Pervaiz Tariq, Haifeng Jia, Salman Haider Muhammad, Cheng Zhang, Cui Mengjie, Wang Mengqi, Cui Liwen, Wang Xicheng, Fang Jinggui
Key Laboratory of Genetics and Fruit development, College of Horticulture, Nanjing Agricultural University, Nanjing, 210095, P. R. China.
Jiangsu Academy of Agricultural Sciences, Nanjing, P. R. China.
PLoS One. 2016 Jan 29;11(1):e0147369. doi: 10.1371/journal.pone.0147369. eCollection 2016.
Proceeding to illumina sequencing, determining RNA integrity numbers for poly RNA were separated from each of the four developmental stages of cv. Summer Black leaves by using Illumina HiSeq™ 2000. The sums of 272,941,656 reads were generated from vitis vinifera leaf at four different developmental stages, with more than 27 billion nucleotides of the sequence data. At each growth stage, RNA samples were indexed through unique nucleic acid identifiers and sequenced. KEGG annotation results depicted that the highest number of transcripts in 2,963 (2Avs4A) followed by 1Avs4A (2,920), and 3Avs4A (2,294) out of 15,614 (71%) transcripts were recorded. In comparison, a total of 1,532 transcripts were annotated in GOs, including Cellular component, with the highest number in "Cell part" 251 out of 353 transcripts (71.1%), followed by intracellular organelle 163 out of 353 transcripts (46.2%), while in molecular function and metabolic process 375 out of 525 (71.4%) transcripts, multicellular organism process 40 out of 525 (7.6%) transcripts in biological process were most common in 1Avs2A. While in case of 1Avs3A, cell part 476 out of 662 transcripts (71.9%), and membrane-bounded organelle 263 out of 662 transcripts (39.7%) were recorded in Cellular component. In the grapevine transcriptome, during the initial stages of leaf development 1Avs2A showed single transcript was down-regulated and none of them were up-regulated. While in comparison of 1A to 3A showed one up-regulated (photosystem II reaction center protein C) and one down regulated (conserved gene of unknown function) transcripts, during the hormone regulating pathway namely SAUR-like auxin-responsive protein family having 2 up-regulated and 7 down-regulated transcripts, phytochrome-associated protein showed 1 up-regulated and 9 down-regulated transcripts, whereas genes associated with the Leucine-rich repeat protein kinase family protein showed 7 up-regulated and 1 down-regulated transcript, meanwhile Auxin Resistant 2 has single up-regulated transcript in second developmental stage, although 3 were down-regulated at lateral growth stages (3A and 4A). In the present study, 489 secondary metabolic pathways related genes were identified during leaf growth, which mainly includes alkaloid (40), anthocyanins (21), Diterpenoid (144), Monoterpenoid (90) and Flavonoids (93). Quantitative real-time PCR was applied to validate 10 differentially expressed transcripts patterns from flower, leaf and fruit metabolic pathways at different growth stages.
进行Illumina测序，使用Illumina HiSeq™ 2000从夏黑葡萄叶片的四个发育阶段分别分离出用于测定多聚RNA完整性数值的样本。从四个不同发育阶段的葡萄叶片中产生了总计272,941,656条读数，序列数据超过270亿个核苷酸。在每个生长阶段，RNA样本通过独特的核酸标识符进行索引并测序。KEGG注释结果显示，在15,614个（71%）转录本中，2,963个（2Avs4A）转录本数量最多，其次是1Avs4A（2,920个）和3Avs4A（2,294个）。相比之下，共有1,532个转录本在基因本体（GO）中得到注释，包括细胞成分，其中“细胞部分”的转录本数量最多，353个转录本中有251个（71.1%），其次是细胞内细胞器，353个转录本中有163个（46.2%），而在分子功能和代谢过程中，525个转录本中有375个（71.4%），生物过程中的多细胞生物体过程，525个转录本中有40个（7.6%）在1Avs2A中最为常见。在1Avs3A的情况下，细胞成分中记录了662个转录本中的476个（71.9%）以及膜结合细胞器，662个转录本中有263个（39.7%）。在葡萄转录组中，叶片发育初期1Avs2A显示单个转录本下调，没有上调的转录本。相比之下，1A与3A的比较显示一个上调（光系统II反应中心蛋白C）和一个下调（功能未知的保守基因）转录本，在激素调节途径中，即SAUR样生长素响应蛋白家族有2个上调和7个下调转录本，光敏色素相关蛋白显示1个上调和9个下调转录本，而与富含亮氨酸重复蛋白激酶家族蛋白相关的基因显示7个上调和1个下调转录本，同时生长素抗性2在第二个发育阶段有单个上调转录本，尽管在侧生生长阶段（3A和4A）有3个下调。在本研究中，在叶片生长过程中鉴定出489个与次生代谢途径相关的基因，主要包括生物碱（40个）、花青素（21个）、二萜类（144个）、单萜类（90个）和黄酮类（93个）。应用定量实时PCR验证了来自花、叶和果实代谢途径在不同生长阶段的10种差异表达转录本模式。
