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Sponsored by Zhejiang University and superintended by Ministry of Education of China
ISSN 1008-9209 CN 33-1247/S

《Journal of Zhejiang University: Agriculture and Life Sciences》 is a bimonthly peer-reviewed scientific journal, which is sponsored by Zhejiang University and superintended by Ministry of Education of China. Its predecessor was Journal of Zhejiang Agricultural University, which started publication in 1956. After the establishment of new Zhejiang University, it was changed to Journal of Zhejiang University: Agriculture and Life Sciences in 1998. The journal has been abstracted and indexed by Chemical Abstracts (CA), Abstract Journal (AJ), Zoological Record (ZR), Food Science and Technology Abstracts (FSTA), CAB Abstracts, Ulrichsweb, Directory of Open Access Journals (DOAJ) and the main periodical databases in China. ... More
, Volume 45 Issue 3 Previous Issue   
Entire Issue
Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(3): 263-390.  
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Reviews
Safe utilization of farmland soil with cadmium pollution: strategies and deliberations.
Long MENG,Tuhai HUANG,Jian CHEN,Fulin ZHONG,Jiachun SHI,Jianming XU
Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(3): 263-271.   https://doi.org/10.3785/j.issn.1008-9209.2018.03.082
Abstract( 50 )   HTML( 6 )   HTML (   PDF(733KB)( 59 )

The cadmium pollution in farmland soils of China has posed a potential threat to food safety and human health. Based on the analysis of sources, status and harms of cadmium pollution in farmland soils, we summarized related technologies and application status of safe utilization of farmland soils with Cd pollution, including screening of low cadmium accumulation crop, in situ immobilization technology, agronomic measures and comprehensive prevention and control patterns. Meanwhile, the safe utilization of farmland soils with cadmium pollution faces many problems including significant regional differences, insufficient pollution source control, incomplete process interception, limited prevention and control technologies and lack of risk evaluation system. Furthermore, corresponding suggestions were proposed towards the control of farmland soils with cadmium pollution in China.

Biological sciences & biotechnology
Straightforward method for preparing a user-customizable DNA marker by improved overlap extension polymerase chain reaction.
Yuanyuan ZHANG,Yan YAN,Yulan JIN,Weiren DONG,Jiyong ZHOU
Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(3): 272-277.   https://doi.org/10.3785/j.issn.1008-9209.2018.06.151
Abstract( 18 )   HTML( 3 )   HTML (   PDF(1508KB)( 50 )

DNA marker is a set of standards that are used to indicate the approximate molecular-mass size of certain DNA fragment which separated by agarose gel electrophoresis. In the present study, a straightforward method for preparing a user-customizable DNA marker by an improved overlap extension polymerase chain reaction (PCR) was established. The DNA fragment, containing the identical sequence in its 5 ' and 3 ' ends, was amplified by a conventional PCR. In the following denaturation and annealing process performed by the overlap extension PCR, two single-stranded DNA in the 3 ' end of which had complementary base sequences could form double-stranded DNA. In the extension process, these single-stranded DNA was utilized as both template and primer. The number of small fragments of DNA decreased and the number of large fragments of DNA increased gradually with the increased number of PCR cycles. The products of different cycles of overlap extension PCR were taken for agarose gel electrophoresis. The optimal PCR cycle or optimal combination of different PCR cycles was chosen according to the results of electrophoresis. Then 100 bp DNA ladder marker was obtained by the overlap extension PCR under the chosen condition. There was no need for purification of PCR products as the amplified fragments could be directly used in the agarose gel electrophoresis. The bands of prepared DNA marker were clear and accurate, and could be used for molecular studies. In sum, this method for DNA marker production is simple, time saving, cost effective, byproduct free and user-customizable in comparison with current ones widely used in most laboratories.

Comparison of simple sequence repeat (SSR) and sequence related amplified polymorphism (SRAP) markers for genetic diversity analysis in strawberry
Ya XIN,Xianping FANG,Shuzhen WANG,Jianxin TONG,Wenguo LAI,Jianrong WANG,Hong YU
Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(3): 278-287.   https://doi.org/10.3785/j.issn.1008-9209.2018.10.261
Abstract( 27 )   HTML( 3 )   HTML (   PDF(809KB)( 56 )

Simple sequence repeat (SSR) and sequence related amplified polymorphism (SRAP) markers were applied to analyze genetic diversity among 43 strawberry (Fragaria×ananassa Duch.) cultivars and effectivenesses of these two kinds of molecular markers were also compared. The results showed that there were 6.43 polymorphic sites per primer pair of 30 SSR primers, and the average polymorphism information content (PIC) of each site was 0.628 4. While there were 14.30 polymorphic sites per primer pair of 20 SRAP primers,and the average PIC of each site was 0.911 4. The correlation coefficient between clustering results based on SSR markers and SRAP markers was 0.817, which was significant. The correlation coefficients between SSR markers, SRAP markers and SSR+SRAP joint markers were 0.938 and 0.966, respectively, which reached extremely significant levels. Both SSR and SRAP markers can be used to analyze the genetic diversity of strawberry, but the effect of SRAP marker is better than that of SSR marker. The analysis of SSR+SRAP joint markers can better evaluate the genetic diversity and genetic relationship of strawberry germplasm.

Crop cultivation & physiology
Effects of agro-photovoltaic integrating system on field illumination and sweet potato growth
Lai WEI,Mingyan YU,Nannan QIN,Chongping HUANG,Ying XIE,Wenbo SUN,Liehong WU,Weizhong WANG,Guoxin WANG
Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(3): 288-295.   https://doi.org/10.3785/j.issn.1008-9209.2018.09.251
Abstract( 81 )   HTML( 5 )   HTML (   PDF(1358KB)( 47 )

In order to investigate the effects of establishment of photovoltaic (PV) panels on field illumination conditions and sweet potato growth in an agro-photovoltaic integrating system, we used wooden boards to simulate the PV panel construction. The simulated PV assembly had a set of standard parameters, which were an average height of 1.6 m, a slope of 22°, a width of 3 m and a length of 9 m. A two-year field experiment was carried out, and three sweet potato cultivars of “Xinxiang” (Xx) , “Zheshu 13” (ZS 13) and “Zheshu 77” (ZS 77) were planted under the PV panel shading and CK (without PV panels) treatments. The results showed that under the sunny weather condition, the illumination intensity was 80-400 μmol/(m2?s), which was higher than the light compensation point of the tested cultivars and slightly lower than the light saturation point of Xx and ZS 13. The variation of field light intensity in cloudy days of summer and autumn was similar to that in the sunny days. The light intensity of observation points under the PV panel shading treatment in rainy days was only slightly higher than the light compensation point of the tested cultivars. At the same light intensity, the net photosynthetic rate and stomatal conductance of sweet potato leaves under the shading treatment decreased significantly, and the intercellular CO2 concentration increased significantly. After the PV panel shading treatment, the maximum leaf area per plant decreased significantly, and the leaf area of Xx, ZS 13 and ZS 77 decreased by 18.94%, 45.54% and 56.26%, respectively. The leaf mass of per area for the three cultivars also decreased significantly. The dry matter accumulation trend of the three cultivars after shading treatment was the same as that of the CK, but the total amount decreased significantly. Compared with the CK, the yield of sweet potato per unit area decreased by 39.25%, 33.70% and 23.60% for Xx, ZS13 and ZS 77, respectively, but the total yield of Xx was still the highest under the shading treatment. In summary, the agro-photovoltaic integrating system formed by the construction of photovoltaic panels in the farmland has some adverse effects on the field light intensity and sweet potato growth, but the economic benefits per unit area are greatly increased. Thus, the crop yield can be increased by increasing density of sweet potato seedlings and reasonable fertilizer, and the agro-photovoltaic integrating system has a good prospect of popularization.

Horticulture
Aseptic germination and bulblet formation of Ixiolirion tataricum (Pall.) Herb. seeds.
Xuesi Lü,Ziming REN,Dong ZHANG,Yiping XIA
Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(3): 296-305.   https://doi.org/10.3785/j.issn.1008-9209.2018.07.041
Abstract( 21 )   HTML( 3 )   HTML (   PDF(1026KB)( 50 )

Ixiolirion tataricum (Pall.) Herb. is a unique and elite plant germplasm resource in China. The distribution of I. tataricum is limited in Xinjiang Uygur Autonomous Region. Ixiolirion. tataricum has strong resistance, high ornamental and medicinal values, which are of great potential in breeding. In this study, the seeds of I. tataricum collected from Xinjiang Uygur Autonomous Region were used as materials to compare the effects of sterilization method, culture conditions, gibberellin A3 (GA3) pretreatment and storage conditions on the aseptic germination and bulblet formation. The results showed that 30 s treatment with 75% ethanol followed by 5 min treatment with 2% NaClO was the best sterilization method for I. tataricum with 100% sterile seedlings. Using this sterilization method, the germination rate was more than 94.0% at 4 ℃, while almost no seed germinated at 25 ℃. Normally, seeds could germinate after storage for six months in the refrigerator at 4 ℃, but completely lost germination capacity at room temperature. We also found that MS+6-benzylaminopurine (6-BA) 1.0 mg/L+1-naphthylacetic acid (NAA) 0.2 mg/L+sucrose 30 g/L was the best germination medium for I. tataricum, which was beneficial to bulblet formation. Germinated in this medium, shoots could eventually expanded into bulbs after being transferred to light culture at 25 ℃ and then transferred to MS medium containing 60 g/L sucrose. This study provides an experimental basis for in vitro conservation of I. tataricum and to enhance the development and utilization of wild resources in China.

Plant protection
Multi-omics reveals the resistance mechanism of grape leaves in response to Botrytis cinerea
Xianping FANG,Yani HE,Xiaojun XI,Qian ZHA,Liqing ZHANG,Aili JIANG
Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(3): 306-316.   https://doi.org/10.3785/j.issn.1008-9209.2018.11.121
Abstract( 17 )   HTML( 2 )   HTML (   PDF(2992KB)( 50 )

Liquid chromatography and mass spectrometry based label-free proteomics and non-target metabolomics technology were used to study the proteome and metabolome change of disease-resistant grape cultivar ‘Shenfeng’ infected with Botrytis cinerea. There were 1 374 proteins and 33 metabolites showing more than 1.5-fold changes in ‘Shenfeng’ leaves infected with B.cinerea, respectively. The differentially expressed proteins and metabolites were analyzed by gene ontology annotation and bioinformatics. The results showed that B. cinerea infection changed the expression level of chloroplast proteins, and mainly focused on plant and pathogen interaction, synthesis pathways of plant hormones and alkaloids. Multi-omics analysis further showed that there was a consistent increase in the expression levels of chorismic acid, salicylic acid, isochorismic pyruvate lyase pchB, transcription factor TGA and pathogenesis-related protein PR-1 in the salicylic acid-mediated disease-resistant signal transduction pathway. The full activation of salicylic acid-mediated disease resistance signaling pathway is an effective means for grape leaves to resist B. cinerea infection. The result is of great benefit to further deeply reveal the molecular mechanisms of plant-pathogen interactions and the breeding of pathogen-resistant grape varieties.

Food sciences
Secretory expression of recombinant human acetylcholinesterase gene in Pichia pastoris and evaluation of its sensitivity to pesticides
Fangfang YANG,Jiahui LI,Sainan ZHANG,Zhen WANG,Zhiyin LIAO,Shoufeng WANG
Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(3): 317-324.   https://doi.org/10.3785/j.issn.1008-9209.2018.09.281
Abstract( 18 )   HTML( 1 )   HTML (   PDF(1647KB)( 44 )

Acetylcholinesterase (AChE) is a key enzyme for the detection of organophosphorus and carbamate pesticide residues by enzyme inhibition method. In this study, human acetylcholinesterase (hAChE) was secreted and expressed by Pichia pastoris expression system, and its sensitivity to eight pesticides was evaluated, which laid the foundation for large-scale industrial production of highly sensitive enzyme sources. Primers F1 and R1 were designed according to the published gene sequence of hAChE in GenBank. Then the hAChE gene coding region sequence was amplified by high fidelity PCR using pReceiver-M02-hAChE-ORF plasmid as the template, and the recombinant expression vector pPIC9K-hAChE was constructed by inserting into the vector pPIC9K through double enzyme digestion of SnaB I and Avr Ⅱ. The transformant was selected to sequence and verify the accuracy of the sequence. The linearized pPIC9K-hAChE by Sal I was electro-transformed into P. pastoris GS115. The recombinant strains were screened by minimal dextrase medium (MD) plate and G418 resistance gradient plate. Finally, the positive strain colony was screened on the yeast extract peptone dextrose medium (YPD) plate with 4.00 mg/mL G418 resistance and was identified by polymerase chain reaction (PCR). The expression of the recombinant strain was induced by 0.5% methanol for 144 h. The sodium dodecyl sulfonate (SDS)-polyacrylamide gel electrophoresis map of the fermentation supernatant showed an obvious protein band at 65 kDa, and the size was consistent with the expected hAChE protein. The purified hAChE was obtained using a Q-Sepharose FF chromatography. The content of hAChE protein in the fermentation supernatant was 1.330 mg/mL and accounted for 5.58% in total protein by Bradford method. The highest enzyme activity reached 1 080 nmol/(min?mL) after purification. By preliminary calculation of the 50% inhibitory concentration (IC50) of the eight common pesticides of the fermentation supernatant, it was found that trichlorfon>dichlorvos>arbofuran>malathion>dimethoate>Methamidophos>Carbaryl>chlorpyrifos, suggesting that hAChE is most sensitive to chlorpyrifos.

Resource utilization & environmental protection
Effects of digested pig slurry application on agronomic trait, yield and forage quality of indica rice
Fuyin HOU,Yingjiang CHEN,Zhiqing YANG,Chongfu JIN,Kai SHI,Changkuan CHEN,Gongneng FENG,Hongshan LI
Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(3): 325-331.   https://doi.org/10.3785/j.issn.1008-9209.2018.07.261
Abstract( 19 )   HTML( 4 )   HTML (   PDF(796KB)( 41 )

In order to promote ecological breeding and reasonable utilization of digested pig slurry from large-scale biogas project, we designed 14 treatments (N1-N14) to explore the effects of biogas slurry application on agronomic trait, yield and forage quality of indica rice. The results showed that: 1) The overall growth, yield and quality were better than no nitrogen fertilizer treatment within the range of biogas slurry applied amount (N 40%-480%); and compared with the conventional chemical fertilizer treatment (N2), except that the plant height of N3 treatment and tiller number of N4 treatment were both significantly lower, the plant height, tiller number and effective panicle number in other biogas slurry applied treatments changed no significance (P>0.05), presented the trend of increasing first and then decreasing. 2) Compared with the N2 treatment, with the increase of biogas slurry applied amount, the rice yield of N5 and N8 treatments increased by 9.5% (P>0.05) and 8.9% (P>0.05), respectively. As for the aboveground biomass, N8 and N9 treatments increased by 21.4% (P<0.05) and 5.4% (P>0.05), respectively. The thousand seed mass of N6-N9 treatments indicated an increasing trend with N7 treatment significantly increasing by 11.3% (P<0.05). However, when the biogas slurry applied amount was above N10 (816 t/hm2), the yield of indica rice decreased, even lower than that of N2 treatment. 3) The protein contents of unpolished rice in N5-N14 treatments were higher than that of N1, with the treatments of N7-N14 markedly higher (P<0.05). The amylose contents of unpolished rice in N3-N14 decreased to a certain extent compared with the N2. All in all, application of proper biogas slurry is more conducive to the growth of indica rice than conventional chemical fertilizers in this test. Besides, biogas slurry can effectively improve the yield and forage quality of indica rice when the application amount of biogas slurry is 612 t/hm2 (N8).

Effects of rice-crab culture on nitrogen leaching in rice fields in the north of China.
Ang WANG,Danchao DAI,Xuzhou MA,Qun MOU,Yongqing YU,Weiqun Lü
Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(3): 332-342.   https://doi.org/10.3785/j.issn.1008-9209.2018.03.061
Abstract( 24 )   HTML( 3 )   HTML (   PDF(1247KB)( 38 )

In order to explore the leaching loss of nitrogen (N) in the rice-crab culture system, a field experiment was carried out in Panjin, Liaoning Province. A split-plot design with two factors was arranged in this experiment, with crab or without crab as main factors, with or without N fertilizer as sub-factors. The treatments included: 1) rice monoculture without N fertilizer (R0M); 2) rice-crab culture without N fertilizer (R0C); 3) rice monoculture with N fertilization (with N of 160 kg/hm2, R1M); 4) rice-crab culture with N fertilizer (with N of 160 kg N/hm2, R1C). The results showed that NH4 +-N was the primary form of N in flooding water, accounting for 50.8% of the total N (TN), while NO3 -N contributed the most to N in leaching water, accounting for 58.5% of TN. Fertilization could significantly increase the concentrations of microbial biomass nitrogen (MBN) in soil, N in flooding and leaching water (P<0.05). The mean concentration of soil MBN in rice-crab field was 17.7% higher than that in rice monoculture. Rearing crab significantly increased the concentration of MBN in soil and reduced leaching losses of NO3 -N (P<0.05), but had little effect on N concentrations in flooding water and leaching losses of NH4 +-N and dissolved organic nitrogen (DON). The concentration of NO3 -N in the leaching water showed positive correlation with NO3 -N in flooding water (P<0.01), while the concentration of DON in the leaching water was negatively correlated with the concentration of soil MBN (P<0.01). The cumulative TN leaching of R1M and R1C treatments was 7.6% and 6.3%, respectively, which indicated that the N leaching was not the predominant fertilizer N losses. Rearing crab in rice fields could reduce the cumulative TN leaching by 15.0% at fertilized plots (P<0.05), and 7.2% at no fertilized plots (P>0.05). All in all, the integration of rice and crab cultivation effectively reduces the leaching loss of N fertilizer in rice field.

Effects of super absorbent polymer with different application rates on soil characteristics and flue-cured tobacco growth in Qinba mountain area
Gangshuan BAI,Wei GENG,Dengfeng HE
Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(3): 343-354.   https://doi.org/10.3785/j.issn.1008-9209.2018.07.171
Abstract( 14 )   HTML( 1 )   HTML (   PDF(805KB)( 40 )

In order to improve soil characteristics, reduce the effect of seasonal drought on the growth of flue-cured tobacco, and promote the application of super absorbent polymer in flue-cured tobacco production, we analyzed the effects of different super absorbent polymer application rates on soil moisture, soil bulk density, flue-cured tobacco growth, conventional chemical composition and economic characteristics. The potassium polyacrylate super absorbent polymer of 45, 60 and 75 kg/hm2 was separately applied to the tobacco field with yellow brown soil in Qinba mountain area, and no super absorbent polymer was applied as a control. The results showed that the application rate of 75 kg/hm2 had a relatively large effect on the soil moisture from seedling survival stage to rosette stage of flue-cured tobacco, while the treatment with application rate of 60 kg/hm2 had a relatively greater effect on soil moisture from flourishing growth stage to after maturity stage. However, different application rates of potassium polyacrylate super absorbent polymer had no significant effect on soil moisture from seedling survival stage to after maturity stage. The soil bulk density of 0-20 and >20-40 cm soil layers was significantly reduced by the treatment with application rate of 60 kg/hm2, but the treatments with application rate of 45 and 75 kg/hm2 had no significant effect on soil bulk density. All application rates of potassium polyacrylate super absorbent polymer promoted the growth of flue-cured tobacco, increased flue-cured tobacco yield, conventional chemical composition and output value. The proportion of superior tobacco significantly increased, while the proportion of inferior tobacco significantly decreased, and the net output value of the flue-cured tobacco significantly increased by the treatment with application rate of 60 kg/hm2. In sum, the suitable application rate of potassium polyacrylate super absorbent ploymer is about 60 kg/hm2 in the tobacco field with yellow brown soil in Qinba mountain area.

Allocation analysis of natural and cultivated grassland resources of eco-family pasture based on the theory of grassland agro-ecosystem discordance
Li FU,Yingzhong XIE,Hongbin MA,Ende XING,Xiumin TIAN
Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(3): 355-364.   https://doi.org/10.3785/j.issn.1008-9209.2018.10.220
Abstract( 16 )   HTML( 3 )   HTML (   PDF(1162KB)( 39 )

In order to achieve sustainable development in pastoral areas, based on the theory of grassland agro-ecosystem discordance, we selected the meteorological factors in the study area of Otog Front Banner in recent 30 years and the water, soil, grass, and livestock in the area in recent years as the objects of study to discuss how to make a grassland resource allocation plan. The result showed that the natural environment in the study area was harsh and the annual precipitation was very limited; the groundwater resources were abundant but unevenly distributed, and the water consumption for agriculture and animal husbandry exceeded the expected limit. The proportion of natural grassland was obvious, but the overgrazing was serious, and the ecosystem was fragile, and the contradiction of herbage resources was prominent. The most important problem was time paradox, followed by space paradox, and finally by species paradox in the development of family ranches. Meanwhile, in the time paradox, it was necessary to reasonable matching cultivation of grassland resources according to the shortage period and surplus period of natural grassland; in the space paradox, on the one hand, developing high-yielding and high-quality cultivated grasslands to replace natural grassland functions in the towns of Aolezhaoqi and Chengchuan, and on the other hand, it was necessary to strictly control over the stock capacity of natural grassland, develop short-term fattening and improve turnover efficiency in the towns of Aangsu and Shanghaimiao.

Determination and ecological risk assessment of phthalic acid esters in marine sediments
Hongmei HU,Tiejun LI,Lu ZHANG,Qing HAO,Xiumei SUN,Yanjian JIN,Zhongzhen YING,Yuanming GUO
Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(3): 365-375.   https://doi.org/10.3785/j.issn.1008-9209.2018.03.282
Abstract( 18 )   HTML( 1 )   HTML (   PDF(1556KB)( 39 )

We proposed a new method for the determination of 16 phthalic acid esters (PAEs) in marine sediments using gas chromatography-mass spectrometry (GC-MS) combined with ultrasonic extraction and dispersive solid phase extraction cleanup. Moreover, the contents of PAEs in marine sediments of Sanmen Bay, Taizhou and waters of Nanji Island, Wenzhou were detected and their ecological risk was assessed. Under the optimized condition, the linearity of this method ranged from 1 to 1 000 μg/L, with regression coefficients ranging between 0.999 1 and 0.999 9. The limits of detection were in the rang of 0.10-0.25 μg/kg. The recoveries of spiked 16 PAEs at different concentration levels were in the range of 75%-116%, with relative standard deviations of 3.1%-7.2%. The results showed that the total concentration of phthalate esters ranged from 297.43 to 550.25 μg/kg. Diisobutyl phthalate (DIBP), di-n-butyl phthalate (DBP) and di (2-ethylhexyl) phthalate (DEHP) were the predominant compounds in all detected seafood, accounted for 88.7%-96.7% of the total PAEs. Marine sediment’s ecological risk was assessed with the methods of sediment quality guidline and risk quotient. The sediment quality guidline results showed the contents of PAEs in the sediments were all lower than the environmental risk limits, which indicated low ecological risk. The risk quotient values of PAEs were in the following order: DBP>DIBP>DEHP, and DBP was high risk to algae and medium risk to crustaceans and fish, and DIBP was medium risk to crustaceans, and DEHP was low risk to aquatic organisms. Overall, the risk of PAEs on algae was the largest, followed by crustaceans, minimal risk for fish, and the health risk of Sanmen Bay, Taizhou was slightly higher than that of waters of Nanji Island, Wenzhou.

Animal sciences & veterinary medicine
Identification for internal reference genes in different periods of granulosa cells of Tianfu meat geese.
Yuanliang MO,Yushi WANG,Jiwen WANG
Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(3): 376-384.   https://doi.org/10.3785/j.issn.1008-9209.2018.03.132
Abstract( 29 )   HTML( 3 )   HTML (   PDF(1963KB)( 46 )

In order to screen out the most stable reference genes in different periods of granulosa cells in goose, we selected 10 candidate reference genes (GAPDH, ACTB, TUB, UBC, HMBS, SDH, 18S, 28S, TBP, HPRT1) to determine the relative expression levels by the real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). The expression stabilities of 10 candidate reference genes in nine different stages of granulosa cells were systematically analyzed by delta-C T, qbase+, NormFinder and BestKeeper, respectively. The results of RT-qPCR melting curve and PCR amplification showed that the 10 candidate reference genes were specifically amplified. By constructing a standard curve, between C q value and the logarithm of relative copy number exhibited a good linear relationship in the serial dilution concentration gradient. Based on the evaluation results of four different algorithms, SDH, HMBS and 18S were found to be three of the most stable reference genes, but UBC, GAPDH and TUB were three of the least stable reference genes in the different periods of granulosa cells. Therefore, the most stable internal reference genes were SDH and HMBS in granulosa cells at different developmental stages, and it could get more accurate normalization of RT-qPCR data by geometric averaging of the most stable reference genes.

Establishment and application of a polymerase chain reaction method for detection of Melissococcus pluton in honeybee (Apis cerana cerana) in Chunan County of Zhejiang Province
Rui GUO,Jie DONG,Haikun GUO,Junzhu LI,Deqian WANG
Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(3): 385-390.   https://doi.org/10.3785/j.issn.1008-9209.2018.07.181
Abstract( 16 )   HTML( 2 )   HTML (   PDF(1355KB)( 42 )

A polymerase chain reaction (PCR) method was developed to detect Melissococcus pluton and an epidemiological investigation of European foulbrood (EFB) was done in the main bee (Apis cerana cerana) breeding area of Chun’an County, Zhejiang Province. Results indicated that the newly developed method was specific, sensitive, repeatable and accuracy. The epidemiological survey results showed that a total of 11 townships were infected by M. pluton among the 298 bee samples in 17 townships of Chun’an County. The total infection rate was 25.2%, of which EFB infection rate of five townships was ≥40%. In a word, the newly established method for PCR detection makes the infection status of EFB clear in Chun’an County, and it lays a scientific basis for formulating rationally prevention measures of EFB.

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