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Study on Silencing of the Carra Gene by RNA Interfetrnce in Filamentous Fungus Blakeslea Trispora

Author: LiZuo
Tutor: YuanQiPeng
School: Beijing University of Chemical Technology
Course: Biochemical Engineering
Keywords: RNA interference shRNA The three spore Brad 's mold carRA gene
CLC: Q78
Type: PhD thesis
Year: 2009
Downloads: 303
Quote: 1
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Abstract

Filamentous fungi is an important industrial production strains, their metabolic regulation has been the focus of attention at home and abroad. Filamentous fungi the three spore Brad's mold (Blakeslea trispora) rapid growth, high biomass, bacteria mevalonate pathway in vivo metabolic flux is the only industrialized production of carotenoids good bacteria for bacteria genetic research lags behind little research about the three spore Brad's the fungal gene functions and metabolic regulation. RNA interference (RNA interference, RNAi) technology is a fast, efficient, powerful used to determine gene function and regulation of the relationship between gene expression as well as the understanding of gene research tools. With traditional gene knockout compared to either silence genes efficiency or experimental cycle, has incomparable advantages. In this study, RNAi technology used in the three spore Brad's mold, RNAi technology applicable to a complete set of the three spore Brad's mold routes and methods to explore the applicability of RNAi technology in filamentous fungi and to deepen the understanding of the function of tomato red cyclase gene (carRA), enrich the understanding of the three spore Brad's fungal metabolic pathways. These results further RNAi technology for genetically engineered fungi provide the experimental and theoretical basis, the main results are as follows: Firstly, for the CDS region of the three spore Brad's mold carRA gene sequence design RNAi interference target by the validity screening and homology comparison identified three targets, designed and synthesized three-targeted carRA gene short hairpin (short hairpin RNA, shRNA) sequences and a negative control sequence, and cloned into the small interfering RNA ( Shortinterfering RNA, siRNA) expression vector mU6 pro, were named as mU6 proshRNA-carRA1 the mU6 pro shRNA-carRA2 mU6 pro shRNA-carRA3 mU6 pro shRNA-control, by double digestion and sequencing identified purpose of the product and expected. The plasmid has simple operation, low cost, targeting, and long duration of action. Protoplast formation and regeneration of the three spore Brad's mold system. Incubation time through exploring the bacterial enzyme type, enzyme concentration, enzymatic hydrolysis pH, enzymatic hydrolysis time, reaction temperature, enzymatic pretreatment methods on three spore Brad's fungal protoplast formation and regeneration of identified optimal conditions for protoplast preparation: the composite snailase 2% lysozyme 3% cellulase enzymatic system pH6.0 of 0.6mol / L NaCl prepared 3% enzymes, reaction temperature of 28 ℃, enzymatic 75rpm oscillation enzymatic hydrolysis time 14h. The protoplast preparation Under these conditions the highest rate, 7.48 × 10 ~ 6 protoplasts / mL. The protoplast regeneration results show in 0.7mol / L NaCl PDA regeneration medium formulated using the double-layer plate culture method for the regeneration of protoplasts, obtained higher regeneration rate, reproducing the tablet can be used for save the strains used. Successfully established mRNA level detection the for DON's mold RNAi effect three Alternaria method, including RNA extraction, PCR optimization, Real time quantitative PCR analysis. First establish a simple, effective for filamentous fungi RNA extraction method - modified guanidine isothiocyanate method, the results show that the benzyl chloride and undermine the fungal cell wall polysaccharide reaction intracellular RNA can be released due to cell rupture; acetic acid potassium and the joint action of the low-concentration ethanol to remove most of the polysaccharide, and the RNA samples were intact. The application of the method from the RNA yields higher to reach 110.7μg / g, and of OD260/OD280 = 1.93 OD260/OD230 = 2.27, description of RNA purity to meet the requirements, further proved by RT-PCR and Northem blot this method to obtain high-quality RNA. Methods based on experimental design and artificial neural networks to optimize the amplification system and conditions, the establishment of the Real Time PCR technology the quantitative analysis carRA gene method to overcome the poor specificity of SYBR Green Ⅰ fluorescent dye, quantitative inaccurate drawbacks. The analysis results: mU6 pro shRNA-carRAl interference plasmid the transfected cells, carRA gene expression not interfere with 43.2%, negative control plasmid transfected cells mU6 proshRNA-control, carRA gene expression as interference 98.7% of the group. MRNA levels proved the interference plasmid mU6pro shRNA-carRAl we built the the carRA gene expression significantly inhibited. Successfully established the protein levels the three spore Brad's mold in RNAi effect a set of methods, including specific antibody preparation, protein extraction and Western blot. Given three spore DON's mold lycopene cyclase gene contains too much rare codons, not the high expression of the antigen in E. coli will, antibodies can not be prepared by conventional methods. In this paper, design and synthetic antigenic peptides prepared three spore Brad's mold lycopene cyclase specific antibody titer as high as 1:32000. Compare the extraction method, of the three proteins by protein yield and SDS-PAGE for analysis to determine the plant kit method for optimal protein extraction methods, protein yield nearly 4mg / g, and by SDS-PAGE pattern of protein bands clear and complete . And established on the basis of the Western blot method of quantitative analysis of lycopene cyclase results show: compared with the undisturbed group, mU6 pro shRNA-carRAl interference plasmid transfection 84h, carRA protein expression was significantly decreased, The negative control group did not significantly change, which proved a the interference plasmid mU6 proshRNA-carRAl. built of carRA expression at the protein level significantly inhibited. The successful establishment of a functional school level detection RNAi effect in the three spore Brad's mold. The shRNA interference gene targeting carRA can be applied in three spore Brad's mold without producing cytotoxic; protoplast morphology and mycelium morphology observed by describing the technology for detection of bacterial biomass after fermentation does not affect cell survival and proliferation; found interference carRA gene expression can also reduce the lycopene, β-carotene synthesis confirmed carRA gene has two functions, i.e. simultaneously encode the production of fermented products by HPLC lycopene ring enzyme phytoene synthase. Ultimately from the the functional school level proved significantly inhibited the interference plasmid we constructed mU6 pro shRNA-carRAl on carRA gene expression.

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