三华李果实发育转录组分析及其花青苷生物合成相关基因表达分析

发布时间：2019-05-11 00:21

【摘要】：中国李(Prunus salicina Lindl.)原产长江流域,经长期栽培已在华南地区形成了一个独特的南亚热带栽培群体,并成为广东省第一大落叶果树,主要有三华李类(红皮红肉)、绿皮白肉类(竹丝李、红线李等)和红皮白肉类(三月李等)等三种类型,其中三华李类品种栽培面积约占广东全省李面积80%以上。本研究以三华李类品种‘华蜜大蜜李’和‘白脆鸡麻李’为材料,通过RNA-Seq技术进行果实、花、嫩茎叶和胚等进行转录组测序,初步建立起三华李果实发育转录数据库,挖掘果实发育成熟过程中差异表达基因,分析果实发育成熟过程中花青苷生物合成、果实成熟软化相关的细胞壁代谢等差异表达基因,为今后果实着色改良、耐储运品种培育提供参考依据。主要研究结果如下:1、以华蜜李大蜜李的叶芽、花朵、胚和不同发育时期的果实(花后75 d、花后100d、花后130 d)为材料建立三华李开花结果时期转录组数据库,获得68,484条Unigene;并对其进行GO、KEGG、NR、PlnTFDB等数据库注释,共有33,630条Unigene获得注释;并对1 kb以上的Unigene进行SSR位点开发,共发现7,984个SSR位点。2、基于4个不同发育时期的华蜜大蜜李果实样品转录组数据进行差异表达基因的筛选,共获得5,394个差异表达基因。对差异表达基因进行转录因子预测、KEGG、表达模式GO富集分析发现一些时期高表达基因模块;蛋白互作网络分析发现果实发育成熟过程中的一些高蛋白互作集中模块。对果实发育差异表达基因预测分析,发现大部分激素相关基因呈下调表达趋势,并发现30个激素合成相关基因。对细胞壁代谢相关差异基因分析,发现41个细胞壁代谢相关基因;其中1个华蜜李幼果期(花后75 d)特异表达、2个华蜜李成熟果(花后130 d)时期特异表达基因。3、对三华李果实花青苷合成相关基因进行分析,发现41个可能花青苷生物合成关键基因、26个可能参与花青苷转运、39个可能花青苷合成相关转录因子、3个果实色素积累而下调的基因。4、以特异引物扩增条带单一、溶解曲线峰值高、最高峰值温度波动小为标准进行筛选内参基因,筛选结果表明HMBS2为华蜜大蜜李果实的基因表达量分析较合适的qRT-PCR内参基因。通过对10个候选花青苷生物合成基因、9个候选MYB转录因子进行qRT-PCR验证表明,相比华蜜大蜜李叶和花,华蜜大蜜李成熟果(花后130 d)果实颜色深可能是受c23975.graph_c0(ANS)和c19863.graph_c0(UFGT)表达的影响;与白脆鸡麻李成熟果(花后130 d)相比较,华蜜大蜜李成熟果(花后130 d)果肉颜色更深可能是受c21951.graph_c0(CHS2)和c19863.graph_c0(UFGT)的影响。并发现c6572.graph_c0可能是影响果实花色苷形成的有效MYB转录因子之一。5、利用TA克隆技术,得到了c6572.graph_c0的cDNA全长;并对cDNA进行结构预测,发现其含有蛋白R2R3结构域。
[Abstract]:Chinese Li (Prunus salicina Lindl.) Native to the Yangtze River Basin, after long-term cultivation, it has formed a unique subtropical cultivation population in South China, and has become the largest deciduous fruit tree in Guangdong Province, mainly Sanhua plum (red skin and red meat), green skin and white meat (bamboo silk plum), There are three types of red plum and red skin and white meat (March plum, etc.). Among them, the cultivation area of Sanhua plum varieties accounts for more than 80% of the total plum area in Guangdong Province. In this study, Sanhua plum varieties' Huami big honey plum 'and' white crispy chicken plum 'were used as materials to sequence the fruit, flowers, tender stems, leaves and embryos by RNA-Seq technique, and the transcription database of fruit development of Sanhua plum was established. The differentially expressed genes during fruit development and ripening were excavated, and the differentially expressed genes such as anthocyanin biosynthesis and cell wall metabolism related to fruit ripening were analyzed, so as to improve the fruit coloring in the future. The cultivation of varieties resistant to storage and transportation provides a reference basis. The main results are as follows: 1. The database of leaf bud, flower, embryo and fruit at different developmental stages (75 d after anthesis, 100 d after anthesis, 130 d after anthesis) was established. Get 68484 Unigene;. GO,KEGG,NR,PlnTFDB and other database comments were carried out, and a total of 33630 Unigene comments were obtained. A total of 7984 SSR loci were found in Unigene with more than 1 kb. 2. 5394 differentially expressed genes were screened based on the data of four different developmental stages of plum fruit samples, and a total of 5394 differentially expressed genes were obtained. the results showed that 5394 differentially expressed genes were obtained by screening the differentially expressed genes based on the data of four different developmental stages of Prunus mandshurica fruit samples. The transcription factor prediction of differentially expressed genes was carried out. GO enrichment analysis of KEGG, expression pattern found some high expression gene modules, and protein interaction network analysis found some high protein interaction concentration modules during fruit development and maturation. Based on the prediction and analysis of differentially expressed genes in fruit development, it was found that most of the hormone-related genes were down-regulated and 30 genes related to hormone synthesis were found. 41 genes related to cell wall metabolism were found by analyzing the differentially related genes of cell wall metabolism. Among them, one was specifically expressed at the young fruit stage (75 days after anthesis) and two genes were specifically expressed at the mature fruit stage (130 days after anthesis). 3. The anthocyanin synthesis related genes in the fruit of Sanhua plum were analyzed. It was found that 41 key genes of anthocyanin biosynthesis, 26 of which may be involved in anthocyanin transport, 39 of which may be involved in anthocyanin synthesis related transcription factors, and 3 genes down-regulated by pigment accumulation in fruit. 4. The bands were amplified with specific primers. The peak value of dissolution curve was high and the maximum peak temperature fluctuation was small as the standard for screening internal reference genes. The results showed that HMBS2 was a suitable qRT-PCR internal reference gene for gene expression analysis of honey plum fruit. The results of qRT-PCR verification of 10 candidate anthocyanin biosynthesis genes and 9 candidate MYB transcription factors showed that compared with Huamei plum leaves and flowers, The color depth of the ripe fruit of plum (130d after anthesis) may be affected by the expression of c23975.graph_c0 (ANS) and c19863.graph_c0 (UFGT). Compared with the ripe fruit of white crispy chicken plum (130 d after anthesis), the darker flesh color of the ripe fruit (130 d after anthesis) was probably affected by c21951.graph_c0 (CHS2) and c19863.graph_c0 (UFGT). It was found that c6572.graph_c0 may be one of the effective MYB transcription factors affecting anthocyanin formation in fruit. The full length of c6572.graph_c0 cDNA was obtained by TA cloning technique, and the structure of cDNA was predicted, and it was found that cDNA contained protein R2R3 domain.
【学位授予单位】：华南农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S662.3

【参考文献】