Table of Content

16 May 2017, Volume 50 Issue 10

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

QTL Mapping and QTL × Environment Interaction Analysis of Salt and Alkali Tolerance-Related Traits in Rice（Oryza sativa L.）

LIANG YinPei, SUN Jian, SUO YiNing, LIU HuaLong, WANG JingGuo, ZHENG HongLiang, SUN XiaoXue, ZOU DeTang

Scientia Agricultura Sinica. 2017, 50(10):  1747-1762.  doi:10.3864/j.issn.0578-1752.2017.10.001

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【Objective】Experiments were carried out in 2014 and 2015 to study the yield related traits in rice under salt and alkaline stress in order to explore the QTL of major genes of salt and alkaline tolerance, and analyze the interaction effects between QTL and environment, thus revealing the genetic mechanism of panicle number per plant, seed setting rate, thousand grain weight and panicle weight per plant in rice under salt and alkaline stress. The results of the present study will provide a scientific basis for the rice genetic mechanism of salt and alkaline tolerance and molecular marker assisted breeding. 【Method】The recombinant inbred line (RIL) population derived from a cross between Dongnong 425 (DN425) with high yielding ability and quality as the female parent and Changbai 10 (CB10) with salt and alkaline tolerance as the male parent. A genetic linkage map was constructed with 120 SSR markers. The panicle number per plant, seed setting rate, thousand grain weight and panicle weight per plant in rice were measured under 6ds·m^-1 NaCl solution of salt stress, Na₂CO₃ solution (pH = 9.0) of alkaline stress and the normal water irrigation as control conditions during the whole growing period in 2014 and 2015. The additive quantitative trait loci (QTL) analysis was conducted by using the complete interval mapping method (ICIM)，the additive and epistatic QTL×environment interaction effects was analyzed by using the mixed composite interval mapping method (MCIM). 【Result】Compared with the salt stress, the alkaline tolerant traits of rice decreased significantly, and were more sensitive to the alkaline stress, the alkaline stress was more restrictive to the high-yield and stable-yield in 2014 and 2015. Under the condition of alkaline stress for two years, no significant correlation was found between the traits of salt stress. There may be genetic differences in rice under salt stress and alkaline stress. By using ICIM, a total of 61 additive QTLs for salt and alkali tolerance-related traits were detected, which were distributed on chromosomes 1, 2, 3, 4, 5, 6, 7, 8, 10, 11 and 12. By using MCIM, a total of 17 the additive QTL×environment interaction effects QTLs for salt and alkali tolerance-related traits were detected, which were distributed on chromosome 1, 3, 5, 7, 8, 9, 11 and 12. By using ICIM, qPN1-1 which was repeatedly detected under both natural and salt stress conditions for two years, qPN11-2 which was repeatedly detected only under alkaline stress conditions for two years, qPN3-3 which was repeatedly detected under both salt and alkaline stress conditions for two years, qRPN1-1 which was repeatedly detected under both natural and salt stress conditions for two years, qGW7 which was repeatedly detected only under natural conditions for two years and qPW11 which was repeatedly detected under salt, alkaline stress and natural conditions for two years, all of them were detected by MCIM. A new salt and alkali tolerance QTL qPW11, can explain 7.94%—20.13% of phenotypic variance. By using MCIM, a total of 13 epistatic QTL×environment interaction effects QTLs for salt and alkali tolerance-related traits were detected. Two pairs of epistatic QTLs which are related to panicle number per plant were detected have significant environmental interaction effects. Two pairs of epistatic QTLs which are related to panicle number per plant under ratio of stress and natural were detected have significant environmental interaction effects. Two pairs of epistatic QTLs which are related to seed setting rate were detected have significant environmental interaction effects, two pairs of epistatic QTLs which are related to seed setting rate under the ratio of stress and natural conditions were detected have significant environmental interaction effects. One pairs of epistatic QTLs which are related to thousand grain weight were detected have significant environmental interaction effects. One pairs of epistatic QTLs which are related to thousand grain weight under the ratio of stress and natural conditions were detected have significant environmental interaction effects. Three pairs of epistatic QTLs which are related to panicle weight per plant were detected have significant environmental interaction effects.【Conclusion】Both the salt stress and the alkaline stress could affect the yield - related traits in rice, but they are two kinds of stresses with different properties. Alkaline stress damage is more severe, and yield reduction is more significantly.

Production and Identification of F₁ Interspecific Hybrid Between Sesamum indicum and Wild Relative S. Indicatum

YANG MinMin, LIU HongYan, ZHOU Ting, QU HongHao, YANG YuanXiao, WEI Xin, ZUO Yang, ZHAO YingZhong

Scientia Agricultura Sinica. 2017, 50(10):  1763-1771.  doi:10.3864/j.issn.0578-1752.2017.10.002

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 【Objective】 The progenies of Sesamum indicum and wild species were obtained by interspecific hybridization in order to improve charcoal rot disease (Macrophomina phaseolina) resistance of sesame cultivars. 【Method】 No.3 wild sesame (S. indicatum) (P₃), Zhongzhi 14 (P₁) and autotetraploid of Zhongzhi 14 (P₂) were used as reciprocal cross parents, the F₁ plants of interspecific hybrids were obtained using the immature embryo culture technique. First, the authenticity of F₁ progenies were confirmed by using phenotypic, cytological and SSR marker methods in order to screen out the true hybrids . Then the three parents of the interspecific hybrids (No.3 wild sesame, Zhongzhi 14 and autotetraploid of Zhongzhi 14 )and hybrids of F₁ lines resistance to Macrophomina phaseolina was identified by artificial inoculations. 【Result】The seedling ratios of immature embryos between reciprocal combinations existed significant difference. The 773 immature embryos were inoculated of which 155 embryos developed into seedlings and the average seedling rate was 20.05%. The seedling ratios of combinations (P₃×P₁ 32.75%, P₃×P₂ 21.11%) were higher than those of reciprocal combinations (P₁×P₃ 8.84%, P₂×P₃ 13.41%). This indicates that the genotype of the parent affects the seedling rate of distant hybridization to a large extent. The number of chromosomes in the F₁ plants of (P₃ × P₁, P₁ × P₃) was 42 and in the F₁ plants of (P₃ × P₂, P₂ × P₃) was 55. The majority of the pollen grains of the F₁ hybrids were regular but no inclusions, all of which were highly sterile. HS142 primer with a better polymorphism could be clearly amplified two specific types in Zhongzhi14 (about 460 bp and 500 bp) and the one in No.3 wild sesame(about 380 bp) .Then, it could be used to identify twelve hybrid progenies and their parents. There are ten of F₁ progenies with three male and female-specific markers of the parents, the other two plants only appeared in the female parent or male parent type which was the fake hybrids. The infected lesion length of progenies from interspecific hybridization P₃×P₁, P₃×P₂, using No.3 wild sesame with high charcoal rot resistance as female parent, was 9.35 cm and 6.65 cm, respectively, while 9.90 cm and 8.90 cm from reciprocal combinations. The resistance to Macrophomina phaseolina of progenies from all combinations was higher than their cultivated parents (P₁ 14.30 cm and P₂ 11.46 cm), but weaker than wild relative (P₃ 4.80 cm). 【Conclusion】 New germplasms with high charcoal rot resistance can be created through interspecific hybridization combined with immature embryo culture, which provides important materials for genetic improvement of sesame with charcoal rot resistance.

Interaction Between TFL1 and GRFs in Arabidopsis thaliana

YUAN Min, XING JiHong, WANG Li, GE WeiNa, GUO Di, ZHANG Lan

Scientia Agricultura Sinica. 2017, 50(10):  1772-1780.  doi:10.3864/j.issn.0578-1752.2017.10.003

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【Objective】The objective of this study is to identify whether the flowering repressor TFL1 interacts with the two GRFs family members GRF4 and GRF7, and to provide a basis for illustrating the mechanism of TFL1 repressing flowering.【Method】The TFL1, GRF4 and GRF7 genes were cloned by specific primers using the Arabidopsis cDNA as the templates. These three genes were linked into pCR8 vector to get the entry vectors. The correct entry vectors TFL1-pCR8, GRF4-pCR8 and GRF7-pCR8 were obtained by colony PCR screening and sequencing. The yeast two hybrid assay vectors, including TFL1-BD, GRF4-AD and GRF7-AD, were obtained by LR reaction between these three entry vectors and the destination vectors pGADT7 or pGBKT7. The yeast competent cells which were co-transformed with TFL1-BD plus GRF4-AD or GRF7-AD vectors were incubated on –Leu/-Trp growth medium under 30℃ for 2-3 days until the yeast colonies show up. The yeast colonies in proper size were chosen and transferred to both –Leu/-Trp and -Leu/-Trp/-His/-Ade growth medium. The interaction between TFL1 and GRFs was determined through observing the growth conditions of those yeast colonies on -Leu/-Trp/-His/-Ade growth medium. The BiFC assay vectors, including TFL1-nYFP, TFL1-cYFP, GRFs-nYFP and GRFs-cYFP, were also obtained by LR reaction between these three entry vectors and the destination vectors px-nYFP or px-cYFP, and were transformed into Agrobacterium competent cells. The tobaccos, which were co-transformed by the Agrobacterium harboring TFL1-nYFP or TFL1-cYFP vector and the Agrobacterium harboring GRFs-nYFP or GRFs-cYFP vector, were grown for more 48 hours before observing YFP fluorescence signals under confocal microscopy. The interaction between TFL1 and GRFs was determined if the fluorescence signals in tobacco cells were observed under confocal microscopy.【Result】The three genes, including 534 bp TFL1, 888 bp GRF4 and 798 bp GRF7, were cloned successfully. The entry vectors (TFL1-pCR8, GRF4-pCR8 and GRF7-pCR8), yeast two hybrid assay vectors (TFL1-BD, GRF4-AD and GRF7-AD), and BiFC assay vectors (TFL1-nYFP, TFL1-cYFP, GRFs-nYFP and GRFs-cYFP) of the three genes were obtained successfully. Compared with the negative controls, the yeast colonies which were co-transformed with TFL1-BD plus GRFs-AD vectors grew well in both -Leu/-Trp and -Leu/-Trp/-His/-Ade media in yeast two hybrid assay. Compared with the negative controls, the obvious nuclear YFP fluorescence signals were observed in the tobacco cells which were co-transformed with the Agrobacterium harboring TFL1-cYFP vector or GRFs-nYFP vector. Meanwhile, the obvious nuclear YFP fluorescence signals were also observed in the tobacco cells which were co-transformed with the Agrobacterium harboring TFL1-nYFP vector or GRFs-cYFP vector.【Conclusion】The flowering repressor TFL1 directly interacts with the two GRFs family members GRF4 and GRF7 in Arabidopsis.

TILLAGE & CULTIVATION·PHYSIOLOGY & ECOLOGY

Retrieval Effects of Remedial Fertilizer After Freeze Injury on Wheat Yield and Its Mechanism at Tillering Stage

LI ChunYan, YANG Jing, ZHANG YuXue, YAO MengHao, ZHU XinKai, GUO WenShan

Scientia Agricultura Sinica. 2017, 50(10):  1781-1791.  doi:10.3864/j.issn.0578-1752.2017.10.004

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【Objective】Freeze injury in wheat often happens and affects wheat yield formation during the wintering stage. There was less study on the remedial measures to recover wheat yield after low temperature stress at wintering stage. Hence, effects of remedial fertilizer after low temperature stress on yield recovery in wheat at tillering stage and its mechanism were studied, which will provide a basis for anti-cultivation technology in wheat. 【Method】The spring wheat cultivar Yangmai16 was treated at -2℃/-6℃(day/night, 2012) and -2℃/-8℃ (day/night, 2013) for 24, 48 and 72 h, respectively, using artificial temperature-controlled phytotron system. Then the different remedial urea (N 46%) amounts of 75, 150 kg·hm^-2 (2012) and 75, 120, 180 kg·hm^-2 (2013) were all used at a time after low temperature stress. The degree and freezing injury proportion of wheat plant under low temperature stress and the changes of soluble sucrose, proline and endogenous hormone contents in the second leaves from the top on the 10th, 20th and 30th day after applying remedial fertilizer were investigated. Plant height and yield at maturity were also recorded. 【Result】The index of freezing injury increased from 0.2 to 0.5 under longer stress at tillering stage. The contents of soluble sugar, proline, abscisic acid (ABA) and zeatin riboside (ZR) in leaves of the treatment increased under longer stress. These parameters in the treatment without fertilizer amendment were higher than those in the treatment with fertilizer amendment on the 10th day after applying remedial fertilizer. The parameters reduced more rapidly with more applying fertilizer under the same duration time. The content of gibberellines (GA₃) decreased gradually with longer stress at tillering stage. The contents of soluble sugar and proline, and the contents of ABA and ZR of these treatments using fertilizer after cold stress gradually declined on the 20th day after applying remedial fertilizer. While the change of GA₃ contents was opposite to ABA and ZR contents. All these parameters reached the levels of the controlled plants in natural environment on the 30th day after applying remedial fertilizer. Wheat yield, the first and second basal internode length and plant height all lowered with longer cold stress. With increased fertilizer applying amount under the same treatment duration, the length of wheat plants were better restored and the loss of grain yield was lessened.【Conclusion】Cold injury wheat will recover growth after using the right urea amount scientifically in time depending on the cold index at tillering stage. Osmotic adjustment substance contents declining and hormone contents becoming balance, new tillers emergency and the basal internode length becoming longer were the main reason for increasing grain yield at tillering stage after applying the urea. At tillering stage, considering recovery effect and nitrogen partial factor productivity, 75 kg·hm^-2 urea would be recommended for nitrogen amendment when wheat plants were damaged slightly and the cold index was about 0.2. When the cold index was about 0.36, 120 kg·hm^-2 urea would be suggested. When the cold index was about 0.50, 180 kg·hm^-2 urea was recommended for recovering wheat growth after severe cold damage.

A Hybrid Yield Estimation Model Based on the Trend Yield Model and Remote Sensing Correction Yield Model

CHEN ChangWei, ZHU XiuFang, CAI Yi, GUO Hang

Scientia Agricultura Sinica. 2017, 50(10):  1792-1801.  doi:10.3864/j.issn.0578-1752.2017.10.005

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【Objective】 This paper analyzed the advantages and disadvantages of current crop yield estimation methods and proposed a novel hybrid yield estimation model which combines statistical yield estimation and yield estimation methods. 【Method】The model consists of three parts, trend yield estimate (Yt), remote sensing correction yield (Ys) and random error. The trend yield estimation was firstly calculated by using the polynomial regression method based on a long time series data of historical yield and then corrected by ARIMA model, which was set up by using the bias between the trend yield estimates and the historical yields. After that, a multiple linear regression model was set up to further reduce the estimation errors by using the bias between the trend yield estimates (Yt) and the reference yields as dependent variable and NDVI in critical growth period of crop as independent variables. In order to verify the feasibility and accuracy of the new hybrid estimation model, this paper estimated the yield of winter wheat in Beijing in 2015 based on three HJ Imagery obtained in winter wheat growing season, winter wheat yield of 30 sampling fields in 2015, and a nearly 30 years time series data of winter wheat yield (1985-2014) of Beijing. The estimation results from the hybrid yield estimation model was then compared with the true yield (2015 statistic winter wheat yield).【Result】The accuracy of winter wheat yield by using novel hybrid yield estimation model was 98.7% at city level and above 90% at country level. Except Fangshan(90.3%), the relative accuracy of yield estimation at the other countries was above 95%. The accuracy of winter wheat yield by using traditional trend yield model in Beijing was 94.75%, but the accuracy by using traditional trend yield model at country level was low, especially was lower 80% in Fangshan. ARIMA model was used for improving the accuracy of the traditional trend yield model. The accuracy of winter wheat yield improved in average by introducing the ARIMA model. For the remote sensing correction model established in this paper, using three remote sensing images for improving the accuracy was better, and this method improved the accuracy of winter wheat yield by 3.55%, especially the accuracy had a significant ascension in Fangshan and Pinggu.【Conclusion】The accuracy of winter wheat yield by using the novel hybrid estimation model is good at city level and county level. The model considers the change of time and spatial and can be used in crop yield estimation.

PLANT PROTECTION

Analysis of Population Distribution and Genetic Variation of Plant Pathogenic Fusarium in Shanxi Province

WANG Lin, LI XinFeng, XU YuMei, CHANG YinDong, WANG JianMing

Scientia Agricultura Sinica. 2017, 50(10):  1802-1816.  doi:10.3864/j.issn.0578-1752.2017.10.006

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【Objective】The objectives of this study are to obtain the sequences of ITS, EF-1α and β-tubulin gene regions from plant pathogen, Fusarium spp., compare the suitable gene sequences for species identification, and to analyze the population distribution and genetic variation of Fusarium spp. in Shanxi province by using the suitable gene sequences. 【Method】 A total of 625 Fusarium strains were collected from 28 counties and 11 cities in Shanxi Province from 2013 to 2015. The ITS, EF-1α and β-tubulin gene fragments of the morphology of clear strains from 625 strains were sequenced and analyzed. The sequences were assembled and edited using Sequencher software, then blasted in Genbank in NCBI and FUSARIUM-ID databases. The sequences from the clearly documented and reference species were downloaded. All the sequences were aligned and edited manually using ClustalX and GelDoc. The inter- and intra-specific variations of Fusarium spp. were analyzed with MEGA, Excel, DNAstar and TaxonGap. All the three gene sequences consisting of ITS, EF-1α and β-tubulin were selected for the taxonomy of the Fusarium species and then the best optimal fragment genes were used in the analysis of the population distributions of Fusarium spp. in Shanxi Province. 【Result】EF-1α was the best gene fragment for identifying Fusarium species among the three candidate gene fragments. Intra-specific pairwise distances was 24 times higher than that of the inter-specific pairwise distances. The intra-specific variation was smaller than those of the inter-specific variation by 73% of the tested species. The accuracy rate for identification was the highest, reached 87%. The phylogenetic relationships derived from the EF-1α sequences showed that different strains in 22 of 27 Fusarium species were monophyly, clustering in a same clade with high supporting values. Among the 27 species of Fusarium, F. oxysporum was the dominant species with 22.1% frequency and covered the largest geographical distribution in the 23 counties or regions in Shanxi province. Followed by F. solani with 13.8% in 14 counties or regions. The results of population distribution of Fusarium in different geographical regions in Shanxi province showed that F. oxysporum was the dominant species in Yuncheng, Linfen, Xinzhou, Changzhi, Lüliang, Jinzhong and Taiyuan city; F. lateritium was the dominant species in Shuozhou, F. solani in Datong, F. verticillioides in Jincheng and F. incarnatum in Yangquan. Among the different regions in Shanxi province, the most abundant Fusarium species was in Jinzhong and Xinzhou, followed by Linfen, and the least distribution was in Shuozhou. The results of the population distribution of Fusarium from different hosts showed that the 15 Fusarium species extracted from Lycopersicon esculentum, 13 species from Solanum tuberosum and 12 from Glycine max. In these hosts, F. oxysporum was the dominant species in Lycopersicon esculentum, Cucumis sativus, Citrullus lanatus, Solanum tuberosum, Solanum melongena, Cucurbita pepo and Brassica oleracea. F. verticillioides was the dominant species in Glycine max and Zea mays. F. avenaceum and F. graminearum were the dominant species in Triticum aestivum. 【Conclusion】There are abundant genetic variation between inter- and intra-specific of Fusarium. Of the three gene regions, EF-1α is the most suitable region for Fusarium species identification. Fusarium morphological taxonomy results are not fully agreed with the molecular phylogenetic results using EF-1α sequences. F. oxysporum is the dominant Fusarium species in Shanxi province. There are obvious genetic variations of Fusarium populations in different districts and hosts. The research results will provide a theoretic and scientific basis for Fusarium taxonomy, DNA barcode screening, quarantine and integrated control.

Expression and Functional Analysis of Endocuticle Structural Glycoprotein Gene LmAbd-5 in Locusta migratoria

ZHAO XiaoMing, JIA Pan, GOU Xin, LIU WeiMin, MA EnBo, ZHANG JianZhen

Scientia Agricultura Sinica. 2017, 50(10):  1817-1826.  doi:10.3864/j.issn.0578-1752.2017.10.007

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【Objective】The objective of this study is to obtain a cDNA sequence of endocuticle structural glycoprotein LmAbd-5based on Locusta migratoria transcriptome, clarify its molecular characteristics and biological function, reveal its role in the formation of cuticle in L. migratoria, and provide a new molecular target for pest control. 【Method】The full length cDNAof LmAbd-5 was searched from transcriptome database of L. migratoria using bioinformatics method. The cDNA was cloned and sequenced. The signal peptide and function domain of deduced amino acid were analyzed by SignalP and SMART, respectively. Phylogenetic tree was constructed using the sequences of amino acid from different insect species by the MEGA 7.0 software with the neighbor-joining (NJ) method. Reverse transcription quantitative PCR (RT-qPCR) was applied to reveal the expression patterns of LmAbd-5 in different tissues on day 2 of 5th instar nymph and different developmental stages of integument. The effects of LmAbd-5 on locust growth development and the structure of cuticle were investigated by using RNA interference (RNAi) and transmission electron microscopy (TEM). 【Result】The full length cDNA of LmAbd-5 was got from transcriptome database, which had 520 bp including ORF 303 bp. The gene structure analysis showed that LmAbd-5 has three exons. The deduced protein contains a signal peptide and one chitin binding domain 4 (ChtBD4) through the BLAST analysis, Abd-5 was highly conserved among insect species, and the sequence identity is as high as 81% between LmAbd-5 and SgAbd-5. Abd-5 belongs to the RR-1 class of CPR family by WebLogo analysis. The result of phylogenetic tree showed that LmAbd-5 has a close genetic relationship with SgAbd-5. RT-qPCR results showed that LmAbd-5 was predominately expressed in the tissues originated from ectoderm, such as the foregut, hindgut, trachea and integument, and lower expressed or not detected in the gastric caecum, midgut, Malpighian tube, fat body and wing pads. The expression at different stages showed that LmAbd-5 mainly expressed at early of 5th instar (0-72 h after ecdysis from 4th instar nymph), and up to the peak at 72 h after molting, then markedly decreased at 96-168 h. The expression pattern is related with the formation of endocuticle. Compared with dsGFP injected control, the nymphs with the injection of dsLmAbd-5 could normally molt, and no visible abnormal phenotypes was found although the expression of LmAbd-5 was decreased significantly after dsLmAbd-5 injection. However, compared to the control group, the lamellar structure from adult cuticle with injection of dsLmAbd-5 was loose, and lamellar became thicker, finally led to the endocuticle thickening. 【Conclusion】LmAbd-5 was obtained from locust transcriptome database, which contains a signal peptide and ChtBD4, belonging to the RR-1 class of CPR family. LmAbd-5 mainly expressed in the tissues derived from ectoderm and in integument at early of 5th instar. Although there was no visible phenotypes after silencing LmAbd-5, but it was found that the lamellar structure of endocuticle is loose and endocuticle becomes thicken from ultrastructure by TEM, suggesting it may be participated in the formation of endocuticle in L. migratoria.

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

A Meta-Analysis of Effects of Biochar Properties and Management Practices on Crop Yield

XIAO Jing, XU Hu, CAI AnDong, HUANG Min, ZHANG Qi, SUN Nan, ZHANG WenJu, XU MingGang

Scientia Agricultura Sinica. 2017, 50(10):  1827-1837.  doi:10.3864/j.issn.0578-1752.2017.10.008

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【Objective】A large number of studies have indicated that application of biochar in cropland has significant effects on crop yield due to its unique physical and chemical properties. It is of important significance to quantify the effects of management practices and biochar quality on crop yield by statistical analysis of large sample numbers.【Method】By collecting global relevant published literatures, 97 relative independent studies with 819 paired datasets on biochar’s effects of crop growth were selected. A meta-analysis was undertaken to quantify the effect of biochar characteristics (e.g., raw material, pyrolysis temperature, C/N, pH etc.) and artificial application management practices (e.g., application amount and duration), soil properties (soil texture and pH) on the crop yield improvement.【Result】Results showed that biochar could improve crop yield significantly by 15.0% in average compared with the control. As for crop types, the effect of biochar on crop yield was significantly different: The yield increase of cash crops (25.3%) was significantly higher than that of grain crops (10.0%). The characteristics of biochar had a significant impact on crop yield. Biochar produced with pyrolysis temperature lower than 600℃, pH over 7, and C/N value between 20-300, obtained significant increase in crop yield ranging from 9.2% to 26.6%. Moreover, the improved percentage of crop yield decreased with increase in pyrolysis temperature and biochar C/N. As for different soil textures and acidities, the order of yield-improving effect was clay soil > sandy soil > loamy soil. The yield-improving effect of biochar application for acid soil (29.2%) was 7.9 and 2.5 times of that for neutral and alkaline soil. Under the condition of management practices, biochar application increased crop yield significantly (by 18.0%) at rates less than 10.0 t·hm^-2. However, there was no significant effect on crop yield when the application rate was more than 80.0 t·hm^-2. The response ratio of biochar application on crop yield decreased with increase in the application duration. Six months to two years after biochar application increased crop yield by about 13.4%-17.5%, whereas after more than 2 years, the response ratio reduced to 9.6%.【Conclusion】The effect of biochar on crop yield varied according to variation in biochar quality and application rate and duration. Choosing biochar in specific quality for application can not only achieve sustainable improvement in crop production, but also minimalize the cost and improve economic efficiency according to crop types and soil texture. This result would provide an option for the development of sustainable agricultural management practices.

Evaluation Analysis of AquaCrop Model in Modeling Winter Wheat Growing Development and Soil Moisture Under Plastic Mulching

LIU Xia, DING DianYuan, ZHANG HaoJie, CHU XiaoSheng, YU Kun, FENG Hao

Scientia Agricultura Sinica. 2017, 50(10):  1838-1851.  doi:10.3864/j.issn.0578-1752.2017.10.009

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【Objective】This study was conducted to evaluate the accuracy of the AquaCrop model in the simulation of winter wheat growth, soil moisture, yield, and water use efficiency under plastic mulching, which can provide a theoretical basis and scientific method for the calibration of the AquaCrop model under plastic mulching.【Method】The experiment was conducted at Yangling, Shaanxi, from 2013 to 2016 including the flat planting under plastic mulching (PM) and a control treatment without mulching (CK). The AquaCrop model was calibrated using the experiment data in 2014-2015 and was validated using the data in 2013-2014 and 2015-2016. 【Result】The determination coefficient (R²) of the simulated and the measured canopy cover was between 0.86 and 0.99. The root mean square error (RMSE) of the simulated and the measured canopy cover was between 2.1% and 8.1%, indicating AquaCrop model a good simulation for canopy cover. The R² of the simulated and the measured biomass was greater than 0.95. Meanwhile, the RMSE of the simulated and the measured biomass was between 0.814 and 1.933 t·hm^-2. The R² of the simulated and the measured soil water content in CK was greater than 0.85, and the R² of the simulated and the measured soil water content in PM was greater than 0.75. The RMSE of the simulated and the measured soil water content in both CK and PM was between 9.2 and 17.6 mm. The normalized root mean square error (NRMSE) of the simulated and the measured soil water content in both CK and PM was lower than 5.5. Moreover, the RE of the simulated and the measured yield in both CK and PM was from -4.4% to 9.0%. The simulated and the measured yield in PM increased 40.5% and 40.3% compared to that in CK, respectively, and there was a significant difference between CK and PM. The RE of the simulated and the measured water use efficiency in both CK and PM was from -10.4% to -1.5%. The simulated and the measured water use efficiency in PM increased by 54.1% and 47.5% compared to that in CK, respectively, and there was also a significant difference between CK and PM. The results showed that the simulated and measured values had the similar trend in wheat canopy coverage, biomass, yield, and water use efficiency. Those indicated a good performance of the AquaCrop model in modeling plastic mulching treatment. 【Conclusion】 The AquaCrop model can be used to model the winter wheat growing development and productivity under plastic mulching. This study provides a scientific method to calibrate the AquaCrop model and a good data support for the application and development of the AquaCrop model.

HORTICULTURE

Cloning and Expression Analysis of Cadmium Tolerance Related Gene CsNAC019 in Cucumber

LI HuiYuan, TIAN ChunYu, ZHENG YuYing, WU Tao

Scientia Agricultura Sinica. 2017, 50(10):  1852-1861.  doi:10.3864/j.issn.0578-1752.2017.10.010

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【Objective】 To understand the molecular mechanism of Cd tolerance in cucumber, a NAC transcription factor CsNAC019 was obtained by using bioinformatics analysis. CsNAC019 was cloned and its expression was analyzed under Cd stress. The results of the present study will provide an experimental foundation for breeding of cucumber cultivars with high capacity for Cd tolerance.【Method】The full length sequence of CsNAC019 was cloned by PCR. Sequence comparison and conserved domain were analyzed by NCBI and DNAMAN. Amino acid composition, stability coefficient, and hydrophilic coefficient were analyzed by online software Expasy and TMHMM. The promoter region was analyzed by online software PlantCARE. Phylogenic tree was constructed by MEGA 6.0 according to the NJ method. The real-time PCR (qRT-PCR) was used to analyze the expression pattern of CsNAC019 under Cd treatments. The variance and significance were analyzed by DPS 7.05.【Result】CsNAC019 contained a typical conserved NAC domain. BLAST analysis revealed that CsNAC019 had 60% homology with NAC019 in the amino acid sequence, and the five relatively conserved regions of its amino acid sequences, A, B, C, D and E, were highly consistent between them from the N-terminal to the C-terminal. The full-length CDS of CsNAC019 was 960 bp, which encoded 319 amino acids with a molecular weight of 35.66 kD. The theoretical isoelectric point is 8.72, the instability coefficient is 68.18, and the average hydrophilic coefficient is -0.483. Besides the eukaryotic promoter TATA and CAAT natural elements, promoter analysis showed that the promoter sequence of CsNAC019 also had cis elements in response to stress, such as G-box, ABRE, W-box, P-box, TCA-element, TC-richrepeats, and HSE, etc. Phylogenetic analysis showed that CsNAC019 had 64%-96% homology with NAC transcription factors of other 15 plants (melon, peach, apple, citrus, grape and soybean, etc). CsNAC019 had the highest homology, 96%, with the NAC sequence of melon. qRT-PCR analysis revealed that the expression of CsNAC019 was significantly increased (8.2 folds) compared with the control under Cd tolerance condition.【Conclusion】CsNAC019 is a Cd tolerance induced gene. It was speculated that CsNAC019 may regulate the cucumber to response to Cd tolerance by regulating the expression of downstream genes.

Effect of Preharvest Acetylsalicylic Acid Treatments on Ripening and Softening of Harvested Muskmelon Fruit

LIU YaoNa, WANG Yi, BI Yang, LI ShengE, JIANG Hong, ZHU Yan, WANG Bin

Scientia Agricultura Sinica. 2017, 50(10):  1862-1872.  doi:10.3864/j.issn.0578-1752.2017.10.011

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【Objective】The experiment was conducted to study the effects of preharvest acetylsalicylic acid (ASA) sprayed for four times during fruit development on ripening and softening of muskmelon fruit (Cucumis melo cv. Agate) at harvest and during storage, and to explore softing mechanism caused by ASA treatments.【Method】The muskmelon, cultivar ‘Agate’, was used as material. The plants were sprayed with ASA at 1mmol·L^-1 for four times at young fruit period (2 weeks after flowering), enlarging period (3 wk after flowering), netting period (4 wk after flowering) and mature period (preharvest 48 h). The changes of physiological and biochemical parameters were determined on respiratory rate and ethylene production, firmness, cell wall component and cell wall-degrading enzymes activity of fruit at harvest and during storage (7℃, RH 55%-60%).【Result】Preharvest spray of ASA significantly decreased respiratory rate and ethylene production of muskmelon fruit at harvest, and delayed climecteric peak and ethylene peak for 1wk during storage. ASA treatments increased the firmness of fruit, the contents of propectin, cellulose, hemicellulose and hydroxyproline-rich glycoproteins (HRGPs) in fruit at harvest, retarded the conversion of propectin to water soluble pectins (WSP), maintained a higher level of cellulose, hemicellulose and HRGPs, kept firmness of fruit during storage. Preharvest spray of ASA noticeably decreased the activity of cell wall degrading enzymes in fruit at harvest and during storage, mainly inhibited the activity of pectin methylesterase (PME), polygalacturonase (PG), cellulase (Cx) and β-gluosidase (β-Glu). The correlation analysis indicated that there was a significant positive correlation between ethylene production and PG activity, respiratory rate and PG activity. And a very significant positive correlation between ethylene production and β-Glu activity, respiratory rate and β-Glu activity in treated fruit. There was a highly significant positive correlation between firmness and PME activity, propectin and hemicellulose content in treated fruit. Moreover, a significant positive correlation was found between firmness and Cx activity, and WSP content in treated fruit, and a significant negative correlation was also observed between firmness and ethylene production, and respiratory rate in treated fruit.【Conclusion】Preharvest ASA treatments promoted the synthesis of cell wall components during fruit development, significantly inhibited the respiratory rate and ethylene production, reduced the activity of cell wall degrading enzyme, such as PME, PG, Cx and β-Glu, prevented the release of cell wall components and maintained higher fruit firmness at harvest and during storage.

FOOD SCIENCE AND ENGINEERING

Isolation and Purification of Polysaccharide from Fermented Apple Pomace and Its Relationship with Processing Characteristics

JIA Feng, GUO YuRong, YANG Xi, LIU Dong, LI Jie

Scientia Agricultura Sinica. 2017, 50(10):  1873-1884.  doi:10.3864/j.issn.0578-1752.2017.10.012

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【Objective】On the basis of dynamic trend of separating and purifying of fermented apple pomace polysaccharides, the authors further investigated the structural properties , and in hoping of further elucidating the separating, purifying, bioactivity and processing properties of fermented apple pomace polysaccharides.【Method】Apple pomace polysaccharides (AP), wine fermented apple pomace polysaccharides (WFP) and vinegar fermented apple pomace polysaccharides (VFP) were used as experimental materials. On the basis of analysis of polysaccharides content, the authors used DEAE-52 cellulose column, NaCl as eluent, to separate polysaccharides according to the polarity difference among polysaccharides components. Through collecting effluent liquid using fraction collector and then the polysaccharides content was determined using with phenol sulfuric acid method, and the elution curve was depicted. Meanwhile, the obtained polysaccharides components with DEAE-52 cellulose column were further separated using Sephadex G-200 gel column, distilled water as eluent. After obtaining the separated polysaccharides components, the structural properties were thoroughly analyzed by using X-ray powder diffraction to observe the crystallographic structure, Thermogravimetric Analyzer was used to analyze thermogravimetric characteristics, Laser particle size analyzer to analyze granularity, and Congo red was used to explore triple helix structure of polysaccharides. Finally, Desktop Scanning Electron Microscope was also used to observe and analyze micro-structure of apple pomace polysaccharides.【Result】The content of original apple pomace polysaccharides was approximately 70%. Because the polysaccharides didn’t include protein and nucleic acid, so the extraction efficiency was excellent. NAP0.1 and NAP0.2 were obtained after AP were purified firstly through DEAE-52 cellulose column and 0.1, 0.2 mol·L^-1 NaCl were respectively used as eluents and then through Sephadex G-200 gel column and distilled water as eluent. Meanwhile, NWFP0, NWFP0.1 and NWFP0.2 were obtained after WFP was purified with DEAE-52 cellulose column and subsequently Sephadex G-200 gel column. After VFP was separated, NVFP0, NVFP0.1 and NVFP0.2 were obtained, too. All the separated constituents included over 92% polysaccharides, suggesting separation effect was satisfactory. Three polysaccharides, AP, WFP and VFP, were non-crystalline material between before and after separation. Concentration temperature of polysaccharides was strictly limited below 65℃, and the processing temperature was below 150℃. Before separation, AP, WFP and VFP all had triple helix structure. After separation, triple helix structure still existed in NWFP0, NVFP0, and NVFP0.1, while NAP0.1 and NAP0.2 had no triple helix structure. Compared with the original apple pomace polysaccharides, the separated apple pomace polysaccharides had better stability and smaller particle size, because lower dispersion coefficient of particle size was observed. Besides, flake structure in polysaccharides was less, and cross-linking effect attenuated after separation, which is beneficial for development of polysaccharides bioactivity.【Conclusion】Fermented apple pomace included over 70% polysaccharides, and polysaccharides content was up to 92% after crude polysaccharides was separated and purified. Besides, according to XRD, TG, LPSA and Congo red as well as DSEM analysis, it was concluded that the changes of solubility, viscosity and physical characteristics facilitated separated apple pomace polysaccharides developing bioactivity and processing characteristics.

ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT

Genetic Parameters Estimation of Test Day Milk Yield in Holstein Heifers in Ningxia Using a Random Regression Test-Day Model

REN XiaoLi, LIU AoXing, LI Xiang, ZHANG Xu, WANG YaChun, SHAO HuaiFeng, QIN ChunHua, WANG Yu, WEN Wan, ZHANG ShengLi

Scientia Agricultura Sinica. 2017, 50(10):  1885-1892.  doi:10.3864/j.issn.0578-1752.2017.10.013

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【Objective】This experiment was conducted to quantify the effect of environmental factors on milk yield of first lactation Holsteins in Ningxia, and to estimate genetic parameters of test day milk yield using test-day model, to provide a theoretical foundation for genetic parameter and breeding value estimation of milk components and somatic cells, and provide basic parameters for optimal breeding scheme which is suited to the Holstein population in Ningxia.【Method】A total of 550 078 test-day milk yield records from Holstein in Ningxia were collected, and the standards of calving month between 22-36 mo, the milk days between 5-305 d, and test day milk yield between 5.9-53.3kg were used to edit data, and finally a total of 127478 test-day milk yield records from 14320 Holstein heifers distributed in 24 herds between 2009 and 2013 in Ningxia were used in lactation curve mimicking and genetic analysis. Pedigree information of three generations were collected (father, mother, grandfather and grandmother from both father side and mother side) to form the pedigree file consisting 24 272 individuals. Microsoft Excel 2013 was used to manage the data to derive average milk production for each test-day, and NLIN procedure of SAS 9.1 was used to fit the Wood model and used to mimic the lactation curve to derive the population characters of milk yield. A random regression test-day animal model was employed and DMU 5.2 software was used for parameter estimation. The model included general fixed effect and fixed regression, random regression. In the present study, herd-test-day was the fixed effects, and a fixed regression were fitted for calving year and calving month combination effects, direct additive genetic, permanent environment were the random effects. Regression curves were modeled using Legendre polynomials of order 4. Based on climate characteristics in Ningxia, four calving seasons were categorized, spring (Mar. 11th to May 20th), summer (May 21^st to Aug. 25^th), autumn (Aug. 26^th to Oct. 15^th) and winter (Oct.16^th to Mar. 10^th).【Result】 The results showed that the average of test-day milk yield in Ningxia was 29.66 kg, milk yield reached its peak at about 90 days and peak yield was 31.84 kg. Through fitting lactation curves to first lactation cows in different seasons, calving years, and farms, the effect of these factors on lactation curves were quantified. Furthermore, lactation curves should be fitted as sub-models in the model for genetic evaluation. The heritabilities of 5-305 day milk yield were from 0.08 to 0.29, and the overall heritability of daily milk yield was 0.16. 【Conclusion】The study mimicked lactation curves for first lactation Holstein cows in Ningxia using WOOD model, the proper model was defined for this population. Results of estimated heritability for daily milk yield was lower than the results in the literatures using similar models. To evaluate the performance of test-day model, the hypothesis for residual variance (e) and integrity of pedigree needs to pay attention. These estimates derived from current study will provide reference for evaluating milk components and somatic cell counts using random regression model, and further establishing breeding value estimation system for performance of Holstein in Ningxia.

Regulation of Related Genes by Lentivirus-Mediated K26/KAP26.1 Gene Overexpression in Mice

JIN Mei, KANG Lin, SUN DongYu, PIAO Jun, PIAO JingAi, ZHAO FengQin

Scientia Agricultura Sinica. 2017, 50(10):  1893-1903.  doi:10.3864/j.issn.0578-1752.2017.10.014

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【Objective】Using the lentivirus-mediated overexpression technique, the aims of this study were at determining the effects of K26 and KAP26.1 gene overexpression on keratin-associated protein genes KAP6.2, KAP7.1, KAP8.2, and KAP11.1, and bone morphogenetic protein genes BMP4 and BMPR1B, and exploring the mechanism by which these genes influence hair fineness in mice, to achieve the goal of improving the quality of animal hair, to realize artificial regulation (such as lentivirus-mediated technology) of overexpression of some genes, and to lay a theoretical foundation for investigating the artificial regulation of mammalian hair fineness.【Method】In October, the experiment was conducted in Liaoning Provincial Key Laboratory of Biotechnology and Drug Discovery. The Kunming species mice aged five weeks were obtained from Experimental Animal Center of Dalian Medical University. The mice gene sequences of K26 (Gene ID: NM_001033397) and KAP26.1 (Gene ID: NM_027105. 2) were retrieved from Genbank, and the primers were designed according to the sequences of target genes. The healthy 293T cells were transfected with the plasmid to establish the vectors of mice K26 and KAP26.1 gene lentivirus overexpression, respectively, which were transfected into mice skin fibroblasts. Transfection efficiency was observed by quantitative fluorescence microscopy. After determining the success of lentivirus overexpression, the total RNA was extracted from the transfected cells, and after reverse transcription, the obtained cDNA was measured with the Eppendorf Realplex florescent quantitative PCR to detect the influence of K26 and KAP26.1 genes overexpression on KAP6.2, KAP7.1, KAP8.2, KAP11.1, BMP-4 and BMPR-IB gene expression 【Result】Proved by RT-PCR detection, mice lentivirus vectors pLenti6.3-K26-IRES-EGFP and pLenti6.3-K26.1-IRES-EGFP were established successfully. Compared by fluorescent field, the highest transfection rate of 293T cell transfected lentivirus vectors was reached after 72hr. Confirmed by PCR detection, packaged K26 and KAP26.1 lentivirus vectors transfected into mice fibroblast successfully after 72hr.Through RT-PCR detection and analyzed by SPSS 19 software, expression levels showed significant difference among the target genes, positive control group (empty plasmid, BLACK) and negative control group (mice skin fibroblasts, NC), indicating that after K26 overexpression, the expression level of KAP26.1 was up-regulated, and vice versa. This finding suggested synergy between K26 and KAP26.1. After K26 and KAP26.1 overexpression, the expression levels of KAP6.2, KAP7.1, KAP8.2, and KAP11.1 were down-regulated. After K26 overexpression, BMP4 gene expression increased, while BMPR1B gene expression decreased. After KAP26.1 overexpression, expression levels ofBMP4 and BMPR1B both were up-regulated.【Conclusion】K26 and KAP26.1 genes had a synergistic effect on the inner root sheath of the hair follicle by influencing the downstream protein synthesis signal of mTOR pathway. The high expression of K26 and KAP26.1 genes could inhibit the expression of KAP6.2, KAP7.1, KAP8.2, and KAP11.1 genes and thereby regulated hair fineness. Both K26 and KAP26.1 overexpression could up-regulate the expression level of BMP-4 gene which is the activator of BMP signaling pathway and could activate the BMP signaling pathway and then affected the growth of hair follicle cycle. K26 gene overexpression could down-regulate BMPR-IB gene expression, while KAP26.1 gene overexpression up-regulate BMPR-IB gene expression. BMPR-IB gene is the receptor I of the BMP signal. When BMPR-IB receptors decreased, the BMP downstream signal transduction will be inhibited, and then restarted hair growth cycle. When BMPR-IB receptors increased, the downstream signaling molecules transcription will be promoted, and then affected hair follicle growth cycle. Both K26 and KAP26.1 overexpression could activate BMP signaling pathway, and the expressions of KAP6.2, KAP7.1, KAP8.2, KAP11.1,BMP-4 and BMPR-IB genes were in turn regulated by the mTOR and BMP signaling pathways. But the opposite regulation effects of K26 and KAP26.1 genes on BMPR-IB gene still need to be further explored.

Identification and Characteristics of Odorant Receptors in Bumblebee, Bombus lantschouensis

WANG Ye, HAN Lei, DONG Jie, HUANG JiaXing, WU Jie

Scientia Agricultura Sinica. 2017, 50(10):  1904-1913.  doi:10.3864/j.issn.0578-1752.2017.10.015

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【Objective】Bumblebee Bombus lantschouensis is one of the most important pollinators for wild plants and crops in North China. Odorant receptors (Ors) gene family from the genome of B. lantschouensis was identified and characterized. Result of this study will help to explore the function of this gene family in foraging, mating and other social behaviors in bumblebee.【Method】Genomic DNA of thorax of B. lantschouensis was extracted and sequenced by the high-output of next generation sequencing. The original sequence of the sequencing was quality controlled and assembled to the genomic sequence. The contigs and scaffolds were used to build local sequence database. Gene sequences of Bombus terresters and Apis mellifera ligustica were used to query the local database. The characteristics and gene structure of Ors were analyzed by EMBOSS1.5 and GSDS2.0 software, respectively. Conservative motif analysis of amino acid sequences was performed using MEME 4.11.2. The phylogenetic analysis of Ors of B. lantschouensis, B. terresters and A. mellifera was studied by ClustaW 2.1, TrimAl 1.2 and PhyML3.0.【Result】One hundred and sixty-five Ors were identified from the genome of B. lantschouensis, including an Orco, 5 pseudogenes and 159 Ors. Gene structure analysis showed that the number of exons of these Ors are varies from 4 to 9. The least number of exons found in Or 47-57 was 4 and the largest number of exons found in Or 128-161 was 9. According to gene structure, Or was classified into 10 groups. Similarity in exon lengths and numbers was identified in the sequence of each group. The numbers of subgroup members were about 10, except in Or 1-38, Or 69-85 and Or 128-164 (which has 38, 17 and 37 members, respectively). The members of each subgroup are arranged in tandem on chromosomes and Or 1-38 have a longer first exon. Conserved motif analysis revealed that 9 motifs are present in Ors conserved domains (7tm_6 domain) except for motif 5 in all 10 motifs. While Or 1-38 and Or 39-46 have all predicted motifs and motifs 2, 3, 4, and 9 are widely present in sequences, which may be the key functional regions of the family. Phylogenetic analysis showed the 5 subfamilies of Ors family. Whereas the subfamily II contains 2 groups (BlOr 97-100 and BlOr 69-85) and subfamily V contains 4 groups (BlOr 1-46, 47-57, 86-95 and 101-107). BlOr 150-155 and AmOr 122-139 were clustered into two branches, and similar phenomena were also found in BlOr 47-57 and AmOr 63-65. It indicates that Ors has species-specific loss or expansion in evolution of Apis and Bombus. Or 115 is located at the base of the tree, suggesting that the sequence may be closer to the ancestor sequence of the odor receptor family.【Conclusion】Numbers, gene structure and phylogenetic relationships of Ors from the genome of B. lantschouensis were clarified in current study. Conserved motif analysis indicated that motif has been missed during the evolution of Ors family among bees. These results would provide an important information for exploring the gene evolution and function of Ors in future.

RESEARCH NOTES

Cloning and Expression Analysis of Fatty Acid Desaturase Gene FAD3 from Oil Peony

HUANG XingLin, LU JunXing, LIAO BingNan, BAI HuiYang, GUAN Li, ZHANG Tao

Scientia Agricultura Sinica. 2017, 50(10):  1914-1921.  doi:10.3864/j.issn.0578-1752.2017.10.016

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【Objective】 The Omega -3 fatty acid desaturase (ω-3 FAD) is a key enzyme in the fatty acid biosynthesis pathway of plant. Through analysis of omega-3 fatty acid desaturase gene structure and its expression in different tissues from Paeonia ostii, this study will lay a foundation for the research of regulating effects of FAD3 gene on fatty acid biosynthesis and provide a theoretical basis for the formation in the process of regulation. 【Method】 The FAD gene from Paeonia ostii (FAD3) was cloned using RT-PCR and RACE-PCR strategies. Nucleotide sequence was analyzed using Vector NTI Advance 11 software. Homology was analyzed using BLAST. A phylogenetic tree was constructed using neighbor-joining of MEGA 7.0. The secondary structure and three-dimensional model of FAD3 were predicted using ExPASy and Phyre2, respectively. Expression profiles of FAD3 at different developmental stages and in different tissues of the P. ostii were assayed using real-time quantitative PCR. 【Result】 The FAD gene in P. ostii was cloned and named as FAD3 (GenBank accession number: KX906966). The full length of FAD3 cDNA is 1 723 bp, contains a 1 308 bp open reading frame (ORF) encoding a putative protein of 435 amino acids with a molecular mass of 49.9 kD and an isoelectric point (pI) of 7.42. The 3′-untranslated region of 287 bp and 5′-untranslated region of 99 bp were obtained from the FAD3 gene of P. ostii. N end without signal peptide, fat coefficient is 83.08, instability index 35.67, the grand average of hydrophobicity value of -0.222. By FAD3 protein secondary structure prediction, FAD3 mainly in the alpha helix and random coil, followed by less extended strand, beta turn content; multiple sequence alignment results showed that it contains two conserved domains of FAD3 gene. Phylogenetic tree analysis showed that the P. ostii has the closest evolutionary relationship with P. lactiflora. Subcellular localization analysis of TMHMM and Target P indicated that it might be targeted to the endoplasmic reticulum with three transmembrane regions. FAD3 gene was expressed in the root, stem, leaf, petal, pistil, stamen and seed of P. ostii by the tissue-specificity expression. The expressive content of FAD3 gene in different tissues of P. ostii was different: The highest was leaf, the next was pistil, and the lowest level was in stamen; In different periods of seeds, the highest expression was seeds of 10d, the next was 20d, in 60d expression was the lowest. 【Conclusion】 The full length cDNA sequence of FAD3 gene was successfully cloned from P. ostii, It shows a variety of expression patterns in different tissues, which laid a foundation for the further study on the function and expression regulation mechanism of FAD3 gene in the process of unsaturated fatty acid biosynthesis.

Effect of Hyperosmotic Stress on Growth and Development of Setosphaeria turcica and Determination of Osmolytes in the Mycelium Cells of the Pathogen

GONG XiaoDong, LIU XingChen, ZHAO LiQing, ZHENG YaNan, FAN YongShan, HAN JianMin, GU ShouQin, DONG JinGao

Scientia Agricultura Sinica. 2017, 50(10):  1922-1929.  doi:10.3864/j.issn.0578-1752.2017.10.017

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【Objective】The objective of this study is to understand the effect of hyperosmotic stress on the growth and development, melanin content in mycelium cells, determine the probable osmolytes in the mycelium cells and to clarify the variation rule of these substances under different hyperosmotic stress conditions in Setosphaeria turcica. 【Method】The effects of hyperosmotic stress on colony growth rate, mycelium morphological characteristics and melanin content in S. turcica mycelium cell were analyzed under different hyperosmotic stress media, in which 0.4, 0.8, and 1.2 mol·L^-1 NaCl were added in PDA medium. High performance liquid chromatography (HPLC) technology was employed to detect the content of polyhydroxy-alcohol including glycerol, erythrol, glucose, mannitol, trehalose and analyzed the change profiles of these substances as time increased. 【Result】Compared to the strain cultured on PDA medium, the strain cultured under hyperosmotic stress treatments appeared with decreased in colony growth rate, shortened septum and swollen cells. Melanin content in mycelium cells cultured under hyperosmotic stress treatments in all time points showed a significant difference compared to the control group, in which melanin content in the samples treated before 12 h were lower than that in control, but there were no obvious differences between the samples treated. Moreover, there were different effects under different times (24, 48 h) and different concentration treatments. Compared to the control group, melanin content in the samples treated with 1.2 mol·L^-1 NaCl was significantly increased, melanin content treated with 0.8 mol·L^-1 NaCl was significantly decreased, melanin content in samples treated with 0.4 mol·L^-1 NaCl was significantly decreased in 24 h, but the content in the samples after 48 h treatment was almost the same with the control. Mannitol contents in mycelium cell under different hyperosmotic stress treatments were increased with the increase in time, especially the mannitol content significantly increased after 36 h treatment. There was a similar change tendency in glycerol and mannitol contents in mycelium cells under different treatments. Glycerol content increased most obviously compared with the control group and the content was significantly increased after 24 h treatment. Trehalose content in mycelium cells showed an increasing tendency as time increased, and the tendency increased with the increase of stress intensity, however, trehalose content after 36 h treatment was significantly lower than that in control group, while no significant difference was found in the changes of erythrol and glucose contents. No mannitol, trehalose and glycerol were detected in culture filtrate, while glucose content had no significant change no matter the fact that the mycelium was cultured in PDA medium or under hyperosmotic stress treatments.【Conclusion】 The colony growth rate was inhibited, mycelium cell was swollen, and septum was shortened under hyperosmotic stress. There was a significant inhibition on mannitol content before 12 h treatment. Mannitol and glycerol were main osmolytes in mycelium cells. Trehalose was also involved in hyperosmotic stress reaction in S. turcica. 

Cloning and Expression Analysis of Carotenoid Cleavage Dioxygenase 1 (CCD1) Gene in Tagetes erecta L.

 

LIU XiaoCong, ZENG Li, LIU GuoFeng, PENG YongZheng, TAO YiWei, ZHANG YaoYue, WANG MengRu

Scientia Agricultura Sinica. 2017, 50(10):  1930-1940.  doi:10.3864/j.issn.0578-1752.2017.10.018

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【Objective】Carotenoid Cleavage Dioxygenase 1 gene of Tagetes erecta L. ‘Scarletade’(TeCCD1) was cloned for bioinformatics and gene expression analysis, which can help clarifying its biological functions in carotenoid degradation pathway and providing a theoretical foundation to further clarify the mechanism of African marigold flower color formation.【Method】According to the transcriptome of African marigold flower bud, the full-length cDNA of TeCCD1 had been obtained, and gene expression profile of ray florets at developmental stages of closed bud, semi-open bud, open flower and fully open flower was studied by Real-time PCR.【Result】The full-length sequence of CCD1 cDNA obtained from African marigold is 1 746 bp (GenBank accession number: KX557488), with a coding region length of 1 626 bp, putatively encoding 541 amino acids. Protein analysis indicated that TeCCD1 is an unstable protein and has no signal peptide, which belongs to the RPE65 superfamily (GenBank accession number is PF03055) having the same conserved domain of CCD family, and it is mainly located in the cytoplasm. CCD1 nucleic acid sequence of African marigold is 89% homologous to that of Pyrethrum. Amino acid sequence analysis suggested that CCD1 of African marigold is 93% homologous to that of Pyrethrum, and 75%-83% homologous to that of 19 different species, indicating that TeCCD1 is highly conserved gene. Phylogenetic analysis showed that the evolution of TeCCD1 is basically in accordance with the evolution law of plant taxonomy and has obvious characteristics of species, which has a closest relationship with that of the species in Compositae. The results of Real-time PCR demonstrated that expression of TeCCD1 increased along with the development of ray floretsand reached the maximum value at S4 stage.【Conclusion】CCD1 homolog was cloned in Tagetes erecta L. ‘Scarletade’ identified to be a typical member of the CCD family, which is a highly conserved gene located in the cytoplasm. The color fading of ray florets during the late development phase is possibly caused by the increase of expression of TeCCD1, which contributes to a decrease in carotenoid content.