生防芽孢杆菌ZA1的GFP基因标记及其定殖动态研究

发布时间：2018-06-09 13:59

本文选题：莫海威芽孢杆菌 + GFP基因　；参考：《甘肃农业大学》2015年硕士论文

【摘要】：本试验运用绿色荧光蛋白表达载体标记拮抗菌株ZA1,测定了标记菌株的功能稳定性,研究标记菌株ZA1-gfp在马铃薯根际及根内的定殖动态,并对ZA1-gfp定殖土壤后对土壤微生物的影响进行安全性评估。主要得到以下结果:1 ZA1的转化及ZA1-gfp的稳定性对外源质粒pGFP78进行甲基化修饰后转化ZA1,得到阳性转化子。转化后菌株ZA1-gfp与野生型菌株ZA1形态一致,在荧光显微镜下呈现明亮的绿色。标记菌株ZA1-gfp遗传稳定性达100%;标记前后的生长速率差异不显著(P0.05);ZA1对马铃薯枯萎病菌、马铃薯炭疽病菌和马铃薯坏疽病菌的抑菌率分别为64.50%、68.35%和66.55%,ZA1-gfp对三种病原真菌的抑菌率分别为67.46%、76.56%和66.75%,表明外源质粒的导入,不影响菌株的抑菌能力;另外,ZA1体内的外源绿色荧光蛋白基因不影响菌株的运动性。2 ZA1-gfp的定殖动态ZA1-gfp在土壤中定殖浓度波动范围为3.68×104~7.31×104 cfu/g。在自然土中,马铃薯根际ZA1-gfp的定殖浓度波动范围为2.97×103~1.46×105 cfu/g,根内定殖浓度为4.86×101~6.3×102 cfu/g;在灭菌土中,马铃薯根际ZA1-gfp的定殖浓度介于2.44×102~2.38×103 cfu/g,根内定殖浓度介于2.42×101~1.16×102 cfu/g。方差分析结果表明,在灌菌后的60 d检测时间范围内,ZA1-gfp在土壤及马铃薯根际、根内的定殖浓度差异不显著(P0.05);同时,马铃薯的根部横切面、根毛和根表皮等部位均观察到发绿色荧光的ZA1-gfp菌体或聚集菌落,其对马铃薯植株的促生作用显著(P0.05)。3 ZA1-gfp对土壤微生物的影响从3大类群土壤微生物总数看,自然土中施入ZA1-gfp,IR值在灌菌后的第15d、第25 d和第35 d分别为1349.1、2163.74和1293.47,表明微生物总数量显著增加(P0.05)。灭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分别为-9.26、53.17和1249.45,微生物总数量相对于灌菌前的数量表现出增长的趋势。从灌菌后土壤中的细菌、真菌和放线菌消长动态看,自然土中的细菌、真菌和放线菌的数量分别在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范围内波动;在灭菌土中,三种微生物的菌量分别介于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之间,动态数据之间均未出现连续的显著性差异(P0.05),可见,ZA1-gfp的施入并未对土壤中的土著微生物群落产生不利影响,即ZA1-gfp不影响土壤中土著微生物的群落稳定性。
[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.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S476.1

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