Scientia Agricultura Sinica

Western Blot Detection of CAS9 Protein in Transgenic Rice

GUO YaLu, MA XiaoFei, SHI JiaNan, ZHANG Liu, ZHANG JianShuo, HUANG Teng, WU PengCheng, KANG HaoXiang, GENG GuangHui, CHEN Hao, WEI Jian, DOU ShiJuan, LI LiYun, YIN ChangCheng, LIU GuoZhen

Scientia Agricultura Sinica. 2017, 50(19): 3631-3639. doi:10.3864/j.issn.0578-1752.2017.19.001

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【Objective】The objective of this study is to generate monoclonal antibodies against CAS9 protein and establish immunological method for the detection of CAS9 protein in transgenic plants, and to understand the characters of the expression patterns of CAS9 protein in transgenic rice. 【Method】The 5′ fragment (810 bp) of Cas9 was amplified using plasmid DNA confers Cas9 as template. The amplican was cloned into expression vector pET30a. Restriction enzyme digestion identified recombinant plasmid were sequencing verified. The recombinant plasmid was transformed into E. coli Condon Plus strain. The induced CAS9 protein was purified and used as immunogene to generate monoclonal antibodies. Positive hybridoma cell lines were identified by western blot analysis. PCR amplifications were carried out to identify positive transgenic rice using specific primers of Cas9. Western blot was carried out to detect CAS9 protein in rice. The recombinant CAS9 protein and protein samples extracted from rice seedling were analyzed in parallel by western blot, and standard curves were drawn based on Image J software extracted signals. CAS9 protein in rice tissues were analyzed quantitatively based on the standard curve. Total protein of single rice grain was extracted and the sensitivity of CAS9 protein detectable by western blot was analyzed using diluted samples. The abundance of CAS9 protein in different tissues, including shoot and root at seedling stage, stem, node, sheath and leaf at tillering stage, were compared by western blot. 【Result】The Cas9 was cloned and the plasmid was transformed into E. coli. Recombinant N-terminal portion of CAS9 protein was obtained and used as immunogen to inject mice. Forty-two positive hybridoma cell lines were obtained after immunization. Among them, cell line #12D2 showed higher specificity and sensitivity for the detection of CAS9 protein in rice tissues. Western blot analysis was carried out for the detection of transgenic rice via the antibody of #12D2-derived hybridoma cell lines. The lowest amount of recombinant CAS9 protein detectable by the established western blot protocol was about 0.25 ng. It was also revealed that the CAS9 protein accounted for about 0.00005% of fresh weight in rice seedling, and CAS9 protein in 8% of single grain rice (about 2 mg) was detectable. It was also found that the abundance of CAS9 protein in rice shoot tissues at seedling stage was higher than that in root tissues, the abundance in stem and leaves at tillering stage was higher than that in root and sheath tissues. 【Conclusion】In this study, anti-CAS9 monoclonal antibodies with satisfied specificity and sensitivity were obtained and western blot protocol for the detection of CAS9 protein in transgenic plants was established. The expression patterns of CAS9 protein in different rice tissues were revealed. Moreover, the data also demonstrated the potential of application in other plants.

QTL Mapping of Yield Associated Traits of Nipponbare ×Zhongjiazao 17 RIL Population

ZHANG YingZhou, LUO RongJian, SHENG ZhongHua, JIAO GuiAi, TANG ShaoQing, HU PeiSong, WEI XiangJin

Scientia Agricultura Sinica. 2017, 50(19): 3640-3651. doi:10.3864/j.issn.0578-1752.2017.19.002

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【Objective】QTL mapping for yield traits were conducted with phenotype data collected from multi-environments, in order to identify stable QTL for yield traits and analyze the mechanism of high yield of super early indica rice Zhongjiazao 17, which will provide useful information for breeding of new varieties with higher yield and better comprehensive characteristics in rice. 【Method】A recombinant inbred lines (RILs) population derived from Nipponbare × Zhongjiazao 17 were used as experimental materials. Then a genetic linkage map was constructed by Mapmaker/EXP 3.0 based on the RILs genotypes which analyzed by polymorphism SSR markers. During 2015-2016, the RILs and two parents were grown in the experimental fields in Hangzhou, Hainan and Hangzhou. The agronomic characters including effective panicles, grain number per panicle, seed setting rate, 1 000-grain-weight, grain length, grain width and grain thickness and yield per plant were investigated. QTL mapping for these traits were detected by Windows QTL Cartographer 2.5 and environment interaction effect was detected by QTLNetwork2.2.【Result】The genetic map was constructed with 163 filtered SSR markers and covered about 1 479.4 cM with an average interval of 9.08 cM. The genotypes of male and female parent of 73.0% makers were segregated as 1﹕1; only 23.0% markers were distorted segregation which was inclined to Zhongjiazao 17. A total of 46 QTLs were mapped on all chromosomes except the 11th with the contribution rate ranged from 3.78% to 25.45%. Ten QTLs, including qEP1, qEP2, qEP4a (QTL for effective panicles), qNGPE1, qNGPE7 (for grain number per panicle), qSRT7 (for seed setting rate), qTGW2 (for 1 000-grain-weight), qGL3, qGL9 (for grain length), qGW2b (for grain width) can be detected on all three environments. The increasing effective allele of qEP4a, qNGPE1, qSRT7, qGL3, qGL9 and most QTL for grain number per panicle, seed setting rate, grain length and yield per plant come from Zhongjiazao 17. Furthermore, some QTLs about different traits exist as clusters on chromosome 1, 2, 7, respectively. Six yield associated traits QTL were also found can significantly interact with environment. 【Conclusion】 The linkage map of RIL derived from Nipponbare × Zhongjiazao 17 has abundant polymorphic makers which cover 93.64% of the rice whole genome, so it is very suitable for QTL mapping for important agronomic traits. Many stable QTLs about rice yield traits were detected using this mapping population, and among them, the alleles which can increase yield of many QTL of grain number per panicle, seed setting rate, grain length and yield per plant were from Zhongjiazao 17. The results were consistent with that the most of these yield traits of Zhongjiazao 17 were better than Nipponbare. These increasing effective alleles of yield associated traits QTL may be the genetic basis of stable high yield of Zhongjiazao 17.

Assessment of the Unintended Effects of Four Genetically Modified Maize Varieties by Proteomic Approach

HAO WenYuan, LI FeiWu, YAN Wei, LI CongCong, HAO DongYun, GUO ChangHong

Scientia Agricultura Sinica. 2017, 50(19): 3652-3664. doi:10.3864/j.issn.0578-1752.2017.19.003

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【Objective】 Unintended effects of genetically modified (GM) crops are defined as the unexpected trait changes in GM breeding and research. Over the last decade, much attention has been taken to GM crops’ intended effects that are attributed to the insertion of foreign gene, leaving the unintended effects of GM crops less investigated. In this study, a comparative proteomic study using two-dimensional electrophoresis and mass spectrometry was conducted to assess the high-abundant protein variation, so as to explore possible occurrence of their unintended effects. 【Method】 Four insect resistant GM maize varieties, i.e SK12-5zd, IE034z, Bt799z and Bt799zd, which were under approval of GM safety assessment by the Ministry of Agriculture, were planted in a condition-controlled greenhouse together with their corresponding CKs, i.e Zheng58 and Zhengdan958. When seedling growing into 5-leaf stage, the top-leaf blades of each maize variety were sampled and their leaf proteins were extracted. After profiling by two-dimensional electrophoresis, on-gel leaf protein spots of each GM maize were compared against their corresponding CKs using Imager Scanner and PD Quest 8.0 (Biored, USA). The resulted unique and differential protein (with a relative volume greater than 2-fold) spots were isolated from the gel and applied for protein identification by mass spectrometry against the relevant protein database. The identified proteins were analyzed in gene ontology (GO) and KEGG for their cellular functionalities and metabolism pathway enrichment. 【Result】 Through comparison of the unique and differential proteins between each GM maize and its corresponding no-GM CKs among 4 insect resistant GM maize varieties, a total of 61 differential proteins were identified in maize PDB (NCBI) alongside the 4 intended insect resistant gene products. The gene ontology analyses of the identified proteins revealed that they are popular cellular enzymes of fundamental metabolism, i.e Rubisco, ATP synthase, pyruvate orthophosphate dikinase, etc. Only a few genes relating to photosynthesis, ATP synthesis and carbon fixation were observed to be up-expressed, possibly due to the genetic background difference between the GM maize and its CK. KEGG analyses uncovered that, in comparison with the corresponding CKs, the differential proteins of SK12-5zd and Bt799zd were enriched similarly into photosynthesis, carbon fixation and metabolism pathways. In comparison with the CK Z58, the differential proteins of both IE034z and Bt799z were enriched into the photosynthetic metabolism pathway. Four GM maize varieties exhibited extensive similarity over their corresponding wild-type controls. It appeared to be no unintended effects observed. 【Conclusion】 The preliminary results revealed no obvious unintended effects in 4 insect resistant GM maize varieties in respect to high-abundant proteomic assessment.

Differential Proteomics Analysis of Cotton Leaf Response to Chemical Topping Based on iTRAQ Technique

HOU XiaoMeng, LIU LianTao, LI Meng, SUN HongChun, ZHANG YongJiang, DU Huan, LI CunDong

Scientia Agricultura Sinica. 2017, 50(19): 3665-3677. doi:10.3864/j.issn.0578-1752.2017.19.004

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【Objective】The objective of this study is to compare the effects of manual topping and chemical topping on physiological changes and proteins differentially expression of cotton. The results of this study would provide a theoretical basis for the mechanism of chemical topping. 【Method】Manual topping, chemical topping and no top pruning were practiced on cultivar GM863 which was planted in large area of the Yellow River basin in 2015-2016. Cotton was treated on July 20th simultaneously. Artificial spraying chemical topping agent and the dosage was 1.125 L·hm^-2. The plant height and hormone contents of functional leaves were determined regularly after topping. The plant height was measured with the height from the cotyledon node to the growth point of the main stem by using ruler. The hormone contents of functional leaves, including IAA, GA₃, ABA and ZR were measured by enzyme-linked immunosorbent assay. The differential proteomics analysis was carried out on the functional leaves of manual topping and chemical topping by using iTRAQ technique. 【Result】 Compared with the cotton of manual topping treatment, chemical topping treatment was significantly higher than manual topping treatment, which was 11.8% and 14.5% higher than others in the two-year trial, but was significantly lower than no top pruning, which was 6.0% and 6.5% lower than others, vertical growth was significantly inhibited after spraying. Different topping treatments greatly affected the GA₃ content of cotton functional leaves, the change of GA₃ content was a single peak curve, the contents of GA₃ were no top pruning＞chemical topping＞manual topping, and reached significant difference. The GA₃ contents of chemical topping and no top pruning were decreased at 30 d after topping and manual topping was decreased at 20 d. There was no significant difference among three treatments at 50 d after topping. The maximum IAA content in chemical topping treatment was significantly lower than that in the other two treatments at 40 d after topping in 2016, and there was no significant difference among the three treatments in 2015. The maximum ABA content in no top pruning treatment was significantly lower than that in the other two treatments at 40 d after topping. There was no significant difference among the three treatments of the contents of ZR. Compared with manual topping treatment, 69 differentially expressed proteins were detected by using iTRAQ in leaves of chemical topping treatment, 29 up-regulated and 40 down-regulated expressions, in which carbohydrate and energy metabolism related proteins were most down-regulated, and it was indicated that the growth of plants was decreased. The up-regulation of most GA regulated positive correlation proteins enhanced the GA effect. 【Conclusion】Chemical topping can effectively control the plant height of cotton, and which has a great effect on the GA content of functional leaves. The contents of GA in chemical topping treated leaves were significantly higher than that of manual topping treatment. Compared with manual topping treatment, the proteins related to plant growth and development in chemical topping treatment were most down-regulated expression, may be the plants reduced carbohydrate synthesis and energy metabolism, and activated GA effect by increasing GA content to control plant height.

Effects of Sub-Soiling in Fallow Period and Phosphorus Fertilizer on Yield and Water Use Efficiency in Dry-Land Wheat

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

Scientia Agricultura Sinica. 2017, 50(19): 3678-3689. doi:10.3864/j.issn.0578-1752.2017.19.005

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【Objective】 To cope with the drought, lower water storage and unreasonable fertilization of dry-land wheat in the Loess Plateau, the best technology way of water conservation of sub-soiling at fallow period and phosphorus fertilization at sowing stage of dry-land wheat was explored. 【Method】 Field experiments were carried out from 2012 to 2016 in Qiujialing village, Wenxi, Shanxi, the main plots were dealt with two tillage methods of sub-soiling and no tillage during fallow period, the subplots were dealt with six pure phosphorus fertilization amount of 0, 75,150, 225, 300, 375 kg·hm^-2,in order to make clear the effect of sub-soiling during fallow period and phosphorus fertilization on yield, water use efficiency of dry-land wheat in different years. 【Result】 The results showed that soil storage efficiency was improved by 20%-86% during fallow period. Under sub-soiling during fallow period, soil water storage of 3 m soil layer was improved by 24-90 mm significantly, spike number was improved by 1%-18%, yield was improved by 3%-25%, water use efficiency was improved by 4%-20% in 2012-2013. The results also showed that phosphorus levels affected dry-land wheat soil water storage of 0-300 cm soil layer, which was the lowest at 150 kg P·hm^-2under the range of 0-225 kg·hm^-2phosphorus amount, and was decreased under the long-term phosphorus application (4 year positioning test), and the difference was significant the fourth year. The difference of soil water because of different water consumption of phosphorus was restored through precipitation during fallow period, in the third and fourth year, soil moisture is not balanced, annual soil water consumption was different between phosphorus of 150 kg·hm^-2and0. In the end, with the phosphorus fertilizer amount (0-225 kg·hm^-2) increasing, average yield and WUE of four years were largest under phosphorus of 150 kg·hm^-2, and the differences of yield were significant among different treatments, differences of WUE were significant between 150 kg·hm^-2and0. In addition, the F value test showed that years had the greatest influence on yield and water use efficiency, the yield increase effect of sub-soiling was higher than that of phosphorus fertilizer. The sowing water storage of 0-300 cm layer with more than 550 mm combined with the phosphorus amount of 75 kg·hm^-2, or below 550 mm combined with the phosphorus amount of 150 kg·hm^-2, made the spike number, yield and WUE higher. 【Conclusion】 Sub-soiling during fallow period is conducive to the accumulation of the precipitation and the improvement of the sowing water storage. Phosphorus fertilizer increased soil water consumption of dry-land wheat, decreased soil water storage of 0-300 cm during growth stages, increased annual water consumption. Yield was increased by 2-31 kg·hm^-2 with increase of 1 mm water storage of sub-soiling during fallow period, yield increased by 2-13 kg·hm^-2 with increase of 1 kg·hm^-2phosphorus amount. The sowing water storage below 550 mm of 0-300 cm combined with the phosphorus amount of 150 kg·hm^-2, or the sowing water storage more than 550 mm combined with the phosphorus amount of 75 kg·hm^-2 could achieve higher yield, water and fertilizer efficiency.

Effect of Nitrogen and Density Interaction on Morphological Traits, Photosynthetic Property and Yield of Maize Hybrid of Different Plant Types

XIAO WanXin, LIU Jing, SHI Lei, ZHAO HaiYan, WANG YanBo

Scientia Agricultura Sinica. 2017, 50(19): 3690-3701. doi:10.3864/j.issn.0578-1752.2017.19.006

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【Objective】The purpose of this study is to elucidate morphological and physiological mutual beneficial mechanism for compact type maize hybrid under nitrogen and density interaction, for further raise overall productivity of density tolerant maize hybrid.【Method】Plant morphological trait, ear leaf photosynthetic ability and yield were determined under field experimental condition in 2014 and 2015. Split-split plot design, 2 plant type hybrids (compact plant type and flat plant type) as the main plot, 3 nitrogen treatments (N1: 0, N2: 90 kg N·hm^-2 and N3: 180 kg N·hm^-2) as the split plot, 3 plant densities (D1: 45 000 plant/hm², D2: 60 000 plant/hm² and D3: 75 000 plant/hm²) as the sub-split plot. 【Result】The effects of nitrogen on internode length, leaf angle, SPAD value, kernel weight per ear and yield were stronger than that of density on those parameters. Stem diameter, Pn and kernel number per ear was sensitive to density increasing. Compared with flat type hybrid, decreased range of stem diameter was small, and response sensitivity from 1 to 3 internode length was slowness with plant density increased for compact type hybrid. However, the 1-3 internode length was shortened significantly with nitrogen input amount increased (P_N2→N3=0.004-0.028), negative response range of 4-5 internode length for compact type hybrid (10.9%) was higher than positive response range of 4-5 internode length for flat type hybrid (3.3%). Leaf angle of compact type hybrid was down to 2.9°±1.1° with nitrogen input. The leaf angle of leaf below ear leaf changed to a relatively lower with plant density increased. Response peak value of SPAD to nitrogen for compact type hybrid (N₃) was higher than that for flat type hybrid (N₂). The negative effect of Pn caused by density increasing was relatively small for compact type hybrid. SPAD and Pn of ear leaf for compact type hybrid were higher than that for flat type hybrid in N₃ and D₃ treatment. Altogether, the effect of nitrogen and density interaction on kernel number and kernel weight per ear for compact type hybrid was smaller than that for flat type hybrid. Harvest index of compact type hybrid was relatively high, which the difference between N×D interaction treatment (P_N1→N3 =0.16，P_D1→D3 =0.12) was no significant, however, that the difference between that (P_N1→N3 =0.03，P_D1→D3＜0.01) of flat type hybrid was very significant. The highest yield record was obtained in N₃D₃ and N₃D₁treatments for compact and flat type hybrid, respectively. And their yield gain ratio for density and nitrogen was 1﹕2.3 and 1﹕4.0, respectively. 【Conclusion】 Compared with flat type hybrid, compact type hybrid had a more adaptable ability of regulating cross and longitudinal growth of basal part of stem. Nitrogen application could reduce leaf angle of leaf above ear leaf, ear leaf and leaf below ear leaf, which could enhance ear leaf light use efficiency. Proper morphophysiological coordinate ability keeps a higher dry matter transfer rate for the compact type hybrid under higher density and higher nitrogen fertilizer condition at kernel weight formation stage, thus achieving a higher population yield.

Productivity and Water Use Efficiency of Maize-Peanut Intercropping Systems in the Semi-Arid Region of Western Liaoning Province

GAO YanLiang, SUN ZhanXiang, BAI Wei, FENG LiangShan, YANG Ning, CAI Qian, FENG Chen, ZHANG Zhe

Scientia Agricultura Sinica. 2017, 50(19): 3702-3713. doi:10.3864/j.issn.0578-1752.2017.19.007

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【Objective】In the experiments, the mechanism of maize-peanut intercropping in increase of land productivity and water use efficiency was studied through comparing yield, land equivalent ratio and water use efficiency, and the optimal maize-peanut intercropping systems in semi-arid region of western Liaoning province were proposed.【Method】Field experiments were conducted in 2015 and 2016 in Fuxin Scientific Observation Research Stations of Agricultural Environment and Cultivated Land Conservation. Four treatments were designed, 2 rows maize and 4 rows peanut (2M:4P), 4 rows maize and 4 rows peanut (4M:4P), sole maize (S-M) and sole peanut (S-P). Yield, land equivalent ratio, water distribution and water use equivalent ratio were calculated to analyze the positive effects on productivity and water use efficiency. 【Result】 Yield of maize and peanut in intercropping system decreased compared with the sole system because of resource competition. But the land equivalent ratio (LER) in 2M:4P and 4M:4P were, respectively, 1.10-1.24 and 1.12-1.23, indicating the advantage of intercropping. The partial land equivalent ratio (LER_p) of peanut in intercropping was 0.41-0.57, suggesting that peanut can decrease the disadvantage of intercropping with cereal crops. Soil water content in peanut strip was higher than that of maize strip but lower than the sole peanut. This finding showed that intercropped maize absorbed water from peanut strip. Water use equivalent ratio (WER) was 1.12-1.23 in 2M:4P intercropping system and 1.16-1.17 in 4M:4P intercropping system. Water use efficiency in intercropping system was increased.【Conclusion】The results indicated that maize-peanut intercropping can increase land and use efficiency in the semi-arid region of western Liaoning province. 2M:4P intercropping system performed better in 2015 with less rain. But in 2016 with more rain, 4M:4P intercropping system had higher land and water use efficiency. And the yield of 4M:4P intercropping system remained stable between two years. So in the semi-arid region of western Liaoning province, 4M:4P intercropping system can decrease climate risk. The results of this study contribute to optimize the field configuration.

The Inhibition Effect of Microbial Volatile Compound Benzothiazole on Botrytis cinerea

CUI KaiDi, HUANG XuePing, HE LeiMing, ZHAI YongBiao, MU Wei, LIU Feng

Scientia Agricultura Sinica. 2017, 50(19): 3714-3722. doi:10.3864/j.issn.0578-1752.2017.19.008

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【Objective】The objective of this study is to evaluate the application potential of microbial volatile compound benzothiazole on Botrytis cinerea, and to provide a valuable information for further exploitation and utilization.【Method】The sensitivity of 73 B. cinerea isolates in Shandong Province to benzothiazole was measured by mycelium growth rate of sealed dishes method. The sensitivity differences among different regions and phynotypes of B. cinerea isolates (resistant and sensitive to carbendazim, procymidone, pyrimethanil or boscalid: Car^R, Car^S, Prc^R, Prc^S, Pyr^R, Pyr^S, Bos^R, Bos^S) were compared. The combination efficiencies on B. cinerea of benzothiazole with carbendazim or boscalid were determined. The protective and curative effect of benzothiazole against cucumber gray mold, and the effects of benzothiazole at different doses and fumigation times on the pathogenicity of B. cinerea were determined on detached cucumber leaves. The mycelial morphology of B. cinerea treated by benzothiazole was observed with scanning electron microscope (SEM). 【Result】 All the tested B. cinerea isolates were sensitive to benzothiazole, range of EC₅₀from 0.38 to 1.08 μL·L^-1, with a mean EC₅₀ value at 0.62 μL·L^-1. There was no sensitivity difference among different regions. All the phynotypes of isolates (Car^R, Car^S, Prc^R, Prc^S, Pyr^R, Pyr^S, Bos^R, Bos^S) were sensitive to benzothiazole, suggesting that there was no cross-resistance between these fungicides and benzothiazole. In the range of 0.1-3 μL·L^-1 of benzothiazole, mycelia were more sensitive than spores to benzothiazole. The inhibition rate of 2 μL·L^-1 benzothiazole for mycelial growth, germ tube elongation and spore germination was 86.12%, 55.15%, and 19.30%, respectively. It is suggested that the sensitivities of different growth stages of B. cinerea to benzothiazole were different. There was no synergism when benzothiazole combined with carbendazim, and which active ingredient performed the main antifungal activity depended on the concentration and proportion of each compound in the mixture. The synergism of benzothiazole combined with boscalid was obvious, but the exact field practical application method, efficacy and mechanism of synergism remains to be further investigated. The result of in vitro leaf test showed that benzothiazole had protective and curative effect against cucumber gray mold, and the curative effect was superior to the protective effect. The curative effect of 2 μL·L^-1 benzothiazole against cucumber gray mold was 91.01%, which was higher than the control fungicide boscalid at 300 mg·L^-1(78.90%) and the protective effect (48.25%). Benzothiazole could significantly reduce the pathogenicity of B. cinerea mycelia on detached cucumber leaves, and the inhibition level was positively correlated with the concentration of benzothiazole. The results of SEM images indicated that benzothiazole caused large alterations in mycelial morphology of B. cinerea, showing a wrinkled surface and anomalous branching of the terminal mycelia. 【Conclusion】Benzothiazole has high toxicity to B. cinerea in different regions of Shandong Province, which indicates that benzothiazole could be applied as a fumigant for controlling gray mold.

Identification and Expression Analysis of a New Pectate Lyase Gene Ha-pel-1 from Heterodera avenae

LI Xin, GU XiaoChuan, LONG HaiBo, PENG Huan, HUANG WenKun, PENG DeLiang

Scientia Agricultura Sinica. 2017, 50(19): 3723-3732. doi:10.3864/j.issn.0578-1752.2017.19.009

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【Objective】The cereal cyst nematode (Heterodera avenae) is one of the important plant parasitic nematodes which seriously threatened cereal crops and caused huge economic losses to agricultural production in China. However, its pathogenic mechanism and effective prevention and control methods still need to be further studied. The objective of this study is to provide a theoretical basis for further study on the gene function of Ha-pel-1 and its interaction with host plants, and to give new ideas for the control strategies of cereal cyst nematode based on the cloning and expression analysis of a new pectate lyase gene Ha-pel-1 from H. avenae.【Method】A novel pectate lyase gene Ha-pel-1 was cloned from H. avenae using homology cloning combined with RACE technology, and its nucleotide sequence and amino acid sequence were analyzed by related bioinformatics softwares and online tools, such as DNAMAN, Clustal, SignalP 4.0 Server and GSDS. a phylogenetic tree was also constructed using MEGA 5.0. the tissue localization and developmental expression characteristics of Ha-pel-1 were analyzed by in situ hybridization and semi-quantitative PCR method. 【Result】 A novel pectate lyase gene (Ha-pel-1, GenBank accession number GQ998895) was cloned successfully from H. avenae. Ha-pel-1 was 1 717 bp in length which contained a 1 563 bp open reading frame (ORF) encoding a protein of 521 amino acid residues. The molecular weight of Ha-pel-1 encoding protein was 57.5 kD and isoelectric point was 8.52. The full length of genomic sequence of Ha-pel-1 was amplified from the nematode genome DNA which contains 7 199 bp. Gene structure analysis showed that the Ha-pel-1 genome contains 14 exons and 13 introns, except for the 3rd intron splice sites are GC-AG, the other 12 introns are in line with the rules of the eukaryotic gene splicing site GT-AG. The results of homologous comparison showed that the C-terminal sequence of the putative Ha-PEL-1 had a 67% identity and a similarity of 83% with that of soybean cyst nematode HG-PEL-1 and beet cyst nematode HS-PEL-1. In addition, after the end of N-terminal signal peptide, the putative Ha-PEL-1 had a sequence of 254 amino acid residues more than other reported plant parasitic nematodes pectate lyases. In this sequence, 184 amino acid residues closing to the N-terminal had no similarity with protein database, while 70 amino acid residues (Lys205-Glu274) closing to the C-terminal had an identity of 32% and a similarity of 47% with the methyltransferase domain of Wesselsbron virus NS5 (Registration No. 3ELD). The amino acid sequence analysis revealed that the predicted protein contained a signal peptide of 20 amino acid residues, as well as 4 highly conserved regions and several conserved cysteine residues characteristic of class Ⅲ pectate lyases (PL3). A phylogenetic analysis revealed that Ha-pel-1 and other nematodes pectate lyase genes are gathered in a large branch with bacterial and fungal sources PEL. In situ hybridization analyses showed that the transcripts of Ha-pel-1 were mainly expressed in the two subventral gland cells of H. avenae. a semi-quantitative RT-PCR analysis confirmed that its transcriptions were highly expressed at the pre-parasitic and parasitic 2nd stage juveniles.【Conclusion】A new pectate lyase gene Ha-pel-1 from H. avenae, closely related to the infection and parasitic process of cereal cyst nematode, was found and analyzed.

Daily Expression of Clock Gene in Compound Eye of Helicoverpa armigera

YAN Shuo, LIU YanJun, ZHANG XinFang, QIN Meng, LIU Hui, ZHU JiaLin, LI Zhen, ZHANG QingWen, LIU XiaoXia

Scientia Agricultura Sinica. 2017, 50(19): 3733-3744. doi:10.3864/j.issn.0578-1752.2017.19.010

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【Objective】The objective of this study is to clone and analyze a circadian clock gene, Clock (Clk) in the compound eyes of Helicoverpa armigera, examine the diurnal changes and determinants of Clk mRNA levels in compound eyes of cotton bollworm, and to determine whether Clk performed circadian functions in compound eyes, which will provide a theoretical reference for understanding the circadian clock machinery in compound eyes. 【Method】The total RNA was isolated from the compound eyes of 2-day-old H. armigera moths, and the Clk was cloned by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The amino acid sequence of CLK from cotton bollworm was analyzed using bioinformatics softwares. The relative mRNA levels of Clk were determined among various adult organs (head, thorax, abdomen, leg, wing, brain, antennae and compound eye) of H. armigera by quantitative real-time PCR (qRT-PCR). The diurnal change of Clk mRNA levels in compound eyes was measured under different photoperiods. Moths were illuminated by UV, blue and green lights, respectively, for 6 h from the beginning of the scotophase, and the expression levels of Clk in compound eyes were determined after light exposure. Female and male moths were paired for mating, and the expression levels of Clk in compound eyes were determined from the moths that had completed mating 0 h and 3 h. The expression levels of Clk in compound eyes were determined from the moths that were not fed. 【Result】An open reading frame of 1 860 bp was cloned, encoding 619 amino acids, designed as “HeCLK” (GenBank accession number KM233158). The molecular mass of the deduced protein was predicted to be 69.32 kD, and the calculated isoelectric point (pI) was 5.71. Deduced amino acids sequence contained three transmembrane topologies, and several conserved domains of insect CLK (PAS and HLH). HeCLK showed high homology with Spodoptera exigua (97% identity) and Danaus plexippus (74% identity), and low homology with Riptortus pedestris (53% identity) and Leptinotarsa decemlineata (52% identity). qRT-PCR revealed that HeCLK showed the lowest mRNA levels in compound eyes, and the highest mRNA levels in antennas among tested adult organs. The mRNA levels of HeCLK in compound eyes increased during the day, and decreased during the night under 14L﹕10D. The cycling of the circadian clock gene mRNA levels persisted for 1 d under dark condition, but did not persist further under constant darkness. HeCLK was up-regulated in compound eyes after light exposure, but there was no significant difference in mRNA levels of HeCLK among different wavelengths of light. The expression levels of HeCLK were tended to be down-regulated in the compound eyes after copulation, and there was a significant difference in mRNA levels of HeCLK between mated males and virgin males. The expression levels of HeCLK in compound eyes were not influenced by starvation. 【Conclusion】HeCLK from the compound eyes of a nocturnal moth, H. armigera, was cloned. Deduced amino acids sequence contained the conserved domains of CLK proteins, and shared high homology with insect CLK. HeCLK showed the lowest mRNA levels in compound eyes among tested adult organs. The expression levels of HeCLK in peripheral tissues (compound eyes) were regulated by the circadian rhythms, light condition and physiological status of moths, confirming that HeCLK played an important role in circadian rhythms in compound eyes. Whether the circadian clock machinery is similar between compound eyes and the central nervous system need to be further studied.

Study of Optimum Potassium Reducing Rate of Rice, Wheat and Oilseed Rape Under Different Soil K Supply Levels with Straw Incorporation

ZHANG Lei, ZHANG WeiLe, LU JianWei, DAI ZhiGang, YI YanRui, CONG RiHuan

Scientia Agricultura Sinica. 2017, 50(19): 3745-3756. doi:10.3864/j.issn.0578-1752.2017.19.011

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【Objective】 The experiments were conducted to study the effect of reducing potassium (K) fertilizer rates with straw incorporation on crop yield of cereal and oil, K uptake and K efficiency under different soil K supply capacities of Hubei, in order to provide a scientific basis for K fertilization and soil K management under straw returning. 【Method】 Field trials in 38 counties (cities) were carried out to study the effect of the substitution of straw incorporation for K fertilizer on rice, winter oilseed rape and wheat. Six treatments were designed: (1) no K fertilization (CK), (2) chemical K fertilization (+K), (3) straw incorporation (+S), (4) straw incorporation with 50% of chemical K fertilization (S+1/2K), (5) straw incorporation with 75% of chemical K fertilization (S+3/4K), and (6) straw incorporation with 100% of chemical K fertilization (S+K). Three soil K supply levels (i.e., HSKS, MSKS, and LSKS) were graded by relative yield (i.e., crop yield in the CK treatment divided by crop yield in the +K treatment) refer to the CK treatment. 【Result】 Both K fertilization and straw incorporation improved crops yield and K uptake under different soil K supply levels. Total straw incorporation only (+S) would satisfy crops K needs to achieve high yield level (i.e., crop yield in the +K treatment) for the HSKS. For the MSKS, 50% of chemical K fertilization with straw incorporation was needed to reach crop yield level of +K treatment. In case of LSKS, chemical K application rate could reduce 25% for rice and 50% for oilseed rape and wheat under straw incorporation condition, respectively. For the apparent K balance, straw incorporation could offset soil K deficit to some extent. For the seasons of oilseed rape and wheat, straw incorporation to the field could surplus 14.1-152.6 kg K₂O·hm^-2 and 25.5-95.9 kg K₂O·hm^-2, respectively. However, soil K balance would still be deficit during the rice season across the sites. The relationship between K fertilization rate and crop yield with straw incorporation was fitted by quadratic and linear-plateau models. The optimum K application rate was obtained from the model under the crop yield of the +K treatment. With straw incorporation, optimum K application rate was 20-33 kg·hm²for the three crops under the HSKS and MSKS levels, where oilseed rape required less K fertilizer than rice and wheat. However, optimum K rate was 45-49 kg K₂O·hm^-2 for the LSKS level, where oilseed rape needed more K fertilizer than rice and wheat. Compared with current K economic fertilization rate (60 kg K₂O·hm^-2), chemical K rate could be reduced by 45.0%-66.7% for the HSKS and MSKS levels, and also chemical K fertilizer could be saved by 18.3%-25.0% for the LSKS level. 【Conclusion】 Chemical K fertilizer could be saved by 18.3%-66.7% while guaranteed crop yields under straw incorporation condition. The reducing rate of chemical K fertilizer should consider soil K supply level.

Effects of Different Fertigation Modes on Tomato Yield, Fruit Quality, and Water and Fertilizer Utilization in Greenhouse

LI YinKun, GUO WenZhong, XUE XuZhang, QIAO XiaoJun, WANG LiChun, CHEN Hong, ZHAO Qian, CHEN Fei

Scientia Agricultura Sinica. 2017, 50(19): 3757-3765. doi:10.3864/j.issn.0578-1752.2017.19.012

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【Objective】Irrigation and fertilization are the key factors that can affect the greenhouse tomato growth and development. The objective of this study was to establish an optimized fertigation mode which can realize water and fertilizer saving and yield increasing in tomato integrative water and fertilizer management in solar greenhouse. 【Method】Based on a negative pressure device and drip irrigation system, the effects of three fertilization modes of conventional application of base fertilizer (CK), drip fertigation (DI) and negative pressure fertigation (NI) on greenhouse tomato growth, yield, quality and water and fertilizer use efficiency were studied. 【Result】The annual soil moisture variation of the negative pressure fertigation mode had a relative stability in the tomato growth, and the 0-20 cm soil water content was 20.8%-25.0%, which was less than the range of 19.7%-28.6% of drip fertigation. Compared to the treatment of CK and DI, the negative pressure fertigation treatment (NI) not only promoted the tomato plants’ growth, increased the tomato yield and improved the fruit quality, but also reduced the application of annual total nutrients (N+P₂O₅+K₂O) by 5.0% and 17.2%, respectively. The tomato biomass and fruit yield of treatment NI had increased by 23.0% (P＜0.05) and 7.5%-10.0% respectively, compared with the treatment CK. The fruit nitrate content of treatment NI also had reduced by 17.3%-21.5% (P＜0.05) compared with the treatment DI. Tomato had the higher water consumption at flowering and fruiting stages, which had accounted for 57.1%-67.3% of the whole growing period. The negative pressure fertigation mode (NI) can reduce irrigation water and fertilizer input and tomato water consumption, and enhance water use efficiency. Compared with the treatments CK and DI, the annual irrigation amount of treatment NI had reduced by 18.4% and 17.2%, the annual water consumption reduced by 12.8% and 12.1% (P＜0.05), the water use efficiency increased by 12.7%-40.1% and 10.0%-30.3% (P＜0.05), and the partial factor productivity of fertilizer increased by 10.4%-19.6% and 14.5%-42.7% (P＜0.05), respectively. The sustainable and steady supply of water is an important reason to achieve the goal of water and fertilizer saving, production increasing and with good quality in the negative pressure fertigation mode. 【Conclusion】The mode of NI, which was based on the negative device to supply nutrient solution, not only can reduce the application of water and fertilizer, but also can promote the growth of greenhouse tomato, ensure the production, improve the fruit quality and make the water use efficiency increased greatly. It can be used as a new mode in integrative management of water and fertilizer in solar greenhouse.

Using Orthogonal Signal Correction Algorithm Removing the Effects of Soil Moisture on Hyperspectral Reflectance to Estimate Soil Organic Matter

HONG YongSheng, YU Lei, ZHU YaXing, LI SiDi, GUO Li, LIU JiaSheng, NIE Yan, ZHOU Yong

Scientia Agricultura Sinica. 2017, 50(19): 3766-3777. doi:10.3864/j.issn.0578-1752.2017.19.013

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【Objective】 Rapid and accurate quantitative analysis of soil organic matter is essential for sustainable development of precision agriculture. Visible and near-infrared (Vis-NIR) reflectance spectroscopy has been widely used for soil properties estimation and digital soil mapping. However, it is less exact in monitoring soil organic matter (SOM) in the field when compared to laboratory-based spectroscopic measurement mainly due to some factors, such as soil moisture, temperature, and soil surface texture. Among these three factors, soil moisture (SM) has the most pronounced effects on spectral reflectance. Therefore, it is urgently significant that a method for removing SM effects from spectral reflectance and improving the accuracy of quantitative prediction of SOM should be proposed. 【Method】 A total of 217 soil samples used in this study were collected at 0-20 cm depth from Gong'an County and Qianjiang City in Jianghan Plain. These soil samples were air-dried, ground, and sieved (less than 2 mm) in the laboratory, and the SOM of each soil sample was analyzed based on potassium dichromate external heating method. These 217 samples were further divided into three non-overlapping data-sets: the model calibration set (S⁰), this set consisted of 122 samples to develop multivariate models for SOM; The orthogonal signal correction (OSC) development set (S¹), this set consisted of 60 samples for OSC development; The validation set (S²), this set consisted of 35 samples for independent OSC validation. Then, sample rewetting (S¹ andS²set) was carried out: each soil sample was weighed 150 g oven-dried soil in a cylindrical black box, and then they were rewetted by 4% SM increment for each level in the laboratory. Total 9 treatments were obtained, corresponding to the following SM levels i.e. 0%, 4%, 8%, 12%, 16%, 20%, 24%, 28%, and 32%. Soil hyperspectral reflectance was measured in the laboratory with an ASD Fieldspec-Pro spectroradiometer for the three data-sets (S⁰, S¹ andS², including the rewetting samples). Savitzky-Golay smoothing with a window size of 11 nm and polynomial order of 2 (SG) were applied to the three data-sets, then external parameter orthogonalization (EPO) and orthogonal signal correction (OSC) were conducted to remove the SM effects on reflectance spectra. In the next, the effect of SM on the reflectance spectra was analyzed, and the scores of the first two principal components from the principal component analysis (PCA) corrected by OSC method and spectral correlation coefficient were used to compare the performance in removing the effects of SM. Finally, the S⁰ data-sets were calibrated using the partial least squares regression (PLSR), and the S² data-sets were then examined as external validation sets. Using the coefficient of determination (R²), root mean squared error (RMSE) and the ratio of prediction to deviation (RPD) between the predicted and measured SOM to compare the performance of PLSR, EPO-PLSR and OSC-PLSR models, high R², RPD and low RMSE were indicators of the optimal model in the removal of SM effects.【Result】SM had an obvious influence on soil spectra reflectance, and the reflectance values across the entire wavelength domain decreased as the SM increased, making it more challenging to identify useful features of SOM by spectra, it dramatically degraded the prediction accuracy of SOM. No overlap before OSC was observed between the wet and dry ground spectra because the wet spectra grouped in an independent space from the dry ground spectra, and the range of the spectral correlation coefficients between different SM levels was large. However, after OSC, the wet spectra had nearly identical positions in the feature space to the corresponding dry ground spectra, which showed the spectral similarity between the two groups of spectra, and the range of the spectral correlation coefficients between different SM levels was small. The validation mean values of R²_pre, RPD for the nine SM levels of EPO-PLSR model were 0.72 and 1.89, respectively. OSC method could effectively remove the effects of SM on SOM estimation, OSC-PLSR model obtained a better performance than the PLSR, EPO-PLSR model, the validation mean values of R²_pre, RPD for the nine SM levels were 0.72 and 1.89, respectively. 【Conclusion】 OSC-PLSR method was recommended for a better quantitative prediction of SOM from the soil samples under different SM levels. In the future, this approach may facilitate the proximally sensed field spectra for rapidly measuring SOM for this study area.

Effects of Endogenous NO on Reactive Oxygen Metabolism and Photosynthetic Characteristics of Cucumber Seedlings Under Salt Stress

YANG JianJun, ZHANG GuoBin, YU JiHua, HU LinLi, LUO ShiLei, NIU Tong, ZHANG Jing

Scientia Agricultura Sinica. 2017, 50(19): 3778-3788. doi:10.3864/j.issn.0578-1752.2017.19.014

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【Objective】 The objective of this study is to investigate the physiological and biochemical regulation mechanism and regulation pathway of endogenous NO in cucumber seedlings under salt stress. 【Method】The effects of nitric oxide synthase inhibitor (L-NAME, Nω-nitro-L-arginine methyl ester hydrochloride), nitrate reductase inhibitor (tungstate) and nitric oxide scavenger (cPTIO, 4-carboxypheny-4,4,5,5-tetramethy limidazoline-1-oxyl-3-oxidepotass-ium salt) on cucumber seedlings under salt stress were studied. The contents of NO, active oxygen metabolism, photosynthetic parameters and chlorophyll fluorescence parameters were used in discussing the results. 【Result】Salt stress increased endogenous NO content in the leaves of cucumber seedlings. The application of 200 μmol?L^-1 L-NAME, 100 μmol?L^-1 tungstate and 200 μmol?L^-1cPTIO inhibited the activities of antioxidant enzymes (SOD, POD and POD) in cucumber seedlings under salt stress. Malondialdehyde (MDA) content, superoxide anion free radical (O₂·^-) production rate and plasma membrane permeability were increased, and the contents of free proline (Pro) and soluble protein were decreased. Peroxidation increased the damage of cucumber seedlings under salt stress. At the same time, the gas exchange parameters Pn, Gs, Tr, Ci, and the chlorophyll fluorescence parameters Fv/Fm, ΦPSⅡ, qP also decreased. However, the NPQ significantly increased, leading to leaf photosynthetic mechanism damage, and the leaf photosynthetic rate decreased. 【Conclusion】Under the salt stress, the change of endogenous NO could directly regulate the antioxidant capacity of cucumber seedlings, alleviate the damage of lipid peroxidation and damage to photosynthetic organs, and the active oxygen metabolism and photosynthetic characteristics of cucumber seedlings are mainly controlled by nitric oxide synthase pathway.

Effect of Three Different Tree Shapes on Growth, Yield and Fruit Quality of ‘Fuji’ Apple Trees on Dwarfing Interstocks

LI MinJi, ZHANG Qiang, LI XingLiang, ZHOU BeiBei, YANG YuZhang, ZHOU Jia, ZHANG JunKe, WEI QinPing

Scientia Agricultura Sinica. 2017, 50(19): 3789-3796. doi:10.3864/j.issn.0578-1752.2017.19.015

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【Objective】 Effect of different tree shapes on growth, yield and fruit quality in ‘Fuji’ apple trees was studied in-depth to provide the theoretical basis and mentoring programs for the selection of tree shapes on dwarfing interstock.【Method】 The ‘Fuji’ apple with SH6 dwarf interstock planted in spring 2010 was used to investigate the effect of three different tree shapes (Spindle 2 m×4.5 m, V-trellis 1.5 m×6 m, Hedgerow 3 m×3 m) on growth, yield and quality in ‘Fuji’ apple trees from 2011 to 2016.【Result】The tree growth, fruit yield and quality of different tree shapes were quite different. Since the first 3 years after planting, the total branches and coverage rate of different tree shapes showed significant differences, spindle maximum, followed by V-trellis, Hedgerow minimum. There was no significant difference on the proportion of different branches among different tree shapes. There was also no significant difference on leaf fresh weight, chlorophyll content and photosynthetic rate among them. All trees began to have production in the fourth year. The cumulative average yield per plant of Spindle was significantly higher than that of V-trellis and Hedgerow. Average fruit weight of Spindle was the biggest, with V-trellis smallest. Firmness of V-trellis was the highest, with Spindle lowest. There was no significant difference on the fruit figure index, soluble solids, titratable acidity and TSS/TA among different tree shapes. 【Conclusion】 According to the survey data of seven years, the ‘Fuji’ apple with SH6 dwarf interstock of Spindle shows large total branches amount, moderate growth, early bearing, high and stable yield, and good fruit quality compared with that of V-trellis and Hedgerow. Spindle is the suitable shape for apple dwarf stock cultivation model.

The Inhibition Effect of Nano-Antibacterial Packages on the Aging of Milled Rice with Embryo During Storage

XIE JunQi, LI Li, SHI You, SHEN Ting, CHEN YuJie, HONG Yang, CAO ChongJiang

Scientia Agricultura Sinica. 2017, 50(19): 3797-3807. doi:10.3864/j.issn.0578-1752.2017.19.016

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【Objective】Milled rice with embryo is easy to deteriorate and difficult to store, which is always a big problem to hinder the promotion. The study was intended to explore the aging mechanism and metamorphism of milled rice with embryo during preservation. The results would provide some references for developing a new packaging material which caters for both market demand and rice preservation.【Method】Milled rice with embryo of WuYou 4 was used as experimental material. Two different kinds of nano-antimicrobial polyethylene (PE) packaging materials were made for the experiment. It was stored at 25℃ and 65% relative humidity for 28 days. And it used ordinary polyethylene (PE) packaging as a comparison. Both of them were sampled once per 7 days. The effect of the nano-packaging material on the quality of stored milled rice with embryo was studied by tracking the changes in the gas proportion in the packages, total colony number, lipase activity, total amount of fatty acids, gelatinization characteristics, fatty acids composition, the cross-section microscopic structure of milled rice with embryo and studying antibacterial property of common plastic packaging film. 【Result】 The results indicate that antimicrobial package material played a part in regulating the gas in the package by decreasing the value of O₂/CO₂, which decreases rice depletion. The data of the bacteriostatic experiment proved that compared with the common packaging materials, the antibacterial effects of two nano-antimicrobial packaging materials are better, whose antibacterial percent to Escherichia coli and rice mold are higher than 99% and 95%. The number of colony preserved in PE packages at room temperature for 28 days is 410 000 cfu/g, while the numbers of those preserved in two kinds of nano-packages in the same condition are just around 170 000 cfu/g and 180 000 cfu/g and the results indicate significant differences (P＜0.05). The decrease of the number of molds in antimicrobial packages reduced the lipase activity and prevented the fatty acids from accumulation. Milled rice with embryo is suitable for human body for its rational acid contents. The initial fatty acid composition consists of 71.22% unsaturated fatty acids. After 28 days of preservation, the fatty acid composition of rice stored in PE packages consists of 65.95% unsaturated fatty acids, while those stored in two kinds of nano-packages in the same condition are closer to the initial data and just around 69.10% and 67.41%. The nano-packages also show special quality in keeping gelatinization characteristics, which affect the taste. As is shown in the microstructure of endosperm section, to some extent, the nano-packaging material makes a difference in delaying the aging of milled rice with embryo.【Conclusion】Milled rice with embryo is easy to aging at room temperature. The growth of the mold, the breakdown of fat and the change of structure show the aging process. The nano-packaging material retards the aging and keeps gelatinization characteristics of rice by stopping the mold from breeding and changing the gas composition.

Production of Transgenic Pigs Over-Expressed PCSK9 D374Y Mutant

ZHANG Xue, HUANG Lei, RUAN JinXue, LIU ZhiGuo, CHENG Ying, FENG ShuTang, MU YuLian, LI Kui

Scientia Agricultura Sinica. 2017, 50(19): 3808-3816. doi:10.3864/j.issn.0578-1752.2017.19.017

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【Objective】 Proprotein convertase subtilisin/kexin type 9 (PCSK9) is the third major gene of autosomal dominant hypercholesterolemia (ADH). Gain-of-function mutations of PCSK9 are directly associated with human familial hypercholesterolemia, such as the D374Y mutant. In vitro detection experimental results on the degradation of LDLR by the PCSK9 D374Y mutant showed that its LDLR degradation ability is ten times stronger than that of the wild-type protein. This ability increases the risk of hypercholesterolemia, and promotes atherosclerotic lesions. The miniature pig is a useful animal model for biomedical research. However the utility of pigs for diseases model preparation is relatively infrequent because of their individual differences. Inbred lines increased the consistency of the genetic background of the experimental animals, thus consequently, would simplify disease progression of the animal model. This study used Wuzhishan minipig intending to make a transgenic pig with gain-of-function D374Y mutation of human PCSK9, and to contribute to the model of atherosclerosis. 【Method】 In this study, D374Y-PCSK9 over-expressing plasmids were transferred into Wuzhishan porcine embryonic fibroblasts by electroporation, and somatic cell nuclear transplantation and embryo transfer were used to make D374Y-PCSK9 over-expressing pig. Using transgenic pig liver, the human PCSK9 expression was assessed by PCR, Southern blot and Western blot in DNA, RNA and protein levels. At the same time, istochemical staining and hematoxylin-eosin (HE) staining were used to identify the pathological changes in liver between D374Y-PCSK9 transgenic pigs and wild-type Wuzhishan pigs. 【Result】 The positive cloned cells appeared for 3 days, and colony point was formed for 7 days. The PCR results showed that the D374Y-PCSK9 fragments could be spliced into a complete fragment. That means the D374Y-PCSK9 over-expressing plasmids were successfully integrated into fibroblasts of Wuzhishan pig. The screened positive cloned cells were used as donor cells for somatic cell nuclear transfer (SCNT) and transplanted into recipient sows, and finally, the transgenic pig was got. PCR and Southern blot detection results of the DNA samples demonstrated that the entire D374Y-PCSK9 gene was integrated into the genomes. RT-PCR and QPCR were performed using primers specifically recognizing human or pig PCSK9 mRNA. The results showed that the human PCSK9 gene could be normally transcribed in the pig liver and did not influence the transcription of the endogenous pig PCSK9. Surprisingly, the expression of the human PCSK9 gene could be detected in other organs including the heart, spleen, lung, and kidney, whereas the expression levels of the endogenous pig PCSK9 in these tissues were very low. The western blot results of the proteins were similar to the RNA detection levels. Therefore, the human D374Y-PCSK9 gene integrated into pig genomes was normally transcribed and translated. Besides, transgenic pig’s liver had pathological changes were detected by HE staining and histochemical staining and the results showed that the LDLR level in the transgenic pigs’ liver was significantly lower than wild-type controls’.【Conclusion】 In conclusion, human PCSK9 protein was successfully over-expressed in Wuzhishan inbred miniature pigs. Compared with wild type pigs, transgenic pigs had a low LDLR level in liver. At the same time, there were obvious pathological changes in transgenic pigs’ liver.

The Fermentation Characteristics and Effects on Nitrogen Excretion Related Indicators of Different Fiber Diets Using an in vitro Method in Pigs

ZHU LiYuan, LU QingPing, XIA Bing, LIU ZhengQun, SUN YongBo, ZHANG HongFu

Scientia Agricultura Sinica. 2017, 50(19): 3817-3827. doi:10.3864/j.issn.0578-1752.2017.19.018

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【Objective】The present study was conducted to investigate and compare the fermentation characteristics and effects on nitrogen excretion related indicators of different fiber diets using an in vitro method in pigs. The study aimed to provide an experimental basis for the rational application of dietary fiber in pig production and for the preparation of diets to reduce nitrogen excretion. 【Method】Wheat fiber (WF), wheat bran fiber (WBF) and oat bran fiber (OBF) were purified from wheat, wheat bran and oat bran, respectively, using the enzyme-gravimetric method (AOAC Method 985.29). WF diet, WBF diet and OBF diet based on maize and soybean were formulated to contain 5% WF, WBF and OBF. The three fiber diets were undergone a stomach-small intestine digestion of pigs by simulative digestion system (SDS-II). A total of six growing pigs, which were in good health and had similar weight, were used to collect fresh feces. The mixed feces and culture solution were compounded in an appropriate proportion to prepare inocula which were quantitatively charged into fermentation flasks for simulating large intestine fermentation. After the stomach-small intestine digestion, the digestive products were pooled. 200 mg digestive products were added into a fermentation flask filled with 30 mL of inocula, placed in an incubator at (39±0.25)℃. The fermentation were stopped at 4, 12, 24, 36, 48 and 72 h of incubation. Then the fermentation residues and fermentation broth were collected, and the relevant indicators were determined.【Result】The results indicated that in the large intestine fermentation stage, there were significant differences in dry matter degradability during the in vitro fermentation (IVDMD) and total short-chain fatty acid (SCFA) concentration, pH, ammonia nitrogen (NH₃-N) concentration and microbial protein (MCP) production in fermentation broth among the three fiber groups (P＜0.05): (1) At 4 h and 12 h, OBF group had the highest IVDMD among the three groups (P＜0.01), and no significant differences at other point in time(P＞0.05). At 4 h, the IVDMD of OBF group was 18.32% and 15.21% higher than WF group and WBF group, respectively (P＜0.01); at 12 h, OBF group was 14.87% and 10.06% higher than WF group and WBF group, respectively (P＜0.01). (2) As for total SCFA concentration in fermentation broth, at 24 h and 36 h, OBF group was significantly higher than other groups (P＜0.05); at 72 h, OBF group and WBF group were significantly higher than WF group (P＜0.01); at 4 h and 12 h, OBF group had a higher trend than other groups (P=0.0599; P=0.0504). At 24 h, the total SCFA concentration of OBF group was 27.32% and 17.11% higher than WF group and WBF group, respectively (P＜0.05); at 36 h, OBF group was 16.65% and 25.96% higher than WF group and WBF group, respectively (P＜0.05). (3) As for pH in fermentation broth, at 4 h and 12 h, OBF group had the lowest values among the three groups (P＜0.01); at 24 h, 36 h and 72 h, OBF group and WBF group were significantly lower than WF group (P＜0.05). (4) As for NH₃-N concentration in fermentation broth, OBF group was significantly lower than other groups at 24 h and 48 h (P＜0.05) and had a lower trend at 12 h (P=0.0559). At 24 h, the NH₃-N concentration of OBF group was 6.86% and 4.59% lower than WF group and WBF group, respectively (P＜0.05); at 48 h, OBF group was 8.44% and 7.09% lower than WF group and WBF group, respectively (P＜0.05). (5) As for MCP production in fermentation broth, OBF group had the highest value among the three groups at 4 h (P＜0.05); at 12 h, 24 h and at 36 h, 48 h, respectively, OBF group was significantly higher than WF group and WBF group (P＜0.05). At 4 h, the MCP production of OBF group was 69.85% and 82.25% higher than WF group and WBF group, respectively (P＜0.05).【Conclusion】In conclusion, oat bran fiber significantly increased IVDMD, total SCFA concentration and MCP production in fermentation broth, and significantly lowered pH and NH₃-N concentration in fermentation broth. Thus, compared to wheat fiber and wheat bran fiber, oat bran fiber had a good potential in promoting microorganism fermentation and reducing nitrogen excretion.

Research and Application of Production Technology of Natural Honeybee Bread

JIANG WuJun,WU XiaoBo, LIU GuangNan, HE XuJiang, YAN WeiYu, ZENG ZhiJiang

Scientia Agricultura Sinica. 2017, 50(19): 3828-3836. doi:10.3864/j.issn.0578-1752.2017.19.019

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【objective】 Honeybee bread is an important food resource for honeybee old larvae and young bees, and contains full nutrition essentially for larval development. It is also edible for humans and has promising commercial prospects. However, the lack of technology has limited the large-scale harvest of honeybee bread. The objective of this study is to develop a device for quick harvesting high quality honeybee bread and provide a technical support for the future mechanized production of honeybee bread.【Method】This study developed a honeybee bread producing instrument (HBPI) including bee bread storage and collecting devices, according to honeybee bread-storing biological characteristics. In the lotus blooming season, 5 strong honeybee Apis mellifera colonies were selected as the experimental materials to explore the practicability of this technology. Each colony was placed with a HBPI. The completely fermented bee bread was collected, and was separated by the bee bread collecting device (HBPI bee bread). Plastic pollen traps were employed for collecting the fresh lotus pollen using the same 5 colonies (fresh pollen). The completely fermented bee bread from the natural frames from these colonies was also excavated (natural bee bread). Furthermore, the quality of fresh lotus pollen and honeybee bread from natural honeybee frames and HBPI was compared, respectively, by analyzing pH, water activity, moisture content, pollen particles activity, enzyme activity and content of amino acids by using pH meter, water activity detector, directly aesiccation method, triphenyl tetrazolium chloride method, Marklund method, ultraviolet spectrophotometry and amino acid analyser, respectively. 【Result】 Honeybees successfully stored honeybee bread into the HBPI and maintained its original shape. The pH, water activity, moisture content, pollen particles activity, catalase and superoxide dismutase activity of fresh lotus pollen were significantly higher than natural and HBPI honeybee bread. There was no significant difference between fresh lotus pollen and two types of honeybee bread in terms of total free amino acids and hydrolysis amino acids. However, the valine, isoleucine, leucine, tyrosine, histidine, arginine in free amino acids, and the aspartate, proline in hydrolysis amino acids were significantly higher in two honeybee bread categories compared to fresh pollen. There was no significant difference between the natural and HBPI honeybee bread in physicochemical property and collectivity nutrient composition. 【Conclusion】 This study developed a honeybee bread producing instrument and confirmed its good practicability, which could be widely applied in further apiculture.

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