本文選題：雨生紅球藻 + 脅迫早期��； 參考：《深圳大學》2017年碩士論文

【摘要】：雨生紅球藻(Haematococcus pluvialis)是一種單細胞真核綠藻,因在強光、高溫、高鹽和氮饑餓等脅迫環(huán)境下大量積累蝦青素而備受關(guān)注。但是,由于蝦青素合成涉及的基因數(shù)目多、調(diào)控網(wǎng)絡(luò)復(fù)雜,至今仍不清楚其分子調(diào)控機制。與此同時,新一代高通量測序技術(shù)的出現(xiàn)大大促進了轉(zhuǎn)錄組學的研究,且不受物種和是否存在參考基因組的限制,能夠快速對復(fù)雜的基因調(diào)控網(wǎng)絡(luò)進行研究。為了弄清雨生紅球藻蝦青素合成調(diào)控的分子機制,本研究擬對脅迫早期的雨生紅球藻192.80進行轉(zhuǎn)錄組測序,通過數(shù)據(jù)分析了解脅迫早期基因表達的特點,挖掘參與基因表達調(diào)控的關(guān)鍵基因,從而初步解析蝦青素合成的調(diào)控網(wǎng)絡(luò)。具體研究內(nèi)容及結(jié)果如下:(1)通過半定量RT-PCR對脅迫早期的雨生紅球藻蝦青素合成關(guān)鍵酶基因HpCrtR-B和Hpbkt1進行轉(zhuǎn)錄分析。在45 mM NaAC和550μmol/m2/s光照強度下脅迫1.5h,已能夠檢測到HpCrtR-B和Hpbkt1基因的轉(zhuǎn)錄,4h后能觀察到藻細胞開始積累蝦青素,表明雨生紅球藻蝦青素合成的調(diào)控發(fā)生在脅迫早期;(2)為了獲得高質(zhì)量的雨生紅球藻192.80的總RNA,實驗中比較了不同培養(yǎng)基、藻細胞破壁方式、提取方法等因素,獲得最優(yōu)的總RNA提取方法:22℃白熾燈連續(xù)光照條件(25μmol/m2/s)下,于MIX培養(yǎng)基培養(yǎng)至對數(shù)中后期,離心收集藻細胞后采用改良Trizol法提取總RNA;(3)提取脅迫早期的雨生紅球藻192.80總RNA進行轉(zhuǎn)錄組測序,經(jīng)過測序、拼接、注釋,共獲得309962820條clean reads,83869個unigenes,unigene序列的平均長度747bp。對轉(zhuǎn)錄組進行GO、KOG、KEEG功能分類,發(fā)現(xiàn)大量unigenes與蛋白質(zhì)轉(zhuǎn)運、催化過程、轉(zhuǎn)錄和翻譯有關(guān),表明在脅迫早期雨生紅球藻細胞內(nèi)代謝旺盛,積極響應(yīng)脅迫。通過與植物轉(zhuǎn)錄因子數(shù)據(jù)庫比對獲得476個轉(zhuǎn)錄因子,包括AP2/EREBP,bHLH,bZIP,C2H2,MYB和WRKY等幾大類轉(zhuǎn)錄因子家族,顯示在脅迫早期的雨生紅球藻產(chǎn)生了大量轉(zhuǎn)錄因子,參與基因表達調(diào)控及轉(zhuǎn)錄激活等活動;(4)數(shù)據(jù)分析獲得4367個差異表達基因,其中上調(diào)表達的基因數(shù)為2050個,下調(diào)表達的基因數(shù)為2317個。經(jīng)過熒光定量RT-PCR的驗證,顯示有75%的差異表達基因與轉(zhuǎn)錄組測序一致。GO和KEEG富集結(jié)果表明差異表達基因中有預(yù)測上調(diào)基因主要集中在催化功能和金屬離子結(jié)合。下調(diào)基因主要集中在小分子代謝方面。同時,獲得71個差異表達的轉(zhuǎn)錄因子,其中上調(diào)28個,主要與蛋白的催化功能相關(guān);下調(diào)43個,與植物的抗逆性相關(guān)。在對差異表達轉(zhuǎn)錄因子進行的轉(zhuǎn)錄水平分析發(fā)現(xiàn),轉(zhuǎn)錄因子對強光和醋酸鈉脅迫的響應(yīng)不一致,轉(zhuǎn)錄活性隨時間變化而變化。其中,HpGNAT-1在醋酸鈉脅迫6h后轉(zhuǎn)錄水平上升300倍,強光脅迫則上升了30倍;(5)最后,對高響應(yīng)強光和醋酸鈉脅迫的HpGNAT-1進行分子克隆及生物信息學分析。該基因開放閱讀框長度為493bp,同源性分析表明HpGNAT-1可能為N-乙�；D(zhuǎn)移酶(NAT)。該基因可翻譯成163個氨基酸,等電點5.92,不存在信號肽和跨膜區(qū),可能定位在細胞質(zhì)。它可能存在4個α-螺旋,7個β-折疊,具有NAT家族保守結(jié)構(gòu)域和RimI結(jié)構(gòu)域,可能通過與輔酶A結(jié)合,并作用于核糖體蛋白S18亞基來對轉(zhuǎn)錄起調(diào)控作用。以上研究結(jié)果表明:在強光和醋酸鈉脅迫早期,雨生紅球藻通過信號傳導(dǎo)、轉(zhuǎn)錄激活、催化等生物過程積極響應(yīng)脅迫,大量與之相關(guān)的基因轉(zhuǎn)錄活性上升,而小分子代謝則受到抑制。轉(zhuǎn)錄因子通過差異性響應(yīng)強光和醋酸鈉脅迫來對轉(zhuǎn)錄起調(diào)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.

【學位授予單位】：深圳大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q943.2

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