Table of Content

16 September 2020, Volume 53 Issue 18

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

Evaluation of Resistance to Stripe Rust and Molecular Detection of Important Known Yr Gene(s) of 152 Chinese Wheat Landraces from the Huang-huai-hai

GUAN FangNian,LONG Li,YAO FangJie,WANG YuQi,JIANG QianTao,KANG HouYang,JIANG YunFeng,LI Wei,DENG Mei,LI Hao,CHEN GuoYue

Scientia Agricultura Sinica. 2020, 53(18):  3629-3637.  doi:10.3864/j.issn.0578-1752.2020.18.001

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【Objective】Stripe rust of wheat (Triticum aestivum L.), caused by Puccinia striiformis f. sp. tritici, has been considered one of the most damaging diseases for wheat production in Huang-huai-hai. Identifying new resistance sources and understanding the distribution of wheat stripe rust-resistance genes in wheat landraces in this region may provide valuable resistance germplasms and molecular basis for predictive resistance rust-breeding program. 【Method】A total of 152 wheat landraces derived from Huang-huai-hai were evaluated at seedling stage in greenhouse with two Chinese predominant stripe rust races CYR32 and CYR34, and at adult-plant stage under field conditions with mixed races of CYR32, CYR33, CYR34, Su11-4 and Su11-5 in Chongzhou and Mianyang, Sichuan, during 2016-2017 and 2018-2019, respectively. The panel of wheat landraces was screened with the closely linked molecular markers of stripe rust resistance genes Yr9, Yr10, Yr18, Yr24/26, Yr30, Yr36, Yr39, Yr41, Yr48, Yr65, Yr67, Yr80, and Yr81. 【Result】Among the 152 accessions, two accessions were resistant to CYR32 and seven to CYR34, and two accessions were resistant to both CYR32 and CYR34 at seedling stage. Thirty-five (23.03%) were stably resistant to at adult-plant stage. Base on the seedling and adult-plant stage reactions, seven (4.61%) shown resistance at the all stage. Molecular detection indicated that 131 accessions carried Yr18, two carried Yr41, thirteen carried Yr48, fifty-seven carried Yr81, including 2 carried both Yr18 and Yr41, eight carried both Yr18 and Yr48, forty-nine carried both Yr18 and Yr81, seven carried both Yr81 and Yr48. All of the tested materials did not contain Yr9, Yr10, Yr24/26, Yr30, Yr36, Yr39, Yr65, Yr67 and Yr80. Simultaneously, no Yr genes were detected in the other five resistant wheat landraces and it indicated that these wheat landraces may carry other known or unknown stripe rust resistance genes. 【Conclusion】Most of wheat landraces from the Huang-huai-hai shown resistance for the current prevalence races of stripe rust at adult-plant stage and the accessions were mainly carried Yr18 and Yr81 which derived from Chinese landraces. These resistant wheat landraces may contain other known or unknown genes/their combinations and could be used as valuable resistance resources for stripe rust in wheat breeding program.

Generation of Marker-Free Transgenic Barley Plants by Agrobacterium-Mediated Transformation

GONG Qiang,WANG Ke,YE XingGuo,DU LiPu,XU YanHao

Scientia Agricultura Sinica. 2020, 53(18):  3638-3649.  doi:10.3864/j.issn.0578-1752.2020.18.002

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【Objective】Genotype dependence is a very serious problem in barley genetic transformation in which Golden Promise has been mainly used. In addition, the transformation efficiency of barley is still very low in China. Moreover, the generation of marker-free transgenic barley plants is very important to the future commercialization of genetically modified barley due to the considerably increased public concerns. It is necessary to establish an efficient genetic transformation system for generating marker-free transgenic barley plants, expand available barley genotypes for genetic transformation, and provide technical support for functional genomics study and breeding and potential commercialization of genetically modified varieties of barley. 【Method】The composition and auxin adding ratio in the medium used for barley transformation as well as culture regime was optimized to establish an efficient Agrobacterium-mediated genetic transformation system for barley using the immature embryos approximately 14 days post anthesis of a good agronomic trait variety Vlamingh. A double T-DNA vector pWMB123 containing the Bar gene and the GUS gene cassette was introduced into barley by Agrobacterium-mediated transformation to obtain marker-free transgenic barley plants in T₁ generation. 【Result】 It is indicated that the shoot induction medium supplemented with 1.0 mg·L^-1 KT, 0.5 mg·L^-1 6-BA and 0.05 mg·L^-1 NAA significantly promoted the differentiation ability of barley transformed calli. The adding of 2.5 mg·L^-1 copper sulfate in shoot induction medium lightened the albinism of transgenic barley plantlets. Through the exploration of different hormone ratios and medium components, it was found that the SM1 supplemented with 1.0 mg·L^-1 IBA without other auxin had the best rooting effect. A total of 138 immature embryos were transformed with Agrobacterium containing pWMB123 vector, and fourteen transgenic barley plants were obtained with a transformation efficiency of 10.14%. Detection by PCR, Quick Stix strips and histochemical staining assays confirmed that all T₀ plants contained Bar gene, among which four plants did not contain GUS gene. The co-transformation efficiency of the two T-DNAs is 71.43%. In the T₁ populations derived from the four T₀ transgenic lines containing Bar and GUS genes, two transgenic plants with GUS gene and without Bar gene were screened out in line BL8, and the efficiency for marker-free transgenic plants was 6.9%. The two foreign genes of Bar and GUS were identified to be integrated in most T₁ transgenic barley plants be multiple copies using Southern blot. It was further confirmed that BL8-15 and BL8-19 were marker-free transgenic plants. 【Conclusion】An efficient genetic transformation system mediated by Agrobacterium-mediated for generating marker-free transgenic plants with healthy roots and less albinism were successfully established in barley.

Gene Expression and Salt-Tolerance Analysis of MsDWF4 Gene from Alfalfa

CUI MiaoMiao,MA Lin,ZHANG JinJin,WANG Xiao,PANG YongZhen,WANG XueMin

Scientia Agricultura Sinica. 2020, 53(18):  3650-3664.  doi:10.3864/j.issn.0578-1752.2020.18.003

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【Objective】 Cloning the Brassinolide (BRS) synthetase gene MsDWF4 of alfalfa (Medicago sativa L.), analyzing the gene characteristics and gene expression pattern, performed the salt-tolerance analysis, therefore to clarify the function of MsDWF4 in abiotic stress resistance and provide a reference for revealing the molecular mechanism of MsDWF4 in regulating abiotic stress resistance in alfalfa. 【Method】 The homologous gene of DWF4 in M. sativa was cloned by using homology-based cloning according to the known DWF4 gene sequence of Arabidopsis. Sequence characteristics were analyzed by bioinformatics tools. Quantitative Real-time PCR (qRT-PCR) was applied for detecting the gene expression in different alfalfa tissues and the expression patterns under multiple abiotic stress (high temperature, cold damage, drought and high salt) and hormones treatment (auxin, brassinolide, abscisic acid and jasmonate). The MsDWF4 overexpression vector was constructed and transformed into alfalfa by Agrobacterium mediated transformation, and MsDWF4 overexpression transgenic alfalfa plants were obtained. The transgenic alfalfa lines were treated with high salt (200 mmol·L ^-1 NaCl) and the activities of antioxidant enzymes were analyzed to study whether MsDWF4 could improve the salt tolerance of alfalfa. 【Result】 The length of MsDWF4 CDS was 1 470 bp, which encoded a protein of 489 amino acids, belonging to the single oxygenases of cytochrome P450 family. The MsDWF4 had 67 kinase phosphorylation sites. Sequence and phylogenetic tree analysis show that MsDWF4 protein was most closely related to DWF4 protein of legume plant M. truncatula and had the farthest relationship with Gramineaeis. Tissue-specific expression analysis illustrated that MsDWF4 had the highest expression in the root tip, followed by flowers and leaves. High temperature, cold, PEG, NaCl, ABA and IAA all positively induced the expression of MsDWF4 gene in the alfalfa. After BR treatment, the expression of MsDWF4 was down regulated in shoot and root. The expression of MsDWF4 was inhibited by JA treatment. A 35S∷MsDWF4 overexpression vector was constructed and transformed into alfalfa by Agrobacterium mediated method. PCR identification showed that MsDWF4 gene has been transferred into alfalfa and 6 positive transgenic lines were obtained. Under salt stress, the expression of MsDWF4 gene and the activity of antioxidant enzymes of alfalfa overexpression MsDWF4 gene are higher than that of the control plants. 【Conclusion】 The cDNA sequence of alfalfa brassinolide synthetase gene MsDWF4 was obtained. It was found that the gene expressed the highest in root tip and its expression is responded to a variety of abiotic stress and exogenous hormone treatment. MsDWF4 gene enhanced the resistance to salt stress in transgenic alfalfa. The results showed that MsDWF4 may participate in many kinds of stress response processes and positively regulate the salt tolerance of alfalfa.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY

Effects of Exogenous 6-BA on Root Growth and Pod Yield of Flooded Peanut at Different Growth Stages

LI Ying,ZHAO JiHao,LI JinRong,QIAN BiChang,LIU ZhaoXin,GAO Fang,YANG DongQing,LI XiangDong

Scientia Agricultura Sinica. 2020, 53(18):  3665-3678.  doi:10.3864/j.issn.0578-1752.2020.18.004

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【Objective】The objective of this study was to investigate the effects of flooding stress and spraying exogenous cytokinin on root respiratory enzymes activities, endogenous hormone content and pod yield at different peanut growth stages, so as to provide a theory base for improving peanut resistance to flooding and using exogenous cytokinin hormone to regulate peanut growth. 【Method】Peanut (Shanhua108) was grown in the pot culture experiments with flooding treatment during 10 days at the seedling stage (V3), the flower needle stage (R3), the pod setting stage (R5), and the full fruit stage (R7),respectively. And the normal water management during the whole growth period (CK) was used as the control. Exogenous 6-benzyladenine (6-BA) (15 mg·L^-1) were sprayed to the whole plants at a rate of 250 mL·m^-2 after waterlogging. A total of 9 spraying combinations, namely, normal water management during the whole growth period (CK), flooding at the seedling stage (V3-W), spraying 6-BA after flooding at the seedling stage (V3-S), and flooding at the flower needle stage (R3-W), spraying 6-BA after flooding at the flower needle stage (R3-S), flooding at the pod setting stage (R5-W), spraying 6-BA after flooding at the pod setting stage(R5-S), flooding at full fruit stage (R7-W), spraying 6-BA after flooding at full fruit stage (R7-S). And then the anaerobic respiratory enzymes, aerobic respiratory enzymes activity, endogenous hormone contents, root dry weight (RDW), and root length density (RLD) were determined every 5 days after treatment. 【Result】The RDW and RLD in 20-60 cm soil layers were significantly decreased by waterlogging treatment. There was no root system in 20-40 cm soil layer under V3-W treatment after waterlogging. Compared with the R3-W treatment, the values of RDW and RLD of the 20-60 cm soil layer under the R3-S treatment was increased by 5.15% and 8.59% in the growing seasons of 2018 and 2019, respectively. Flooding stress increased the activities of Alcohol dehydrogenase (ADH), Lactate dehydrogenase (LDH), and decreased the activity of Malate dehydrogenase (MDH). For example, the activity of ADH and LDH was increased under V3-W treatment by 12.49 and 18.99 times, respectively. Whereas, the activity of MDH decreased by 65.15%. In addition, compared with CK treatment, ABA content in the two growing seasons was increased by 22.51%, 15.81%, 10.57% and 5.64% under V3-W, R3-W, R5-W and R7-W, respectively. However, spraying 6-BA significantly reduced the ABA content during R3 stage, which was 7.60% lower than that of R3-W treatment. On the contrary, endogenous ZR content was reduced by flooding stress at all the growth stages. Compared to CK treatment, ZR content under V3-W, R3-W, R5-W and R7-W treatment was decreased by 16.84%, 15.61%, 15.35%, and 8.51%, respectively. While application of exogenous 6-BA decreased the ABA content, but increased the ZR content. Flooding significantly reduced the number of fruit per plant and the yield per plant in the R5 period, which decreased by 38.39% and 30.43% in 2018, respectively, and decreased by 31.60% and 25.06% in 2019. The R3 period was sprayed with 6-BA in the growth season of 2018 and 2019, increased production by 5.38% and 6.91%, respectively. 【Conclusion】 Application of exogenous 6-BA after flooding increased peanut yield due to increasing root respiration performance and the leaf photosynthetic productivity resulting from reducing the root ABA content and increasing the ZR content to enhance root ADH and MDH activity, and to increase leaf chlorophyll content and photosynthetic rate.

Using Canopy Time-Series Vegetation Index to Predict Yield of Winter Wheat

XIANG FangLin,LI XinGe,MA JiFeng,LIU XiaoJun,TIAN YongChao,ZHU Yan,CAO WeiXing,CAO Qiang

Scientia Agricultura Sinica. 2020, 53(18):  3679-3692.  doi:10.3864/j.issn.0578-1752.2020.18.005

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【Objective】The research elaborated the dynamic change trend of canopy time series vegetation index in winter wheat. The yield prediction model was constructed based on time series vegetation index in wheat, which provided effective technical support for obtaining crop yield information timely and accurately. 【Method】 From 2017 to 2019, the field experiments involving different nitrogen (N) rates and varieties were conducted in Ten Thousand Acres Grain Industrial Park located in Xinghua, Jiangsu province. The normalized difference red edge (NDRE) and normalized difference vegetation index (NDVI) were obtained from the active canopy sensor RapidSCAN CS-45. The curve of time-series vegetation index was fitted based on the double logistic function, and the characteristic parameters of curve were extracted. The correlation between each characteristic parameters and yield were analyzed. The yield estimation models were verified with independent test data. 【Result】 The results of the study indicated that the relationship between NDRE and yield was performed well at booting and heading stage, and R² of them was 0.86 and 0.85, respectively. The results of multiple stepwise linear regression showed that the yield prediction models could be improved using NDRE of two or more growth stages, compared with using single growth stage information. The first and second selected periods were jointing and booting stage, respectively. Based on the relative NDRE (RNDRE) and relative NDVI (RNDVI) of the whole growth period, the time-series curve was constructed and the yield prediction models were developed using the characteristic parameters of the curve. The maximum value, accumulative value and growth rate of RNDRE and RNDVI time series curve had a good relationship with the yield. The yield prediction models based on the maximum and accumulative values of RNDRE performed satisfactorily with validation using independent data, the R² was greater than 0.80, and the relative root mean square error and relative error were less than 10%. The validation effect was better than NDRE-based prediction model with the single-period or multi-period, which was better than NDVI-based yield predicted model. 【Conclusion】 The maximum and the accumulative RNDRE extracted from the canopy time series vegetation index had a good potential to estimate the yield, which provided technical support for real-time and accurate yield prediction in the field.

PLANT PROTECTION

Surveillance and Genetic Diversity Analysis of Puccinia striiformis f. sp. tritici in Gansu and Qinghai Provinces

HUANG MiaoMiao,CHEN WanQuan,CAO ShiQin,SUN ZhenYu,JIA QiuZhen,GAO Li,LIU Bo,LIU TaiGuo

Scientia Agricultura Sinica. 2020, 53(18):  3693-3706.  doi:10.3864/j.issn.0578-1752.2020.18.006

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【Objective】In order to clarify the relationship about migration and inoculum source exchange of Puccinia striiformis f. sp. tritici (Pst) in spring epidemic time in Gansu and Qinghai provinces, and provide a theoretical basis for the epidemic prediction of wheat stripe rust and tracing the initial over-summering inoculum source and benefiting the effective disease management, the population genetic diversities and the potential spring epidemic migration of Pst were investigated. 【Method】The regions where stripe rust occurred frequently in the past years were selected as the investigation and studying regions. Four test sites were included in Gansu Province: Wenxian in Longnan City; Kongtong District in Pingliang City, Longdong; Linxia County, and Lintao County in Dingxi City, Longzhong; Two test sites were included in Qinghai Province: Chengbei District, Xining City; Huzhu County, Haidong City. In autumn of 2017, 82 wheat materials for trap nurseries were seeded sequentially in six regions according to the local wheat planting date in Gansu and Qinghai provinces. Field investigation was performed and molecular genotyping for 551 Pst samples collected from 82 materials with known resistant genes in six test sites of Gansu and Qinghai provinces from April to August 2018 was carried out by using 15 pairs of primers simple sequence repeat (SSR) markers. The GenAlEx and POPPR v2.5.0 software were used to analyze the data and the non-significant rbarD value might indicate linkage equilibrium was used to infer whether sexual recombination does occur or not. 【Result】The severity of the 82 wheat materials for trap nurseries of Pst in Gansu was more severe than that in Qinghai Province. The 15 SSR markers produced a total of 81 polymorphic alleles, ranging from 2 to 12 for each of the locus. A total of 505 unique multi locus genotypes (MLGs) were identified after clone correction cross the 551 Pst individuals, 32 MLGs were cloned and resampled between 2 and 6 times at six test sites. The entire genetic diversity (G) was 0.917 in six test sites from two provinces, the G value of Pingliang population (0.974) from Gansu was the highest, then it was Huzhu from Qinghai (0.957), and the Lintao population from Gansu was the lowest (0.841). The genetic variations of Pst mainly existed within the population. In spring epidemic, the migration occurred frequently among the populations. The gene flow was higher between Qinghai and Pingliang/Linxia than that was between Qinghai and Wenxian. The minimum spanning networks (MSN) and non-parametric discriminant analyses of principal components (DAPC) showed that the Huzhu and Xining populations from Qinghai had the closest relationship for the source of inoculum with the Pingliang and Linxia populations from Gansu, and the differences were the smallest. The genetic distance from the Lintao population was relatively far and the Lintao population was relatively independent. The Wenxian population was a completely independent population, with the largest difference from the other five populations. The analysis of linkage disequilibrium showed the presence of insignificant rbarD values in the Wenxian and Linxia populations from Gansu, and Xining population from Qinghai. The Wenxian population (rbarD=0.0139, P=0.186) showed obvious sexual recombination characteristics with a significant rbarD value. 【Conclusion】In spring epidemic season, the Pst source in Qinghai might originate from both Pingliang/Linxia and Wenxian, but it was mainly from Pingliang/Linxia. Signs for sexual reproduction were found in populations from Linxia and Wenxian of Gansu and Xining of Qinghai. The sexual reproduction might contribute to the richness of genetic diversity of Pst in Gansu and Qinghai.

Construction of Genome-Length cDNA of Citrus Vein Enation Virus and Identification of Its Infectivity

XU JianJian,WANG YanJiao,DUAN Yu,MA ZhiMin,BIN Yu,ZHOU ChangYong,SONG Zhen

Scientia Agricultura Sinica. 2020, 53(18):  3707-3715.  doi:10.3864/j.issn.0578-1752.2020.18.007

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【Objective】The objective of this study is to construct infectious clone of citrus vein enation virus (CVEV), and to lay a foundation for analyzing its pathogenic mechanism at the molecular level. 【Method】5′ RACE was performed using SMARTer^?RACE Kit to confirm the exact 5′ sequences of CVEV. A specific primer pairs for amplification of CVEV genome-length cDNA was designed according to the sequence analysis result and the conservative sequence of isolate VE-1. Genome cDNA was amplified with EV25-F/EV5983-R primers using total RNA of CVEV infected fragment. The full-length cDNA of CVEV and linearized pXT1 were connected by In-Fusion recombination. Genome-length cDNA clones of CVEV were identified by PCR and sequencing analysis, and then subjected to agrobacterium-mediated inoculation. Citrus aurantium, C. paradisi, C. limon, C. paradisi × Poncirus trifoliata, P. trifoliata × C. sinensis, P. trifoliata were inoculated by agrobacterium-mediated vacuum infiltration. The infectivity of the cDNA clone was further identified by RT-PCR detection and symptom observation. 【Result】An RT-PCR amplification system for full-length genome of CVEV was established, and 10 full-length cDNA clones of CVEV genome based on the pXT1 were obtained. Six clones, namely CVEV1901-CVEV1906, were randomly selected and sequenced. The nucleotide sequence identity of them was 99.35%. Among them, the CVEV1901 is 5 983 nt in length and consists of five open reading frames (ORF), two untranslated regions (UTR) at the 5′ end (207 nt) and 3′ end (198 nt), and an intergenic region (IR) of 122 nt between ORF2 and ORF3. The nucleotide sequence analysis results showed that the identity of CVEV1901 and CVEV isolates from China was the highest, and that of Zhejiang isolate XZG and Sichuan isolate SM was 99.98% and 99.11%, respectively. The nucleotide sequence identity of CVEV1901 and CVEV isolate VE-1 from Spain, VE701 from California, America and IBK from Japan was 96.89%-98.61%. CVEV1901 showed much lower nucleotide sequence identity with pea enation mosaic virus and alfalfa enamovirus (about 90%) in the same genus. CVEV1901 was inoculated onto 6 different citrus varieties by agrobacterium-mediated vacuum infiltration. RT-PCR detection results at 120 days after inoculation showed that the positive plant/inoculated plant (positive rate) of C. aurantium, C. paradisi, C. limon, C. paradisi × P. trifoliata, P. trifoliata × C. sinensis, P. trifoliata was 16/17 (94.12%), 12/14 (85.71%), 16/21 (76.19%), 15/19 (78.95%), 13/14 (92.86%) and 0/18 (0), respectively. Among them, some C. aurantium showed typical symptoms of CVEV infection, with small auricular-shaped protrusions in the lateral veins of the leaves, and corresponding depressions in the back of the leaves. Some C. paradisi and C. limon had shrunken leaves. 【Conclusion】An RT-PCR amplification system for the full-length CVEV genome was established, and an infectious full-length cDNA clone of CVEV was obtained. The full-length cDNA clone can induce typical CVEV infection symptoms on C. aurantium, C. paradisi, C. limon by agrobacterium-mediated vacuum infiltration.

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

Research on Spatial Distribution of Soil Particle Size Distribution in Loess Region Based on Three Spatial Prediction Methods—Taking Haiyuan County in Ningxia as an Example

SHEN Zhe,ZHANG RenLian,LONG HuaiYu,WANG Zhuan,ZHU GuoLong,SHI QianXiong,YU KeFan,XU AiGuo

Scientia Agricultura Sinica. 2020, 53(18):  3716-3728.  doi:10.3864/j.issn.0578-1752.2020.18.008

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【Objective】 This study was oriented to explore for a model that was capable of predicting spatial distribution of soil particle size distribution in the region with complicated terrain and single parent material, like Haiyuan County, Ningxia. 【Method】 Empirical Bayesian Kriging with the symmetry logratio transform (SLR-EBK), regression Kriging with the symmetry logratio transform (SLR-RK) and random forest with the symmetry logratio transform (SLR-RF) were used to predict spatial distribution of soil particle size distribution in Haiyuan County based on the training set of 100 samples, combined with topographic factors, normalized difference vegetation index and soil types, and then the predictions were validated with a validating set of 24 validation points in the study area for comparison of prediction accuracy. 【Result】 (1) The auxiliary variables that came into the linear regression equator for prediction included elevation (Ele) and soil types. The auxiliary variables that came into the RF model included Ele, soil type, slope (Slo) and wind exposition index (WEI), of which Ele was the most important auxiliary variable, followed by soil types. Slo and wind exposition index (WEI) were less important. (2) Results predicted by three methods showed a spatial distribution trend that the sand content was lower in the southwest and higher in the northeast of the county, while the silt content and clay content were higher in the southwest and lower in the northeast. SLR-RK and SLR-RF could better describe local variation of different soil particle size contents. (3) The mean absolute error (MAE) and root-mean-square error (RMSE) of SLR-RF were lower than those of the other two methods. As for mean Aitchison distance (MAD), the sequences of MAD were obtained as following: SLR-RF (0.208)

Regional Variation in the Effects of No-Till on Soil Water Retention and Organic Carbon Pool

LI JingYu,LI Qian,WU XuePing,WU HuiJun,SONG XiaoJun,ZHANG YongQing,LIU XiaoTong,DING WeiTing,ZHANG MengNi,ZHENG FengJun

Scientia Agricultura Sinica. 2020, 53(18):  3729-3740.  doi:10.3864/j.issn.0578-1752.2020.18.009

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【Objective】Soil tillage is an important factor which affecting soil organic carbon, but the impact of tillage on soil organic carbon and the driving factors in different regions are still unknown. In this study, the regional variation in the effects of no-till on soil water retention and organic carbon pool were studied to improve regional soil tillage in the north of China. 【Method】The four typical long-term tillage experiments were selected, which was located in Shouyang (SSY) and Linfen (SLF) of Shanxi Province, Gongzhuling (GZL) of Jilin Province and Langfang (HLF) of Hebei Province. Two tillage treatments, including conventional tillage (CT) and no-till (NT) , were selected to study the effects of tillage on organic carbon. 【Result】 (1) Soil bulk density and penetrometer resistance were significantly affected by no-tillage in different regions. Compared with CT, NT significantly increased the bulk density in black soil (GZL) and sandy soil (HLF), which was increased by 12.1% and 0.7%, respectively. But CT reduced the bulk density in fluvo-aquic soil (SLF) and cinnamon soil (SSY), which was decreased by 1.5% and 8.2%, respectively. (2) The soil water content of 0 and 10 kPa under NT in black soil (GZL) was significantly different, which were increased by 40.4% and 30.1%, respectively; the soil water content of 0, 10 and 500 kPa under NT in cinnamon soil (SSY) was reduced by 6.4%, 4.3%, and 5.9%, respectively, and which was increased by 2.1% at 350 kPa. The soil water content of 10 kPa, 350 kPa, and 500 kPa under NT in sandy soil (HLF) were increased by 0.6%, 5.6%, and 2.6%, respectively. The soil water content of 0 and 10 kPa under NT in fluvo-aquic soil (SLF) decreased by 7.1% and 5.5%, respectively, and the soil water content of 350kPa and 500kPa was increased by 2.9% and 8.9%, respectively. (3) Soil organic carbon was affected by no-tillage in different regions. Compared with CT, NT significantly increased the organic carbon storage in 0-80 cm layer in black soil (GZL); in cinnamon soil (SSY), fluvo-aquic soil (SLF) and sandy soil (HLF), NT decreased soil organic carbon storage by 26.8%, 31.3%, and 23.5%, respectively. (4) Soil organic carbon stocks were positive correlated with altitude and saturated moisture, which was negatively correlated with annual average temperature, annual precipitation, and penetrometer resistance. This showed that organic carbon storage was significantly affected by climatic factors, soil water retention and penetrometer resistance. 【Conclusion】No-tillage changed soil organic carbon stocks by affecting penetrometer resistance and soil water retention. However, due to differences in climate, crop and soil factors in northeast and north of China, the increase in soil organic carbon stocks was different. In general, conservation tillage was an effective way to increase the surface soil organic carbon storage.

The Characteristics of Soil Ammonia Volatilization Under Different Fertilizer Application Measures in Corn Field of Liaohe Plain

ZHAO XinZhou,ZHANG ShiChun,LI Ying,ZHENG YiMin,ZHAO HongLiang,XIE LiYong

Scientia Agricultura Sinica. 2020, 53(18):  3741-3751.  doi:10.3864/j.issn.0578-1752.2020.18.010

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【Objective】This study was performed to explore the characteristics of ammonia (NH₃) volatilization from corn field affected by different fertilization measures, to understand the contribution of different fertilization measures to NH₃ emission, and to obtain the localized NH₃ emission factors of chemical fertilizer application in Liaohe plain, northeastern China, so as to provide reference for relevant research in the fields of atmospheric environment and ecology. 【Method】 A field experiment of NH₃ emission responses under different fertilization measures was carried out in the south experimental field of Shenyang agricultural university, Liaoning province from May to October 2018, which was set up with 5 treatments: no nitrogen treatment (T0), half-amount conventional fertilization (T1), conventional fertilization + biochar (T2), one-time conventional fertilization (T3), conventional fertilization (T4). The base fertilizer was coated with slow-release fertilizer and urea was applied at jointing stage. From May to October 2018, NH₃ gas was collected by aeration method, ammonium concentration was analyzed by continuous flow analyzer, and NH₃ emission flux was calculated. Meanwhile, ammonium nitrogen (NH₄⁺-N) content in soil was measured. 【Result】 The NH₃ volatilization rate showed a bimodal trend after the application of base fertilizer, and the maximum NH₃ volatilization rates occurred on the 1st-2d or 5th-7d after the application of base fertilizer, respectively. The maximum NH₃ volatilization rates in the treatments of base fertilizer were as follows: T1>T2>T3>T4>T0. All treatments reached the maximum NH₃ volatilization rates at the 1st - 2d after applying top dressing, and the maximum NH₃ volatilization rates at the top dressing stage were as follows: T4>T2>T1>T3>T0. The accumulation of NH₃ volatilization loss was shown as T2)>T4>T3>T1>T0. There was no significant difference in soil NH₄ ⁺-N content between different treatments in different periods, but the soil NH₄ ⁺-N content and NH₃ volatilization rate in the same period showed a similar change trend, and the correlations after applying top fertilizer were more significant than that after applying base fertilizer. Due to the application of urea under T1, T2 and T4 at top dressing period, urea released NH₄⁺-N more rapidly than slow-release fertilizer, and NH₃ volatilization was relatively fast. Overall, a 50% reduction in nitrogen application resulted in a 20% reduction in NH₃ volatilization loss accumulation. The accumulation of NH₃ volatilization loss was significantly different among the treatments during the growth season. T2 had the largest accumulation of NH₃ volatilization loss. Under the same nitrogen application amount, the cumulative ammonia volatilization loss of biochar treatment increased by 22%. Under the condition of the same nitrogen application amount in the whole growth season, the NH₃ volatilization accumulation was reduced by 12% in the one-time application of slow-release fertilizer without urea topdressing than that with urea topdressing. 【Conclusion】Ammonia volatilization showed a marginal decreasing effect with the increase of nitrogen application. Biochar promoted ammonia volatilization in farmland, while corn straw biochar was alkaline, resulting in increased accumulation of ammonia volatilization. However, it had the characteristics of large porosity and specific surface area, strong adsorption effect, and could improve soil and reduce emissions of other greenhouse gases. The ammonia volatilization was significantly reduced by applying slow-release fertilizer at one time without urea topdressing.

Nitrogen and Phosphorus Loads and Their Environmental Effects of Animal Manure in Hainan Island from 1988 to 2018

DING Shang,FU Yang,GUO HaoHao,SONG ChenYang,LI BoLing,ZHAO HongWei

Scientia Agricultura Sinica. 2020, 53(18):  3752-3763.  doi:10.3864/j.issn.0578-1752.2020.18.011

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【Objective】The objective of this study is to reveal the flow characteristics of nitrogen (N) and phosphorus (P) in manure and their effects to the environment during the development of livestock and poultry breeding in Hainan, and to provide references for regional agricultural development planning and adjustment of livestock and poultry breeding structure. 【Method】Based on the statistical data and literature data, the nutrient yield of animal manure and nutrient carrying capacity per unit cultivated area in Hainan Island from 1988 to 2018 were estimated, and their effects to the environment were deeply analyzed by using alarm value and environmental risk index. When the alarm value is less than 0.4, it can be considered as no threat to the environment, and when the environmental risk index is below 1.00, the risk to the environment can be ignored, above these safety values will have different degrees of impacts on the environment. 【Result】From 1988 to 2018, the changes of the animal manure amount in Hainan Island experienced three stages. The total amount of animal manure increased from 1988 to 2005, and decreased a lot from 2006 to 2008. Since then it developed slowly and stabilized, and the amount of animal manure produced in 2018 was 10.50×10⁶t. In terms of nutrient yield, the annual variation trends of nitrogen and phosphorus were similar, with the largest yield in 2005 and a relatively large decline in 2006. To 2018, the yields of nitrogen and phosphorus in manure were 7.41×10⁴ and 1.01×10⁴ t, respectively. The nitrogen and phosphorus loads per unit cultivated land area also experienced a stable trend after continuous increasing. To 2018, cultivated land nitrogen load was 168.80 kg·hm^-2, and cultivated land phosphorus load was 23.05 kg·hm^-2. In 2018, regional variations were relatively large. The nitrogen and phosphorus loads per unit cultivated area were higher in eastern and northern regions, while lower in western and central regions. The highest nitrogen and phosphorus loads were Dingan (288.50 and 40.35 kg·hm^-2, respectively), and the lowest were Dongfang (61.66 and 8.83 kg·hm^-2, respectively). In terms of environmental effects, the alarm values of animal manure per cultivated land from 1988 to 2018 were always at high levels, most years between 1.0 to 1.5, which belonged to the classification standard of IV level and had a serious threat to the environment. In 2018, the alarm values in Dingan and Chengmai were 2.66 and 4.59 (reached the VI level), which had a serious threat to the environment, while only Dongfang was at a safe level with the alarm value of 0.36, which reached the I level. According to the environmental risk assessment results, the environmental risk of animal manure in cultivated land in Hainan Island was relatively high. In recent years, with the total amount of animal breeding reduction, the environmental risk indexes in 2018 dropped to 1.99 (N) and 1.32 (P), respectively. At the regional level, for nitrogen pollution, the environmental risk index of Dingan was the highest (3.39), while the lowest (0.73) was Dongfang; for phosphorus pollution, the environmental risk index in Dongfang (0.50) was the lowest in 2018, which can be considered that phosphorus in Dongfang had a low potential threat to the environment, while Dingan had a serious impact on the environment, with a value as high as 2.31. In general, both the alarm value and the environmental risk index indicated that the potential environmental risk of animal manure pollution in Hainan Island was serious, from the perspective of spatial distribution, environmental risks in eastern cities and counties such as Dingan and Wanning were higher than those in western regions such as Lingao and Dongfang, and most cities and counties posed a higher degree of threat to the environment. 【Conclusion】Due to the influence of animal breeding scale and cultivated land area, the nitrogen load per cultivated land area in Hainan Island is relatively high, and the potential environmental risks of the region scales cannot be ignored. Therefore, the future development plan of livestock and poultry breeding in Hainan Island should focus on the control of pollutants, regional distribution and management, meanwhile, realize the utilization of animal manure resources and the green development of Hainan Island by reducing the animal manure discharge to the environment and adopting resource recycling modes.

HORTICULTURE

Cloning and Analysis of Folate Synthesis Key Genes in Cucumber

ZHOU Qi,LIU XiaoPing,BO KaiLiang,MIAO Han,DONG ShaoYun,GU XingFang,ZHANG ShengPing

Scientia Agricultura Sinica. 2020, 53(18):  3764-3776.  doi:10.3864/j.issn.0578-1752.2020.18.012

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【Objective】This study analyzed the quantity, location and expression pattern of folic acid metabolization-related genes in cucumber, cloned and made bioinformatics analysis of the key enzyme genes, aiming to lay a foundation for the study on the regulation mode of folic acid synthesis in cucumber. 【Method】The reported folic acid metabolism related genes in Arabidopsis thaliana was blasted in the cucumber genome database 9930 _V3 to obtain the folic acid metabolism related genes in cucumber. These genes were mapped onto the cucumber chromosome by using Mapchart, and their expression pattern was examined in different materials and at different developmental stages. Bioinformatics analysis of key enzyme genes was conducted by MEGA, Web LOGO, and ExPASy. Key enzyme genes were cloned by PCR amplification, and sequence differences were analyzed. 【Result】A total of 19 genes related to folate metabolism were blasted in cucumber, which were distributed non-uniformly on seven chromosomes, mostly on Chr.4 and Chr.5. The expression levels of 11 folic acid synthetic genes in fruits of sequenced material 9930, inbred line with low folic acid 65G, and inbred line with high folic acid 02245 at different developmental stages were analyzed. It was found that the expression pattern of CsFPGS, CsHPPK/CsDHPS, and CsDHNA were consistent with the changes of folic acid content, and there were significant differences in the expression levels of CsADCS, CsADCL, CsDHNA, CsHPPK/CsDHFS, CsFPGS and CsDHFS between 65G and 02245. CsGCHI and CsADCS were two key enzymes regulate folate synthesis in rate-limiting steps, and then their amino acid sequences and protein domains were analyzed. The result showed that it turned out that CsGCHI homologs all had two GTP_cyclohydroI domains, and CsADCS homologs all had two GATase domains, including one Anth_synt_I_N domain, and one Chorismate_bind domain. The domains were highly conserved in different species, evolutionary tree analysis clustered the proteins of closely related species together. The GCHI and ADCS gene were cloned from 65G and 02245, respectively. Sequence analysis showed that the full length of CsaV3_1G041250 was 3012 bp, the length of CDS sequence was 1 413 bp, and the mutations in the three SNP sites led to the variation of amino acid sequence. The full length of CsaV3_7G026240 was 3 047 bp, and the CDS length was 1 407 bp with no sequence variation. The total length of CsaV3_5G036360 was 7 941 bp, and the length of CDS sequence was 2 706 bp. 【Conclusion】It was identified that 19 genes were related to folate metabolism in cucumber. These genes were distributed unequally on seven chromosomes. CsFPGS, CsHPPK/CsDHPS, CsDHNA and CsADCS affected folic acid content and trend in cucumber fruit mostly, while CsGCHI and CsADCS were the Key enzyme genes regulating rate-limiting steps in folic acid synthesis, which was relatively conservative in function, and 3 SNP mutations led to variations in protein sequences in CsGCHI between 65G and 02245.

Screening of Polyphenol Oxidase Interaction Proteins from Nelumbo nucifera and Their Verification

YUAN XinBo,CHENG TingTing,XI XiaoHan,CHEN ZhangYu,WANG RuiHong,KE WeiDong,GUO HongBo

Scientia Agricultura Sinica. 2020, 53(18):  3777-3791.  doi:10.3864/j.issn.0578-1752.2020.18.013

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【Objective】The protein interacting with NnPPO1 (GenBank: ADC92563.1), a member of the polyphenol oxidase family of China Lotus (Nelumbo nucifera Gaertn.), was screened and verified, which laid the foundation for further studying of the molecular mechanism of PPO and precise inhibition of its activity in Nelumbo nucifera. 【Method】The interaction between Nelumbo nucifera antioxidant enzyme and NnPPO1 was verified by yeast double hybridization (Y2H). The toxicity and self-activation activity of the interacting protein catalase isoenzyme NnCAT1 (GenBank: XP_010242894.1) were detected by the transformed yeast experiment. The expression vectors of 35S-NnPPO1-SPYNER173 and 35S-NnCAT1-SPYCEM used in the BiFC experiment were constructed. The recombinant plasmid was transformed into tobacco by an agrobacteria-mediated method to further verify the interaction between NnPPO1 and NnCAT1. The proteins were truncated based on the domains of NnPPO1 and NnCAT1 to find the key domains of interaction. PlantCARE and real-time fluorescent quantitative PCR (qRT-PCR) were used to analyze the promoter-related components of NnPPO1 and NnCAT1 as well as their expression levels in different tissues of Nelumbo nucifera. The 35S-NnCAT1-GFP fusion protein expression vector was constructed by homologous recombination method to analyze subcellular localization. DNAMAN software was used to perform multi-sequence alignment between the NnCAT1 sequence and other species sequences. The basic character and structure of NnCAT1 were analyzed by using bioinformatics website. 【Result】NnCAT1 interacts with NnPPO1, had no self-activation or toxicity to yeast. The interaction between NnPPO1 and NnCAT1 was further confirmed by the yellow fluorescence signal observed on the cell membrane and nucleus. The tyrosinase domain of NnPPO1 played a major role in protein interactions. NnPPO1 and NnCAT1 promoter sequences contained a variety of cis-regulatory elements, such as photoresponse element (Box 4, GT1-motif, and TCT-motif), stress response element (ARE), and hormone response element (CGTCA-motif and TGACG-motif). The expression patterns of NnPPO1 and NnCAT1 were almost the same. They were expressed in all tissues, with the highest expression in leaf and lowest expression in stem tip. Subcellular localization showed that NnCAT1 was localized in the cell membrane and nucleus. NnCAT1 had a molecular weight of 57.0 kD and a theoretical isoelectric point (PI) of 6.93. This protein was a homologous tetramer hydrophilic protein without transmembrane structure and signal peptide, and its chloroplast transport peptide was located in the former 21 aa. The secondary structure was composed of 27.64% α-helix, 15.65% extended chain, 6.30% β-turn and 50.41% random coil. 【Conclusion】Firstly, NnCAT1 interacted with NnPPO1, and the authenticity of the interaction was further verified by the BiFC test. Secondly, the conserved tyrosinase domain of NnPPO1 played a major role in the interaction. NnPPO1 and NnCAT1 had almost the same expression pattern, and they might lead to browning of fruits and vegetables such as Nelumbo nucifera through a synergistic effect.

FOOD SCIENCE AND ENGINEERING

Microstructure and Rheological Behavior of Mixed Konjac Glucomannan and Xanthan Induced by Thermo-Alkali Treatment

LI PeiYuan,LI XiaoFei,LI AnQi,YU WenYan,GUO Chuo,YANG Xi,GUO YuRong

Scientia Agricultura Sinica. 2020, 53(18):  3792-3804.  doi:10.3864/j.issn.0578-1752.2020.18.014

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【Objective】This study intended to investigate the gelation mechanism and gel properties of the alkaline-induced gelation of mixed konjac glucomannan and xanthan sol systems under heating treatment, in order to provide a theoretical basis for konjac glucomannan and xanthan-related gel food design. 【Method】At a fixed total polysaccharide concentration of about 2.0%, the mixed sol systems with different konjac glucomannan to xanthan ratios were prepared by altering the mixing volume ratio of konjac glucomannan to xanthan. Then, the composite gels were prepared by heating the sol systems at 90℃ for 2 h with Na₂CO₃ as alkaline regulator, followed by cooling to room temperature. The effect of alkaline treatment on the rupture strength of the composite gel was investigated by determining the gel rupture strength with and without alkali addition and at room temperature (20℃). In order to investigate the properties of the composite gel, the gel rupture strength was tested, and microstructure was observed by scanning electron microscope (SEM) after deionized water immersion, 2.0% citric acid solution immersion and freeze-thaw treatment, respectively. Furthermore, rheological analysis was adopted to study the formation mechanism of the composite gel. 【Result】At room temperature, the optimum synergistic ratio of konjac glucomannan to xanthan was 5:5 for without hot alkali treated composite gels. However, the optimum synergistic ratio shift towards 7:3 after hot alkali treatment, because part of the acetyl group was removed from the molecular chain of konjac glucomannan after alkalization, forming a three-dimensional network structure between the molecules. In the subsequent cooling process, the synergistic binding sites of konjac glucomannan and xanthan were reduced. Therefore, more konjac glucomannan molecules were needed to participate in the maximum synergistic ratio. Furthermore, after immersing the gels in deionized water and 2.0% citric acid solution, a slightly decrease in gel rupture strength was observed, and 2.0% citric acid solution immersion caused a more pronounced decrease in gel rupture strength compared with deionized water immersion. Syneresis phenomenon of all composite gels was also observed after freeze-thaw treatment. The higher the ratio of konjac glucomannan to xanthan, the more significant the syneresis phenomenon was. The gelation process of the composite sol system was further revealed under the condition of 2.0% alkali concentration and at 90℃. The results showed that the gelation rate was decreased with increasing xanthan addition. G' of the gels was decreased when the temperature decreased from 90℃ to 60℃, but the G' showed a continuously upward trend as the temperature further decreased. 【Conclusion】 Konjac glucomannan and xanthan could form a thermal irreversible gel under alkaline treatment at 90℃ followed by cooling to room temperature. When the temperature of the system was lowered, xanthan began to combine with konjac glucomannan network at 60℃ and increased the elastic modulus of the whole gel. When the ratio of konjac glucomannan to xanthan was 7:3, the alkaline-treated composite gel showed the highest gel strength. After immersion in deionized water and 2.0% citric acid solution, the gel strength decreased. Compared with alkaline-induced konjac glucomannan gels, the composite gel presented higher gel strength, better freeze-thaw stability and lower syneresis ratio.

ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT

Codon Optimization of Human Lysozyme and High-Efficiency Expression in Bovine Mammary Cells

TIAN Yuan,WANG Li,LONG Feng,ZAN LinSen,CHENG Gong

Scientia Agricultura Sinica. 2020, 53(18):  3805-3817.  doi:10.3864/j.issn.0578-1752.2020.18.015

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【Objective】The aims of present study were to investigate the codon usage bias of seven milk protein genes in cattle mammary gland, to optimaize human lysozyme gene partially and totally based on codon usage bias of milk protein genes, and to evaluate the codon optimized effect of human lysozyme in multiple cells, so as to provide theoretical basis for increasing recombinant human lysozyme expression and developing new high-effect and safe recombinant human lysozyme. 【Method】The codon usage bias and high/low frequency codons of bovine major milk protein genes were confirmed through bioinformatics analysis by CodonW and EMBOSS software. Human lysozyme gene was partially (22 codons in the translation start region, named LYZop22) and totally (named LYZop) optimized according to the codon usage frequency of bovine major milk protein genes. The human lysozyme-luciferase fusion expression vector (pGL3-LYZcw/op22/op) and overexpression vector (pcDNA-LYZcw/op22/op) were constructed and transfected to cattle mammary epithelial cells (BMEC), cattle fibroblasts (BFFC) and mouse mammary epithelial cells for codon optimization effect evaluation by luciferase, real-time qPCR and western blot methods. 【Result】 The cattle milk protein genes prefer GC end, which the GC3s content was 0.537±0.062. However, the human lysozyme gene prefer AT end, and the GC3s content was 0.407. Moreover, differences in codon usage bias between casein protein genes and whey protein genes were found by cluster analysis. Five high frequency codons (RSCU>1.5) and seven low frequency codons (RSCU<0.5) were found in the bovine milk protein genes.The human lysozyme gene was optimized according to the bovine milk protein codon usage bias and evaluated by multiple cells transfection. The luciferase result showed that, the codon optimization could significantly increase the human lysozyme expression, which were 1.48-fold (P<0.01) and 1.30-fold (P>0.05) increased in BMEC and BFFC of LYZop22, respectively. While, 2.2-fold and 2.44-fold increase in the BMEC and BFFC of LYZop, respectively. Real-time qPCR result showed that, the mRNA levels of human lysozyme were increased in BMEC (2.08-fold, P<0.05) and BFFC (1.5-fold, P>0.05) of LYZop22. While the significantly increase in mRNA level of human lysozyme were found in the BMEC (17.8-fold , P<0.01) and BFFC (22-fold, P<0.01) of LYZop, the totally codon optimized type. In addition, there was a positive correlation between mRNA level and mRNA second structure stability. Western-blot result showed that, the expression of human lysozyme was effectively increased both in partially and totally codon optimized human lysozyme gene in the BMEC. Moreover, the effect on human lysozyme expression in totally codon optimization type was better than partially codon optimization type. 【Conclusion】 The codon usage bias and high/low frequency codons of bovine major milk protein genes were obtained, and the codon optimization of human lysozyme gene according bovine major milk protein gene codon usage bias could significantly improve the mRNA and protein level of human lysozyme, which would lay the foundation for producing recombinant human lysozyme effectively by bioreactor.

Cloning and Function Analysis of MsNST in Lignin and Cellulose Biosynthesis Pathway from Alfalfa

JIANG Xu,CUI HuiTing,WANG Zhen,ZHANG TieJun,LONG RuiCai,YANG QingChuan,KANG JunMei

Scientia Agricultura Sinica. 2020, 53(18):  3818-3832.  doi:10.3864/j.issn.0578-1752.2020.18.016

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【Objective】Lignin and cellulose, one of the main components in the secondary cell wall of higher planta, are one of the important factors affecting the quality and digestibility of legume forage alfalfa. The model plant, Arabidopsis thaliana, NST genes belong to the NAC transcription factor family were shown to have a key role in regulating secondary cell wall biosynthesis. However, the function of MsNST in alfalfa was still elusive. In this study, the aim was to investigate the expression patterns of MsNST gene induced by exogenous hormones and its function in lignin and cellulose biosynthesis pathway. 【Method】MsNST was isolated from alfalfa by homology-based cloning. Bioinformatic analysis was conducted by online tools. In order to investigate the functions of MsNST in cell wall cellulose and lignin synthesis pathway, overexpression of MsNST in transgenic alfalfa plants was programed. In this study, qRT-PCR was applied to reveal the expression pattern of genes related to lignin and cellulose biosynthesis pathway, and expression level under gibberellin (GA3), salicylic acid (SA) and paclobutrazol (PCB) treatments. 【Result】 An open reading frame (ORF) of 945 bp encoding MsNST with 314 amino acids was cloned. The similarity analysis of amino acid showed both MsNST and AtNST1-3 shared high sequence homology (from 49% to 55.9%) containing five typical NAC conservation subdomains. Bioinformatic analysis indicated that MsNST was mainly constituted by the random coil (60.83%), and a comparative protein modeling implied that homodimer might promote protein-protein interaction. Phylogenetic analysis revealed that MsNST widely expressed in higher plant species from monocot to dicot. The increased MsNST expression levels were detected with GA3, SA, and PCB (2.19- 3.67 times at 12h) treatment. Overexpression of MsNST in Arabidopsis resulted in shortening of hypocotyl axis, semi-dwarf and interfascicular fiber cell wall thickening and the accumulation of lignin (11.7%), cellulose (13%) and total sugar (7%) compared with wide type. Otherwise, the expression level of other genes in lignin and cellulose biosynthesis pathway, including PAL1, 4CL1, and CesAs were detected by qRT-PCR and the results determined these genes were up-regulated in transgenic plants compared with the control. 【Conclusion】MsNST was inducible by GA3, SA, and PCB, and the overexpression of MsNST in transgenic plants triggered the expression of key genes related to secondary cell wall synthesis, and led to shortened hypocotyl, semi-dwarf and thickened interfascicular fiber cell wall. Additionally, overexpression MsNST accumulated the content of lignin, cellulose and total sugar. These findings suggested that MsNST might play a crucial role in cell wall lignin and cellulose synthesis pathway.

Evaluation and Optimization of Metabolite Extraction Protocols for Royal Jelly by High Resolution Mass Spectrometry and Metabolomics

ZHANG LiCui,MA Chuan,FENG Mao,LI JianKe

Scientia Agricultura Sinica. 2020, 53(18):  3833-3845.  doi:10.3864/j.issn.0578-1752.2020.18.017

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【Objective】Royal jelly, a natural product with health-promoting effect, is rich in small-molecule bioactive compounds. The systematic analysis of the metabolite extraction from royal jelly is still lacking. This study performed metabolic profiling of royal jelly with an aim to compare the metabolite extraction efficiency using different solvents, optimize the metabolite extraction protocol, and globally identify the small-molecule compounds in royal jelly. 【Method】Metabolites in royal jelly were extracted separately using six different solvents including 50% and 80% methanol, ethanol, and acetonitrile. They were analyzed by reverse phase liquid chromatography (RPLC) and hydrophilic interaction chromatography (HILIC) each combined with Quadrupole-Exactive Orbitrap high resolution mass spectrometry (HRMS). The amount and relative standard deviation (RSD) values of metabolite features among the solvent groups were compared. Principal component analysis (PCA) was conducted on the basis of these metabolite features. The compound identification was based on mass spectrometry databases and was validated with standards. Both the RSD value comparison and clustering heatmap analysis were performed for the identified compounds. Significantly differential metabolites among the solvent groups were screened out by orthogonal partial least squares-discriminant analysis (OPLS-DA), univariate analysis (Student’s t-test) and fold change (FC). 【Result】Highly polar metabolites, including two isomeric sugars (glucose and fructose), and medium and weakly polar metabolites such as lipids were sufficiently separated using the HILIC and PRLC approach, respectively. The combination of the two separation methods enabled comprehensive detection of metabolites with different polarity in royal jelly. A total of 70 high-abundance compounds were identified, including carbohydrates, amino acids, lipids, and vitamins, with the highest difference of 8 340 times in abundance. Among them, 17 compounds were reported for the first time. The lowest number of metabolite features was obtained with acetonitrile, and a worse coverage was obtained with 80% than 50% acetonitrile. For methanol and ethanol, the number of metabolite features was higher at higher solvent concentrations. The RSD values of most metabolite features were lower than 20% in all solvent groups, and the 80% acetonitrile group had the lowest proportion within 10% RSD values compared with other solvents. The RSD values of the 70 identified compounds provided further evidence for the poor repeatability of 80% acetonitrile. The PCA score plots indicated a similar metabolic profiling from the same solvent. Differences in metabolic profiling were observed among the solvents, and the largest difference existed between 80% acetonitrile and the five other solvents. The clustering heatmap showed a lower abundance of medium and weakly polar metabolites obtained from 50% solvents and a lower abundance of highly polar metabolites including, in particular, fructose, glucose, sucrose, lysine, adenosine, choline, phosphorylcholine, and gluconic acid from 80% acetonitrile. 【Conclusion】The combination of RPLC-HRMS and HILIC-HRMS enables comprehensive and accurate detection of small-molecule compounds in royal jelly. 80% methanol or 80% ethanol is an optimal solvent for metabolite extraction from royal jelly.

RESEARCH NOTES

Development of New Varieties of ZHONGJIAO Pepper Resistant to Pepper Mild Mottle Virus Using L ³Gene

ZHANG BaoXi,WANG LiHao,ZHANG ZhengHai,CAO YaCong,YU Hailong

Scientia Agricultura Sinica. 2020, 53(18):  3846-3855.  doi:10.3864/j.issn.0578-1752.2020.18.018

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Hot (sweet) pepper is the largest area vegetable crop in China, with an annual planting area of 2.2×10⁶ hm ², including 5×10⁵ hm² of sweet pepper. Beside traditional diseases, in recent years, pepper milt mottle virus (PMMoV) as a new popular disease broke out, which seriously restricted the production of sweet pepper. At the same time, consumers put forward higher requirements for the quality and diversity of varieties. Our research group has introduced more than 1 400 accessions of hot (sweet) pepper germplasm resources from all over the world. Through identification and evaluation, more than 120 accessions with good characteristics such as antiviral disease, powdery mildew, large fruit, thin skin, good glossiness and high quality have been screened out. The molecular markers closely linked with TMV (L³, L⁴), TSWV (Tsw) and other important characters were constructed. The accuracy of assisted breeding was over 90%. Through the combination of conventional breeding techniques and molecular markers, the core backbone parental sweet pepper inbred line ‘0516’ was created with good combining ability, resistant to TMV, ToMV, PMMoV (P_0,1,2), CMV and Phytophthora capsici, large fruit, uniform and good glossiness, which contained L³ gene resistant to PMMoV (P_0,1,2). Four new varieties Zhongjiao 105, 106, 107 and 108 with high quality, multi resistance and adaptability to different ecological areas were developed. These new varieties contained L³ gene, which was resistant to TMV, PMMoV (P_0,1,2), CMV and disease; the shape, size, color, uniformity and other commodity quality, vitamin C and other nutritional quality of fruits were significantly improved. It was important to improve the disease resistance and quality of sweet pepper.