Table of Content

16 August 2017, Volume 50 Issue 16

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

Molecular Regulation Mechanism of Leaf Development Mediated by Ath-miR169d in Arabidopsis thaliana

ZHANG Min, ZHU Ming, LI WenZong, MA Jie, LIU YuePing, JIANG HaiYang, WANG Lei, XU MiaoYun

Scientia Agricultura Sinica. 2017, 50(16):  3063-3070.  doi:10.3864/j.issn.0578-1752.2017.16.001

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【Objective】The aims of this study are 1) to analyze the role of ath-miR169d in the progress of leaf development in Arabidopsis, 2) to illuminate the mechanisms of molecular regulation mediated by ath-miR169d, and 3) to provide a new insight into improving the photosynthetic performance and the genetic engineering of vegetables biomass and crop productivity.【Method】Ath-miR169d over-expression transgenic plants, STTM ath-miR169d knockdown transgenic plants, and WT plants were grown in a controlled culture room at 22℃ with a relative humidity of 60% and 16 h/8 h photoperiod. Number and size of rosette leaf were measured; pmiR169d::GUS vector were constructed and transformed into Agrobacterium tumefaciens EHA105, and then infected the buds of wildtype Arabidopsis (Columbia, Col-0), the pmiR169d::GUS transgenic Arabidopsis plants were used to observe the expression profile of ath-miR169d in different organs and tissues by GUS staining method. Using 8-week-old seedlings of ath-miR169d over-expressing transgenic Arabidopsis thaliana and wild type plants as materials, morphology of leaf epidemic cells were observed by scanning electron microscope (SEM). Whole shoots were harvested from 4-week-old seedlings of ath-miR169d over-expressing transgenic A. thaliana and wild type plants, subsequently the total endogenous IAA were detected and quantified as methyl esters by gas chromatography-mass spectroscopy (GC-MS). The mRNA expression profiles were detected by microarray analysis to screen differentially expressed genes. The expression of key genes in plant auxin signal pathway were verified by real-time fluorescent quantitative PCR (RT-qPCR). 【Result】In contrast to WT, ath-miR169d overexpression plants exhibited fewer and smaller rosettes, STTM ath-miR169d knockdown plants showed more and larger rosettes. Moreover, STTM ath-miR169d plants could generate new rosette leaves incessantly even after bolting and seeds harvest, whereas the leaves of ath-miR169d overexpression and WT plants generally decayed after harvest. The epidermal cells in the leaves of ath-miR169d over-expression plants were smaller than that in the WT leaves by SEM detection. Ath-miR169d was highly expressed in SAM and leaf vasculature, and expression profile was stronger in new leaves than in old ones. Endogenous IAA content reduced by 38.6% in ath-miR169d over-expressing plants compared to wild-type plants, showing Ath-miR169d was potentially involved in auxin signal pathway by microarray analysis. The auxin biosynthesis gene YUC2 and transporter gene PIN1 were downregulated in ath-miR169d over-expressing plants whereas, auxin response factors 1 and 2 (ARF1 and ARF2) genes were upregulated. Expression profile of YUC2, PIN1, ARF1 and ARF2 in STTM miR169d plantswere opposite to over-expressing plants. 【Conclusion】Results of the experiment showed that the leaf development was regulated by ath-miR169d through the auxin-signaling pathway. Number and size of rosette could be alerted by ath-miR169d expression level, and further affected biomass of the whole plant.

Transcription Factor SiNAC18 Positively Regulates Seed Germination Under Drought Stress Through ABA Signaling Pathway in Foxtail Millet (Setaria italic L.)

DOU YiNing, QIN YuHai, MIN DongHong, ZHANG XiaoHong, WANG ErHui, DIAO XianMin, JIA GuanQing, XU ZhaoShi, LI LianCheng, MA YouZhi, CHEN Ming

Scientia Agricultura Sinica. 2017, 50(16):  3071-3081.  doi:10.3864/j.issn.0578-1752.2017.16.002

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【Objective】 Foxtail millet (Setaria italica L.) has strong tolerance to drought stress. The objective of this research is to screen key regulatory factors affecting the germination process under drought conditions in plants through reverse genetics method, which will contribute for further research of regulation mechanism of seed germination under drought condition. 【Method】 Multiple sequence alignment of SiNAC18 protein sequences and millet homologous sequences were made by using ClustalX 2 and MEGA 5.05 softwares, and the phylogenetic tree was constructed. The expression patterns of SiNAC18 under the stress condition were analyzed using the real-time PCR method. SiNAC18 protein subcellular localization was analyzed by transient transfection method. Biological function of SiNAC18 was analyzed by using overexpression of SiNAC18 in Arabidopsis thaliana. The expression of downstream genes of SiNAC18 was analyzed by real-time PCR. 【Result】 The length of SiNAC18 is 1 074 bp encoding a hydrophilic protein with polypeptide of 357 amino acids, and its molecular weight is about 38.8 kD. Phylogenetic tree analysis indicated that SiNAC18 belongs to NAP subgroup of group I in the NAC transcription factors family and has the highest homology with Arabidopsis gene AtNAC29. The amino acid sequence alignment results show that the N-terminal of SiNAC18 and the highest homology transcription factors of SiNAC18 in other species, including rice, Arabidopsis thaliana, soybean and maize, has A, B, C, D and E five conserved domains. The C-terminal of the protein has a high degree of polymorphism, demonstrating that the N-terminal sequence of SiNAC18 is associated with its downstream promoter. Real-time PCR results showed that SiNAC18 were induced by drought (PEG), high salt (NaCl) and hydrogen peroxide (H₂O₂) treatment. Subcellular localization results showed that SiNAC18 protein is localized in the nucleus. Gene function analysis showed that in the ABA and PEG stress treatments, the germination rate of SiNAC18 transgenic Arabidopsis thaliana and wild type seeds was significantly different. Under normal growth conditions, germination rate of the wild type Arabidopsis WT and SiNAC18 transgenic Arabidopsis was the same, and when the concentration of PEG was increased to 10% and 15% on MS medium, the germination rate of SiNAC18 transgenic Arabidopsis was significantly higher than WT. Under the conditions of 2 and 5 μmol·L^-1 of ABA treatment, the germination rate of SiNAC18 transgenic Arabidopsis was significantly lower than that of WT. Analysis results showed that the expression of downstream genes of ABA related genes AtRD29A, proline synthesis related genes AtP5CR and AtPRODH and peroxidase gene AtPRX34 in SiNAC18 transgenic plants was higher than that in WT, which suggesting that SiNAC18 affects the germination rate of transgenic plants under drought conditions by affecting the expression of those downstream genes. 【Conclusion】 NAC like transcription factor gene SiNAC18 positively regulates the germination of plants under drought conditions through ABA and oxidative stress response signaling pathway in foxtail millet.

Cloning and Functional Analysis of the CTR1 in Soybean

ZHANG NanNan, XUE Dong, CUI XiaoXia, ZHAO JinMing, GUO Na, WANG HaiTang, XING Han

Scientia Agricultura Sinica. 2017, 50(16):  3082-3091.  doi:10.3864/j.issn.0578-1752.2017.16.003

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【Objective】The plant hormone ethylene involved in plant growth, development, biotic and abiotic stress processes. The CONSTITUTIVE TRIPLE RESPONSE1 gene acts as a key negative regulator of ethylene receptors and participates in ethylene signal transduction pathway by combining ethylene receptor. In order to conduct a preliminary study on the function of GmCTR1, GmCTR1 was cloned and its expression was analyzed, and then transformed into soybean hairy roots for analyzing resistance to Phytophthora root rot of soybean.【Method】Based on the sequence of AtCTR1 (AT5G03730), Glyma.13G151100 which with the highest homology was selected by BLAST, and it was named GmCTR1. The coding sequence of GmCTR1 was isolated from Williams82. Sequence alignment, phylogenetic analysis were performed. The expression level of GmCTR1 uponinfection of Phytophthora sojae wasanalyzed by qRT-PCR. The plant overexpression vector pBinGFP2:GmCTR1 was constructed. Then the biological function of GmCTR1 against P. sojae was explored through transforming soybean hairy roots in agrobacterium-mediated method. The transgenic overexpressing hairy roots and empty-vector hairy roots were obtained by GFP fluorescence screening. Then the lesion length, accumulation of P. sojae andthe relative expression level of resistance related genes were measured.【Result】 Based on the sequence of AtCTR1 (AT5G03730), the gene Glyma.13G151100 which with the highest homology was selected by BLAST, and it was named GmCTR1. The coding sequence of GmCTR1 was isolated from Williams82. The CDS of GmCTR1 is 2 511 bp in length. GmCTR1 is a serine/threonine protein kinase encoding 836 amino acids. The molecular weight is 92.35 kD, and the isoelectric point is 6.51. Multiple sequence alignment of GmCTR1 and other CTR1s showed that GmCTR1 contains the typical domain of CTR1 protein. Phylogenetic analysis indicated that the CTR1s from Phaseolus vulgaris, Medicago truncatula were highly similar to GmCTR1 which located in the same branch. qRT-PCR analysis showed that the expression level of GmCTR1 was up-regulated upon infection by P. sojae. There was the highest expression level at 48 h post infection, and then the expression level decreased slightly. The CDS of GmCTR1 was constructed into the overexpression vector pBinGFP2. The recombinant plasmid was confirmed by bacteria PCR and enzyme digested. After inoculation of P. sojae, the resistance was significantly decreased in overexpressed hairy roots. Compared with the empty-vector hairy roots, there are longer lesion length at 36 hpi. Expression analysis showed that there are more accumulation of P. sojae inoverexpressing hairy roots. Besides, the expression level of resistance related genes is significantly reduced in overexpressing hairy roots.【Conclusion】GmCTR1 may act as a negative regulator in the interactions between soybean and the P. sojae.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY

Effect of Nitrogen Application Rates on Quercetin and Hesperetin Exuded by Roots in Wheat and Faba Bean Intercropping System

LIU YingChao, XIAO JingXiu, TANG Li, ZHENG Yi

Scientia Agricultura Sinica. 2017, 50(16):  3092-3100.  doi:10.3864/j.issn.0578-1752.2017.16.004

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【Objective】The dynamic changes and accumulation characteristics of quercetin and hesperetin at different nitrogen levels and different growth stages of wheat and faba bean intercropping system in order to provide a basis for further investigation of the mechanism of increasing yield and controlling diseases.【Method】 In a pot experiment, mesh barrier (MB) and, polythene barrier (PB) were used to determine the secretion amount of quercetin and hesperetin exuded by roots of wheat and faba bean at different nitrogen levels (1/2N﹕half of the normal application rate; N: conventional application rate; 3/2N: 1.5 times rate of the normal application rate) in intercropping system.【Result】N levels and root separations affected biomass and root-shoot ratio of crops in wheat and faba bean intercropping system. With the increase of nitrogen application, the biomass of wheat and faba bean increased by 45%-62.5% and 3.2%-18.9%, and the root-shoot ratio decreased by 33.8%-47.3% and 11.8%-26.9%, respectively; Compared with the plastic separation, the biomass of nylon separated wheat and faba bean increased by 4.2%-25% and 19%-38.6% at the same nitrogen levels at 60 d. With the growth stages, the differences were not significant. Root separation and nitrogen levels affected quercetin and hesperetin exuded by roots of wheat and faba bean in intercropping system. With the increase of nitrogen application, the secretion amount of quercetin and hesperetin were decreased, compared with low nitrogen conditions, under conventional nitrogen application rate and high nitrogen application rate, quercetin exuded by wheat root decreased by 23.4% and 62.3%, hesperetin decreased by 32.2% and 64.5%, quercetin exuded by faba bean root decreased by 35.4% and 44.1%, hesperetin decreased by 11.9% and 23.9%. At the same nitrogen level, quercetin and hesperetin exuded by nylon separated roots of wheat and faba bean were higher than that of plastic partition. Under the condition of low nitrogen and conventional nitrogen application rates, the secretion of quercetin in nylon-separated wheat root were higher 15.3% and 27.1% than that in plastic, the secretion of hesperetin in nylon-separated wheat root were higher 21% and13.7% than that in plastic; the quercetin secreted by nylon-separated faba bean were higher 34.6% and 56.6% than that of plastic, hesperetin was higher than plastic separated by 16.9% and 5.1%; there was no significant difference between the two root systems under high nitrogen condition.【Conclusion】Root separation affected quercetin and hesperetin secretion amount exuded by wheat and faba bean roots, but this effect was controlled by nitrogen application levels, under the conditions of low nitrogen and conventional nitrogen application rates, quercetin and hesperetin that exuded by nylon-separated root in wheat and faba bean intercropping were higher than that of plastic partition, the difference was not significant under high nitrogen condition.

Comparison of the Methods for Predicting Wheat Yield Based on Satellite Remote Sensing Data at Anthesis

TAN ChangWei, DU Ying, TONG Lu, ZHOU Jian, LUO Ming, YAN WeiWei, CHEN Fei

Scientia Agricultura Sinica. 2017, 50(16):  3101-3109.  doi:10.3864/j.issn.0578-1752.2017.16.005

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【Objective】With the advantages of wide coverage, high speed, large amount of information and strong dynamics, satellite remote sensing technology can obtain crop yield timely and accurately, reflect the spatial change trend of field crop yield. The remote sensing technology has become a hot research topic in agricultural production to estimate crop yield. Through improving the method of establishing remote sensing estimation yield models, this research aims to make further efforts to improve the accuracy of predicting crop yield and provide an intuitive and reliable reference for the macro understanding of crop yield formation and changes in different regions.【Method】In this paper, based on experimental data obtained from 2011-2012 in the fixed-point observation experiment in 5 counties of Jiangsu province (Dafeng, Xinghua, Jiangyan, Taixing, Yizheng), remote sensing data of HJ-1A/1B satellite images were used to analyze the quantitative correlations between the remote sensing vegetation index, key growth index and wheat yield per unit area at anthesis in order to further enhance the mechanism and reproducibility of remote sensing inversion models. The direct model building method was used to analyze the correlation between satellite remote sensing variables and wheat yield directly. While the indirect model building methods needed to choose remote sensing variables which closely related with yield, and choose growth indices which closely related with the remote sensing variables. Firstly, the corresponding wheat growth indices were monitored by using the sensitive remote sensing variables. Then, the indirect estimation model could be established and worked for the indirect remote sensing estimation in wheat yield. Based on the remote sensing vegetation index and the highest relationship, sensitive remote sensing variables were selected to estimate wheat yield, and the wheat yield estimation model, which was built and analyzed with ground measuring results in 2012, was analyzed with the linear regression analysis method and established by using direct and indirect model building methods. Based on the evaluation indexes: R² and RMSE, the accuracy of the two models was validated and compared using the observed data in 2011 in order to increase the quantitative level and reliability of remote sensing inversion models.【Result】Single factor models based on difference vegetation index (DVI) or ratio vegetation index (RVI) extracted from HJ-1A/1B image could predict the yield directly with root mean square error (RMSE) of 918 kg·hm^-2 and 1 399.5 kg·hm^-2. A two-factor model based on DVI and RVI could predict the yield directly with RMSE of 1 036.5 kg·hm^-2. The RMSE of the indirect yield model based on normalized difference vegetation index (NDVI) and leaf nitrogen accumulation was 805.5 kg·hm^-2. It was concluded that the HJ-1A/1B image at flowering stage was feasible and the precision was high. The accuracy of the indirect yield estimation model based on NDVI and leaf nitrogen accumulation was significantly higher than that of the direct estimation model. The RMSE decreased by 112.5 kg·hm^-2, 594 kg·hm^-2 and 231 kg·hm^-2 with the comparison of DVI direct estimation model, RVI direct estimation model and two-factor model, respectively. 【Conclusion】It was confirmed that HJ-1A/B, the satellite made in China, can meet the requirement of wheat yield estimation. Compared to the direct method, it is more feasible to predict field crop yield through remote sensing model based on the indirect method. The results provide a new way to accurately estimate field wheat yield using remote sensing technology.

Effect of Partial Water Resupply at Seedling Stage on Maize Growth, Water Absorption Capacity and Anatomical Structure

NIU XiaoLi, HU TianTian, ZHANG FuCang, DUAN AiWang, LIU ZhanDong, SHEN XiaoJun

Scientia Agricultura Sinica. 2017, 50(16):  3110-3121.  doi:10.3864/j.issn.0578-1752.2017.16.006

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【Objective】 Response of crops to partial water resupply has attracted more attention. It is necessary to investigate soil moisture condition previous partial root-zone irrigation when the technology of partial root-zone irrigation is applied. This study was aimed to identify the dynamics of maize growth and water absorption capacity under partial water resupply and the physiological mechanism of compensation effect. 【Method】 With the split-root technology, a hydroponic experiment was conducted to analyze non-stressed and stressed sub-root under partial water resupply, where the water stress was simulated by the osmotic potential of a nutrient solution (PEG-6000). There were three water stress levels, i.e., -0.2 MPa, -0.4 MPa, -0.6 MPa and a control treatment (no PEG). The maize growth, root hydraulic conductance and leaf water potential were measured on the 0, 0.25, 0.5, 1, 3, 5, 7 and 9 day after partial water resupply (DAT). 【Result】 Root growth rate and hydraulic conductance in non-stressed sub-root were higher than that in stressed sub-root under partial water resupply. Compared to control treatment, root dry weight growth rate in non-stressed sub-root was significantly enhanced during 0-5 DAT, 0.25-0.5 DAT and 0.5-1 DAT in -0.2, -0.4 and -0.6 MPa treatments, respectively. Average increase rate of root hydraulic conductance in non-stressed had no significant difference at 5 DAT in -0.2 MPa treatment if compared to control treatment, whereas it was significantly reduced in the whole treatment period in -0.4 and -0.6 MPa treatments, indicating that the threshold of water stress previous partial water resupply for the compensatory effect of water uptake in non-stressed sub-root system was ≥-0.2 MPa. Moreover, root diameter and vessel diameter in non-stressed sub-root was significantly reduced at 5 DAT compared with that of 1 DAT in -0.2 MPa treatment, but it was maintained or higher than the level of control treatment. There was no significant difference in root cortex thickness/diameter ratio in non-stressed sub-root at 5 DAT between -0.2 MPa and control treatments. At 9 DAT, compared to control treatment, root diameter and vessel diameter in non-stressed sub-root was significantly decreased by 19%, but cortex thickness/diameter ratio showed a reverse trend. 【Conclusion】The compensatory effects of root growth and water uptake in non-stressed sub-root under partial water resupply were closely related to water stress severity and water resupply duration, which depended on root anatomical structure in non-stressed sub-root. Thus, the above conclusion provides theoretical support for regulating the interaction between plants and soil environment and making use of the potential plant response to soil water stress.   

PLANT PROTECTION

Evaluation of Stripe Rust Resistance and Molecular Detection of Yr Genes of 79 Wheat Varieties (Lines) in China

HUANG Liang, LIU TaiGuo, XIAO XingZhi, QU ChunYan, LIU Bo, GAO Li, LUO PeiGao, CHEN WanQuan

Scientia Agricultura Sinica. 2017, 50(16):  3122-3134.  doi:10.3864/j.issn.0578-1752.2017.16.007

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【Objective】The objectives of this study are to understand the resistance to Puccinia striiformis of wheat cultivars and the distribution and application of Yr genes in the main wheat growing regions, to give some suggestions for the farmers and governors when they choose cultivars, and to prolong the planting years of cultivars based on the harbored Yr genes. 【Method】 There were four races, designating CYR32, CYR33, G22-9 and G22-14 used to identify stripe rust resistance of 79 wheat cultivars or lines from main wheat producing areas in China at seedling stage in greenhouse. The seedlings were put into barrels for keeping humidity in dark for 12-18 h at (10±1)℃ after inoculation, then they were put into greenhouse at 16 to 18℃ in the day time and 14 to 16℃ in the nighttime with 16 hours’ light and 8 hour’s darkness. The investigation was carried out 15 days after inoculation as 0-9 scales. And CYR32 and CYR33 were used for further resistance evaluation at the adult stage at Langfang Station of Hebei Province. In order to keep soil humidity, enough water should be irrigated before the inoculation of adult plants was carried out at jointing stage. The urediniospore suspensions were adjusted to 1 g urediniospores﹕300 mL mineral oil and sprayed the susceptible control cv. Mingxian 169. After the mineral oil evaporated thoroughly, the plants were sprayed water by hand-hold sprayer and covered with plastic film for 12-16 h for keeping moisture. The incidence, severity and infection type were recorded at least twice to keep the severer records as the final data. Then the authors screened these wheat cultivars with the closely linked molecular markers of wheat stripe rust resistance genes Yr5 (Wmc175F/R), Yr10 (SC₂₀₀F/R), Yr18 (csLv34 F/R), Yr26 (We173) and 1B/1R (AF1/AF4) translocation lines. With all data were integrated, such as pedigree of cultivars (lines), resistance to the 4 races, and molecular detection, the yellow rust resistance genes were postulated. 【Result】Among all tested cultivars or lines at seedling stage, 16 (20.3%) were resistant to CYR32 and CYR33, 4 (5.1%) were resistant to G22-9 and G22-14, only 4 (5.1%) cultivars showed resistance to 4 races of Puccinia striiformis f. sp. tritici (Pst). However, there were 24 (30.4%) showed resistance to CYR32 and CYR33 at adult stage. As for wheat cultivars at all growth stages, 12 (15.2%) were resistant to CYR32, 16 (20.3%) were resistant to CYR33 and only 11 (14.0%) were resistant to both CYR32 and CYR33. Only 4 (5.1%) cultivars showed resistance to CYR32, CYR33, G22-9 and G22-14 at seedling stage and CYR32 and CYR33 at adult stage, respectively. After all the information integrated, such as pedigree of cultivars (lines), resistance to the 4 races, and molecular detection, it showed that 4 (5.1%) tested cultivars contained Yr5, 8 (10.1%) contained Yr10, 3 (3.8%) contained Yr18, 1 (1.3%) contained Yr26, 35 (44.3%) contained 1B/1R, and 4 cultivars contained 2 Yr genes, and Yuanfeng 139 contained Yr5, Yr10 and Yr26.【Conclusion】The 79 cultivars (lines) have low resistance to the prevalent 4 races and the usage of 1B/1R translocation line was still high. In the future breeding, the utilization of effective resistance genes such as Yr5 and Yr18 should be increased. Pyramiding multi-gene varieties with effective durable resistance should be strengthened and the use of 1B/1R translocation line should be decreased.

Cloning and Expression Pattern Analysis of cAMP Phosphodiesterase Coding Genes in Setosphaeria turcica

SHEN Shen, LI ZhenYang, ZHAO YuLan, LI Pan, HAN JianMin, HAO ZhiMin, DONG JinGao

Scientia Agricultura Sinica. 2017, 50(16):  3135-3144.  doi:10.3864/j.issn.0578-1752.2017.16.008

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【Objective】The objective of this study is to clarify the function of cAMP phosphodiesterase (PDE) and cAMP signal pathway in regulating the pathogenicity of Setosphaeria turcica, the genes encoding cAMP phosphodiesterase were cloned and the expression pattern of these genes were analyzed during the development of infective structures and the early stage of infecting the host. 【Method】Based on conserved sequence of PDE genes from Saccharomyces cerevisiae, Candida albicans, Botrytis cinerea, Magnaporthe oryzae, Metarhizium anisopliae and Aspergillus niger, the homologous fragments were amplified by degenerate primers PCR, and the full length PDE genes and flanking sequences were obtained by combining RACE and Genome Walking. Multiple sequence alignment for prediction coding product of the protein was constructed by MEGA 5.0 software and phylogenetic tree was constructed by adjacent method. Gene structure was analyzed by GSDS. Physical and chemical properties were analyzed by ProtParam. Secondary structure was predicted by SOMPA. Conserved domains were analyzed by SMART on line. The material of different development periods from S. turcica was collected under the induction on artificial hydrophobic medium and on the susceptible host surface. Quantitative real-time PCR (qRT-PCR) was used to analyze the gene expression patterns.【Result】There were one high-affinity phosphodiesterase gene (StH-PDE) and one low-affinity phosphodiesterase gene (StL-PDE) in the genome of S. turcica. Full-length of StH-PDE was 3 208 bp, which consisted of 5 introns and 6 exons, the coding region was 2 898 bp. The full length of StL-PDE was 5 054 bp, which contained 4 introns and 5 exons, with the coding region of 3 090 bp. StH-PDE protein contained conservative type I cAMP phosphodiesterase, and StL-PDE contained L-PDE specific type II cAMP phosphodiesterase catalytic domain. PDE homologous genes in different plant pathogenic fungi presented a high degree of similarity. StH-PDE and the high affinity phosphodiesterase gene from Magnaporthe grisea, Cordyceps militaris, Metarhizium acridum were clustered in the same evolution. StL-PDE and the low affinity phosphodiesterase gene from Ascochyta rabiri, Scedosporium apiospermum, Fusarium oxysporum, Metarhizium album were clustered in the same evolution. Compared with the mycelia, the expression levels of StL-PDE and StH-PDE in conidia were significantly up-regulated, and StL-PDE and StH-PDE were up-regulated by about 52-fold and 2-fold under the induction on artificial hydrophobic medium, respectively. The expression of both genes at the early stage of spore germination was significantly down-regulated. With the germination process, the expression level gradually increased, and the second peak appeared at the appressorial formation stage, and then the expression level was down again. However, in the whole process, the highest expression level of StH-PDE appeared in the appressorial formation period, reaching nearly 7-fold and 2-fold of that in the mycelia and the conidia, respectively, and the expression level of StL-PDE was always lower than that in conidia. During the interaction of S. turcica with the susceptible host, the expression of StH-PDE and StL-PDE in the process of pathogen infection was consistent with that under the induction of artificial hydrophobic material. The expression of both genes in the early stage of spore germination was significantly down-regulated. With the germination process, the expression level gradually increased. The expression of StH-PDE was down-regulated in 18 and 24 h post-inoculation beyond the early stage of spore germination. 【Conclusion】StH-PDE and StL-PDE showed a down-regulation-up-regulated-down-regulated expression pattern in the development of pathogen infection. The relative expression level of StH-PDE was the highest during the appressorial formation, and the expression level of StL-PDE in conidia was the highest.

Sequence Analysis and Induced Expression of Three Novel Small Heat Shock Proteins Mediating Cold-Hardiness in Harmonia axyridis

WANG HuiJuan, ZHAO Jing, SHI ZuoKun, QIU LingYu, WANG Su, ZHANG Fan, WANG ShiGui, TANG Bin

Scientia Agricultura Sinica. 2017, 50(16):  3145-3154.  doi:10.3864/j.issn.0578-1752.2017.16.009

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【Objective】 Harmonia axyridis is an important predator insect, and it is widely used in agricultural and forestry production. In this study, three small heat shock proteins (sHSP) were screened out and cloned using high throughput sequencing techniques. Also the expression levels of these three sHSP (sHPSs/sHSP genes) were detected under cold stress in order to explore the improvement of H. axyridis cold resistance role under low temperature stress conditions. 【Method】 Based on H. axyridis gene sequences in the transcriptome, several specific primers were designed to obtain the full-length open reading frame (ORF) sequence of the three sHSP genes. The expression of these sHSP genes was detected by quantitative real-time PCR (qRT-PCR) under short-time cooling or short-time cooling recover conditions, and between the black and yellow females of experimental and overwintering populations under low-temperature storage.【Result】 HaHSP47.74 and HaHSP21.53 were highly expressed at the pupal and adult stages during different developmental stages. And the expression of HaHSP21.52 in 4th instar larvae on the 4th day was significantly higher than that in control group. In short-term cooling treatment, the expression of HaHSP47.74 was significantly higher than that of the control group when the temperature was reduced to 0℃ and -5℃. The expression of HaHSP21.53 and HaHSP21.52 decreased significantly during cooling. There was no significant difference in the expression of the three sHSP genes in short-term heating treatment. The HaHSP47.74 expression of black female was significantly higher on the 5th day, while the yellow female was significantly higher from the 5th to 15th day in the experimental population under the condition of low temperature storage. The expression of HaHSP21.53 in black females was significantly higher from the 5th day to 20th day, while in the yellow females was significantly higher on the 15th day. Moreover, there was no significant expression of HaHSP21.52 in black or yellow female, and HaHSP47.74, HaHSP21.53, HaHSP21.52 in black females and yellow females were not significantly higher in the low temperature storage treatment in overwintering population. 【Conclusion】 sHSP may play a role in developmental stages of H. axyridis. HaHSP47.74 expression increased significantly under short-term cooling conditions, suggesting that sHSP in H. axyridis may play a key role under low temperature stress. While higher expression of HaHSP47.74 and HaHSP21.53 was found in the experimental population under low temperature storage, which indicated that these two genes played an important role in the cold acclimation. In addition, the expression level of black and yellow females of H. axyridis was different, it was speculated that the resistance and mechanism of different colors of H. axyridis are different.

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

Response of Anammox Bacteria Community Structure and Vertical Distribution to Different Long-Term Fertilizations in Calcareous Purple Paddy Soil

WANG YingYan, LU ShengE, LI YueFei, TU ShiHua, ZHANG XiaoPing, GU YunFu

Scientia Agricultura Sinica. 2017, 50(16):  3155-3163.  doi:10.3864/j.issn.0578-1752.2017.16.010

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【Objective】The impacts of long-term fertilizations on the anaerobic ammonium oxidation bacteria (AAOB) community structure and vertical distribution were studied in order to deeply understand the microbial mediated mechanism of anammox reaction, develop a sound fertilization regime and provide theoretical foundations for maintaining the soil quality in this region.【Method】Chemical analysis, real-time PCR and terminal restriction fragment length polymorphism (T-RFLP) were used to analyze the soil physico-chemical properties, the 16S rRNA gene abundance and composition of AAOB community, respectively.【Result】The results showed that compared to no fertilizer application control (CK), all fertilizers decreased the soil pH and nitrate content, while increased soil organic matter (SOM), total nitrogen (TN) and ammonia content. With increasing of soil depth, soil parameters including soil pH, TN, and nitrate content were decreased, while the ammonia content was not obviously changed. The qPCR results showed: the abundance of AAOB 16S rRNA gene peaked in the 0-20 cm layer, while being the lowest in the 20-40 cm depth. Mere nitrogen treatment (N) had an evident positive effect on the abundance of AAOB. The T-RFLP results indicated that in the 0-20 cm layer, the community compositions of AAOB were the most complex, which were also supported by the highest Shannon-wiener diversity index. Compositions of AAOB community in the soil fertilized with N only were the simplest, while those in the soil amended with CK were the richest. Besides, the predominant AAOB were phylogenetically affiliated to Candidatus Brocadia. Redundant gradient analysis (RDA) showed that pH was the key factor in shaping the AAOB community in calcareous purple paddy soil amended with different fertilizer treatments.【Conclusion】This study suggested that mere N fertilizer would decrease the compositions of AAOB community in calcareous purple paddy soil but increase their abundances. The topsoil (0-20 cm) was the main distribution soil depth in calcareous purple paddy soil for AAOB.

Effect of Long-Term Fertilization on Temperature Sensitivity of Soil Respiration During Fallow Season

ZHANG YanJun

Scientia Agricultura Sinica. 2017, 50(16):  3164-3174.  doi:10.3864/j.issn.0578-1752.2017.16.011

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【Objective】The objective of this experiment is to study the mechanism of changes in the temperature sensitivity of soil respiration (Q₁₀) during the fallow season under different agricultural management measures in the crop-fallow rotation system as it is of great importance for scientific regulation of greenhouse gas emissions of agricultural ecosystem in rain-fed areas of the Loess Plateau. 【Method】An experiment of long-term fertilization experiment was carried out in “Changwu Agricultural Ecological Experiment Station”, and the soil respiration rate, soil temperature, soil moisture, the quantity and quality of substrate under different fertilization treatments (CK, N, NP, M, and NPM) were determined during the fallow season after winter wheat was harvested for studying the mechanism of changes in the Q₁₀ during fallow season under different agricultural management measures.【Result】During the fallow season, the soil respiration rate was statistically significant under different fertilization treatments (P＜0.05), with soil respiration rate was increased by 6%-127% under different fertilization treatments. Soil temperature, soil moisture, the quantity and quality substrate were important factors affecting soil respiration rate (P＜0.05) in the experiment. The effect of soil temperature on soil respiration rate was fitted by the exponential relationship model (P＜0.05), and the soil temperature could explain 40%-57% of the variability of soil respiration rate. The response of soil respiration rate to soil moisture was fitted with a parabolic model (P ＜0.05), and the soil moisture could explain 56%-74% of the variability of soil respiration rate. In addition, the effect of the quantity and quality of the substrate on the soil respiration rate was simulated by the linear relationship model (P＜0.05), and the quantity and quality of the substrate could explain up to 66%-94% of the variability of soil respiration rate. The effect of long-term fertilization on soil organic carbon was not significant (P＞0.05) under N treatment, whereas increased soil organic carbon by 12%-36% under NP, M, and NPM treatments, respectively. At the same time, long-term fertilization reduced root stubble C by 34% under N treatment, whereas increased root stubble C by 15%-63% under NP, M, and NPM treatments, respectively. The effect of long-term fertilization on soil C﹕N was not significant (P＞0.05) under N and NP treatments, whereas increased soil C﹕N by 12%-13% under M and NPM treatments, respectively. At the same time, long-term fertilization decreased root stubble C﹕N by 8%-38%. During the fallow season, long-term fertilization decreased Q₁₀ by 12%-56%, which was closely related with substrate quantity (soil organic carbon and root stubble C) and quality (soil C﹕N and root stubble C﹕N) or their interplay (P＜0.05). Q₁₀ under different fertilization treatments increased with decreasing of soil organic carbon, root stubble C, and soil C﹕N (P＜0.05), with substrate quantity and soil C﹕N could explain 61%-95% of the variability of Q₁₀. Q₁₀ under different fertilization treatments increased with root stubble C﹕N (P＜0.05), with root stubble C﹕N could explain 72% of the variability of Q₁₀. Meanwhile, the contribution of substrate quantity and quality to Q₁₀ showed the ordered of root stubble C＞ root stubble C﹕N＞ soil organic carbon ＞ soil C﹕N (2.16 vs. 1.22 vs. 0.48 vs. 0.03).【Conclusion】It was concluded that in the crop-fallow rotation system, long-term fertilization affects the variation in Q₁₀ through the quantity and quality of the substrate during the fallow season in the agro-ecosystems are of great importance for scientific regulation of the greenhouse gas emissions in rain-fed areas of the Loess Plateau.

Winter Wheat Grain Yield and Zn Concentration Affected by Long-Term N and P Application in Dryland

HUI XiaoLi, WANG ZhaoHui, LUO LaiChao, MA QingXia, WANG Sen, DAI Jian, JIN JingJing

Scientia Agricultura Sinica. 2017, 50(16):  3175-3185.  doi:10.3864/j.issn.0578-1752.2017.16.012

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【Objective】 Wheat is one of the main cereal crops in northern China, and mainly grown in calcareous soils with low available zinc (Zn) , thus its grain Zn concentration generally is low. Nutrient biofortification of Zn in wheat grain has attracted great attention in recent years. Zn absorption and utilization of wheat are affected by the interaction between Zn and nitrogen (N) or phosphorus (P). Present study was based on a long-term fertilization experiment initiated in 2004 and on the potentially Zn-deficient and calcareous soil in dryland areas of northwestern China. Changes of yield and Zn concentration in grain of wheat affected by N and P application were investigated. 【Method】 The field experiment was carried out in a completely randomized block design with four treatments: control (CK, no fertilizer added), mono N application (N160, 160 kg N·hm^-2), mono P application (P100, 100 kg P₂O₅·hm^-2) and combined of N and P fertilization (N160P100, 160 kg N·hm^-2, 100 kg P₂O₅·hm^-2). Plant samples were collected to analyze the wheat biomass, grain yield and yield components, and Zn concentration, Zn uptake and distribution in wheat during four cropping seasons from 2012-2016.【Result】 Compared with the control, the N mono-application decreased the spike number of wheat by 9% and the grain yield and shoot biomass both by 12%, but increased the grain Zn concentration from 29.4 mg·kg^-1 to 42.8 mg·kg^-1 and by 46%, enhanced Zn uptake in grain and shoot, respectively, by 29% and 37%, and reduced the N /Zn and P/Zn ratios in shoot, respectively, by 13% and 45%. The P mono-application increased spike number, grain yield and shoot biomass, respectively, by 18%, 15% and 16%, but decreased grain Zn concentration and Zn uptake in grain and shoot, respectively, 31%, 19% and 17%, with the N/Zn and P/Zn ratios in shoot increased, respectively, by 19% and 83%. The N and P application also significantly increased the spike number, grain yield and shoot biomass, respectively, by 40%, 46% and 38%, enhanced Zn uptake in grain and shoot, respectively, by 36% and 34%, but decreased grain Zn concentration by 8%, with the N /Zn and P/Zn ratios in shoot increased, respectively, by 43% and 27%. Compared with the mono P, the N and P application not only increased grain yield, but also elevated the grain Zn concentration, as the result of the enhanced wheat Zn uptake and the weakened P inhibition on Zn uptake. 【Conclusion】 Although N application could increase the grain Zn concentration of winter wheat to achieve crop nutrient biofortification, the long term of mono N application is not a conducive way to sustain and increase the wheat grain production due to the nutrient imbalance in soil. The mono P application could increase the wheat grain yield, but suppress the Zn uptake, then depress the grain Zn accumulation and decrease the grain Zn concentration. Therefore, it is suggested that the N and P fertilizers should be applied together to ensure the wheat production with high yield and high quality in dryland of the Loess Plateau.

HORTICULTURE

Effects of Exogenous Melatonin on Photosynthesis of Tomato Leaves Under Drought Stress

YANG XiaoLong, XU Hui, LI TianLai, WANG Rui

Scientia Agricultura Sinica. 2017, 50(16):  3186-3195.  doi:10.3864/j.issn.0578-1752.2017.16.013

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【Objective】Melatonin is a small molecules widely exist in higher plants which is regarded as a new plant growth regulator and biological stimulant. Melatonin plays an important role in improving plant resistance, but research about melatonin involved in regulation of plant photosynthesis under adversity stress is rarely reported. The objective of this study is to explore the influence of exogenous melatonin on photosynthesis of tomato leaves under drought stress. 【Method】Tomato cultivar ‘LiaoYuanDuoLi’ was used as the experimental materials, concentration screening tests were firstly carried out: CK: Leaf spray with water and root application with 50 mL water. R5, R50, R100, R150, R250: leaf spray with water and root application with 50 mL 5, 50, 100, 150, 250 µmol?L^-1 melatonin. L5, L50, L100, L150, L250: root application with 50 mL water and leaf spray with 5, 50, 100, 150, 250 µmol?L^-1 melatonin. This process was repeated in the morning and afternoon for three consecutive days and the drought stress treatment was conducted for subsequent three days (CK0: normal water after leaf spray with water and root application with 50 mL water, CK1: drought treatment after leaf spray with water and root application with 50 mL water). The optimal melatonin concentration was screened by comparing the maximum photochemical quantum yield of PSII (Fv/Fm) and a parameter representing the quantity of efficient PSI complex (Pm). Then the influence of root application and leaf spray with exogenous melatonin on gas exchange parameters, light energy distribution and electron transfer rate of PSI and PSII and the integrity of the thylakoid membrane and ATP enzyme activity of tomato leaves under drought stress were analyzed by using photosynthetic fluorescence synchronous measurement technology. 【Result】Root application and leaf spray with different concentrations of melatonin both increased the Fv/Fm and Pm of tomato leaves under drought stress, and all showed a trend of increase at first and then decreased with the increase of concentration, the values of Fv/Fm and Pm were the highest under L100 and R100, both significantly higher than that of control, thus it was determined that L100 and R100 were the optimal concentration treatments for leaf spray and root application, respectively. L100 and R100 significantly alleviated the inhibition of drought stress on the gas exchange parameters, and the leaf net photosynthetic rate (Pn) was 2.04 µmol?m^-2?s^-1 and 1.71 µmol?m^-2?s^-1, respectively, which both significantly higher than the control (CK1) (0.52 µmol?m^-2?s^-1); transpiration rate (E) was 0.66 mmol?m^-2?s^-1 and 0.54 mmol?m^-2?s^-1, respectively, and both significantly higher than that of CK1 (0.25 mmol?m^-2?s^-1). L100 and R100 treatments significantly increased the stomatal conductance (GH₂O) and the maximum water use efficiency (WUE) and significantly reduced stomatal limitation (Ls) of tomato leaves under drought stress, it was also found that L100 treatment was superior to R100. The results of rapid light response curve showed that L100 and R100 enhanced Fv'/Fm' and qP of tomato leaves, indicated melatonin is beneficial to improve photochemical reaction efficiency of PSII of tomatoes under drought stress. Cyclic electron flow of tomato seedlings under drought stress was significantly enhanced, while melatonin treatment reduced on cyclic electron flow, but strengthened ETRI and ETRII, and they were both higher under L100 compared with R100. Y (I) and Y (II) both improved under L100 and R100 compared with CK1, indicate melatonin treatment strengthened light energy distribution to the direction of photochemical reaction of PSI and PSII under drought stress. P515 induction curves of L100 and R100 were higher than that of CK1 after dark adaptation, and after illumination, P515 signal of CK0 fell fast, followed by L100 and R100, CK1 treatment decreased slowest, showed that exogenous melatonin protected the thylakoid membrane from damage caused by drought stress and strengthened the ATP-synthase activity. 【Conclusion】Root application and leaf spraying with exogenous melatonin can relieve the inhibition of drought stress on photosynthetic performance of tomato leaves, strengthen the photosynthetic efficiency, leaf spraying is a more simple and efficient measure compared with root application. Melatonin can enhance crop photosynthesis adaptability to the environment stresses and has an regulatory role in crop growth and development.

Effects of Overexpression of Apple Cytokinin Response Factor Gene MdCRF6 on Anthocyanins Accumulation and Salt Stress Tolerance

AN JianPing, SONG LaiQing, ZHAO LingLing, YOU ChunXiang, WANG XiaoFei, HAO YuJin

Scientia Agricultura Sinica. 2017, 50(16):  3196-3204.  doi:10.3864/j.issn.0578-1752.2017.16.014

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【Objective】The objective of this study is to clone apple cytokinin response factor gene MdCRF6, to analyze its homology with Arabidopsis AtCRF6, its expression of exposing to cytokinin and salt stress as well as its binding to DRE motif (ACCGAC), to identify its role in regulating anthocyanins accumulation and salt stress, and to provide a theoretical basis for studying cytokinin signaling pathway and regulating fruit tree growth and development. 【Method】 MdCRF6 gene was cloned by homology sequence alignment and PCR technique. The phylogenetic tree of MdCRF6 and Arabidopsis CRFs was constructed using MEGA5.0. The conserved domains of MdCRF6 were analyzed using SMART software and DNAMAN software. Real-time fluorescent quantitative PCR (qRT-PCR) was performed to detect the gene expression of MdCRF6 exposing to cytokinin and salt stress. The electrophoretic mobility shift assay (EMSA) was conducted to verify whether MdCRF6 could bind to the DRE motif. A plant over-expression vector of MdCRF6 was constructed and used to transform apple calli by Agrobacterium-mediated method. The phenotypes of wild-type and transgenic apple calli on anthocyanins accumulation and salt stress tolerance were characterized to investigate the function of MdCRF6 in regulating anthocyanins accumulation and salt stress tolerance in apple. 【Result】 A cytokinin response factor gene named MdCRF6 (MDP0000783818) was cloned from Malus domestic ‘Gala’. Sequence analysis showed that its open reading frame (ORF) was 1 047 bp, which encoded 348 amino acids. The results of phylogenetic tree and amino acid sequence alignment indicated that MdCRF6 contained a CRF domain in its N-terminal side and an AP2/ERF domain in its C-terminal side. qRT-PCR analysis indicated that MdCRF6 was responsive to cytokinin and salt stress, and the expression levels peaked at 3 h and 6 h exposing to 10 μmol?L-1 BA and 100 mmol?L-1 NaCl, respectively. EMSA assay showed that MdCRF6 could bind to the DRE motif. The MdCRF6-overexpressing apple calli exhibited reduced anthocyanins content and decreased salt stress tolerance. The gene expression analysis showed that overexpression of MdCRF6 significantly repressed the expression levels of anthocyanins biosynthetic genes and salt response related genes. 【Conclusion】The apple MdCRF6 exhibited high similarity to AtCRF6, and it was involved in the response to cytokinin and salt stress. Overexpression of MdCRF6 in apple calli inhibited anthocyanins accumulation and decreased salt stress tolerance. It is speculated that MdCRF6 down-regulated anthocyanins accumulation and salt stress tolerance by repressing the expression of anthocyanins biosynthetic genes and salt response related genes.

FOOD SCIENCE AND ENGINEERING

Phenolic Profiles and Antioxidant Activity of Fruit Pulp from Different Types of Peaches

LU JuanFang, LIU ShengYu, LU Wang, XI WanPeng

Scientia Agricultura Sinica. 2017, 50(16):  3205-3214.  doi:10.3864/j.issn.0578-1752.2017.16.015

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【Objective】 The aim of this study was to provide reference for the quality breeding and scientific utilization of peach by analyzing the difference of the content and composition of phenolic compounds and antioxidant activity of fruit pulp from different types of peaches.【Method】In this study, phenolic compounds of 15 peach cultivars, including five honey peach cultivars, flat peach cultivars and nectarine cultivars, were determined by high performance liquid chromatography (HPLC), and the antioxidant activities of their extracts were evaluated by DPPH, FRAP and ABTS⁺ antioxidant index. And the relationship between phenolic compounds and antioxidant activity were also analyzed by correlation analysis.【Result】The total phenolic content of 15 peach cultivars ranged from 0.35 to 2.54 mg chlorogenic acid equivalent (CHA)·g^-1 fresh weight (FW), total flavonoid ranged from 0.08 to 3.32 mg of rutin equivalent (RE)·g^-1 FW. On the whole, the total phenolic and total flavonoid contents in flat peach were significantly higher than those in honey peach and nectarine, Zaolupantao had the highest content of total phenolic and total flavonoid. A total of 6 phenolic acids (gallic acid, vanillic acid, protocatechuic acid, ferulic acid, neochlorogenic acid and chlorogenic acid) and 4 flavonoids (catechin, epicatechin, rutin, and quercetin) were identified from the tested peaches. Epicatechin, catechin, chlorogenic acid and neochlorogenic acid were the most abundant phenolic compounds in peach fruits. The composition and content of phenolics in three types of peaches were different. Epicatechin and chlorogenic acid were rich in honey peach cultivars, varying from 37.57 to 105.49 and 40.19 to 49.8 µg·g^-1 FW. Epicatechin, chlorogenic acid, neochlorogenic acid and catechin were rich in flat peach cultivars, ranging from 35.94 to 297.32, 36.14 to 80.57, 1.45 to 29.26 and 0 to 44.64 µg·g^-1 FW, respectively. Chlorogenic acid and catechin were rich in nectarine, ranging from 30.97 to 48.05 and 9.22 to 53.73 µg·g^-1 FW, respectively. The value of DPPH inhibition, ABTS and FRAP in tested fruits were 0.21-7.01, 0.66-8.57 and 0.59-5.60 µmol trolox equivalents (TE)·g^-1 FW, respectively. The overall antioxidant potency composite index (APCI) revealed that flat peach presented the highest antioxidant capacity, followed by honey peach and nectarine. Among these tested cultivars, Zaolupantao not only contained the highest phenolic content, but also presented the highest antioxidant activity. There was a significantly positive correlation between total phenolic, total flavonoid, neochlorogenic acid, epicatechin and DPPH inhibition, ABTS, FRAP values (P<0.01).【Conclusion】 These results indicated that there are significant differences in phenolics and antioxidant capacity in different types of peaches. Epicatechin and chlorogenic acid are the main phenolic compounds of honey peach, epicatechin, catechin, chlorogenic acid, neochlorogenic acid are the main phenolic compounds of flat peach, chlorogenic acid and catechin are the main phenolic compounds of nectarine. Overall, flat peach presented higher phenolic content and antioxidant capacity than honey peach and nectarine. Epicatechin and chlorogenic acid may play an important role in antioxidant capacity of peach fruit.

ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT

Polymorphisms of DGAT1 Gene and Their Association with Milk Quality Traits in Yak

GAO XiaoLi, HU Jiang, GUO ShuZhen, SHI BinGang, XIE JianPeng, LUO YuZhu, WANG JiQing, MU YongJuan

Scientia Agricultura Sinica. 2017, 50(16):  3215-3225.  doi:10.3864/j.issn.0578-1752.2017.16.016

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【Objective】In this study, polymorphisms of yak DGAT1(diacylglycerol acyltransferase 1) gene were investigated and their effects on some milk quality traits were estimated so as to enrich the molecular genetic data for yak. 【Method】 Mutations in intron5-exon7 and intron15-exon17 of DGAT1 gene were checked by PCR-SSCP in 4 yak populations (Gannan yak, Tianzhu white yak, Qinghai yak and Wild-blood yak), and the effects of mutations on the milk quality traits were analyzed in Gannan yak.【Result】The c.562+32_c.562+33insCCGCCC was found in intron5-exon7 of yak DGAT1 gene ,and allele M and genotype MM were the dominant allele and genotype, and each yak population was moderately or low polymorphic (PIC＜0.5). The 2 SNPs (c.1249-23C＞T and c.1336C＞T) were found in intron15-exon17, and nucleotide substitution c.1336C＞T at exon 17 resulted in p.Arg450Cys change. Allele A was the dominant allele, the genotype AA in Gannan yak, Tianzhu white yak and Qinghai yak and AB in Wild-blood yak were the dominant genotype, respectively. Each yak population was moderately polymorphic (0.25＜PIC＜0.5). Total 6 haplotypes and 11 haplotype combinations were constituted by mutations in these two regions, and there was a linkage relationship between 3 mutations and closed to linkage equilibrium. The association analysis showed that the genotype in intron5-exon7 was not significantly correlated with the milk quality traits in Gannan yak. The milk quality traits of genotype BB were the lowest, and the milk protein rate, fat rate, total solids percentage were lower than those of other genetypes (P＜0.05), and individuals with possessing allele A had higher milk protein rate, fat rate, total solids percentage and non-fat solids percentage than those no-possessing (P＜0.05), and yaks with allele C also showed higher milk fat rate than those no-possessing (P＜0.05), but individuals with allele B showed lower milk fat rate and total solids percentage than those no-possessing (P＜0.05) in intron15-exon17 of DGAT1 gene in Gannan yak. Individuals with haplotype combination H1H3 had higher milk protein rate, fat rate, total solids percentage and non-fat solids percentage, and the milk fat rate and total solids percentage were higher than those of other haplotype combinations (P＜0.05), while those with haplotype combination H2H2 were lower, in addition to the lactose rate, the other traits were lower than other combinations (P＜0.05). 【Conclusion】The intron5-exon7 and intron15-exon17 of yak DGAT1 geneare low and moderately polymorphic, and the c.562+32_c.562+33insCCGCCC in intron6 and 2 SNPs (c.1249-23C＞T, c.1336C＞T) in intron15 and exon17 were detected, respectively. The mutations in intron15-exon17 of DGAT1 gene may affect some milk quality traits of Gannan yak, and selecting yak with allele A and haplotype H1H3, or eliminating ones with allele B and haplotype H2H2 can improve the milk quality of Gannan yak.

Study on Fetal Skin Hair Follicle Structure and Morphological Development of Subo Merino Sheep

Ablat Sulayman, TIAN YueZhen, XU Qin, HE JunMin, ZHAO BingRu, XU XinMing, FU XueFeng, Ranagul Anwarjan, HUANG XiXia, TIAN KeChuan

Scientia Agricultura Sinica. 2017, 50(16):  3226-3235.  doi:10.3864/j.issn.0578-1752.2017.16.017

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【Objective】The main purpose of this study was to study the tissue structure and morphological development process of hair follicles in Subo Merino sheep, and results of the study will lay a histological basis for further resolving the molecular regulation mechanism of hair follicle development of fine-wool sheep. 【Method】The histological characteristics of ten different parts of skin, such as (the poll, midside of neck, up side of neck, wither, back, midside of body, shoulder, belly, sacrum and femur, respectively) of Subo Merino sheep fetuses on the 65, 85, 105 and 135 day of gestational age were analyzed using paraffin section methods. 【Result】 The results showed that the hair follicle structure of Subo Merino sheep was in the same manner compared with other animals, which was composed of hair bulb, connective tissue sheath, outer root sheath, inner root sheath and hair shaft. At the embryonic age of 65 days，primary follicles has been formed hair germs, with the extension of the hair germs, and continue depth into the dermis, the columnar structure was formed. The cap-like structure was formed by the aggregation of a large number of dermal cells at the end parts; at this stage, the epidermis of each part were formed a complete structure. At the embryonic age of 85 days，the upper part of the hair follicle formed bulge, formed hair cone structure above the dermal papilla, hair bulb was basically formed, and the length of the hair papilla was larger than the width; the vesicle structure of the secondary follicle could be found at the bottom of the primary hair follicle, and also the original sebaceous cell were found. A great quantity of secondary follicles were formed at the embryonic age of 105 days; along with the extension of the primary follicles, secondary follicles inserted into the dermis layer, the diameter of the dermal papilla was gradually increased and sebaceous glands began to form; the secondary-derived follicles began to differentiate from the neck carina of the original secondary follicles, sweat gland and hair canal could also be found, and formed a complete hair follicle group structure, trio group was dominant; each hair follicle group was composed of 1-3 primary follicles and several secondary hair follicles surrounding it. A large number of secondary-derived follicles were formed at the embryonic age of 135 days, formed mature sweat glands, most of primary follicles and some of secondary hair follicle had matured at this stage, hair follicle formed a keratinizated hair shaft and emerged through the epidermis.【Conclusion】 It can be inferred from the experiment that primary follicles of embryonic skin in Subo Merino sheep began to form about 50-55d; gestational age of 65-75 days are an important period for forming of primary follicle; gestational age of 80-85 days are the formation period of secondary follicle, in this period, a great number of primary follicles were formed; a great deal of secondary follicles were differentiated at the gestational age of 105-135 days, this is the critical period of the formation and development of secondary follicles. This study described the hair follicle structure and the whole formation process of hair follicles from occurrence to maturity of Subo Merino sheep and provide a reference information for the related field researchers.

Identification, Expression, and Functional Analysis of Cathepsin L in Silkworm (Bombyx mori)

PAN GuangZhao, ZHANG Kui, LI ChongYang, ZHAO YuZu, SHEN Li, XU Man, SU JingJing, LIN Xi, CUI HongJuan

Scientia Agricultura Sinica. 2017, 50(16):  3236-3246.  doi:10.3864/j.issn.0578-1752.2017.16.018

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【Objective】 The objective of this study is to identify and clone the BmCathepsin L (BmCat L) from silkworm (Bombyx mori), investigate its sequence features and expression profiles, and to clarify the response of BmCat L under E. coli stimulate in B. mori. Results of the study will provide a theoretical basis for further studying the function of BmCat L in B. mori.【Method】The primers was designed based on the sequence of BGIBMGA006893 in SilkDB database. The full length cDNA of BmCat L was acquired by RACE (rapid-amplification of cDNA ends) technology. The protein structure and molecular weight was predicted online. About the homologous sequences of Cathepsin L from other species were downloaded from NCBI, and the deduced amino acid sequence of putative BmCat L was aligned using the Clustal X (1.83) software, phylogenetic tree was constructed using MEGA 6.0. Its expression profile was performed by RT-PCR and qRT-PCR. Specific primers were designed to amplify the high specificity fragment, and then the PCR product was ligated to the PET-28 vector, which was transformed into Rosetta E.coli. The recombinant protein was induced by IPTG and purified by Ni⁺ affinity chromatography. Finally, the immune response was challenged by E. coli. Its relative mRNA level was detected by qRT-PCR.【Result】BmCat L was clustered on nscaf2860 which was located on chromosome 10, and the gene number in silkDB was BGIBMGA006893. Full length of its ORF is 687 bp, encoding 228 amino acids, there is a conservative Pept_C1 domain structure. The molecular weight and isoelectric point of the deduced protein is 26.132 kD and 4.57, respectively. Phylogenetic analysis showed that it is close to its homologous protein from Spodoptera frugiperda and Danaus plexippus. RT-PCR results showed that BmCat L was specifically and highly expressed in haemocyte. The recombinant protein of BmCat L was produced and purified, and finally verified by His-antibody. The immune challenge experiments results revealed the relationship between Cathepsin L gene and its immune system in B. mori.【Conclusion】 The Bmcat L cDNA was cloned. It was specific highly expressed in haemocytes. The recombinant proteins were obtained through prokaryotic expression and protein purification. More importantly, the expression of BmCat L was significantly increased after treating with E. coli in haemocytes. It is speculated that BmCat L might be involved in the immune response of B. mori.