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16 June 2018, Volume 51 Issue 12

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Scientia Agricultura Sinica. 2018, 51(12):  0-0. 

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

Functional Analysis of AtTGA4 Transgenic Wheat Tolerance to Low Phosphorus Stress in Field

LING BingQi, BAI XingXuan, ZHOU YongBin, WANG ChunXiao, XU ZhaoShi, MA YouZhi, CHEN Ming, ZHANG XiaoHong

Scientia Agricultura Sinica. 2018, 51(12):  2225-2234.  doi:10.3864/j.issn.0578-1752.2018.12.001

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【Objective】 In previous work, we had proven that a bZIP type transcription factor gene, AtTGA4 can improve the drought resistance and low nitrogen tolerance of transgenic Arabidopsis. In this study, AtTGA4 was transformed into wheat to identify the tolerance of transgenic wheats to low phosphorus stress in field. At the same time, the physiological mechanism of AtTGA4 gene to improve the stress resistance of transgenic wheats was analyzed, which laid the foundation for the molecular breeding of wheat tolerant to low phosphorus stress. 【Method】Used particle bombardment method of minimal expression box to co-transform AtTGA4 and marker gene Bar into wheat variety Shi4056. After transformation, we screened many transgenic wheat lines without Bar gene and with AtTGA4 gene through PCR assay. In field experiment, base on fertilizer nutrient content in soil we applied different levels of phosphate, which led to low phosphorus stress in soil. AtTGA4 transgenic wheat lines were tested under low phosphorus. During the flowering stage some physiological indexes were identified such as light efficiency of the light system Ⅱ (Fv/Fm), the relative content of chlorophyll (SPAD) and crown the temperature difference (CTD), and other agronomic traits were investigated, during mature stage such as plants height, tiller number, grains per spike in mature period, and some values such as the grain yield and phosphorus concentration and phosphorus uptake in different components in wheat (root, stem, leaf and grain) were measured and data statistic analysis were completed. 【Result】 PCR analysis showed that AtTGA4 gene had been stable heritability for T₄ generation in Shi4056 and four stable transgenic lines were obtained. According to the results of soil nutrient content, 812.39 kg·hm^-2 of superphosphate was applied to normal plots, and no phosphorus was applied in low phosphorus treatment plots. The statistical results of yield and agronomic characters showed that the grain yield of AtTGA4 transgenic lines L1 and L2 increased compared to wild type (WT) significantly under normal condition and low phosphorus stress. Under normal conditions the yield of transgenic wheat increase of 5.3%-8.6%, and under low phosphorus stress grain yield of transgenic wheat increased of 4.4%-7.7%. Thousand seed weight of AtTGA4 transgenic wheat increased significantly than WT. The results of physiological indexes assay in field showed that the Fv/Fm and CTD of transgenic lines L1 and L2 were significantly better than those of WT under the condition of low phosphorus, while SPAD had no significant difference. We found that under low phosphorus stress WT were in early grain filling than the transgenic wheat, and the ears turn yellow early in WT than that in transgenic wheat plants. The phosphorus content assay during the late mature stage showed that the phosphorus concentration in the stems of L1 and L2 of transgenic lines increased significantly compared with that of WT under the condition of low phosphorus, but there was no significant difference in other tissues. Under the condition of low phosphorus, the total phosphorus content in stems and leaves and grains of two transgenic lines was higher than that of WT, and the total phosphorus content in the aerial part increased by 6.38%-17.47%. qRT-PCR results of AtTGA4 in transgenic wheat showed that the expression of AtTGA4 in Line 2 (L1) was 0.69 times lower than that of Line 1 (L1). 【Conclusion】 The above results showed that under the condition of low phosphorus stress, AtTGA4 can improve the uptake and transportation of phosphorus in transgenic wheat plants, and increase the yield of transgenic plants, and further enhance the tolerance of transgenic wheat to low phosphorus stress.

Screening Method for Salt Tolerance in Sesame (Sesamum indicum L.) and Identification of Candidate Salt-tolerant Genes

ZHANG YuJuan, YOU Jun, LIU AiLi, LI DongHua, YU JingYin, WANG YanYan, ZHOU Rong, GONG HuiHui, ZHANG XiuRong

Scientia Agricultura Sinica. 2018, 51(12):  2235-2247.  doi:10.3864/j.issn.0578-1752.2018.12.002

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【Objective】In order to evaluate salt tolerance of sesame germplasms and reveal the genetic basis of salt tolerance in sesame at the germination stage, optimum NaCl concentration and best salt tolerance indexes were determined, salt-tolerant sesame genotypes as well as candidate genes were identified in this study. 【Method】 Eight sesame genotypes with different salt tolerance were germinated under different NaCl concentrations (0, 50, 100, 150, 200 and 250 mmol·L^-1 ). Seven germination indexes including germination potential, germination index, vigor index, seedling rate, radicle length, embryo length and fresh weight of seedling were measured. The optimum NaCl concentration and salt tolerance indexes of sesame were determined using variance analysis, principal component analysis, membership function and correlation analysis based on the relative values of multiple indexes. In addition, genome-wide association analysis (GWAS) of salt tolerance related trait in 71 sesame germplasms was performed to find the SNP loci and candidate genes related to salt tolerance in sesame. Gene function annotation, transcriptome analyses and real-time quantitative reverse transcriptase PCR (qRT-PCR) were used to identify important candidate genes involved in salt tolerance. 【Result】The larger standard value deviations of germination indexes were observed at 100 mmol·L^-1 NaCl concentration, implying that 100 mmol·L^-1 is the optimum NaCl concentration for salt-tolerance screening in this study. The seven indexes were highly correlated with salt tolerance at germination stage, suggesting that, these indexes could be targeted for effective screening of salt-tolerance in large sesame germplasm. The genome-wide association analysis identified 7 SNP loci peaks (LG5:688003, LG7:9582027, LG10:5274091, LG10:10788493, LG11:11924186, LG14:2128695 and LG16:3930301) significantly associated with salt tolerance and 34 functional candidate genes. The transcriptome analysis and qRT-PCR showed that 21 candidate genes respond strongly to salt stress.【Conclusion】In this study, 100 mmol·L^-1 is the optimum NaCl concentration for salt-tolerance screening and seven indexes including relative seedling rate could be targeted for effective screening of salt-tolerance in large sesame germplasm. In addition, 7 SNP loci peaks significantly associated with salt tolerance were identified and 21 candidate salt-tolerant genes were discovered.

SSR Analyser：A Special Software Suitable for SSR Fingerprinting of Plant Varieties

WANG FengGe, LI Xin, YANG Yang, YI HongMei, JIANG Bin, ZHANG XianChen, HUO YongXue, ZHU Li, GE JianRong, WANG Rui, REN Jie, WANG Lu, TIAN HongLi, ZHAO JiuRan

Scientia Agricultura Sinica. 2018, 51(12):  2248-2262.  doi:10.3864/j.issn.0578-1752.2018.12.003

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【Objective】Develop software tools for plant variety identification by SSR fingerprinting to realize the automatic and standardized analysis of plant variety identification, solving problems of low efficiency of data collection and hard for data sharing et al for SSR markers in practice. 【Method】Based on commercialized software GeneMarker®, develop and optimize algorithms to deal with the specialty of SSR fingerprinting analysis for data analysis, panel design, database synchronization et al. SSR Analyser is generated as personalized software and tested on maize and other crops for its effectiveness. 【Result】From the perspective of processing function, the software is able to first weakly eliminate pull-up peak using matrix by system calculation. Then, use matching algorithm with single peak removal method to completely remove pull-up peak automatically and correctly. By optimizing the reading algorithm of N+1 peak, stutter peak et al, unrecognized/inaccurate/mis-reading of special peak is solved. Therefore, it is more accurate for Dinucleotide SSR markers’ fingerprinting. By completing the filtering algorithm of neighboring peak, high and low peak, and diploid crops, it solves the problem of effective peak collection for blended samples. From the perspective of panel design, the software balances the flexibility, convenience and uniformity. On the premise of standardized data collection, the software is more suitable for complicated experiment: enhances the flexibility of marker parameter settings, which include setting parameters at one time for specific species using panels, or setting parameters individually for electrophoresis gel for each primer locus; realizes that generating new panels from existing markers; ensures the unification by calling panels and its synchronous updating. By seamless connecting the software and fingerprinting database management system, it realizes the automation and standardization from sample preparation to fingerprint collection, which makes the database more intuitive and traceable. Using the SSR Analyser for building database of maize indicates that the software is more efficient than the original software, which is 10 times of efficiency; After expanding the SSR Analyser’s application scope to rice, soybean, cucumber, watermelon, Chinese cabbage and other diploid crops, and polyploidy crops such as wheat and cotton, suggests that it is more applicable to diploid crops for polyploidy crops, markers developed based on diploid principle have better application. However, other types of markers need optimization on filtering algorithm. 【Conclusion】SSR Analyser is simple and effective in data analysis, which can accurately collect special peak for SSR markers. It is suitable for fingerprint collection of mixed samples and seamless connects with fingerprint database system, which greatly improves the application of SSR markers in plant variety identification.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY

Effects of Low Temperature on Maize Superior and Inferior Kernels Development During Grain Filling in Vitro

ZHANG Xun, HAO JianPing, WANG Pu, ZHANG Ping, CHEN LuJie

Scientia Agricultura Sinica. 2018, 51(12):  2263-2273.  doi:10.3864/j.issn.0578-1752.2018.12.004

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【Objective】In this study, the physiological mechanism of kernel weight reduction under low temperature had been uncovered through grain filling dynamic, carbon metabolism, and phytohormone balance, and reveal the effect of low temperature on maize superior and inferior kernels during grain filling, which could provide a theoretical guidance for crop production. 【Method】 Hybrid maize Zhengdan 958 was chosen for the experimental material, which was grown in the filed condition. Kernel was collected at 3 days after pollination, and was divided into superior and inferior kernel according to its position at the ear. Afterward, kernels were separately incubated with solid medium under normal (25°C) and low (16°C) temperature in artificial climate incubators. Superior and inferior kernels were sampled with 10-day interval since pollination for analyzing dry weight dynamic, hormone contend, starch accumulation and its related enzymes. 【Result】Compared with CK, Final kernel weight decreased by 47.58% for superior kernel and 50.95% for inferior kernels under low temperature, respectively. A longer active grain-filling period had been observed, caused by slower grain filling rate increase during lag period and slower decrease during maturation drying period. But, the grain-fill rate during active grain-filling period decreased 55.39% for superior kernel and 54.72% for inferior kernel, respectively. The increase of grain-filling rate in lag periods and the decrease of grain-filling rate in maturation drying periods of maize kernels were reduced significantly, and the time of active grain-filling periods was prolonged by 5-7 days under low temperature stress. The content of IAA, ZR and ABA decreased significantly in both superior and inferior kernels after 10 days low temperature incubation, while it was opposite for GA3. Moreover, decreased content of IAA and ZR in inferior kernels and increased ABA in superior kernels had been found after 30 days low temperature treatment. Low temperature decreased activity of soluble acid invertase (SAI), sucrose synthase (SS), soluble starch synthase (SSS) and ADP-glucose pyrophosphorylase (APGase) in both superior and inferior kernel during early grain filling periods, resulting in decreased starch accumulation in kernel. Furthermore, the decrease of SAI in inferior kernels was greater than that of superior kernels, while it was opposite for other enzymes.【Conclusion】The disturbed hormone balance during earlier grain filling period greatly weakened the sink capacity under low temperature stress, while the decreased activity of enzyme during mid-grain filling period limited the starch accumulation. The grain filling rate decreased in both superior and inferior kernels, resulting in kernel weight reduction. The grain filling process had been affected in both superior and inferior kernels under low temperature stress. Furthermore, the inferior kernels was more sensitive to low temperature than superior kernels.

Effects of Different Film Mulching-Patterns on Soil Thermal-Moisture and Broomcorn Millet Water Consumption Characteristics in Semiarid Region on Northwest Loess Plateau

DONG KongJun, LIU TianPeng, HE JiHong, REN RuiYu, ZHANG Lei, XU Yan, YANG TianYu

Scientia Agricultura Sinica. 2018, 51(12):  2274-2287.  doi:10.3864/j.issn.0578-1752.2018.12.005

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【Objective】The objective of this paper was to explore effect of soil thermal-moisture and the yield-increasing mechanism of broomcorn millet under different plastic mulching-patterns in the dry farming regions of Loess Plateau.【Method】 In this study, “Longmi 10” was used as trial material with different plastic mulching modes, including ridge-furrow planting with whole film mulching under no tillage (A1), ridge-furrow planting with whole film mulching (A2), flat planting with whole film mulching beyond covering soil (A3), flat planting with whole film mulching (A4), uncovered and row-planting (CK) to explore the effect of them on soil thermal-moisture, water consumption characteristics, water use efficiency, and yield of broomcorn millet in rainfed agriculture area during 2015-2016.【Result】 Compared with CK, soil average temperature, and average temperature daily variation of 5 to 25 cm soil layer under A1, A2, A3, and A4 treatments showed different degrees of warming effect. During the growth period of broomcorn millet, in the rainy year (2015), the water storage amount of A1, A2, A3, and A4 treatments were 94.7, 67.9, 58.0, and 21.0 mm higher than that of CK, respectively. The water consumption was CK＞A1＞A4＞A2＞A3, and its contents in each treatment was more balanced in different stages of fertility and different soil layers. In the severe drought year (2016), treatments of A1 and A2 were 83.9 mm and 57.4 mm higher than that of CK, respectively. However, A3 and A4 treatments reduced 27.1 mm and 25.3 mm, respectively. The water consumption, the use efficiency of 80-100 cm deep soil moisture were A3＞A4＞A1＞A2＞CK, and the water consumption of the 4 kinds of film mulching was lower than that of the CK from sowing to the seedling stage. The water consumption intensity of the jointing to mature period was controlled by a more balanced approach. Based on the different soil thermal-moisture environment in 4 kinds of mulching methods and the water consumption characteristics of broomcorn millet under treatments of A1, A2, A3 and A4 were increased by 1.3-3.9, 5.2-5.5, 3.4-3.5, and 4.1-4.2 kg·hm^-2·mm^-1, the yield of broomcorn millet were increased production by 9%-43.3%, 34.8%-58.2%, 20.8%-49.4%, and 29%-52.9%, respectively. Furthermore, the increase in yield was higher in arid years.【Conclusion】 Four kinds of mulching could improve the soil thermal environment of soil water in the broomcorn millet growing period, adjust the water consumption rate of the different growth stage, and significantly improve the water utilization efficiency, yield and related traits. The yield-increasing potential was A2＞A4＞A3＞A1.

Genetic Variations and Cluster Analysis of Photosynthetic Gas Exchange Parameters in Exotic Sugarcane Cultivars

LI ChunJia, QIN Wei, XU ChaoHua, LIU HongBo, MAO Jun, LU Xin

Scientia Agricultura Sinica. 2018, 51(12):  2288-2299.  doi:10.3864/j.issn.0578-1752.2018.12.006

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【Objective】To identify genotypes with high photosynthesis capacity and to optimize methods for further screening, genetic variations of photosynthetic gas exchange parameters among exotic sugarcane cultivars was investigated. 【Method】Using a LI-6400 portable photosynthesis system, gas exchange measurements were conducted on the youngest fully-expanded leaves of 50 cultivars introduced from abroad at their grand-growth stage. Six parameters were measured, namely photosynthesis (A), stomatal conductance (g_s), intercellular CO₂ concentration (C_i), transpiration rate (E), intrinsic water use efficiency (WUE_intr), and instantaneous water use efficiency (WUE_inst). Key statistics were determined including broad sense heritability, correlations among parameters. Besides, variation in responses among cultivars was characterized using principal component analysis, while cluster and discriminant analysis were conducted for elite screening. 【Result】 Significant genetic variation was found for every parameter, with the order (highest to lowest variation) being g_s, A, E, C_i, WUE_intr, to WUE_inst. Broad heritability was high (>70%) for all parameters except WUE_inst (which was 58.8%). There were significant correlations between all the parameters except that between WUE_inst and E. Stomatal conductance had strong nonlinear relationships with other parameters, consistent with its pivotal role in regulating leaf gas exchange. Two principal components were extracted through principal component analysis, which could be interpreted as being predominately related to "carbon assimilation" and "water use efficiency" respectively. These two components varied independently among the clones, indicating the possibility of screening for elites harboring both high carbon assimilation and high water use efficiency. Cluster analysis identified a group of six genotypes which had both high carbon assimilation and water use efficiency. 【Conclusion】 Significant variation due to genetic differences in gas exchange parameters exists among exotic sugarcane cultivars. An elite group of cultivars with both high carbon assimilation and water use efficiency were identified, including B4362, B51-410, US67-22, BH10-12, C323-87, and Co685. Methods to optimize large-scale screening in sugarcane breeding programs for favorable photosynthetic capacity were also determined and discussed.

PLANT PROTECTION

New Functions of Ceriporia lacerate in Phytophthora Blight   Control and Growth Promotion of Eggplants

YIN Jie, FAN Qian, HUANG JianGuo

Scientia Agricultura Sinica. 2018, 51(12):  2300-2310.  doi:10.3864/j.issn.0578-1752.2018.12.007

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【Objective】 Bio-pesticides and bio-fertilizers are safe and environment friendly, but need to find more effective strains to increase efficiencies in biological control and fertilization practice. Eggplant (Solanum melongena) is an indispensable vegetable in our country. It is an urgent production problem to effectively control diseases, improve plant nutrition, increase yield and quality. Ceriporia lacerate has been used in human disease treatment, environmental protection, bioenergy and other field. The objective of this study is to explore the potential of this fungus further in plant disease control and nutrition improvement. 【Method】 A new self-isolated C. lacerate (strain HG2011) was grown in Bonnet liquid medium and mixture made of rice husk, vermiculite, and maize powder, respectively, to produce culture broth and solid inoculant. By pure culture, antagonism, pot culture and field experiment, a several experiments were performed to detect extracellular enzyme activity in culture broth, antagonism against Phytophthora capsici on culture plate, biological control of phytophthora blight in greenhouse, nutrient uptake, including nitrogen, phosphorus, and potassium, and changes in yield and quality of eggplant in field as affected by this new fungal strain. 【Result】 C. lacerate HG2011 strain could produce cellulase, β-1,3-glucanase, protease and phosphatase, with the activities of 46.11, 63.02, 199.33, and 27.25 U·mL^-1, respectively, in the culture broth. Moreover, the culture broth C. lacerate HG2011 inhibited the hyphal growth of P. capsici, with inhibition rate of 36.13%-60.59% on antagonism plate. In duel culture, this antagonistic fungus could cover P. capsici colonies and make the hyphae deformed, broken and melted. In greenhouse pot experiment, P. capsic was successful to infect eggplants through inoculation, with phytophthora blight incidence over than 50% and disease index of 64.50. After application of C. lacerate HG2011 culture broth, the disease incidence decreased to 10.50%-18.52%, the disease index was 13.46-20.60 and control efficiency was 68.06%-79.13%. The preventive effect was better than that of therapy. Seedlings supplied with the fungal solid inoculant in combination with chemical fertilizers showed the highest nutrient absorption efficiency, followed by only chemical fertilizer and control (without fertilizer). Compared with sole chemical fertilizer, C. lacerate solid inoculant increased nitrogen, phosphorus, and potassium accumulation in eggplant seedlings by 30.99%-47.72%, 19.97%-43.40%, and 11.21%-41.34%, absorption efficiency by 31.01%-47.74%, 19.80%-43.40% and 11.21%-41.34%, fertilizer partial productivity by 5.88%-18.43%, 5.91%-18.44%, and 5.88%-18.43%, plant biomass by more than 30.00%, and fruit yield by over than 16.06%. Nitrogen, vitamin C, soluble protein, and free amino acids in the fruit were enhanced by 13.86%-20.79%, 62.46%-65.30%, 36.30%-37.67%, and 25.46%-33.08%, respectively, under C. lacerate solid inoculant treatments. 【Conclusion】 C. lacerate HG2011 strain inhibited hyphal growth of P. capsici, made it deform, break and melt, and reduced the incidence and disease index of eggplant phytophthora blight. The preventive effect was better than that of therapy. The application of the fungal inoculant promoted the nutrient absorption, stimulated the seedling growth, increased the yield, and improved fruit quality of eggplants in the field. Therefore, the new C. lacerate HG2011 strain and the founding of its new roles in plant disease control and growth promotion extended its biological functions, enriched the members in microbe source bank, and showed a promising potential in plant protection and fertilization.

Compositive and Inductive Expression Patterns of Protease Inhibitor Genes OsLTPL164 and OsLTPL151 in Rice (Oryza sativa)

HE YuJuan, JU Di, WANG Yue, YANG XueQing, WANG XiaoQi

Scientia Agricultura Sinica. 2018, 51(12):  2311-2321.  doi:10.3864/j.issn.0578-1752.2018.12.008

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【Objective】Protease inhibitors (PI) is a kind of protein widely existed in plants. It has the function of resisting herbivorous insects feeding. However, the expression patterns of rice (Oryza sativa) PI genes in the feeding process of the Chilo suppressalis are not clear. The objective of this study is to analyze the expression patterns of rice protease inhibitor genes OsLTPL164 and OsLTPL151 under C. suppressalis infestation and mechanical damage, which will lay a theoretical foundation for confirming the function of OsLTPL164 and OsLTPL151 in rice against C. suppressalis infestation and constructing transgenic rice strains using these two genes in the future. 【Method】 Three rice lines, Liaoyan 2 (1654), Liaoxing 17 (1665) and Changbai 17 (1688), which were conventionally planted in Northeast China, were selected as the research materials. Artificial infestation and mechanical injury treatments were performed at the tillering stage of rice, which is the key period of C. suppressalis infestation. The root, stem and leaf of three rice lines were sampled after different times (0, 3, 6, 12, 24, 48, 72 h) of treatments, then the expression level of OsLTPL164 and OsLTPL151 of three rice lines was determined by RT-qPCR. 【Result】The tissue expression pattern of the OsLTPL164 was consistent in three rice lines. The expression level of OsLTPL164 in leaf and stem was higher than that in root. The tissue expression pattern of the OsLTPL151 was not consistent in three rice lines. The expression level of OsLTPL151 in the stem and leaf was significantly higher than in root in the 1654 line, but in the 1688 and 1665 lines, the expression level of OsLTPL151 in root was significantly higher than that in stem and leaf. In addition, the two genes in the same tissue among three rice lines had different expression patterns. The expression level of the OsLTPL164 was the highest in 1665 line, followed by 1688 line, and the lowest in 1654 line. The OsLTPL151 exhibited different expression patterns in different tissues among three rice lines: in the root, the expression level of OsLTPL151 in the 1665 line was significantly higher than that in the 1688 and 1654 lines; in the stem, the expression level of OsLTPL151 in the 1654 and 1665 lines was significantly higher than that in the 1688 line; in the leaf, the expression of OsLTPL151 in the 1654 line was significantly higher than that in the 1665 and 1688 lines. Up-regulation of OsLTPL164 and OsLTPL151 in leaf was more obvious than that of stem and root in the three lines by C. suppressalis infestation, and with a higher inducing effect in 1665 line than that in 1688 and 1654 lines. Both OsLTPL164 and OsLTPL151 showed a rise -decrease-rise expression tendency at different time points after artificial infestation and mechanical injury treatments. The expression level of OsLTPL164 and OsLTPL151 was significantly induced after 6 h of mechanical damage treatment, while the expression level of OsLTPL164 and OsLTPL151 could be significantly induced after 3 h of C. suppressalis infestation. 【Conclusion】The expression of OsLTPL164 and OsLTPL151 in three conventionally grown rice lines can be induced by C. suppressalis infestation and mechanical damage treatment, suggesting that both the two genes are involved in resistance to C. suppressalis. However, other specific functions of the two genes in different rice lines might be different.

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

The Characteristics and Evaluation of Wheat Fertilization in Shandong Province

LI JianMin, ZHAO GengXing, LI Tao, XIAO Yang, ZHOU Xue, YUE YuDe

Scientia Agricultura Sinica. 2018, 51(12):  2322-2335.  doi:10.3864/j.issn.0578-1752.2018.12.009

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【Objective】Entering the 21st century, the contradiction of population, resources and environment is becoming more and more prominent, and Chinese agriculture is facing a serious challenge in many aspects. Shandong province is a typical intensive agricultural area of high investment and high output in northern China. The agricultural development in Shandong province has set a template for other provinces. The research and analysis of the fertilization status in this province can play a certain role of predictive and early warning in the management of crop fertilization in the whole country. This study analyzed the fertilization status of winter wheat in Shandong province, aiming to provide scientific basis for the macro management of winter wheat fertilization, which has practical and wide significance.【Method】The fertilization status and characteristics of wheat were clarified by using investigation analysis and statistics analysis based on the data and statistics of Soil Testing Formula Applying Fertilizer Project in Shandong province. Then the optimal fertilization model was established and the fertilization parameters of wheat were identified with MATLAB modeling and analysis.【Result】In 2015, fertilizer expend coefficient (Fec) of winter wheat in Shandong Province declined 5.71% compared to 2010. The average fertilizer amount of nitrogen, phosphorus and potassium in winter wheat was higher than the national average. The proportion of phosphate fertilizer was excessive and the proportion of potassium fertilizer was inadequate. The multi-element fertilizer ratio of basal and topdressing in winter wheat was increased, while the straight fertilizer ratio was decreased. For the total provincial fertilization, nitrogen and phosphorus were declined from west to east; the western and northern plain of Shandong were the highest; the eastern hilly area of Shandong Province was the lowest; the potassium was on the contrary. The nitrogen and phosphorus fertilization were the highest in fluvo-aquic soil, followed by Shajiang black soil, cinnamon soil and brown soil; saline-alkali soil was at a lower rate; potassium fertilization was the highest in brown soil, followed by Shajiang black soil and cinnamon soil; saline-alkali soil and fluvo-aquic soil were at a lower rate. There is a mismatching situation between NPK fertilization and total N, available P, rapidly available K. It could appropriately increase the amount of nitrogen in the eastern hilly area, reduce fertilization input in the high-yield area, and increase fertilization input in low-yield area. With the target of yield, the optimal fertilization application of nitrogen, phosphorus and potassium for winter wheat was 182.02, 82.58 and 83.22 kg·hm^-2, respectively,in Shandong province. By contrast, the nitrogen and phosphorus were overstepped 25.60 kg·hm^-2 and 37.77 kg·hm^-2, respectively. And potassium was short of 3.84 kg·hm^-2.【Conclusion】The present situation of winter wheat fertilization in Shandong province is making the benign developments. But there still exist many problems, such as the amount of fertilizer is too high and the way and proportion of fertilization are not reasonable. The results of this research have positive significance to rational fertilization, resources conservation and environmental protection of winter wheat in Shandong province.

Effects of Long-Term Additional Application of Organic Manure or Straw Incorporation on Soil Nitrogen Leaching Risk

GAI XiaPu, LIU HongBin, ZHAI LiMei, YANG Bo, REN TianZhi, WANG HongYuan, WU ShuXia, LEI QiuLiang

Scientia Agricultura Sinica. 2018, 51(12):  2336-2347.  doi:10.3864/j.issn.0578-1752.2018.12.010

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【Objective】In order to provide a basis for promoting soil fertility, increasing crop yield and reducing non-point source pollution for rotation of winter wheat and summer maize in North China Plain, it is necessary to study the effects of long-term additional application of organic manure/straw incorporation on crop yield and soil nitrogen leaching risk.【Method】Considering a 27-year long-term fertilizer experiment in Fluvo-aquic soil in Changping County, Beijing, China as the research platform, five treatments were set up, including namely control (CK), chemical fertilizers (NPK), NPK + organic manure (NPKM), NPK+ 50% more organic manure (NPKM+) and NPK + straw incorporation (NPKS). And then, crop yield of wheat and maize, soil fertility, nitrogen leaching risk and soil nitrogen distribution characteristics under different fertilization treatments were analyzed. 【Result】Results showed that (1) long-term additional application of organic manure or straw incorporation could increase crop yield and improve soil fertility during the past 27-year. Among these different fertilizer treatments, additional application of organic manure was particularly excellent. Compared with NPK, the yield of wheat and maize could increase by 41%-50% and 30%-32% under NPKM and NPKM+ treatments, respectively. Meanwhile, NPKM and NPKM+ treatments could increase soil organic carbon (SOC) and total nitrogen (TN) in 0-20 cm soil by 62%-121% and 107%-187%, respectively. However, the partial factor productivity (PFP_N) in wheat and maize season was reduced by 22%-32% and 27%-41%, respectively. Contrarily, the effects of straw incorporation on yield enhancement and soil nutrients improvement was lower than that of additional application of organic manure. The yield of wheat and maize and SOC and TN contents were increased by 24%, 6%, 9%, 97% under straw returning, respectively, relative to NPK. However, the PFP_N in wheat season was increased by 216% while was reduced by 40% in maize season. (2) SOC, TN, nitrate nitrogen (NO₃^--N), soil dissolved organic carbon, soil dissolved organic nitrogen contents and the microbiological processes of soil nitrogen mineralization rate and nitrification potential in 0-20 cm soil depth were all higher than that of 20-200 cm soil depth, which indicated that the effects of long-term additional application of organic manure or straw incorporation on soil nutrients and microbiological processes mainly occurred in the surface soil. Compared with NPK, NPKM treatment could significantly increase NO₃^--N content in the 100-200 cm soil layer and the average value of NO₃^--N was 17.8-26.1 mg·kg^-1. NPKS treatment could increase soil NO₃^--N content in the 0-100 cm soil depth to some extent and the average content of NO₃^--N was 3.6-13.4 mg·kg^-1. This indicated that additional application of organic manure could promote the downward migration of soil NO₃^--N and straw returning could retain soil NO₃^--N. As introducing additional nitrogen from manure or straw, the nitrogen surplus was increased by 312%, 1 037%, 953% in NPKM, NPKM+, NPKS treatments compared with NPK treatment, suggesting a relatively high nitrogen leaching risk.【Conclusion】Based on the conventional fertilization, long-term additional application of organic manure and straw incorporation could increase crop yield and soil fertility, but also promote soil nitrogen surplus and nitrogen leaching risk. Especially, long-term additional application organic manure could increase nitrogen leaching risk.

Nitrogen Volatilization Characteristics of Different Materials Coated Nitrogen Fertilizer and Its Effect on Rape Yield

JIANG Yifei, ZHANG YanMing, YANG Ming,YU Na, ZHANG YuLing, ZOU HongTao, ZHANG YuLong

Scientia Agricultura Sinica. 2018, 51(12):  2348-2356.  doi:10.3864/j.issn.0578-1752.2018.12.011

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【Objective】The application of coated slow/controlled release fertilizer is an important method for reducing nitrogen loss and increasing nitrogen use efficiency. A new type of organic-inorganic materials coated fertilizer has advantages of excellent release performance and environmentally friendly, etc. Studies on nitrogen volatilization characteristics of organic-inorganic materials coated fertilizer could provide scientific basis for the development and application of coated slow/controlled release fertilizers. 【Method】 In our study , the coated fertilizers were prepared by encapsulating urea granules with blend films of modified polyvinyl alcohol (PVA) and inorganic material including diatomaceous earth (Ag)，zeolite powder (Af), biochar (Ac), phosphate rock (Ap) and sulfur (As). A laboratory culture experiment was conducted to demonstrate the characteristics of ammonia volatilization and nitrogen oxide emissions from soil treated with various coated fertilizers through determining soil ammonia volatilization rate and nitrogen oxides emission rate in 60 days. A pot culture experiment was also carried out to study the effects of different coated fertilizer applications on growth and yield of rape.【Result】Ammonia volatilization began at the first day of incubation after fertilizer incorporating into soil. Moreover, Ammonia volatilization rate for different treatments reached maximum values within 10 days after incubation. The peak values for CK, Ag, Af, Ac, Ap and As were 1.132 mg·L^-1·d^-1, 0.373 mg·L^-1·d^-1, 0.508 mg·L^-1·d^-1, 0.696 mg·L^-1·d^-1, 0.347 mg·L^-1·d^-1, 0.304 mg·L^-1·d^-1, respectively. The peak values of soil ammonia volatilization treated with coated fertilizers appeared later than urea treatment, indicated that the blend film encapsulated urea granules could prolong urea dissolved time and subsequently slow urea dissolution rate through effectively preventing the external which could effectively prevent the external water from contacting urea. Generally, ammonia volatilization rate exhibited a steady trend after dramatic variation. The total ammonia volatilization for treatments of CK, Ag, Af, Ac, Ap and As were 104.0 mg, 88.2 mg, 93.4 mg, 95.6 mg, 81.9 mg, 79.4 mg respectively. The total ammonia volatilization from these five coated fertilizer treatments were 15 % (Ag), 10 % (Af), 8 % (Ac), 21 % (Ap) and 23 % (As) lower than that of urea treatment (CK). The emission characteristics of nitrogen oxide for different coated fertilizers were similar with ammonia volatilization. Compared with ammonia volatilization, the peaks of NOx emission obviously shifted rearward, which were observed between 6 and 23 days. The peak values of NOx emission for CK, Ag, Af, Ac, Ap and As were 0.092 mg·L^-1·d^-1, 0.033 mg·L^-1·d^-1, 0.039 mg·L^-1·d^-1, 0.051 mg·L^-1·d^-1, 0.027 mg·L^-1·d^-1, 0.022 mg·L^-1·d^-1, respectively. NOx emission accumulation for CK, Ag, Af, Ac, Ap and As were 15.8 mg, 11.1 mg, 12.4 mg, 13.2 mg, 10.3 mg, 8.5 mg, respectively. The overall nitrogen oxides emission from coated fertilizer treatment was lower than that of uncoated urea treatment. Ammonia volatilization from different treatments accounted for 80-90 % of nitrogen gaseous loss. The rape yield of coated fertilizer treatment was higher than that of CK. The yield increase for treatments were diatomaceous earth (Ag) 47%, zeolite powder (Af) 37%, biochar (Ac) 31%, phosphate rock (Ap) 52%, sulfur (As) 63%, respectively.【Conclusion】In our study, ammonia volatilization was the main form of nitrogen gaseous loss from soil treated with coated fertilizers, which mainly occurred in the first two weeks after fertilization. The organic-inorganic materials coated fertilizers could inhibit ammonia volatilization and nitrogen oxides emission from soil. Ultimately, these coated fertilizers increased rape yield.

HORTICULTURE

Full Length Cloning, Expression and Correlation Analysis of P450 CYP79F1 Gene with Sulforaphane Content in Different Broccoli Organs

LI ZhanSheng, LIU YuMei, FANG ZhiYuan, YANG LiMei, ZHUANG Mu, ZHANG YangYong, Lü HongHao

Scientia Agricultura Sinica. 2018, 51(12):  2357-2367.  doi:10.3864/j.issn.0578-1752.2018.12.012

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【Objective】The aim of this study was to clone the full length of P450 family CYP79F1 gene from broccoli, investigate its sequence characteristic and analyze its expression and correlation with sulforaphane content in different developmental broccoli organs. These findings will provide a scientific basis for further revealing the mechanism of sulforaphane metabolism in broccoli and lay a practical foundation for the genetic improvement and breeding of broccoli varieties. 【Method】 The full length sequence of CYP79F1 gene was obtained by RACE cloning (5′ and 3′). According to the software of DNAMAN 6.0, gene splicing, amino acid sequence analysis and protein secondary structure prediction of CYP79F1 gene, at the same time, bioinformatics information was also predicted by online program and software. The quantitative real-time PCR (qRT-PCR) method was used to study the expression of CYP79F1 gene in different developmental organs of broccoli. The sulforaphane contents of different developmental organs were detected by high performance liquid chromatography (HPLC) method, and they were roots, stems, leaves, developmental buds at bolting stage (top buds, mature buds, buds one day before flowering and flowers), and seeds. Pearson correlation analysis was carried out by the software SPSS 10.0.【Result】Full length sequence of CYP79F1 gene were cloned, and the gene of CYP79F1 was 2 014 bp in length, containing a 1 620 bp opening reading frame (ORF), encoded a polypeptide of 540 amino acids. They shared over 95% identity with the homologous proteins from Brassica plants, such as cabbage, Chinese cabbage, Chinese kale and rape. The bioinformatics analysis showed that the CYP79F1 protein was hydrophilic protein with two signal-peptides and two transmembrane regions, and the Wolf Psort protection indicated that they were located in the cytoplasm. The expression of CYP79F1 gene was higher in the roots and the stems, and the lowest in the leaves. During developmental stage of buds, the expression of CYP79F1 gene was up-regulated in the early (young buds), and then decreased (mature buds to flowers). There was no difference significance in gene expression between roots, stems, leaves, developmental buds at bolting stage and seeds. Correlation analysis showed that the expression level of CYP79F1 gene showed an extremely significant positive correlation with sulforaphane content in developmental buds at bolting stage (R=0.96，P<0.05), and the expression of CYP79F1 gene might influence the generation of sulforahane in different organs of broccoli, especially in developmental buds.【Conclusion】The CYP79F1 gene were comprehensively obtained and characterized from broccoli, CYP79F1 may play an important role in sulforaphane metabolism and revealing the diversity of sulforaphane content in different organs of broccoli.

The Regulations of the MYBA1 in UFGT and DFR from the Grape Berries

NIU TieQuan, DONG YanMei, LIU HaiXia, ZHANG XiaoJun, GAO Yan, ZHANG PengFei, LIANG ChangMei, WEN PengFei

Scientia Agricultura Sinica. 2018, 51(12):  2368-2377.  doi:10.3864/j.issn.0578-1752.2018.12.013

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【Objective】MYBA1 transcription factor plays an important role in the biosynthesis of anthocyanins. To further explore the synergistic regulatory mechanism of anthocyanins, we analyzedthe mutual mechanism and expression pattern of VvMYBA1, VvUFGT, VvDFR and content of anthocyanins during grape berry development.【Method】The real-time PCR, spectrophotometer and SAS8.0 were used to analyze the expression patterns of VvMYBA1, VvUFGT, VvDFR content of anthocyanins and correlation in different time periods, respectively. The transcriptional activity of VvMYBA1 and the interactions between the VvMYBA1 and VvUFGT, VvDFR were determined by yeast hybrid system. 【Result】The expression pattern analysis showed that the expression of VvMYBA1, VvUFGT and VvDFR was increased until veraison (60-80 d after full bloom) and then declined. The content of anthocyanins was fist increased until veraison (80 d after full bloom) and then stabilization. Correlation analysis showed that the expression of MYBA1 with expression ofthe DFR, UFGT had positive correlation, and the content of anthocyanins with the expression of MYBA1, DFR, UFGT had positive correlation. Yeast hybrid assay indicated that VvMYBA1 had transcriptional activation functions which could specifically combine promoter of VvUFGT, VvDFR and had no interaction with VvUFGT and VvDFR encoded proteins.【Conclusion】The content of anthocyanins was significantly correlated with expression of VvMYBA1, VvDFR, and VvUFGT. The VvMYBA1 had transcriptional activation functions. All results indicated that VvMYBA1 could regulate the accumulation of anthocyanins in grape berry development by regulating VvUFGT and VvDFR expression combined with VvUFGT and VvDFR promoter specifically.

Leaf Nutritional Diagnosis of Powell Navel Orange at Flowering Stage in Chongqing Three Gorges Reservoir Area

ZHENG YongQiang, WANG Ya, YANG Qiong, JIA XueMei, HE ShaoLan, DENG Lie, XIE RangJin, YI ShiLai, Lü Qiang, MA YanYan

Scientia Agricultura Sinica. 2018, 51(12):  2378-2390.  doi:10.3864/j.issn.0578-1752.2018.12.014

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【Objective】 The objective of this study is to evaluate leaf nutritional status by investigating the mineral elements content in the leaves of Powell navel orange orchard at flowering stage in Jiangjin and Fengjie regions of Chongqing in the Three Gorges Reservoir Area, and to provide a basis for formulating fertilization scheme of Powell navel orange. 【Method】 Field experiments were conducted in the forty representative 12-year-old Powell navel orange plots from Jiangjin and Fengjie regions in Chongqing Three Gorges Reservoir. The plot yield and content of nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), iron (Fe), copper (Cu), manganese (Mn), zinc (Zn) were measured. The compositional nutrient diagnosis (CND), diagnosis and recommendation integrated system (DRIS), and deviation from optimum percentage (DOP) methods were used to diagnose the leaf nutritional status for the low-yield groups. 【Result】 The leaf analysis of CND indicated that the yield cutoff value of high-yield orchard was 330 t·hm^-2, only 6 plots from Fengjie fulfilled this criterion, accounting for 15.0% of 40 plots. The leaf nutrition optimum values at flowering stage were N (2.0±0.1) %, P (0.12±0.01) %, K (2.1±0.5) %, Ca (3.1±0.4) g·kg^-1 DW, Mg (0.31±0.03) g·kg^-1 DW, Fe (36.6±13.1) mg·kg^-1 DW, Mn (51.4±21.6) mg·kg^-1 DW, Cu (2.2±0.7) mg·kg^-1 DW and Zn (12.3±1.5) mg·kg^-1 DW according to the leaf nutrition concentration in Fengjie high-yield group. The CND norms had been established with V_N*=3.62±0.07, V_P*=0.78±0.08, V_K*=1.36±0.21, V_Ca*=1.74±0.14, V_Mg*=-0.55±0.10, V_Fe*=-2.74±0.36, V_Mn*=-2.40±0.39, V_Cu*=-5.55±0.32, V_Zn*=-3.78±0.10, respectively. Meanwhile, there were 30 (N/K, N/Fe, N/Cu, P/K, P/Fe, P/Cu, K/Fe, K/Cu, Ca/N, Ca/P, Ca/K, Ca/Fe, Ca/Mn, Ca/Cu, Mg/N, Mg/P, Mg/K, Mg/Fe, Mg/Cu, Mn/N, Mn/P, Mn/K, Mn/Mg, Mn/Fe, Mn/Cu, Mn/Zn, Zn/Ca, Zn/Mg, Zn/Fe, Zn/Cu) and 14 (Ca/K, Mg/K, Mg/Zn, Mn/N, Mn/P, Mn/K, Mn/Ca, Mn/Mg, Mn/Fe, Mn/Cu, Cu/P, Cu/K, Cu/Mg, Cu/Fe) important DRIS norms in Jiangjin and Fengjie regions by DRIS method, respectively. Moreover, CND, DRIS, and DOP were used for the leaf nutrition diagnosis in producing the low-yield subpopulation of Fengjie plots and Jiangjin region. Among them, the fertilization order was confirmed with Ca＞Mg＞N＞P＞Mn in Jiangjin region and N＞Ca＞P＞Zn＞Fe＞Mn in Fengjie region by CND method; Ca＞Mg＞＞Mn＞N＞P in Jiangjin region and Zn＞＞Fe＞Ca＞Mn＞N＞Mg in Fengjie region by DRIS method; Ca＞Mn＞Mg in Jiangjin region and Mn＞＞Zn＞Fe＞Ca＞Mg＞N in Fengjie region by DOP method.【Conclusion】The average nutrient balance index of CND, DRIS and DOP was 166.5, 4 291.0, and 117.5 in Jiangjin region, respectively. the average nutrient balance index of CND, DRIS and DOP was 37.2, 570.0, and 14.1, respectively. The leaf nutritional status in Fengjie region was superior to that in Jiangjin region, and both areas had a great potential of production.

FOOD SCIENCE AND ENGINEERING

The Characters and Influence Factors of Stable Isotope Fingerprints in Yak Muscle

GUO BoLi, WEI YiMin, WEI Shuai, SUN QianQian, ZHANG Lei, SHI ZhenQiang

Scientia Agricultura Sinica. 2018, 51(12):  2391-2397.  doi:10.3864/j.issn.0578-1752.2018.12.015

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【Objective】the characters of carbon, nitrogen and hydrogen isotopic composition in yak (Bos grunniem) muscle and the effect of geographical origin, pasture and drinking water were analyzed, which would provide the technical support for tracing and identifying the yak meat. 【Method】The yak muscle, pasture and water samples were collected from Haibei, Hainan and Yushu of Qinghai province, and longitude, latitude and altitude of sampling site were recorded by using GPS. The carbon, nitrogen and hydrogen stable isotopic ratio in samples were tested by using EA-IRMS. 【Result】The average value of δ¹³C in yak de-fatted muscle and fat was (-23.99±0.25)‰ and (-28.77±0.50)‰, respectively, and the average value of δ¹⁵N and δ²H in yak muscle was (4.04±0.91)‰ and (-107.99±11.08)‰, respectively. The δ¹³C and δ¹⁵N values in yak muscle were closely related to the pasture of the Tibetan Plateau, and the region had certain effect on δ¹³C values of yak muscle, indicating the δ¹³C values increased with the altitude increasing. The δ²H values of pasture and water samples among three regions of Haibei, Hainan and Yushu of Qinghai province showed significant differences. The order of δ²H values in pasture was Hainan＞Haibei＞Yushu, and the order of δ²H values in water was Haibei＞Hainan＞Yushu. The order of δ²H values in yak muscle was Hainan＞Haibei＞Yushu, and it was the same order as the pasture. The results indicated that the δ²H values in yak muscle were closely related to the drinking water and pasture, and they were decreased with altitude increasing, and the pasture probably had more effect on δ²H values in yak muscle than water. But it needs to be further researched and confirmed.【Conclusion】The stable isotopic fingerprints in yak muscle were closely related to the pasture, drinking water and terrain of high altitude, which had the unique fingerprints character.

ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT

Breeding by Molecular Writing (BMW): the Future Development of Animal Breeding

LIU ZhiGuo, WANG BingYuan, MU YuLian, WEI Hong, CHEN JunHai, LI Kui

Scientia Agricultura Sinica. 2018, 51(12):  2398-2409.  doi:10.3864/j.issn.0578-1752.2018.12.016

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With the rapid development of genomics and genome editing techniques, and the extensive application of techniques such as microinjection and somatic cell nuclear transfer, a new set of breeding strategies and methods has gradually formed, which we named breeding by molecular writing (BMW). Using BMW, directional breeding of new varieties can be achieved by molecular-level genome editing, which can not only break down reproductive barriers separating different taxa, allowing the cross-species introduction of new traits, but also enable single-nucleotide insertion, deletion, and substitution in individual genomes. This can include the precise integration of exogenous genes; precise deletion and substitution of endogenous genes; and replication, deletion, and substitution of SNP loci. The main advantage of BMW is the rapid and efficient gathering of several beneficial traits into one species, while significantly reducing unintended effects. Molecular writing can be used for the following tasks: (1) identification and verification of new breeding markers; (2) cross-species molecular writing; (3) base sequence deletion in genomes; and (4) molecular writing within species. The BMW technique allows the introduction of only one or several target genes or SNPs and the rapid acquisition of new stable genetic varieties with pronounced target characters without sexual hybridization, and can then create new varieties in combination with conventional breeding methods. BMW can achieve genetic (or molecular) hybridization breeding at the individual and group levels, acquiring molecular heterosis, efficiently resolving several long-standing difficulties in breeding, and significantly improving breeding efficiency. It has strong potential for application in livestock and poultry breeding, and is a key part of the future of breeding. This paper discusses the basic concepts, research methods, research contents, and current research status of breeding by molecular writing in detail, and presents the prospects of applying BMW, providing references for researchers and practitioners in fields such as animal breeding and livestock and poultry reproduction.

Genetic Parameter Estimation of Cashmere Yield and Body Weight at Different Staple Types of Inner Mongolian Cashmere Goats

LI XueWu, LIU Yan, WANG RuiJun, WANG ZhiYing, NA Qing, LI HongWei, WANG ZhenYu, XU BingBing，SU Rui, ZHANG YanJun, LIU ZhiHong, LI JinQuan

Scientia Agricultura Sinica. 2018, 51(12):  2410-2417.  doi:10.3864/j.issn.0578-1752.2018.12.017

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【Objective】 Inner Mongolian Cashmere Goats (IMCGs) is an excellently breeding which keeps for both cashmere and meat production by long-term natural selection and artificial selection. And IMCGs Distributed in Erlang, Arbas and Alxa. The cashmere yield (CY) and body weight (BW) were important economic traits, which belonged to quantitative traits and were controlled by the microscopic polygene. The genetic parameters of the CY and the BW of IMCGs were evaluated by quantitative genetic methods. The aim of this study was to investigate the effects of different staple length types on the phenotypic characteristics and genetic parameters of CY and BW for Inner Mongolia cashmere goats, so as to provide a theoretical basis for Cashmere goat breeding. 【Method】 In this study, the repetitive data of CY, BW and staple length (SL) of 54 044 Inner Mongolia White Cashmere goats from 1990 to 2014 were collected in a breeding farm. The staple length of IMCGs were divided into three types: short-staple length (≤13cm, SSL), intermediate-staple length (＞13cm and ≤22cm, ISL), and long-staple length (＞22cm, LSL). Excel software was used to analyze the phenotypic of CY and BW. Then, the REG program of SAS 9.2 was employed to calculate the regression coefficient of CY and BW at different staple length that determined the effects of different staple length types on CY and BW. At last, the average information restricted maximum likelihood (AIREML) method in a multivariate animal model on wombat software was used to analyze variance components and estimate the genetic parameters. 【Result】According to the basic statistical analysis of different types of CY and BW, it was found that the average of staple length at LSL increased 6.20 cm and 13.40 cm than that at ISL and SSL, respectively. The CY of was 105.03 g and 59.85 g higher than that at ISL and SSL, respectively. And the BW of LSL was 8.78 kg and 10.06 kg higher than that at ISL and SSL, respectively. According to the basic statistical analysis CY and BW at different staple types, it was found that the CY (721.15 g) was highest and BW (41.98 kg) was highest weight in three types. The variation coefficient of SL and CY decreased with increasing of staple length, but the coefficient of variation of BW increased with the increasing of staple length. Coefficient of variation of CY and BW were above 27%, which indicated that CY and BW had higher potential for improvement. The result showed that the heritabilities of CY were 0.14, 0.22 and 0.33 in SSL, ISL and LSL, respectively. And the heritabilities of CY in SSL belong to the low heritability, while the heritability of CY in ISL and LSL was the moderate heritability. The heritabilities of BW were 0.10, 0.11 and 0.12 in SSL, ISL and LSL, respectively, which belonged to the low heritability. The heritabilities for CY and BW of LSL were highest in three types. The genetic correlation between CY and BW (-0.20 ~ 0.26), CY and SL (-0.11 ~ 0.37) and BW and SL (0.21-0.43) were increased with increasing of staple length. 【Conclusion】The individuals of LSL had the highest phenotypic value of CY and BW, and the heritability of CY and BW were highest, and had the highest genetic correlation among the traits. Therefore, selecting of LSL could accelerate genetic progress for CY and WT and improve the CY and BW, so this study laid the theoretical foundation for the indirect selection in other important economic traits.

RESEARCH NOTES

The antibody preparation and expression analysis of Chitinase 5-1 in Locusta migratoria

ZHANG TingTing, LIU WeiWei, GAO Lu, LI RenJian, FU SuiYe, LIU XiaoJian, LI DaQi, LIU WeiMin, DONG Qing, ZHANG JianZhen

Scientia Agricultura Sinica. 2018, 51(12):  2418-2428.  doi:10.3864/j.issn.0578-1752.2018.12.018

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【Objective】 The objective to this study is to prepare polyclonal antibody of Locusta migratoria chitinase5-1 (LmCht5-1), establish method for detection of LmCht5-1 protein, understand the expression pattern of LmCht5-1 protein in the 4thinstar nymph and finally confirm its location and function in vivo.【Method】LmCht5-1 antigen structure area, which was selected by sequence alignment between LmCht5-1 and LmCht5-2 with MEGA software and antigen epitope prediction analysis with the website of Expression, was amplified using cDNA of L. migratoria as the template. The LmCht5-1 antigen sequence was cloned into expression vector pET32a-OVA, which contains ovalbumin to improve immunogenicity, to generate the antigen expression recombinant plasmid pET32a-OVA-LmCht5-1. pET32a-OVA-LmCht5-1 was then transformed into E. coli strain BL21 (DE3). The IPTG induced OVA-LmCht5-1 was purified through Ni-NTA system and used as the antigen to generate polyclone antibody by rabbit immunization. The titer and specificity of OVA-LmCht5-1 were detected by ELISA and Western blot analysis, respectively. Then the expression pattern of LmCht5-1 in cuticle of the 4th instar nymph was analyzed in parallel by Western blot. Finally, the intracellular localization and function of LmCht5-1 in the 4thinstar nymph cuticle were detected by immunohistochemical analysis after the dsLmCht5-1 injection. 【Result】The 5′ fragment (471-533AA) of LmCht5-1, which was selected as the antigen area with the sequence alignment between LmCht5-1 and LmCht5-2 and the epitope prediction analysis, was inserted into pET32a-OVA to obtain recombinant plasmid pET32a-OVA-LmCht5-1. The 71.34 kD recombinant protein OVA-LmCht5-1 was expressed, purified and immunized with rabbit to generate polyclone antibody. The titer of anti-serum of OVA-LmCht5-1 was 1﹕102 400 by ELISA detection. Antibody OVA-LmCht5-1 could accurately distinguish the LmCht5-1 but not LmCht5-2 in Western blot analysis. Further, it was found that the protein expression level of LmCht5-1 increased gradually with the age in the 4th instar nymph of L. migratoria and reached to the highest at the same day of molting, while it declined to the lowest after molting. Injection dsLmCht5-1 into the 4th instar nymph led to the expression of LmCht5-1 significantly declined to 70.0% in mRNA level and 73.6% in protein level. The immunohistochemical analysis with the epidermis of L. migratoria after dsLmCht5-1 injection, showed that LmCht5-1 was located in old cuticle and epidermis cell. Finally, it was found that the inhibition of protein expression of LmCht5-1 led to the chitin degradation blocking in old cuticle. 【Conclusion】 High titer and specificity of LmCht5-1 antibodies, which could be used for Western blot and immunohistochemistry analysis were obtained. The protein expression of LmCht5-1 reached to the highest level at the day of molting in 4th instar nymph. LmCht5-1 was located in the epidermis cell and old cuticle in the 4th instar nymph. Moreover, the injection of dsLmCht5-1 to L. migratoria could inhibit the protein expression of LmCht5-1 in epidermis cells and old cuticle, and finally caused the chitin degradation deficiency in old cuticle.