發(fā)布時(shí)間：2018-06-21 08:30

  本文選題：鴨源巴氏桿菌 + 鴨源沙門氏菌��； 參考：《四川農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：細(xì)菌原生質(zhì)體融合是一個(gè)隨機(jī)的過(guò)程,通過(guò)標(biāo)記雙親本菌株來(lái)篩選陽(yáng)性融合子。本試驗(yàn)利用了親本菌株在培養(yǎng)特性及耐藥性兩方面存在的差異,標(biāo)記親本菌株對(duì)融合菌株進(jìn)行篩選。通過(guò)藥敏紙片試驗(yàn)和最低抑菌濃度試驗(yàn)(MIC)篩選出兩親本菌株鴨源沙門氏菌(S6)和鴨源巴氏桿菌(CBa),確定親本菌株藥物標(biāo)記,藥敏試驗(yàn)結(jié)果表明鴨源沙門氏菌(S6)對(duì)壯觀霉素敏感,抑菌圈為26 mm,鴨源巴氏桿菌(CBa)對(duì)壯觀霉素耐藥,抑菌圈為0mm。通過(guò)測(cè)定最低抑菌濃度(MIC),確定32 μg/ml的壯觀霉素作為選擇培養(yǎng)基的抗生素濃度。壯觀霉素應(yīng)用濃度試驗(yàn)結(jié)果表明,在含32μg/ml壯觀霉素的麥康凱培養(yǎng)基中S6完全不能生長(zhǎng),在含32μg/ml的新霉素的血瓊脂培養(yǎng)基中CBa能良好生長(zhǎng)。篩選出的親本菌株通過(guò)紫外分光光度計(jì)測(cè)量OD600值繪制CBa和S6的生長(zhǎng)曲線,確定原生質(zhì)體制備時(shí)間：CBa為12-14 h,S6為14-16 h。在溶菌酶的作用下制備原生質(zhì)體,確定制備CBa原生質(zhì)體的最佳溶菌酶濃度為3 g/L,可得到95.25%的原生質(zhì)體制備率以及33.52%的再生率；制備S6原生質(zhì)體的最佳溶菌酶濃度為2.5g/L,可得到95.32%的原生質(zhì)體制備率以及32.19%的再生率。通過(guò)聚乙二醇(PEG)法進(jìn)行原生質(zhì)體融合,利用抗藥性結(jié)合培養(yǎng)條件對(duì)融合子進(jìn)行篩選,再對(duì)融合菌株進(jìn)行培養(yǎng)特性、菌落形態(tài)、菌體形態(tài)、血清學(xué)試驗(yàn)、穩(wěn)定性檢測(cè)等初步鑒定最終得到5株能夠在32μg/ml壯觀霉素的麥康凱平板上穩(wěn)定遺傳的融合菌株。所得融合子進(jìn)行血清學(xué)試驗(yàn)均能凝集雙親菌株陽(yáng)性血清,菌體形態(tài)為中等大小兩端頓圓的革蘭氏陰性桿菌,能夠在麥康凱培養(yǎng)基長(zhǎng)出圓形淡黃色菌落,生化試驗(yàn)結(jié)果表明氧化酶為陽(yáng)性,吲哚、枸櫞酸鈉、蔗糖、乳糖、側(cè)金盞花、硫化氫、氰化鉀均為陰性。根據(jù)CBa的外膜蛋白H (OmpH)基因和S6的外膜蛋白A (OmpA)基因的保守區(qū)域設(shè)計(jì)兩對(duì)引物。利用雙重PCR法檢測(cè)5株融合菌株,結(jié)果表明3株能夠同時(shí)檢測(cè)到雙親本的基因,其余2株則只能檢測(cè)到單一親本基因,雙重PCR產(chǎn)物測(cè)序結(jié)果表明與親本菌株同源性為100%。
[Abstract]:Bacterial protoplast fusion is a random process. In this experiment, we used the differences in culture characteristics and drug resistance of parent strains to screen the fusion strains. Two parent strains, Salmonella duck-origin S6) and Pasteurella duckelis CBaA, were screened by drug sensitive disk test and minimal inhibitory concentration test (MICM). The drug markers of parent strains were determined. The results of drug sensitivity test showed that Salmonella duck-derived Salmonella S6) was sensitive to spectinomycin. The antimicrobial circle was 26 mm, the resistance of CBato to spectinomycin was 0 mm. The antibiotic concentration of 32 渭 g/ml spectinomycin as the selective medium was determined by the determination of the minimum inhibitory concentration (MIC). The results of spectinomycin concentration test showed that S6 could not grow completely in wheat Kang Kai medium containing 32 渭 g/ml spectinomycin, but it could grow well in blood Agar medium containing 32 渭 g/ml neomycin. The growth curves of CBa and S6 were plotted by measuring OD600 value by UV spectrophotometer, and the protoplast preparation time was determined to be 12-14 hS6 and 14-16 h. Protoplasts were prepared under the action of lysozyme. The optimum concentration of lysozyme was 3 g / L to obtain 95.25% protoplast preparation rate and 33.52% regeneration rate. The optimum concentration of lysozyme for preparation of S6 protoplast was 2.5 g / L, and the protoplast preparation rate and regeneration rate were 95.32% and 32.19% respectively. The protoplast fusion was carried out by polyethylene glycol (PEG) method. The fusants were screened by the combination of drug resistance and culture conditions. The culture characteristics, colony morphology, mycelial morphology, serological tests of the fusion strains were carried out. Finally, five fusion strains with stable inheritance on the wheat Kang Kai plate with 32 渭 g/ml spectinomycin were obtained. In serological tests, the fusion could agglutinate the positive sera of the parent strains, and the bacteria were of medium size and round Gram-negative bacilli, and could grow round yellowish colonies in wheat Kang Kai medium. The results of biochemical tests showed that oxidase was positive, indole, sodium citrate, sucrose, lactose, lateral calendula, hydrogen sulfide and potassium cyanide were all negative. Two pairs of primers were designed according to the conserved region of the outer membrane protein (H) gene of CBa and the outer membrane protein A (OmpA) gene of S6. The double PCR method was used to detect 5 fusion strains. The results showed that 3 strains could detect parental genes at the same time, the other 2 strains could only detect single parent genes. The sequencing of double PCR products showed that the homology was 100% with parent strain.
【學(xué)位授予單位】：四川農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.61

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本文編號(hào)：2047901