Abstract:To identify the optimal internal reference genes for qRT-PCR analysis in loach (Misgurnus anguillicaudatus), six commonly used reference genes, including ACTB, TUBA, EF1A, GAPDH,TUBB and 18S rRNA were selected as candidates, and their expression stability during different developmental stages, acrosstissues and betweenploidy were evaluated by using BestKeeper, geNorm, NormFinder, ΔCt and RefFinfer software. The results showed that, during the process of embryonic development, the expression levels of TUBB and TUBA were more stable than the other genes, suggesting that they can be used as reference genes. Besides, the expression level of TUBB showed no significant difference between diploids and tetraploids (P＞0.05), suggesting it is the most suitable internal reference genes for qRT-PCR analysis during embryonic development between ploidy. Among different tissues of diploids and tetraploids, the expression levels of ACTB and 18S rRNA were more stable than the other genes, thus they can be used as internal control genes. Of which,18S rRNA was considered as the most suitable internal control genes, because the expression level of it exhibited no significant difference in different tissues between diploids and tetraploids (P＞0.05).

Abstract:To understand the expression difference of IGFBP-1 between diploid and tetraploid Misgurnus anguillicaudatus,and their relationship with DNA methythylation,we cloned the CDS and promoter sequence of the IGFBP-1 gene,and analyszed the expression level and DNA methylatyion rate in diploids and tetraploids using qRT-PCR method and bisulfite sequencing technology (BSP),respectively. The results showed that,the CDS of IGFBP-1 was 789 bp,encoding 262 amino acid,and 5 basepair difference but no amino acid difference were found between diploids and tetraploids. We cloned 2 341 bp and 2 331 bp promoter sequences in diploids and tetraploids,respectively,with the sequence similarity of 99%. The core transcription element TATAbox,and transcription factor binding sites such as SP1,CREB,C/EBP,POU2F2,GATA2/3/4 and SRY were observed in the promoter. The IGFBP-1 gene was mainly expressed in the liver,and the expression level was significantly higher in tetraploids than in diploids (P＜0.01). The methylation level of promoter and 1st exon were lower in teterploids than in diploids,expecially in the liver (P＜0.01). In conclusion,in the liver,the major expression tissue of the IGFBP-1 gene,the mRNA expression level and DNA methylation level were negatively correlated,suggesting that the higher DNA methylation rate in promoter may suppressed the expression of IGFBP-1 in the diploid M. anguillicaudatus.

Abstract:Trim63(tripartite motif containing 63),also known as muscle RING finger protein 1 (MuRF1),plays an important role in the development and differentiation of the muscles in mammals.Partial cDNA sequence of the Misgurnus anguillicaudatus trim63a gene (Matrim63a) was cloned in this study.Sequence analysis showed that the fulllength Matrim63a CDs was 1 035 bp,encoding 344 amino acids,which shared high similarity with those of other teleosts.The Matrim63a mRNA expression in different tissues of the adults and during embryonic developmental stages in the diploid and tetraploid M. anguillicaudatus were examined by qPCR. Matrim63a showed highest expression in the muscle and followed by the heart.During embryonic development,Matrim63a was examined in all the 13 stages with different expression levels.

Abstract:Six different polyploid loaches (DD×PP-0,3n=74; DD×DD0,3n=75; DD×TT-0,4n=100; TT×PP-0,5n=124; TT×DD-0,5n=125 and TT×TT-0,6n=150. Female in the former) were induced using large scale loach (Paramisgurnus dabryanus,PP),diploid (DD),and tetraploid (TT) dojo loach (Misgurnus anguillicaudatus) by cold shock. The cold shock was maintained for 30-35 min using 23 ℃ icewater from 5 min after fertilization. The ploidy of induced progenies at different development stages were measured by flow cytometry. The results showed that the ratios of induced triploid progenies were 87.50% of DD×PP-0,53.85% of DD×DD-0,90.91% of DD×TT-0,84.96% of TT×PP-0,76.92% of TT×DD-0,and 12.5% of TT×TT-0 at 1 month of age. The ratios of induced polyploidy progenies were 91.43% (DD×PP-0,3n=74),60% (DD×DD-0,3n=75),85.19% (DD×TT-0,4n=100),83.33% (TT×PP-0,5n=124),87.50% (TT×DD-0,5n=125) and 6.06% (TT×TT-0,6n=150) at 12 months of age,respectively. The ploidy composition of each group at 12 months was similar to that at 1 month of age. The ratio of induced polyploid progenies decreased with increasing ploidy levels. Growth performance of each group was measured and analyzed by statistical analysis. Little difference was observed at early development stage,while significant difference was observed at 12 months of age. The progenies of TT×PP-0 had the highest total length and body weight value at 12 months of age. Furthermore,the fertilization cytology observation confirmed that the second polar body was retained in fertilized ovum by cold shock to produce triploidtype progenies.

Abstract:In order to find the morphological differences between hybrid 1 (Misgurnus anguillicaudatus♀×Paramisgurnus dabryanus♂),hybrid 2 (Paramisgurnus dabryanus♀×Misgurnus anguillicaudatus♂) and their parents，the morphological differences,countable traits and measurable traits were analyzed by combining morphological measurements with statistical analysis．The results showed that the two hybrids were similar in appearance,integrating their parental characteristics. For example,the welldeveloped caudal fold at the caudal peduncle of the two hybrids was similar to P. dabryanus,while the pattern on the body was more similar to that of M. anguillicaudatus,most of which were round spots with a small number of stripes. The body shape of hybrids was between their parents，which was rounder than P. dabryanus. Cluster analysis and principal component analysis indicated that the morphology of the two hybrids were similar to each other,and were more similar to P. dabryanus．The comprehensive accuracy of discriminant analysis reached 94.1%,which can effectively identify the two loach and their hybrids. There was no significant difference of hatching rate,deformity rate and early survival rate between the hybrids and selfcrossing group (P>0.05).

Abstract:A high quality BAC library of the oilseed Brassica juncea variety Sichuan Yellow Seed was constructed to accelerate the study and utilization of the yellowseed rapeseed genome.The library contains 82 944 clones and is stored in 216×384 microtiter plates.The results of restriction enzyme digestion of plasmid showed that the average insert size of the library was about 140 kb and the emptyvector rate was about 2.5%.Based on the Brassica juncea genome size of 1 000 Mb,the library covers about 11.6× genomes.The results of Blast analysis of the BAC end sequences showed that the clones were evenly mapped onto the 18 chromosomes of the Brassica juncea genome.No chloroplast,mitochondria and bacterium genome DNA contamination was found.

Abstract:A PutNaKR3 gene was screened and identified from the cDNA library of Puccinellia tenuiflora under NaCl treatment. Bioinformatics, tissuespecific expression and abiotic stress response expression of the PutNaKR3 gene and its encoded amino acid sequence were performed. The results showed that PutNaKR3 gene in Puccinellia tenuiflora had a tissue expression specificity, and its expression in roots and leaves was significantly higher than that in other tissues. Overexpression of PutNaKR3 gene enhanced the viability of yeast strain under salt, alkali, oxidation and osmotic stress, and improved the adaptability of yeast under partial metal stress. It is indicated that the PutNaKR3 gene plays a certain role in plant stress response, especially in enhancing plant salt tolerance.

Abstract:Three kinds of biochar including straw charcoal,husk charcoal and charcoal were compared by pot experiment.Four levels of straw charcoal including 5 t/hm2,10 t/hm2,20 t/hm2,40 t/hm2,and two levels of husk charcoal and charcoal including 20 t/hm2,40 t/hm2 were set up to study the effects of different treatments on rice growth and soil nutrients.The results showed that three kinds of biomass charcoal promoted dry matter accumulation and yield formation of rice in a certain range,and straw charcoal had the best effect.Compared with the control,the dry matter and yield of straw increased at first and then decreased,reaching the highest at 20 t/hm2.The biomass and grain yield increased by 51.45% and 72.55% ,respectively.40 t/hm2 was the best application of husk charcoal,and the yield was increased by 60.52%.The application of 20 t/hm2 charcoal increased biomass and yield,but application increased to 40 t/hm2 decreased biomass and yield significantly.Appropriate application of biomass charcoal could increase the content of available nitrogen,phosphorus,potassium and total nutrients in soil,with straw charcoal as the best,followed by husk charcoal and charcoal.Carbon content of charcoal was up to 74.28%.Excessive application can significantly reduce the content of available nitrogen,phosphorus and potassium in soil,which is unfavorable to rice growth.

Abstract:Simple sequence repeat (SSR) markers were used to study the genetic relationship among different strains of Citrus grandis ‘Tomentosa’ and other representative germplasms of Citrus. 175 alleles were amplified with 25 pairs of SSR primers and 15 samples. The results of cluster analyses showed that ‘Tomentosa’ and pomelos were closely related. Some ‘Tomentosa’ materials with homonym are found to be genetically different. High performance liquid chromatography (HPLC) was used to determine the content of naringin in young fruit of different germplasm and fruit tissue of ‘Tomentosa’ with varied diameters. The results showed that the content of naringin in the young fruit of ‘Tomentosa’ was significantly higher than that of Huazhou local pomelo ‘Guangqing’ and common pomelo from other areas,affected by the growing season and environmental factors. With the increase of fruit diameter,the content of naringin in pericarp and pulp of ‘Zhaomao(Wu)’ and ‘Jiaxiyang(Wu)’ decreased,and the content of naringin in pulp was higher than that in pericarp. The results of comprehensively analyzing fruit size and active ingredient content showed that the fruit diameter of 4-8 cm was the suitable harvesting period of ‘Tomentosa’,and the fruit with diameter of 6 cm was optimal for harvesting.

Abstract:WRKY transcription factor plays a vital role in response to cold stress in plants. To reveal the function of WRKY gene in Prunus mume,the PmWRKY40 gene was cloned from P. mume ‘Xuemei’ by RTPCR. The fulllength cDNA sequence of PmWRKY40 was 1 072 bp,containing a complete open reading frame (ORF) of 972 bp encoding 323 amino acids. The deduced amino acid sequence alignment of PmWRKY40 contained a conserved WRKY domain and C2H2 zincfinger structure. The results of phylogenetic analyses showed that the PmWRKY40 was most closely related to WRKY protein from P. avium. The results of quantitative PCR showed that PmWRKY40 was induced by cold and H2O2 stresses,but the induction levels of the gene expression were different between cold and H2O2 stress. Meanwhile,PmWRKY4 was significantly triggered by SA treatment and its expression was inhibited by ABA. It is indicated that PmWRKY40 from P. mume may play a key role in response to cold stress.

Abstract:To investigate the metabolic effects of NOX related genes on Coniothyrium mintans，LC MS based metabolomics analysis was applied to detect the metabolites of mutant Qnox1-6 and the wild strain zs-1.The study shows that fourteen substances are significantly different between the two strains, most of which are carboxylic acids and glycosides. And seven metabolites only exist in the mutant strain,including hydroquinone,coniferine,saponin,three carboxylic acids and one other substance. These metabolites,which are closely related to the parasitism of C. mintans againstS. sclerotiorum.

Abstract:To establish an in vivo transposition mutagenesis system of Saccharopolyspora,the transposition plasmids pJTn1pJTn6 were constructed by enhancing the expression of Tn5 transposase gene tnp (5) with strong promoters originated from Sa. spinosa and optimizing the Streptomyces Tn5based transposition plasmid pHL734. The results showed that plasmids pJTn1-pJTn6 transposed efficiently in Sa. erythraea. Plasmid pJTn1 and pJTn5 worked well in Sa.spinosa. Canonical events of Tn5 transposition were detected on 30 genomes out of 31 Sa.spinosa transposition mutants. There was a significant decrease in the yield of spinosad in eight mutants. It demonstrated that the pJTn plasmids are valuable tools for the transposition mutagenesis in the genus of Saccharopolyspora,which will facilitate studying the regulation of spinosad production and screening target genes for the high yield breeding.

Abstract:To reveal the changes of fungal community during fermentation of soybean meal and provide basis for the stability and safety evaluation of fermented soybean meal,MiSeq highthroughput sequencing technique was used to detect the fungal community composition of two batches of fermented soybean meal at 0 h, 24 h and 48 h,respectively.The results showed that Saccharomyces was the only dominant fungal genus(≥1%) shared by two batches of fermented soybean meal at 24 h and 48 h.The average relative abundance was less than 1% at 0 h,but 98.30% at 24 h and 89.56% at 48 h.And no noxious fungi were detected in the fermented soybean meals,which proved that the fermented soybean meal had good safety.The changes of fungal community in the two batches of fermented soybean meal were similar at the three timepoints.The relative abundance of Saccharomyces cerevisiae increased continuously firstly,and then maintained at a high level.During the fermentation process,Saccharomyces cerevisiae continuously consumed and inactivated antinutritional factors in soybean meal,and produced various beneficial metabolites which improved the quality of fermented soybean meal.

Abstract:To realize the rapid identification of freshwater fish species,near infrared reflectance spectroscopy was employed to establish the identification models of fish species. 772 samples of 7 freshwater fish species (silver carp,grass carp,snakehead,crucian carp,common carp,black carp,bighead carp) were prepared to collect near infrared spectra data. The effects of preprocessing methods including standard normalized variate (SNV),multiple scattering correction (MSC) and the feature extraction methods including kernel principal component analysis (KPCA) and principal component analysis (PCA) on the discrimination models of support vector machine (SVM) were investigated,respectively. The results showed that the overall accuracy rate was 92.68% for the unknown sample after the SNV preprocessing and KPCA extraction of characteristic variables. Therefore,the SVM model constructed by near infrared spectroscopy combined with chemometric methods is feasible for rapid identification of freshwater fish species.

Abstract:The compositions of metal ions,free amino acids,total amino acids and nucleotides of four different yeast extracts (KA66,FA28,KU012,FA01) were analyzed. Subsequently,the contribution of each taste component to the taste of yeast extract was evaluated using taste activity value (TAV),and their synergistic interactions were explored through the equivalent umami concentrations (EUC). The effect of yeast extracts on the flavor of the surimi product was explored by sensory evaluation and electronic nose. The results showed that the yeast extract KU012 had the highest content of glutamic acid,alanine,5′IMP and 5′GMP with the biggest TAV,which were the lowest in FA01. The content of total amino acids in FA28 was the highest and the glutamic acid was the main component. The EUC of KU012 was 125.22 gMSG/g,namely,the umami intensity produced by 1 g KU012 was equal to that of 125.22 g single monosodium glutamate. Sensory results showed that yeast extracts could significantly improve the taste of surimi products,and also had a certain masking effect on fishyodors. KU012 had the highest sensory score. The results of the electronic nose showed that the volatile components of the surimi products were significantly different after the addition of different yeast extracts.

Abstract:This study aimed to examine the effects of sodium glutamate and alcohol on the preservation quality of surimi during freezethaw cycles. In this experiment，surimi product was stored at -22℃ and tested for total volatile basic nitrogen(TVB-N) value，pH value，2thiobarbituricacid (TBA) value and the total number of bacterial colonies and chroma (L*，a*，b*)，water holding capacity and textural properties at different storage time (0，7，14，21，28 d). The addition of sodium glutamate and alcohol could improve the water holding capacity，whiteness and springiness of surimi gel. Compared with the control，the addition of sodium glutamate and alcohol could (P＜0.05) prevent not only the decrease in water holding capacity，whiteness and gel strength but also the increase of the total number of bacterial colonies，TBA and TVB-N. Results indicated that sodium glutamate and alcohol had a positive effect on surimi product during freezethaw cycles. The effect in three experimental groups was in the order of MIX＞Alcohol＞MSG.

Abstract:The changes of cathepsin activity in myofibrils and sarcoplasm,degradation of myofibrillar and sarcoplasmic protein by SDSPAGE,myofibrillar microstructure by scanning electron microscopy (SEM) and transmission electron microscopy (TEM),instrumental texture,waterholding capacity (WHC) by drip loss and LFNMR for bighead carp fillets were measured during -20℃ storage.The results showed that the cathpsins were still active for thawed fillets stored at -20℃.The degradation of myosin,actin,troponin T and tropomyosin were observed.A 20 ku sarcoplasmic protein band which might be a proteolysis product of cathepsin L was found.The results of SEM and TEM showed that the muscle fibers were severely damaged at 112 d,which expressed by indistinguishable A band and I band in the sarcomere and almost disappeared M line.The hardness and elasticity of the fish were significantly decreased.The drip loss and T21,T22 and P22 of fillets increased,but P21 decreased significantly with the extension of frozen storage time.Overall the proteolysis of cathepsins on myofibrillar and sarcoplasmic protein during frozen storage led to the great destruction of integrity of myofibrillar microstructure and weakeness of fish texture and WHC.

Abstract:Qingtuan is a traditional glutinous rice product with problems of high viscosity and easy collapse. The effects of adding four kinds of starch materials including pueraria,sweet potato,wheat and rice on the sensory index and texture characteristics of Qingtuan were investigated. The Pearson method was used to analyze the correlation between starch raw materials,texture indicators and the sensory sense of Qingtuan. Then the second rotation orthogonal combination method was used to obtain the best formula of Qingtuan. The results showed that effect of texture characteristics on the sensory score of Qingtuan was in the order of adhesiveness>hardness>springiness>chewiness. The four kinds of starch materials were significantly and positively correlated with the hardness index(P<0.01).The ratio of amylose/total starch and amylose/amylopectin could be used as a preferred index for evaluating Qingtuan hardness (r=0.503,P<0.05) and adhesiveness (r=-0.468,P<0.05). The best formula of Qingtuan with the highest sensory score was 17.1% pueraria flour,5.0% sweet potato flour,10.0% wheat flour,and 30.0% rice flour. Adding amyloserich raw materials based on glutinous rice flour improved the sensory quality of Qingtuan. It will provide further reference for developing Qingtuan.

Abstract:The naringenin phospholipid complex was prepared by solvent evaporation method. The structure of naringenin phospholipid complex was characterized by ultraviolet spectroscopy,Fourier transform infrared spectroscopy,and Xray diffraction. The thermal properties of the complex were analyzed by differential scanning calorimetry.The solvent residues in phospholipid complex were determined by gas chromatography. The equilibrium solubility in water,noctanol and PBS (pH 2.5,pH 5.0,pH 6.5,pH 7.0,pH 7.8-8.0),oil/water partition coefficient and in vitro dissolution were determined by high performance liquid chromatography. The results showed that the drug loading efficiency of naringenin phospholipid complex was 31.79%±0.99% under the condition of naringenin mass fraction 5 g/L,naringenin and phospholipid mass ratio 1∶1.5,reaction time 2 h and reaction temperature 40℃. The residual amounts of methylene chloride and ethyl acetate in the naringenin phospholipid complex were 0.016 3%±0.002 5% and 0.040 8%±0.001 9%,respectively,which all met the standard of Chinese Pharmacopoeia. The equilibrium solubility of the naringenin phospholipid complex was significantly higher than that of naringenin in water,noctanoland and PBS with different pHs. The logarithmic value of oilwater partition coefficient of naringenin phospholipid complex was 1.11±0.09. The in vitro dissolution of naringenin phospholipid complex was significantly higher than that of naringenin,and easier to be absorbed and utilized by human.

Abstract:An oblique taper holetype wheel centralized metering device for rapeseed and wheat without cleaning and protection device was developed to satisfy the requirements of planting rapeseed and wheat in the middle and lower reaches of the Yangtze River in China,and to improve the efficiency of the planter and simplify the structure of the planter.The key component of the metering device was the holetype wheel designed with two rows of staggered holes for rapeseed and three rows for wheat.An oblique tapered hole structure was developed to prevent seed from plugging holes and reduce the damage rate of seed.The mechanical model of filling,carrying,throwing,and delivering seed were established.The results showed that the minimum height of the seed filling was 10 mm lower than the reference height.The rationality of the device structure was verified.The maximum seeding angle of rapeseed and wheat reached 40.8° and 34.4° when the back angle of the hole ranged from 0° to 30° and from 0° to 15°,respectively.The angle between the seed tube and the horizontal plane should be no less than 33°.The results of orthogonal test showed that the back angle of the hole was 20°,the filling height was the reference height,the rotation speed was 20 r/min for rapeseed.The back angle of the hole was 5°,the filling height was 20 mm,the rotation speed was 15 r/min for wheat.The variation coefficient of the consistency of each line displacement was less than 6.00% for rapeseed and 6.32% for wheat.The coefficient of variation of total displacement stability was less than 1.25% for rapeseed and 1.00% for wheat.The damage rate of both rapeseed and wheat was less than 0.5%.The results of field test showed that both rapeseed and wheat had good sowing.It will provide a reference for designing the structure of mechanical seed metering device for rapeseed and wheat.

Abstract:A 2BYML-4 ridge notillage maize planter was developed,which can complete the maize stubble smashing and returning to the field,ridging,fertilizing and precision seeding at one time. The structure and working principles of the machine were analyzed. The results of the prototype in field test showed that the qualified rate of ridge height,ridge top width,ridge spacing,the qualified index of grain spacing,the standard deviation,the coefficient of variation,the missing sowing index,the replaying index,the qualified rate of seed covering depth,the qualified rate of fertilization under sowing,and the coefficient of variation was 94.73%,90.64%,90.13%,92%,11.99,5.24%,4%,4%,88.3%,89.7%,9.71% and 10.32%,respectively. The machine has regular ridge shape,with uniform and reasonable distribution of seeds and fertilizers in the field. Its combination of structural parameters and working parameters can better meet the requirements of conservation tillage in dryland agriculture and effectively improve the ecological environment.

Abstract:The static calculation and modal analyses of the frame plate of a hammer feed grinder rotor system were performed with ANSYS Workbench software. The results showed that under the actual working conditions,the maximum total deformation of the frame plate was 0.035 mm,occurrin at the mounting holes of the two pin rolls with eight hammers on the intermediate support plate, and the maximum deformation of the frame plate along the X axis Y axis and Z axis was 0.035 mm,0.001 mm and 0.016 mm,respectively. All deformations were small and consistent with the actual load condition of the frame plate. The maximum stress on the frame plate was 165.72 MPa,and the parts with large stress were only concentrated near the mounting holes of the two pin rolls with eight hammers on the intermediate support plate. The stress values at most other positions on the frame plate were below 36.862 MPa. The firstorder natural frequency of the frame plate was 339.79 Hz,and its first six order natural frequencies are much higher than the excitation frequency of the hammer feed grinder calculated as 58.33 Hz. But there is a serious material redundancy problem. Based on this,the topology optimization of the frame plate was conducted. The frame plate structure was optimized on the basis of calculating material removal rate as 50%. The quality of the new frame plate was reduced from the initial 46.66 kg to 25.85 kg after optimization. The results of static calculation and modal analyses of the optimized frame plate showed that both the strength and stiffness of the new frame plate were sufficient,and its material redundancy was greatly reduced. The frame plate did not resonate during the working process. It is indicated that the optimized design of the frame plate has desirable effects.