Analysis Of Genetic Diversity In Rhizosphere Soil Microbes Of Transgenic Bt Rice

Safety of soil environment is one important item in safety evaluation of transgenic plants. Transgenic plants can release transgenic genes and their expression products into the soil by different ways, resulting in selective pressure on soil microbes and possibly affecting them. In this dissertation two kinds of new technologies suitable to genetic diversity analysis of soil microbes were established and applied to study the effects of two transgenic Bt rice lines on the genetic diversity of rhizosphere soil microbes.Followings are the main results:1. By optimization of reaction constituents, the suitable SRAP(Sequence-related amplified polymorphism)-PCR system was established, totally 25μl reaction mixture containing 1×PCR buffer,2.0 mmol·L-1 MgCl2, DNA template 30ng, dNTP mixture 0.2 mmol·L-1, each primer 0.4μmol·L-1 and Taq DNA polymerase 1U. By test on annealing temperature, the amplification program were determined,94℃5 min; 94℃45 s,35℃1 min and 72℃90 s for 5 cycles; 94℃45 s,50℃1 min and 72℃90 s for 35 cycles; and 72℃10 min. Twenty-two SRAP primer combinations were further employed to analyze genetic diversity among rhizosphere soil microbes from 20 plants species. A total of 237 scorable fragments were identified, of which 221 were polymorphic loci (93.2%). The average percentage of polymorphic loci (PPL), polymorphic information content (PIC), allele haplotype (Ah) and expected heterozygosity (He) for each primer combination were 93.78,0.94,18.05 and 0.92, respectively. These parameters with higher value not only indicate the high resolving ability of SRAP to genetic bases of rhizosphere soil microbes, but also reflect the rich genetic diversity in rhizosphere soil microbes from 20 plants in the experiment. GD (genetic distance) differences of rice rhizosphere microbes both between 2 different planting locations and among 4 different developmental stages were significant at 0.01 level, but not significant between 2 different varieties. Shannon index indicates that the genetic diversity in rhizosphere soil microbes of rice was the lowest and that of lettuce was the highest. At GD=0.454 the rhizosphere soil microbes from 20 plants species could be classified into three groups clustered by UPGMA, the first from rice, the second from celery planted in plastic green house and the third from other 18 plants species cultivated in dry lands. The results suggest that SRAP is an efficient method to analyze the genetic diversity in soil microbes.2. Twenty-seven SRAP primer combinations were used to analyze microbes from rhizosphere of transgenic Bt rice TT51, Minghui 63(CK for TT51), transgenic Bt rice Huachi B6 and Jiazao 935 (CK for Huachi B6), respectively. The genetic distance difference of microbes between transgenic rice TT51 and Huachi B6 was not significant at same planted location, but significant at 0.01 level at different locations. The differences between them among 4 different developmental stages also varied with locations. No differences were observed in the genetic diversity of rhizosphere microbes between TT51 and Minghui 63, Huachi B6 and Jiazao 935 at different developmental stages and different locations, respectively. The results suggest that the plantation of transgenic rice TT51 and Huachi B6 do not affect the genetic diversity of soil microbes.3. Enzymatic activity for mismatch cleavage of CEL I crude extract from celery was characterized by using DNA hexteroduplexes containing a single mismatch as zymolyte and an enzymatic method based on the CEL I crude extract for detection of point mutations was developed after optimization of reaction conditions for its mismatch cleavage. The results showed that the mismatch in DNA hexteroduplexes could be effectively recognized and cleaved by the self-made CEL I crude extract. Effect of the amounts of CEL I crude extract used within a certain range was not significant when the quantity of DNA hexteroduplexes was definite, and usually the amount of CEL I crude extract containing total protein about 900ng could be used for effective cleavage of the mismatch in DNA hexteroduplexes formed by 12μL PCR products. Common PCR buffer was similar to CEL I buffer reported for the mismatch cleavage and the former would be feasible in practice. Effect of buffer pH on the mismatch cleavage was significant and the near neutral pH was suitable for the mismatch cleavage by CEL I crude extract. Zn2+ could promote the mismatch cleavage with a suitable concentration about 0.25mmol/L, but not necessary. The feasible reaction time was 20min at 42℃for the mismatch cleavage by CEL I crude extract. Comparison test showed that the result for detecting point mutations by our method was almost same with that by the kit from Transgenomic Company. This technique was combined into Eco-TILLING and was applied to detect the variation of the single nucleic acid in 16S rDNA genes of rice rhizosphere microbes and the results suggest that the Eco-TILLING established in the experiments is an efficient method to analyze the genetic diversity of specific genes of soil microbes.4. Differences in 16S rDNA genes of rhizosphere soil microbes between transgenic rice TT51 and its control Minghui63, transgenic rice Huachi B6 and its control Jiazao935 are analysed using fluorescent labeled and none labeled primers, respectively. After treatments with CEL I nuclease, heteroduplex DNA formed from application of rhizosphere soil microbes of TT51, mixture of TT51 and its control showed new cleavage fragments, indicating that there are 4 same single nuclear acid variations located at 134nt,423nt,911nt and 1163nt, respectively. The variation at 4 different developmental stages between the transgenic rice and its control is also same. The results suggest that there is no difference between them and no effects on the 16S rDNA gene diversity of rhizosphere soil microbes.