Abstract：
The Chinese white wax scale insect (CWWSI), Ericerus pela, can secret an amount of wax equivalent to their body weight. Previous studies demonstrated the fatty acyl-CoA reductase (far3) plays a pivotal role in wax secretion of CWWSI. The high expression of far3 is crucial for the massive wax secretion. However, the transcription regulation of far3 was not clear. To identify regulatory factors that control the expression of far3, the assay for transposase-accessible chromatin (ATAC) and yeast one-hybrid (Y1H) were carried out in this study. The ATAC sequencing of the CWWSI at the early wax-secretion stage ATAC-seq resulted in 22.75 GB raw data, generated 75,827,225 clean reads and revealed 142,771 peaks. There was one significant peak in the 3 kb upstream regulation regions. The peak sequence is located between -1000 and -670 bp upstream of the far3 transcription start site, spanning a length of 331 bp. This peak sequence served as bait for creating the pAbAi-peak recombinant vector, used in Y1H screenings to identify proteins interacting with far3 gene. The results indicate a successful CWWSI cDNA library construction with a capacity of 1.2 × 107 colony forming unit, a 95.8% recombination rate, and insert sizes between 1,000 and 2,000 bp. Self-activation tests established that 100 ng/mL of AbA effectively inhibited bait vector self-activation. Finally, a total of 88 positive clones were selected. After sequencing and removal of duplication, 63 unique clones were obtained from these screened colonies. By aligning the clone sequences with full-length transcriptome and genome of CWWSI, the full-length coding sequences of these clones were obtained. BlastX analysis identified a transcription factor, nuclear transcription factor Y beta, and two co-activators, cAMP-response-element-binding-protein-binding protein and WW domain binding protein 2. Reverse transcription quantitative polymerase chain reaction analysis confirmed that their expression patterns were consistent with the developmental stages preceding wax secretion and matched the wax secretion characteristics during ovulation periods. These results are beneficial for further research into the regulatory mechanisms of wax secretion of CWWSI.
摘要：
中国白蜡鳞虫（CWWSI），Ericeruspela,可以秘密量的蜡相当于他们的体重。先前的研究表明，脂酰辅酶A还原酶（far3）在CWWSI的蜡分泌中起关键作用。far3的高表达对于大量的蜡分泌至关重要。然而,far3的转录调控尚不清楚。为了鉴定控制far3表达的调节因子，在这项研究中进行了转座酶可接近的染色质（ATAC）和酵母单杂交（Y1H）的测定。在早期蜡分泌阶段ATAC-seq的CWWSI的ATAC测序得到22.75GB的原始数据，产生75,827,225个干净的读数，并显示142,771个峰。在3kb的上游调节区域有一个显著的峰值。峰序列位于far3转录起始位点上游的-1000和-670bp之间，跨度为331bp。此峰序列用作创建pAbAi峰重组载体的诱饵，用于Y1H筛选，以鉴定与far3基因相互作用的蛋白质。结果表明成功的CWWSIcDNA文库构建，具有1.2×107个集落形成单位的能力，95.8%的重组率，和插入大小在1,000和2,000bp之间。自激活测试确定100ng/mL的AbA有效地抑制诱饵载体自激活。最后,共筛选出88个阳性克隆。测序和删除重复后，从这些筛选的菌落获得63个独特克隆。通过将克隆序列与CWWSI的全长转录组和基因组进行比对，获得这些克隆的全长编码序列。BlastX分析确定了一种转录因子，核转录因子Yβ，和两个助活化剂，cAMP反应元件结合蛋白结合蛋白和WW结构域结合蛋白2。逆转录定量聚合酶链反应分析证实，它们的表达模式与蜡分泌之前的发育阶段一致，并且与排卵期的蜡分泌特征相匹配。这些结果有利于进一步研究CWWSI蜡质分泌的调控机制。
