Scientia Agricultura Sinica

Response of Wheat Zinc-Finger Transcription Factor TaDi19A to Cold and Its Screening of Interacting Proteins

RU JingNa, YU TaiFei, CHEN Jun, CHEN Ming, ZHOU YongBin, MA YouZhi, XU ZhaoShi, MIN DongHong

Scientia Agricultura Sinica. 2017, 50(13): 2411-2422. doi:10.3864/j.issn.0578-1752.2017.13.001

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【Objective】Zinc-finger transcription factors play an important role in stress signal transduction and abiotic stress response in plants. In this study, the function of TaDi19A was identified under low temperature stress and its interacting proteins were screened by yeast two-hybrid system to explore the regulation mechanism of TaDi19A.【Method】TaDi19A gene was isolated from the cold-treated wheat transcriptome profile. Bioinformatics method was used to analyze the molecular properties of the TaDi19A gene, SMART online tools were used for protein structure analysis; GSDS and PHYRE2 online tools were used to analyze gene structure and tertiary structure of TaDi19A protein; NetPhos 2.0 Server database was used to predict phosphorylation sites of TaDi19A protein. The quantitative real-time PCR (qRT-PCR), conducted using the cold-treated wheat cDNA based on SYBR Green technology, was used to analyze the expression pattern of TaDi19A under cold temperature stress treatment in different time periods. TaDi19A was fused with PBI121 to transform into wild-type (WT) Arabidopsis plants (Columbia-0) mediated by the floral dip method, homozygous T3 seeds of transgenic lines and WT were used for cold tolerance analysis which the root length, fresh weight, and survival rate were measured before and after cold treatment. The expressions of four stress-response genes were investigated in transgenic lines and WT under normal and low temperature conditions to analyze the cold-resistant regulation mechanism of TaDi19A. Bait plasmid pGBKT7-TaDi19A was constructed and the self-activation was detected. The pGBKT7-TaDi19A and wheat cDNA library was co-transformed into yeast cell AH109 by two-hybrid system, and the positive clones were screened via SD/-Trp/-Leu/-His/-Ade and SD/-Trp/-Leu/-His/-Ade /X-α-gal plate and these single clones were sequenced and analyzed by BLAST to obtain the interaction candidate proteins.【Result】The full length of TaDi19A gene was 747 bp with 4 exons, encoding 248 amino acids, and the protein molecular weight and isoelectric point of were 28.03 kDa and 4.74, respectively. TaDi19A protein included Zinc-finger binding domain, Di19 domain and the predicted tertiary structure contained 2 alpha helix. Phosphorylation site analysis showed that there were 12 serine, 9 threonine, and 3 tyrosine phosphorylation sites in TaDi19A protein. qRT-PCR analysis showed that TaDi19A was induced by low temperature. The root length and cold tolerance assays revealed that TaDi19A transgenic Arabidopsis increased the cold tolerance. The expression of several cold-stress-responsive genes was monitored through PCR analysis, the expression of genes CBL1, CBL2, CBL3 and KIN1 showed elevated levels in both WT and transgenic Arabidopsis plants under cold-stress condition, and the expression levels in transgenic plants were significantly higher than those in WT. Analysis of candidate proteins screened by yeast two-hybrid system revealed that those proteins mainly affected the signal transduction and abiotic stress response, which demonstrated that TaDi19A is critical to stress signal transduction and abiotic stress response in plants. 【Conclusion】Cold-inducible TaDi19A improved cold tolerance in transgenic Arabidopsis; TaDi19A might work via interacting with other proteins.

Response of Root Morphology and Leaf Osmoregulation Substances of Seedling in Barley Genotypes with Different Heights to PEG Stress

DU Huan, MA TongTong, GUO Shuai, ZHANG Ying, BAI ZhiYing, LI CunDong

Scientia Agricultura Sinica. 2017, 50(13): 2423-2432. doi:10.3864/j.issn.0578-1752.2017.13.002

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【Objective】The aim of this study was to assess the relationship between the semi-dwarf gene uzu of barley and the drought resistance and to provide a theoretical basis for drought resistance and high quality breeding of barley.【Method】A hydroponic experiment was carried out to study the plant height, root-shoot ratio, root morphology and leaf osmoregulation substances of seedling of two pairs barley genotypes with different heights under different drought stress levels resulted by PEG6000 at different concentrations (0, 5%, 15%, 25%). Samples were taken during seedlings grew to four leaves and a heart. Plant height was measured by ruler. Root-shoot ratio was calculated by root biomass to aboveground biomass ratio multiplied by 100%, which were obtained by drying and weighing. Total root length, root surface area and volume were measured and analyzed by root system scanner and the WinRHIZO software. The soluble sugar, soluble protein and proline content were determined by sulfuric acid-anthrone colorimetry, coomassie brilliant blue G-250 dyeing method and acidic indene three ketone chromogenic method, respectively.【Result】Analysis showed that barley seedling growth was significantly inhibited, and the plant height decreased gradually, while the opposite trend showed in the root-shoot ratio with the increase of stress concentration. In terms of root morphology, as the stress concentration increased, two pairs near-isogenic line showed different trends. The change range of 3D and 15T was less than that of 3T and 15D, respectively, under the mild and moderates tress, while the change range of 3D and 15T showed an opposite trend under severe stress. Furthermore, soluble sugar content showed an increase at first and then a decrease and an increase again with the increase of the stress concentration, while the opposite trend showed in soluble protein content. The change range was characterized by 3D < 3T, 15D < 15T under the mild and moderate stress, and the change range of 3D and 15D also showed an opposite trend under severe stress. But the proline content presented a tendency of increasing with the increase of the stress concentration, and the small variations were characterized in 3D and 15D under different treatments. Correlation analysis indicated that plant height showed significant correlation with other traits in addition to the soluble protein content. There was also a significant relationship between root-shoot ratio and root morphology under different treatments. Meanwhile, the strains with higher soluble sugar content also had higher proline content, while the soluble protein content was low. Moreover, root morphology also had certain correlation with leaf osmoregulation substances, namely, the total root length and root surface area had significant negative relationship with soluble sugar content and proline content, respectively.【Conclusion】In the matter of root morphology and leaf osmoregulation, there are different responses under drought stress and influence with each other at seedling stage of barley genotypes with different heights. Under the mild and moderate stress, the semi-dwarf gene in the near-isogenic lines 3T and 3D can resist drought stress.

Construction of Core Collection of Sesame Based on Phenotype and Molecular Markers

LIU YanYang, MEI HongXian, DU ZhenWei, WU Ke, ZHENG YongZhan, CUI XiangHua, ZHENG Lei

Scientia Agricultura Sinica. 2017, 50(13): 2433-2441. doi:10.3864/j.issn.0578-1752.2017.13.003

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【Objective】The objective of this study was to manage, research and utilization of sesame (Sesamum indicum L.) germplasm resources more effectively, and to provide excellent genetic resources for sesame breeding.【Method】In this study, 5 020 accessions of sesame germplasm resources were systematically identified. Firstly, the primary core collections were constructed by using proportion strategy and UPGMA clustering sampling method within subgroups according to geographical origins. Then using an allele preferred sampling strategy and stepwise UPGMA clustering sampling approach according to SSR molecular data, these accessions were further screened to form core collections. The Nei’s gene diversity (He) and the Shannon-Wiener index (I) of the core collection and the primary one were measured by t-test. The cluster sampling was terminated until the genetic diversity of the core collection begun to have a significant difference with the primary one. Then the core collections without a significant difference with the primary core collection were chosen as the best core collections. The representativeness of the core collections was assessed by the Nei’s diversity index, Shannon-Wiener diversity index, percentage of polymorphic bands, polymorphic band retention, variable rate of coefficient of variation, coincidence rate of range, variance difference percentage and mean difference percentage.【Result】The primary core collections containing 816 accessions and core collections with 501 accessions were constructed, accounting for 16.25% and 9.98% of the total germplasm resources, respectively. The core collections consist of 442 Chinese landraces and 59 foreign germplasm resources. The core collection with 0.2989 in Nei’s diversity index and 0.4243 in Shannon-Wiener diversity index, and did not have a significant difference in molecular diversity with primary core collections (He=0.2791, I=0.4302) at P＜0.05. The percentage of polymorphic loci and reserved rate of number of polymorphic loci, variable rate of coefficient of variation and coincidence rate of range were 91.25%, 95.23%, 99.14%, 86.85%, respectively. Variance difference percentage and phenotypic indexes of mean difference percentage was 0. Results of t-test showed that no significant difference was found in genetic diversity indexes between the core collections and original collections. Compared with the random sampling strategy, allele preferred sampling strategy could construct more representative core collections with higher values of genetic diversity indexes and fewer loss of allele. The Shannon-Wiener index performed higher identifying efficiency than Nei’s diversity index. 【Conclusion】The primary core collections were constructed by using proportion strategy and clustering sampling method within subgroups according to geographical origin, and then using an allele preferred sampling strategy and stepwise UPGMA clustering sampling approach according to SSR molecular data to form core collections, which is a suitable method for constructing sesame core collections. The core collections of sesame are well representative of the original collections in the phenotypic and molecular genetic diversity.

The Expression of Genes Related to Cell Wall Formation at Fast Growth Stage in Flax (Linum usitatissimum L.)

JIANG HaiXia, GUO DongLiang, LI YuHuan, YAN WenLiang, YANG LiangJie, XIE LiQiong

Scientia Agricultura Sinica. 2017, 50(13): 2442-2450. doi:10.3864/j.issn.0578-1752.2017.13.004

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【Objective】 The development of flax bast cells is separated into two non-overlapping phases, cell elongation and secondary cell wall thickening phases which are splitted by the snap point at rapid-growing phase. Genes of β-glycosyl hydrolyse (LuBGALs) family and Cellulose synthase (LuCESAs) family focused on the role of cell wall formation in flax (Linum usitatissimum) fibres were analyzed in order to gain a deeper insight into the development of cell walls in rapid growth of flax.【Method】The microstructure of the cell walls at snap point (SP) of stem was studied by transmission electron microscope. LuBGALs and LuCESAs were analyzed by quantitative real-time PCR (qRT-PCR) in the bast fiber of stem, leaves and root of flax at 45 days after germination. 【Result】The width of cell walls at top of SP (TOP) was 110 nm. Though they at MID were about 500 nm and BOT about 650 nm under the SP were thicker than theirs in TOP, it was difficult to find different layers in the thicken cell wall. It showed that the cell walls above the SP were undergone cell elongation and below were thickening but not secondary cell walls thickening. The expression of LuBGAL1 at TOP was significantly lower than it’s in MID and BOT, indicating that it was mainly involved in cell wall thickening of fiber cell in the stem. While the expressions of LuBGAL3, LuBGAL6, and LuBGAL9 at TOP were the highest and the followed was MID zone, which showed that those genes mainly worked in cell wall reconstructure and bast fiber elongation. LuBGAL5 was higher at TOP and lower at MID than other genes, which demonstrated that LuBGAL5 played a role in cell wall formation. And the expression of other BGALs genes were low. LuCESA1, LuCESA3, LuCESA7, LuCESA8, LuCESA9, and LuCESA10 highly expressed in TOP zone and were significantly higher than their expression in MID and BOT. The expression of LuCESA3 and LuCESA10 in MID was lower than theirs in BOT and there was no significant difference between MID and BOT of other CESAs genes. The results suggested that these CESA genes played an important role in cell elongation of flax bast fiber combining their expression in different organs. The character of LuSuSy expression indicated that a lot of UDPG was required in cell elongation and thickening of bast fiber. LuXTH4 played a role in cell wall development of flax.【Conclusion】The cell walls of bast fiber had not processed secondary thickening in the stem at fast growth stage. LuBGAL3, LuBGAL5, LuBGAL6, LuBGAL9, LuCESA1, LuCESA3, LuCESA9, and LuCESA10 mainly promoted cell elongation of cell wall in flax. LuBGAL1 played an important role in cell wall thickening. LuSuSy and LuXTH4 also took part in the development of cell wall.

Effects of Incremental Seeding Rate Under Sub-Soiling During the Fallow Period on Nitrogen Absorption and Utilization, Yield and Grain Protein Content in Dryland Wheat

XUE LingZhu, SUN Min, GAO ZhiQiang, WANG PeiRu, REN AiXia, LEI MiaoMiao, YANG ZhenPing

Scientia Agricultura Sinica. 2017, 50(13): 2451-2462. doi:10.3864/j.issn.0578-1752.2017.13.005

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【Objective】The objective of these experiments is to clarify the effects of sub-soiling during the fallow period(SS) on soil water storage, explore the optimum seeding rate for constructing rational community of dryland wheat, and find out the best tillage and seeding technique, which could improve yield and quality coordinately. 【Method】 Field experiments were carried out from 2012 to 2014 in Wenxi, Shanxi province, with or without SS as the main plots and three seeding rates (67.5, 90, 112.5 kg·hm^-2) as subplots. Soil water storage during the fallow period, community tiller amount of plants before winter, dry weight and nitrogen content in different organs of plant, the yield and its components were measured to assess effects of seeding rate under SS on plant nitrogen absorption and utilization, yield, and grain protein content. 【Result】 The soil water storage efficiency during the fallow period was improved by more than 60% under SS compared with CK. Plant community tiller amount before winter, dry weight and nitrogen accumulation in plant at wintering stage, pre-anthesis accumulated nitrogen translocation amount from leaf and glume+spike to grains and nitrogen accumulation in the whole plant after anthesis were significantly increased under SS compared with CK. Plant community tiller amount before winter, dry weight at wintering stage and pre-anthesis accumulated nitrogen translocation amount from vegetative organs to grains and the contribution of pre-anthesis accumulated nitrogen translocation amount from leaf and glume+spike to grains were increased with the increase of seeding rate under SS while no significant difference between the seeding rate of 90 kg·hm^-2 and 112.5 kg·hm^-2 was presented. Spike number and grain number per spike were significantly increased under SS compared with CK, meanwhile, the yield was improved by 26%-66% and 17%-34% in two growing years respectively. Grain protein content was decreased under SS compared with CK, however, no significant difference was observed between them under 90 kg·hm^-2 condition. Spike number, 1000-grain weight and the yield were increased with the increase of seeding rate under SS, however, there was no significant difference between seeding rate of 90 kg·hm^-2 and 112.5 kg·hm-2. In addition, the highest grain protein content and its yield were observed under the seeding rate of 90 kg·hm^-2 under SS. Water use efficiency was significantly improved by 13%-22% and 9%-16% in two growing years, respectively, under SS compared with CK. Nitrogen uptake efficiency and nitrogen productive efficiency were significantly improved, and nitrogen use efficiency was significantly improved under the seeding rate of 67.5 kg·hm^-2 and 90 kg·hm^-2 condition under SS compared with CK. The highest water use efficiency was significantly observed under the seeding rate of 90 kg·hm^-2 condition under SS. Nitrogen uptake efficiency and nitrogen productive efficiency were improved with the increase of seeding rate under SS, while nitrogen productive efficiency had no significant difference between the seeding rate of 90 kg·hm^-2 and 112.5 kg·hm^-2 treatments. Moreover, under the condition of SS combined with different seeding rates, the yield and grain protein yield have significant and very significant correlation with pre-anthesis accumulated nitrogen translocation amount from vegetative organs to grains after anthesis, especially with the glume + spike under the condition of more precipitation. And nitrogen accumulation after anthesis was more significantly correlated with the yield under the condition of more precipitation. 【Conclusion】 Sub-soiling during the fallow period plus the seeding rate of 90 kg·hm^-2 was not only beneficial to form the strong seedlings before winter for dryland wheat, increase nitrogen accumulation of various organs at anthesis, promote pre-anthesis accumulated nitrogen translocation from leaf and glume + spike to grains, form the effective spike number, construct plant rational community of dryland wheat, and improve yield, water use efficiency, nitrogen uptake efficiency and nitrogen productive efficiency, and ultimately achieve the coordinated improvement of yield and grain protein content.

Effects of Different Planting Densities on Photosynthetic Characteristics and Yield of Different Variety Types of Spring Maize on Dryland

XU ZongGui, SUN Lei, WANG Hao, WANG ShuLan, WANG XiaoLi, LI Jun

Scientia Agricultura Sinica. 2017, 50(13): 2463-2475. doi:10.3864/j.issn.0578-1752.2017.13.006

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【Objective】Effects of different planting densities and different variety types of spring maize on physiological characteristics and yield in Weibei highland were investigated. The aim of this study was to determine the suitable maize varieties and their planting densities in dryland. 【Method】Field experiments were carried out by using spring maize cultivar Yuyu22, Zhengdan958, Xianyu335, and four planting density treatments including D1(52 500 plants/hm²), D2(67 500 plants/hm²), D3(82 500 plants/hm²) and D4(97 500 plants/hm²) were designed. The changes of photosynthetic characteristics, leaf area index(LAI), biomass yield and yield related traits of spring maize at different growth stages were observed.【Result】(1)Photosynthetic rate (Pn) and transpiration rate (Tr) decreased with the increase of planting density, while LAI increased. Pn decreased by 1.32 μmol CO₂·m^-2·s^-1, Tr decreased by 0.297 mmol·m^-2·s^-1 and LAI increased by 0.181 for each increase in density of 10 000 plants/hm². (2)The number of productive ear was remarkably increased with the increase of planting density, but the kernels per ear and 1 000-kernel weight were significantly decreased(P＜0.05). The kernels per ear decreased by 45 and the 1 000-kernel weight decreased by 12 g for each increase in density of 10 000 plants/hm². The yield of three maize varieties was the highest at D2 density. In treatment D2, the yield of Yuyu 22, Zhengdan 958, and Xianyu 335 were 10.52, 9.59, and 9.14 t·hm^-2 in 2015, and 11.37, 9.73, and 9.77 t·hm^-2 in 2016, respectively. The average yield of three maize varieties was increased by 21.9%, 19.5%, and 7.5%, respectively, within two years, when the density was increased from 52 500 plants/hm² to 67 500 plants/hm². The average yield of three maize varieties was decreased by 19.8%, 15.4%, and 7.7%, respectively, within two years, when the density was increased from 67 500 plants/hm² to 97 500 plants/hm². (3) The stem diameter, and ear length of spring maize decreased with the increase of planting density. The ear length decreased by 0.86 cm, the stem diameter decreased by 0.09 cm for each increase in density of 10 000 plants/hm². The lodging rate of Yuyu 22 and Zhengdan 958 rather than Xianyu 335 increased with the decrease of stem diameter. (4)The harvest index varied greatly in two years, the average performance was 2015＞2016, and the varieties showed that Xianyu 335＞Zhengdan 958＞Yuyu 22. Water use efficiency (WUE) and radiation use efficiency (RUE) were increased with the increase of planting density and then decreased.【Conclusion】The optimum planting density of different variety types of spring maize in Weibei dryland was different. Yuyu 22 was 72 500 plants/hm², Zhengdan 958 was 74 000 plants/hm², and Xianyu 335 was 73 200 plants/hm². Yuyu 22 had high stability and high yield. The optimum planting density of different variety types of spring maize was 72 600-74 000 plants/hm². Low density was recommended for thin planting varieties, and high density was recommended for close planting varieties.

Effects of Climate Change on Cropping Pattern and Yield of Summer Maize-Winter Wheat in Huang-Huai-Hai Plain

SUN XinSu, LONG ZhiWei, SONG GuangPeng, CHEN ChangQing

Scientia Agricultura Sinica. 2017, 50(13): 2476-2487. doi:10.3864/j.issn.0578-1752.2017.13.007

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【Objective】Global climate is changing significantly characterized by warming. Exploring the influence of climate warming on winter wheat-【Method】 The meteorological data, phenological data and yield data of HHH during 1992-2013 were used to analyze the effect of climate change on summer maize-winter wheat system by the mathematical statistics analysis method. The meteorological information including the temperature and precipitation data were obtained from China Meteorological Administration. Climate change of HHH area during 1992-2013 were analyzed by monadic linear regression. Through the weighted average of phenological data, the growth period and stubbles changes of summer maize-winter wheat were analyzed. The summer maize-winter wheat yields during 1992-2013 were analyzed by monadic linear regression. What’s more, nonlinear regression analysis and panel data sensitivity analysis were used to analyze the effect of climate change on annual yield of summer maize-winter wheat.【Result】The temperature of HHH showed a rising trend while precipitation generally remained unchange in the 1992-2013, but the regional difference was obvious. Climate change significantly changed the summer maize and winter wheat planting system: The whole growth stages of winter wheat observably shortened by 2 to 5 days in different regions with sowing time delayed. Sowing time of maize showed a difference of southern delay but northern advance while mature time delayed in general. What’s more, there was no significant change of summer maize growth period in the whole area. Stubbles time of summer maize-winter wheat was postponed because of the change of growth period, which led to the waste of climatic resources and land resources. In recent years, the anniversary yields of summer maize and winter wheat showed a significant rising trend in HHH. Climate sensitivity analysis showed that the impact of low temperature, high temperature and average temperature on winter wheat yield was alike, the yield in Jiangsu and Shandong yield decreased while that in Hebei and Henan increased, which the minimum temperature showed a significant effect. In the whole HHH plain, summer maize yield was reduced in different degrees when temperature rising except Henan province because of the difference of temperature rise range and precipitation. There were regional difference of annual yield affected by climate changes due to the regional difference of precipitation. Effect of climate change on annual yield was different that yield in north was increased but decreased in south, so choosing appropriate summer maize varieties is meaningful to guarantee the annual yield. 【Conclusion】Global warming delayed the winter wheat sowing time and shortened winter wheat growing period. It also made summer maize growth period changed that is extended in the north but that is shortened in the south. The prolonged stubbles owing to the shortened growth period led to the waste of climatic resources and land resources. Actual anniversary yield showed a significantly increasing trend in recent years. Effect of increasing temperature and precipitation had regional differences. Generally speaking, temperature rise made maize yield reduced and wheat yield increased, while precipitation made maize yield in northern HHH increased and maize yield reduced in the southern HHH as well as wheat production decreased in different degrees.summer maize cropping system in Huang-Huai-Hai Plain (HHH) to provide a theoretical foundation for formulating reasonable counter measures.

Gene Cloning and Expression Analysis of Canker-Related Transcription Factor CsBZIP40 in Citrus

JIA RuiRui, ZHOU PengFei, BAI XiaoJing, CHEN ShanChun, XU LanZhen, PENG AiHong, LEI TianGang, YAO LiXiao, CHEN Min, HE YongRui, LI Qiang

Scientia Agricultura Sinica. 2017, 50(13): 2488-2497. doi:10.3864/j.issn.0578-1752.2017.13.008

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【Objective】 The objective of this study is to annotate the BZIP family and clone the citrus canker related transcription factor CsBZIP40. It is also aimed to confirm the subcellular localization and the expression profile reduced by exogenous hormone and mechanical damage and the relations between CsBZIP40 and Xanthomonas citri subsp. citri (Xcc) infection. 【Method】Based on the public genome databases, the BZIP gene family was expertly and comprehensively annotated and named based on the chromosomal localization of all the members of BZIP; the motifs of the BZIPs were analyzed by MEME online tool. the phylogenetic tree of BZIPs in Citrus and Arabidopsis thaliana was constructed using software Mega 6.0 based on which the category of BZIP family was obtained. Canker-related transitional factor CsBZIP40 obtained from transcriptome data was also detected by qRT-PCR. Elements in the promoter and the nuclear localization signal were analyzed with database plantCARE and online tool cNLSmapper, respectively. And then the subcellular localization was confirmed by GFP fusion experiments in onion to confirm the prediction of nuclear localization analyzed with softwares. Expression profiles induced by salicylic acid (SA), jasmonic acid methyl ester (MeJA), ethylene (ET) and mechanical damage of CsBZIP40 were checked with qRT-PCR. 【Result】A total of 47 BZIP genes were annotated from the whole genome of Citrus sinensis and all the BZIPs are located on every chromosome except the 9th one. The BZIP concentration on chromosome 3 are 4.5×10^-7/Mb which is the highest while chromosome 2 is the lowest, contains only 2% of all BZIPs in citrus. There were fewer gene duplication events detected from BZIP family of citrus compared with other plants, such as Arabidopsis, grapevine and so on. That is why citrus has a smaller BZIP family size. The full-length of CsBZIP40 is 5 756 bp with a 1 530 bp open reading frame which codes a protein containing 509 amino acids. It is closely related to AT1g08320 in A. thaliana based on the evolutionary analysis. In citrus, the BZIPs have been annotated which can be divided into 10 different sub-families. CsBZIP40 belongs to sub-family D, which is always take part in the pathogen resistance in plants. The gene promoter contains multiple cis involved in plant adversity or hormone response, such as Box-W1, HSE, ERE and so on. Subcellular localization results confirmed the prediction of protein localization in nucleus. Based on the qPCR data, the exogenous salicylic acid cannot induce the different expression of CsBZIP40, in contrast, jasmonic acid methyl ester, mechanical damage and ethylene can induce significant differences in gene expression level. Xcc attack can significantly increase the expression level of CsBZIP40 in Calamondin but no difference in Newhall navel orange. 【Conclusion】CsBZIP40 would be an important transcription factor which is closely associated with the resistance of citrus canker. This gene should be a potential candidate in the molecular breeding to improve the canker resistance of citrus.

Molecular Characterization and Biological Function of Chitin Deacetylase Genes in Locusta migratoria

YU RongRong, DING GuoWei, LIU WeiMin, ZHANG Min, ZHAO XiaoMing, HAN PengFei, MA EnBo, ZHANG JianZhen

Scientia Agricultura Sinica. 2017, 50(13): 2498-2507. doi:10.3864/j.issn.0578-1752.2017.13.009

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【Objective】 Chitin deacetylase (CDA) is a key enzyme involved in chitin metabolism. The objective of this paper is to study the molecular characteristics and biological function of chitin deacetylase genes in Locusta migratoria, and to provide a theoretical basis for screening novel targets for pest control.【Method】Based on the transcriptome of L. migratoria, three cDNA sequences putatively encoded LmCDAs were obtained, and the gene structure was analyzed by comparing with the locust genome database. Alignment was performed with the CDAs from Tribolium castaneum and the functional domains were predicted by BlastP and SMART software. The homologous sequences from T. castaneum, Drosophila melanogaster, Anopheles gambiae, Bombyx mori, Oxya chinensis and Choristoneura fumiferana were selected to perform clustering analysis with LmCDA4, LmCDA5a and LmCDA5b, and phylogenetic tree was constructed by using MEGA 5.02 software with the neighbor-joining (NJ) method. The RT-qPCR was applied to detect the relative expression of LmCDA4 and LmCDA5 in different tissues and developmental stages of the 5th instar nymphs. The RNA interference (RNAi) was performed to study the biological function of LmCDA4 and LmCDA5 during the molting process of L. migratoria. The chitin content was detected by chemical method. The transmission electron microscope (TEM) was applied to study whether the LmCDA4 and LmCDA5 have effects on the cuticle structure.【Result】The three full length cDNA sequences putatively encoded chitin deacetylase were identified from locust transcriptome database. The bioinformatics analysis showed that they possess the signal peptide, the open reading frame contains two functional domains: chitin binding domain (ChBD) and chitin deacetylase catalytic domain (CDA). Alignment with the CDAs from T. castaneum showed that partial sequences of two CDAs from L. migratoria were different, and the splicing sites are similar to two alternatively spliced variants of TcCDA5 from T. castaneum, which indicated two CDAs were alternatively spliced variants. The phylogenetic analysis showed that three LmCDAs were closely grouped with the CDA4 and CDA5 from other six insect species with high bootstrap value, and named as LmCDA4, LmCDA5a and LmCDA5b, respectively. Tissues expression pattern showed that both LmCDA4 and LmCDA5 were mainly expressed in the integument, foregut and hindgut. Besides, LmCDA5 also had a high expression in the fat body. Expression of CDAs in integuments of different days of 5th instar nymph showed that both of LmCDA4 and LmCDA5 were highly expressed in the 1st and 2nd days of 5th instar nymph, then decreased gradually. After injection of dsLmCDA4 and dsLmCDA5, the expression of target gene was significantly reduced at 24 h compared with dsGFP injected controls, and the silence efficiency of gene was 98.1% and 95.6%, respectively. Phenotypic analysis showed no visible phenotypes were observed and both treatment and control insects could molt to adult successfully. Chitin content detection and TEM observation suggested that silencing the expression of both LmCDA4 and LmCDA5 did not affect the chitin content and chitin organization.【Conclusion】LmCDA4 and LmCDA5 are highly expressed in the integument, foregut and hindgut. Biologic functional analysis suggested both of them are not essential in the molting process of L. migratoria, silencing of their expression showed no effect on locust development and cuticle structure.

Effects of Brassinolide on Photosynthesis, Chlorophyll Fluorescence Characteristics and Carbohydrates Metabolism in Leaves of Foxtail Millet (Setaria italica) Under Sigma Broad Stress

YANG HuiJie, YUAN XiangYang, GUO PingYi, DONG ShuQi, ZHANG LiGuang, WEN YinYuan, SONG XiE, WANG HongFu

Scientia Agricultura Sinica. 2017, 50(13): 2508-2518. doi:10.3864/j.issn.0578-1752.2017.13.010

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【Objective】 The effects of brassinolide (BR) on photosynthesis, chlorophyll fluorescence characteristics and carbohydrates metabolism of foxtail millet (Setaria italica) under Sigma Broad stress wereexplored to provide some theoretical bases and technological approaches for security application of sulfonylurea herbicide and the alleviation of herbicide phytotoxicity in foxtail millet field. 【Method】Completely randomized design and repeated 3 times, using high-yielding hybrid Zhangza 5 and Jigu 21 with general high quality as materials, through pot experiments, applicating 7.5 mg·L^-1 of Sigma Broad at three-leaf to five-leaf stage, and after 1 d foliar spraying water (CK), 0.05, 0.1, 0.2 and 0.4 mg·L^-1of BR. the effect of BR on growth parameters (plant height, leaf area, fresh mass), photosynthetic pigment content (Chla, Chlb, Car, Chl (a+b)), gas exchange parameters, chlorophyll fluorescence parameters, sugar content, activities of key enzyme of sucrose metabolism were determined and analyzed to reveal the effects of BR on modulation of photosynthetic capacity and carbohydrates metabolism by Sigma Broad of foxtail millet after 7 d.【Result】 Sigma Broad significantly decreased plant height, leaf area, fresh mass, photosynthetic pigment content, net photosynthetic rate (Pn), transpiration rate (Tr), stomatal conductance (Gs), the maximum photochemical yield (F_v/F_m), apparent photosynthetic electron transport rate (ETR), quantum yield dissipated by non-regulatory energy (Y(NO)), activity of neutral invertase (NI), sucrose synthetase (SS) and sucrose phosphate synthase (SPS), but significantly increased intercellular CO₂ concentration (Ci), and the quantum yield dissipated by regulatory energy (Y (NPQ), reducing sugar, sucrose and starch content. One day after Sigma Broad treatment, spraying BR dramatically increased plant height, leaf area, fresh mass, photosynthetic pigment content, Pn, Tr, Gs, F_v/F_m, ETR, Y (NO), NI, SS and SPS of foxtail millet, but decreased Ci, Y (NPQ), reducing sugar, sucrose and starch content. BR at 0.05-0.1 mg·L^-1 had a better effect on relieving the Sigma Broad of Zhangza 5, BR at 0.1-0.2 mg·L^-1 had a better effect on relieving the Sigma Broad of Jingu 21. BR at ≥0.4 mg·L^-1 did not alleviate the phytotoxicity of Sigma Broad.【Conclusion】Results showed that one of the reasons that 7.5 mg·L^-1 of Sigma Broad made obvious damage to foxtail millet is that the photosynthetic pigment content was reduced, and photosynthetic pathways and sucrose metabolism were hindered, the PSⅡ photochemical activity was inhibited, and the normal operation of sucrose metabolism was affected. treatment with BR at 0.1 mg·L^-1 could alleviate the inhibition of photosynthesis of Sigma Broad in foxtail millet through improving the photosynthetic pigment content, increasing the stomatal conductance, raising PSⅡ photochemical activity, promoting the discharge of carbohydrates and maintaining balance of sucrose metabolism.

Integrated Analysis of Liming for Increasing Crop Yield in Acidic Soils

ZENG TingTing, CAI ZeJiang, WANG XiaoLi, LIANG WenJun, ZHOU ShiWei, XU MingGang

Scientia Agricultura Sinica. 2017, 50(13): 2519-2527. doi:10.3864/j.issn.0578-1752.2017.13.011

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【Objective】 Application of lime is one of the traditional and effective methods for improving soil acidity to obtain crop yield, so it is very important to integratedly analysis of the effect and cause during liming to increase yield of crops by scientific and rational application of lime to maintain crop yield. Sustain crop production in acidic soils by rational application of lime.【Method】 The published data from 76 studies were collected, a relational database of soil pH-crop yield/biomass was developed. Then the crop growth rate (CGR) was assessed under initial soil pH (3.1-6.6), crop species (cereal crops, cash crops), application rate of lime (＜750, 750-1 500, 1 500-3 000, 3 000-6 000,＞6 000 kg·hm^-2) and lime type (calcium lime, slacked lime, limestone powder). 【Result】Compared to control (without liming), crop yield was promoted by liming in acidic soils, with CGR ranging from 2% to 255%, where CGR was 42% and 47% for cereal crops and cash crops, respectively, and where the order for cereal crops was maize (149%)＞sorghum (142%)＞wheat (55%)＞bean (32%)＞rice (4%)＞tuber (2%), whereas the order for cash crops was vegetable (255%)＞pasture (89%)＞rape (26%)＞fruit (23%)＞tobacco (7%). Under application of lime, with increasing initial soil pH, CGR first increased, reached a maximum where 99% at pH 4.3 was found, and then decreased, even less than zero at pH 5.8. In common acidic soils (pH 4.5-5.5), the optimal application rate of lime ranged from 3 000 to 6 000 kg·hm^-2, where CGR could reach 55%-173%. In addition, slacked lime showed the best effect on increasing crop yield, where CGR was 100%, higher than that of calcium lime (32%) and limestone powder (64%). Application of lime to improve soil pH and exchangeable calcium, decreased exchangeable aluminum content, which was the main cause of increased crop yield under application of lime to acidic soils. However, CGR reached the maximum at ΔpH was 1.5 and exchangeable calcium was 6.2 cmol·kg^-1, suggesting that a moderate amount of lime should be applied during ameliorating soil acidity. 【Conclusion】 The highest priority should be given to vegetable and maize when application of lime to acidic soils, and slacked lime would be employed as the first choice. The application rate of lime was recommended to be 3 000-6 000 kg·hm^-2, and no lime was applied at pH more than 5.8. That is, the pH target 5.8 should be set and correspondingly lime requirement be determined for amelioration of soil acidity.

Effect of Soil Surface Mulching on the Maize Source-Sink Relationship of Nitrogen Accumulation and Dry Matter Transfer

ZHU Lin, LI ShiQing

Scientia Agricultura Sinica. 2017, 50(13): 2528-2537. doi:10.3864/j.issn.0578-1752.2017.13.012

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【Objective】A field study on dryland spring maize in three treatments was conducted to explore the source-sink relationship between nitrogen accumulation and dry matter transfer in different mulching treatments on the Changwu Tableland of the Loess Plateau in northwest China. 【Method】The light energy capture and soil temperature in treatments of plastic film mulching, gravel mulching, and no mulching (CK) were observed, and dry matter accumulation and nitrogen accumulation were analyzed through two years.【Result】The effective accumulated temperature of the plastic film mulching was significantly higher than that of the CK treatment, but no significant differences compared with gravel mulching treatment. The radiation utilization efficiency of the plastic film mulching treatment was significantly higher than that of the other treatments. The accumulated temperature production efficiency of plastic film mulching treatment was significantly higher than that of other treatments, the difference of its radiation production efficiency with other treatments was not obvious in 2010, and however, its radiation production efficiency was significantly higher than that of CK treatment and lower than that of gravel mulching treatment in 2011. The dry matter transfer amount of stem and leaf sheath reached the maximum. The contribution rate of soil surface mulching to dry matter transfer and its impacts on dry matter transfer rate were not significant. At reproductive growth stage, the single grain weight of corn grain increased with growth of corn. However, the number of single grain decreased instead. Under the condition of plastic film mulching, the weight and number of single grain in harvest time were higher than that in other treatments. Within 30 days after silking, the average nitrogen content of corn grain in plastic film mulching was significantly higher than that in gravel mulching and CK treatments; within 30 days after grain filling and up to mature period, the difference of nitrogen content in corn grains was not significant and the nitrogen content in plastic film mulching treatment was slightly higher than that in gravel mulching and CK treatments. As the difference in dry weight of corn grains, the nitrogen accumulation amount in plastic film mulching was significantly higher than that in grave mulching and CK treatments. The mulching treatment had effectively increased the nitrogen accumulation of grains in upper, middle and lower parts of corn ear; the nitrogen accumulation amount of grains in each parts of corn ear in plastic film mulching treatment was significantly higher than that in gravel and CK treatments. The dry matter transfer amount and contribution rate to dry matter transfer were positively related to the volume and weight of single grain and effective accumulated temperature, which reached at a significant level; however, although the dry matter transfer amount and contribution rate to dry matter transfer were positively related to the number of single grain, the fractional interception of photosynthesis active radiation and production efficiency of accumulated temperature and radiation, it did not reach at a significant level. The plastic film mulching increased the dry matter accumulation capacity based on the number of single grains, and dry weight of ear grain to facilitate nitrogen accumulation of corn grains, improve the nitrogen use efficiency and increase production.【Conclusion】The plastic mulching promoted the accumulation of nitrogen and improved the utilization efficiency of nitrogen, thus improved the yield of maize on the Loess Plateau.

Transcriptome and Cytological Researches on the Anther Abortion of a Thermo-Sensitive Genic Male Sterile Line GS-19 in Cauliflower

TAO XingLin, HOU Dong, ZHU HuiXia, LIU MingXia, ZHANG JinWen, HU LiMin

Scientia Agricultura Sinica. 2017, 50(13): 2538-2552. doi:10.3864/j.issn.0578-1752.2017.13.013

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【Objective】 In order to clarify the mechanism of cellular and molecular sterility characteristics, the flower morphology, anther abortion period and molecular mechanism of the thermos-sensitive male sterile line of cauliflower were studied, thus laying a theoretical foundation for further study of the male sterility. 【Method】 The thermo-sensitive male sterile line GS-19 was used as the experimental material, using morphological, cytological and high-throughput sequencing techniques, the morphological, cytological and molecular mechanism were studied with treatment at different temperatures (15℃ and 20℃). 【Result】 The fertility conversion of the thermo-sensitive male sterile line GS-19 was controlled by temperature, sterility at high temperature (20℃), and fertility at low temperature (15℃). The flower difference between sterile and fertile plants reached a significant level in GS-19, the flower was significantly smaller than that of fertile plants. The anthers abortive stages and manners were different between sterile and fertile plants of GS-19 by the microstructure observation. The anther development of GS-19 had differentiation of pollen mother cell, formed normal pollen sac, did not produce pollen grains or produce trace of pollen grains without viability, the development of anthers was blocked from pollen mother cell to the tetrad stage. The pollen mother cells could not pass the meiosis, with no dyads and tetrads formed in GS-19, insteading of some “pseudo microspores”, belong to an abortive type of pollen mother cell. The pollen exines of the “pseudo microspores” were abnormal. The “pseudo microspores” were decayed gradually and only empty shells remained at last. A total of 67 930 unigenes were generated in GS-19 alabastrum by sequence, Nt database compared to the largest number of similar sequences, for the 52 191, Nr database to similar sequence of times (46 447), KOG database than similar sequence of the least (13 198). By GO database the 25 336 unigenes were divided into 3 categories containing 55 function groups; by KOG databases the 13 198 unigenes were grouped into 26 functional categories; by KEGG database the 14 778 unigenes were divided into 19 metabolism pathways. Gene differential expression analysis revealed that there were 2 170 differentially expressed genes in the bud of GS-19 male sterile line and fertile plants, 1 078 genes up-regulated and 1 092 genes down regulated. 【Conclusion】 The fertility conversion of the thermo-sensitive male sterile line GS-19 is controlled by temperature, sterility at high temperature. The flower is significantly smaller than that of fertile plants. The filaments are significantly shortened, the anthers shrank, and dried. The development of anthers is blocked from pollen mother cell to the tetrad stage, belonging to an abortive type of pollen mother cell. A total of 67 930 unigenes were generated in GS-19 alabastrum by transcriptome analysis. Gene differential expression analysis reveals that there are 2 170 differentially expressed genes in the bud of GS-19 male sterile and fertile plants.

The Difference of Anthocyanin Accumulation Pattern and Related Gene Expression in Two Kinds of Red Flesh Peach

DING TiYu, CAO Ke, FANG WeiChao, ZHU GengRui, CHEN ChangWen, WANG XinWei, WANG LiRong

Scientia Agricultura Sinica. 2017, 50(13): 2553-2563. doi:10.3864/j.issn.0578-1752.2017.13.014

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【Objective】The objective of this experiment is to investigate the coloring mechanism of Chinese red-flesh peach germplasms and lay a theoretical foundation for excellent gene identifying and molecular marker assisted breeding in blood-flesh peach accessions.【Method】Eight red flesh peaches and one white flesh peach were selected as samples. The samples were collected one month after full bloom, and then collected every 10 days until the mature period. The anthocyanin of pulp of different germplasms was extracted with 2% formic acid methanol, determining the absorbance at 510 nm and 700 nm. The expression of 13 key structural genes and regulatory genes associated with anthocyanin synthesis was determined by real-time quantitative PCR (qRT-PCR).【Result】According to the accumulation of anthocyanin at middle-late stages of fruit, eight red-flesh peaches could be divided into two categories. The peak of anthocyanin biosynthesis appeared at mature stage, which was referred to as mature period accumulation type, including Zheng Yin 82-9, Da Hong Pao, Hei Bu Dai, Hong Tao and Tianjin Shui Mi; The peak of anthocyanin synthesis occurred at the middle stage of fruit development, and the content of anthocyanin decreased at mature stage, which was referred to as the mid-developmental accumulation type, including Harrow Blood, Da Guo Hei Tao and Wu Hei Ji Rou Tao. In the structural genes associated with anthocyanin, the expression of PpCHS and PpUFGT was consistent with the trend of the variation of the anthocyanin content of mature period accumulation type germplasms, which were the key speed-limiting genes of these kinds of germplasms. In the mid-developmental accumulation type, the accumulation of anthocyanin in the mesocarp was consistent with the trend of expression of all structural genes. Among the four regulatory genes, only the expression level of PpMYB10.1 was high and the expression pattern was similar to the pattern of anthocyanin accumulation in the two types of red flesh germplasms during fruit developmental stages. 【Conclusion】According to the coloring pattern of peach fruit and the pattern of anthocyanin accumulation, eight red-flesh peaches could be divided into two types, namely mature period accumulation type and mid-developmental accumulation type. The PpCHS and PpUFGT genes were the key structural genes of mature period accumulation type accessions, while PpMYB10.1 played a key role in the synthesis of anthocyanin in all red-flesh peach germplasms.

Compositions and Physicochemical Properties of Sweet Almond Isolate Proteins

ZHANG QingAn, ZHANG XinYun, FENG YuLin, SHI FangFang

Scientia Agricultura Sinica. 2017, 50(13): 2564-2575. doi:10.3864/j.issn.0578-1752.2017.13.015

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【Objective】The aim of this study is to investigate the physical and chemical properties of sweet almond albumin, globulin, gliadin and glutelin. The relationship between the change of protein’s properties and product processing features was also studied to promote the development of high-quality sweet almond products. 【Method】Alkali-solution and Acid-isolation was employed upon the skimmed sweet almond to extract crude protein, and, subsequently, Osborne classification method was adopted to extract the albumin, globulin, gliadin and glutelin. The amino acid composition was determined by Automatic Amino Acid Analyzer. ESEM was used to observe the morphological characteristics of above mentioned proteins; CD spectra scanning was utilized to measure its secondary structure; thermal properties were determined by using DSC and TGA; Rheometer was applied to determine the influence of shear rate, moreover, the linear equation between temperature and viscosity of proteins was determined, and, therefore, the value of Ea and K₀ could be calculated.【Result】Various amino acids were detected in isolated proteins derived from sweet almond, containing 17 kinds of amino acids which the human need. It should be noticed that eight kinds of essential amino acids (including histidine) are completely embraced, among which the content of glutamic acid is the highest. Albumin, globulin and gliadin show a state of close aggregation structure, while glutelin shows a state of loose and porous plate. As for the constitution of the secondary structure, alpha helix and random curl are dominant in the isolated proteins compared with other forms of secondary structures. The denaturation temperatures for albumin, globulin, gliadin and glutelin are 62.84, 72.98, 78.33 and 45.70℃, respectively. Therefore, the thermal stability should coincide with the following order: gliadin ＞ globulin ＞ albumin ＞ glutelin. Combined DSC with ESEM, the aggregation degree should conform to the following order: albumin ＞ gliadin ＞globulin ＞ glutelin protein. The aqueous solution of the four kinds of isolated proteins derived from sweet almond are non-newtonian fluid and, furthermore, the relationship between temperature and viscosity is conform to the Arrhenius index equation, and the linear equation matches the parameters in processing well.【Conclusion】Sweet almond isolated protein can be regarded as a high-quality source for plant protein. Results of this study will make greater contributions to the development of sweet almond protein products. Besides, this study definitely do contribution to the function and property control of the protein products derived from sweet almond both in development and processing of the products, which could enhance the additional value and promote the development of sweet almond industry.

Effects of Different Packages on Edible Quality and Volatile Components of Rice During Storage

WANG LiFeng, WANG HongLing, YAO YiJun, ZHANG YiYi, CHEN JingYi, WANG HaiFeng, SHI JiaYi, JU XingRong

Scientia Agricultura Sinica. 2017, 50(13): 2576-2591. doi:10.3864/j.issn.0578-1752.2017.13.016

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【Objective】 To provide data support and a theoretical basis for small packaging rice preservation, rice was packaged in different ways. Edible quality (texture quality, pasting properties) and volatile components of rice were measured during storage, through judging the changes of rice quality, the suitable packaging method was used to carry on the preservation of rice. 【Method】In this study, “No. 99japonica rice” was used as the experimental materials. Rice were packaged in woven, natural airtight anoxia and vacuum and stored in artificial climate chamber at temperatures of 15℃, 25℃, 30℃ under 60% RH for 180 d. Edible quality and volatile components were analyzed per month. 【Result】With the extension of storage time, the elasticity and recovery of cooking rice decreased gradually, and the hardness and adhesion showed a trend of first increasing and then decreasing. The setback value increased gradually while the breakdown value first increased and then decreased, thermal paste stability became worse and easy to aging with the extension of storage time under three types of packing. There were 41 kinds of volatile compounds in fresh rice were detected, including hydrocarbons, alcohols, aldehydes, ketones, esters, organic acids and heterocyclic compounds. The content of aldehydes could be used to evaluate the taste and freshness of rice. The content of aldehydes in rice by woven bag was the highest after 180 d of storage, which accounted for more than 20% of the volatile constituents, followed by natural airtight hypoxia packaging, and there was minimum content of aldehydes in rice by vacuum packaging. 2-pentyl furan was oxidation product of linoleic acid which was only 0.30% in fresh rice, however, the content rose sharply by woven bag with the extension of storage time. The contents of aldehydes and 2-pentyl furan were greatly influenced by packaging methods, the order of the preservation effect was vacuum＞natural sealed hypoxia＞woven bag. However, storage quality of rice had also deterioration in high storage temperature even with vacuum packaging. 2-pentyl furan was detected in every period under three types of packing, hexaldehyde content in woven bag, natural sealed hypoxia and vacuum packaging was 1.82%, 1.55%, 1.16%, respectively. aldehyde C-9 content in woven bag, natural sealed hypoxia and vacuum packaging was 4.75%, 3.94%, 2.77%, 2-pentyl furan content in woven bag, natural sealed hypoxia and vacuum packaging was 2.27%, 1.85%, 1.43%, respectively. Thus it was deduced that woven bag packaging of rice produce more apparent the peculiar smell of the oil oxidation and aging is the worst, followed by natural airtight hypoxia packaging. Vacuum can effectively delay the rate of aging rice quality.【Conclusion】Deterioration is inevitable during the storage process. In the respect of hardness, elasticity, viscosity, resilience and gelatinization properties, rice quality after storage was inferior than fresh rice. Vacuum packaging was beneficial to rice for rice storage can effectively delay the rice deterioration. The content of aldehydes can be used to evaluate the freshness of rice. Aldehyde content in three types of packing both grew after 180 d of storage, with aldehyde content increased significantly in woven bag packaging. Aldehydes volatile components in rice, hexanal, nonanal and 2-pentyl furan can reflect the rice storage quality.

Genetic Effect Analysis of SNPs from 6 Genes on SCS and Milk Production Traits in Xinjiang Brown Cattle

LIU LiYuan, ZHOU JingHang, ZHANG MengHua, LI JinXia, FANG JiQing, TAN ShiXin, WANG AiFang, HUANG XiXia, WANG YaChun

Scientia Agricultura Sinica. 2017, 50(13): 2592-2603. doi:10.3864/j.issn.0578-1752.2017.13.017

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【Objective】The association between polymorphisms of ERAP2, Cwc27, PPFIBP2, THADA, ZNF804B and AMHR2 genes and somatic cell score (SCS) and milk production traits in Xinjiang brown cattle were studied in order to find out molecular markers association with SCS and/or milk production traits.【Method】Taking 169 Xinjiang brown cattle cows as experimental animals from Xinjiang Urumqi cattle farm and Xinjiang Tianshan Animal Husbandry and Biotechnology Co., Ltd. The experimental data including Dairy Herd Improvement records (including milk fat percentage, milk protein percentage, lactose percentage, total solid content and SCS) and milk yield of 305d, 10 SNPs were selected from exon area based on the results of DNA sequencing of 15 Chinese Holstein cows in an initial study and genotyped by Sequenom MassARRAY Genotype technology, the association between SNPs or haplotype and SCS and milk yield traits were analyzed by the least squares method in the GLM procedure of SAS8.1.【Result】The results showed that the 10 SNPs were polymorphic, and 9 SNPs were in Hardy-Weinberg equilibrium. Association analysis results showed that two SNPs in ERAP2 gene (T98741711C and G98736141A) had a highly significant association with milk yield of 305d in Xinjiang brown cattle(P＜0.001), the milk yield of TT and GG genotype was the highest in T98741711C locus and G98736141A locus, respectively; C45667492G locus in PPFIBP2 gene had a significant association with milk lactose percentage (P＜0.005), GG genotype was the highest; Cwc27 (T14533269A) had a significant association with milk fat percentage and total solid content(P＜0.005) and had a highly significant association with SCS(P＜0.001), milk fat percentage of TT genotype was the highest and SCS of AA genotype was higher than AT and TT genotypes; AMHR2 (C26758055G) had a highly significant association with milk protein percentage and total solid content(P＜0.001), GG genotype was higher than CC and GC genotypes. The results of linkage disequilibrium analysis and haplotype construction revealed that the 10 SNPs constructed two haplotype blocks, among them, SNP1 and SNP2 were in linkage disequilibrium state(0.3＜r²＜0.6), SNP4 and SNP10 in strong linkage disequilibrium state(r²＞0.6). Then the correlation between haplotype and SCS and/or milk production traits was analyzed, and the results showed that haplotype had no significant association with SCS and milk production traits(P＞0.001).【Conclusion】In this study, it was found that ERAP2, Cwc27, PPFIBP2 and AMHR2 genes had a significant association with SCS and milk production traits, and the results of the study will provide a theoretical basis and reference for the molecular marker-assisted selection in Xinjiang Brown Cattle.

Cloning and Expression Analysis of CCND2 Gene in Yak Ovaries During Different Periods of Estrus

SHI Xian, XIONG XianRong, LAN DaoLiang, CHEN WeiMing, HU JiaJia, CAI WenYi, LI Jian

Scientia Agricultura Sinica. 2017, 50(13): 2604-2613. doi:10.3864/j.issn.0578-1752.2017.13.018

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【Objective】The aim of the study was to investigate the sequence characteristics and expression level of cyclin D2(CCND2) in yak ovary during different periods of estrus. 【Method】 Total RNA of yak ovary were extracted and reverse transcribed into cDNA. Specific primers were designed according to mRNA sequence of cattle CCND2 gene in the GenBank(GenBank accession No: NM_001076372.1) by software Primer 5.0. The CDs region and part of 5′UTR and 3′UTR in yak CCND2 gene were cloned from yak ovary by polymerase chain reaction(PCR) amplification and sequencing technology. The physicochemical properties, secondary structure, tertiary structure, domain, homology were analyzed and phylogenetic tree of CCND2 was constructed by online software like Protparam, SOPMA, SWISS-MODEL and SMART. Semi-quantitative PCR was used to examine the expression profiles of CCND2 gene in different tissues. Quantitative real-time PCR (qRT-PCR) was used to detect the expression level of CCND2 gene in the ovary during different periods of estrus (ovarian follicular phase, red body phase and luteal phase).【Result】The 909 bp length sequences of CCND2 gene in yak, including 870 bp length CDS region encoding 289 amino acids, were obtained. Alignment with cattle CCND2 gene CDS and amino acid, five bases mutations and one amino acid mutation were found. The CDs homology of CCND2 compared yak to wild yak, cattle, Indian buffalo, sheep, wild boar, horse, dog, cat and human was 99.54%, 99.31%, 99.31%, 98.16%, 94.81%, 90.46%, 90.80%, 91.49% and 88.62%, respectively. There was a high homology among different species, and phyletic evolution was the same as their genetic relationship. The research indicated that the CCND2 gene CDs was conservative in the course of evolution. The formula of protein encoded by CCND2 gene in yak was C₁₄₄₅H₂₃₁₅N₃₇₇O₄₄₀S₁₈, and the molecular weight was 32.59 kD, the theory isoelectric point was 4.75, the grand average of hydroparthicity was -0.107, and the instability index was 44.56. It was an unstable, soluble and acidic protein without transmembrane regions and signal peptide. Random coil and α-helix were mainly in the secondary structure of CCND2, which were the same as the tertiary structure of CCND2 protein. The amino acids sequence of CCND2 contains Cyclin_N domain in the 26-152 amino acids and Cyclin_C domain in the 154-257 amino acids. The CCND2 gene was expressed in various tissues of yak, and relatively higher in stomach, ovary and brain. qRT-PCR results showed that the mRNA expression level of CCND2 in ovary of follicular phase was the highest (P＜0.01), and no difference between the red body phase and luteal phase (P＞0.05).【Conclusion】The complete CDs of CCND2 gene was successfully cloned and the tissues expression profiles were clarified, and its bioinformatics and encoded protein function and structure were investigated. These provide a theoretical basis for further study of CCND2 gene in regulation of yak reproduction. The different expressions of CCND2, in different periods of estrus in yak, might be related to proliferation and differentiation of granulose cells, development process of follicular wave, and ovarian endocrine. These indicated that CCND2 plays an important role during the reproductive cycle, and these results of study will provide basic information for further study on the ovarian development mechanism and improving reproductive competence in yak.

Transcriptome of Apis cerana cerana larval gut Under the Stress of Ascosphaera apis

CHEN DaFu, GUO Rui, XIONG CuiLing, LIANG qin, ZHENG YanZhen, XU XiJian, ZHANG ZhaoNan, HUANG ZhiJian, ZHANG Lu, WANG HongQuan, XIE YanLing, TONG XinYu

Scientia Agricultura Sinica. 2017, 50(13): 2614-2623. doi:10.3864/j.issn.0578-1752.2017.13.019

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【Objective】So far, no study on application of next-generation sequencing technology for the research of chalkbrood disease was reported. In the present research, RNA-seq technology was utilized to deep sequence of normal and Ascosphaera apis-treated 4th instar Apis cerana cerana larval gut to mine larvae’s responses to A. apis challenge.【Method】AcCK (un-treated group) and AcT (A. apis-treated group) were sequenced on Illumina HiSeq 2500 platform. After evaluation and filtration of raw data from RNA-seq, differentially expressed gene (DEG) analysis was performed using edgeR software, further, Gene Ontology (GO) and KEGG pathway enrichment analyses were carried out, and finally, real-time quantitative PCR (qRT-PCR) was conducted to validate the RNA-seq data.【Result】 In total, RNA-seq produced 188 457 338 raw reads, and after filtration, 182 088 448 clean reads were obtained, Q20 and Q30 of each sample were above 97.96% and 94.97%, respectively, indicating that RNA-seq data in this research were with high quality. principle component analysis (PCA) was performed on all genes level and the result showed PC1 and PC2 were able to account for 75.8% and 10.7% of the expressed genes’ overall differences, respectively. DEG analysis result displayed that there were 344 up-regulated genes and 239 down-regulated genes in AcCK VS AcT. GO enrichment analysis result showed that the DEGs were enriched in 36 GO terms, among them, the mostly enriched ones were cell (106 unigenes), cell part (106 unigenes) and metabolic process (104 unigenes). KEGG pathway enrichment analysis result suggested that up- and down-regulated genes were enriched in 72 and 45 pathways, respectively, and the mostly enriched pathways for up-regulated genes were ribosome (72 unigenes), carbon metabolism (16 unigenes) and glycolysis/gluconeogenesis (14 unigenes), while the mostly enriched pathways for down-regulated genes were carbon metabolism (9 unigenes), glyoxylate and dicarboxylate metabolism (8 unigenes) and amino acids biosynthesis (7 unigenes). further analysis demonstrated that the immune-related genes in A. c. cerana larval gut were activated, while the metabolism-related genes were greatly inhibited.【Conclusion】The findings of the study not only uncovered the A. c. cerana larval gut’s responses to A. apis during the early stage of invasion, but also provided key information for clarifying the mechanism underlying the host’s responses to A. apis, thus laying a foundation for functional investigation of key responding genes.

Effects of Stubble Returning to Soil and Fertilization on Soil Nitrogen Availability and Root Biomass of Cotton in Arid Region

ZHANG GuoJuan, PU XiaoZhen, ZHANG PengPeng, ZHANG WangFeng

Scientia Agricultura Sinica. 2017, 50(13): 2624-2634. doi:10.3864/j.issn.0578-1752.2017.13.020

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【Objective】The objective of this paper was to study the cotton soil nitrogen transformation process in arid region and it’s influence on the root biomass of cotton. The availability of cotton soil nitrogen which responds to agriculture management measures was clarified which respond to agriculture management measures, made high yield and high efficiency management measures for cotton, which implement high yield, good quality and low cost, serve for environmental friendly production of cotton.【Method】The main plot treatment consisted of two stubble management measures: stubble-removed (S0) and stubble returning to soil (S1). The split-plot treatment was composed of four fertilizer treatments: no fertilization (F0), NPK fertilizer (F1), organic fertilizer (F2) and combined application of NPK fertilizer and organic fertilizer (F3). The influences of stubble returning to soil and fertilization on soil nitrogen availability were studied and the process of cotton soil nitrogen transformation, including the change of net mineralization, net nitrification, gross nitrification and denitrification was discussed and the response of soil available nitrogen content and root biomass of cotton to stubble returning to soil and fertilization was clarified. 【Result】Stubble returning to soil and fertilization increased the net mineralization, gross nitrification and denitrification of soil in cotton field significantly, each index among different fertilization treatments and at different growth stages had different variations, there was no significant difference among the three fertilizer treatments, but the maximum rate was observed at full-bloom stage. Stubble returning to soil and fertilization increased ammonium, nitrate and inorganic N significantly, but there was no significant difference among the three fertilizer treatments, the content of inorganic N at full-bloom stage and full-boll stage was higher than at harvesting stage. Stubble returning to soil decreased the root-shoot ratio significantly, had no significant effect on root biomass and fine/coarse root biomass ratio. Fertilization increased the root-shoot ratio, root biomass and fine root biomass, there was no significant difference among the three fertilizer treatments. In summary, stubble returning to soil increased soil net mineralization, net nitrification, gross nitrification, denitrification, nitrate nitrogen, ammonium nitrogen and absorbable inorganic nitrogen content and root biomass. Combined application of NPK fertilizer and organic fertilizer had the largest soil net mineralization, net nitrification, gross nitrification, denitrification, nitrate nitrogen and absorbable inorganic nitrogen content. Combined application of NPK fertilizer also has the largest root biomass and fine/coarse root biomass ratio.【Conclusion】Stubble returning to soil and fertilization are beneficial to promotion of soil nitrogen transformation and available nitrogen content, thus affecting cotton root growth and biomass. Adoption of stubble returning to soil and different fertilization measures in cotton field in arid areas can promote soil nitrogen availability. Implementation of stubble returning to soil in arid areas, combined with application of NPK fertilizer and chicken manure, are helpful to accelerate the transformation of soil nutrient, improve efficiency of fertilizer, increase the effective content of nutrient, and promote the root growth and carbon assimilation ability of crop aboveground part.

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