Scientia Agricultura Sinica

The Safety Management of Genome Editing Technology

SHEN Ping, ZHANG QiuYan, YANG LiTao, ZHANG Li, LI WenLong, LIANG JinGang, LI XiaYing, WANG HaoQian, SHEN XiaoLing, SONG GuiWen

Scientia Agricultura Sinica. 2017, 50(8): 1361-1369. doi:10.3864/j.issn.0578-1752.2017.08.001

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Genome editing technologies using sequence-specific nuclease (SSN) creates DNA double-strand breaks (DSBs) in the genomic target sites, and the DSBs can be repaired by the non-homologous end joining (NHEJ) or homologous recombination (HR) pathways with the help of artificially engineered nucleases, which can be employed to achieve targeted genome modifications such as gene mutation, gene insertion, gene replacement or chromosome rearrangement. There are three major artificially engineered nucleases including zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) systems. The genome editing techniques have been widely used in the fields of plant gene function research, molecular breeding, and etc. The genome editing crops (GEC) show promising application prospects compared with traditional genetically modified organism (GMO). The GECs with good traits have been gradually transferred from labs to fields. Herein, the principles, characters, and applications of these three mainly used techniques in animal and plants genome editing have been described. The advantages and disadvantages compared with conventional transgenic technique were also discussed, including the safety came from the off-target effects. The management regulations of GECs of different countries in global area were elaborated. Finally, the safety management of GECs and their products in China were discussed combined with Chinese current regulations on the safety management of GMOs, so as to facilitate the development and commercialization of GECs and their products.

Effects of TaLox-B Alleles on Lipoxygenase Activity and Flour Color in Wheats

ZHANG FuYan, CHEN Feng, CHENG ZhongJie, YANG BaoAn, FAN JiaLin, CHEN XiaoJie, ZHANG JianWei, CHEN YunTang, CUI Long

Scientia Agricultura Sinica. 2017, 50(8): 1370-1377. doi:10.3864/j.issn.0578-1752.2017.08.002

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【Objective】Lipoxygenase (Lox) is closely related to flour color. The objective of this study was to investigate the influence of different allelic variations at TaLox-B loci on Lox activity and flour color in wheat to provide a theoretical basis for improving flour color and related quality traits in breeding programs.【Method】A total of 122 wheat cultivars and advanced lines from Henan province were used to measure the Lox activities and flour color by spectrophotometer and colorimeter. The functional markers Lox16, Lox18 and Lox-B23 were used to identify the polymorphism of the Lox genes on chromosome 4BS in current Henan wheat cultivars and advanced lines.【Result】There were significant differences of Lox activities, L^*, a^*and b^*values as well as Wht data among different cultivars. Genotyping results indicated two alleles TaLox-B1a and TaLox-B1b at TaLox-B1 locus in these wheat varieties, with 63.9% and 39.1%, respectively. At TaLox-B2 locus, two alleles TaLox-B2a and TaLox-B2b were identified by the functional marker Lox-B23, with 57.4% and 42.6%, respectively. It was also found two alleles TaLox-B3a and TaLox-B3b at the TaLox-B3 locus, with 41.8% and 48.2%, respectively. Six allele combinations of Lox genes, i.e., TaLox-B1a/TaLox-B2a/TaLox-B3a, TaLox-B1a/TaLox-B2a/TaLox-B3b, TaLox-B1a/TaLox-B2b/TaLox-B3b, TaLox-B1b/TaLox-B2a/TaLox-B3a, TaLox-B1b/TaLox-B2a/TaLox-B3b and TaLox-B1b/TaLox-B2b/TaLox-B3b were found with 41.8%, 15.6%, 6.6%, 28.7%, 5.7% and 1.6%, respectively. Analysis of association of Lox alleles at different TaLox-B loci and Lox activities and flour color parameters indicated that the allelic variation at a single locus showed diverse effects on Lox activities and flour color. For a^*value, the allelic effect was not significant. For Lox activities, the order of effect was TaLox-B2a > TaLox-B2b (P＜0.05), TaLox-B3a > TaLox-B3b (P＜0.01). The Wht value of TaLox-B2a was significantly lower than that of TaLox-B2b (P＜0.01), and TaLox-B3a showed also a significant lower Wht value than TaLox-B3b (P＜0.05). These results suggest that TaLox-B2 and TaLox-B3 significantly affect Lox activities and flour color. Further analysis found that the genotype TaLox-B1a/TaLox-B2a/TaLox-B3a had the highest Lox activity and b^* and the lowest a^* and Wht value, and TaLox-B1b/TaLox-B2b/TaLox-B3b had the lowest Lox activity and b^* and the highest L^*, a^* and b^* values. 【Conclusion】Among the 6 different combinations of three Lox genes in Henan wheat varieties, the genotype TaLox-B1a/TaLox-B2a/TaLox-B3a possess the highest Lox activity and the lowest Wht value (P＜0.05), and wheat cultivars with TaLox-B1b/TaLox-B2b/TaLox-B3b have the lowest Lox activity and the highest Wht value (P＜0.05). Therefore, the genetic control of Lox activities plays a key role in the improvement of wheat quality.

Karyotypic Analysis of Aegilops speltoides Revealed by FISH

DONG Lei, DONG Qing, ZHANG WenLi, HU XiaoLong, WANG HongGang, WANG YuHai

Scientia Agricultura Sinica. 2017, 50(8): 1378-1387. doi:10.3864/j.issn.0578-1752.2017.08.003

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【Objective】The objective of this study is to reveal the karyotypic polymorphism of Aegilops speltoides (Aegilops short for Ae. hereafter) and the karyotypic difference between common wheat and Ae. speltoides via the establishment of FISH karyotype of Ae. speltoides.【Method】Multicolor fluorescence in situ hybridization (mc-FISH) was employed to detect the distribution of Oligo-pSc119.2 and Oligo-pTa535 in chromosomes of Ae. speltoides; centromere-specific oligonucleotide CCS1 was used to identify the location of centromeres on chromosomes; FISH karyotype comparison was conducted to show the karyotypic differences between Ae. speltoides and wheat.【Result】In wheat, oligo-pTa535 signals were observed mainly on chromosomes in A and D genomes, and only very sporadic signals were found in B genome. However, oligo-pTa535 signals were absent in Ae. speltoides of five accessions. Oligo-pSc119.2, compared to a little distribution in A and D genomes, shined plentiful fluorescence in the whole genome B in wheat, and especially in S genomes in Ae. speltoides of five accessions used in this study. Different pairs of chromosomes in wheat could be distinguished from each other according to the distribution of Oligo-pSc119.2 and Oligo-pTa535 on chromosomes of wheat. FISH patterns produced by Oligo-pSc119.2 in wheat showed similarity among wheat materials whether of different ploidy or of different varieties of same ploidy, and that in Ae. speltoides varied depending on accessions. Even homologous chromosomes in one cell in Ae. speltoides exhibited differences in FISH pattern. Oligo-pSc119.2 FISH patterns of five accessions each showed obvious differences from that of B genomes in wheat. Four of five Ae. speltoides accessions possess six pairs of metacentric chromosomes except for homologous pairs 4S of submetacentric chromosomes, which were involved in the karyotype formula 2n = 14 = 12m + 2sm, and the rest one, PI542238, however, houses seven pairs of metacentric chromosomes which resulted in the karyotype formula 2n = 14 = 14m. 【Conclusion】 Chromosomes of Ae. speltoides house rich repetitive DNA sequences highly homologous to pSc119.2 and lack that highly homologous to pTa53. The distribution of pSc119.2 on chromosomes of Ae. speltoides showed differences between accessions, between plants of one accession and even between homologous chromosomes in one plant. FISH patterns produced by Oligo-pSc119.2 on Ae. speltoides chromosomes exhibited significant difference from that on chromosomes of B genomes in wheat. FISH analysis, using Oligo-pSc119.2 and Oligo-pTa535 as probes, not only could differentiate the chromosomes in wheat from that in Ae. speltoides, but also could dishtinguish the chromosomes from each other whether in wheat or in Ae. speltoides.

Drought Resistance Identification and Drought Resistance Indices Screening of Liquor-Making Waxy Sorghum Resources at Adult Plant Stage

WANG Can, ZHOU LingBo, ZHANG GuoBing, XU Yan, ZHANG LiYi, GAO Xu, GAO Jie, JIANG Ne, SHAO MingBo

Scientia Agricultura Sinica. 2017, 50(8): 1388-1402. doi:10.3864/j.issn.0578-1752.2017.08.004

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【Objective】Drought is one of the major problems for liquor-making waxy sorghum production. Drought resistance identification, indices screening, and cultivar breeding of liquor-making waxy sorghum resources are important for sorghum industrial development.【Method】The plant height, spike length, culm diameter, tiller number, grain number per spike, 1000-grain weight, grain weight per plant, and yield of 50 liquor-making waxy sorghum materials were measured in the normal irrigation and drought stress treatments in field experiments in rainprotection shed during 2015 and 2016. Drought resistance comprehensive evaluation value (D value), comprehensive drought resistance coefficient (CDC value), weight drought resistance coefficient (WDC value), correlation analysis, frequency analysis, principal component analysis, grey relational analysis, subordinate function analysis, clustering analysis, and stepwise regression analysis were used to identify the drought resistance and to screen drought resistance indices of tested liquor-making waxy sorghum materials at adult plant stage.【Result】Drought stress had significant effects on all indices. Correlation analysis showed that the yield was significantly and positively correlated with tiller number, grain number per spike, grain weight per plant, spike length, culm diameter, and 1000-grain weight but not with plant height. Frequency analysis showed that the sensitive degrees of all indices response to drought stress in turn for yield, grain number per spike, tiller number, grain weight per plant, 1000-grain weight, culm diameter, spike length, and plant height. Principal component analysis showed that 5 principal components could represent 84.26% of the original data information of liquor-making waxy sorghum drought resistance. The ranks of drought resistance based on the D value, CDC value, and WDC value were similar. Grey relational analysis showed that the correlation degree between DC value of all indices and D value in turn for yield, tiller number, grain number per spike, grain weight per plant, 1000-grain weight, spike length, culm diameter, and plant height, which was similar to the correlation degree between DC value of all indices and WDC value. According to D value clustering analysis, tested liquor-making waxy sorghum materials were divided into 5 drought resistance grades, 2 belonged to grade I, 16 belonged to grade II, 28 belonged to grade III, 2 belonged to grade IV, and 2 belonged to grade V. The subordinate function values of tested indices except for culm diameter and tiller number, CDC value, D value, and WDC value were increased with increase of drought resistance grades. Stepwise regression analysis showed that the tiller number, grain number per spike, and grain weight per plant were closely related to the D value.【Conclusion】Drought stress had significant effects on all indices of liquor-making waxy sorghum resources at adult plant stage. D value was determined as the appropriate method for drought resistance identification. Liangfeng 141-3 and Liangfeng 247-3 were identified as drought resistance liquor-making waxy sorghum materials at adult plant stage, which could provide basic materials for the researches on cultivar breeding, mechanism, and regulation and alleviation mechanism of drought resistance in liquor-making waxy sorghum. The tiller number, grain number per spike, and grain weight per plant could be used as the simple and intuitive identification indices of drought resistance in liquor-making waxy sorghum resources at adult plant stage.

Differences of Double-Cropping Late Rice in Yield, Growth Stage and Utilization of Temperature and Illumination in Different Latitudes of Jiangxi Province

CHEN Bo, ZHOU NianBing, GUO BaoWei, SHU Peng, ZHANG HongCheng, HUO ZhongYang, CHENG FeiHu, HUA Jin, HUANG DaShan, CHEN ZhongPing, CHEN Heng, LIU YunFa, LIAO ShiLiang

Scientia Agricultura Sinica. 2017, 50(8): 1403-1415. doi:10.3864/j.issn.0578-1752.2017.08.005

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【Objective】This study was conducted to clarify the differences of different-types of late rice in yield, growth period and the utilization of temperature and illumination in different latitudes of Jiangxi province, which would provide grounds and references for scientific selection of Japonica rice varieties in Jiangxi. 【Method】The yield, growth stage and utilization of temperature and illumination of double-cropping late rice were comparatively studied by using four kinds of rice varieties as test materials namely indica-japonica hybrid rice, japonica hybrid rice, japonica conventional rice and indica hybrid rice. The experiment was carried out in such three cities indicating latitude discrepancy as Leping (29.00°N, 117.12°E), Shanggao (28.27°N, 115.12°E）and Ganzhou (25.81°N, 114.96°E) in Jiangxi province.【Result】The yield of three-types of late japonica rice increased with the increasing latitudes. In the 2013 (2014), when the latitudes rose by one unit, the yield of indica-japonica hybrid rice, japonica hybrid rice, japonica rice and indica hybrid rice increased by 0.36 (0.32), 0.18 (0.22), 0.25 (0.20) t·hm^-2, respectively. Both indica-japonica hybrid rice and japonica hybrid rice yield significantly higher than indica hybrid rice in the same latitude, and with the exception of Ganzhou, the yields of conventional japonica rice were significantly higher than indica hybrid rice. The three types of japonica rice at each experimental site could mature in safety. With the increasing latitude, the stage from seeding to elongation of each type rice lengthened noticeably and its whole growth period was extended. As shown in the extensions of stages from heading to mature, the whole growth periods of three types japonica rice were extremely noticeably longer than indica hybrid rice at the same experimental site. Utilization efficiency of light and temperature of the three types of Japonica rice showed such a trend as indica-japonica hybrid rice > japonica hybrid rice > japonica conventional rice > indica hybrid rice, therefore a conclusion could be drawn that the growth period was in direct proportion to utilization rate of temperature and light in terms of double cropping late rice maturing in safety. 【Conclusion】 “Indica Rice to Japonica Rice” project in different latitudes in double cropping late rice area of Jiangxi could significantly increase yield, temperature and illumination resource utilization. Compared with indica hybrid rice in local area, growing indica-japonica hybrid rice, japonica hybrid rice and japonica conventional rice in Leping and Shanggao has advantages in yield and temperature and illumination resource utilization, while growing indica-japonica hybrid rice in Ganzhou has obvious advantages. “Indica Rice to Japonica Rice” may be the main way to increase the production in double cropping rice area.

Effects of Supplemental Irrigation Based on Soil Moisture Measurement on Flag Leaf Chlorophyll Fluorescence and Senescence Characteristics in Two Wheat Cultivars

YAN LiXia, YU ZhenWen, SHI Yu, ZHAO JunYe, ZHANG YongLi

Scientia Agricultura Sinica. 2017, 50(8): 1416-1429. doi:10.3864/j.issn.0578-1752.2017.08.006

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【Objective】This study investigated the effects of supplemental irrigation based on soil moisture measurement on flag leaf chlorophyll fluorescence and senescence characteristics in two wheat cultivars. 【Method】Under field conditions, the experiments were conducted in the growth seasons of 2013-2015 using the cultivars Jimai 22 (J22) and Tainong 18 (T18). Three treatments were applied, namely, no-irrigation during whole growth season (W0); an average relative soil water content of 65% in 0-40 cm soil layers at jointing and anthesis, respectively, by supplemental irrigation based on measured soil moisture (W1); and irrigation with 60 mm each at jointing and anthesis (W2, local supplemental irrigation practice). 【Result】W1 regulated the amount of irrigation at jointing and anthesis, maintained the relative water content of soil by 65% after irrigation, and prevented excessive or too low irrigation to create suitable soil moisture environment for wheat growth and development. On the 14, 21, and 28 day after anthesis, the electron transport rate, photochemical quenching of chlorophyll fluorescence, actual photochemical efficiency, and sucrose content of flag leaves were significantly higher under W1 than those under W2. The activity of sucrose phosphate synthase in flag leaves of W1 on 14 and 21 day after anthesis was significantly higher than that of W2. On the 14, 21, and 28 day after anthesis, the activities of superoxide dismutase and catalase in flag leaves in W1 were significantly higher than those in W2 for two cultivars. In addition, the MDA content in flag leaves was significantly lower than that of W2, and a high level of soluble protein content was maintained in flag leaves. During the two growing seasons, the grain yield, water use efficiency, and irrigation efficiency of W1 treatment for T18 and J22 were significantly higher than that of W2. Comparison among cultivars, on the 21, and 28 day after anthesis, the electron transport rate, photochemical quenching of chlorophyll fluorescence, actual photochemical efficiency, and sucrose content of flag leaves were significantly higher of T18 under W1 and W2 than those of J22. The activity of sucrose phosphate synthase in flag leaves of T18 on the 7, 14 and 21 day after anthesis was significantly higher than that of J22. On the 21, and 28 day after anthesis, the activities of superoxide dismutase and catalase in flag leaves of T18 were significantly higher than those of J22 in W1 and W2. In addition, the MDA content in flag leaves of T18 was significantly lower than that of J22, and a high level of soluble protein content was maintained in flag leaves. In the same growing season under the same treatment, no significant difference in total water consumption and water use efficiency was observed between T18 and J22. Grain yield and irrigation efficiency of T18 in W1 and W2 treatments were significantly higher than those of J22. However, the yield of T18 in W0 was significantly lower than that of J22. 【Conclusion】Supplemental irrigation based on soil moisture measurement (W1) could regulate the relative water content in soil to the targeted level at jointing and anthesis. W1 was suitable for two wheat varieties and effective in achieving both high yield and saving water. Under irrigation conditions, the yield potential of T18 was higher than that of J22. Under drought conditions, however, the sensitivity to moisture and yield of T18 were lower than those of J22.

Gene Cloning and Functional Analysis of GcAP1 Complex Beta Subunit in Glomerella cingulata

ZHANG JunXiang, JI ZhiRui, WANG Na, XU ChengNan, CHI FuMei, ZHOU ZongShan

Scientia Agricultura Sinica. 2017, 50(8): 1430-1439. doi:10.3864/j.issn.0578-1752.2017.08.007

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【Objective】 The objectives of this study are to determine the function of β subunit of adaptor protein GcAP1 complex in growth and pathogenicity of Glomerella leaf spot of apple pathogen Glomerella cingulata, investigate expression patterns of the GcAP1β in the fungal growth and pathogenicity, decipher whether or not GcAP1β regulate the expression of endopolygalacturonase genes CgPG1 and CgPG2, pectin lyase genes pnl-1 and pnl-2, pectate lyase genes pelA and pelB, and to lay a foundation for further studies of adaptor protein in pathogenic signal transduction pathways of G. cingulata. 【Method】 Based on the GcAP1β deletion vector and GcAP1β-gfp fused expression vector, the Δgcap1β mutant and the GcAP1β complementation strain Δgcap1β-GcAP1β were structured using ATMT, respectively, verified by RT-PCR and Southern blot analysis. Colony growth rate, sporulation, germination rate, appressorial formation rate and pathogenicity of the Δgcap1β mutant and the GcAP1β complementation strain Δgcap1β-GcAP1β were assayed, compared with the wild-type strain W16. GcAP1β subcellular localization was carried out with the bioinformatics softwares ProtComp 9.0 and TMHMM, along with signal observation of GcAP1β-GFP. The GcAP1β expression levels in hyphae, conidia, appressoria and pathogenicity stage were identified by qRT-PCR. Moreover, the expression levels of CgPG1, CgPG2, pnl-1, pnl-2, pelA and pelB in the wild-type strain W16 and the Δgcap1β mutant were detected, respectively. 【Result】 GcAP1β is 2 321 bp in length, including 3 introns, which encodes a 720 amino acids. Compared with the wild-type strain W16, the Δgcap1β mutant showed a rill-like fold colony and decreased growth, while sporulation, germination rate and appressorial formation rate were unaffected. Virulence of the Δgcap1β mutant reduced significantly, which induced tiny spots on the leaves. Moreover, the GcAP1β complementation strain Δgcap1β-GcAP1β fully restored the phenotype flaws by reintroducing GcAP1β to the Δgcap1β mutant. Fluorescent signal showed that the fused protein GcAP1β-GFP was distributed to the cytoplasm. qRT-PCR analysis showed that GcAP1β expresses through the lifecycle of G. cingulata, and the highest expression level of GcAP1β occurred at the post-invasion to leaves. Compared with WT, the Δcgap1β mutant showed a drastic reduction of CgPG1 transcripts (20.3%), CgPG2 transcripts (16.5%), pnl-1 transcripts (8.2%), pnl-2 transcripts (14.4%), pelA transcripts (4.4%) and pelB transcripts (0.8%). 【Conclusion】 The adaptor protein GcAP1 complex is distributed to the cytoplasm and is necessary for growth and development of G. cingulata; GcAP1 regulates the expression of CgPG1, CgPG2, pnl-1, pnl-2, pelA and pelB and is a vital virulence factor of G. cingulata.

Effects of Sublethal Doses of Chlorantraniliprole on the Detoxification Enzymes Activities and the Growth and Reproduction of Spodoptera exigua

CHEN YiQu, XIANG Xin, GONG ChangWei, WANG XueGui

Scientia Agricultura Sinica. 2017, 50(8): 1440-1451. doi:10.3864/j.issn.0578-1752.2017.08.008

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【Objective】 Spodoptera exigua is a polyphagous pest and shows different characteristics of growth and development under the selective pressure of different environments, drugs and so on. Chlorantraniliprole, a novel insecticide that acts on ryanodine receptors, has highly activity to those insects in Lepidopteran. The objective of this study is to explore the effects of S. exigua larvae treated by the sublethal doses of chlorantraniliprole on the toxicities, the activities of three main detoxifying enzymes, including carboxylesterase (CarE), glutathione S-transferase (GSTs), and mixed-function oxidase (MFOs), and population breeding.【Method】 The toxicities of chlorantraniliprole on SE-Lab and SE-Sel strains were detected by incorporation bioassay and the SE-Sel strain was achieved with continuous selecting with LC₂₅ sublethal doses for six generations from SE-Lab strain, then the synergistic effects of enzyme inhibitors (TPP, DEM, PBO) with chlorantraniliprole on the SE-Lab and SE-Sel strains were assayed using the dip-leaf method. The toxicities of chlorantraniliprole on the insects which were fed the leaves soaked with the solution of enzyme inhibitor or 0.1% TritonX-100 as blank control before 12 h were assayed by the dip-leaf method. The midgut and fatbody of tested insects were dissected on the ice and the effects of the sublethal doses of chlorantraniliprole and enzyme inhibitors on three metabolic detoxification enzyme activities were analyzed by the determination of detoxification enzyme activities. According to the age-stage two-sex life tables theory, the growth, mortality, fecundity data of the tested insects were also recorded to analyze the differences of the age-stage two-sex life table parameters between the strains of SE-Lab and SE-Sel. 【Result】 The synergistic effect of the PBO was the strongest among the three enzyme inhibitors and the synergic ratios on the strains of SE-Sel and SE-Lab reached 1.58- and 1.69-fold, respectively. The activities of the three detoxification enzymes, which were induced by the continuous selection of the sublethal doses of chlorantraniliprole, were promoted and the MFOs activities were the most significant, which of the SE-Sel strain in the midgut and fatbody were enhanced by 2.07- and 2.10-fold, meanwhile, the MFOs activities of the insects of SE-Sel induced by the sublethal dose of chlorantraniliprole again were also promoted by 4.02- and 3.44-fold in the midgut and fatbody compared to those in SE-Lab strain, respectively. The enzyme activities of three detoxifying enzymes were decreased when the tested insects were treated with enzyme inhibitors and the descend range of MFOs activity among the three enzymes was maximum, which was only 42.3%-44.8% compared to the treatment not treated with the enzyme inhibitor. The adult preoviposition period and total preoviposition period of F₁ generation of SE-Sel strains became shorter and spawning quantity of became higher compared to those in F₁generation of SE-Lab. SE-Sel strains had the longest mean generation time and least amount of eggs. The intrinsic rate of increase (r), finite rate of increase (λ) and net reproductive rate (R₀) of SE-Sel strain were significantly lower than those in SE-Lab strain. The r, λ and R₀ values of SE-Lab and SE-Sel strains were 0.18 and 0.16 d^-1, 1.20 and 1.17 d^-1, 358.42 and 203.12 d^-1, respectively. Even though the mean generation times of SE-Sel strain was longer than SE-Lab strain, there was no significant difference between the two strains.【Conclusion】MFOs may be the major detoxication enzyme on the metabolic detoxification of chlorantraniliprole in the S. exigua and involved in the formation of resistance. A longer generation period, lower fecundity and slower population growth of S. exigua were demonstrated when it was treated by a continuous selection of lethal dose of chlorantraniliprole. The sublethal doses of chlorantraniliprole have a continuous control effect on S. exigua.

Identification of the Pathogen Causing Pepper Anthracnose in Shandong Province and Screening of Highly Effective Fungicides

GAO YangYang, HE LiFei, LI BeiXing, LIN Jin, MU Wei, LIU Feng

Scientia Agricultura Sinica. 2017, 50(8): 1452-1464. doi:10.3864/j.issn.0578-1752.2017.08.009

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【Objective】The objective of this study is to identify the pathogen species of pepper anthracnose in major pepper growing areas in Shandong Province, and to screen the highly effective fungicides by inhibitory activity test in vitro and relative control efficacy trials on detached pepper fruits inoculated with conidial suspension. 【Method】 The isolates of pepper anthracnose were collected from the major pepper producing areas in Jining, Heze, Weifang, Taian and Dezhou. After isolation and purification, the isolates were identified by using the methods of morphological characteristics analysis, its pathogenicity test on detached pepper fruits and molecular diagnostics. Morphology parameters mainly include the colony and conidial morphology, conidial size. The internal transcribed spacer (ITS) rDNA region of the four typical isolates was amplified by using the fungal universal primers ITS4 and ITS5, and then the amplified product was recycled and sequenced. MEGA 5.1 was used to generate phylogenetic tree to confirm the pathogen species. The inhibitory activity in vitro of 11 fungicides to Colletotrichum acutatum isolate was determined by the inhibition of the 11 fungicides against mycelial growth and spore germination of the pathogen. Control efficacies of the selected fungicides against pepper anthracnose were determined on the detached pepper fruits.【Result】All of the twenty-seven isolates exhibited similar morphology, which produced white to pale grey colonies. The oval conidia were single-celled with a sub-cute end, (7.48-14.69) μm × (2.52-5.64) μm in size. Both of the mature and immature pepper fruits with or without wound could be infected by this pathogen, and disease spot expanded rapidly on wounded pepper fruits. Typical symptom of pepper anthracnose was sunken and necrotic lesion that covered with massive orange conidia. The rDNA-ITS sequence length of the four representative isolates was 562, 541, 557 and 553 bp (GenBank Accession No KX830854-KX830857), respectively. Phylogenetic tree analysis indicated that the four representative isolates and C. acutatum were divided into one group, and the bootstrap value was 100%. The inhibitory activity test in vitro indicated that pyraclostrobin, fludioxonil, and pyrisoxazole exhibited strong inhibitory activity against mycelial growth and spore germination of C. acutatum. Moreover, pyraclostrobin, fludioxonil and pyrisoxazole at the dose of 400 μg·mL^-1 also showed high preventive and curative activity against C. acutatum on detached pepper fruits, with the efficacy greater than 60%, which showed significantly higher efficacy than the preventive and curative activity of azoxystrobin.【Conclusion】 The major pathogenic fungus of pepper anthracnose in Shandong Province was identified as C. acutatum. Pyraclostrobin, fludioxonil and pyrisoxazole showed excellent inhibitory activity against C. acutatum both in vitro and in vivo, make these fungicides become potential candidates for the control of pepper anthracnose in the field.

The Role and Interaction of Soil Water and Organic Matter on Hyper-Spectral Reflectance

SHANG Xuan, LI XiCan, XU YouYou, LIU ShaSha

Scientia Agricultura Sinica. 2017, 50(8): 1465-1475. doi:10.3864/j.issn.0578-1752.2017.08.010

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【Objective】The objective of this study is to reveal the rule of soil moisture and organic matter on the high spectrum quantitatively and to provide a basis for improving the accuracy of spectral estimation of soil moisture and organic matter.【Method】Ninety brown soil samples collected from the Tai'an city, Shandong province were used as research materials, and their outdoor spectrum, soil moisture and organic matter were obtained. At the same time, smoothing denoising pretreatment of the spectral curves were carried out by Savitzky-Golay filter. Soil samples were divided into 9 groups according to water and organic matter contents. By using comparative method, 9 groups of original spectral data were analyzed, and the effect of soil water and organic matter on the spectrum was discussed. Then, correlation analysis was used to analyze the correlation between water, organic matter and raw spectral reflectance (R), first order differential reflectance (D (R)) and grouping spectra. Under the basic assumption that other factors were identical, two factor variance interaction analysis was used to analyze the degree of water, organic matter and their interaction to the soil spectral reflectance and spectral derivative quantitatively. According to the law of interaction between soil water and organic matter, characteristic factor was selected on the basis of the principle that the correlation coefficient is relatively large and the interaction is small. Finally, partial least squares regression model was established to predict the soil organic matter content of hyperspectra, and the effectiveness of the model which established with the factors selected according to the interaction was analyzed.【Result】 The results showed that water had the main effect on the reflectance of soil spectral reflectance in the range of field capacity, and the interaction between water and organic matter was exist objectively. When the soil moisture content was less than 10%, the original spectra of 600-1 800 nm could better reflect the effect of organic matter. And when the soil moisture content was more than 15%, the role of organic matter was almost covered by the action of water. The role and interaction of soil water and organic matter reached significant level in 360-1 800 nm, 410-1 800nm and 509-1 800 nm, And all of them reached significant level in 1 951-2 450 nm (α=0.05). The effect on soil spectrum from large to small was water, organic matter and interaction. The effect of water on soil spectrum was about 5 to 8 times in 425-1 800 nm and 8 to 12 times in 1 950-2 300 nm than that of organic matter. The effect of organic matter on soil spectrum was about 2 times as much as interaction in 350-2 500 nm. After the first-order differential transformation, the effect of soil water at 450-530 nm, 600-790 nm, 1 019-1 027 nm, 2 000-2 020 nm and 2 045-2 075 nm was enhanced, but weakened in other bands. The effect of soil organic matter was enhanced at 471-824 nm, 851-949 nm, 967-1 140nm, 1 172-1 340nm, 1 379-1 428 nm, 1 450-1 770 nm, 1 953-2 122 nm, 2 174-2 199 nm, and 2 271-2 342 nm, and weakened at other bands. The interaction between water and organic matter also changed at different wavelengths, but change was relatively weakened than that of the soil water and organic matter. Based on the characteristics selected by the interaction between soil water and organic matter, the accuracy of the hyperspectral model of soil organic matter was improved. The determination coefficient R2 of the 16 test samples increased from 0.6764 (without considering the interaction) to 0.7934.【Conclusion】The researches showed that the effect of organic matter on the spectral reflectance may not be considered in the inversion of soil water content. In the inversion of organic matter content, not only the influence of water on the reflectance should be eliminated, but the interaction between water and organic matter on the spectrum should be considered. The accuracy of spectral estimation of soil organic matter can be effectively improved when considering the interaction of water and organic matter on soil spectrum.

Effect of the Inherent Soil Productivity on High, Stable and Sustainable Yield of Grain and Oil Crops in Yellow Soil Region

HUANG XingCheng, SHI XiaoJun, LI Yu, ZHANG YaRong, LIU YanLing, ZHANG WenAn, JIANG TaiMing

Scientia Agricultura Sinica. 2017, 50(8): 1476-1485. doi:10.3864/j.issn.0578-1752.2017.08.011

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【Objective】Yellow soil is an important zonal soil in China. High, stable and sustainable yield of grain and oil crops are fundamental to food security, economic development and people's livelihood for yellow soil region. This paper studied the characteristics of inherent soil productivity for grain and oil crops in yellow soil region in order to evaluate the effect of inherent soil productivity on high, stable and sustainable yield of grain and oil crops. The results of this study will provide a theoretical basis for cultivating land conservation and producing green crops in yellow soil region. 【Method】The assessment was conducted based on data-set derived from National Soil Test and Fertilizer Recommendation projects during 2006 to 2013 in yellow soil region. Yields of unfertilized control (CK) and NPK fertilization (N2P2K2) treatments in 3 515 on-farm trials (434 potato trials, 525 rapeseed trials, 1 318 maize trials, and 1 238 rice trials) were surveyed. Characteristics of inherent soil productivity and its contribution rate to fertilization yield under on-farm conditions were assessed by using a plant-based agronomic approach. The effects of inherent soil productivity on fertilization yield and yield gap were also assessed by using linear fitting and boundary line analysis. Meanwhile, the effect of stable indexes and sustainable indexes of grain and oil crops under different inherent productivities were also assessed. 【Result】The average yield based on inherent soil productivity of potato, rapeseed, maize and rice were 10.8, 1.13, 4.57 and 5.73 Mg·hm^-2, and the average contribution rates of inherent soil productivity were 50.8%, 49.0%, 59.0% and 70.8%, respectively. It was found that the contribution rate of inherent soil productivity was increased with improvement of inherent soil productivity. There was a significant and positive correlation between fertilization yield and inherent soil productivity, R² of linear fitting with fertilization yield and inherent soil productivity of potato, rapeseed, maize and rice were 0.476, 0.284, 0.382 and 0.366 (P<0.001), respectively. Boundary line analysis showed that the attainable yield of potato, rapeseed, maize and rice were 42.8, 4.07, 11.8 and 12.4 Mg·hm^-2, respectively. Meanwhile, it found that stability and sustainable of grain and oil crops yield were increased with improvement of inherent soil productivity. 【Conclusion】It was concluded that improving inherent soil productivity of farmland can decrease yield gap while promote high, stable and sustainable yield of grain and oil crops in yellow soil regions.

Cloning and Function Analysis of Heat-Shock-Protein LsHsp70-2711 Gene Under High Temperature Stress in Leaf Lettuce (Lactuca sativa L.)

LI YaBo, LI Ting, HAN YingYan, FAN ShuangXi

Scientia Agricultura Sinica. 2017, 50(8): 1486-1494. doi:10.3864/j.issn.0578-1752.2017.08.012

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【Objective】Through cloning of genes related to Hsp70, and using VIGS to analyze Hsp70 expression and morphological change of leaf lettuce under heat stress to lay a foundation for analysis of the response mechanism and molecular mechanism of heat shock protein under heat stress.【Method】The full-length cDNA gene of LsHsp70-2711 gene was obtained by homologous cloning and RACE technology. By real-time fluorescence quantitative PCR (qRT-PCR), the expression of the gene from P-S11and G-S59 was analyzed. According to the VIGS technology, the pTRV-LsHsp702711 vector was constructed and transformed into Agrobacterium GV1301. The leaves of lettuce were infected by the injection method. After three weeks, the positive plants were identified by PCR. The gene expression and morphology were compared between the control group and positive group, and the expression characteristics of LsHsp70-2711 were analyzed again after heat stress and drought treatment. 【Result】The sequence analysis indicated that the full-length cDNA was 2226 bp, the open reading frame was 2154 BP, encoding 718 amino acids, and its homology compared with that of Arabidopsis (NP_187864.1), Tomato (NP_001266213.1), Saussurea Medusa (AAB99745.1), and other species. qRT-PCR results showed that the expression of the gene in the two cultivars was up-regulated, and the expression level of heat resistant cultivars was significantly higher than the heat sensitive cultivar, and the gene expression of P-S11 was inhibited by the increase of stress time, and the expression level of G-S59 was higher in the heat resistant variety. In heat resistant variety, the maximum expression of LsHsp70-2711 appeared at 42℃, 60 min, and the maximum expression of heat sensitive variety reached at 37℃, 60 min. Prediction of subcellular localization showed that LsHsp70-2711 was mainly in the cytoplasm. The constructed vector was used to invade the leaves of lettuce, and the positive plants were obtained after identification. LsHsp70-2711 expression decreased after it was silenced by VIGS, compared with the control, the stem length of the positive plants was significantly increased. After heat stress and drought treatment, the expression of LsHsp70-2711 was significantly lower than the control, and the influence of high temperature treatment on LsHsp70-2711 was greater than drought stress.【Conclusion】 LsHsp70-2711 gene belongs to Hsp70 gene family, and is related to the heat resistance in lettuce. The research results of the study will provide theoretical supports for the analysis of function of high temperature in bolting of lettuce.

Evaluation of Flavor Quality and Antioxidant Capacity of Apple Fruits from Three Xinjiang Red-Flesh Lines

LIU Yu, LIU ShengYu, LU JuanFang, YU QingFan, XI WanPeng

Scientia Agricultura Sinica. 2017, 50(8): 1495-1504. doi:10.3864/j.issn.0578-1752.2017.08.013

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【Objective】In order to characterize the flavor quality of Xinjiang red-flesh apple, the composition and content of flavor compounds were analyzed and a preliminary evaluation on nutritional quality of these fruits was made, which will provide useful information for exploitation and utilization of these apple fruits.【Method】High performance liquid chromatography (HPLC) and gas chromatography-mass spectrometer (GC-MS) were employed to detect and analyze sugars, organic acids and volatile compounds in the peels and pulps of three Xinjiang red-flesh apple lines, and compared with the control Fuji apple ‘Changfu 2’. The contents of total phenols (TP), total flavonoids (TF) and antioxidant activity were also measured and evaluated. 【Result】The titratable acid (TA) of apple tested were 21.01-27.71 mg·mL^-1, which were 3.68-4.85 times of the control. The pH values were 3.12-3.19, which was significantly lower than the control (P<0.05). Fructose, glucose and sucrose were identified in these fruits, fructose was the dominant sugar (56.71%-64.07%), while sucrose content was the least and it only accounted for 8.89%-31.12% of total sugar. The contents of soluble sugars in three Xinjiang red-flesh apple were significantly lower than those in the control. Fructose in pulps was higher than those in peels, while no significant differences of glucose and sucrose were found between them (P<0.05). Five organic acids, including oxalic acid, tartaric acid, malic acid, citric acid, and quinic acid, were identified from the lines tested. Malic acid (56.51%-95.11%) was the richest component, followed by citric acid (2.07%-40.72%). The contents of other acids were less than 0.6 mg·g^-1 fresh weight (FW). Quinic acid was only detected in ‘13-3’. The content of malic acid in peels and pulps was 6.56-8.99 times and 5.58-6.61 times as much as that in control, and the content of citric acid was 16.80-117 times and 4.50-16.17 times of that in control, respectively. The content of malic acid in pulps was significantly higher than that in peels, while citric acid in pulps is significantly lower than that of peels (P<0.05). Totally, 85 volatile compounds were identified from the fruits tested. Aldehydes, esters and terpenes were the most abundant components, accounting for 92.32%-97.84% of the total volatile compounds. Similar with the control, ‘13-3’ was characterized by esters and terpenes. Ethyl butyrate, ethyl 2-methylbutyrate, methyl hexanoate and hexyl acetate were the main esters, while styrene, D-limonene and α-farnesene were the dominant terpenes. ‘P3’ and ‘Xinnong’ were mainly characterized by aldehydes and terpenes, hexanal and (E)-2-hexenal were the main aldehydes. The content of volatile components in peels were significantly higher than those in pulps (P<0.05). The contents of TP and TF in peels and pulps of three Xinjiang red-flesh apple lines were 2.31-2.65 times and 1.23-1.61 times as that of the control, and as 5.53-16 times and 1.43-3.49 times as that of the control, respectively. Free radical scavenging ability of extraction from these fruits were also significantly higher than the control. The contents of TP and TF in peels were higher than those in pulps, and its antioxidant capacity were significantly higher than those in pulps. Among the tested lines, ‘13-3’ presented the highest TP and TF contents and antioxidant capacity. 【Conclusion】 The apple lines tested belong to the high-acid type of Xinjiang red-flesh apple. Low soluble sugar, high malic acid and citric acid, low ratio of total sugar to malic acid and the characteristic aroma components, such as hexanal, (E)-2-hexenal, ethyl butyrate, ethyl hexanoate, hexyl acetate and ethyl 2-methylbutyrate, play an important role in flavor quality decision. ‘13-3’ belongs to “fruity odor” fruit, while ‘P3’ and ‘Xinnong’ are the “grassy odor” type. The three lines tested presented characteristic flavor and high nutritional quality, which could be good materials for studying flavor quality and breeding functional processing apple, and ‘13-3’ is the optimal.

SNP Marker Tightly Linked to Tssd for Peach Using High Resolution Melting Analysis

LU ZhenHua, NIU Liang, ZHANG NanNan, CUI GuoChao, PAN Lei, ZENG WenFang, WANG ZhiQiang

Scientia Agricultura Sinica. 2017, 50(8): 1505-1513. doi:10.3864/j.issn.0578-1752.2017.08.014

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【Objective】 SNP marker is characterized as highly distribution, high resolution and co-domination in plant organism, which is considered as a promising molecular marker. The achievements of peach whole genome sequencing generated massive amounts of SNPs. How to establish a sensitive and effective method for identifying genotyping of different SNP genotypes is a base of further research on gene mapping, cultivar identification and linkage map construction. 【Method】 The aim of this study was to determine if HRM can detect the genotyping of different SNP genotypes and identify a SNP marker tightly linked to PpTssd gene. The segregation population of semi-dwarf progeny was selected with parents 97-32-46 (ST) × 03-94-2 (Tssd). According to gene mapping, different types of SNP markers within mapping region were developed, and the HRM analysis was employed to conduct SNP genotyping and the SNP marker linked to desired traits were generated.【Result】As the key factors for genotyping, the proper concentrations of temperate DNA and Mg²⁺ were established. In the 15 µL PCR reaction system, genotyping could not be complete when template DNA was less than 5 ng and Mg²⁺ was less than 1.6 µmol·L^-1. The primers were designed based on the phenotype and genotype, spanning each desired SNP to amplify DNA fragments shorter than 150 bp. HRM analysis could discriminate four types of SNPs (A/T, A/G, A/C, and C/G) occurred via single nucleotide mutation and the result was validated by Sanger sequence. HRM analysis divided temperature-sensitive semi-dwarf and standard type individuals into two groups. A SNP tightly linked to Tssd gene was identified in 96 individuals consisting of 48 Tssd and 48 ST, respectively. The HRM technique distinguished the Tssd and ST into two groups except for one individual with null amplification. Ultimately, the homozygous A/A and heterozygous A/T were identified, and generated a SNP tightly linked to Tssd gene with 2.11cM with two recombinants.【Conclusion】SNP genotyping of different SNPs were established based on HRM analysis. Although HRM could not distinguish two types of homozygote, HRM analysis still can be a effective method for SNP genotyping and can be used for gene mapping, genetic diversity and cultivar identification in peach based on this study.

Freeze-Thaw Stability of Waxy Corn Starch Gel

NING JiYing, GU FengYing, GAO PingPing, CAO JingJing, LUO QiQi, WANG Feng

Scientia Agricultura Sinica. 2017, 50(8): 1514-1524. doi:10.3864/j.issn.0578-1752.2017.08.015

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【Objective】The objective of this study was to research on the changes of the physical properties of waxy corn starch gel by multiple freeze-thaw cycles (FTC), which has relation with freeze-thaw stability. Results of his research will provide scientific supports for popularization and application of waxy corn starch in frozen foods. 【Method】 An ordinary corn starch samples (YM) and two waxy corn starch samples (WCS) were taken as the control and research materials, respectively. The syneresis of starch gels was determined by centrifugation-filtration. The gelatinization enthalpy, retrogradation enthalpy, and ice melting enthalpy of starch gels were determined by DSC. The pasting properties of starches were determined by Brabender Viscograph. The texture of starch gels were determined by Texture Analyser. The dynamic rheological properties of starch gels were determined by dynamic rheometer. The microstructure of starch gels were observed by SEM.【Result】The result of syneresis rate showed that after the first FTC, WCS displayed ultra-strong ability of holding water. WCS2 had the strongest ability to hold water, and the syneresis rate was 5.75%. However, YM had the weakest ability to hold water, and the syneresis rate was more than 50%. The results of the retrogradation rate and ice melting enthalpy showed that they gradually increased with the increase of freeze-thaw cycles. And there was a significant difference in the retrogradation rate and ice melting enthalpy among 1, 3, and 5 FTCs, which showed the retrogradation degree and freezable water increased because of multiply freeze-thaw cycles. After the first FTC, the retrogradation rate of WCS and YM were 17%-18% and 40%-50%, respectively. And after the first FTC, ice melting enthalpy of WCS and YM was about 540 J·g^-1 and 555 J·g^-1, respectively. The result of hardness showed that the hardness of starch gels increased obviously after five FTCs, and the hardness of WCS and YM was 45-100 g and 440 g, respectively. The result of dynamic rheological properties showed that tanδ of starch gels gradually decreased with the increase of freeze-thaw cycles. However, tanδ of WCS was always higher than tanδ of YM, which showed WCS gels were softer and more sticky. The result of pasting properties showed that WCS was not easy to aging in the process of cooling. The result of microstructure showed that the structure of starch gels changed obviously after five FTCs. The matrix surrounding pores of WCS gels were thin and atypical. However, the matrix surrounding pores of YM gel were thick and typical.【Conclusion】The ability of WCS gels resisting undesirable physical changes caused by temperature fluctuations is strong when WCS gels undergone FTC treatment. And its freeze-thaw stability is better than YM gel. This research has important reference values for application of waxy corn starch in frozen foods.

Effects of Different Rotational Grazing Ways on Grazing Characteristics, Weight and Reproductive Performance of Tan Sheep in Desert Steppe

ZHOU JingJing, MA HongBin, ZHOU Yao, WANG Li, NIU JiaWei, SHEN Yan, XU DongMei

Scientia Agricultura Sinica. 2017, 50(8): 1525-1534. doi:10.3864/j.issn.0578-1752.2017.08.016

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【Objective】It is significantly important to maintain grassland ecosystem balance by reasonable grazing method. The study of the effects of rotational grazing on grazing characteristics, weight and reproductive performance of Tan sheep could provide a basis for grassland grazing management.【Method】The desert steppe in Ningxia was used as the research object, and under the stocking rate of 0.75·hm^-2, 4 treatments was designed which were continuous grazing (CG), two-area rotational grazing (TG), four-area rotational grazing (FG) and six-area rotational grazing (SG). The feeding habits, intake amount and nutrient intake of Tan sheep were studied by using all-day observation method, difference method and conventional nutrient determination method, respectively. Then the weight of tan sheep at the end of each rotational period was observed and the reproductive performance during grazing period was counted.【Result】The results are as follows: All the days of grazing, Tan sheep feeding time occupied the main part, the proportion of feeding time was 64% or more. With the increase of area number in rotational grazing, the ingestion time, standing and wandering time of Tan Sheep reduced（P＜0.05）, the rumination and resting time increased correspondingly（P＜0.05）. The differences of individual behavior had a greater impact on excretion, fighting, itching, eating foreign body and other acts , the proportion of time was less than 1.4%. The pasturing amount of four-area rotational grazing and six-area rotational grazing showed no significant differences and were both higher than the continuous grazing and two-area rotational grazing significantly, continuous grazing intake was the lowest among them（P＜0.05）. The grassland feeding rate showed a trend of CG＞TG＞FG＞SG , among them, the continuous grazing was 39.06%, and the six-area rotational grazing was 27.9%. The intake rate declined with the increase of rotational grazing area number; there was hardly significant difference in Tan sheep daily intake among three kinds of rotational grazing patterns（P＞0.05）, the continuous grazing was the last（P＜0.05）. The contents of crude protein, crude fat and phosphorus in grass taken by Tan sheep were higher than those in grass group, and crude ash and nitrogen-free extract were close, but crude fiber and calcium were lower. With the increase of area numbers in rotational grazing, the crude protein content of feeding forage had a downward trend, the crude fiber showed an opposite trend, but the total intakes of dry matter, crude protein and phosphorus were increased. The nutrient intakes of all the treatments were higher than that of NRC sheep feeding standards in the United States, which indicated that Tan sheep did not need supplementary feeding after grazing in the warm season. During grazing, the body weight and average daily gain of Tan sheep in continuous grazing area was lower than the other three areas which had hardly significant differences among them. The reproductive performance of Tan Sheep was not significantly affected by the differences of grazing patters. The reproductive performance of each treatment was similar, such as lambing rate, lamb average birth weight and so on, which indicated that the grazing method had little effect on the reproductive performance of Tan sheep at an appropriate stocking rate.【Conclusion】Rotational grazing patterns had an impact on the feeding characteristics and production performance of Tan sheep. Compared with the continuous grazing, with the increase of area number in rotational grazing, the pasture intake rate, feeding time and the intake of protein content were decreased, but the total amount of daily intake, dry matter, crude protein and other nutrients were increased. Rotational grazing did not improve the reproductive performance of Tan sheep, but more conducive to Tan sheep weight gain. The two-area rotational grazing is the best pattern for Tan sheep farming at warm season in Ningxia desert steppe if take consideration of the feeding characteristics, production performance of Tan sheep and convenience of production management.

Characteristics of Hc-daf-22 Gene from Haemonchus contortus: Crokaryotic Expression and Its Enzymatic Activity

ZHENG XiuPing, DING HaoJie, GUO XiaoLu, YANG Yi, HUANG Yan, CHEN XueQiu, ZHOU QianJin, DU AiFang

Scientia Agricultura Sinica. 2017, 50(8): 1535-1542. doi:10.3864/j.issn.0578-1752.2017.08.017

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【Objective】 Haemonchus contortus is one of the major gastrointestinal nematodes infecting millions of ruminants, the disease (haemonchosis) was nationally epidemic in China. In order to analyze biochemical properties of Hc-DAF-22 in Haemonchus contortus, the enzyme activity of renatured Hc-DAF-22 was measured in this study. By the results of enzyme activity assay, the role of Hc-DAF-22 protein is understood in peroxisomal β-oxidation. 【Method】 First, the full-length of open reading frame of Hc-daf-22 was amplified by PCR from total cDNA of adult H. contortus ZJ Strain （GenBank：HQ738470.1）. A recombinant pET-22b-Hc-daf-22 was constructed and transformed into E. coli BL21 strain; and its prokaryotic expression was induced for four hours by IPTG (0.1 mmol·L^-1). Then bacterial liquid were centrifuged and cells were resuspended in PBS solution (50 mmol·L^-1). After verification by SDS-PAGE and Western Blot, the prokaryotic Hc-DAF-22 was purified by Ni-chelating affinity chromatography and concentrated by ultrafiltration tube. The concentration of protein was measured according to protein concentration assay kit. Natural state of Acetoacetyl CoA can occur keto - enol tautomerase then enol compound were formed. Using this property, taking acetoacetyl coenzyme A as the substrate, the enzyme activity of renatured Hc-DAF-22 was measured in the reaction system (50 mmol·L^-1 Tris-Cl pH 8.1, 20 mmol·L^-1 MgCl₂, 60 μmol·L^-1 CoA, 10 μmol·L^-1 AcAc-CoA, 0.1 μg protein). Different temperatures and pH were set in order to determine the optimum reaction conditions. 【Result】 The results showed that Hc-daf-22 gene was successfully cloned and gene similarity was 99.9% compared with H. contortus ZJ strain. Recombinant Hc-daf-22 could be expressed in E. coli BL21 and it could be detected both in the lysate supernate and precipitates, and molecular weight of the fusion protein was about 59 kD. The enzyme assay indicated that it could catalyze the substrate acetoacetyl coenzyme A, suggesting the activity of thiolase. And the optimal pH of this reaction was 8 and the optimal temperature was 37ºC. Under the optimal reaction conditions, the Michaelis constant Km and the maximum reaction velocity Vmax were 33.765 μmol·L^-1 and 1 784 nmol·L^-1·min^-1, respectively. 【Conclusion】 H. contortus DAF-22 protein is a key enzyme of peroxisome β-oxidation, this experiment is successfully determined of Hc-DAF-22 protein activity in vitro activity test. It proved that Hc-DAF-22 have certain thiolase activity, but is low compared with the Caenorhabditis elegans homolog protein.

A Modified Paraffin-Section Technique for Ovine Cumulus-Oocyte Complexes

LI HaiJun, YU BoYang, DUAN YunJiao, LIU XingYu, WENG YaZheng, DU ChenGuang, WANG XiuMei

Scientia Agricultura Sinica. 2017, 50(8): 1543-1550. doi:10.3864/j.issn.0578-1752.2017.08.018

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【Objective】 During the ovine antral follicle development, the multilayer cumulus cells closely surround the oocyte to form a special structure named cumulus-oocyte complex (COC), existing in the follicular cavity. The ovine COC will be discharged tube from the mature follicle into the fallopian to be fertilized. The existing technologies for protein identification in COC choose the ovarian tissue containing follicles to be sliced up or to immunostain the whole COC after the COC isolated from the follicle. However, there are significant defects when using the two methods to detect protein expression in ovine COC from the antral follicle. In the present study, the conventional paraffin section technology was improved so as to meet the demand of ovine COC protein identification. 【Method】 The COCs were obtained from the antral follicles by aspiration, and the special structure containing multiple ovine COCs was created by paraffin embedding. Together with the healthy ovarian tissue, the target protein expression, urokinase-type plasminogen activator (uPA) and urokinase-type plasminogen activator receptor (uPAR), were identified in the COCs structure and the immunostaining effects and procedures were compared between the modified and conventional paraffin immunohistochemical technologies. 【Result】 The samples of ovine COCs and ovaries were obtained by paraffin embedding. The single COC sheets distributed in glass slides and the thin ovarian layer (5µm) was formed following sectioning, plastering and dewaxing. The indirect immunostaining results showed that, ? The target protein expression was consistent in the two types of COCs, which revealed that uPAR was expressed in both bovine cumulus and oocyte, while uPA was only expressed in cumulus. ? In the COC section, a well-preserved bovine COC structure with well-defined layers and easily identified protein location in either the oocyte or cumulus was observed. In the ovarian section, the follicle remained intact, and the clear protein localization in oocyte could be observed; however, cumulus layers and its protein localization were unclear. ?A comparison of the staining procedures between the both methods described herein showed that ovine COCs paraffin-sectioning technique provided a more simplified process and significantly shorter handling for fixation, dehydration, transparent processing, waxing and dewaxing. For example, fixation was shortened from 24 h to 2 h; dehydration was converted from the granual steps of 10 h to the two-step of 1 h; transparent processing was shortened from 30 mins to 10mins; waxing and dewaxing were simplified from 6 h to 1 h, and from the granual steps of 25 mins to the two-step of 10 mins, respectively. 【Conclusion】 The modified COCs paraffin sectioning technique showed a better immunostaining effect and featured the higher generation rate for COCs, the clearer outline of oocyte and cumulus cells, and the more simplified operation process. Based on the complete COCs structure, this new technology effectively detected the target protein expression in ovine COCs, with great methodological significance to reveal the developing mechanism of ovine follicles.

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