雨生红球藻的转录组测序及乙酰转移酶基因的克隆和分析

发布时间：2018-04-11 11:35

本文选题：雨生红球藻 + 胁迫早期　；参考：《深圳大学》2017年硕士论文

【摘要】：雨生红球藻(Haematococcus pluvialis)是一种单细胞真核绿藻,因在强光、高温、高盐和氮饥饿等胁迫环境下大量积累虾青素而备受关注。但是,由于虾青素合成涉及的基因数目多、调控网络复杂,至今仍不清楚其分子调控机制。与此同时,新一代高通量测序技术的出现大大促进了转录组学的研究,且不受物种和是否存在参考基因组的限制,能够快速对复杂的基因调控网络进行研究。为了弄清雨生红球藻虾青素合成调控的分子机制,本研究拟对胁迫早期的雨生红球藻192.80进行转录组测序,通过数据分析了解胁迫早期基因表达的特点,挖掘参与基因表达调控的关键基因,从而初步解析虾青素合成的调控网络。具体研究内容及结果如下:(1)通过半定量RT-PCR对胁迫早期的雨生红球藻虾青素合成关键酶基因HpCrtR-B和Hpbkt1进行转录分析。在45 mM NaAC和550μmol/m2/s光照强度下胁迫1.5h,已能够检测到HpCrtR-B和Hpbkt1基因的转录,4h后能观察到藻细胞开始积累虾青素,表明雨生红球藻虾青素合成的调控发生在胁迫早期;(2)为了获得高质量的雨生红球藻192.80的总RNA,实验中比较了不同培养基、藻细胞破壁方式、提取方法等因素,获得最优的总RNA提取方法:22℃白炽灯连续光照条件(25μmol/m2/s)下,于MIX培养基培养至对数中后期,离心收集藻细胞后采用改良Trizol法提取总RNA;(3)提取胁迫早期的雨生红球藻192.80总RNA进行转录组测序,经过测序、拼接、注释,共获得309962820条clean reads,83869个unigenes,unigene序列的平均长度747bp。对转录组进行GO、KOG、KEEG功能分类,发现大量unigenes与蛋白质转运、催化过程、转录和翻译有关,表明在胁迫早期雨生红球藻细胞内代谢旺盛,积极响应胁迫。通过与植物转录因子数据库比对获得476个转录因子,包括AP2/EREBP,bHLH,bZIP,C2H2,MYB和WRKY等几大类转录因子家族,显示在胁迫早期的雨生红球藻产生了大量转录因子,参与基因表达调控及转录激活等活动;(4)数据分析获得4367个差异表达基因,其中上调表达的基因数为2050个,下调表达的基因数为2317个。经过荧光定量RT-PCR的验证,显示有75%的差异表达基因与转录组测序一致。GO和KEEG富集结果表明差异表达基因中有预测上调基因主要集中在催化功能和金属离子结合。下调基因主要集中在小分子代谢方面。同时,获得71个差异表达的转录因子,其中上调28个,主要与蛋白的催化功能相关;下调43个,与植物的抗逆性相关。在对差异表达转录因子进行的转录水平分析发现,转录因子对强光和醋酸钠胁迫的响应不一致,转录活性随时间变化而变化。其中,HpGNAT-1在醋酸钠胁迫6h后转录水平上升300倍,强光胁迫则上升了30倍;(5)最后,对高响应强光和醋酸钠胁迫的HpGNAT-1进行分子克隆及生物信息学分析。该基因开放阅读框长度为493bp,同源性分析表明HpGNAT-1可能为N-乙酰基转移酶(NAT)。该基因可翻译成163个氨基酸,等电点5.92,不存在信号肽和跨膜区,可能定位在细胞质。它可能存在4个α-螺旋,7个β-折叠,具有NAT家族保守结构域和RimI结构域,可能通过与辅酶A结合,并作用于核糖体蛋白S18亚基来对转录起调控作用。以上研究结果表明:在强光和醋酸钠胁迫早期,雨生红球藻通过信号传导、转录激活、催化等生物过程积极响应胁迫,大量与之相关的基因转录活性上升,而小分子代谢则受到抑制。转录因子通过差异性响应强光和醋酸钠胁迫来对转录起调控作用。
[Abstract]:Haematococcus pluvialis (Haematococcus pluvialis) is a unicellular eukaryotic green algae, because in light, high temperature, high salinity and nitrogen starvation stress under the environment of a large number of astaxanthin accumulation and concern. However, because the number of astaxanthin biosynthesis genes involved in more complex regulatory networks, still not clear regulation of the molecular mechanisms. At the same time, a new generation of high-throughput sequencing technology has greatly facilitated the study of transcriptomics, regardless of species and the existence of the reference genome, can quickly research the complex gene regulatory networks. In order to clarify the molecular mechanism of astaxanthin synthesis regulation, this study intends to stress Haematococcus the early 192.80 algae transcriptome sequencing, analyze the stress characteristics of early gene expression through the data mining, the key genes involved in the regulation of gene expression, and preliminary analysis of astaxanthin synthesis The regulation of network. The detailed research contents and results are as follows: (1) by semi quantitative RT-PCR stress on Astaxanthin biosynthesis key enzyme genes HpCrtR-B and Hpbkt1 in early transcriptional analysis. In 45 mM NaAC and 550 mol/m2/s light intensity under the stress of 1.5h, has been able to detect the transcription of HpCrtR-B gene and Hpbkt1 gene. 4H can be observed after algal cells began to accumulate astaxanthin, show that astaxanthin synthesis regulation occurs in the early stress stage; (2) in order to obtain high quality haematococcuspluvialis total RNA 192.80, we compared the different culture medium, algae cell wall breaking method, factor extraction method, extraction method of total RNA get the best C: 22 incandescent lamp continuous illumination (25 mol/m2/s), in MIX culture medium to late logarithmic, algal cells collected by centrifugation after using improved Trizol method to extract total RNA; (3) extracting stress early Haematococcus 192.80 of the total algae RNA transcriptome sequencing, after sequencing, splicing, annotation, received a total of 309962820 clean reads, 83869 unigenes, the average length of 747bp. UniGene sequences of GO, KOG KEEG on the transcriptome, functional classification, and found a large number of unigenes protein transport, catalytic process, transcription and translation, indicates that the stress in the early stage h. pluvialis cell metabolism, positive response to stress. With the plant transcription factor database 476 transcription factors, including AP2/EREBP, bHLH, bZIP, C2H2, several transcription factor families MYB and WRKY, displayed on the stress of haematococcuspluvialis early produced a large number of transcription factors involved in gene expression. The regulation of transcriptional activation and other activities; (4) data analysis of 4367 differentially expressed genes, including genes up-regulated at 2050, down the number of gene expression was 2317. After the experiment of fluorescence quantitative RT-PCR Display card, gene and transcriptome sequencing.GO and KEEG enrichment showed that differentially expressed genes are up-regulated gene prediction mainly concentrated in combining catalytic function and metal ions. 75% differentially expressed genes were down regulated mainly in small molecule metabolism. At the same time, obtain the differential expression of 71 transcription factors, which are increased by 28 that is mainly related to protein catalytic function; by 43, related with plant resistance. At the transcriptional level of transcription factor analysis of differences in transcription factors in response to high light stress and sodium acetate is not consistent, the transcriptional activity varies with time. Among them, HpGNAT-1 in sodium acetate stress after 6h transcription a 300 fold increase in light stress increased by 30 times; (5) finally, on the high response of HpGNAT-1 light and sodium acetate stress by molecular cloning and bioinformatics analysis. The ORF gene The degree of 493bp, homology analysis showed that HpGNAT-1 may be N- acetyltransferase (NAT). The gene can be translated into 163 amino acids. The isoelectric point of 5.92, there is no signal peptide and transmembrane regions may be localized in the cytoplasm. It may have 4 alpha helix, 7 beta folding, with the family of NAT domain and RimI domain, possibly by binding A, and the role of the ribosomal protein S18 subunit to effects on transcription. The above results show that: in the light of sodium acetate and stress early haematococcuspluvialis through signal transduction, transcriptional activation, catalysis and other biological processes actively respond to stress a large number of genes, transcription activity associated with the rise, and the small molecule metabolism is inhibited. The transcription factor through the difference in response to light and sodium acetate stress on transcription regulation.

【学位授予单位】：深圳大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2

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