Abstract:genes including RbohA(Lj5g3v1497840),RbohB(Lj6g3v1549190) and RbohC (Lj5g3v1497820) were found to be primarily expressed in nodules based on the transcriptions of Rboh genes in symbiosis in Lotus japonicus. Their function involved in symbiosis were further studied in Lotus japonicus.The subcellular location of proteins,the expression pattern of Rboh genes,the proteins function and the promoter activity of Rboh genes in Lotus japonicas were studied with methods of molecular biology,cell biology and genetics. The results showed that LjRbohA,LjRbohB and LjRbohC proteins were localized at the plasma membrane.The transcript of LjRbohA,LjRbohB and LjRbohC accumulated abundantly in roots and nodules. The activity of LjRbohA and LjRbohB promoters were detected at vascular tissue,nodule,and the lateral root primordia.The numbers of nodules were reduced in LjRbohB,LjRbohALjRbohC  and LjRbohALjRbohBLjRbohC knockout transgenic roots compared with control transgenic roots. It is indicated that these three Rbohs might have an overlapping function in regulating nitrogen fixation symbiosis in L. japonicus.

Abstract:Yeast twohybrid assay and bimolecular fluorescence complementation assay (BiFC) were performed to identify the interaction of these transcription factors IPN2,NSP2,CYCLOPS,and DELLA involved in regulating the expression of the symbiotic gene during nodulation in the model legume Lotus japonicus. The results showed that Lotus DELLA interacted with IPN2,NSP2,and CYCLOPS,indicating that IPN2 and DELLA may form a large protein complex to regulate the spatialtemporal expression of the nodule inception gene in legumes.

Abstract:In order to explore the correlation between cysteine proteases and the senescence of determinate nodules,LjCyp1 and LjCyp5,associated with root nodule senescence were identified from Lotus japonicus by sequence alignment analysis with cysteine protease AsNODf32.The protein sequence and structure showed that the homology of LjCyp1 and LjCyp5 was as high as 96.2%,both of which contained two typical cysteine protease domains and vacuolar localization signal peptides,belonging to the papain family. In vitro enzyme activity assays showed that both proteins had cysteine protease activity. Realtime PCR and histochemical localization analysis showed that two protease genes were mainly expressed in mature and aged root nodules. The results indicated that LjCyp1 and LjCyp5 cysteine proteases may play an important role during nodule senescence in Lotus japonicus.

Abstract:Fourteen genes encoding E2(LjE2) in Lotus japonicus genome were identified based on the sequence of E2 in Arabidopsis to study the function and mechanism of ubiquitination in the process of symbiotic nitrogen fixation in legumes. Among them,eleven LjE2s were cloned to construct the fusion expression vectors pETLjE2s:His. Ten LjE2 proteins were successfully expressed and purified under the prokaryotic expression system. The results of ubiquitination in vitro and Western blotting showed that nine LjE2s had ubquitinconjugation enzyme activity and one had no detectable ubquitinconjugation enzyme activity.

Abstract:An ACC deaminase gene from Sinorhizobium meliloti1021 was introduced into Sinorhizobium fredii HH103 and Sm1021 (providing extra copy of ACC deaminase gene) using the conjugation of Escherichia coli S171 and Rhizobium.These genetic engineering strains were inoculated to host plants to study the effect of ACC deaminase on symbiotic nitrogen fixation and competitive nodulation ability. The results showed that overexperssion of ACC deaminase in Sm1021 increased the nodule number and fresh weight in alfalfa. In soybean,inoculated with HH103 containing ACC deaminase also increased the nodule number,the dry weight and competitive nodulation ability. But the nodule fresh weight and nodule nitrogenase enzyme activity had no significant change both in alfalfa and soybean. 

Abstract:The GlnK protein is a PⅡ family protein existing widely in bacteria,archaea and plants and involved in regulating nitrogen metabolism. The insertion mutant of glnK homologue gene SfHH103_03182 in Sinorhizobium fredii HH103 was constructed. The results of PCR and sequence analyses confirmed that the mutant was correctly constructed. The results of pot plant experiments showed that the nodule number of the plant inoculated with the mutant,the fresh weight of plants and the fresh weight of root nodules increased compared with inoculated with wildtype strain HH103. There was no significant difference in root nodule nitrogenase activity. The results of symbiotic phenotype tested on host plant soybean at different concentrations of nitrogen levels showed that there was no significant changes in the number of root nodules in the mutant inoculated plants.

Abstract:Phosphorus is an important element required for plant growth. The absorption and transportation of Pi are carried out by phosphate transporter in legumes. In this paper,phosphate transporter AsPT6,a new member of pht1 family,was identified in Astragalus sinicus. AsPT6 was expressed in leaves,stems and roots. The expression of AsPT6 was increased significantly in the roots and nodules when inoculated with Mesorhizobium huakuii 7653R. The results of tissue localization showed that AsPT6 was expressed in the vascular tissue of roots,nodules,and the junctions between root and nodule,indicating that AsPT6 may play a role in transporting phosphate from the root to nodule. Overexpression and RNA silence of AsPT6 significantly affected the nodulation and nitrogenase activity of nodules. It is indicated that AsPT6 is involved in the transportation of phosphorus during the formation and development of nodules.

Abstract:Phosphorus is an important element required for plant growth. The absorption and transportation of Pi are carried out by phosphate transporter in legumes. In this paper,phosphate transporter AsPT6,a new member of pht1 family,was identified in Astragalus sinicus. AsPT6 was expressed in leaves,stems and roots. The expression of AsPT6 was increased significantly in the roots and nodules when inoculated with Mesorhizobium huakuii 7653R. The results of tissue localization showed that AsPT6 was expressed in the vascular tissue of roots,nodules,and the junctions between root and nodule,indicating that AsPT6 may play a role in transporting phosphate from the root to nodule. Overexpression and RNA silence of AsPT6 significantly affected the nodulation and nitrogenase activity of nodules. It is indicated that AsPT6 is involved in the transportation of phosphorus during the formation and development of nodules.

Abstract:Degenerated primers were designed according to the conserved regions of NBSLRR domain from known plant resistance genes. Then these primers were used for amplification of the NBSLRR analogs from Lycopersicon esculentum. Sequence analysis showed that 5 fragments contained the resistance gene analogs,two of which have the ORF region and conserved NBSLRR domain. F58 (MG860907) shared 94% identity with the resistance genes of Mi1, and F520 (MG860908) shared 96% identity with the resistance genes of Hero. The quantitative realtime PCR results showed that expression of F58 and F520 was changed after Meloidogyne incognita infection,suggesting that F58 and F520 might be related with nematode infection.

Abstract:Degenerated primers were designed according to the conserved regions of NBSLRR domain from known plant resistance genes. Then these primers were used for amplification of the NBSLRR analogs from Lycopersicon esculentum. Sequence analysis showed that 5 fragments contained the resistance gene analogs,two of which have the ORF region and conserved NBSLRR domain. F58 (MG860907) shared 94% identity with the resistance genes of Mi1, and F520 (MG860908) shared 96% identity with the resistance genes of Hero. The quantitative realtime PCR results showed that expression of F58 and F520 was changed after Meloidogyne incognita infection,suggesting that F58 and F520 might be related with nematode infection.

Abstract:329 soil samples were collected from the citrus growing areas in Honghuatao Town,Yidu City,Hubei Province.Based on the principle of spatial stratified heterogeneity,the top five major impact factors having the greatest correlation with soil organic matter (SOM) were selected with the GeoDetector software.Using the interpolation results of ordinary Kriging as control,the global model multiple linear regression (MLR),partial least squares regression (PLSR) and local model geographical weighted regression (GWR) were established by the soil organic matter and its main environmental factors.After analyzing the structure of the model residuals,GWRMLR and GWRPLSR were constructed as the extensions of GWR model.The results showed that the mean square error (MSE),root mean square error (RMSE),relative analysis error (RPD) and the correlation coefficient (r) between measured and predicted values of GWRPLSR were 9.834,3.136,1.468,0.743,respectively.The GWRPLSR model had the highest prediction accuracy,followed by GWRMLR.In summary,except for the spatial correlation between SOM and its major impact factors,analyzing model residuals can further eliminate the predicted instability.Therefore,taking the model residual terms into consideration is more suitable to predict the regional SOM spatial distribution and digital soil mapping.

Abstract:Eight characteristic values of asteriscus from 810 individuals in four culters species,including Culter alburnus,C. mongolicus,C. dabryi and Ancherythroculter nigrocauda,from 9 sampling points,were measured,and the regression analysis between culter’s body growth and otolith growth,and multivariate statistical analysis of morphological characters,six shape factors and six shape indices were analyzed using Statistica software. The results showed that:(1) There were significant differences of the asteriscus morphology among the four culters. (2) Power function regression equation could better describe the relationship between the culter’s body growth and otolith growth than linear regression equation. The regression equations between culter’s body quality and otolith quality,and between standard length and otolith diameter were approximate to linear regression relation,with the exponential value of regression equation between body quality and otolith diameter and standard length being 3.5 and 3,and between otolith quality and otolith diameter being 2. The growth rate of otolith mass was similar to that of body mass,but the growth rate of otolith mass relative to otolith length was slower than that of body mass relative to standard length or otolith length. (3) Discriminant function analysis of culter’s morphological characters had an accuracy of 71.11%,and the characteristic values that had great influence on the identification were OW,OA,OL and BL,etc. Through principal component analysis of the shape factors and shape indices,F2,F1,F6,F5 and CF,CR,EL,etc. had a greater influence on the recognition of shape. (4) Through otolith morphological characters discriminant analysis and cluster analysis among culter populations,it could be better distinguished from three Changjiang groups of C. alburnus (from Taihu Lake,Poyang Lake,and Danjiangkou Reservoir),the Lijiang group,and five Poyang Lake groups of C. mongolicus (from Xinmiao Lake,Nanbeigang Lake,Qilang Lake,Lushan West Sea Reservoir,and Zhangjiang River),with the group clustering distance associated with geographic distance among the populations. In short,asteriscus shape analysis in this study provides a basis for correlation analysis among culter species or populations.

Abstract:To explore the function of the bmp2a and bmp4 genes and regulatory networks of the related transcription factors in yellow catfish Pelteobagrus fulvidraco, RLM5′RACE method was used to identify the transcription start site (TSS) of bmp2a and bmp4. Then, the promoters of bmp2a and bmp4 were cloned by hiTAILPCR method and a cluster of putative binding sites of several transcription factors were identified by bioinformatics analysis. The results showed that the TSS of bmp2a and bmp4 were located at 391 bp and 351 bp upstream of the coding sequence, respectively. Then 1 830 bp and 1 962 bp upstream of the TSS of bmp2a and bmp4 were cloned and key binding sites of several transcription factors, such as AP1, SP1, GATA1, CREB, PPARγ, SOX5, SOX6 and SOX9, were predicted, suggesting that these transcription factors may play crucial roles in the transcriptional regulation of the bmp2a and bmp4 genes．

Abstract:The depletion rules of enrofloxacin and its metabolite residues were studied in channel catfish (Ietalurus punetaus) feeding with 20 mg/kg and 40 mg/kg enrofloxacin for 5 d under pond conditions. The results showed that the peaking time (tmax) of enrofloxacin in the liver was 3 d at low dose, and 4 d at high dose during the period of treatment, and the corresponding maximal drug concentration (Cmax) was 12.24 mg/kg and 15.97 mg/kg, respectively. The equations of elimination curve of enrofloxacin in the liver, muscle and blood were C=0.25e-0.136t, C=0.31e-0.082t and C=0.23e-1.37t, and the elimination halflives were 5.10 d, 8.43 d and 5.06 d at dose of 30 mg/kg, respectively. The equations of elimination curve of the drug were C=0.70e-0.173t, C=0.50e-0.096t and C=1.08e-0.182t, and the elimination halflives were 4.00 d, 7.22 d and 3.81 d at dose of 60 mg/kg, respectively. Enrofloxacin was changed into ciprofloxacin in channel catfish. The concentration of enrofloxacin in the muscle was 0.013 mg/kg after 138 d at low dose, and 0.019 mg/kg after 138 d at high dose. The elimination rate of the drug in various tissues was in the order of blood, liver and muscle.

Abstract:The single factor and orthogonal experiments were performed to optimize the dissolution of selenium from Enshi Seenriched tea. The effects of brewing times using tap water and ultrapure water on the antioxidant activity of the tea and the dissolution characteristics of selenium and polyphenols were studied.The results showed that the ratio of tea and water 2∶50 (m/V,g/mL),brewing temperature 100℃,brewing time 6 min,tea particle size 150 μm was the optimal brewing condition of Enshi Seenriched tea. Under the optimal brewing condition,the dissolution rate of selenium was 385.57 ng/g. The selenium leaching rate of the first tea soup was about 12%,nearly 3 and 67 times higher than that of the second and third brewing tea soup,respectively. The general dissolution rate of selenium in tea soup using the tap water was higher than that in ultrapure water. The proportion of tetravalent selenium was 70% to 90% in each tea soup,and the selenium was mostly distributed in the small molecules of tea soluble substance. Similar trend on polyphenols was found with 60 mg/g of extraction rate in the first tea soup,2 and 3 times higher than that in the second and third tea soup,respectively. Seenriched tea soup had excellent antioxidant activity,and the antioxidant activity of tea soup brewed by ultrapure water was slightly higher than that of tap water. The antioxidant activity of tea soup decreased with the increase of the time of brewing. Brewing three times can achieve greater use of active ingredients.

Abstract:Xuxiang kiwifruit was used to determine the content of its total acid,total sugar and vitamin C.Polyphenols from Xuxiang kiwifruit were extracted with ultrasonic extraction.Its total reducing capacity and scavenging activities against four free radicals including hydroxyl radical,superoxide radical,DPPH radical and ABTS radical were identified to evaluate the antioxidant activity of kiwifruit polyphenols using vitamin C as a control.The results showed that the content of total acid,total sugar,and vitamin C in Xuxiang kiwifruit was 1.13%±0.16%,10.03%±0.78% and （78.71±7.60） mg per 100 g,respectively.The content of polyphenols in kiwifruit extract was 0.123 mg/mL.The total reducing capacity of kiwifruit polyphenols was stronger than that of vitamin C.The IC50 value of the scavenging rate for four free radicals of kiwifruit polyphenols was lower than that of vitamin C.It is indicated that kiwifruit polyphenols had high antioxidant activity.The kiwifruit polyphenols analyzed with HPLC contains 6 kinds of polyphenols:gallic acid,fenugreek acid,catechin,chlorogenic acid,caffeic acid and ferulic acid.Caffeic acid was the highest among them.It will provide a theoretical basis for further studying kiwifruit polyphenols.

Abstract:To study the effects of plant polyphenols including tea polyphenols (TP),apple polyphenols (AP) and grape polyphenols (GP) on the preservation of surimi quality during cold storage,surimi product was stored at 4℃ and tested for whiteness,waterholding capacity(WHC),gel strength,TBA,TVBN and aerobic plate count at different storage times including 0,5,10,15,20 days. The results showed that the whiteness of surimi products was decreased with adding polyphenols,while the whiteness value of GP group changed insignificantly during the preservation process. The whiteness value of TP and AP groups showed a tendency of decreasing slightly first and then tending to be gentle. The addition of polyphenol improved the water holding capacity and gel strength of surimi products. Compared with the control group,the addition of polyphenols significantly inhibited the decrease of WHC and gel strength and the increase of TBA and TVBN. It inhibited or killed spoilage bacteria,indicating that polyphenols had a positive effect on surimi products during cold storage. The effect order of three experimental groups was TP>AP>GP.

Abstract:A mountain orchard transport vehicle with single crawler was designed.The main body of that dimension is 1 540 mm×600 mm×815 mm,assembled by a single crawler walking device,frame,transmission,power systems and other equipment.The vehicle centroid analysis and ergonomics were used to determine the structure and movement parameters of vehicle.Creo was operated to build a model to calculate the vehicle’s centroid position.Based on the vehicle’s centroid position,the stability of the vehicle on the lateral and longitudinal slopes was analyzed.With full load,the prototype under different working environment was tested.Results showed that the maximum load capacity was 75 kg,with the ability to go up 10 degrees and go down 30 degrees of the longitudinal slope.It can run across the slope of 20 degrees and run through various complex roads.The vehicle can well meet the requirements of mountain orchard lateral transport.

Abstract:Animal manures (cow dung,pig manure) and cornstraw were used to investigate the effects of different temperatures(200,300,400 and 500℃) and different amount of straw (20%,40%,60%,80%) on yields and physicochemical properties of biochar produced in a closed lowoxygen reactor using pyrolysis process. The results showed that biochar yields,volatile content decreased gradually as pyrolysis temperature increased,while ash content,pH and total content of P,K and nutrient contents increased,N content first increased and then decreased. The pore characteristics of cow dung biochar were best at 400℃,while the pore characteristics of pig manure were poor. Adding straw improved pH of biochar and regulated nutrient content. Compared with pig manure biochar,cow dung biochar had better carbon yield,pH and pore characteristics. The nutrient content of cow dung was less than that of pig manure. Cow manure and straw mixed biochar is more suitable for using its pore characteristics as adsorbent and so on. Pig manure and straw mixed biochar is more suitable for using its nutrient characteristics as phosphate fertilizer production auxiliary or soil conditioner. The influence of mixed pyrolysis on the charcoal characteristics needs to be further studied.