Effects Of High Lysine Transgenic Maize On Soil Enzyme Activities And Bacterial Community Structure

As the largest crop in China, maize is not only an important source of food, but also an important feed and industrial raw materials. But, normal maize lacks of essential amino acids such as lysine, which is necessary for human and animals’health. Therefore, breeding high lysine maize has great significance not only in easeing the food shortage and malnutrition problem in China, but also can promot the development of the feed and stock industry. Compared to the traditional maize, the nucleic acids of transgenic maize containing different exogenous gene products of transgenic maize, maybe affect the physiological and biochemical characteristics of transgenic maize. And direct impact on its soil surroundings by biological gene transfer and diffusion, the expression products from root exudates, shedding and roots and leaves residues can enter into the soil environment, and further affect the soil environment. Therefore, evaluation of the environmental impacts of transgenic maize is precondition of application and large-area promotion, effects of transgenic maize on soil enzyme activity and microbial community structure are important part of environmental assessment.As compared with the non-genetically modified receptor maize, in four growth stages (seedling stage, silking stage, maturing stage and after maturing stage), effects of high lysine transgenic maize on soil enzyme activity and bacterial community structure were characterized by the method of traditional siol activity determination and plate count method combining with DGGE-cloning and QPCR.1). Urease, invertase and hydrogen peroxide enzyme activity in maize rhizosphereb soil were determined by the method of traditional soil activity determination.2). Fungi, bacteria and actinomycetes in maize rhizosphere soil were counted with the method of traditional plate count; the total soil DNA was extracted by the direct lysis method, bacterial universal primers were used to amplify16S rDNA V3region fragments, then DGGE method was applied to study microbial communities structure in different soil samples.3). NifH gene was amplified with specific primers, then DGGE method and QPCR technology were applied to study communities structure and biomass of nitrogen fixation microbe in different soil samples.The results demonstrated,1). By the method of traditional soil activity determination, urease, invertase and hydrogen peroxide enzyme activity in maize rhizosphereb soil were determined. Rhizosphere soil invertase activity was up then down, urease and hydrogen peroxide enzyme activities were relatively stable in the entire growth period of soybean; compared to non-genetically modified maize, three soil enzyme activities of high lysine transgenic maize had no significant change in the same growth phase.2). By plate count method and with DGGE-cloning technology, microbial biomass and microbial communities’structure in different soil samples were studied. Results show,(1). There was a certain impact of genetically modified corn on rhizosphere soil microbial biomass, impacts on different microbial populations in different growth stages of corn also varied. Compared to non-genetically modified maize, biomass of bacteria in rhizosphere soil of high lysine transgenic maize had no significant change in four growth stages of soybean; but the biomass of soil fungi and actinomycetes were influenced by high lysine transgenic maize, the number of actinomycetes was relatively increased in transgenic maize soils in seedling stage, and the number of fungi had been significantly decreased in transgenic maize soils in the silking stage and after mature.(2). The results of DGGE-cloning showed, impacts of genetically modified corn on bacterial community structure in rhizosphere soil were different in different growth stages of maize. a). In the seedling stage of maize growth, the results showed that types of bacterial communities in different maize soil were abundant. Genetically modified high lysine maize did not have a significant impact on bacterial community structure in rhizosphere soil. And the main bacterial populations were attributed to Proteobacteria, TM7, Acidobacteria, Bacteroidetes, Actinobacteria and Firmicutes. Some bacterial groups were inhibited in some degree, and attributed to TM7, Proteobacteria and Bacteroidetes. b). In the silking stage of maize growth, communities structure in soybean rhizosphere soil were not influenced by high lysine transgenic maize apparently. And the main bacterial populations were attributed to Proteobacteria, Acidobacteria. Bacteroidetes, TM7and Nitrospirae; some bacterial groups were motivated, and attributed to Acidobacteria, Bacteroidetes and Nitrospirae. c). In the mature stage, high lysine transgenic maize did not have a significant impact on bacterial community structure in rhizosphere soil. And the main bacterial populations were attributed to Proteobacteria, Acidobacteria, Bacteroidetes, Chloroflexi, Actinobacteria, TM7, Firmicutes and Verrucomicrobia. d). After the mature stage, high lysine transgenic maize also did not have a significant impact on bacterial community structure in rhizosphere soil. And the main bacterial populations were attributed to Proteobacteria, Acidobacteria, Bacteroidetes, TM7and Firmicutes.3). Used the total soil DNA as template, and by the analysis of QPCR and DGGE-cloning, the results showed,(1). In seedling, silking and maturing stage, there was not a certain impact of genetically modified corn on the nitrogenase microbial biomass in rhizosphere soil, but after maturing stage, the nitrogenase microbial biomass of high lysine transgenic maize was significantly increased in rhizosphere soil.(2). DGGE analysis of nifH gene revealed, in four growth stages of maize, the cultivation of genetically modified maize had some influence on nitrogen-fixing microbial community structure in rhizosphere soil. But, its influence on nitrogen-fixing microbial community diversity was not significant. The specific bands were cut, cloned and sequenced, the results displayed, the main nitrogen fixation microbial groups in rhizosphere soil were attributed to Bradyrhizobium, Burkholderia, Azoarcus, Pelomonas, Desulfovibrio and Unknown. Compared to non-genetically modified maize, in the seedling stage, weakened bands were O (Unknown) and P (Unknown), disappearanced bands were H (Azoarcus) and I (Burkholderia); enhanced bands were C (Bradyrhizobium) and F (Unknown), added band was K (Pelomonas). In the silkling stage, weakened bands were C (Bradyrhizobium), E (Bradyrhizobium), L (Burkholderia), M (Burkholderia) and U (Unknown), disappearanced band was F (Unknown); enhanced bands were K (Pelomonas), O (Unknown) and P (Unknown), added bands were I (Burkholderia) and J (Unknown). In the mature stage, enhanced bands were B (Azoarcus), C (Bradyrhizobium) and R (Unknown), added band was E (Bradyrhizobium), other bands no obvious changed. After the mature stage, weakened bands were G (Unknown), H (Azoarcus), and I (Burkholderia); enhanced bands were E (Bradyrhizobium) and V (Desulfovibrio). added band was A (Bradyrhizobium).