本文選題：莫海威芽孢桿菌 + GFP基因　； 參考：《甘肅農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：本試驗(yàn)運(yùn)用綠色熒光蛋白表達(dá)載體標(biāo)記拮抗菌株ZA1,測定了標(biāo)記菌株的功能穩(wěn)定性,研究標(biāo)記菌株ZA1-gfp在馬鈴薯根際及根內(nèi)的定殖動態(tài),并對ZA1-gfp定殖土壤后對土壤微生物的影響進(jìn)行安全性評估。主要得到以下結(jié)果:1 ZA1的轉(zhuǎn)化及ZA1-gfp的穩(wěn)定性對外源質(zhì)粒pGFP78進(jìn)行甲基化修飾后轉(zhuǎn)化ZA1,得到陽性轉(zhuǎn)化子。轉(zhuǎn)化后菌株ZA1-gfp與野生型菌株ZA1形態(tài)一致,在熒光顯微鏡下呈現(xiàn)明亮的綠色。標(biāo)記菌株ZA1-gfp遺傳穩(wěn)定性達(dá)100%;標(biāo)記前后的生長速率差異不顯著(P0.05);ZA1對馬鈴薯枯萎病菌、馬鈴薯炭疽病菌和馬鈴薯壞疽病菌的抑菌率分別為64.50%、68.35%和66.55%,ZA1-gfp對三種病原真菌的抑菌率分別為67.46%、76.56%和66.75%,表明外源質(zhì)粒的導(dǎo)入,不影響菌株的抑菌能力;另外,ZA1體內(nèi)的外源綠色熒光蛋白基因不影響菌株的運(yùn)動性。2 ZA1-gfp的定殖動態(tài)ZA1-gfp在土壤中定殖濃度波動范圍為3.68×104~7.31×104 cfu/g。在自然土中,馬鈴薯根際ZA1-gfp的定殖濃度波動范圍為2.97×103~1.46×105 cfu/g,根內(nèi)定殖濃度為4.86×101~6.3×102 cfu/g;在滅菌土中,馬鈴薯根際ZA1-gfp的定殖濃度介于2.44×102~2.38×103 cfu/g,根內(nèi)定殖濃度介于2.42×101~1.16×102 cfu/g。方差分析結(jié)果表明,在灌菌后的60 d檢測時間范圍內(nèi),ZA1-gfp在土壤及馬鈴薯根際、根內(nèi)的定殖濃度差異不顯著(P0.05);同時,馬鈴薯的根部橫切面、根毛和根表皮等部位均觀察到發(fā)綠色熒光的ZA1-gfp菌體或聚集菌落,其對馬鈴薯植株的促生作用顯著(P0.05)。3 ZA1-gfp對土壤微生物的影響從3大類群土壤微生物總數(shù)看,自然土中施入ZA1-gfp,IR值在灌菌后的第15d、第25 d和第35 d分別為1349.1、2163.74和1293.47,表明微生物總數(shù)量顯著增加(P0.05)。滅菌土中施入生防菌株,其IR值在灌菌后的第15 d、第25 d和第35 d分別為-9.26、53.17和1249.45,微生物總數(shù)量相對于灌菌前的數(shù)量表現(xiàn)出增長的趨勢。從灌菌后土壤中的細(xì)菌、真菌和放線菌消長動態(tài)看,自然土中的細(xì)菌、真菌和放線菌的數(shù)量分別在3.66×108~2.63×109 cfu/g,2.38×104~2.50×105 cfu/g和8.11×104~6.32×105 cfu/g范圍內(nèi)波動;在滅菌土中,三種微生物的菌量分別介于3.15×108~4.22×109 cfu/g,1.46×105~5.23×105 cfu/g和1.41×104~3.65×105 cfu/g之間,動態(tài)數(shù)據(jù)之間均未出現(xiàn)連續(xù)的顯著性差異(P0.05),可見,ZA1-gfp的施入并未對土壤中的土著微生物群落產(chǎn)生不利影響,即ZA1-gfp不影響土壤中土著微生物的群落穩(wěn)定性。
[Abstract]:Using green fluorescent protein expression vector to label antagonistic strain ZA1, the functional stability of labeled strain ZA1-gfp was determined, and the colonization dynamics of labeled strain ZA1-gfp in rhizosphere and rhizosphere of potato were studied. The effect of ZA1-gfp colonization on soil microorganism was evaluated. The main results were as follows: 1) the transformation of ZA1 and the stability of ZA1-gfp methylated the exogenous plasmid pGFP78 into ZA1, and the positive transformants were obtained. The transformed strain ZA1-gfp had the same morphology as wild strain ZA1, and showed bright green under fluorescence microscope. The genetic stability of the labeled strain ZA1-gfp was 100, and the growth rate of ZA1-gfp was not significantly different from that of P0.05 and ZA1 against Fusarium wilt. The inhibitory rates of anthracnose and gangrene were 64.50% and 66.55%, respectively. The inhibition rates of ZA1-gfp to the three pathogenic fungi were 67.4676% and 66.75%, respectively, indicating that the introduction of exogenous plasmids did not affect the bacteriostatic ability of the strains. In addition, the exogenous green fluorescent protein gene in ZA1 did not affect the colonization dynamics of strain 2.ZA1-gfp in soil. The range of colonization concentration was 3.68 脳 10 ~ 4 ~ (4) ~ 7.31 脳 10 ~ (4) cfur ~ (-1) 路g ~ (-1) 路min ~ (-1) ~ (-1) 路min ~ (-1) ~ (-1) ~ (-1). In natural soil, the colonization concentration of ZA1-gfp in potato rhizosphere was 2.97 脳 10 ~ 3 ~ 1.46 脳 10 ~ 5 cfup / g, and that of root colonization was 4.86 脳 10 ~ (1) C ~ (6.3) 脳 10 ~ (2) cfup / g. In sterilized soil, the colonization concentration of ZA1-gfp in potato rhizosphere was 2.44 脳 10 ~ (2) U _ (2.38) 脳 10 ~ 3 ~ (3) cfup / g, and that of root colonization was 2.42 脳 10 ~ (11) 1.16 脳 10 ~ (2) cfup 路g. The results of variance analysis showed that there was no significant difference in colonization concentration of ZA1-gfp in soil and potato rhizosphere within 60 days after inoculation, and there was no significant difference in colonization concentration in rhizosphere of potato. In root hair and root epidermis, green fluorescent ZA1-gfp bacteria or aggregative colonies were observed. The effects of ZA1-gfp on soil microbes were significant (P0.053.ZA1-gfp) from the total number of soil microbes in three groups. The IR values of ZA1-gfpN in natural soil were 1349.1 ~ 2163.74 and 1293.47 on the 15th day, 25th day and 35th day after inoculation, respectively, indicating that the total number of microorganism increased significantly (P0.05). The IR values of biocontrol strains applied in sterilized soil were -9.2653.17 and 1249.45 on the 15th day, the 25th day and the 35th day after inoculation, respectively. The total number of microorganism showed an increasing trend compared with the number before irrigation. From the dynamic changes of bacteria, fungi and actinomycetes in the soil after irrigation, the number of bacteria, fungi and actinomycetes in natural soil fluctuated in the range of 3.66 脳 10 ~ 8 ~ 2.63 脳 10 ~ 9 ~ (10 ~ 9) C _ (10 ~ 3) C _ (10 ~ (3) cfu/g and 8.11 脳 10 ~ (4) N ~ (6.32 脳 10 ~ 5) cfu/g, respectively. The microbial biomass of the three microbes ranged from 3.15 脳 10 ~ 8 to 4.22 脳 10 ~ 9 ~ (10 ~ 9) cfup / g ~ (1.46 脳 10 ~ 5) ~ 5.23 脳 10 ~ 5 cfu/g and 1.41 脳 10 ~ (4) C ~ (3.65) 脳 10 ~ 5 cfu/g, respectively. There was no significant continuous difference among the dynamic data (P _ (0.05), which indicated that the application of ZA1-gfp had no adverse effect on the indigenous microbial community in soil. That is, ZA1-gfp did not affect the community stability of indigenous microorganisms in soil.
【學(xué)位授予單位】：甘肅農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S476.1

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相關(guān)期刊論文 前1條

1 李軼;唐佳妮;呂緒鳳;張鎮(zhèn);張玉龍;;施用沼肥對設(shè)施土壤真菌動態(tài)變化的影響[J];中國沼氣;2013年05期

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本文編號：1999964