Munich Personal RePEc Archive China-Bulgaria Rural Revitalization Development Cooperation Forum Bachev, Hrabrin and Ivanov, Bojidar and Terziev, Dimitar and Toteva, Dessislava and Che, Shengquan and Koleva, Lyubka Institute of Agricultural Economics, Sofia, University of National and World Economy, Shanghai Jiao Tong University, Agricultural University-Plovdiv 23 April 2018 Online at https://mpra.ub.uni-muenchen.de/86908/ MPRA Paper No. 86908, posted 22 May 2018 06:13 UTC PROCEEDINGS China-Bulgaria Rural Revitalization Development Cooperation Forum Honorary Chair Professor Ivan Dimov, Vice Minister of Education and Science of Bulgaria Organizers Shanghai Jiao Tong University, Institute of Agricultural Economics-Sofia, Agricultural University-Plovdiv, University of National and World Economy- Sofia SOFIA 23 April 2018 Supported by China Ministry of Science and Bulgarian Ministry of Education and Technology Science Embassy of China in Bulgaria Bulgarian Science Fund Association for the Promotion of Bulgarian Ministry of Agriculture, Food Agricultural Cooperation between and Forestry Bulgaria and China and CEEC Agricultural Academy Organization Committee Shengquan Che, Hrabrin Bachev, Bozhidar Ivanov, Dimitar Terziev, Lyubka Koleva The Forum is organized and the current book is published by the exclusive financial support of the Bulgarian Science Fund under the Bilateral project “Comparative Study of Environmental Conditions, Cultural Resources, Conservation and Sustainable Development of Tourism in Rural Areas of China and Bulgaria” on the Agreement ДНТС/Китай 01/7 and “The Management of Agricultural Environment Sustainability and Food Supply Chain Safety” on the Agreement - ДНТС/Китай 01/14 from 2016. Institute of Agricultural Economics - Sofia ISBN: 2 CONTENT Page 1. Bioremediation of secondary salinization soil by a novel isolated strain Bacillus megaterium NCT-2 and its nitrate assimilation related enzymes 4 DanZhang,ShaohuaChu, WeiweiShi, PeiZhou 2. Tourism factors and rural development in Bulgaria Bozhidar Ivanov, Hrabrin Bachev, Dilyana Mitova, Daniela Dimitrova and 16 Vasil Stoychev 3. Grape Breeding in China Jiang Lu1, Yu Gao1 29 4. Development of the Viticulture and Wine Industry in Bulgaria - Problems and Perspectives LyubkaKoleva, AdelinaHarizanova, DesislavaToteva, SvetlaYancheva, 49 HristinaYancheva 5. Effects of inhaling ylang-ylang essential oil on anxiety, social interaction and sleep behaviors in mice 63 Nan Zhang, Lei Yaoa, Linyin Feng 6. Development of the production of aromatic oil crops in Bulgaria 71 Mariana Petkova1, Nurettin Tahsin, Svetla Yancheva, Hristina Yancheva 7. Evaluation of rural landscape recreation quality in metropolis: Case study in Shanghai 87 Shengquan CHE, Zhijie YANG, Anze LIANG 8. The Sustainability of farming enterprises in Bulgaria Hrabrin Bachev 109 9. Research on the Landscape of Shanghai Rural Settlements YANG Zhi-jie, CHE Sheng-quαn, DU Yu-yu 128 10. Economic Viability: Sustainability and Safety through Human Values and Economic Modes Dimitar Terziev, Donka Radeva 141 11. The straw organic fertilizer workshop in the container: Reearch and application of integrated technology for distributed straw cleaning and fertilizer fermentation 149 Dan Zhang, Haiwei Feng, Jie Feng, Minglei Yu, Pei Zhou 12. Study on the Spatial Structure of Shanghai Urban Agriculture Tourism 157 WANG Ling, CHE Shengquan, LIANG Anze, LIU Xiao 13. History, Challenge and Opportunity of Grape Wine in China Yu Gao1#, Jiang lu1 169 3 Bioremediation secondary salinization soil by a novel isolated strain Bacillus megaterium NCT-2 and its nitrate assimilation related enzymes DAN ZHANG1,2, SHAOHUA CHU1,2, WEIWEI SHI1,2, PEI ZHOU1,2* 1School of Agriculture and Biology, Shanghai Jiaotong University, Shanghai, 200240, PR China 2Key Laboratory of Urban Agriculture, Ministry of Agriculture, Shanghai Jiaotong University, Shanghai, 200240, PR China Abstract Large accumulation of nitrate in soil has resulted in “salt stress” and soil secondary salinization. Bacillus megaterium NCT-2 which was isolated from secondary salinization soil showed high capability of nitrate reduction. The resting cells could remove nitrate over 200mg/L within 48h. In order to further investigate the nitrate assimilation function of the NCT-2, genome sequencing was performed and submitted to the NCBI GenBank (AHTF00000000). The genes encoding assimilatory nitrate and nitrite reductase from NCT-2 were indentified, cloned and over-expressed in Escherichia coli. The putative 3D protein structures of these genes were modeled by SWISS MODEL, and shown to be highly similar to the nitrate assimilation related genes in the PDB database. The molecular mass of nitrate reductase was 87.3 kDa and 80.5 kDa for electron transfer and catalytic subunit, respectively. The large and small subunit of nitrite reductase was 88 kDa and 11.7 kDa, respectively. The purified recombinant enzymes showed broad activity range of temperature and pH. The effects of metal ions and electron donors on enzyme activities were analyzed. Moreover, the kinetic parameters of enzymes were measured. These results revealed adaption of NCT-2 to secondary salinization condition and shed light on the role played by the nitrate assimilatory pathway in NCT-2. Keywords: Bacillus megaterium NCT-2; secondary salinization soil; nitrate reductase; nitrite reductase 1. Introduction Since nitrogen fertilizers increase agricultural productivity [1], nitrate has been used more than is actually required for crop and vegetable cultivations [2, 3]. Large accumulation of nitrate in soil not only contributes to air pollution and climate change [4], but also affects the plant growth negatively, and this is known as “salt stress” [5, 6]. Furthermore, high concentration of soil nitrate has resulted in soil secondary salinization [7], which is considered to be one of the major factors that limits sustainable development of agricultural production in the protected farmland of China [8]. Therefore, excess nitrate should be removed from agricultural soils for the cultivation and production of safe products [2]. Bioremediations using microbes have been successful for the removal of various pollutants from diverse environments [2, 9, 10], so that similar strategies may be applicable to the nitrate removal from secondary salinization soil. Nitrate is a significant nitrogen source for microorganisms [11]. It can be converted to the biologically useful form during assimilation that provide for nitrogen incorporation into the carbon backbone of organic compounds [12]. The nitrate assimilatory pathway is mediated by two enzymes, nitrate reductase and nitrite reductase, which catalyse the stepwise reduction of nitrate to nitrite and nitrite to ammonia, respectively [13]. Nitrate reductase is the first enzyme involved in the chain of nitrate reduction. Three classes of nitrate reductases can be identified in prokaryotes according to their cellular location and function: membrane-bound respiratory (Nar), periplasmic dissimilatory (Nap), and cytoplasmic assimilatory (Nas) nitrate reductases [12, 14]. In comparison with Nar and Nap in both heterotrophic and phototrophic bacteria, Nas have been scarcely studied, especially in heterotrophic bacteria [15]. Nas in the anabolic pathway of nitrate assimilation catalyses the reduction of nitrate to nitrite and has been considered the rate-limiting step for the conversion of nitrate to ammonium [16]. Two classes of Nas are have been found in bacteria: ferredoxin (flavodoxin)-dependent Nas and NADH-dependent Nas [12]. The ferredoxin- dependent Nas consists of one catalytic subunit (70-85 kDa) [17]. The NADH-dependent Nas is a heterodimer containing a catalytic subunit (90-105 kDa) and a subunit that is responsible for the electron transport (45-50 kDa) [18]. The researches on nitrite reductase are focused on dissimilatory pathway, especially the denitrification process. Assimilatory nitrite reductase contains sirohaem and iron-sulfur cluster [11]. There are also two types of this enzyme: the ferredoxin-dependent enzyme typical of photosynthetic organisms and the NAD(P)H-dependent enzyme of most heterotrophic organisms [14, 19]. In comparison with assimilatory nitrate reduction in fungi, algae and plants documented for a long time, data on nitrate assimilation in bacteria are relatively scarce [20]. The genes coding for assimilatory nitrate reduction systems which are normally clustered have been sequenced in some bacteria, however, biochemical studies on bacterial assimilatory nitrate reductases have not kept pace with genetic characterization, and this situation needs to be redressed in future studies [14]. Most nitrate and nitrite reductases were generally isolated from organisms inhabiting “normal environments, only a few papers were available on this two enzymes from microorganisms living in stressful environments [16, 21-25]. However, this information is extremely important both for revealing general mechanisms of adaption to stress and for applied 5 investigations with due regard to their roles in nitrogen turnover in nature [12]. To our knowledge, there are few studies related to their properties of bacteria isolated from secondary salinization soil. A novel bacterium B. megaterium NCT-2 which was isolated and identified from secondary salinization soil from the greenhouses in China, can utilize nitrate as its only nitrogen source for growth [26]. Thus, B. megaterium NCT-2 would be applied as biological components to eliminate excessive amounts of nitrate in soil. In the present