The major royal jelly proteins (MRJPs) as the alternative of fetal bovine serum (FBS) were used to culture Chinese hamster ovary cell line (CHO-K1). The effects of mixture media with different MRJPs/FBS (M/F) proportions on the proliferation of CHO-K1 cell were compared. The result of methyl thiazolyl tetrazolium (MTT) assay showed that the cells in the medium with M/F 50/50 possessed the highest relative proliferation activity. Compared to the CK (M/F 0/100), the relative cell proliferation activity in the medium with M/F 50/50 was increased by 25.1% (48 h) and 13.6% (72 h), respectively. Cell imager showed that the cell density and diameter in the medium with M/F 50/50 were significantly higher than those in the CK. The detection of cell cycle with flow cytometry showed that the cell proliferation index (PI) in the medium with M/F 50/50 was increased by 19.4% compared to that in the CK, indicating that MRJPs might promote the synthesis of DNA, proteins and enzymes. The Raman spectrum of cells in the medium with M/F 50/50 was similar with those in the CK and bovine serum albumin (BSA) medium, demonstrating that the complex serum maintains the cell type stable. In conclusion, the results suggest that MRJPs could partially replace FBS in the cultivation of CHO-K1 cell line and possess industry prospect.

A full-length sequence of phytase gene (phyA) from Aspergillus niger ZJUY was cloned and modified based on polymerase chain reaction (PCR)-directed mutagenesis, aiming at addressing phytase denaturalization and deactivation during the process of pelleting. The mutation types included codon optimization for key sites, the introduction of hydrogen bond (T295S, Q296R and V43N) and the deletion mutation of disulfide bond (Cys196-Cys446). The anchored-Flo1p (FS) and target-phyA were successfully inserted into the yeast expression vector pPICZαC by seamless cloning. Eight kinds of expression vector pPICZαC- FS/phyA were integrated into the Komagataella phaffii GS115 genome by homologous recombination using lithium chloride transformation method, respectively. Phytases were successfully displayed on the surface of K. phaffii GS115, verified by immunofluorescence staining and flow cytometry. The resulted recombinants A31, A61 and A84 all showed a high phytase activity of 7 000 U/g. In addition, phytase-displaying exhibited better thermostability and pH-stability than those of secretory phytase. Among them, A61 remained 18% phytase activity after water bath at 90 ℃ for 1 h, and still maintained more than 80% of phytase activity at a pH range of 1.6 to 4.0. This characteristic of strain A61 can overcome deactivation during the process of granulating at 60 to 90 ℃ and better meet the demand in animal feed and food processing.

In order to clarify the influence of different hill spacings and seedling numbers on dry matter accumulation and yield formation of machine-transplanting hybrid rice, two different rice varieties, F you 498 and Yixiangyou 2115 were selected as plant materials, and a randomized block experiment with three replicates was conducted to analyze the effects of field collocation pattern on tillers, dry matter production, yield and its component factors of rice. Based on the production practice, the reasonable seedling rate of hybrid rice is 0.42 million per hectare in Sichuan Province, and three field collocation patterns were set as: 1) high hills with low seedlings, 1.5 seedlings transplanted at 30 cm × 12 cm spacing; 2) middle hills with middle seedlings, 2.1 seedlings transplanted at 30 cm × 17 cm spacing; 3) low hills with high seedlings, 2.9 seedlings transplanted at 30 cm × 23 cm spacing. The results showed that the tiller numbers of single hill and population increased first and then decreased with the rice development process. The tiller number of single hill in every development period improved as the hill spacing increased, and the earring- tiller percentage of Yixiangyou 2115 was higher in low hills with high seedlings. The leaf area of single hill and dry matter mass of rice increased as the spacing enlarged, and Yixiangyou 2115 had larger leaf area and dry matter mass of single hill in low hills with high seedlings. Under the three different field collocation patterns, the differences of tiller numbers, dry matter accumulation and transportation, and yield were not significant, which indicated that the decrease of hill numbers per unit area did not influence the productive ear numbers and yield with the same basic seedlings. Reduce hills but not reduce seedlings, and low hills with high seedlings can also achieve high and stable yield.

Two new recorded species of genus Longidorus (L. pius and L. diadecturus) were recovered and collected from rhizosphere of deciduous trees in Hangzhou of Zhejiang Province and Wuhan of Hubei Province, China, respectively. Morphological identity was confirmed using the revised polytomous key of Loof & Chen, 1999. The codes for identifying the species of L. pius and L. diadecturus are A56-B4-C3-D1-E1-F3-G1-H1-I1 and A4-B23-C5-D3-F2-G2-H1-I1, respectively. L. pius was characterized by medium body size (L=4.7-6.3 mm), anterior region abruptly tapering with rounded lip continuous from body profile, odontostyle long (130-143 μm) and short rounded tail. Meanwhile, L. diadecturus could be distinguished by short body size (3.2-4.6 mm), short odontostyle (108-120 μm), posterior position of guide ring (33-38 μm), wide lip region and a relatively anterior vulva. Phylogenetically, the rDNA internal transcribed spacer 1 (ITS1) region of L. pius from China showed 99% supported clade with L. pius topotype (AM743178) from Macedonia, and the similar relationship
was obtained in L. diadecturus from China with 99% well supported clade to L. diadecturus type population (AF511416) from Canada. In addition, the 28S region of L. diadecturus from China showed 99% supported clade with L. diadecturus type specimen (AY601584), whereas L. pius noticeably clustered to L. raskii (AJ549983) and Longidorus sp. FDL-2011 (FR775757). The two species, L. pius and L. diadecturus were recorded for the first time in China, and the morphological description and phylogenetic relationships of the given species were provided in this study.

Sunflower Verticillium Wilt is a soil-borne disease caused by Verticillium dahliae, which can infect a variety of crops, resulting in a serious decline in both yield and quality. The conidia and microsclerotia in sunflower debris are the principal sources of infection. A nested polymerase chain reaction (PCR) was used to detect the contamination of V. dahliae in sunflower seed coats. Seven sunflower varieties were selected to isolate DNA from their seed coats, and then performed a nested PCR by using specific primers of V. dahliae and the universal primers of fungi (ITS1/ITS4). The PCR products were sequenced then BLAST-searched in GenBank. In order to confirm the PCR results, green fluorescent protein (GFP) labeled V. dahliae was used to inoculate sunflower roots. The seed coats were then removed from inoculated sunflower seeds and detected GFP signal under a fluorescence microscope. The results showed that all the amplified PCR bands belonged to V. dahliae isolated from the seed coats of the tested sunflower cultivars. Meanwhile, variable seed contamination ratios were found among different sunflower varieties. The lowest and highest contamination ratios were observed in 3638C (10.0%) and Chi029X115R (25.0%) varieties, respectively. Moreover, the GFP signals obtained on the seed coats of inoculated sunflowers further supported the PCR results. Based on the above results, we confirm that the sunflower seed coat is an important tissue for V. dahliae colonization, and it is the main carrier for the long distance transmission of the sunflower Verticillium Wilt. Additionally, we conclude that the nested PCR method is fast and highly reliable to detect V. dahliae contamination in sunflower seed coats.

By high-throughput sequencing on Illumina Miseq platform and bioinformatics, changes of soil bacterial communities in a small- to medium-diameter Phyllostachys edulis plantation subjected to different soil management practices (mulching, alternate operation, and long-term mulching) were analyzed to investigate the mechanism of soil bacterial community variation and its effect on soil degradation of mulched P. edulis plantation. The results showed that soil subjected to four different soil management practices (CK, mulching, alternate operation, and long- term mulching) contained a wide diversity of bacterial communities covering 26 phyla, 69 classes, 118 orders, and 197 genera. Among them, Acidobacteria were the dominant in all four plots (30.85%- 44.22%), followed by Proteobacteria (8.23%-25.41%), Chlorobacteria (2.48%-7.91%), Verrucomicrobia (2.39%-6.82%), AD3 (1.05%-15.18%), Actinobacteria (3.99%-8.39%) and TM7 (0.61%-6.68%). Soil total nitrogen and total organic carbon of all the three treatment sample plots were higher than those of the CK. The soil pH of the sample plots of mulching, alternate operation, and long-term mulching significantly decreased, and the soil became more acidic as the mulching period was prolonged. α-diversity and principal co-ordinate analysis showed that mulching had a greater impact on the diversity and structure of the soil bacterial community, and increased the number, species and abundance of bacteria. The shallow tillage of soil surface under the mulching prevented mechanical disturbance of the soil, thus forming a 0-20 cm thick layer of semi-decomposed organic matters within a short period of time to provide energy sources for bacterial reproduction. The alternate operation plot showed the greatest number, species and abundance of bacteria. However, the long-term mulching plot showed a decrease in the number, species and abundance of bacteria, as well as a significantly lower Chao1 index than the CK. This study provides technical support and data reference for research on orientation cultivation of low-yield bamboo and ecological restoration technology of bamboo forest.

Based on the hydrologic data of East Tiaoxi watershed from 2011 to 2014, taken ammonia nitrogen as control criterion, we calculated the ammonia assimilative capacity of four areas divided from upstream to downstream by load duration curve, and analyzed spatial and temporal variation of assimilative capacity in the basin. Combined with the actual water quality data, we measured the residual assimilative amount. The results showed that the assimilative capacity fluctuated dramatically with time, and the high assimilative capacity was mainly from June to September and March. The summer had the maximal assimilative capacity while the winter had the minimal one. The assimilative capacity of wet season was 1.7 times and 2.4 times that of the normal season and the dry season, respectively. Moreover, the assimilative capacity in downstream area was higher than that in upstream area, especially in dry condition interval when the assimilative capacity in downstream was 4.8 times of that in upstream. Except Luoshezha station, the residual assimilative capacity of other stations had the same variation, which showed as high flow interval＞moist condition interval＞mid- range flow interval＞dry condition interval＞low flow interval. The residual assimilative capacity in the Luoshezha station was low in the high flow interval and moist condition interval because of non- point source pollution. In conclusion, the load duration curve is effective in calculating and analyzing assimilative capacity, and this study provides valuable principles for total capacity control in watershed scale.

In order to provide scientific supports for watershed sustainable development, operation and management, Nanxi River was selected as a typical red-soil region, and ecosystem health assessment methodology and pressure analysis combining remote sensing (RS), geographic information system (GIS) and multi-index were applied. The ecosystem health assessment was classified into two categories: water area and terrestrial area, and the indices were divided into six sections including habitat structure, aquatic organisms, ecological pressure of water area and ecological patterns, ecological function, ecological pressure of terrestrial area, which consisted of 16 sub-indicators. The results showed that the health condition of the study area was good, and only the aquatic organisms and ecological function were in ordinary condition. Among them, 83.3% of sub-basins were in good condition, while the others were in ordinary condition, and all of them were located in the downstream areas. Human disturbance was the major reason of the degradation of watershed ecosystem health, such as rapid growth of urban land areas, excessive livestock breeding and fishing. In order to improve the health conditions, more attention should be paid to adjusting the scope of the conservation areas, controlling pollution from point and nonpoint sources as well as protecting the biodiversity. The proposed assessment methodology can comprehensively indicate the health condition of Nanxi River. This case study could make a contribution to guiding the watershed management in ecologically fragile areas, and ensuring the water ecological health of the basin and its downriver.

Soil and water assessment tool (SWAT) was employed to analyze the main sources and patterns of eutrophic pollutants in Laohutan reservoir, located in Huzhou City, Zhejiang Province from Jan. 2010 to Apr. 2015. The situation of hydrology, water quality and economic benefit in the reservoir area under six management measures was simulated with scenario analysis. The results showed that the accuracy of daily runoff simulation was pretty good, with the percent bias (BIAS) below 20% (－17.57%-－0.63%), coefficient of determination (R²) greater than 0.75 (0.79-0.85), Nash-Sutcliffe coefficient of efficiency (NSCE) greater than 0.70 (0.71-0.86). The simulation of total nitrogen was relatively good (NSCE=0.39-0.58, R²=0.58-0.74, BIAS=－14.08%-7.18%), which met the requirements of the model. Therefore, the simulation results can reflect the hydrology and water quality changing rule of Laohutan reservoir. Compared to various hypothetical scenarios, the best water quality safety management measure was found to be promoting scientific fertilization. In conclusion, the SWAT model is applicable to simulate the runoff and agricultural nonpoint-source pollution in hilly area of western Zhejiang, and it is of scientific significance for hydrological simulation and nonpointsource pollution control in this area.

Based on the concentrations of SO_², NO_², CO, O_³, PM_^2.5 and PM_¹⁰ measured by Xixi monitoring station, meteorological data such as temperature, relative humidity, wind speed, visibility and rainfall, variation characteristics and correlations of various air pollutants in Hangzhou City from December 2014 to November 2016 were analyzed, and the influence of meteorological conditions on pollutants was discussed. The results showed that the concentrations of SO_² and CO reached the first grade standard of air quality during the study period. NO_² and particulate matter were severely over-standard and the average concentrations of NO_², PM_^2.5 and PM_¹⁰ were 1.21, 1.40 and 1.04 times of the second grade standards, respectively. The concentrations of all pollutants were high in winter and low in summer. However, the concentration of O_³ was high in summer and low in winter. Atmospheric oxidative O_^x remained at a relatively stable level throughout the year and was dominated by NO_² in winter and O_³ in other seasons. The concentration of PM_^2.5 was positively correlated with that of NO_² and CO, followed by SO_² and O_^x (in summer), which indicated that the local vehicle exhaust emission was an important source of atmospheric particulates. The temperature was positively correlated with O_³, and negatively correlated with other pollutants. Relative humidity, wind speed and rainfall were negatively correlated with SO_², NO_², CO, PM_^2.5 and PM_¹⁰, which showed that the pollutants were easy to diffuse under strong wind and wet deposition condition. However, CO was less affected by humidity and rainfall. This study provides a theoretical foundation for controlling atmospheric pollution in Hangzhou City.

To realize pollination mechanization for hybrid rice seed production, a collision-pneumatic hybrid rice pollination machine was designed and manufactured. The airflow velocity in the air hole of pollination tubes with diameter of 50, 55, 60, 65 and 70 mm was simulated by computational fluid dynamics (CFD). The results showed that the average airflow velocity simulated in the air hole of pollination tube with the diameter of 60 mm was similar to the measured value. The field pollination experiments at the average airflow velocity of 9.35, 11.82 and 15.07 m/s were conducted with the spacing between the air hole and collision rope of 50, 100 and 150 mm respectively. The pollen amount received by four line female parents beside male parents was super to that of the artificial pollination and distributed uniformly. The pollen amount distributed on the four line female parents increased with the distance between the air hole and collision rope. However, the pollen amount was not obviously increased when the distance between the air hole and collision rope increased to 100-150 mm, and the average airflow velocity of the air hole increased to 11.82-15.07 m/s. In conclusion, the optimal pollination parameters for the collision-pneumatic hybrid rice pollination machine are obtained when the airflow velocity is 11.82 m/s, and the distance between the air hole and collision rope is 100-150 mm.

Parameters of wheat crop growth model WOFOST (world food studies) were discriminated, and the sensitivity of crop and soil parameters in WOFOST model was analyzed using OAT (one-at-a-time) analysis method. Meanwhile, the simulation results of wheat growth were explained. Results showed that the relative sensitivities of seven parameters of WOFOST model, including TSUM1, FRTB, CVO, CVS, DVSI, SPAN, SLATB, were all greater than 0.5, which presented strong sensitivity and had greater influence on the model simulation results. The parameters which had greater influence on the WOFOST model based on different levels of yield and production were almost the same, and the relative sensitivity had no significant difference. Simulation results of leaf area index (LAI) showed that the days of wheat from sowing to flowering were 137, and the days of wheat from flowering to maturity were 66, which was in accordance with the actual days. This study provides a basis for the application and parameter adjustment of WOFOST model.

Focusing on the defects in fiber structure of corn stalk feed, we proposed a novel production method of ruminant animal fodder by co-treatment with ultrasound and alkalization. By using central composite design with four factors at five levels and response surface analysis, we investigated the effects of ultrasonic power, ultrasonic time, liquid-to-solid ratio and ultrasonic power density in the container on extraction of cellulose and removal of hemicellulose and lignin in corn stalk. In addition, the two-phase regression model was established by taking the contents of lignin, cellulose, and hemicellulose as response values, respectively. The model explained the factor contribution rate of ultrasonic factors to response value and the significant influence on response surface model by the interaction between ultrasonic power, ultrasonic time, liquid-to-solid and ultrasonic power density in the container. The results showed that the determination coefficients of lignin, cellulose and hemicellulose models were 0.69, 0.79 and 0.73, respectively. The process parameters were optimized by the response surface model, and the optimal working parameters of each ultrasonic factor were obtained as ultrasonic power 99 W, ultrasonic time 20 min, liquid-to-solid ratio 7.8:1, and ultrasonic power density in the container 2.05 W/mL. The relative errors of actual values and predicted values were less than 13%, 12% and 14%, respectively. It is indicated that reasonable matching of ultrasonic parameters is helpful to reduce the content of lignin and cellulose of corn stalk feed and increase livestock digestibility, and provides scientific basis for the design of ultrasonic alkalization device and selection of working parameters.