红白果肉颜色火龙果果肉转录组分析和基因功能注释

Gene Functional Annotation and Transcriptomic Analysis of Pitaya Fruit with Red and White Pulp Color

为探明红肉和白肉火龙果果肉转录组差异,挖掘影响火龙果色素代谢的候选基因。利用Illumina HiSeq测序平台对不同颜色火龙果果肉组织中所有m RNA进行高通量测序,所得序列经质控、组装后比对到Nr (NCBI non-redundant protein sequences)、SWISS-PROT (an annotated protein sequence database)、Nt(NCBI nucleotide sequences)、KEGG (kyoto encyclopedia of genes and genomes)、COG (clusters of orthologous groups)和GO (gene ontology)数据库,并进行差异表达基因聚类分析和KEGG通路分析,对基于高通量测序拼接获得Unigenes进行SSR (simple sequence repeats)位点分析。结果表明,测序共获得74 033个Unigenes,其中有42 606个得到注释,‘紫红龙’和‘晶红龙’果肉差异表达基因11 958个,在‘紫红龙’果肉中分别有2 674个基因上调表达、9 284个下调表达,找到与甜菜苷代谢相关的差异表达基因25个,DODA、CYP76AD和GT7基因可能在火龙果甜菜苷调控中发挥正调控作用。通过SSR位点信息分析,从74 033个Unigene中共检测到15 770个SSR位点,含SSR标记序列12 988个,出现频率最高的复基元为AG/CT,共获得6 562条SSR引物,按照SSR序列长度大于20 bp的标准筛选得到1 291条SSR引物。本研究结果可为火龙果果肉颜色形成机制的阐明及其遗传育种提供理论依据。

This study proved the transcriptome difference in pitaya pulp with different color(red and white), and revealed candidate genes of pigment metabolism related to the pitaya. All the m RNA in the different color pitaya pulp was sequenced and analyzed using Illumina Hi Seq sequencing platform. After quality control and assemble,the sequences acquired were compared to Nr, SWISS-PROT, Nt, KEGG, COG and GO databases, and then the cluster analysis of differential expressed genes and KEGG pathway analysis were performed. And SSR locus analysis of Unigenes based on high-throughput sequencing splicing was performed. The results showed that 74 033 Unigenes were acquired and 42 606 Unigenes were annotated in all database. There were 11 958 DEGs(differential expressed genes) in the pulp of‘Zihonglong’ and ‘Jinghonglong’, among which 2 674 up-regulated and 9 284down-regulated, twenty-five genes involved in betalains biosynthesis were identified in DEGs. DODA, CYP76 AD and GT7 genes might play important roles in the regulation of pitaya betalains. A total of 15 770 SSR loci were detected in 12 988 SSR labeling sequences, AG/CT was the most frequent one among all the repeat types. A total of 6 562 primer pairs were designed and 1 291 SSR primers were selected according to the standard SSR sequence length greater than 20 bp. The results listed here would provid a theoretical basis for elucidation of the mechanism of pitaya puip pigment formation mechanism and its genetic breeding.