【作者】 李晔；

【导师】 袁其朋；

【作者基本信息】 北京化工大学 ， 生物化工， 2009， 博士

【摘要】 丝状真菌是一种重要的工业生产菌种,对其代谢调控的研究一直是国内外关注的焦点。丝状真菌三孢布拉氏霉菌(Blakeslea trispora)生长迅速,生物量高,菌体内甲羟戊酸途径代谢流量大,是目前唯一可以工业化生产类胡萝卜素的优良菌种,但对于该菌的遗传学研究较为滞后,有关三孢布拉氏霉菌基因功能和代谢调控的研究很少。RNA干扰(RNA interference,RNAi)技术是一种快速、高效、强有力的用于确定基因功能、调节基因表达以及了解基因之间相互关系的研究工具。与传统的基因敲除相比,无论是沉默基因的效率还是实验周期,都有着无可比拟的优势。本研究将RNAi技术应用于三孢布拉氏霉菌中,建立了一套完整的适用于三孢布拉氏霉菌的RNAi技术路线和方法,探讨了RNAi技术在丝状真菌中的适用性,并加深了对番茄红环化酶基因(carRA)功能的了解,丰富了对三孢布拉氏霉菌代谢途径的认识。这些结果为进一步将RNAi技术应用于真菌的基因工程改造提供了实验基础和理论依据,主要结果如下:本文首先针对三孢布拉氏霉菌carRA基因序列的CDS区设计RNAi干扰靶点,通过有效性筛选和同源性比对确定了3个靶点,设计并人工合成了三条靶向carRA基因的短发卡状(short hairpin RNA,shRNA)序列及一条阴性对照序列,并定向克隆到小干扰RNA(Shortinterfering RNA,siRNA)表达载体mU6 pro上,分别命名为mU6 proshRNA-carRA1,mU6 pro shRNA-carRA2,mU6 pro shRNA-carRA3和mU6 pro shRNA-control,通过双酶切与测序法鉴定得到的目的产物与预期一致。该质粒具有操作简单、成本低廉、靶向性好、作用时间长等优点。对三孢布拉氏霉菌的原生质体制备及再生进行了系统研究。通过摸索菌体培养时间、酶种类、酶浓度、酶解pH、酶解时间、酶解温度、酶解方式、预处理方式等对三孢布拉氏霉菌原生质体形成和再生的影响,确定了原生质体制备的最佳条件:酶解体系为pH6.0的0.6mol/L NaCl配制的2%溶菌酶+3%纤维素酶+3%蜗牛酶的复合酶系,酶解温度为28℃,酶解方式采用75rpm振荡酶解,酶解时间为14h。在此条件下得到的原生质体制备率最高,为7.48×10~6原生质体/mL。原生质体再生结果显示,在0.7mol/L NaCl配制的PDA再生培养基上,采用双层平板培养法进行原生质体再生,得到的再生率较高,再生平板可作保存菌种之用。成功建立了mRNA水平检测三孢布拉氏霉菌中RNAi效果的方法,包括RNA提取、PCR优化、Real Time PCR定量分析。首先建立了一种简单、有效的适合丝状真菌的RNA提取方法—改良异硫氰酸胍法,结果表明氯化苄能与多糖反应破坏真菌细胞壁,胞内RNA因细胞破裂而得以释放;乙酸钾和低浓度乙醇共同作用可去除绝大部分的多糖,且RNA样品完好。应用该方法所得的RNA产量较高,达到110.7μg/g,且OD260/OD280=1.93,OD260/OD230=2.27,说明RNA纯度符合要求,进一步通过RT-PCR和Northem blot证明此方法得到了高质量的RNA。在此基础上结合实验设计与人工神经网络的方法优化了扩增体系及条件,建立了Real Time PCR技术定量分析carRA基因的方法,克服了SYBR GreenⅠ荧光染料特异性差、定量不准的弊端。分析结果显示:mU6 pro shRNA-carRAl干扰质粒转染细胞后,carRA基因的表达量为未干扰组的43.2%,而mU6 proshRNA-control阴性对照质粒转染细胞后,carRA基因的表达量为未干扰组的98.7%。因此从mRNA水平证明了我们构建的干扰质粒mU6pro shRNA-carRAl对carRA基因的表达有明显的抑制作用。成功建立了蛋白水平检测三孢布拉氏霉菌中RNAi效果的一套方法,包括特异性抗体制备、蛋白提取和Western blot检测。鉴于三孢布拉氏霉菌番茄红素环化酶基因中含有太多稀有密码子,不能在大肠杆菌中将抗原高表达,无法用常规方法制备抗体。本文利用设计并合成抗原多肽的方法制备得到了三孢布拉氏霉菌番茄红素环化酶专一性抗体,效价高达1:32000。比较了三种蛋白提取方法,通过对蛋白产量和SDS-PAGE图谱进行分析,确定植物试剂盒法为最适蛋白提取方法,蛋白产量将近4mg/g,SDS-PAGE图谱中蛋白条带清晰且完整。并在此基础上建立了Western blot技术定量分析番茄红素环化酶的方法,结果显示:与未干扰组相比,mU6 pro shRNA-carRAl干扰质粒转染84h后,carRA蛋白表达量明显下降,而阴性对照组没有显著变化,从而在蛋白水平证明了我们构建的干扰质粒mU6 proshRNA-carRAl对carRA表达有明显的抑制作用。成功建立了功能学水平检测三孢布拉氏霉菌中RNAi效果的方法。通过对原生质体形态及菌丝体形态的观察发现靶向carRA基因的shRNA干扰技术可以应用在三孢布拉氏霉菌中而不产生细胞毒性;通过对发酵后菌体生物量的检测说明该技术不影响细胞的存活与增殖;通过HPLC检测发酵产品的产量发现干扰carRA基因的表达可同时减少番茄红素、β-胡萝卜素的合成,证实carRA基因有两个功能,即同时编码番茄红素环化酶和八氢番茄红素合成酶。最终从功能学水平证明了我们构建的干扰质粒mU6 pro shRNA-carRAl对carRA基因的表达有明显的抑制作用。更多还原

【Abstract】 The filamentous fungus Blakeslea trispora is the best strain used for the production of carotenoids.However,the study on gene function and metabolic regulation of Blakeslea trispora is limited.The traditional approach,homologous recombination,is both time-consuming and labor-intensive compared with RNA interference(RNAi) in filamentous fungi.In this paper,RNAi method was tried to silence lycopene cyclase gene carRA in Blakeslea trispora.The relevant techniques suitable for filamentous fungi and the effect of shRNA-mediated RNAi in Blakeslea trispora were investigated.The results obtained in this paper will make us understand the metabolic pathway more thoroughly and provide a new powerful genetic tool for strain improvement and genome-wide analysis of this biotechnically important filamentous fungus.In our investigation,four shRNA-expressing plasmids(shRNA-carRA1, shRNA-carRA2,shRNA-carRA3,shRNA-control) were constructed using the mU6 pro vector.Firstly,the oligonucleotide sequences of three different carRA shRNAs and a control shRNA were designed and synthesized.These oligonucleotide sequences encoding shRNAs for targeted gene carRA coding region were taken from GenBank,and they were evaluated for sequence specificity by a BLAST search and did not show homology to other known genes.The shRNA expression cassette driven by U6 promoter was engineered into the XbaI and BbsI sites of plasmid mU6 pro vector,resulting in the following plasmids:mU6 pro shRNA-carRA1,mU6 pro shRNA-carRA2,mU6 pro shRNA-carRA3 and mU6 pro shRNA-control.The mU6 pro shRNA-control was designed as an unspecific shRNA which has no homology to carRA or other known genes.To generate plasmids for cloning shRNAs,mU6 pro vector was linearized with XbaI and BbsI restriction endonuclease and the annealed oligonucleotides were ligated with the linearized mU6 pro vector at XbaI-BbsI sites using T4 DNA ligase.All the constructed plasmids were confirmed by restriction endonuclease digestion and DNA sequencing.The effects of key parameters on the preparation and regeneration of protoplast from theβ-carotene-producing fungus Blakeslea trispora were discussed in our study,including combination of various enzymes, mycelial age,digesting time and temperature,pH value,osmotic stabilizers,pretreatment,culture medium and culture method.Under the condition of mixed enzymes in osmotic stabilizer(0.6 mol/L NaCl) combined with 2%lysozyme,3%cellulase and 3%snailase,the highest protoplast yield as high as 7.48×10~6 protoplasts/mL was obtained when mycelial age was 60 h at pH 6.0 with digesting for 14h at 28℃.After purification of the obtained protoplasts,they were regenerated in PDA regenerative medium using bilayer plate culture method.To investigate the activity of protoplasts and usability of the mU6 pro vector,we assayed genetic transformation of these protoplasts with the mU6 pro vector.It is clear that the transformants with mU6 pro-GFP showed strong GFP expression,which can prove that the protoplasts were active and applicable in further gene manipulation experiments,the method of transformation is befitting and mU6 pro vector was suitable for Blakeslea trispora.Monitoring of downregulation of carRA gene expression at mRNA level was conducted.Isolation and purification of biologically active RNA from filamentous fungi is difficult because of the complex cell wall and the high level of polysaccharides which bind to or co-precipitate with RNA.Using benzyl chloride and guanidine thiocyanate,RNA was successfully isolated from Blakeslea trispora in which other RNA extraction methods and commercially available kits failed to deliver suitable results.The integrity of the RNA was further substantiated by RT-PCR and Northern hybridization,respectively.This procedure should be useful for isolating RNA from other filamentous fungi and therefore, will serve as an important tool for the molecular analysis of these organisms.Then Real-time quantitative RT-PCR was carried out for mRNA expression level assay.Two plasmids(mU6 pro shRNA-carRA1 and mU6 pro shRNA-control) were respectively transfected into protoplast of Blakeslea trispora and their effects on carRA mRNA levels were determined by comparison with the untreated cells by Real-time quantitative RT-PCR analysis.The results showed that carRA mRNA levels were decreased by 56.8%in the mU6 pro shRNA-carRA1 transfected group at 84h after transfection,compared with nontransfected (untreated-control) group.The results obtained in this paper indicated carRA mRNA expression was effectively suppressed by mU6 pro shRNA-carRA1.Monitoring of downregulation of carRA gene expression at protein level was conducted.For production of specific anti-carRA antibodies,a polypeptide(KC-15)antigene was designed and synthesized.KC-15 is a kind of hapten that must conjugate to carrier protein KLH with MBS methods.Highly titer and specifc antiserum is prepared.At the same time, the system of protein extraction suitable for Blakeslea trispora was established.Then to determine whether the decreased mRNA expression could be translated into decreased carRA protein levels,Western blot hybridization analysis was employed in the carRA.The results showed carRA protein expression was significantly reduced by mU6 pro shRNA-carRA1,compared with nontransfected(untreated-control) cells, whereas mU6 pro shRNA- control transfection could not affect the carRA protein expression.These decreases in carRA protein levels correlate well with the reduction in its mRNA levels.Monitoring of downregulation of carRA gene expression at performance level was conducted.To further evaluate whether silencing carRA gene in Blakeslea trispora may inhibit cell growth and proliferation,the morphology of protoplasts and mycelia nontransfected and transfected with mU6 pro shRNA-carRA1 and mU6 pro shRNA-control were detected by microscope.It was found that shRNA-mediated carRA gene silencing had no distinct effect on cell growth and viability. Furthermore,the carotenoid production of Blakeslea trispora nontransfected and transfected with mU6 pro shRNA-carRA1 and mU6 pro shRNA-control was analyzed by HPLC.Our results revealed that the production of lycopene andβ-carotene of Blakeslea trispora in fermentation was reduced remarkably,after treatment with mU6 pro shRNA-carRA1,compared with nontransfected(untreated-control) group, whereas mU6 pro shRNA-control transfection could not affect their production.The results were well consistent with recent report that carRA gene encodes lycopene cyclase and phytoene synthase.Because phytoene is the first carotenoid in the biosynthesis pathway of Blakeslea trispora, inhibition of phytoene synthase results decrease of production of both lycopene andβ-carotene.Our results demonstrated that carRA RNAi could effectively down-regulate carRA expression with great specificity. The shRNA-expressing plasmids could successfully suppress the expression of carRA at different levels.更多还原