Scientia Agricultura Sinica

Development and Application of the Functional Marker for the Broad-Spectrum Blast Resistance Gene PigmR in Rice

WANG FangQuan,CHEN ZhiHui,XU Yang,WANG Jun,LI WenQi,FAN FangJun,CHEN LiQin,TAO YaJun,ZHONG WeiGong,YANG Jie

Scientia Agricultura Sinica. 2019, 52(6): 955-967. doi:10.3864/j.issn.0578-1752.2019.06.001

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【Objective】Rice blast is one of the most serious rice diseases in the world. The objective of this study was to develop the functional marker for broad-spectrum blast resistance gene PigmR in rice. The marker improved the application of PigmR in blast resistance rice breeding. 【Method】The characteristics of PigmR were analyzed by Snapgene 2.3.2, and the specific function markers were designed with Oligo 7. To avoid mistake results of the PCR amplification failure, the functional marker was optimized by the specific primers of Actin1 as a reference. The parent materials, the monogenic lines of Lijiangxintuanheigu (LTH), the bridge materials, and the BC₁F₃ population lines of Nangeng 53045/Gumei 4 were identified by the functional markers. The blast isolates in this study were the mixed representative strains of rice blast in Jiangsu Province (2018-4, 2018-65, 2018-102, 2018-222, and 2018-241). The tested isolates were transplanted into RCA medium, cultured at 25 ℃ for 7 d, and irradiated for 72 h. After spores were produced, they were washed with sterile water and then formulated 30-40 spores per field in 10×10 microscope. The mixed spores were injected into each panicle with 1 mL of blast isolates solution at 3-4 days before heading. The resistance was investigated after the rice grains were matured.【Result】Eight pairs of molecular markers were designed, according to the sequence difference of PigmR and PigmS, Pigm-R4. By molecular detected, the function marker of PigmR, GMR-3, could specifically amplify PigmR from Gumei 4 with 98 bp fragment, and no fragment was amplified in the samples without PigmR. The functional marker was optimized by different concentrations of GMR-3 and Actin1-1 (a marker for the internal reference gene Actin1), results shown that the marker consist of 0.4 μmol·L ^-1 GMR-3 and 0.1 μmol·L ^-1 Actin1-1 had the best effect. The functional marker was named GMRA. Samples carrying PigmR were amplified the expected size of 146 and 98 bp by GMRA. By contrast, samples without PigmR were only amplified a 146 bp fragment. The 229 rice materials were detected with GMRA, only Gumei 4 amplified the size of 146 and 98 bp, others only amplified a 146 bp fragment. Furthermore, the results detected the monogenic lines of LTH by GMRA suggested that the marker had a strong specificity and could effectively distinguish PigmR from homology genes, such as Pi9, Piz, and Piz-t. Moreover, three donor materials carrying PigmR were obtained from 240 bridge materials by GMRA. By molecular marker-assisted selection (MAS) of GMRA, PigmR was transferred to the good eating quality rice cultivar Nangeng53045 by backcrossing. The BC₁F₃ plants with PigmR showed resistant/middle resistant to the panicle blast, while others showed high susceptible. It was suggested that PigmR observably improved the resistance of Nangeng53045-Pigm lines in the panicle blast.【Conclusion】In conclusion, the functional marker of PigmR can be effectively used for genetic improvement in breeding and germplasm screening.

Effects of Different Red and Blue Ratios on the Somatic Embryogenesis and Plant Regeneration of Cotton

WEI Xi,WANG QianHua,GE XiaoYang,CHEN YanLi,DING YanPeng,ZHAO MingZhe,LI FuGuang

Scientia Agricultura Sinica. 2019, 52(6): 968-980. doi:10.3864/j.issn.0578-1752.2019.06.002

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【Objective】Different light quality combinations were set up to clarify the effects of different light quality on cotton somatic embryogenesis, and to provide basis for improving cotton somatic embryogenesis ability and accelerating cotton genetically modified process. 【Method】The light quality combinations B﹕R (blue﹕red) = 1﹕1, B﹕R (blue﹕red) = 3﹕1, B﹕R (blue﹕red) = 1﹕3 and DL (fluorescent lamp) with different red and blue ratios were set respectively. The hypocotyl segments of CCRI12 cotton were cultured on the callus induction medium (CIM), embryogenic callus induction medium (ECDM), somatic embryo induction medium (EIM) and root induction medium (RIM) in turn. Under four different light quality ratios, the corresponding indicators of somatic embryogenesis at different stages were counted, including callus proliferation rate (CPR), callus proliferation, embryogenic callus differentiation rate, somatic embryo number, rooting rate, hypocotyl number, regenerated cotton plant number, chlorophyll concentration, etc.【Result】Compared with B﹕R=1﹕3 and DL light quality combinations, B﹕R=1﹕1 and B:R=3﹕1 light quality combinations significantly promoted callus proliferation. Compared with B﹕R=1﹕1 and B﹕R=3﹕1 light quality combinations, B﹕R=1﹕3 light quality combination significantly increased weight proliferation of callus without EC differentiation ability. B﹕R=1﹕3 and DL light quality combinations had significantly higher embryonic callus differentiation rate than B﹕R=1﹕1 and B﹕R=3﹕1 light quality combinations. The number of somatic embryos induced by B﹕R=1﹕3 treatment was significantly higher than that of B﹕R=1﹕1 and B:R=3﹕1 light quality combination, but there was no significant difference compared with DL; the number of hypocotyls induced by B﹕R=1﹕1 and B﹕R=3﹕1 light quality combination was significantly lower than that induced by B﹕R=1﹕3 and DL light quality combination, and the number of somatic embryos induced by B﹕R=1﹕1 light quality combination and B﹕R=3﹕1 light quality combination was significantly lower than that induced by B﹕R=1 light quality combination and B﹕R=3﹕1 light. The plant height of B﹕R=1﹕1 and B﹕R=1﹕3 combinations was significantly higher than that of B﹕R=1﹕3 and DL combinations; the chlorophyll content of B﹕R=1﹕3 treatment was higher than that of B﹕R=1﹕1 treatment, but the chlorophyll content of B﹕R=1﹕3 treatment was significantly higher than that of B﹕R=3﹕1 and DL light quality combination.【Conclusion】The regulation of different light quality combinations in different stages of somatic embryogenesis was clarified. Optimal light quality combinations could be selectively applied in different stages of somatic embryogenesis. The combination of red and blue B﹕R=1﹕3 can promote the somatic embryogenesis of cotton.

The Daily Yield of Medium Hybrid Rice in Machine Transplanting and Its Relationship with Plant Type

HE LianHua,CHEN Duo,ZHANG Chi,TIAN QingLan,WU ZhenYuan,LI QiuPing,ZHONG XiaoYuan,DENG Fei,HU JianFeng,LING JunYing,REN WanJun

Scientia Agricultura Sinica. 2019, 52(6): 981-996. doi:10.3864/j.issn.0578-1752.2019.06.003

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【Objective】To explore the feasibility of daily yield as a screening of medium-indica hybrid rice varieties by machine transplanted, systematically study the plant type characteristics of medium-indica hybrid rice varieties and their relationship with daily yield, and screen out high-yield varieties suitable for machine-planting in Sichuan, the theory and practice basis were provided for the high-yield cultivation and breeding of medium-indica hybrid rice.【Method】The 34 planting hybrid rice varieties were used as materials, and the data were collected by single-factor randomized block field experiment, and then the plant type and daily yield of the hybrid rice varieties under machine transplanting were studied by cluster analysis and analysis of variance analysis. 【Result】(1)The whole rice growth period, daily yield and yield of different varieties of machine-planting cultivation were different. The whole growth period of the tested rice varieties was 141-168 d, mainly 154-164 d, accounting for 82.4% of the tested varieties; The yield variation was 10 261.05 -13 099.34 kg·hm ^-2, of which Tianyouhuazhan accounted for the highest yield, Yixiang 3728 was the lowest, the yield in the range of 11 110.90-12 827.15 kg ·hm ^-2 accounted for 76.5% of the tested varieties; The daily yield of the tested varieties was mainly distributed in the range of 67-80 kg·hm ^-2·d ^-1, accounting for 82.4% of the tested varieties. The daily yields of Tianyouhuazhan, Fanyou 609 and Jingliangyou 534 were higher than 80 kg·hm ^-2·d ^-1, but only 8.8% of the tested varieties. (2) Cluster analysis showed that 29.4% of the total varieties were high-daily yield, 38.2% were middle-daily yield and 32.4% were low-daily yield. (3) Compared with the low-yield varieties, the high-yield varieties had higher grain per panicle and group spikes, and the effective panicles were sufficient, and the seed setting rate was higher, so the yield was higher. (4) There were significant differences in plant type characteristics of different varieties. Correlation analysis showed that plant height, stalk length, N3 internode length, N4 internode length, panicle length and daily yield were significantly or significantly negative correlation, grain density was significantly positively correlated with daily yield.【Conclusion】The daily yield could be used as one of the important indicators for screening of Chinese cultivars for hybrid rice cultivars, and the varieties with higher daily yields had stronger adaptability. Plant height and stalk length were suitable, and the third and fourth internode lengths were short, and high grain density was an important plant type characteristic of high daily yield varieties. In addition, the medium-indica hybrid rice varieties suitable for machine transplanting in Sichuan also had the basic characteristics of moderate growth period, more spikelets, higher group spikelets and higher seed setting rate. On the whole, Tianyouhuazhan, Fanyou 609, Jingliangyou 534, Y Liangyou 1, C Liangyouhuazhan and F You 498 were suitable for planting in Sichuan.

Pixel-Based and Object-Oriented Classification of Jujube and Cotton Based on High Resolution Satellite Imagery over Alear, Xinjiang

JI XuSheng,LI Xu,WAN ZeFu,YAO Xia,ZHU Yan,CHENG Tao

Scientia Agricultura Sinica. 2019, 52(6): 997-1008. doi:10.3864/j.issn.0578-1752.2019.06.004

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【Objective】Jujube and cotton are widely cultivated in Xinjiang. In this study, we had an insight into the planting area and distribution area of jujube and cotton quickly and accurately by crop identification based on remote sensing technology, which was helpful to policies making and crops precise management.【Method】This paper evaluated the pixel-based and object-oriented classification methods for crop mapping using several mono-temporal (date of image acquisition: May 10, 2016; September 7, 2016; and October 8, 2016) high spatial resolution images of Alear city, Xinjiang. This research involved four different machine learning algorithms, including Spectral Angle Mapping (SAM), Support Vector Machine (SVM), CART Decision Trees (DTs) and Random Forest (RF). 【Result】 The results showed that it had the highest crop classification accuracy when using the satellite images acquired on May, followed by the satellite images acquired on October. Crop classification accuracy was the lowest when using the satellite image acquired on September. In addition, compared to pixel-based classification methods, the classification accuracies were improved when using object-oriented classification methods. The classification accuracies of each period were improved by 4.83%, 7.77%, and 7.22%, respectively. The highest classification accuracy was 93.52% (May 10, 2016), 85.36% (September 7, 2016), and 88.88% (October 8, 2016), respectively.【Conclusion】 The research results suggested that using the image acquired in May, which covers cotton seedling stage, could improve crop classification accuracy for our study area. The cotton in this period was covered by plastic film, and the jujube trees showed obvious spectral characteristics of vegetation. The two crops showed different spectral characteristics in the early stage of growth. Therefore, crop classification should be executed at the early growth stage. What’s more, spectral, texture and spatial features can be combined when using object-oriented classification methods, especially the addition of texture information, so that the overall accuracy of crop classification in each period was improved (except SAM). We can delineate the field boundaries efficiently by this method, which is important for the improvement of local crop field management. Additionally, texture features were more important than spectral and spatial features. Green and red bands had a greater contribute on crop classification.

Development of 10% Diethofencarb·Procymidone Micropowder and Its Control Efficacy to Cucumber Corynespora Leaf Spot

CHUAI HongYun,SHI YanXia,CHAI ALi,YANG Jie,XIE XueWen,LI BaoJu

Scientia Agricultura Sinica. 2019, 52(6): 1009-1020. doi:10.3864/j.issn.0578-1752.2019.06.005

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【Objective】 The objective of this study is to obtain the optimal mixing ratio of diethofencarb and procymidone, which had high inhibitory activity against Corynespora cassiicola, the micropowder was prepared and its control efficacy on cucumber leaf spot was determined in the field. 【Method】 The inhibitory activity of the mixtures at different mixing ratios to C. cassiicola was determined by mycelial growth rate method. The synergistic effect was evaluated according to the Wadley formula and the best synergism combination was defined. Through single factor test, orthogonal test, the carrier, additives and optimal ratio were screened, the best formula was determined and then micropowder was processed by using air jet mill. The control efficacy of micropowder on cucumber leaf spot was determined. 【Result】 When the mass ratio of diethofencarb and procymidone was 1﹕1 and 1﹕4, the virulence synergistic effect against C. cassiicola strains HG09112606, FQ07091401 and HG11011509 was observed, with the synergistic ratios of 3.19, 2.53, 1.68; 1.99, 1.77, 1.98, respectively. Among which, the ratio of 1﹕1 had a significant synergistic effect. Active ingredient diethofencarb and procymidone account for 10%, dispersant NNO accounts for 3%, surfactant K12 accounts for 3%, stabilizer polyoxyethylene alkyl ether accounts for 1%, carrier carbon-white is 15% and diatomite make up to 100%. The results showed that the above formulation had excellent characteristics, and the particle size is 6.18 μm, the dispersion index is 95.18%, the planktonic index is 86.26, the water content is 1.24%, the slope angle is 67°, and the thermal storage decomposition rate is 4.12%. All above test results are in accordance with standard. In the pot experiment, the control efficacy of 10% diethofencarb and procymidone micropowder on the cucumber leaf spot by powder injection method was 89.82% when the dosage was 100 g a.i./hm ², which was significantly higher than that of the control agent 35% difenoconazole·prochloraz emulsion in water (EW) and 43% fluopyram·trifloxystrobin suspension concentrate (SC) agent spray at the recommended dosage. In the field test, the control efficacy of 10% diethofencarb and procymidone micropowder on the cucumber leaf spot by powder injection method was 84.39% when the dosage was 100 g a.i./hm ², which had no significant difference with that of spray with 35% difenoconazole·prochloraz EW in the amount of 300 g a.i./hm ², 43% fluopyram·trifloxystrobin SC in the amount of 90 g a.i./hm ². 【Conclusion】Diethofencarb and procymidone used in combinations had different degrees of synergistic interaction against cucumber Corynespora leaf spot. The 10% diethofencarb·procymidone micropowder prepared according to the selected formula has the advantages of small particle size, high dispersion index and planktonic index, and storage stability. It has a broad application prospect in the control of cucumber Corynespora leaf spot.

Comparison of Metabolic Profile Between Diapause-Destined and Non-Diapause-Destined Pupae of Bactrocera minax

WANG Jia,WANG Pan,FAN Huan,LIU YingHong

Scientia Agricultura Sinica. 2019, 52(6): 1021-1031. doi:10.3864/j.issn.0578-1752.2019.06.006

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【Objective】The objective of this study is to determine the metabolic profile differences of diapause-destined (D) and non-diapause-destined (ND) pupae of Bactrocera minax at different time points, and between D and ND pupae at the same time point, and to identify metabolites with significant difference in different comparisons.【Method】Within 1 d after pupation, 20E solution was injected to release diapause and acquire ND pupae; 10% ethanol was injected to acquire D pupae. Both types of pupae were collected at 1, 15, and 30 d after injection, and the samples were detected by nuclear magnetic resonance (NMR). All the spectra were analyzed with the Chenomx Compound Library one by one to determine the metabolite species and the corresponding concentration. Then, the partial least squares discriminant analysis (PLS-DA) was conducted to determine the metabolomic variations in 5 comparisons, D1 vs D15 vs D30, ND1 vs ND15 vs ND30, D1 vs ND1, D15 vs ND15, and D30 vs ND30. Lastly, the variable importance in projection (VIP) and one-way ANOVA/t test were performed to find out the metabolites with significant concentration difference in different contrast combinations.【Result】In all samples, a total of 50 metabolites and their concentrations were obtained, including 20 kinds of amino acids and their derivatives, 11 kinds of organic acids, 4 kinds of sugars, 5 kinds of nucleic acid components, and 10 kinds of other metabolites. PLS-DA analysis indicated that the metabolic profile of D pupae was significantly different among the three time points. The metabolic profile of ND pupae was not significantly different between 15 d and 30 d after injection, but both of them were significantly different with that of 1 d after injection. At three different time points, the metabolic profiles of D pupae were all significantly different from those of ND pupae. VIP analysis and one-way ANOVA/t test collectively indicated that the concentrations of 21 metabolites were significantly different in at least one comparison. Of these 21 metabolites, 10, 12, 6, 12, and 8 species were found in D1 vs D15 vs D30, ND1 vs ND15 vs ND30, D1 vs ND1, D15 vs ND15, and D30 vs ND30 comparisons, respectively. Citrate, glucose, maltose, proline, trehalose, glutamate, asparagine, and N-acetylglutamate showed significantly different level in most comparisons.【Conclusion】Among the metabolites with the most significant differences, citrate, glucose, maltose, proline, trehalose, glutamate, and asparagine can directly or indirectly enter the tricarboxylic acid (TCA) cycle, indicating that the energy metabolism and substance transformation have significant changes during B. minax diapause. In addition, the significant differences of trehalose, proline, N-acetylglutamate, and inositol contribute to the regulation of freezing tolerance during B. minax diapause. The results can provide a basis for further investigation of mechanisms of important metabolites and relevant physiological pathways during B. minax diapause.

Spatial and Temporal Variation of Soil Organic Matter and Cause Analysis in Hainan Island in Resent 30 Years

JIANG SaiPing,ZHANG RenLian,ZHANG WeiLi,XU AiGuo,ZHANG HuaiZhi,XIE LiangShang,JI HongJie

Scientia Agricultura Sinica. 2019, 52(6): 1032-1044. doi:10.3864/j.issn.0578-1752.2019.06.007

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【Objective】Soil organic matter (SOM) is an important index for evaluating soil fertility and soil carbon pools. SOM usually has strong spatial and temporal variability for the complicated soil forming process and human activity. Studying the temporal and spatial variability of SOM could provide an important reference for the adjustment of agricultural planting structure and response to global climate change.【Method】The contents of SOM of 0-20 cm soil layer in Hainan Island in 1980s and 2012 were collected and measured by field investigation and soil sampling. RandomForest (RF) model was used to predict spatial distribution of SOM based on the training set of 410 and 128 samples, and then the predictions were validated with validating set of 103 and 32 samples, respectively. Then the spatial and temporal variability and driving factors of different land use types in Hainan Island in resent thirty years were studied by using statistical method combined agricultural statistical data.【Result】The mean value of SOM contents in 1980s was 20.57 g·kg ^-1, showing a distribution that SOM contents gradually decreased from southwest to northeast parts of the island. And SOM contents were mainly in two levels of 15-20 and 20-30 g·kg ^-1, which occupied 75.29% area of the whole island. The mean value of SOM contents in 2012 was 15.89 g·kg ^-1, showing a distribution that SOM contents were higher in the southwest and northeast parts, lower in the west and south parts. And the contents of SOM were mainly in two levels of 10-15 and 15-20 g·kg ^-1, which occupied 78.28% area of the whole island, but the ratio of 15-20 and 20-30 g·kg ^-1 levels was 66.04%, which lowered 9.45 percentage points compared with 1980s. The order of SOM contents in different land use types was: paddy field > garden plot > dry land. And there was a significant difference in SOM contents among three land use types in 1980s. In 2012, there was a significant difference in SOM contents between paddy fields and dry land, dry land and garden plot, while the difference in SOM contents between paddy fields and garden plot was not significant. 【Conclusion】 After nearly 30 years, SOM contents of Hainan Island showed a decreasing trend overall. In 2012, the mean value of SOM contents decreased by 4.68 g·kg ^-1 compared with 1980s, with a reduction of 22.75%. The mean value of SOM contents of paddy field decreased by 6.42 g·kg ^-1, with a reduction of 27.34%, which was most obvious among three land use types. And the mean value of SOM contents of garden plot decreased by 2.65 g·kg ^-1, with a reduction of 14.25%, while the mean value of SOM contents of dry land decreased by 1.28 g·kg ^-1, with a reduction of 8.84%. Rice crop rotation, land reclamation and increasing multiple crop indexes were the main reasons that caused the decrease of SOM contents in Hainan Island.

Comprehensive Effects of Organic Materials Incorporation on Soil Hydraulic Conductivity and Air Permeability

ZHAO LiLi,LI LuSheng,CAI HuanJie,SHI XiaoHu,XUE ShaoPing

Scientia Agricultura Sinica. 2019, 52(6): 1045-1057. doi:10.3864/j.issn.0578-1752.2019.06.008

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【Objective】 The comprehensive analysis of soil water and gas transport properties in response to different types of organic amendments is important for optimizing water and air environment of root-zone soil and improving the soil productivity. 【Method】 For this purpose, a two-year (from June 2014 to June 2016) field experiment was conducted with a fixed plot test on a Lou soil (Eum-Orthic Anthrosols) in the Guanzhong Plain. There were five treatments: application of mineral fertilizer both alone (control, CK) and along with wheat straw (MWS), wheat husk (MWH), farmyard soil (MFS), and bioorganic fertilizer (MBF). This experiment was used to study the effects of combined use of mineral fertilizer and organic materials on soil porosity, hydraulic conductivity and air permeability in the 0-30 cm soil layer. These soil parameters were comprehensively evaluated by using the principal component analysis method.【Result】 Integrated application of organic materials and mineral fertilizer improved soil pore size distribution and promoted the increase in macro-porosity compared to the CK treatment. This was especially true at the 0-10 cm and 20-30 cm soil depths, where incorporation of organic materials significantly (P<0.05) increased soil macro-porosity by 12.3%-136.4% compare to the CK treatment. The significant (P<0.05) increase in the macro-porosity was also recorded in the MWS treatment at the 10-20 cm depth soil layer compared to the CK treatment. Combination with organic materials and mineral fertilizer significantly (P<0.05) enhanced soil hydraulic conductivity at the 0-10 cm and 10-20 cm soil depths compared to the CK treatment, such as increasing initial infiltration rate, steady infiltration rate, average infiltration rate, 90 min cumulative infiltration, as well as saturated hydraulic conductivity. These parameters were highest in the MWS treatment (greater by 5.3-8.8 times compared to the CK treatment) at the 0-10 cm soil depth and in the MBF treatment (greater by 2.0-4.5 times compared to the CK treatment) at the 10-20 cm soil depth, respectively (P<0.05). Meanwhile, the MBF treatment also significantly (P<0.05) improved soil hydraulic conductivity relative to the CK treatment at the 20-30 cm soil depth. With regard to soil air permeability, the MWS and MWH treatments led to significantly (P<0.05) better soil pore continuity and hence higher soil air permeability at the 0-10 cm soil depth, compared to the CK treatment, while the MBF treatment yielded significantly (P<0.05) higher soil air permeability at both the 10-20 cm and 20-30 cm soil depths. The principal component analysis results indicated that the MWS treatment had the strongest improvement on soil water-gas transport properties at the 0-10 cm soil depth, and the MBF treatment had the strongest improvement at 10-20 cm and 20-30 cm soil depths. 【Conclusion】 To alleviate sub-surface soil compaction and improve soil water-gas transport properties, application of bioorganic fertilizer was highly recommended. The MBF treatment exhibited the best improvement in soil water-gas transport properties at 10-30 cm soil depth.

Physiological Mechanism of S-adenosylmethionine on Alleviating Chlorothalonil Residue in Tomato

GU ChaoHeng,YAN YanYan,WEI XiYa,SHI QingHua,GONG Biao

Scientia Agricultura Sinica. 2019, 52(6): 1058-1065. doi:10.3864/j.issn.0578-1752.2019.06.009

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【Objective】 In order to provide basic mechanism for vegetable safe production, this study aimed at revealing the physiological mechanism of S-adenosylmethionine (SAM)-induced pesticide metabolism. 【Method】 The fresh-eating tomato ‘QianXi’ and broad-spectrum pesticide ‘chlorothalonil, CHT’ were selected as the experimental materials. Then, we studied the effects of exogenous SAM on the metabolism of pesticide, reactive oxygen species and glutathione. 【Result】 The results showed that the CHT residues were significantly reduced by exogenous spraying 0.5-2 μmol·L ^-1 SAM. However, no significantly changes could be observed when the concentrations of SAM arrived at 0.5 μmol·L ^-1. Compared with control, the activities of glutathione S-transferase (GST), glutathione reductase (GR) and dehydroascorbate reductase (DHAR) were significantly increased under CHT treatment. And similar results could be obtained with the contents of glutathione (GSH), oxidized glutathione (GSSG) and total glutathione (GSH+GSSG). The GSH/GSSG were induced by CHT firstly, and then decreased. At the same time, superoxide anion ($\mathop{{O}}_{2}^{{\mathop{}_{\ ·}^{-}}}$) and hydrogen peroxide (H₂O₂) were significantly induced by CHT. However, no significant changes were observed in malondialdehyde (MDA) content. Importantly, exogenous application of 0.5 μmol·L ^-1 SAM had significant effects on improving the activities of GST, GR and DHAR, as well as contents of GSH and GSH+GSSG. Similarly, no significant changes were observed in MDA contents among different treatments. 【Conclusion】 Taken together, exogenous application of 0.5 μmol·L ^-1 SAM could active the key enzymes of GR and DHAR to improve the glutathione cycle and GSH regeneration, which induced the CHT metabolism of tomato fruit depending on GST, $\mathop{{O}}_{2}^{{\mathop{}_{\ ·}^{-}}}$, and H₂O₂ pathway.

Effects of Shading on Main Quality Components in Tea (Camellia Sinensis (L) O. Kuntze) Leaves Based on Metabolomics Analysis

CHEN QinCao,DAI WeiDong,LIN ZhiYuan,XIE DongChao,LÜ MeiLing,LIN Zhi

Scientia Agricultura Sinica. 2019, 52(6): 1066-1077. doi:10.3864/j.issn.0578-1752.2019.06.010

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【Objective】Shading treatment is widely used to improve the quality of teas in tea yielding. However, the effects of dark shading on the quality of teas remain unclear. This study focused on the effects of dark shading on the main quality components in teas to understand the relationship between shading and tea quality in detail.【Method】Moderate (65.0%) and dark (99.7%) shading treatments were applied to tea plants by using black shading net, with non-shading as control. Ultraviolet spectrophotometry was employed to determine the contents of total polyphenols, total amino acids and total flavonoids, and ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) was employed to investigate the quality components in tea leaves.【Result】Compared with the control group, moderate shading treatment significantly reduced the contents of total amino acids and total flavonoids (P<0.05), and slightly reduced the content of total polyphenols, and therefore increased the ratio of polyphenols to amino acids. Dark shading treatment significantly reduced the contents of total amino acids and total flavonoids, while significantly increased the contents of total polyphenols and the ratio of phenol to ammonia (P<0.05). Principal component analysis showed that the two shading treatments significantly changed the metabolite pattern in tea leaves. A total of 87 compounds were identified, including 2 alkaloids, 18 amino acids, 12 catechins, 8 dimeric catechins, 19 flavone glycosides and flavonol glycosides, 5 glycosidically bound volatiles (GBVs), 6 nucleosides and nucleotides, 9 phenolic acids, and 8 other compounds. The contents of 82 compounds in two shading groups showed significant differences comparing with the control (P<0.05). After shading, the contents of alkaloids significantly increased; amino acids showed various change trends, with the contents of more than half of amino acids significantly decreased. The contents of several catechins and dimeric catechins significantly decreased in moderate shading group, while the contents of most catechins and their dimers significantly increased in dark shading group. The content of most flavone glycosides and flavonol glycosides showed decreased trend along with the increase of shading degree. The contents of most GBVs significantly increased. The contents of most of nucleosides and nucleotides significantly decreased; the content of most phenolic acids significantly increased.【Conclusion】After dark shading treatment, the contents of alkaloids, catechins and dimeric catechins significantly increased, while the contents of amino acids significantly decreased, and therefore the ratio of phenol to ammonia significantly increased, which indicated that dark shading might not contribute to improve the tea quality.

Chromium (III) Stress Alleviation by Sulfur Compounds During Chromium Bio-enrichment by Saccharomyces cerevisiae

LI HanTong,JIA ChengLi,ZHANG ShuWen,LU Jing,PANG XiaoYang,LIU Lu,LÜ JiaPing

Scientia Agricultura Sinica. 2019, 52(6): 1078-1089. doi:10.3864/j.issn.0578-1752.2019.06.011

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【Objective】The objective of this study was to investigate the antioxidative mechanism and role of sulphur during chromium (Ⅲ) enrichment by Saccharomyces cerevisiae. The mechanisms of alleviation chromium (Ⅲ) toxicity against yeast by sulphur were revealed. 【Method】Saccharomyces cerevisiae YSI-3.7 was used in this study. Various incubation conditions were investigated, such as various concentrations Cr(Ⅲ) and sulfate. And the corresponding biomass, total chromium content, organic chromium content and oxidative stress markers (including malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT) and so on), were measured and analyzed. 【Result】 Low Cr(Ⅲ) concentration (0-200 μg?mL ^-1) could stimulate the growth of yeast, while high Cr(Ⅲ) concentration (>500 μg?mL ^-1) inhibited its growth. After Saccharomyces cerevisiae YSI-3.7 was incubated with 500 μg?mL ^-1 Cr(Ⅲ) for 44 h, the content of organic Cr in yeast was found to be 725.55±55.08 μg?g ^-1 DCW and that of total Cr was 1255.53±43.75 μg?g ^-1 DCW. After Saccharomyces cerevisiae YSI-3.7 was incubated with 800 μg?mL ^-1 Cr(Ⅲ) for 44 h, the content of organic Cr in yeast was found to be 536.25±36.89 μg?g ^-1 DCW and that of total Cr was 1812.22±38.24 μg?g ^-1 DCW. The content of MDA increased (from 11.83 nmol?mL ^-1 to 18.04 nmol?mL ^-1) with the increasement of Cr(Ⅲ) concentration (0-800 μg?mL ^-1), while the activity of SOD and CAT decreased. The content of GSH, total sulfhydryl and T-AOC increased at lower Cr(Ⅲ) concentration (≤500 μg?mL ^-1), and decreased at the high concentration (800 μg?mL ^-1). The supplementation of 1 mmol?L ^-1 Na₂SO₃during incubation could alleviate the stress of Cr(Ⅲ) against yeast. The protein content increased and MDA content decreased (12.83%) with the addition of 1 mmol?L ^-1 Na₂SO₃ during incubation. The activity of CAT was almost unaffected. The activity of SOD was increased to 4.41%. GSH, T-AOC and GSH-Px content increased to 28.83%, 14.29% and 18.80%, respectively. 【Conclusion】During the Cr(Ⅲ) bio-enrichment process by yeast, Cr(Ⅲ) stress could aggravate the lipid peroxidation of cell membrane. At low Cr(Ⅲ) concentration(0-500 μg?mL ^-1), yeast could protect itself from this stress by its own antioxidant enzymes, among which glutathione and its related enzymes played an important role. At high concentration of Cr(Ⅲ) (800 μg?mL ^-1) , the degree of membrane lipid peroxidation was aggravated and the yeast’s own antioxidant capacity was not enough to protect itself from Cr(Ⅲ) stress. Supplementation of S (1 mmol?L ^-1 Na₂SO₃) could mitigate membrane lipid peroxidation caused by Cr(Ⅲ)by improving SOD activity, GSH, T-AOC and GSH-Px content in yeast, improving the antioxidant capacity of yeast itself and Cr(Ⅲ) bio-enrichment by yeast.

Differential circRNA Analysis in the Spleen of Hu-sheep Lambs Infected with F17 Escherichia coli

ZOU ShuangXia,JIN ChengYan,BAO JianJun,WANG Yue,CHEN WeiHao,WU TianYi,WANG LiHong,LÜ XiaoYang,GAO Wen,WANG BuZhong,ZHU GuoQiang,DAI GuoJun,SHI DongFang,SUN Wei

Scientia Agricultura Sinica. 2019, 52(6): 1090-1101. doi:10.3864/j.issn.0578-1752.2019.06.012

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【Background】Sheep colibacillosis is an acute infectious disease by E. coli that is characterized by severe diarrhea and sepsis, which is one of the most common bacterial diseases in large-scale sheep farms. Especially the newborn lamb is infected by enterotoxin-producing Escherichia coli (ETEC), causing lamb diarrhea, also known as lamb white diarrhea. Sheep colibacillosis let the farm to suffer serious economic losses, but the traditional antibiotic treatment program has many defects. 【Objective】In this study, we obtained oral diarrhea and diarrhea lambs by oral administration of E. coli F17 strain, and screened out circRNAs that were differentially expressed in individuals with diarrhea and diarrhea after taking E. coli F17 pili, and then explored circRNA for sheep in anti-diarrhea to find candidate genes associated with resistance to E. coli disease traits. From the circRNA level, the understanding of sheep antagonizing E. coli F17 fimbriae was deepened, and the functional genes of sheep antagonizing E. coli F17 fimbriae were determined.【Method】We used CIRI software to predict circRNA from scratch, and firstly screened differentially expressed (DE) circRNA in spleen of Escherichia coli F17 fimbriae without diarrhea and diarrhea by using RNA-seq technology. Then we performed GO on differentially expressed transcripts, and its function was described in combination with GO annotation results. The number of differential transcripts included in each GO entry was counted and the significance of differential transcript enrichment in each GO entry was calculated by using Fisher's exact test. Then, 6 DE circRNAs were randomly selected, and the relative expression levels of the 6 DE circRNAs in the spleen of lambs without diarrhea and diarrhea were verified by q-PCR, and the miRNAs and miRNA target genes were predicted by Miranda software. The function of this part of circRNA was elucidated based on the functional annotation of the miRNA target gene, and the circRNA-miRNA-mRNA interaction was analyzed. Finally, the relative expression level of mRNA in the non-diarrhea group and the diarrhea group lamb was verified by q-PCR.【Result】After mapping the reference sequence, we identified 7730 circRNAs, and DE circRNA was compared with the GO database. A total of 60 circRNAs were annotated and classified into 297 functional subclasses. Using RNA-seq to screen 60 DE circRNAs in spleens of diarrhea-free and diarrhea lambs, 31 of which were up-regulated, and 29 of which were down-regulated. Relative expression levels of randomly selected 6 DE circRNAs in non-diarrhea and diarrhea lambs by q-PCR and found to be consistent with RNA-seq results. Using Miranda to analyze circRNA-miRNA-mRNA interactions, we found a certain target relationship between 6 circRNAs, 5 miRNAs and 8 mRNAs. q-PCR was used to verify the relative expression of mRNA in lambs without diarrhea and diarrhea, and the results were found to be consistent with RNA-seq results.【Conclusion】 This study explored the expression profile of circRNA in the spleen of lambs without diarrhea and diarrhea, and further understood its regulation in the process of disease resistance in sheep. The discovery of circRNA, which was differentially expressed in the spleen of lambs without diarrhea and diarrhea, could help to find out how the lambs resist the mechanism of diarrhea, and to provide a scientific basis for improving the lamb's ability to resist diarrhea.

Construction of Chicken TIGAR Gene Eukaryotic Expression Plasmid and Evaluation of Its Anti-Apoptotic Function

LI YongHua, CHE LuPing, QIU XuSheng, TAN Lei, SUN YingJie, LIU WeiWei, SONG CuiPing, LIAO Ying, DING Chan, WANG JinQuan, MENG ChunChun

Scientia Agricultura Sinica. 2019, 52(6): 1102-1109. doi:10.3864/j.issn.0578-1752.2019.06.013

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【Background】TP53-induced glycolysis and apoptosis regulator (TIGAR) is a target gene downstream of p53, which can regulate glycolysis level, remove reactive oxygen species (ROS) and reduce apoptosis induced by reactive oxygen species (ROS). 【Objective】The objective of this study was to construct the eukaryotic expression plasmid contain of chicken TIGAR gene, and to evaluate its resistance apoptosis effect in DF1 cells, in order to establish the cell line with stable expression of the chicken TIGAR gene. 【Method】According to design primers based on the predicted TIGAR gene published in Genbank (accession number: XM_417232.6), the TIGAR gene from SPF chicken spleen was amplified by RT-PCR, and then amplified products were cloned into expression vector (Flag-CMV14), finally the positive clone was verified by DNA sequencing. Then the phylogenetic tree of chicken TIGAR genes with other mammals and aquatic animals TIGAR gene was constructed to do homology analysis. The recombinant plasmid was transfected into DF1 cells, and infected with Newcastle disease virus to induce apoptosis. The expression of the TIGAR gene and the cleavage level of PARP protein were detected by Western Blot. At the same time the recombinant plasmid (Flag-TIGAR) was transfected into DF1 cells and incubated with apoptosis inducer staurosporine for 2 hours before sample collection, then cells were collected at 24 hours and 48 hours after transfection to evaluate apoptosis level by flow cytometry. 【Result】The TIGAR gene was amplified by RT-PCR, and a band appeared at 843 bp, which was consistent with the prediction. The constructed TIGAR eukaryotic expression plasmid (Flag-TIGAR) was sequenced, and the result showed that the sequence of amplified TIGAR gene were almost consistent with the predicted sequence published on GenBank. Western Blot results showed: cleavaged PARP bands were existed, and the expression level in transfected recombinant plasmid (Flag-TIGAR) group was significantly different (P<0.05) from that in non-transfected plasmid (MOCK) group and transfected empty vector (Flag-CMV14) group. Flow cytometry results showed that the total apoptotic rate was 11% (early apoptosis 7.8%, late apoptosis 3.2%) after transfection with Flag-CMV14 for 24 h, while the total apoptotic rate was decreased to 4% (early apoptosis 3.7%, late apoptosis 0.3%) in Flag-TIGAR transfected group. The early apoptotic rate of Flag-CMV14 transfected group was much higher than that of Flag-TIGAR transfection group, and the difference was very significant (P<0.01), while the late apoptotic rate was also higher than that of Flag-TIGAR transfected group and the difference was significant (P<0.05). The total apoptotic rate was 20.3% (early apoptosis 14.3%, late apoptosis 6.0%) after transfection with Flag-CMV14 for 48 h, while the total apoptotic rate was only 6.4% (early apoptosis 4.8%, late apoptosis 1.6%) after transfection with Flag-TIGAR for 48 h. Both the early apoptotic rate and late apoptotic rate of Flag-CMV14 transfected group were much higher than that of Flag-TIGAR transfected group and the difference was very significant (P<0.01). 【Conclusion】The chicken TIGAR gene was amplified and the eukaryotic expression plasmid was constructed successfully. It was also confirmed that overexpression of chicken TIGAR could obviously reduce the degree of apoptosis and increase cell survival rate.

Infection Status and Genetic Relationship of Crithidia bombi in Different Regions of China

TANG YuJie,WANG LiuHao,LI Kai,LI JiLian

Scientia Agricultura Sinica. 2019, 52(6): 1110-1118. doi:10.3864/j.issn.0578-1752.2019.06.014

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【Objective】The objective of this study is to clarify the prevalence of Crithidia bombi, an intestinal parasite of bumblebees, and its genetic relationship among different species in different regions.【Method】In this study, the infection status of 1 007 bumblebees from 25 species in four provinces (autonomous region) including Inner Mongolia, Gansu, Qinghai, and Sichuan was investigated, and the infection rate of C. bombi in different species and regions was analyzed by chi-square test (SPSS 22.0). PCR amplification, cloning and sequencing were carried out based on the ITS sequences of C. bombi. The specific ITS primers were used to amplify the total DNA in the intestines of bumblebees, and gel electrophoresis was performed. The infection status of bumblebee colony was determined by whether 675 bp of ITS gene fragment of C. bombi was amplified or not, and further sequencing was carried out to analyze the genetic relationship of C. bombi in different species and regions. 【Result】Out of all the samples investigated, 262 bumblebees of 20 species were infected by C. bombi, with an infection rate of 26.0%. Among them, Bombus festivus and B. pyrosoma had the highest infection rates, and B. sibiricus had the lowest infection rate. The infection rate of bumblebee was the highest in Qinghai Province and lowest in Inner Mongolia. The infection rate of male bee was significantly higher than that of worker bee and queen bee. The genetic relationship of most C. bombi was relatively close, except for B. melanurus and B. difficillimus in Gansu Province, and B. remotus, B. impetuosus, B. friseanus in Sichuan Province.【Conclusion】The infection of C. bombi is widespread in China, and the infection rate is different among different species, regions and castes of bumblebee. After a long-term of population evolution, the dominant species of bumblebee are relatively fixed in different regions, so the bumblebee species collected in different regions are different. The statistical analysis of the infection of C. bombi in different regions of China focused on the sample size ≥30, which are the dominant species in each region. Some bumblebees have not collected enough samples, but the infection rate is high according to the sample that we already get. There is host specificity in C. bombi, and C. bombi are adaptive to the infection of the host. In some regions of China, the genetic relationship between the C. bombi is relatively strong on the whole, but it is also affected by the geographical location and the species of bumblebee.

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