Table of Content

01 February 2017, Volume 50 Issue 3

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

Stripe Rust Resistance and Genes in Chongqing Wheat Cultivars and Lines

LI Bei, XU Qi, YANG YuHeng, WANG QiLin, ZENG QingDong, WU JianHui, MU JingMei, HUANG LiLi, KANG ZhenSheng, HAN DeJun

Scientia Agricultura Sinica. 2017, 50(3):  413-425.  doi:10.3864/j.issn.0578-1752.2017.03.001

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【Objective】China has one of the largest stripe rust epidemic areas in the world. Chongqing, as an important overwintering region, plays a key role in wheat stripe rust epidemic. Understanding of resistance levels of the wheat cultivars (lines) and the application of Yr genes in this region may provide valuable recommendations for managing the disease.【Method】A total of 18 varieties and 89 advanced lines of wheat were tested at seedlings stage with four Chinese predominant stripe rust races CYR32, CYR33, V26/G22-9 and V26/CM42 of Puccinia striiformis f. sp. tritici. In field tests, wheat entries were evaluated for stripe rust resistance in Yangling, Shaanxi, artificially inoculated with mixture of CYR32 and CYR33. In Tianshui, Gansu Province, an over-summering region, the entries were evaluated under natural infection in 2015 and 2016, based on the seedling and field reactions, the resistance of the germplasms was classified and assessed. Based on the reactions of Yr single-gene lines, molecular markers for Yr5, Yr9, Yr10, Yr15, Yr17, Yr18, Yr26were used to detect the corresponding genes.【Result】Among the 107 entries, 57 (53.27%) were resistant to both CYR32 and CYR33, 11(10.28% ) were resistant to CYR32, CYR33 and V26 / CM42, 9 (8.41%) were resistant to CYR32, CYR33 and V26/G22-9. 8 (7.48%) lines were resistance both at seedling and adult stages, 9 (8.41%) were resistant at adult plant stage, 90 (84.11%) were susceptible. Among the tested materials, 21 germplasms may contain Yr9 gene, 39 lines(varieties) may contain Yr26 gene, 17 materials may contain Yr17 gene, and 3 may contain Yr18 gene. Other materials controlled by unknown resistance genes. And Yr5, Yr10 and Yr15 were absent in the test lines. The 8 all-stage resistant materials were not detected the presence of the Yr5, Yr9, Yr10, Yr15, Yr17, Yr18 and Yr26 genes, and they may contain other resistance genes.【Conclusion】The level of resistance of wheat varieties (lines) in the region of Chongqing to tested races is extremely low, and Yr26 materials were used with high frequency especially as the rise of the V26/G22-9 and V26/CM42Yr26-virulent races. It was recommended that in order to improve the diversity of resistance gene, use of resistant varieties is important.

Molecular Cloning and Functional Analysis of GhNAC7 in Upland Cotton (Gossypium hirsutum L.)

ZHENG XueWei, SHAH Syed Tariq, FAN ShuLi, WEI HengLing, PANG ChaoYou, LI HongBin, YU ShuXun

Scientia Agricultura Sinica. 2017, 50(3):  426-436.  doi:10.3864/j.issn.0578-1752.2017.03.002

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【Objective】The primary objectives of this experiment are to clone GhNAC7 gene, analyze its structure, detect its expression in different tissues of cotton and at different developmental leaf senescence stages. Furthermore, its function in cotton leaf senescence was further studied through transforming GhNAC7 gene into Arabidopsis. 【Method】Based on the cotton senescent leaves cDNA library, which was built by state key laboratory of cotton biology of institute of cotton research of CAAS, this gene was cloned from upland cotton using an expressed sequence tag (EST) containing NAM domain after designing primer using Oligo 6.71. Gene Structure Display Sever was conducted to analyze its structure, PlantCARE was used on-line to study its promoter sequence, and GenScan was simultaneously performed to translate amino acid on-line. Meanwhile, NAC family genes with higher scores were chosen after aligning sequence from Arabidopsis in TAIR. Afterwards MEGA 6.06 was used to display evolutionary relationships of the gene and GeneDOC was conducted to perform sequence alignment of amino acids. Via constructing 35S::GhNAC7-GFP fusion expression vector with XbaⅠand SacⅠrestriction sites, subcellular localization of GhNAC7 was studied by transient expression analysis of onion epidermal cells. Expression profiles of GhNAC7 in various tissues, in response to 200 μmol·L^-1 ABA treatment and developmental leaf senescence stages were investigated through quantitative real-time PCR (qRT-PCR). Its promoter specificity was conducted by transforming into Arabidopsis thaliana after constructing pGhNAC7-GUS fusion expression vector. Simultaneously, using pBI101 and pBI121 with EcoRⅠand SalⅠrestriction sites were used to construct fusion expression vectors, and then over-expression analysis was performed by transforming GhNAC7 into Arabidopsis thaliana.【Result】 In this study, a novel gene GhNAC7 was successfully cloned from upland cotton (Gossypium hirsutum L.). Its full-length was 1 064 bp with three exons and two introns. Results of bioinformatics analysis exhibited that its open reading frame (ORF) was 834 bp, which encoding 277 amino acids. The molecular weights of GhNAC7 encoding protein were 31.35 kD and isoelectric point was 9.22. Domain analysis of GhNAC7 showed that it belongs to the NAM subgroup of NAC family, moreover phylogenetic tree analysis showed that GhNAC7 has the closest genetic relationship with ANAC041 and ANAC083, and GhNAC7 displayed the same domain positions with ANAC083 at 17-58 aa. Its core promoter elements were also predicted, which contained a series of aging, hormone, stress-related cis-acting elements. Subcellular localization elucidated the protein of GhNAC7 encoding is a nucleoprotein. Tissue-specific analysis showed that this gene was significantly expressed in true leaves, cotyledons, flowers, anthers and senescent leaves, but displayed the highest expression in senescent leaves. And in promoter specificity analysis, it was also exhibited the strongest GUS activity in senescent leaf. Over-expression in transgenic Arabidopsis verified evident symptoms of aging compared to the wild type. Fluorescence quantitative PCR analysis showed that the gene expression was significantly up-regulated after 6 h of ABA treatment and displayed the highest at 48 h, so it was hypothesized that ABA could regulate GhNAC7 gene expression to mediate cotton leaf senescence.【Conclusion】It was concluded that GhNAC7 gene could promote cotton leaf senescence and be regulated by ABA.

Construction of EST-SSR Fingerprinting Based on Fluorescence Detection Technology for Italian Ryegrass

LIU Huan, ZHANG XinQuan, MA Xiao, ZHANG RuiZhen, HE GuangWu, PAN Ling, JIN MengYa

Scientia Agricultura Sinica. 2017, 50(3):  437-450.  doi:10.3864/j.issn.0578-1752.2017.03.003

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【Objective】In this study, a Italian ryegrass (Lolium multiflorum Lam.) variety identification system based on fluorescently labeled ETS-SSR markers was developed to provide a high-throughput DNA profiling means for identification of Italian ryegrass varieties, which can provide valuable information for the use of Italian ryegrass production and an effective method of protecting farmers’ benefits and breeders’ rights.【Method】Using three Italian ryegrass varieties (Tetragold, ChangjiangNo.2 and Aubade) with high difference of phenotypic traits, 30 primers were screened from the original 200 EST-SSR primers by polyacrylamide gel electrophoresis, which had clear amplification bands, rich polymorphism and stable amplification. Markers selected were labeled at the 5′ end of forward primer using fluorescent tags FAM, DNA analyzer was employed to detect different allelic variations of 200 individual PCR-amplified fragments by capillary electrophoresis. After further screening, 25 out of 30 fluorescent markers were chosen based on stable amplification to construct a high throughput identification system for Lolium multiflorum L..【Result】DNA fingerprint of ten Italian ryegrass materials were constructed using 25 EST-SSR primers for variety identification. A total of 127 alleles were amplified by 25 primer pairs, and the amplification fragment length ranged from 51 to 249. The effective number of alleles ranged from 2 to 11 on each pair of primers among them, primer N101 with 11 specific alleles was the most of all primers and each pair of primers had 4.00 specific alleles on average. The ratio of polymorphism sites ranged from 33.33% to 100.00%. The average value of polymorphic information content (PIC) was 0.702, the largest Shannon’s value (I) was 3.322 (N101) and the average value was 1.929, the Nei’s genetic diversity (H) ranged from 0.159 to 0.500 and the average value was 0.318. The amount of identified materials was from 0 to 10, and 14 of 25 primer pairs had 25 specific amplification sites among ten varieties (strains). Taken together, the results showed that the N101 fluorescence primer had the highest identification capability, which can identify these varieties (strains) directly. In addition, specific alleles were detected in primer ‘N101’ in three varieties such as Changjiang No.2, Ganxuan No.1 and Chuannong No.2. But due to high variation exists in same variety and different varieties of Lolium multiflorum L., in order to identify other materials, 6 EST-SSR primers (N54, N101, N146, N151, N154, N156) with good amplification and detection effect were chosen from 25 primers, the numbers of stable alleles were no less than 19, and the varieties “Double Barrel” and “Abundant” with 22 alleles were the most of all materials. Six high-efficiency primers were used for construction of DNA fingerprint spectrum including the standard model, fingerprint code and QR encodes of fingerprint spectrum. In this study, the fingerprint code and the unique QR encode for ten varieties of Italian ryegrass were firstly obtained using EST-SSR molecular marker with the aid of capillary electrophoresis and fluorescence labeling technology.【Conclusion】In this study, a SSR high-throughput identification system was constructed according to six pairs high efficiency primers, in which the ‘N101’ fluorescence primer having the most polymorphism can directly apply to identification of 10 Italian ryegrass varieties (strains).

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY

Three Dimensional Fractal Characteristics of Wheat Root System for Rice-Wheat Rotation in Southern China

CHEN XinXin2, DING QiShuo, LI YiNian, XUE JinLin, HE RuiYin

Scientia Agricultura Sinica. 2017, 50(3):  451-460.  doi:10.3864/j.issn.0578-1752.2017.03.004

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【Objective】Root system architecture (RSA) has a significant effect on water uptake and nutrient absorption. However, relevant indices for the quantification of crop RSAs are limited to 2D fractal analysis. Analytical tools for 3D fractal analysis on crop RSAs are lacking. Thus there is a need to investigate the related parameters and operational procedures suitable for the analysis of the 3D characteristics of crop RSAs.【Method】A self-fabricated digitizer for crop RSAs was used to measure the topological parameters of the field-grown wheat root, and the spatial dimensions of wheat RSAs were obtained. Virtual wheat RSAs were then modeled and reconstructed with Matlab programming, which guaranteed a realization of the real-world wheat RSAs with virtual reality. The fractal theory was then introduced into the computing software to calculate the fractal parameters of the modeled virtual wheat RSAs, including 3D fractal dimension, 3D fractal abundance, 2D fractal dimension, 2D fractal abundance and total root length. These parameters were used to quantify the dynamics of wheat RSAs, in both the 2 experimental years and the 2 tillage treatments. Correlations among 3D fractal dimension, 3D fractal abundance, 2D fractal dimension, 2D fractal abundance and total root length were also analyzed.【Result】It was found that all the RSA-related parameters were steadily increased along wheat developmental stages, in either different years or under different tillage treatments. Differences between the 2 years appeared as the 2010-2011 crop season revealed a steady increase of RSA-related parameters, while the 2011-2012 crop season observed a more radical increase of root elongation rate. A comparison between the 2 years revealed that tillage treatment had a contrasting effect from year to year, with a better crop performance under rotary till than no-till in the first year, whereas the no-till treatment in 2011-2012 outperformed the first year. At the early stage (0-98 d), the crop season had pronounced influences on wheat RSAs, as compared with tillage treatments. At the ensuing stage (98-112 d), however, annual difference of wheat RSA parameters was as similar as the tillage treatments. A comparison between 3D fractal parameters with the 2D parameters revealed that 3D parameters were markedly contrasted with the 2D parameters, indicating that introducing the 3D parameters for crop RSA analysis is necessary. Disregard annual difference and tillage treatment, all the dynamics of 3D fractal dimension, 3D fractal abundance, 2D fractal dimension, 2D fractal abundance and total root length satisfied power law functions and were all co-related significantly. This means that the effects of crop season and tillage treatment were only related to the coefficients of the power law models. 【Conclusion】 It was concluded that the visualization and analytical tools developed with hardware and software integration and combined with fractal theory was a guarantee for precise quantification of crop root system architectures. Such an analytical tool allows recasting the spatio-and-temporal dynamics of field crop RSAs with modeled virtual roots. 3D fractal parameters could be used as a precision analytical tool for crop RSAs. In selecting root elongation tactics and optimizing the root-soil interactions an important consideration should be taken to match the crop root with its soil environment and the tillage system.

Meta Analysis on Impact of No-Tillage and Subsoiling Tillage on Spring Maize and Winter Wheat Yield and Water Use Efficiency on the Loess Plateau

WEI HuanHuan, WANG ShiWen, YANG WenJia, SUN HaiNi, YIN LiNa, DENG XiPing

Scientia Agricultura Sinica. 2017, 50(3):  461-473.  doi:10.3864/j.issn.0578-1752.2017.03.005

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【Objective】The objective of the experiment was to clarify the adaptability of no-tillage and subsoiling tillage to spring maize and winter wheat planting in different areas of Loess Plateau, and the effects of no-tillage and subsoiling tillage on crop yield-increasing in Loess Plateau.【Method】A total of 209 databases were obtained from 45 published literatures, and then the effects of no-tillage and subsoiling tillage on the yield and water use efficiency of spring maize and winter wheat were quantified using a meta-analysis method in different areas, annual precipitations and annual average temperatures of Loess Plateau.【Result】Compared with those under conventional tillage, the yield and water use efficiency of spring maize under no-tillage conditions increased by more than 10% in north and middle of Loess Plateau; the yield and water use efficiency of spring maize under no-tillage conditions significantly increased by 13.4% and 13.6% (P＜0.05) in areas with annual precipitation ≤500 mm and significantly increased by 7.6% and 9.3% (P＜0.05) in areas with annual average temperature ≤10℃. Moreover, the yield and water use efficiency of winter wheat under subsoiling tillage conditions significantly increased in southeast and northwest of Loess Plateau. In areas with annual precipitation of 500-600 mm, the yield and water use efficiency of winter wheat under subsoiling conditions significantly increased by 14.5% and 12.2% (P＜0.05), and the yield and water use efficiency of winter wheat under subsoiling conditions also increased significantly in areas with annual average temperature of ≤10℃ or ＞10℃. In various areas, annual precipitation, annual average temperature, the increases of the yield and water use efficiency of winter wheat under subsoiling tillage conditions were higher than that under no-tillage conditions.【Conclusion】In different areas of Loess Plateau, the adaptability of no-tillage and subsoiling tillage were different. No-tillage was beneficial to the yield and water use efficiency of spring maize in north and middle of Loess Plateau. The yield and water use efficiency of spring maize under no-tillage conditions increased more significantly in areas with annual precipitation of 500-600 mm and annual average temperature ≤10℃. Subsoiling tillage was beneficial to the yield and water use efficiency of winter wheat in southeast and northwest of Loess Plateau, and the effects of subsoiling tillage were better than that of no-tillage.

Sensitivity of Different Spectral Vegetation Index for Estimating Winter Wheat Leaf Nitrogen

ZHANG XiaoYuan, ZHANG LiFu, ZHANG Xia, WANG ShuDong, TIAN JingGuo, ZHAI YongGuang

Scientia Agricultura Sinica. 2017, 50(3):  474-485.  doi:10.3864/j.issn.0578-1752.2017.03.006

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【Objective】Nitrogen is one of the most important nutrients in crop growth and development. The objective of this paper is to study the setting of effective index of leaf nitrogen content inversion in order to provide an important basis for the application of hyperspectral vegetation index of leaf nitrogen content estimation, and for real-time monitoring and accurate diagnosis of crops.【Method】A total of 225 groups of canopy reflectance and leaf nitrogen content data which covering the whole winter wheat growth period and under different levels of coverage, were collected to simulate different spectral index like different central wavelengths, SNR and band width indicators, and to analyze the influence of different observation pattern on quantitative models. And then, the indicators of accuracy evaluation, coefficient of determination, root mean square error, mean absolute error, mean relative error and P＜0.01 were used to select the optimal model and the best indicators, and the sensitivity and effectiveness of leaf nitrogen content quantitative models inversion were analyzed with different spectral indicators.【Result】MTCI_B was the best vegetation index for leaf nitrogen content inversion with the center wavelengths of 420 nm, 508 nm and 405 nm, band width of 1nm, SNR greater than 70 DB; the correlation with measured nitrogen content was preferably (R²=0.7674, RMSE=0.5511% , MAE=0.4625%, MRE=11.11 percentage points and P＜0.01). RVI_{inf_r} was the best index for inversion of high coverage with the optimal center wavelengths 826 nm and 760 nm (R²=0.6739, RMSE=0.2964%, MAE=0.2851%, MRE=6.44 percentage points and P＜0.01). MTCI was the best index for inversion of low coverage nitrogen (R²=0.8252, RMSE=0.4032%, MAE=0.4408%, MRE=12.22 percentage points and P＜0.01), corresponding to the optimal center wavelengths 750 nm, 693 nm and 680 nm. Using hyperspectral vegetation indexes RVI_{inf_r} and MTCI to build a joint inversion model, the model accuracy evaluation result (R²=0.9286, RMSE=0.3416%, MAE=0.2988%, MRE=7.16 percentage points and P＜0.01) was significantly better than the best single index MTCI_B. When the optimal model was used to simulate Hyperion and HJ1A-HSI data, the accuracy of the joint model (R² reached 0.92-0.93, RMSE were between 0.37%-0.39%) was better than the single vegetation index (R² were 0.79-0.81, RMSE were between 0.63%-0.66%).【Conclusion】A good estimation of crop leaf nitrogen content could be realized by using hyperspectral vegetation index, quantitative inversion of crop leaf nitrogen content had a strong sensitivity with different spectral indexes, center wavelength, SNR, and band width. Application of multi-exponential joint inversion model significantly improved the accuracy of the inversion. And the joint inversion model had a certain degree of universality in different hyperspectral sensors.

PLANT PROTECTION

The Lethal Mechanism of Trap Plant Vetiveria zizanioides Against the Larvae of Chilo suppressalis

LU YanHui, GAO GuangChun, ZHENG XuSong, Lü ZhongXian

Scientia Agricultura Sinica. 2017, 50(3):  486-495.  doi:10.3864/j.issn.0578-1752.2017.03.007

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【Objective】 Vetiver grass (Vetiveria zizanioides) can effectively attract female adults of Chilo suppressalis to lay eggs, while the larvae can not complete their life cycles by feeding on vetiver. The objective of this study is to illuminate the lethal mechanism of trap plant-vetiver against the larvae of C. suppressalis and provide a theoretical basis for the development of sustainable control technology for C. suppressalis based on the application of trap plant vetiver in rice based ecosystem. 【Method】 The toxic substances were extracted from vetiver by the methods of extraction and silica gel column chromatography, and the biological activities of these extracts against 3rd instar larvae of C. suppressalis were determined by mixing them in the artificial diet. The nutrient contents of rice and vetiver, and the detoxifying and digestive enzymes activities of C. suppressalis larvae fed on rice and vetiver were analyzed using biochemical methods. 【Result】 The mortalities of the C. suppressalis 3rd instar larvae after 9, 12, 15 days treated by sherwood oil extract of vetiver were 38.89%, 57.41%, and 85.19%, respectively, which were significantly higher than the mortalities of larvae treated by ethyl acetate extract, n-butyl alcohol extract and other fractions. The fraction 1 (Fr1) and fraction 5 (Fr5) were isolated and obtained from sherwood oil extract of vetiver, and the mortalities of C. suppressalis the 3rd instar larvae to Fr1 and Fr5 (0.05 g·mL^-1) were 85.00% and 67.67% after 3 days treatment, respectively. These results indicated that vetiver contains toxic substances, which have lethal effect on C. suppressalis larvae. Results of nutrient contents determination showed that the contents of total protein, cellulose, total sugar and amino acids in vetiver were 9.45 μg, 1.61%, 1.45%, 4.00%, respectively, significantly lower than those in rice, and the methionine (MET) content in rice was 7-fold higher than that in the vetiver. However, the content of tannin was 1.31-fold higher than that in rice. In addition, the protease, amylase, trehalase and sucrase enzyme activities of C. suppressalis larvae on 3rd day after fed (DAF) on vetiver were significantly lower than those in the larvae fed on rice; and on the 6th DAF, the activity of P450 enzyme in the larvae fed on vetiver was significantly lower than that in the larvae fed on rice; and the esterase activity of C. suppressalis larvae on the 9th DAF on vetiver was significantly lower than that in the larvae fed on rice. Compared to glutathione S-transferase (GSTs) activity of the 3rd instar larvae feeding on rice, the enzyme activity of the larvae feeding on vetiver was also decreased, however, there was no significant difference by statistical analysis. 【Conclusion】 Vetiver as a dead-end trap plant of C. suppressalis, there were probably two lethal mechanisms of vetiver against C. suppressalis. Firstly, vetiver contains toxic substances, which have lethal effect on C. suppressalis and these substances inhibited the esterase and cytochrome P450 enzyme activities, leading to the function loss of larvae in detoxification and metabolism. Secondly, compared to rice, lack of nutrients in vetiver can not meet the demand of the larvae, and led to digestive function and physiological activity disorder and ultimately death. 

Sublethal Effects of Sulfoxaflor on the Growth and Reproduction of the Green Peach Aphid Myzus persicae

WANG ZeHua, FAN JiaMin, CHEN JinCui, GONG YaJun, WEI ShuJun

Scientia Agricultura Sinica. 2017, 50(3):  496-503.  doi:10.3864/j.issn.0578-1752.2017.03.008

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【Objective】 Sulfoxaflor is the fourth generation of neonicotinoids. In this study, the sublethal effects of this pesticide on the development and reproduction of the F₀ (parental) and the F₁ (first generation) green peach aphid Myzus persicae were investigated to provide a theoretical basis for proper usage of this insecticide. 【Method】 The sublethal concentrations of LC₁₀ and LC₂₅ were determined by the bioassay method of Potter spray tower. The agar was placed at the bottom of the glass dish, while leaf discs were placed with their adaxial surface downward onto agar. Fifteen adult aphids were transferred onto each leaf disc. Insecticide were sprayed to the leaf disc with aphids by using the Potter spray tower under seven concentrations. Mortality of the aphids was recorded 48 h later. LC₁₀ and LC₂₅ were estimated using POLO-Plus10.0 software. Sublethal effects of sulfoxaflor on the development and reproduction of the green peach aphid was evaluated by the method of establishing a life table. For the F₀ aphid, sulfoxaflor was sprayed on the adult aphids at the concentrations of LC₁₀ and LC₂₅. After the application of the insecticide for 48 h, the adults were moved to fresh leaves without insecticide and reared separately until death. For the F₁ aphid, sulfoxaflor was sprayed on the adult aphids at the concentrations of LC₁₀ and LC₂₅. After the application of insecticide for 48 h, the adults were moved to fresh leaves without insecticide. When the adult aphid produced nymphs for 24 h, one nymph was randomly selected and reared separately until death. The survival and reproduction of each aphid were recorded. The statistical differences of the development duration of nymphs, the adult longevity, the number of nymph per aphid and the life table parameters of F₀ and F₁ were analyzed using SPSS 16.0.【Result】 According to the bioassay, the LC₁₀ and LC₂₅ of sulfoxaflor on the green peach aphid after 48 h were 0.012 and 0.041 mg·L^-1. Treatments with sublethal concentrations of sulfoxaflor significantly reduced the adult longevity, the number of nymph per aphid and the reproductive period of F₀ and F₁. The values reduced with the increase of the concentration of insecticide. After being exposed to the sublethal concentrations LC₁₀ and LC₂₅ of sulfoxaflor, for the F₀ aphid, the average longevity of aphid adult was 20.89 and 15.47 d, respectively, shorter than that of control (25.41 d). The nymph number per aphid after treatment with LC₁₀ and LC₂₅ of sulfoxaflor was 56.51 and 27.33, respectively, significantly less than that of control (71.02), while the reproductive period was 20.74 and 14.37 d, respectively, significantly shorter than that of control (25.27 d). For the F₁ aphid, the average longevity of adult was 14.80 and 9.76 d, the reproductive period was 12.03 and 8.59 d, the nymph number per aphid after treatment with sulfoxaflor at LC₁₀ and LC₂₅ was 46.20 and 28.23, respectively. Compared with the control, treatment with sulfoxaflor at LC₁₀ significantly extended the development duration of 1st instar nymph (1.73 and 2.21 d), while treatment with sulfoxaflor at LC₂₅ significantly extended the development duration of 2nd instar nymph (1.43 and 1.58 d). However, there was no significantly difference in the other instar development duration and the total nymph period. Life table analysis showed that the net reproductive rate R₀ was decreased significantly after treatment with sulfoxaflor at LC₁₀ and LC₂₅ with the values of 47.15, 24.55, respectively, compared with the control with a value of 64.47. 【Conclusion】Sublethal concentrations of sulfoxaflor have inhibitory effects on adult longevity and fecundity of the F₀ and F₁ M. persicae.

Molecular Cloning, Prokaryotic Expression and Binding Functions of Pheromone Binding Protein 2 (PBP2) in the Ectropis obliqua

FENG YiLu, FU XiaoBin, WU Fan, CUI HongChun, LI HongLiang

Scientia Agricultura Sinica. 2017, 50(3):  504-512.  doi:10.3864/j.issn.0578-1752.2017.03.009

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【Objective】The larva of tea geometrid (Ectropis oblique) is a major lepidoptera pest in tea plantation, which can cause enormous economic losses every year due to its violent foraging to tea leaves. Although pheromone communication is widely involved in the process of courtship of male moths towards females, the intrinsic pheromone cognitive mechanism has not been figured out yet in tea geometrid. This experiment aims to provide a theoretical basis of chemical communication and olfactory cognition and transportation for E. oblique, by means of the molecular characterization and functions for the binding protein involved in the process of sex pheromone cognition. 【Method】A pheromone binding protein gene, EoblPBP2, was amplified and cloned by RT-PCR (Genbank number KX421383), and then subcloned into pET32a vector. Then the recombinant plasmid pET32a/EoblPBP2 was transformed into E. coli BL21 (DE3) and induced to express recombinant protein with IPTG at the concentration of 1 mmol·L^-1. Furthermore, the protein was separated by Ni²⁺-NTA agarose FF and bacterial supernatant buffer solution. The purified recombinant protein was purified by PBS buffer solution. Finally, for the investigation of molecular binding functions of EoblPBP2 with test ligands, the fluorescence competitive assay was applied to measure the binding profile of EoblPBP2 recombinant protein with tea geometrid sex pheromone and candidate tea leaves volatiles. With the help of the fluorescence reporterN-phenyl-1-naphthylamine (1-NPN), the binding constants of EoblPBP2 protein binding with the candidate ligands, including a sex pheromone component (Z, Z, Z)-3, 6, 9-octadecatriene and 10 plant volatiles molecules, were measured and calculated. 【Result】EoblPBP2 has 492 base pairs, encodes 163 amino acid residues with 6 conservative cysteines. The relative molecular mass is 15.9 kD and the isoelectric point is 4.983. The fluorescence experiment results indicated that there were 10 volatiles could quench relative fluorescence intensity below 50% of 1-NPN and EoblPBP2 recombinant protein complex system. Meanwhile, 5 tea volatiles, dibutyl phthalate, phenyl acetaldehyde, β-ionone, decanal, and trans-2-decenal exhibit strong binding affinity (the dissociation constant K_D is 7.923, 14.830, 30.368, 28.068, and 27.597 μmol·L^-1, respectively) with the EoblPBP2 recombinant protein. While the dissociation constant K_D of (Z, Z, Z)-3,6,9-octadecatriene is only 172.591 μmol·L^-1, which was evidently weaker than the candidate plant volatiles except for benzyl alcohol (the dissociation constants K_Dis 230.880 μmol·L^-1). 【Conclusion】 It was concluded that EoblPBP2 may be a PBP protein with complex biological functions, due to the multiple binding capability with test sex pheromones and plant volatiles. This study will be helpful for the further elucidation of the sex pheromone recognition and transportation in tea geometrid.

SOIL & FERTILIZER·WATER-SAVING IRRIGATION·AGROECOLOGY & ENVIRONMENT

Starch Physico-Chemical Properties and Their Difference in Three Sweetpotato (Ipomoea batatas(L.) Lam) Genotypes Under Low Potassium Stress

TANG ZhongHou, ZHANG AiJun, CHEN XiaoGuang, JIN Rong, LIU Ming, LI HongMin, DING YanFeng

Scientia Agricultura Sinica. 2017, 50(3):  513-525.  doi:10.3864/j.issn.0578-1752.2017.03.010

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【Objective】Sweetpotato (Ipomoea batatas (L.) Lam) is a typical K-preferred starchy root crop. Potassium (K) deficiency (namely low potassium), an important abiotic stress in plants，has become a major limiting element for obtaining crop high yield and good quality. However, in China, K deficiency in soils exists commonly in sweetpotato-growing areas. Sweetpotato starch utilization is closely related with its physico-chemical properties. The influence of low potassium on starch physic-chemical properties was investigated in an experiment conducted by using three sweetpotato cultivars with different potassium utilization efficiencies (KUE) in response to low potassium(K) field condition, aiming at providing reference for improving sweetpotato root properties and potassium utilization efficiency. 【Method】Three sweetpotato cultivars with different potassium utilization efficiencies (KUE) were chosen and cultivated under different potassium field condition, including two treatments (low potassium treatment, LK, and normal potassium treatment, CK).【Result】The results showed that under low K condition, the main components (amylose, amylopectin, nitrogen, total phosphorus and total lipid) in sweetpotato storage root starch changed to a certain extent. There were some differences among the three cultivars in the main components. Three distribution peaks in ≤2.50 μm, 2.50-5.00 μm and 5.00-30.00 μm were observed in starch granule size obviously. The slight decrease was observed in the whole average granule size under low K, which of Xushu32 had the least decrease in volume distribution. However, there was a significant difference in decrease of average granule size of Ningzishu1. The percentage, average granule size and peak value of different granule sizes in three distributions had some differences among three cultivars. Starch T₀ (gelatinization temperature), Tp (peak temperature) and ΔH (heat enthalpy) were basicially lower than that in CK treatment, and Ningzishu1 had significant differences in the three characteristic values. Peak viscosity and breakdown viscosity in three genotypes had an obvious decrease. Hot paste viscosity, cool paste viscosity in Xushu18 and Ningzishu1 significantly increased, but Xushu32 was on the contrary. Moisture absorption degree of starches in three genotypes was reduced differently. There was a significant difference in the decline of moisture absorption in Xushu18 and Ningzishu1. In addition, swelling power of starch increased differently in three storage root starchs. There was a significant difference in a rise of swelling power of starch in Ningzishu1. Xushu32 had high starch swelling power, but low K had a little effect on it. Correlation analysis showed that the main physico-chemical indicators of sweetpotato starch are closely related. The low K changes starch compositions and particle sizes in sweetpotato root, which led to different degrees of influence on starch gelatinization properties and thermal properties.【Conclusion】The results of the study findings indicated that potassium is a key plant element influencing sweetpotato starch physico-chemical properties. Some obvious effects on physico-chemical properties and main components exist in storage root starch under low K stress. The responses of sweetpotato genotypes differing in potassium utilization efficiency to low K stress are different.

Effects of Canal Well Water Ratios on Root Layer Soil Desalination and Groundwater Hydrochemical Characteristics

LI Ping, Magzum Nurolla, LIANG ZhiJie, HUANG ZhongDong, QI XueBin

Scientia Agricultura Sinica. 2017, 50(3):  526-536.  doi:10.3864/j.issn.0578-1752.2017.03.011

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【Objective】The effects of water utilization patterns on root soil desalination and groundwater hydrochemical characteristics were studied in well-canal combined irrigation areas in North China. 【Method】A representative area was selected in the Renmin Shengli Canal District from 2013 to 2015. Precipitation of the area, surface irrigation amount, groundwater irrigation amount, canal well water ratio (ratio of surface to groundwater irrigation amount, CWWR), salinity in 0-100 cm soil layer and groundwater total dissolved solids were analyzed for different irrigation patterns. 【Result】CWWR of 1^st, 2^nd, 3^rd branch canals was between 0.72 and 1.03, 2.50 and 2.63, 0.65 and 1.26 in 2013 to 2015, respectively. Soil saline contents of 0-100 cm layer under water utilization patterns were high in topsoil and lower layers, but low in middle layers. Salt accumulation occurred especially in the 0-20 cm soil layer. The ratio of the area with soil salinity greater than 0.32 mS·cm^-1 to 1^st branch canal irrigation areas was 60.38%, 25.99% and 41.16% in 2013 to 2015, for 2^nd branch canal, was 59.61%, 0.94% and 8.81%, for 3^rd branch canal, was 84.40%, 41.87% and 52.49%, respectively. The area with topsoil salinity greater than 0.32 mS·cm^-1of branch canals irrigated area was negatively correlated with CWWR, that is to say, the larger CWWR, the less areas with topsoil salinity greater than 0.32 mS·cm^-1 of irrigation areas. Salt accumulation in topsoil was suppressed under the water utilization pattern of larger CWWR. Compared with 2013, desalination rate in 0-20 cm layer was between 15.61% and 25.85% in 2014, and between 13.33% and 23.15% in 2015. According to the desalination rate in 0-100 cm layer from 2013 to 2015, it was found that the desalination rate of root layers was improved under larger CWWR. Hydrochemical characteristics of groundwater was obvious alkaline because cation changed from Ca²⁺/Na⁺ type in dry period to Na⁺/Ca²⁺ type in normal period. Specifically increase of total soluble solid of 2^nd branch canal irrigation area was higher than that of 1^st and 3^rd branch canal irrigation areas by 1.23-fold and 3.48-fold in normal period, respectively. Compared to sodium adsorption ratio (SAR) in dry period in 2014, SAR of 1^st, 2^nd, and 3^rd branch canal irrigation areas in 2015 decreased by 23.58%, 36.82%, and 55.47%, respectively. Furthermore, SAR of groundwater was lower than 18 in 2015.【Conclusion】Larger ratio of surface water to groundwater irrigation amount would promote desalination of soil root layer, suppress salt accumulation in topsoil, and furthermore alleviate alkaline trend of groundwater in short term.

HORTICULTURE

Molecular Cloning and Functional Characterization of the α-Subunit of Heterotrimeric G Protein Gene MdGPA1 of Apple

LI Rui, AN JianPing, YOU ChunXiang, WANG XiaoFei, HAO YuJin

Scientia Agricultura Sinica. 2017, 50(3):  537-544.  doi:10.3864/j.issn.0578-1752.2017.03.012

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【Objective】As an important signal transduction molecule in plant biology, heterotrimeric G protein plays an important role in the stimulation of the external environment, the functions in regulation of responses to biotic and abiotic stresses and transmembrane signal transduction. A α-subunit of the apple heterotrimeric G protein named MdGPA1 was cloned from Malus ×domestica ‘Royal Gala’. Its basic biological functions were identified in transgenic tobacco. It provides a reference for the study of the molecular mechanism of perennial woody plants in response to environmental factor signal transduction. 【Method】MdGPA1 gene was cloned by homology sequence alignment and PCR technique. The phylogenetic tree of GPA1 homologous species was constructed using MEGA5.0. The induced expression and tissue-specific expression profiles of MdGPA1 gene in apple with abiotic stress were detected by real-time fluorescent quantitative PCR (qRT-PCR). A plant over-expression vector of MdGPA1 was constructed and used to transform tobacco by Agrobacterium-mediated method. The phenotypic and physiological performance of the transformants were characterized under drought stresses to investigate the function of MdGPA1 on stress resistance in tobacco.【Result】A α-subunit of the apple heterotrimeric G protein named MdGPA1 (MDP0000309677) was cloned from Malus ×domestic ‘Royal Gala’. Sequence analysis showed that the length of MdGPA1 gene is 1 173 bp, which encoded 390 amino acids. A phylogenetic tree indicated that the apple MdGPA1 exhibited the highest sequence similarity to Pyrus bretschneideri PbGPA1. The qRT-PCR analysis indicated that MdGPA1 has the highest expression levels of expression in apple leaves and response to drought stress, low temperature and salt abiotic stress. Expression levels were significantly down regulated at 150 mmol·L^-1NaCl, 150 mmol·L^-1 mannitol, 10% PEG and 4℃ abiotic stress, and the expression level was significantly increased under 5% H₂O₂ stress treatment. The MdGPA1 transgenic tobacco showed a drought sensitive phenotype. The fresh weight, chlorophyll content of the leaves and proline content of MdGPA1 transgenic tobacco were significantly lower than that of the wild type tobacco. Compared to the wild type, the root morphology of MdGPA1 transgenic tobacco was lower than that of the wild type, and the dry weight was significantly lower than that of the wild type.【Conclusion】The MdGPA1 is involved in the process of plant environment stimulation, and has different responses to abiotic stresses such as drought, low temperature and salt. Transgenic tobacco was more sensitive to drought stress than the wild type tobacco. The experimental results indicate that MdGPA1 plays a negative role in response to drought stress in plants.

The Composition and Content of Polyphenols in 16 Parts of ‘Zaosu’ and ‘Nanguoli’

ZHANG XiaoShuang, ZHENG YingChun, CAO YuFen, TIAN LuMing, DONG XingGuang, ZHANG Ying, QI Dan, HUO HongLiang

Scientia Agricultura Sinica. 2017, 50(3):  545-555.  doi:10.3864/j.issn.0578-1752.2017.03.013

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【Objective】 In order to find out the best parts for polyphenol extraction and also provide the basic information for further utilization of pear germplasm, the composition and contents of polyphenols in 16 different tissues of two pear cultivars (crisp-fleshed cultivar, ‘Zaosu’, and soft-fleshed cultivar, ‘Nanguoli’) were analyzed. 【Method】 UPLC method was applied to determine the composition and contents of polyphenols in 16 parts of ‘Zaosu’ and ‘Nanguoli’, and 35 standard phenolic compounds were used for the detection. Namely, arbutin, gallic acid, 3,4-dihydroxybenzoic acid, neochlorogenic acid, procyanidins B1, DL-catechin, chlorogenic acid, cryptochlorogenic acid, 3,4-dihydroxycinnamic acid, procyanidins B2, syringic acid, epicatechin, pro-cyanidins C1, cyclohexanecarboxylic acid, P-hydroxycinnamic acid, quercetin-arab-glucoside, rans-4- hydroxy-3-met-hoxycinnamic acid, rutin, quercetin-3-galactoside, quercetin-3-glucoside, luteolin-7-O-glucoside, isochlorogenic acid B, kaempferol-3-O-rutinoside, avicularin, isochlorogenic acid A, isorhamnetin-3-rutinoside, isorhamnetin-3-O-galactoside, isorhamnetin-3-O-glucoside, phlorizin dihydrate, resveratrol, cinnamic acid, qutercetin dihydrate, phloretin, apigenin and kaemferol.【Result】Twenty-three polyphenols, including two simple polyphenols, one benylidene, five kinds of caffeoylquinic acid, five flavanols, nine flavonols and one dihydrochalcone were detected in ‘Zaosu’. Twenty-one polyphenols, including two simple polyphenols, one phenolic acid, two kinds of benylidene, four kinds of caffeoylquinic acid, four flavanols and nine flavonols were detected in ‘Nanguoli’. For ‘Zaosu’, young leaves had the highest contents of polyphenols (93.82 mg·g^-1), while flesh had the least (0.63 mg·g^-1). Arbutin and chlorogenic acid were observed in all tested tissues, and the former was analyzed to be the most important polyphenol in all detected tissues except the flesh, with the contents ranging from 0.08 mg·g^-1 to 64.86 mg·g^-1. Arbutin, syringic acid, chlorogenic acid, epicatechin, luteolin-7-O-glucoside and phlorizin dihydrate were the primary polyphenols in the six categories of detected polyphenols, with the contents of 403.5, 0.04, 79.83, 16.16, 9.07 and 0.13 mg·g^-1, respectively. Different varieties of polyphenols were also detected in each tissue, 14 kinds of polyphenols were detected in mature leaf stalk and three kinds in xylem. For ‘Nanguoli’, young leaves had the highest contents of polyphenols (104.04 mg·g^-1), while seeds had the least (2.66 mg·g-1). Arbutin and chlorogenic acid existed in all tested tissues, and the former was analyzed to be the most important polyphenol in all detected tissues except flesh and core, with the contents ranging from 0.87 mg·g^-1 to 51.75 mg·g^-1. Arbutin, 3, 4-dihydroxybenzoic acid, chlorogenic acid, epicatechin, luteolin-7-O-glucoside and phlorizin dehydrate were the primary polyphenols in the six categories of detected polyphenols, with the contents of 357.29, 0.01, 0.08, 122.15, 12.89 and 4.87 mg·g^-1, respectively. Up to 13 different polyphenols were detected in mature leaf stalk and skin and at least three in the core. Among the 16 detected tissues, young leaves contained the highest contents of polyphenols in both ‘Zaosu’ and ‘Nanguoli’. Fruit skin showed higher contents and more diversity of polyphenols than fruit core, flower stalk had higher contents of polyphenols than flower and phloem showed higher contents and more diversity of polyphenols than xylem. 【Conclusion】 Arbutin and chlorogenic acid were detected in all parts of ‘Zaosu’ and ‘Nanguoli’, and arbutin was the polyphenol with the highest content. The kinds of polyphenols detected in this research were unanimous except phlorizin and protocatechuic acid. Young leaves were relatively abundant in the composition and contents of polyphenols and therefore could be used as extraction resources for further development and utilization.

STORAGE·FRESH-KEEPING·PROCESSING

Characteristics of Stable Carbon and Nitrogen Isotopic Ratios in Wheat Milling Fractions

LIU HongYan, GUO BoLi, WEI Shuai, JIANG Tao, ZHANG SenShen, WEI YiMin

Scientia Agricultura Sinica. 2017, 50(3):  556-563.  doi:10.3864/j.issn.0578-1752.2017.03.014

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【Objective】 It remains unclear for several points when identifying the geographical origin of wheat. Is there any fractionation for the stable isotopic fingerprints of milling fractions by comparing with whole wheat flour, and whether the stable isotopic fingerprints in milling fractions can be used for identifying the geographical origin of the milling fractions as well as the whole wheat flour? These problems need to be resolved. This study was conducted to reveal the characteristics and correlations of stable carbon (δ¹³C) and nitrogen (δ¹⁵N) isotopic ratios in different milling fractions by analyzing the difference in stable isotopic ratios among milling fractions, regions or genotypes, which could provide a theoretical and technical basis for geographical traceability of wheat and its milling fractions.【Method】 In 2014, three genotypes of wheat (Han 6172, Heng 5229 and Zhoumai 16) were grown in three regions of China which were Huixian (Henan Province), Yangling (Shaanxi Province) and Zhaoxian (Hebei Province). Three plots were conducted in each region, the typical size of plot was 10 m², recommended local agricultural practices were adopted. Totally 27 wheat samples were collected from three regions in 2015, whole wheat flour were obtained by grinding, and flour, wheat shorts and bran were obtained by milling. δ¹³C and δ¹⁵N were measured for whole wheat flour and milling fractions (flour, wheat shorts and bran) by an element analysis-isotope ratio mass spectrometer. One-way analysis of variance combined with Duncan’s multiple comparison was employed to identify the significant differences among different regions, genotypes and milling fractions at isotopic levels, and Pearson correlation analysis and linear regression analysis were used to test the correlations of δ¹³C and δ¹⁵N among different categories of samples.【Result】Significant differences were observed among different regions in δ¹³C and δ¹⁵N in whole wheat flour and milling fractions, and the δ¹³C in wheat from three regions decreased in the following order: Huixian＞Zhaoxian＞Yangling. No significant difference was found between different genotypes in δ¹³C in whole wheat flour, bran and flour, and in δ¹⁵N in each category of wheat samples, significant differences were found in δ¹³C between wheat genotypes of Han 6172 and Heng 5229. Significant differences were also found in δ¹³C among different categories of wheat samples (P＜0.05), δ¹³C was relatively enriched in flour and depleted in wheat shorts and bran, while no significant difference was found in δ¹⁵N among different categories of wheat samples. Significant correlations were found in δ¹³C and δ¹⁵N between different kinds of wheat samples (P＜0.01). 【Conclusion】There were significant differences in δ¹³C among different wheat milling fractions, but no significant differences in δ¹⁵N among different wheat milling fractions. Significant correlations were observed between different categories of wheat samples in δ¹³C and δ¹⁵N. Both δ¹³C and δ¹⁵N of whole wheat flour and milling fractions were characterized by geographical features, which could be used for identifying the geographical origin of wheat and its milling products.

Effects of pH on the Non-Covalent Forces and Structure of Myofibrillar Protein and Heat Induced Gel

ZHANG Xing, YANG YuLing, MA Yun, WANG JingYu

Scientia Agricultura Sinica. 2017, 50(3):  564-573.  doi:10.3864/j.issn.0578-1752.2017.03.015

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【Objective】The influence of pH on non-covalent forces and structure of myofibrillar protein heat-induced gel was studied. The relationship between gel non-covalent forces and gel structure was revealed. 【Method】 AA type broilers were slaughtered. The myofibrillar proteins were extracted from breast muscle. The myofibrillar protein solution and heat-induced gel with different pH values (5.0, 5.5, 6.0, 6.5, 7.0) were prepared. The potential on myofibrillar protein gel molecule presents the electrostatic interaction was measured by zeta potential instrument. The I760/I1003 showing the hydrophobic interaction of gel, the I850/I830 showing the hydrogen bonding of gel, and the secondary structure contents were calculated by analyzing the amide I Raman spectrum region, these were measured using Raman spectrometer. The particle size distribution was measured by a particle size analyzer. The microstructure was measured using scanning electron microscope.【Result】 From pH 7.0 to 5.0, Zeta potential value of the gel changed from -17.87 to -0.263 (P＜0.05), which show that the surface negative charges and the electrostatic interaction of myofibrillar protein gel had significant decline. The normalized intensity of 760 cm^-1 increased from 0.86 to 0.927, which show more Trytophan were buried and a general increase in hydrophobic interactions of myofibrillar protein gel. The normalized intensity of I850/I830 ratio decreased from 1.039 to 0.927, which indicated hydroxyl groups on the phenyl ring of tyrosine are to form hydrogen bonds with water molecules change to generate hydrogen bonds with other protein molecule residues. The interactions between myofibrillar protein molecules increased, and the interactions between myofibrillar protein and water therefore declined. From pH 7.0 to 6.5, the α-helix content of myofibrillar protein gel abruptly decreased from 59.96% to 55.24% (P＜0.05). The β-sheet content significantly increased from 15.83% to 19.44% (P＜0.05). β-turn and random coil content both significantly increased (P＜0.05). From pH 6.5 to 6.0, all structure content had no obvious change (P＞0.05). From pH 6.0 to 5.0, the α-helix content of myofibrillar protein gel significantly decreased from 51.61% to 16.76% (P＜0.05). The β-sheet content significantly increased from 22.23% to 48.93% (P＜0.05). β-turn and random coil content both significantly increased (P＜0.05). As the pH decrease, the α-helix content of myofibrillar protein gradually decreased, the β-sheet, β-turn and random coil content significantly increased (P＜0.05). From pH 7.0 to 5.0, particle size of myofibrillar protein gradually increased. D₁₀ increased from 13.4 μm to 48.4 μm, D₅₀ increased from 38 μm to 253 μm, D₉₀ increased from 236 μm to 805 μm. As the pH far away from neutral condition, the microstructure of gel changed to unordered and had smaller pore. Gel has disordered microstructure in pH 5.0, when has ordered structure at pH 7.0. The largest gel pore ware found at pH 5.0, the least were found at pH 7.0. pH had a highly negative significant correlation with electrostatic interaction and hydrophobic interactions (P＜0.01), and had a positive significant correlation with hydrogen bonding and α-helix content (P＜0.05). pH also led to negative significant change of β-sheet content (P＜0.05). These show that pH had a significant impacts on electrostatic repulsion, hydrophobic interactions, intermolecular hydrogen bonding and secondary structure. Electrostatic interaction, hydrophobic interactions and hydrogen bonding had significant correlation with secondary structure (P＜0.05), which indicated non-covalent forces had significant effects on secondary structure.【Conclusion】 Non-covalent forces and secondary structure content are significantly correlated with the pH valves. The reasons of gel α-helix reduction and β-sheet increases are the decreases of electrostatic interaction, and the increase of the hydrophobic interaction and the intermolecular hydrogen bonding of myofibrillar protein gel, as the pH far away the neutral conditions.

ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT

Growth and Expression of NOD2 mRNA in Bovine Mammary Epithelial Cells Treated with Different Concentrations of MDP in Vitro

XU DanDan, WANG JianFa, ZHANG Xu, LIU DongYu, XU XiaoNan, WANG Le, CHEN Jia, SHAN XuFei, WANG XiaoYa, WU Rui, YANG Bin

Scientia Agricultura Sinica. 2017, 50(3):  574-581.  doi:10.3864/j.issn.0578-1752.2017.03.016

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【Objective】 Dairy cow mastitis is one of the most common diseases causing serious economic losses in dairy-farming industry. Bacterial infection is the main cause of mastitis. Innate immunity is the first line of defense against the invasion of pathogenic bacteria in mammary gland. NOD2 is an important member of the innate immune pattern recognition receptor of nucleotide-binding oligomerization domain (NOD) family, which defenses against various microbial invasions by recognizing its specific ligand-muramyl dipeptide (MDP), a component widely existing in gram positive bacteria and gram negative bacteria cell wall. Bovine mammary epithelial cell (BMEC) is the immune barrier of dairy cow mammary gland other than secreting milk. Here, the effect of MDP on the in vitro growth state of BMEC and the expression ofNOD2 in the BMEC was explored in this experiment.【Method】 Mammary gland tissue of healthy and lactating Holstein cows was chosen as raw materials. Collagenase digestion method combined with concentration gradient of trypsin was used to separate BMEC. Cytokeratin-18 specific expression in epithelial cells and vimentin specific expression in fibroblasts were used to identify the obtained cells by immunofluorescent techniques. BMEC was set to 6 treatment groups, including MDP stimulating concentrations of 0 (control group), 1, 5, 10, 15 and 20 μg·mL^-1. Twenty-four hours of poststimulation, BMEC status were observed under a microscope, meanwhile total RNA was extracted from BMEC and reverse transcribed to cDNA. Real time fluorescent quantitative PCR method was used to detect the expression of NOD2 in BMEC.【Result】Those cells separated by collagenase digestion method combined with concentration gradient of trypsin, immunofluorescence results of CK-18 reaction was positive and vimentin reaction was negative. All cells were in a good growth condition. In the control group and in the groups of MDP stimulating concentration at 1, 5 and 10 μg·mL^-1, BMEC grew well without any visible abnormalities. There was a small amount of BMEC detached from bottom in the group of MDP stimulating concentration at 15 μg·mL^-1. However, the group of MDP stimulating concentration at 20 μg·mL^-1 showed a large number of BMEC detached and floated from bottom. Even though those BMEC were still attached to the bottom, their morphology had already changed. Compared with the control group, the expression of NOD2 mRNA in BMEC was positively correlated with the stimulating concentrations of MDP. In other words, 24 h of poststimulation, the expression of NOD2 mRNA in BMEC gradually increased along with the stimulating concentrations of MDP.【Conclusion】High purity BMEC was successfully obtained. The obtained cells grew well and could be used in the following experiments. Although the expression of NOD2 mRNA was positively correlated with the stimulating concentrations of MDP, the stimulating concentrations of MDP in vitro culture BMEC should be controlled below 10 μg·mL^-1 in order to maintain the normal growth condition. These results suggested that BMEC could participate in the immune defense response of bovine mammary gland through the NOD2 receptor pathway. But this defense capability was influenced by the number of bacteria or the intensity of bacterial virulence. In a certain number or virulence of bacteria, the immune defense response of bovine mammary gland was enhanced along with the increasing number of bacteria or the enhancement of virulence to eliminate intramammary pathogens. While the number or virulence of bacteria exceeded to a certain range, bovine mammary gland tissue would be seriously damaged, so the immune defense barrier would be collapsed. Under this condition, the local bovine mammary gland or even all over the body would present obvious clinical symptoms.

RESEARCH NOTES

Construction and Activity Analysis of the Promoter of Purple Acid Phosphatase Gene GmPAP4 in Soybean

KONG YouBin, LI XiHuan, ZHANG CaiYing

Scientia Agricultura Sinica. 2017, 50(3):  582-590.  doi:10.3864/j.issn.0578-1752.2017.03.017

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【Objective】GmPAP4 promoter (PAP4-pro) was cloned, and its expression character was analyzed, thus providing a basis for research of regulatory mechanism of GmPAP4.【Method】On the basis of GmPAP4 (GenBank No. HQ162477) cDNA sequence and blasting the soybean reference genome, GmPAP4 promoter sequence was cloned with specific primers. The promoter regulatory elements were predicted through the online database PLACE and PlantCARE. PAP4-pro-GUS was constructed and transformed into Agrobacterium tumefaciens GV3101, which subsequently was transformed into Arabidopsis thaliana with Floral dip method. The T₃ transgenic plants were obtained by being screened with kanamycin (kan) and PCR amplification.【Result】GmPAP4 promoter was cloned, and its regulatory elements were analyzed using online databases PLACE and PlantCARE. The results showed that the promoter of GmPAP4 not only contained the core elements, but also contained the following elements: (1) tissue specific regulatory elements including as1 (root specific expression element), Skn-1_motif (endosperm specific expression element); (2): Response elements including TC-rich repeats (stress responsive elements), Box-W3 (fungal response related regulatory element); (3) binding sites including MBS (MYB binding sites of transcription factors), etc. The results of GUS staining showed that GmPAP4 promoter was mainly expressed in roots of transgenic Arabidopsis. The expression and activity of GUS measurement showed that GUS expression and GUS activity in roots of transgenic plants under phytate condition was, respectively, 1.3-fold (P＜0.05) and 1.9-fold (P＜0.05) than that under normal phosphorus condition.【Conclusion】Soybean GmPAP4 promoter was obtained in this study. According to GUS staining in different tissues and activity analysis under different phosphorus conditions, it was found that the promoter of GmPAP4 was mainly expressed in roots and significantly induced by low phosphorus signal.

Changes in the Contents of Proteins and Free Amino Acid in haemolymph of Delia antique Adult Infected by Beauveria bassiana

 

ZHANG Hui, WU ShengYong, WANG XiaoQing, LEI ZhongRen

Scientia Agricultura Sinica. 2017, 50(3):  591-598.  doi:10.3864/j.issn.0578-1752.2017.03.018

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【Objective】Beauveria bassiana is an important biological agent for control of Delia antiqua. The objective of this study is to assess the changes of proteins and free amino acid in haemolymph of D. antiqua adult infected by B. bassiana at different times, understand the relationship between the contents of proteins and free amino acids and the infection of B. bassiana.【Method】Based on the result of dose-response bioassay against D. antiqua adults infected by B. bassiana,individual newly enclosed D. antiqua adults were inoculated with 5 µL 1×10⁸ conidia/mL conidial suspension of the GZGY-1-3 strain by micro-drop method. Then the contents of proteins and free amino acid of at different times (12, 24, 36, 48, 60 and 72 h) were measured in infected and uninfected D. antiqua adult using BCA and HPLC-MS/MS. Finally, the relationship between the infection and the contents of the key free amino acid was analyzed. 【Result】The content of proteins declined first and then increased at 12-36 h. The treatment group and control group showed similar changes. However the contents of proteins of the treatment group were higher than the control group. The content of proteins increased to the maximum in 24 h after inoculation, was 1.20 times greater than the control group. The content of proteins of the treatment group declined to the minimum at 48 h, was only 0.83 lower times than the control group, and increased at 60-72 h. The results of amino acids analysis showed that there were no different of free amino acid between the treatment and control groups of D. antiqua adult. The content of free amino acids decreased in 12-36 h, increased in 48-60 h and decreased in 72 h again after inoculation. The content of free amino acids declined to the minimum in 36 h after inoculation, was 0.63 times lower than the control group and increased to the maximum in 60 h after inoculation, was 2.01 times greater than the control group. After infection, there was no change in the type of free amino acids in the hamolymph, free amino acid content of various fluctuations was quite different. The infection of B. bassiana had significant influence on the contents of important amino acids in the haemolymph. 【Conclusion】 The B. bassiana invaded into the insect body affected the balance of the proteins and amino acids in the haemolymph. It seriously hampered the normal physiological activity of the D. antiqua adult. The results of the experiment indicated that B. bassiana strains couldeffectively control D. antiqua adult.