是花青苷合成速率降低和花青苷降解速率增加共同导致,但两者相比花青苷合成速率的降低对‘红巴梨’的褪色作用更大。3.RT-qPCR分析与花青苷合成有关的结构基因及转录因子在‘红星’和‘红巴梨’不同时期的表达情况,结果表明CHS、ANS、UFGT、MYB10和bHLH33是引起‘红巴梨’果实发育后期花青苷含量降低的重要基因。4.转录组数据分析鉴定出947个差异表达基因(Differentially Expressed Genes:DEGs),其中471个基因在‘红巴梨’花后75天表达上调,476个表达下调;KEGG代谢通路分析将947个DEGs聚集94条KEGG代谢通路上,显著性排名前五位依次为:光合作用、植物昼夜节律、过氧化物酶体、类黄酮物质的合成、次生代谢产物的合成,其中,“次生代谢产物的合成”通路中富集的DEGs最多;利用Entrez数据库对947个DEGs进行功能注释,筛选出61个可能与花青苷含量积累有关的DEGs,其中包括5个参与类黄酮物质的合成,3个参与花青苷的降解,8个参与花青苷的转运,其余全为转录因子MYB,bHLH,WRKY,NAC,ERF和zinc finger;另外还鉴定出7个与光信号有关的基因;RT-qPCR检测12个候选基因的表达情况,结果与RNA-Seq一致。5.推测红皮西洋梨‘红巴梨’后期褪色与花青苷合成的减少、降解的增加、转运受阻及转录因子的调控作用关系密切,光信号也有可能参与其中。
[Abstract]:Color is an important appearance quality of pericarp. Due to its excellent quality, red pear is popular among consumers, but the problem of red fading of ripe fruit skin seriously affects its commodity value. Anthocyanin is the material basis for the formation of red pericarp. In order to determine the molecular mechanism of discoloration of red peel pear peel, the key genes of anthocyanin synthesis and extinction must be clarified. In this study, 'Pyrus communis L.', which is not fading in fruit development, was used in this study. And 'Pyrus communis L.' (Pyrus communis L.) In order to determine the anthocyanin content in different development stages of fruit, to analyze the changes of anthocyanin synthesis rate and degradation rate in fruit peel, and to select the peel material of 35 days after anthesis for transcriptional sequencing according to the changing pattern of anthocyanin content, and to analyze the changes of anthocyanin synthesis rate and degradation rate of anthocyanin in fruit peel. According to the results of fluorescence quantitative expression analysis, the candidate genes for red regression of 'Hongba pear' fruit peel were identified. The results are as follows: 1. At 55 days after anthesis, the content of anthocyanin reached the highest level, the content of anthocyanin in 'Hongba pear' decreased significantly but not significantly in the later stage of fruit development, and the content of anthocyanin in 'Hongba' was always lower than that of 'Hongxing' in the whole development period. Consistent with the color change of pericarp. 2. Before the peak period of anthocyanin (35-55 days after anthesis), the synthetic rate of anthocyanin was degraded, the accumulation of anthocyanin increased and maintained a high level, and after the peak of anthocyanin (55-75 days after anthesis), the synthesis rate of anthocyanin decreased significantly. The degradation rate of anthocyanin changed from accumulation mode to consumption mode and the content of anthocyanin decreased obviously. Therefore, the red regression of 'Hongba pear' fruit skin is caused by the decrease of anthocyanin synthesis rate and the increase of anthocyanin degradation rate. However, compared with the decrease of anthocyanin synthesis rate, the decolorization of 'Hongba pear' was more obvious. 3. RT-qPCR was used to analyze the expression of structural genes and transcription factors related to anthocyanin synthesis at different stages of 'Hongxing' and 'Hongba pear'. The results showed that CHSANSU UFGTMYB10 and bHLH33 were the important genes causing the decrease of anthocyanin content in 'Hongba pear' in the later stage of fruit development. 947 differentially expressed genes (genes: degs) were identified by transcriptome data analysis. Among them, 471 genes were up-regulated in 476 down-regulated KEGG metabolic pathways at 75 days after anthesis, and 947 degs were aggregated into 94 KEGG metabolic pathways. The top five were: photosynthesis, circadian rhythm of plants, peroxisome, synthesis of flavonoids, synthesis of secondary metabolites, among which, the most abundant DEGs were found in the pathway of "secondary metabolite synthesis". Using Entrez database to annotate 947 DEGs, 61 DEGss, including 5 involved in the synthesis of flavonoids, 3 involved in the degradation of anthocyanin, 8 involved in the transport of anthocyanin, were screened out. The rest were all transcription factors MYBB BHLHN WRKYYNACU ERF and zinc finger.The expression of 12 candidate genes was detected by RT-qPCR, and the results were in agreement with RNA-Seq .5.At the same time, 7 genes related to optical signal were detected by RT-qPCR. It is inferred that the fading of 'Hongba pear' is closely related to the decrease of anthocyanin synthesis, increase of degradation, inhibition of transport and regulation of transcription factors, and the light signal may also be involved in it.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S661.2

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