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01 August 2017, Volume 50 Issue 15

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

Genetic Diversity Analysis of Pi-ta Gene 3′-UTR in Rice Landraces

LIU LiNa, YANG Jing, XU LiuYan, LI ChengYun

Scientia Agricultura Sinica. 2017, 50(15):  2851-2860.  doi:10.3864/j.issn.0578-1752.2017.15.001

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【Objective】 In order to understand the mechanism of Pi-ta resistance and provide a basis for the development of more durable resistant varieties, the relationship between the genetic diversity in 3'-UTR and the resistance function of the Pi-ta gene were studied. 【Method】 A total of 137 Yunnan rice landraces were collected. DNA from seedling at the three- to four-leaf stage was extracted and the DNA coding sequences of Pi-ta gene 3'-UTR were amplified with designed primers in Yunnan rice landraces which possess high genetic diversity. The sequence from functionally key nucleoside 6 640 to the stop codon 6 675 was also amplified. Through bidirectional sequencing, DNA sequences in 3'-UTR region of all genetic varieties in Yunnan rice landraces were obtained and submitted to the GenBank. The genetic diversity of Pi-ta 3'-UTR region in Yunnan rice landraces was analyzed based on detecting polymorphic sites. The distribution of different haplotypes was analyzed in combination with the key resistance locus 6640 G in coding region. The network diagram of the relationship between different haplotypes was constructed based on maximum parsimony. 【Result】 The Pi-ta gene 3'-UTR of Yunnan rice landraces shows a high degree of genetic diversity. A total of 12 SNPs were found in the 3'-UTR with a length of 1.1 kb. The 137 rice landraces could be divided into 7 haplotypes by these SNPs. No recombination signal was found among haplotypes. The length of Pi-ta 3'-UTR coding region is 1 120 bp, 5 times more than the average length of plant gene 3'-UTR (200 bp). The content of G+C is relatively low, 40.43%, and there is no insertion or deletion polymorphism in the 3'-UTR coding region. There are many non-conservative potential polyA sites in the 3'-UTR sequence of Pi-ta, in addition, there is a very high frequency TTTT sequence in the 3'-UTR region of Pi-ta, which suggests that Pi-ta has a complex regulatory mechanism when terminate transcription. Analysis of the different transcripts of Pi-ta also showed that the 3'-UTR has complex and variable transcripts as contrasted to the coding sequence, indicating that the selective splicing of 3'-UTR may be related to resistance determination. The analysis of the haplotype distribution indicated that the 3'-UTR high polymorphism appeared only in the susceptible rice landraces, and all the resistant individuals share one haplotype. Interestingly, the only 3'-UTR resistant haplotype corresponds to the only resistant haplotype in the Pi-ta coding region, that is, the haplotype containing 6640 G is also the 3'-UTR resistant haplotype. This indicates that 3'-UTR is closely linked to the coding region, and is consistent and continuous in terms of function and selection pressure. The Pi-ta resistant haplotype range has been extended from the coding region to the 3'-UTR region, and when introducing Pi-ta into broad-spectrum-resistant varieties, it is necessary to ensure that the 3'-UTR region must have no other SNPs, all should be 3'-UTR resistant-haplotype-specific SNPs. 【Conclusion】 The 3'-UTR is closely linked to the encoding region in Pi-ta gene. In resistant rice landraces, the 3'-UTR suffered purifying selection, and maintained a single haplotype. Results of the study demonstrate that the 3'-UTR is indispensable for the resistance function of Pi-ta gene.

CRISPR/Cas9 System-Based Editing of Phytochrome-Interacting Factor OsPIL15

JI Xin, LI Fei, YAN Yun, SUN HongZheng, ZHANG Jing, LI JunZhou, PENG Ting, DU YanXiu, ZHAO QuanZhi

Scientia Agricultura Sinica. 2017, 50(15):  2861-2871.  doi:10.3864/j.issn.0578-1752.2017.15.002

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【Objective】 As an important environmental signal, light can regulate gene expression, affect the activity of enzymes and plant morphogenesis. Phytochrome interacting factors play an important role in the signal transduction of light. Therefore, constructing the expression vector of CRISPR/Cas9 containing rice phytochrome-interacting factors OsPIL15 and creating the ospil15 mutants have important significance to exploit functional genes and enrich light signal regulation of the molecular mechanism of rice. 【Method】 According to the principle of CRISPR/Cas9 technology, the sgRNA was designed. To exclude non-specific target sites, the sgRNA was analyzed by sequence alignment in the rice genome database. At the same time, the target sequence contained the common restriction site to identify mutants. The oligonucleotides of sgRNA were chemically synthesized and inserted into linearized plasmid pBUN411 to construct the expression vector. Transgenic rice plants harboring sgRNA:Cas9 were obtained by A. tumefaciens-mediated stable transformation and the positive transgenic plants were screened by herbicide resistance. The PCR products of T₀ transgenic plants were digested by restriction enzyme to judge whether they were mutants. Then, the mutated genotypes of these mutants were analyzed by DNA sequencing. After searching rice genome using sgRNA sequence, five highly identical sites with less 4 mismatching bases were selected to assess off-target efficiency and specificity of sgRNA.【Result】The recombinant vector succeeded in oriented editing of OsPIL15. The restriction enzyme analysis results indicated that 15 mutants from the 25 randomly selected T₀ transgenic lines were obtained. They included 5 homozygous mutants, 6 biallelic mutants and 4 heterozygous mutants, and a total of 10 different genotypes and 11 mutant lines. The mutant types were mainly insertions or deletions of single base, besides, two mutant types of large deletions with 56 and 66 bp were obtained. Analysis of T₁ transgenic plants from some T₀ mutants indicated that the genotypes in T₀ mutants could descend stably into the next generation. The progeny of homozygous mutants in T₀ were homozygous mutants. However, only one wild type with no mutation was detected in a homozygous mutant progenies of line 14. Two homozygous mutations and one biallelic mutations were obtained in the progeny of biallelic T₀ lines. Three mutation types including homozygous mutants, heterozygous mutants and wild type with no mutation were detected in the progenies of heterozygous T₀ mutants. Restriction enzyme analysis was used to detect the engineered target site of T₁ positive transgenic plants which had no mutation in T₀. No mutation in 62 T₁ plants was detected. The results showed that CRISPR/Cas9 system played no role in gene editing in T₁ positive transgenic plants. After searching the rice genome using the target sequence with PAM, five highly identical sites were found. However, any mutations at these sites in T₀ and T₁ generations were not observed by restriction enzyme, which indicated the sgRNA was highly specific. Three groups of different genotypes were selected representative ospil15 mutants in T₁ generation to observe phenotypes, the investigation result showed that the growth stage and tiller number were not obviously changed, the plant height decreased significantly (P＜0.01), and the grain length was significantly increased compared with the wild type, up 5.69%. 【Conclusion】CRISPR/Cas9 system succeeded in oriented editing of OsPIL15, the ospil15 mutants with 10 different genotypes were obtained and observed that the plant height decreased significantly and the grain length increased significantly compared with those of wild type.

Drought Resistance Identification and Drought Resistance Indices Screening of Job’s Tears (Coxi lacryma-jobi L.) Germplasm Resources at Seedling Stage

WANG Can, ZHOU LingBo, ZHANG GuoBing, ZHANG LiYi, XU Yan1, GAO Xu, JIANG Ne, SHAO MingBo

Scientia Agricultura Sinica. 2017, 50(15):  2872-2887.  doi:10.3864/j.issn.0578-1752.2017.15.003

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【Objective】Drought is the important abiotic stress factor for Job’s tears production. Drought resistance identification, indices determination, and germplasms screening of Job’s tears germplasm resources are important for Job’s tears industrial development.【Method】The seedling drought survival rate, plant height, culm diameter, leaf length, leaf width, shoot fresh weight, shoot dry weight, root length, root diameter, root fresh weight, and root dry weight of 50 Job’s tears germplasms were measured at the normal water supply and repeated drought treatments in pot experiments in rainprotection shed. Drought resistance comprehensive evaluation value (D value), comprehensive drought resistance coefficient (CDC value), weight drought resistance coefficient (WDC value), correlation analysis, frequency analysis, principal component analysis, grey relational analysis, subordinate function analysis, clustering analysis, and stepwise regression analysis were used to identify the drought resistance and screen drought resistance indices of tested Job’s tears germplasms at seedling stage.【Result】Drought stress had significant effects on all indices. Correlation analysis showed that the seedling drought survival rate was significantly and positively correlated with culm diameter, leaf width, root diameter, leaf length, shoot fresh weight, shoot dry weight, root length, root fresh weight, and root dry weight, but not with plant height. Frequency analysis showed that the sensitive degrees of all indices response to drought stress in turn for root dry weight, root fresh weight, leaf width, root diameter, leaf length, seedling drought survival rate, shoot dry weight, culm diameter, shoot fresh weight, leaf length, and plant height. Principal component analysis showed that 6 principal components could represent 91.40% of the original data information of Job’s tears drought resistance. The ranks of drought resistance of tested Job’s tears germplasms based on the D value, CDC value, and WDC value were similar. Grey relational analysis showed that the correlation degree between DC value of all indices and D value in turn for root dry weight, leaf width, root fresh weight, culm diameter, root diameter, seedling drought survival rate, root length, plant height, shoot fresh weight, shoot dry weight, and leaf length, which was similar to the correlation degree between DC value of all indices and WDC value. According to D value clustering analysis, tested Job’s tears germplasms were divided into 5 drought resistance grades, 3 belonged to grade I, 17 belonged to grade II, 9 belonged to grade III, 20 belonged to grade IV, and 1 belonged to grade V. The subordinate function values of tested indices except for leaf length, shoot fresh weight, shoot dry weight and root dry weight, CDC value, D value, and WDC value were increased with increase of drought resistance grades. Stepwise regression analysis showed that the seedling drought survival rate, plant height, leaf width, root length, and root fresh weight were closely related to the D value.【Conclusion】yy18-1, yy14-3, and yy13-1 were identified as drought resistance Job’s tears germplasms at seedling stage, which could be used as materials for the researches on cultivar breeding, mechanism, and regulation and alleviation mechanism of drought resistance in Job’s tears. The seedling drought survival rate, plant height, leaf width, root length, and root fresh weight could be used as the simple and intuitive identification indices of drought resistance in Job’s tears germplasm resources at seedling stage.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY

Effects of Mulching During the Fallow Period and Nitrogen Fertilizer on Soil Water and Plant Nitrogen Use of Dry-Land Wheat

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

Scientia Agricultura Sinica. 2017, 50(15):  2888-2903.  doi:10.3864/j.issn.0578-1752.2017.15.004

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【Objective】For the problem of less rainfall and uneven distribution, lower water and nitrogen use efficiency of dryland wheat in the Loess Plateau, the best technological way for water conservation through mulching and nitrogen fertilization of dryland wheat was studied.【Method】Field experiments were carried out in Qiujialing village, Wenxi, Shanxi from 2010 to 2013, the main plot was mulched with plastic film or no mulched after deep tillage during the fallow period, the subplot was applied with nitrogen fertilizer at 75 kg·hm^-2, 150 kg·hm^-2, and 225 kg·hm^-2 in order to make clear the effect of mulching during the fallow period and nitrogen fertilization on soil moisture, plant nitrogen utilization and its contribution to yield of dryland wheat in different years. 【Result】The results showed that the soil moisture, plant nitrogen accumulation, the N translation amount before anthesis and its contribution proportion to grain at every growth period were the highest in humid year and the lowest in dry year. Compared with the dry year, the yield of grain in humid and normal year, respectively, increased by 80% and 69%, the water use efficiency, respectively, increased by 7% and 20%, the N use efficiency, respectively, increased by 6% and 5%. Compared with no mulching during the fallow period, the soil water storage in 0-300 cm depth was significantly increased by 52-60 mm at sowing stage under the condition of mulching, and the soil water storage was significantly increased before anthesis, plant nitrogen accumulation was increased in every growth period, the N accumulation of grain was significantly increased. Compared with no mulching during the fallow period, the N accumulation was significantly increased after jointing stage under the condition of mulching in humid and normal years, the contribution rate to grain of the N translation amount of leaf and spike before anthesis was increased; the N accumulation and its proportion at different growing stages were increased in dry year, the contribution rate to grain of the N translation amount of stem and sheath before anthesis was significantly increased. Compared with no mulching during the fallow period, the grain yield was significantly increased by 23%-41%; water use efficiency increased by 3%-15%, the N use efficiency was significantly increased in humid and normal years by 14%-26% and significantly increased by 10% under the condition of low N fertilization in dry year. Combined with high N fertilization in humid year, combined with middle N fertilization in normal year and dry year under the condition of mulching during the fallow period, combined with low N fertilization in dry year under the condition of no mulching during the fallow period, the soil water storage, yield and water use efficiency were the highest before booting stage. Combined with high N fertilization in humid year, the translation amount of N before anthesis and the N accumulation after anthesis were significantly the highest, especially promoted the translation from leaf and spike to grain before anthesis. Combined with middle N fertilization in normal year and dry year under the condition of mulching during the fallow period, the N translation amount before anthesis and the N accumulation of grain were significantly the highest, and promoted the translation from leaf and spike to grain in humid year, especially the spike. And promoted the translation from stem and sheaths and spike to grain in dry year under the condition of mulching, especially the stem and sheaths. Combined with low N fertilization in dry year under the condition of no mulching during the fallow period, the N accumulation of grain was significantly the highest, the N translation amount before anthesis and its contribution rate to grain was the highest, the N translation amount of stem and sheaths and its contribution rate to grain was the highest. 【Conclusion】 Mulching during the fallow period of dry-land contributed to accumulating precipitation, promoting N accumulation at middle and late growth stages in humid and normal years, promoting the N translation from leaf and spike to grain, contributed to promoting the N accumulation at early and middle growth stages in dry year, promoting the N translation from stem and sheaths to grain. Humid year combined with high nitrogen at 225 kg·hm^-2, normal year and dry year under the condition of mulching during the fallow period combined with middle nitrogen at 150 kg·hm^-2, and dry year under the condition of no mulching during the fallow period combined with low nitrogen at 75 kg·hm^-2 could achieve synchronous improvement of yield and water use efficiency.

Effects of Subsoiling During the Fallow Period and Timely Sowing on Water Storage and Wheat Yield of Dryland

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

Scientia Agricultura Sinica. 2017, 50(15):  2904-2915.  doi:10.3864/j.issn.0578-1752.2017.15.005

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【Objective】In order to solve the practical productive problems that sowing only to wait for the rain decreased the yield of dryland wheat conventionally cultivated as a single crop per year in the Loess Plateau in most of the year, the study of effects of subsoiling during the fallow period (SS) with great soil water storage and timely sowing on yield and its component was implemented to improve the precipitation use efficiency, and to build the rational community structure for achieving high yield and efficiency.【Method】The field experiments were conducted in a two-factor split-block design with SS or traditional farming (CK) in the main plots and three sowing dates (Sep 20^th, early sowing date, T₁; Oct 1^st, timely sowing, T₂; Oct 10^th, late sowing date, T₃) in the subplots at Qiujialing village, Wenxi county, Shanxi province from 2012 to 2014, to study the effects of SS on yield formation of dryland wheat and the adjusting effects of sowing dates. 【Result】 The soil water storage in the 0-300 cm soil profile was increased by 59-71 mm and 34-52 mm under SS compared with CK in 2012-2013 and 2013-2014 at sowing stage, respectively. Adoption of SS obviously improved the group tillers before wintering stage and plant dry matter at different growth stages, which increased by 8%-18% and 8%-15% of the number of spike, 19%-36% and 17%-22% of yield, 6%-21% and 10%-12% of water use efficiency compared with CK during 2012-2013 and 2013-2014, respectively. Soil water storage in T₂ treatments was the highest from wintering to booting stage under SS treatments. The highest tiller number before wintering stage and plant dry matter before anthesis stage were obtained in T₂ treatment, however, no significant difference was observed with T₁ treatment. The largest post-anthesis plant dry matter was attained in T₃ treatment under SS, no significant difference was observed with T₂ treatment. The significant highest spike number, grain number per spike and yield were observed in T₂ treatment, while 1000-grain weight increased with the delay of sowing date. Under traditional farming in the dry year (2012-2013), the highest soil water storage from wintering to booting stage, spike number, grain number per spike and yield were attained in T₁ treatment. In addition, in three sowing dates and SS treatments, spike number, grain number per spike, plant dry matter at mature stage and yield were more significantly correlated with soil water storage of different soil layers before anthesis than post-anthesis, especially with soil water moisture in deeper layers before anthesis. Wheat yield was increased by 17 and 23 kg·hm^-2 with an increase of 1 mm soil water storage at sowing stage under SS matched with T₂ treatment during 2012-2013 and 2013-2014, respectively. 【Conclusion】 Subsoiling during the fallow period was conducive to store precipitation during the fallow period into the soil resulting in the increase of soil water storage before sowing. Adoption of early sowing and timely sowing in SS treatments were favor to build the strong seedling before wintering, however, the number of spike, grain yield and water use efficiency were the highest under timely sowing. In conclusion, adoption of subsoiling during the fallow period matching sowing on October 1^st could optimize the yield composition and increase yield and efficiency of dryland wheat.

Response of Dry Matter Accumulation and Yield Components of Maize Under N-Fertilizer Postponing Application in Oasis Irrigation Areas

WEI TingBang, HU FaLong, ZHAO Cai, FENG FuXue, YU AiZhong, LIU Chang, CHAI Qiang

Scientia Agricultura Sinica. 2017, 50(15):  2916-2927.  doi:10.3864/j.issn.0578-1752.2017.15.006

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【Objective】In oasis irrigation agricultural region, the traditional fertilizer application in maize production has caused serious problems, such as soil available nitrogen hunger, premature-senescence and yield reduction in the late growth stages under plastic-film mulching. The aim of this study was to provide academic and practical evidence for enhancing dry matter accumulation and yield parameters of maize through the optimization of nitrogen fertilizer management. 【Method】The three-year field experiment was carried out in Hexi Oasis irrigation region of Gansu province from 2012 to 2015. Each of the treatment in this study received the same amount of total nitrogen and with 10% as basal N and 40% as pre-tasseling N. Three N application treatments were set with application of different N amounts postponed from jointing N to post-flowering N: 20% of N postponing applied (M₁), 10% of N postponing applied (M₂), and traditional nitrogen application (M₃). 【Result】Postponing of application of nitrogen fertilizer increased the highest dry matter accumulation rate, the average dry matter accumulation rate and advanced the days of emergence of the highest dry matter accumulation rate. Compared with traditional nitrogen application, the maximum dry matter accumulation rate and the average rate of maize in the treatment with postponing application of 20% of N was increased by 15.6% and 6.6%, respectively. Meanwhile, the days of emergence of the highest dry matter accumulation rate was advanced by 2.9 days. Nitrogen fertilizer application had significant influence on biomass yield, grain yield, harvest index, number of productive ear, seed number per ear, and 1000-grain weight. Compared with traditional nitrogen application treatment, biomass yield of maize in the treatment postponing of application of 20% of N was increased by 6.6%, but not significant between the treatment with postponing of application of 10% of N and the traditional treatment. Similarly, grain yield of maize in the treatment with postponing of application of 20% and 10% of N was respectively, increased by 14.1% and 5.5%. The harvest index of maize in treatment with postponing of application of 20% of N was increased by 7.5%, but not significant between the treatment with postponing of application of 10% of N and the traditional treatment. Compared with traditional nitrogen application, the number of productive ear, seed number per ear, 1000-grain weight of maize in the treatment with postponing of application of 20% of N were increased by 8.9%, 12.9%, 5.8%, respectively, however, 1000-grain weight of maize in the treatment with postponing of application of 10% of N was not significant. The facts showed that the treatment with postponing of application of 20% of N has regulated grain yield with the improvement of number of productive ear and the enhancement of seed number per ear and 1000-grain weight. 【Conclusion】The treatment with postponing of application of 20% of N, (i.e. N application with 45 kg·hm^-2 at maize jointing, 180 kg·hm^-2 at pre-tasseling stage, and 135 kg·hm^-2 at 10 days post-flowering stage of maize) could be considered as the best feasible nitrogen fertilizer management for optimizing dry matter accumulation and obtain high yield for maize in oasis irrigation region.

PLANT PROTECTION

Effects of STK1 on Glycogen and Lipid Accumulation During the Appressorium Development of Setosphaeria turcica

ZHANG YunFeng, ZHANG ShuHong, WU QiuYing, FAN YongShan

Scientia Agricultura Sinica. 2017, 50(15):  2928-2935.  doi:10.3864/j.issn.0578-1752.2017.15.007

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【Objective】 The objective of this paper is to study the relationship between STK1 and appressorium development, clarify the regulation of STK1 on the glycogen and lipid accumulation, and to make a foundation for elucidation of the molecular mechanism of appressorium development of Setosphaeria turcica.【Method】The appressoria were induced with hypha tips of wild type (WT) isolate and STK1 knock-out mutant (ΔSTK1) on the hydrophobic surface of glass slide by the method of “hypha separation with slide insertion” and incubation in a petri-dish moist chamber at 22℃ and under alternative changes of 14 h lightness and 10 h darkness, to observe the course of appressrium development under the microscope at intervals of 12 h. The glycogen in mycelia and appressoria of WT andΔSTK1 on the slides were stained 48 h with I₂/KI before and after 48 h of appressorium induction, and the changes of glycogen metabolism during appressorium development were microscopically observed. The lipid in mycelia and appressoria of WT andΔSTK1 on the slides were placed in a -70℃ ultra-low temperature freezer refrigeration for 30 min before stained for 24 h with Oil Red O, and the changes of lipid metabolism during appressorium development were microscopically observed. The expression of key genes in glycogen and lipid synthesis during appressorium development was detected by real-time PCR. 【Result】The appressoria from hypha tips could be induced on the hydrophobic surface of glass slide for both WT isolate and ⊿STK1 mutant. However, the appressoria of ΔSTK1 were differentiated into several different abnormal types, which were obviously different from WT isolate. The appressorium of WT was single cell within 48 h of appressorium induction and only a few multicellular appressoria were found after 48 h of induction. Nevertheless, the twisted appressoria were found after only 24 h of appressorium induction of ΔSTK1, and some other abnormal appressorium forms, such as double-bifurcation, multi-bifurcation and “O” type, were found after 48 h of induction. After the glycogen and lipid staining in hypha and appressoria of WT and ΔSTK1, it was found that the accumulation of glycogen and lipid was equally distributed in the mycelia and appressoria of WT isolate, but there was almost no accumulation of glycogen and lipid in the appressoria of ΔSTK1. The glycogen accumulation was significantly reduced in the mycelium of ΔSTK1 and the lipid was mainly distributed in the septum parts of hyphal cells, which were significantly different from WT. The gene expression of glycogen synthase (GS) and diacylglycerol acyltransferase (DGAT) increased by 6.6% and 40.3%, respectively, after the appressoria of WT isolate were induced after 48 hours. However, the GS gene expression decreased by 9.0% and the DGAT gene expression increased by only 24.5% in the ΔSTK1 mutant.【Conclusion】The loss of STK1 function resulted in abnormal appressorium development, decreases of glycogen accumulation, uneven distribution of lipid, and a significant reduction in the expression of key genes of glycogen and lipid synthesis, indicating the accumulation of glycogen and lipid is closely related to the appressorium development of S. turcica.

Cloning, Expression Analysis of Solanum lycopersicum SYTA

PAN Qi, LIU XuXu, PENG HaoRan, PU YunDan, ZHANG YongZhi, YE SiHan, WU GenTu, QING Ling, SUN XianChao

Scientia Agricultura Sinica. 2017, 50(15):  2936-2945.  doi:10.3864/j.issn.0578-1752.2017.15.008

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【Objective】 The objective of this study is to clone Solanumlycopersicum SYTA (S.l SYTA), to analyze its bioinformatic characteristics, tissue expression and subcellular localization, the effect of green fluorescence protein (GFP) labeled Tobacco mosaic virus (TMV) on the expression level of S.l SYTA, its effect on the infection and movement of TMV and to provide a theoretical basis for further investigation of the S.l SYTA influence on the process of the infection and movement of the plant viruses. 【Method】The primer pairs of S.l SYTA were designed using Primer Premier 5.0 software based on the sequence of homologous SYTA in the genome ofthe S. lycopersicum. A complete coding sequence of S.l SYTAwas isolated and obtained by RT-PCR method. The sequence characteristics ofS.l SYTA was analyzed by bioinformatics tools. Multiple sequence alignments between S.l SYTAand its homologous ones from other species and a phylogenetic tree of homologous species were made by MEGA 7.0. The subcellular localization of S.l SYTA was analyzed through fusing with GFP protein. The real-time fluorescent quantitative PCR (qRT-PCR) was used to analyze the expression of S.l SYTAin different tissues and the change of its expression level in the TMV-GFP infected S. lycopersicum. The S.l SYTA was transiently expressed in the Nicotiana benthamiana by agroinfiltration, and then ELISA was used to detect the accumulation and movement of TMV-GFP. 【Result】A full-length SYTA of 1 620 bp was cloned from S. lycopersicum. Sequence alignment and bioinformatics analysis revealed that the deduced amino acids ofS.l SYTA had common characteristics of SYTs family with an N-terminal transmembrane region, a linker of variable size, and two C-terminal C2 domains. Furthermore, SYTA has the closest phylogenetic relationship to that of the N. sylvestris and N. tomentosiformis in amino acids sequences, and distanced from that of Cucumis sativus. Subcellular localization results showed that S.l SYTA was distributed at plasma membrane. The expression level of S.l SYTAin different tissues of S. lycopersicum was root＞leaf＞stem. The expression of S.l SYTAin S. lycopersicum obviously increased on the 1st day and decreased to the normal level on the 7th day after inoculation of TMV-GFP.Following the transient expression of S.l SYTA in N. benthamiana, TMV-GFP was inoculated. After 5 days, TMV-GFP had moved to the new leaf of the N. benthamiana. But no TMV-GFP was found in the new leaf of the control N. benthamiana in which the empty vectors were transiently expressed. ELISA assay results showed that the accumulation of the TMV-GFP in the samples under treatment was obviously higher than that in the control group after 5 days of TMV-GFP inoculation.【Conclusion】S.l SYTAhas the typicalcharacteristics of the SYTs family. S.l SYTAlocated on the plasma membrane of epidermal cell of N. benthamiana. The S.l SYTA expressed highest in root of S. lycopersicum. Under the stress of TMV-GFP, the expression of S.l SYTA in S. lycopersicum obviously increased firstly and then decreased to the normal level. In the S.l SYTA transient expression of N. benthamiana, the S.l SYTA could promote the initial accumulation and movement of TMV-GFP.

Function Analysis of Flagellar Gene fliS in Acidovorax citrulli

YANG BingYe, FU Dan, HU FangPing, CAI XueQing

Scientia Agricultura Sinica. 2017, 50(15):  2946-2956.  doi:10.3864/j.issn.0578-1752.2017.15.009

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【Objective】 Bacterial fruit blotch (BFB) caused by Acidovorax citrulli (Ac) is one of the most serious diseases in the world. Flagella are always considered as movement organ of bacteria and play an important role in their infection, and they have been reported that they could be controlled by flagellar protein gene fliS. The function of gene fliS in A. citrulli is still unclear. The objective of this study is to investigate the function of gene fliS of A. citrulli ineffecting on flagellum formation and pathogenicity. 【Method】 A set of primes were designed based on the genomic DNA No.1 of A. citrulli wild strain. The upstream and downstream fragments knocked out gene fliS were amplified by PCR, respectively. The gene knockout and complementary vectors were constructed through PCR amplification, recovery, digestion, connection and transformation. The strains with knockout gene fliS and complementation were constructed with the method of triparental hybridization. Morphological characteristics of flagella, pathogenicity, hypersensitive response, motility, quorum sensing, biofilm formation, growth rate, colonial morphology, etc have been tested among the wild type, the mutant and the complementary strains. In addition, the total RNA of the bacteria was extracted, and a real-time quantitative PCR (qRT-PCR) was carried out using glnA as an internal control for normalization. Then the expression of genes, flhD, fliE, fliC, flgK, flgM, fliD and fliA in the wild type, the mutant and the complement strains were compared.【Result】The deletion mutant and complementary strain were obtained successfully by screening of gentamicin resistance and PCR verifying, named as mutant 1-fliS and complementary strain 1-fliShb. The results showed that the deletion mutant had weakened motility and biofilm formation, and the complementary strain was almost recovered. Compared with the wild strain, the pathogenicity of the mutant on watermelon and melon was reduced and the complementary strain was recovered completely. The length of flagellum of the deletion mutant was 1/3-1/4 of that of the wild strain, and the complementary strain almost recovered and it was about 4/5 of that of the wild strain. The wild strain could form typical haloes obviously by bacteria migrating via twitching on NA medium, but the deletion mutant did not form haloes on NA medium and the complementary strain recovered partially. The growth rate of deletion mutant was slower than the wild strain and the complementary strain was not recovered. There were no difference among the wild, mutant and complement strains on hypersensitive response and quorum sensing. The results of qRT-PCR showed that in the mutant the expressions of gene flhD decreased obviously compared with the wild strain. In addition, the expressions of fliE, fliC and flgK increased obviously, the expressions of flgM and fliD increased slightly, the expressions of fliA was constant compared with the wild strain, the expressions of genes flhD, fliE and fliC recovered partially in complementary strain and, the expressions of flgK, flgM and fliD did not recover. 【Conclusion】 The flagellar gene fliS could regulate the flagellum formation, motility, biofilm formation, growth rate, colony morphology and pathogenicity of A. citrulli.

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

Effects of Different Nitrogen Fertilization Treatments on Soil Nitrogen Supply and Yield in Rice-Rape Rotation

HE Jie, LI Bing, WANG ChangQuan, LI YuHao, YANG BangFeng, ZHANG JingSheng, XIANG Hao, YIN Bin, LI HongHao

Scientia Agricultura Sinica. 2017, 50(15):  2957-2968.  doi:10.3864/j.issn.0578-1752.2017.15.010

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【Objective】 In order to study the effects of different nitrogen fertilization treatments on soil nitrogen supply and yield in rice- rape rotation system, the differences of effects in different treatments in the rice and rape rotation system were analyzed. 【Method】In 2014 and 2015, field experiments were carried out to study the effects of different N treatments on the inorganic nitrogen, enzyme activity, yield and nitrogen use efficiency. Four four fertilization patterns were designed, including CK (non- N fertilization), UR (100% urea), 40%CRU (40% controlled- release urea+ 60% urea), CRU (100% controlled- release urea). 【Result】（1）Compared to UR, the soil inorganic nitrogen at the mid-late stage of rice in CRU treatment was increased significantly. With the increasing of N in CRU, the soil inorganic nitrogen in the mid-late stage of rape was all increased. 40%CRU and CRU had a little influence on the soil nitrogen in the mid-late stage of rape. (2) The highest urease activity and protease activity in the early stage of rice and rape were obtained in UR. With the development of growth period, the enzyme activity was higher than UR by controlled- release fertilizers with different patterns. 40%CRU and CRU had a little influence on the soil enzyme at the mid-late stage of rice and rape. The enzyme activity of rice increased at beginning and then decreased afterwards, with the peak at booting stage. With the development of rape growth period, the urease activity reduced gradually. After adding controlled- release fertilizer, the protease activity increased at beginning and then decreased afterwards. (3) The yields of rice and rape were the highest in 40%CRU treatment, compared with UR treatment, the yield of rice increased by 597.04 kg·hm^-2 (2014) and 582.61 kg·hm^-2 (2015), respectively, improved by 7.50%-7.83%; the yield of rape increased by 391.19 kg·hm^-2 (2014) and 378.49 kg·hm^-2 (2015), improved by 15.39%-16.70%, respectively. The regression equation of yield and component factors showed that grain number and seed setting rate were positively correlated with rice yield, compared with UR treatment, 40%CRU treatment increased grain number by 15.17% (2014) and 17.72% (2015), the seed setting rate increased by 4.49% (2014) and 4.44% (2015). The rape yield showed a significant correlation with seed number per pod and total silique number, 40%CRU treatment had the biggest seed number per pod, total silique number increased by 8.98% (2014) and 13.80% (2015) compared with the UR treatment. (4)Nitrogen application significantly increased the overground nitrogen accumulation at the mature stage of rice and rape, and 40%CRU treatment was the highest. Compared with other nitrogen treatments, the nitrogen accumulation in the mature stage of rice in 40%CRU treatment increased by 6.21%-21.83% (2014), 6.51%-20.74% (2015), and the nitrogen accumulation at the mature stage of rape increased by 8.42%-24.74% (2014), and 9.39%-22.77% (2015). Nitrogen application could effectively improve the NAUE, NPP, NAE, and the optimal treatment was 40%CRU, followed by CRU treatment. 【Conclusion】 Controlled-release fertilizer could improve the soil nitrogen and enzyme activity, increase significantly yield of rice and rape, enhance nitrogen use efficiency. And controlled release blend bulk urea will enhance the nitrogen supply of soil at the mid-late growing stage of rice and rape, which can effectively promote the absorption and utilization of nitrogen, thus increase the yield effectively.

Optimal Scale Analysis of Soil Salinity Prediction in Oasis Irrigated Area of Arid Land Based on Landsat OLI

WEI Yang, DING JianLi, WANG Fei

Scientia Agricultura Sinica. 2017, 50(15):  2969-2982.  doi:10.3864/j.issn.0578-1752.2017.15.011

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【Objective】 Based on the multi-scale remote sensing indicators and measured soil conductivity data, the objective of this study is to exploit the appropriate scales and indicators for inferring soil salinity in irrigation area of arid oasis, and to provide alternative schemes for rapid assessment of soil salinization in the study area. 【Method】Weigan-kuqa oasis located in southern Xinjiang was selected as the study area. Soil electrical conductivity of multiple soil layers (0-10 cm, 10-20 cm, 20-40 cm, 40-60 cm), as inference object, were analyzed. Raster resampling (30-1 000 m) and neighborhood extent (3×3 to 31×31) were employed as the method of scale transformation to generate multiple scales of environmental factors (with help of principal component analysis, tasseled cap, vegetation index, soil wet index) based on Landsat OLI image data, then, a total of 1 078 environmental variables (the former produced 352 variables, the latter produced 726 variables). On this basis, linear and nonlinear curve models were used to fit the correlation between soil salinity and environmental variables under the above mentioned method of scale transformation, and then the optimal prediction scale and environmental factor were explored. 【Result】Nonlinear curve models showed a significance between soil salinity and environmental factors compared to linear model under multiple scale (raster resampling model). Secondly, the fitting accuracy decreased when the spatial resolution become coarse. In this model, the best inference scale was 30 m, and the sensitive variable was three-band maximal gradient difference (TGDVI) at depth of 40-60 cm, and the remaining depths were implied by Extended Normalized Difference Vegetation index (ENDVI). The optimal inference scale in model of neighborhood extent was 180 m (filtering scale 3×3), and the best fit variables for each layer was EEVI. Compared with the grid resampling mode, the fitting accuracy of neighborhood extent was improved by 14.60%, 34.40%, 32.10% and 21.70% (from top layer to bottom one) compared to fomer model, respectively. 【Conclusion】Based on the model of neighborhood extent, the nonlinear model constructed by Extented Enhanced Vegetation Index(EEVI) (180 m, 3×3) is more suitable for the prediction of soil salinity variation in the study area.

Evaluation of Maize Waterlogging Disaster Using UAV LiDAR Data

GAN PING, DONG YanSheng, SUN Lin, YANG GuiJun, LI ZhenHai, YANG Fan, WANG LiZhi, WANG JianWen

Scientia Agricultura Sinica. 2017, 50(15):  2983-2992.  doi:10.3864/j.issn.0578-1752.2017.15.012

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【Objective】Unmanned aerial vehicle (UAV) remote sensing technology is a hot research topic in the remote sensing sector, which is also one of the forces driving the development of modern agriculture. The objective of this study is to quickly and precisely measure the area of maize waterlogging disaster and evaluate disaster levels by analyzing maize canopy height derived from UAV LiDAR point cloud data. Thus it can provide a guideline for disaster prevention and mitigation, high and stable yield, agricultural insurance claims, etc. The aim of this study is to expand the application of UAV LiDAR data in agriculture, and provide a guarantee for agriculture field to quickly and effectively master agricultural information.【Method】The experiment was carried out and the UAV LiDAR data were obtained in Changping District, Beijing, where suffered a heavy rainstorm which led to a large-scale maize waterlogging on July 19-20, 2016. LiDAR point cloud data were classified and extracted, and canopy height of maize was obtained by LiDAR point cloud data from canopy height model (CHM). A double threshold partition strategy based on the normal statistics theory was adopted to determine the thresholds and a remote sensing monitoring model for maize waterlogging was built by analyzing the differences of canopy heights to evaluate the disaster levels of the maize waterlogging. Finally, the accurate assessment of the model was conducted by comparing the in-field measured data with predicted results from the built model.【Result】(1) After the occurrence of waterlogging of maize, there was a significant difference of maize growth between pre and post the disaster, and maize height showed the most obvious difference after the disaster. The maize canopy heights of final severe waterlogging, medium waterlogging, and the slight waterlogging were 0.30-0.84 m, 0.84-1.70 m, and above 1.70 m, respectively. (2) The confusion matrix analysis on the results estimated using the airborne LiDAR data was performed via ground survey samples; the overall classification accuracy of waterlogging degree reached 72.15%, and the Kappa coefficient was 0.44. (3) In general, remote sensing mapping was consistent with the monitoring data from the digital images.【Conclusion】The maize canopy height inversion can be achieved by UAV LiDAR data, and the waterlogging levels can be effectively reflected by the differences in maize plant heights. UAV LiDAR data can measure the area of maize waterlogging and evaluate disaster levels at regional scale, providing a convenient and efficient way to acquire the disaster information.

HORTICULTURE

Response of Photosynthetic Characteristics and Water Use Efficiency of Cucumber to Soil Water Content

LI ShengPing, WU XuePing, GAO LiLi, LONG HuaiYu, LI Jing, WANG BiSheng, DANG JianYou, PEI XueXia

Scientia Agricultura Sinica. 2017, 50(15):  2993-3005.  doi:10.3864/j.issn.0578-1752.2017.15.013

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【Objective】 Water deficit is the principal factor affecting cucumber production. However, the mechanisms for the decline of photosynthesis in leaves of cucumber and the synergistic action between carbon assimilation and transpiration water consumption under different water stresses were not very clear. The objective of this study was to investigate the processes and mechanisms of drought stress on photosynthesis, to study the impact of soil water content on carbon assimilation and transpiration water consumption, and to analyze the optimum range making cucumber grow better, which would supply the reference basis about cucumber water-saving physiology. 【Method】The pot experiments were conducted in the rain-proofing solarium and two factors were designed: irrigation and nitrogen fertilization. There were five irrigations levels (W1: 0, W2: -5 kPa, W3: -10 kPa, W4: -15 kPa). 【Result】During cucumbers growth period, soil water content could keep stability essentially under the same negative hydraulic head supplied by the negatively pressurized irrigation system. The relative soil water content (RSWC) controlled by W1, W2, W3 and W4 were (103.8±1.2)%, (88.7±3.7)%, (77.4±4.5)% and (61.8±3.2)%, respectively. When RSWC was between 61.8% and 88.7%, both the daily mean value of stomatal conductance (Gs) and transpiration rate (Tr) significantly decreased with reducing soil water content, which could maintain leaf water use efficiency (LWUE) at a relatively high level to accommodate water stress. The daily mean values of net photosynthetic rate and LWUE at 3 growth stages were parabolic correlated to the value of relative soil water content. Nevertheless, the values of RSWC were different when the values of P_n and LWUE were maximum at different growth stages. When RSWC was 88.7%, plant dry weight and yield were the maximum, while water use efficiency (WUE) was the highest as RSWC was 77.4%. 【Conclusion】 Appropriate ranges of relative soil water content were between 70% and 93% at flowering stage, between 78% and 103% at full fruit stage and between 73% and 104% at last fruit stage. In these ranges, cucumber leaf could maintain a preferable synergistic relationship between carbon assimilation and transpiration water consumption, which resulted in the improvement of amount of dry matter and yield.

Development of DNA Molecular Markers for the Dwarf Trait in Pear Through the Method of 2b-RAD Sequencing and HRM Analysis

XIAO YuXiong, WANG CaiHong, TIAN YiKe, YANG ShaoLan, LI DingLi, ZHANG HaiYue

Scientia Agricultura Sinica. 2017, 50(15):  3006-3012.  doi:10.3864/j.issn.0578-1752.2017.15.014

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【Objective】As an important agronomic trait, the dwarf character of tree is significant to fruit intensive culture. The dwarf traits of pear originated from variety ‘Le Nain Vert’, a chance seedling of Pyrus communis, is determined by a dominant gene PcDw, which sequence information is still unknown. Development of DNA molecular markers tightly linked to the PcDw locus could provide important information for identifying of this gene.【Method】Using 2 different F₁ populations obtained from ‘Aishengli’בChili’ and ‘2-3’בLvbaoshi’, respectively, as plant materials, 2 pairs of bulks for dwarf type/standard type were sequenced through IIB restriction association site DNA (2b-RAD) technology, and then single nucleotide polymorphism (SNP) markers were detected between the dwarf and standard bulks based on the principle of bulked segregant analysis. After that, those SNPs located on the PcDw mapped chromosome were selected and tested on the whole population to confirm their linkage relationship to the PcDw gene by high-resolution melting (HRM) analysis.【Result】A total of 67 186 260 reads were produced from the 2b-RAD sequencing of the 4 samples (2 pairs of bulks). That means the average reads per sample was 16 796 565. Statistical analysis of the filtered raw reads showed that the average unique tags per sample were 86 810, and the average sequencing depth was 77× which was enough for accurate genotyping. For the 4 libraries, mapping through SOAP software indicated that the high quality reads containing the restriction site accounted for more than 70% of the raw reads, which means the high quality of sequencing. Totally 1 317 polymorphic SNP markers were screened between the dwarf bulks and standard bulks derived from the 2 different populations, and 8 of them were mapped on the PcDw-localized chromosome scaffold00074. Further detection of the 8 SNPs through HRM analysis showed that 2 and 4 SNP markers co-segregated with the PcDw locus were identified from population ‘Aishengli’בChili’ and population ‘2-3’בLvbaoshi’, respectively. According to the different shapes of the melting curves of amplicons, the dwarf/normal phenotype could be distinguished effectively. The recombinants for each marker and the target trait were not found in both of the two populations, which contained 215 and 168 progenies, respectively. 【Conclusion】The combination of 2b-RAD sequencing and HRM analysis technology is an efficient method for exploiting molecular markers of important agronomic traits in fruit trees. In this study, a total of 4 SNP markers co-segregated with the PcDw locus were identified based on this strategy.

FOOD SCIENCE AND ENGINEERING

Effects of Hydroxyl Radicals Oxidation on Structure and Hydration Properties of Bovine Serum Albumin

WANG Ce, LI Xia, DENG ShaoYing, WANG Hang, ZHANG ChunHui

Scientia Agricultura Sinica. 2017, 50(15):  3013-3023.  doi:10.3864/j.issn.0578-1752.2017.15.015

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【Objective】The effects of oxidation on protein structure and its hydration properties were investigated. The purpose of this study was to explore the changes in protein and water interactions mediated by oxidation.【Method】Ferric ion-ascorbic acid-hydrogen peroxide (FeCl₃-Vc-H₂O₂) hydroxyl radical oxidation system was used in this study. Bovine Serum Albumin (BSA) was suspended in 15 mmol·L^-1 piperazine-1,4-bisethanesulfonic acid (PIPES) buffer (pH 6.0) and incubated at 4℃ for 12 h with ferric ion (Fe³⁺) and ascorbic acid (Vc) at different concentrations of hydrogen peroxide (0, 0.5, 1.0, 5.0, 10.0, and 20.0 mmol·L^-1 H₂O₂). Amino acid content was measured by automatic amino acid analyzer. Carbonyl content and sulfhydryl content were detected to evaluate the degree of proteins oxidation. The changes of hydratability of BSA was evaluated by measuring the surface hydrophobicity, the content of ionic bond and the content of hydrogen bond. Electrochemical change rule under different oxidation levels was studied by measuring Zeta potential. In addition, the protein secondary structure was analyzed by Fourier Transform Infrared (FT-IR) spectroscopy. 【Result】 The content of amino acids in BSA declined with the increasing H₂O₂ concentration. However, the selective effects of ·OH on amino acids were observed. Compared to the control group (0 H₂O₂ treatment), threonine (Thr) and lysine (Lys) decreased significantly by 4.30% and 4.22%, respectively, at 1 mmol·L^-1 of the H₂O₂ concentration. When the concentration of H₂O₂ increased to 20 mmol·L^-1, the contents of methionine (Met), tyrosine (Tyr) and histidine (His) showed the highest reduction, which was reduced by 35.32%, 19.19% and 11.15%, respectively. Then the content of leucine (Ile), glycine (Gly) and cysteine (Cys) also showed their higher reduction. It was observed that the carbonyl content increased significantly (P＜0.05) with increasing H₂O₂ concentration, while sulfhydryl content significantly declined (P＜0.05), i.e. Carbonyl content in 20 mmol·L^-1 H₂O₂ treatment group (0.723 nmol·mg ^-1 protein) increased by 57.52% when it was compared to control (0.459 nmol·mg ^-1 protein), and sulfhydryl content in 20 mmol·L^-1 H₂O₂ treatment group (19.853 nmol·mg ^-1 protein) decreased by 74.04% when it was compared to control (76.471 nmol·mg ^-1 protein). Both indicated that BSA protein oxidation became more severe with the extension of H₂O₂ concentration. The result of secondary structure analysis indicated thatα-helix, random coil decreased significantly with the increase of ·OH (P＜0.05), while β-sheet, β-turn increased significantly (P＜0.05), which demonstrates BSA oxidation induced the α-helix structure become a linear structure. The exposure of hydrophobic residues led to the significant increase of protein surface hydrophobicity (P＜0.05). The absolute value of Zeta potential was significantly decreased (P＜0.05) with the increase of H₂O₂ concentration, which demonstrated that the protein oxidation could significantly affect the surface charge of protein. Chemical interactions results showed that hydrogen bond and ionic bond were weakened significantly (P＜0.05), with reduction of interaction between protein and water molecules and the protein hydration capacity. 【Conclusion】BSA oxidation is H₂O₂ concentration-dependent in hydroxyl radical oxidation system. Protein surface electrostatic charge decreased and protein secondary structure changed significantly as affected by protein oxidation, the interaction between protein and water molecules were reduced.

ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT

Change Regularity and Correlation Analysis of Hainan Special wild Boar Taint Substances

CAI KeQi, YANG XuanKe, WANG Peng, WU KeBang

Scientia Agricultura Sinica. 2017, 50(15):  3024-3032.  doi:10.3864/j.issn.0578-1752.2017.15.016

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【Objective】 The aim of this study is to reveal the change regularity of boar taint substances by comparing differences in androstenone, skatole and indole in F₁, F₂, F₃ different generations and boar taint substances effects on sex hormone levels to provide a theoretical basis for breeding of low-lying boar taint pigs. 【Method】 Eighteen individuals each of the different generations (boar♂×Tunchang pig♀) F₁, F₂, and F₃ were used as the research objects. Then blood was collected and sera were extracted from the precaval vein of the experimental pigs, and then the fat and muscle samples were collected from the neck after slaughtering with standard method. ELISA kit and high performance liquid chromatography were used to detect and analyze the sex hormones, skatole and indole levels of each sample. The correlation and significance analysis of data were conducted by using SAS 9.0 software. 【Result】 The contents of skatole, androstenone and indole showed different correlation levels in different tissues. Skatole and androstenone contents of fat and muscle showed a significant positive relationship (P＜0.05 or P＜0.01) with serum, and the maximum correlation coefficient of skatole in fat with that in serum was 0.94. Skatole, androstenone and indole contents of pork showed significant differences among different hybrid generations. With the increase of wild boar blood, androstenone levels were significantly increased, while the skatole levels were significantly reduced, but the indole showed no regularity in change. The sex hormone levels also had significant differences among different hybrid generations, the testosterone levels were significantly increased with wild boar blood, estradiol levels of F₁ were significantly lower than that of F₂ and F₃. Skatole, androstenone and indole contents of fat, muscle and serum in different hybrid generations had different correlations with sex hormone levels, and androstenone of them had significant positive (P＜0.01) correlation with testosterone. Skatole had a significant negative (P＜0.05 or P＜0.01) correlation with testosterone. The maximum correlation coefficient between androstenone and testosterone in serum was 0.95. 【Conclusion】 With the increase of the content of the Hainan special wild boar blood, the testosterone levels increased significantly, the androstenone sedimentary volume increased significantly, and the skatole deposition quantity significantly reduced. So the changes of testosterone levels of Hainan special wild boar affect the contents of skatole and androstenone.

Validation of Reference Genes for Quantitative RT-PCR Analysis in Porcine Testis Tissues

PENG FuZhi, RAN MaoLiang, WENG Bo, LI Zhi, DONG LianHua, CHEN Bin

Scientia Agricultura Sinica. 2017, 50(15):  3033-3041.  doi:10.3864/j.issn.0578-1752.2017.15.017

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【Objective】Reference gene is the key to obtain the accurate analysis results when apply quantitative RT-PCR (qRT-PCR) technique to identify the gene expression level. However, reference gene and microRNA (miRNA) which are used in researching the molecular biology of porcine testis are not well characterized. Screening suitable reference gene will provide a reliable evidence for the quantitative analysis of target gene in different periods of porcine testis tissues.【Method】Porcine testis tissues at 8 different developmental stages (E 90, D 1, D 30, D 60, D 90, D 120, D 150, and DM) were selected as materials in this study. Trizol method was used to extract the total RNA and the concentration and purity of total RNA were detected by ND-2000 NanDrop spectrophotometer. The primers of 5 commonly used protein coding reference genes (GAPDH, TBP, β-actin, SDHA and B2M) and 5 miRNA reference genes (U6, ssc-miR-17-5p, ssc-miR-26a, ssc-miR-27a and ssc-miR-103) were designed and synthesized to reverse transcription. cDNA templates were diluted to 7 concentration gradients with 1:10 serial dilution to construct standard curves. The expression of 10 candidate reference genes in porcine testis tissues was systematically analyzed at specified times, then the Genorm 3.5 was used to analyze the results. The most stable reference genes were picked up according to the stability value which called M value, the smaller the value of M, the better the stability of the reference gene; otherwise, the stability would be worse.【Result】The melting curves of 10 candidate reference genes (GAPDH, TBP,β-actin, SDHA, B2M, U6, ssc-miR-17-5p, ssc-miR-26a, ssc-miR-27a, and ssc-miR-103) indicated that the expression of all the candidate reference genes was of some specificity, no primer-dimers and nonspecific amplification. The relationship between Ct value and the logarithm of relative copy number was coincidence with linear relation according to the standard curves which were made with Ct value as ordinate and the logarithm of relative copy number as abscissas in a series of diluted concentration gradients. Analysis showed that the most stable reference gene of protein encoding gene was TBP and the most unstable was GAPDH. U6 was the most suitable gene for miRNA expression analysis and ssc-miR-26a was the most unsuitable. 【Conclusion】 This study has screened the most suitable reference genes (TBP and U6) to identify porcine testis gene expression level successfully. The highly ranked reference genes identified from this study can provide a theoretical basis for the selection of the most suitable reference gene to detect differences in expression rates of genes and miRNAs in porcine testis tissues.

Plasma Metabolic Profiling of Postpartum Dairy Cows with Inactive Ovaries Based on GC/MS Technique

FAN ZiLing, XU ChuChu, SHU Shi, XIAO XinHuan, WANG Gang, BAI YunLong, ZHANG Jiang, ZHAO Chang, XIA Cheng

Scientia Agricultura Sinica. 2017, 50(15):  3042-3051.  doi:10.3864/j.issn.0578-1752.2017.15.018

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【Objective】This trial was designed to screen plasma differential metabolites between postpartum dairy cows with inactive ovaries and estrous cows and to clarify the changes of metabolites in dairy cows suffering from inactive ovaries by gas chromatography/mass spectrometry technique (GC/MS).【Method】Dairy cows which had similar age, parity and body condition score were selected at 60-90 d postpartum from an intensive dairy farm in Heilongjiang Province. According to clinical manifestations, B-ultrasound scan, rectal palpation and hormone tests, 22 cows were divided into estrous group (A) and 20 to the inactive ovaries group (B). All plasma samples were detected by GC/MS to obtain plasma metabolic profiles between the groups, and ChromaTOF software was used to analyze peak figures and obtain raw data. The standardized GC/MS data matrix was imported into SIMCA-p＋14 to conduct multivariate statistical analysis including principal component analysis (PCA) to observe the overall distribution between the samples and the stability of the whole analysis process, and (orthogonal) signal correction-partial least squares-discriminant analysis (O)PLS-DA to distinguish the overall differences in metabolic profiles between groups and find differential metabolites between groups. In order to prevent the model from over-fitting, the quality of the model was examined by seven cycles of reciprocal verification and 200 response sequencing tests. Differential metabolites between groups were screened by (O)PLS-DA and t-test. In the PLS-DA analysis, the variable weight value VIP＞1 was considered as the difference variable; in the t-test, the variable with P＜0.05 was considered as the difference variable. The metabolites with VIP＞1 and P＜0.05 were screened as differential metabolites. Finally, plasma differential metabolites between two groups were analyzed by KEGG pathway database including channel enrichment and interaction network construction.【Result】Compared to estrous cows, there were 20 differential metabolites in cows with ovarian inactivity, of which 17 differential metabolites were closely related to cows with ovarian inactivity, including level increased cholic acid and decreased vanillylmandelic acid, nicotinoylglycine, 6-hydroxynicotinic acid, beta-alanine, L-Tyrosine, phenylpyruvate and so on. These metabolites were involved in biosynthesis of phenylalanine, tyrosine and tryptophan, and metabolism of ethyl propionate, nicotinic acid, nicotine, phenylalanine and tyrosine. The normal growth of follicle in cows was interfered with these differential metabolites by a single or comprehensive pathway. The other three compounds, Iminodiacetic acid, N-Methyl-L-glutamic acid and 3-Aminoisobutyric acid, may be related to amino acid metabolism and cell energy transport, and their biological role in dairy cows’ inactive ovaries remains to be further confirmed.【Conclusion】The plasma differential metabolites between cows with inactive ovaries and estrous cows were identified effectively by GC/MS technology. These differential metabolites suggested that the occurrence of ovarian inactivity may be related with metabolic disorders of various substances. Results of this study will lay a foundation for further study on the pathogenesis, prevention and treatment of postpartum inactive ovaries in dairy cows.

Functional Mode and Immunocytochemical Localization of Chemosensory Protein 1 (CSP1) in Apis cerana cerana

TAN Jing, SONG XinMi, FU XiaoBin, TANG MingZhu, WU Fan, HUA QiYun, LI HongLiang

Scientia Agricultura Sinica. 2017, 50(15):  3052-3062.  doi:10.3864/j.issn.0578-1752.2017.15.019

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【Objective】The objective of this study is to research the functional mode with candidate semiochemicals and immunocytochemical localization of the recombinant chemosensory protein1 (CSP1) in Chinese honey bee, Apis cerana cerana. It has an important theoretical significance for clarifying the function of CSP1, which is specifically expressed in antennae, and enriching the chemosensory mechanism of Chinese honey bee to semiochemicals. 【Method】 CSP1 was constructed into the pET-32a (+) vector and transferred into BL21 (DE3) competent E. coli. The single positive clone was inoculated on LB medium and cultured overnight (followed by 1% (v/v) transfer to the OD₆₀₀≈0.4), then added the IPTG with a final concentration of 1 mmol·L^-1 and continued to be induced for 5 h. The recombinant CSP1 protein existing in the supernatant was purified by Ni²⁺-NTA affinity chromatography. The soluble recombinant CSP1 protein with biological activity was finally obtained after dialyzed by PBS (pH 7.4). When the excitation wavelength of the fluorescence spectrophotometer was 281 nm, the interaction between the fluorescent probe 1-NPN and CSP1 was determined. The dissociation constant was calculated by Scatchard equation, and the binding affinities of CSP1 with various candidate semiochemicals were also measured. Based on the CSPMbraA6 crystal structure (PDB entry code: 1n8v) as template, the binding model of CSP1 protein and semiochemicals was analyzed by homology modeling and molecular docking. In order to obtain the binding mode of pheromone and CSP1, according to MolDock Score, the best mechanism of the docking model was analyzed, and the hydrogen bonds between the ligand and the CSP1 residues were obtained. Finally, the polyclonal antibody of CSP1 was obtained by immunizing rabbits, and the antennae of worker bee were immobilized at low temperature, dehydrated and embedded. The subcellular distribution of CSP1 on the antennal sections samples were then immunogolded by colloidal gold and finally observed with the electron microscopy. 【Result】 After the soluble recombinant CSP1 protein was successfully induced and purified, the fluorescence quenching assay was used to obtain the dissociation constant K_1-NPN between CSP1 and 1-NPN as 2.1 μmol·L^-1. The number of binding sites n was 0.99, indicating the bind ratio of CSP1 and 1-NPN was 1﹕1. In the candidate 9 semiochemicals, CSP1 and two queen pheromone components: p-hydroxybenzoic acid methyl ester (HOB) and (E)-9-Oxodec-2-enoic acid (9-ODA), and plant volatile components, 3-carene had a strong ability to bind, the [IC₅₀] and dissociation constant K_D of the most strongly bound HOB was 10.1 and 7.68 μmol·L^-1, respectively. The results of molecular docking showed that the binding of various ligands to CSP1 was due to some specific amino acid residues (or by means of the hydrogen bonds) in the hydrophobic cavity of CSP1. Typically, the interaction between CSP1 and HOB is predicted by the contribution of eight amino acid residues, including four hydrophobic residues (Phe30, Phe44, Leu70 and Phe85), three polar neutral residues (Tyr26, Tyr27 and Ser41) and one acidic residue (Asp40). The two hydrogen bonds were produced between the oxygen atoms on the two carboxyl groups in Asp40 and the oxygen atoms on the hydroxyl group in the HOB benzene ring, respectively. The results of immuno-electron microscopy showed that CSP1 was mainly expressed in the ancillary supporting cells around the sensilla placodea, while only slightly expressed in the inner area of sensilla placodea. It was significantly different from the localization of odorant binding proteins.【Conclusion】 CSP1 of A. c. cerana has a strong ability to bind two queen pheromone components and some plant floral scents, and concentrates the function of pheromone binding protein and general odorant-binding protein. Although their functional modes are similar, there are significant differences in the antennal subcellular localization between CSP1 and odorant-binding proteins, showing the physiological characteristics variation of chemoreceptive proteins.