RFLP Analysis On Microbial Diversity Of Dry Land Soils Under Different Fertilizer Level

The growth of microorganism in soil depends greatly on the fertilizing level and environmental situation of soil, the relationship between microorganism and fertilizing level is co-promotive and they both develop in cooperation. Of the entire microorganism in soil, bacteria dominate in both species and quantity. Study on bio-diversity of bacteria in soil will deepen the understanding and cognition of the correlation between bacteria and the soil environment.Earth-cumuli-Orthic Anthrosols in the north west of China was taken as object in this study, after natural drying by wind and grinding and screening, the soil sample was used to cultivate little cabbage to consume its nutrition. The soil was collected after 40d, dried by wind again, then grinded and screened, the property of the soil was also measured in the end. Fertilizing treatment was set up on the prepared soil: F1 represented soil sample of which fertilizing level was 1/2 times as many as normal quantity; F2 represented soil sample of which fertilizing level was the same as normal quantity; F4 represented soil sample of which fertilizing level was 5 times as many as normal quantity; the soil without fertilizing was take as CK. The nutrition N, P and K were fertilized seperately as urea, KH2PO4 and KCl. Soils of different fertilizer were incubated without light for 15d, under condition of 80% moisture of max water holding by soil and constant temperature of 20℃. Total DNA of microorganism in soil were extracted directly, and to eliminate impurity such as humic acid, all the DNA samples were purified by agar gel electrophoresis. To establish the clone library of 16S DNA of bacteria in soil, process concluded amplifying the partial sequence of 16S rDNA by PCR using universal primer pair 63F/1387R for bacteria, connecting the partial sequence of 16S rDNA into pMD19-T vector, transforming into E. coli JM109, screening positive clone by white bacterial colony. By bacteria colony PCR, about 200 positive clone had been got randomly, then amplified the inserted partial sequence of 16S rDNA using universal primer M13 of pMD19-T vector, purified the PCR production and digested them separately by restrict endonuclease Hha I and Rsa I.The data got by PCR-RFLP was analyzed byα-measurement to figure out their bio-diversity character; the clustering analysis was determined by software NTsys 2.10 with Bray-Curtis similarity as distance index which based on the distribution of bacteria. Results show that:(1) Restriction endonuclease types of these samples were got as 146, 133, 187 and 170 after counting. The coverage (C value) of the clone libraries respectively were 0.2527, 0.3480, 0.1845 and 0.145. The data are proper to describe the character of sampling from uncountable bacterial family in soil.(2) All the diversity data was analyzed byα-measurement indices, which show that: according to different samples ordering as CK, F1, F2 and F3, we got 10.705, 9.3119, 13.029, 12.465 for Menhinck indices, 0.9483, 0.3724, 1.0003 and 0.9905 for Pielou evenness indices, combined with other important indices such as Probability of Inter specific Encounter(PIE) and indices of species abundance, the order based on value is identical as proper fertilization sample,excessive fertilization sample, CK, low fertilization sample, which shows that the species of the bacteria in proper fertilization soil sample has the biggest species abundance, the least species predominance, the most equally distributing, and microbial community with the most sound functional system.(3) Sequencing analysis show that most of the bacteria were uncultured and the distribution of bacterial evolution was more even in CK and sample with normal fertilizing level than that in samples with fertilizing level of 1/2 of normal and excessive dosage. The bacteria community here distributed in proteobacterium, Firmicute, Gemmatimonadetes, and Acidobacteri, which, on some level, could represented the construction of bacteria community in soil. By short-term fertilizing of different level, the distribution of dominant bacteria in different soil had changed, which argued that the bacterial diversity in soil was sensitive and obvious in responding to the change of soil environment.