| Aflatoxin is a class of mycotoxins which produce by Aspergillus flavus andAspergillus parasiticus after infection of susceptible host, commonly found in soil,crops and a variety of foods, and thus endanger the lives and health of animals andhumans.it is considered one of the most serious food security problems. Peanuts isthe most serious crop affectioned by Aspergillus flavus that has become a majorlimiting factor in the global peanut industry. In recent years, scholars from variouscountries committed to addressing the problem of aflatoxin contamination inpeanuts, has raised a lot of preventive measures. Due to excessive weatherconditions, differences in agronomic and processing methods, as well as thelimitations of the various regions of prevention techniques are not an effectivesolution peanuts contamination problems by aflatoxin. Currently, scholarsgenerally believe that the most feasible and cost-effective way to solve peanutaflatoxin contamination is through genetic engineering to cultivate resistantvarieties. By studying the mechanism of peanut resistance to aspergillus flavus,digging resistance-related genes and markers, using genetic engineeringtechniques to cultivate good agronomic characters of new peanut varieties resistant.The difference between pre-lab proteomic and genomic studies found thatpre-harvest aflatoxin infection under drought stress and anti-there are somedifferences in gene expression between the sense of aflatoxin peanut resources,including stress iso-ARah3gene drought and aflatoxin induced expression of themost significant differences, suggesting that it has an important functional role inthe anti-peanut aflatoxin reaction. Studies on iso-ARah3gene mainly in proteomicand genomic level, aflatoxin its direct study of molecular function andanti-reaction mechanism of action has not been reported.Based on the previous research, this thesis:1) Through gene chip technology,we compared gene expression differences of resistance varieties (Y20)-Sensitivevarieties (H23) under drought and aspergillus flavus processing conditions beforeharvest,screened1-2significantly in response to aflatoxin-related genes, detectedits express amount and analysised its temporal expression pattern by fluorescencequantitative PCR;2) cloned iso-ARah3full-length genes by PCR, bioinformatic analysis, build prokaryotic expression vector, fusion protein expression andpurification in vitro, obtained polyclonal antibodies through immunization rabbit,and detected iso-ARah3expression of tissue proteins by WesternBlot;3) theiso-ARah3gene into a plant expression vector by cloning ultra usingAgrobacterium-mediated positive vector containing iso-ARah3transformed intopeanut gene in vivo overexpression of analysis;4) Using genomic DNA frompeanut as template, cloned iso-ARah3gene promoter upstream sequence bygenomic walking and bioinformatic analysis.The results showed that:1.comparative anti-sense of aflatoxin varieties under normal conditions (CK),drought stress, drought and aflatoxin treatment conditions differentially expressedgenes by microarray hybridization, found that resistant varieties iso-ARah3significantly upregulated under arid and Aflatoxin stress,the results prove thatpeanut iso-ARah3gene releted to the invasion of aflatoxin of resistant varieties insome way. Analyzing the Tissue expression differences under different stress andtime of iso-ARah3gene Tissue, the expression levels of iso-ARah3is not alike indifferent developmental stages of seeds, the maximum amount of its expression isthat just at the begin of seed development. the expression of the gene is not thesame in different tissues, there is no expression in flower, the similarly traceexpression level in leaves and shoots, The expression of the stem is relativelyhigh,it has the highest expression in the seed. its expression pattern is not thesame under different disease stress,the expression under Rust infection is200times than the normal leaf. The expression under mosaic infection is not onlyincreased, but declined.2. we cloned the gene with the cDNA as a template and make bioinformaticsanalysised after sequenced. The sequencing results showed that the gene has a1531bp open reading frame, encoding510amino acids. The predicted secondarystructure of the protein contains eight α-helices and22β-fold. The sequencecontains two relatively conserved region named Cpuin-s which belong to cupinprotein superfamily. Through homologous sequence alignment, there are twodifferent nucleotide sequences to the published sequences in NCBI. constructingpET28a-isoARah3prokaryotic expression vector, Expressioning in E. coli BL21ang Purificationing by Ni affinity chromatograpHy, we obtainedprokaryotic expression of fusion proteins which the size is approximately54kD. Functionesstudy in vitro showed that the fusion protein can inhibited the growth ofAspergillus flavus spores, but the effect is not very obvious. Antibody wasdetectioned successfully by WesternBlot and has the purity of84.5%.3. The plant expression vector was constructed.we inserted promoter35S andiso-ARah3gene to the pCAMBIA1301by Double digestion.We infested plantseeds by EHA105to make the gene express in penut plant. After obtaining peanutplant, we extracted plant DNA, Hygromycin primers PCR results show that wetotally obtain seven transgenic peanut plants. It provides a foundation for furtherunderstaning of the function and expression mechanism of iso-ARah3genes inpenut peanuts.4. To clone the promoter, a DNA library was constructed and successfullycloned upstream sequence of iso-ARh3gene.we obtained776bp length promotersequence by Nested PCR. The results through online software showed that: thereare a lot of TATA-boxs and CAAT-box which is the promoter features. Thepromoter has multiple components and abscisic acid response element Stressresponse. According to the study, there will be a further understanding of the geneexpression and regulatory mechanism. |
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