Scientia Agricultura Sinica

Screening of High-Efficient sgRNA for Targeted Knockout of GhAGL16 Gene in Cotton

LEI JianFeng, YOU YangZi, ZHANG JinEn, DAI PeiHong, YU Li, DU ZhengYang, LI Yue, LIU XiaoDong

Scientia Agricultura Sinica. 2024, 57(6): 1023-1033. doi:10.3864/j.issn.0578-1752.2024.06.001

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【Objective】 As an important negative transcriptional regulator in cotton MADS-box gene family, AGL16 plays an important role in resisting drought and salt stress. Virus-induced gene editing (VIGE) was used to screen sgRNAs that knockout the cotton GhAGL16 and verify the specificity of these sgRNAs, which laid a foundation for the creation of cotton agl16 mutants.【Method】 Three sgRNAs could knockout GhAGL16 were predicted based on the actual GhAGL16 genomic sequence cloned on subgroup A and D in cotton YZ-1; Three CLCrV-AtU6-26::GhAGL16-sgRNAs vectors were constructed based on the cotton leaf crumple virus (CLCrV)-mediated VIGE system; The expression of Cas9 in Cas9 over-expression (Cas9-OE) plants was detected by qPCR to determine whether Cas9 was stably genetically expressed; Three CLCrV-AtU6-26::GhAGL16-sgRNAs vectors were transformed respectively into Cas9-OE cotton cotyledons and detected the mutations of the three targets by PCR/RE; The secondary structures of three GhAGL16-sgRNAs were analyzed by bioinformatics; Hi-TOM high-throughput sequencing was performed on mutant plants to determine the efficiency of gene editing. Meanwhile, the off-target rate of GhAGL16-sgRNA2 mutant plants were identified to detect the specificity of gene editing.【Result】 Three sgRNAs capable of simultaneously knocking out GhAGL16-A and D subgroups were successfully constructed. The detection results of Cas9 expression showed that Cas9 was stably expressed in different Cas9-OE cotton plants. PCR/RE mutation detection results showed that GhAGL16-sgRNA2 could be effectively used for the knockout of GhAGL16. Different mutation types with base deletions appeared at the target sites of cotton subgroups A and D, while GhAGL16-sgRNA1 and GhAGL16-sgRNA3 were two invalid sgRNAs. The secondary structure analysis results of three GhAGL16-sgRNAs indicated that GhAGL16-sgRNA1 and GhAGL16-sgRNA3 might have a phenomenon that the guide sequence was easy to pair with other sequences and difficult to unwind, which interfered with the recognition of the target site by the guide sequences and lead to the invalid sgRNA. To further quantify the editing efficiency of GhAGL16-sgRNA2 on GhAGL16, the mutation detection results of each Cas9-OE plant transformed with CLCrV-AtU6-26::GhAGL16-sgRNA2 showed that six of the nine Cas9-OE plants were mutated, with a mutation efficiency of 66.67%. In addition, Hi-TOM high-throughput sequencing results showed that the editing efficiency of GhAGL16-sgRNA2 for GhAGL16 was 13.69%-54.42%. The off-target identification results showed that no off-target phenomenon was detected at the four predicted off-target sites, indicating that GhAGL16-sgRNA2 not only has high gene editing efficiency, but also has specific gene editing specificity.【Conclusion】 A sgRNA that can effectively knocking out the GhAGL16 was obtained by transforming Cas9-OE cotton using the CLCrV-mediated VIGE system, providing an ideal sgRNA for creating cotton agl16 mutants.

Analysis of Transposable Element Associated Epigenetic Regulation under Drought in Maize

GAO ChenXi, HAO LuYang, HU Yue, LI YongXiang, ZHANG DengFeng, LI ChunHui, SONG YanChun, SHI YunSu, WANG TianYu, LI Yu, LIU XuYang

Scientia Agricultura Sinica. 2024, 57(6): 1034-1048. doi:10.3864/j.issn.0578-1752.2024.06.002

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【Objective】 Drought is one of the serious abiotic stresses influencing maize production worldwide. Understanding the molecular mechanisms underlying drought tolerance is of great importance in maize improvement. 【Method】 In the present study, representative maize inbred lines were selected for field drought experiment and the drought tolerance was estimated based on leaf relative water content and anthesis-silking interval. Two inbred lines with contrasting drought tolerance was used for genome resequencing and transposable element insertions were identified. The DNA methylation level of leaf and root tissues under different water treatments of the two lines were measured using Whole Genome Bisulfite Sequencing (WGBS). And the gene expression profiles of these samples were detected by RNA sequencing. The integrative atlas of transposable element insertion/deletion variants, differentially methylated regions and differentially expressed genes in the two lines were constructed. In addition, the transposable element insertion/deletion variant mediated epigenetic regulation of ZCN7, which has been conferred the drought tolerance function in our previous study, was analyzed. 【Result】 The field experiment showed inbred line H082183 showed highest drought tolerance, in which the leaf relative water content and anthesis-silking interval had no significant difference between drought and well-watered treatments. While the Lü28 displayed lowest leaf relative water content and largest anthesis-silking interval under drought. Thus, these two lines were selected for further analysis. A total of 333 754 and 333 296 transposable element insertions were identified in the genome of H082183 and Lü28, respectively. And 89 954 transposable element insertions were polymorphism between two lines. The transposable element insertions, introns and promoters showed higher CG and CHG methylation level than exons and untranslated regions. Furthermore, 41 352 differentially methylated regions were identified between H082183 and Lü28. And 60% of the differentially methylated regions were located in the transposable element insertion\deletion variants and 5 kb flanking regions. The gene expression level showed negatively correlated with CG and CHG methylation. Differentially expression analysis between H082183 and Lü28 obtained 4 196 and 3 500 differentially expressed genes in leaf and root under drought, respectively. The 19.5% and 19.7% of these differentially expressed genes were located in differentially methylated regions. Three LTR transposable element insertions were identified in the 34 kb region of ZCN7 in Lü28 but absent in the genome H082183. And the DNA methylation levels of CG and CHG in this genomic region were significantly higher in Lü28 than H082183 under both drought and well-watered environments, which conferred higher ZCN7 expression in the drought tolerant line H082183. 【Conclusion】 Our results highlight the important role of interplay of transposable element insertions, DNA methylation and gene expression under drought. And gene expression regulation mechanism of ZCN7 relied on the transposable element insertion/deletion variants mediated DNA methylation was proposed.

Response of Wheat Grain Yield and Water Use Efficiency to Ploughing Time and Precipitation and Its Distribution in Dryland

DANG JianYou, JIANG WenChao, SUN Rui, SHANG BaoHua, PEI XueXia

Scientia Agricultura Sinica. 2024, 57(6): 1049-1065. doi:10.3864/j.issn.0578-1752.2024.06.003

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【Objective】 Aiming to solve the problems of low precipitation and uneven distribution of precipitation during the growth period, significant inter-annual variability, large proportion of precipitation during fallow period, and low water use efficiency (WUE) of precipitation, the objective of this study is to investigate the impact of deep ploughing time during fallow period in different precipitation years on soil water storage and wheat yield, and to provide a theoretical basis for revealing the yield depending precipitation years in southern Shanxi.【Method】 The field experiment was laid out following a randomized complete block design from 2010 to 2021, with three deep ploughing times set on August 10th (P₁), August 20th (P₂), and August 30th (P₃) to investigate the effects of ploughing time, precipitation, and its distribution on wheat yield, soil water storage characteristics during fallow periods, water consumption during growth periods, and their correlations.【Result】 (1) The precipitation year type exhibited a strong correlation with the average yield of dryland wheat, which ranged from 2 400.0-6 298.0 kg·hm^-2, with the coefficient 29.8%. The average yields for wet years (2012, 2014 and 2015), normal years (2011 and 2013), and dry years (2010, 2016, 2019, 2020 and 2021) were 5 524.6, 3 048.2, and 4 088.7 kg·hm^-2, respectively. The impact of precipitation year type on yield followed a descending order: annual precipitation, precipitation in the fallow period, and precipitation in the growth period. Both precipitation in the fallow period and the growth period primarily affected yield through their effect on spike number and kernels per spike. Additionally, an increase in both annual precipitation and March to April’s precipitation led to a significant increase in yield. The effect of deep ploughing time on wheat yield in dryland was closely related to the precipitation distribution in fallow period, which showed that P₁ yield was higher with more precipitation in July-August, and P₂ or P₃ yield was higher with more precipitation in September. (2) Precipitation, its distribution, and ploughing time had an impact on soil storage and consumption characteristics before sowing and after harvest. Precipitation during the fallow period mainly affected the soil water storage capacity in the lower layer (100-200 cm) before sowing and after harvest. The water storage capacity of the upper layer (0-100 cm) and the lower layer was similar or slightly low before sowing under the water-rich type in the fallow period. The water storage capacity of the lower layer was 6.17% and 24.66% lower than that of the upper soil under normal water and low water in the fallow period, respectively. More precipitation in July led to greater water storage in the lower soil, while more precipitation in August and September resulted in greater water storage in the upper soil. During growth periods, precipitation primarily impacted water storage capacity of the upper soil at harvest time, especially precipitation occurring in May. The average soil water storage capacity in the upper layer was the highest in P₂ before sowing and at harvest time, and the water storage capacity of subsoil and the average water storage capacity of 0-200 cm soil was the highest in P₂or P₃ at harvest time. (3) The effects of ploughing time and precipitation on water consumption and WUE were relatively small. The water consumption of dryland wheat during the growing period was affected by the annual precipitation, especially the precipitation in March.【Conclusion】 Precipitation and its distribution during the fallow period affect the soil water storage capacity of 0-200 cm before sowing of dryland wheat, and affect the yield of dryland wheat through spike number, kernels per spike together with the precipitation in March and April. There is a significant positive correlation between WUE and yield. In dryland wheat fields in south Shanxi Province, when there is more precipitation in July-August, deep ploughing is done on August 10th, and when there is less precipitation, deep ploughing is properly delayed from 10 to 20 d, which can effectively maintain soil water content during fallow period and achieve high yield.

Inversion of Chlorophyll Content of Film-Mulched Maize Based on Image Segmentation

ZHOU ZhiHui, GU XiaoBo, CHENG ZhiKai, CHANG Tian, ZHAO TongTong, WANG YuMing, DU YaDan

Scientia Agricultura Sinica. 2024, 57(6): 1066-1079. doi:10.3864/j.issn.0578-1752.2024.06.004

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【Objective】 In order to quickly and accurately monitor chlorophyll content of film-mulched maize, explore whether the removal of film and shadow background pixels can improve the accuracy of chlorophyll content inversion with spectral and texture features.【Method】 This study was based on multi-spectral remote sensing image data of unmanned aerial vehicle (UAV) and took chlorophyll content of film-mulched maize at seedling stage, jointing stage, tasseling stage and filling stage as objects. The support vector machine supervised classification was used to segment image background pixels and maize pixels, analyze the influence of background pixels on the spectra of maize canopy, the vegetation index and texture features of all pixels and maize pixels images were calculated and the better variable input was screened, and the inversion model of leaf chlorophyll content was established by using three machine learning algorithms, partial least squares, support vector machine and BP neural network.【Result】 (1) Background pixels in the multispectral images at seedling stage, jointing stage, tasseling stage and filling stage had significant effects on the spectra of maize canopy. (2) The inversion accuracy of vegetation index, texture feature and vegetation index + texture feature as variable input based on maize pixels image extraction was better than that of all pixels image (R² for optimal model was increased by 0.078, RMSE and MAE were decreased by 0.060 and 0.055 mg·g^-1, respectively, and R² for verification was increased by 0.109, RMSE and MAE were reduced by 0.075 and 0.047 mg·g^-1, respectively. (3) The modeling accuracy based on maize pixels image with spectral features + texture features as variable inputs was significantly improved over the modeling accuracy using only spectral features or texture features as variable inputs; The BP neural network model with spectral features + texture features as variable inputs had the highest accuracy (R², RMSE and MAE were 0.690, 0.468 mg·g^-1 and 0.375 mg·g^-1, respectively).【Conclusion】 The multispectral image spectral and texture feature data of UAV with removing background pixels and combined with BP neural network can better realize the inversion of chlorophyll content of film-mulched maize. The results can provide theoretical reference for quick and accurate retrieval of leaf chlorophyll content of film-mulched maize by UAV remote sensing.

Autophagy-Related Gene FpAtg3 Involves in Growth and Pathogenicity of Fusarium pseudograminearum

DONG ZaiFang, DING TengTeng, SHAN YiXuan, LI HongLian, CHEN LinLin, XING XiaoPing

Scientia Agricultura Sinica. 2024, 57(6): 1080-1090. doi:10.3864/j.issn.0578-1752.2024.06.005

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【Background】 Fusarium crown rot (FCR), caused by Fusarium pseudograminearum, is one of the newly-occurred destructive diseases in the main wheat-growing region of China. Autophagy is an evolutionarily conserved cellular process that regulates the growth, development and infection of different phytopathogenic fungi. However, the role of autophagy in F. pseudograminearum is still unknown.【Objective】 To clarify the role of the autophagy gene FpAtg3 in the growth and pathogenicity of F. pseudograminearum, understand the mechanisms of the fungal infection, and to provide a theoretical basis for FCR prevention.【Method】 The known Atg3 proteins of different fungi were downloaded from NCBI, and the MEGA 5.05 was used to construct the phylogenetic tree of Atg3 proteins. The Split-PCR approach was used to generate FpAtg3 gene-replacement constructs, and then the FpAtg3 deletion mutants (ΔFpAtg3) were constructed by polyethylene glycol (PEG)-mediated protoplast fungal transformation, and obtained by hygromycin resistance screening and PCR detection. The sequence of FpAtg3 and its native promoter was amplified and fused in pKNTG vector. The plasmid of pKNTG-FpAtg3 was then introduced into ΔFpAtg3 protoplasts for the complementation assay. The hyphal growth and colony morphology of the wild type, ΔFpAtg3 and the complementary (FpAtg3-C) strains were assayed on PDA, CM and MM plates. The hyphal blocks were cultured on PDA plates for hyphal morphology and hyphal fusion test; hyphal blocks were introduced into liquid CMC medium to assess conidiation and conidia morphology; conidia were cultured on Petri dishes to explore conidial anastomosis tube (CAT)-mediated fusion. The hyphal blocks were inoculated on wheat coleoptiles and barley leaves to explore pathogenicity, and the pot-culture experiment was performed for FCR assay. Congo red, sodium dodecyl sulphate (SDS) and H₂O₂ reagents were added to PDA plates to determine the F. pseudograminearum responses to cell wall, cell membrane and oxidative stresses, respectively.【Result】 Atg3 homologous proteins from different fungi were highly conserved and consistent with the direction of species evolution. The FpAtg3 had a close relationship with Atg3 of Fusarium graminearum and Fusarium oxysporum. The ΔFpAtg3 and FpAtg3-C strains were obtained. The phenotypic measurements showed that compared to wild type and FpAtg3-C strains, ΔFpAtg3 exhibited significantly reduced colony growth rates and aerial hypha, curved hypha, reduced conidiation, shorter in length and fewer in septa; ΔFpAtg3 also showed significantly reduced hyphal fusion rate and CAT-mediated fusion rate. The pathogenicity of ΔFpAtg3 on wheat coleoptiles and barley leaves was significantly reduced comparing to that of the wild type and FpAtg3-C strains. The reduced pathogenicity of ΔFpAtg3 was further examined on FCR. Furthermore, ΔFpAtg3 displayed more sensitive to cell wall, cell membrane and oxidative stress than that of the wild type and FpAtg3-C strains.【Conclusion】 Autophagy-related gene FpAtg3 plays important roles in growth, conidiation, hyphal fusion, pathogenicity and response to abiotic stresses of F. pseudograminearum.

Effects of Extreme High Temperature on Trehalose Content and Trehalose Transporter Gene in Tetranychus truncatus

LUO LiDan, CHEN JiaMing, AN Qi, LIU Lei, SUN QinZhe, LIU Huan, WANG SenShan, SONG LiWen

Scientia Agricultura Sinica. 2024, 57(6): 1091-1101. doi:10.3864/j.issn.0578-1752.2024.06.006

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【Objective】 The objective of this study is to clarify the effect of high temperature on the trehalose transporter gene of Tetranychus truncatus, and to provide a theoretical basis for the green control of pests.【Method】 According to the transcriptome data of T. truncatus induced by high temperature, the CDS sequences and protein amino acid sequences of two trehalose transporter genes TtTret1-like and TtTret1 were obtained. Bioinformatics analysis of TtTret1-like and TtTret1 was carried out using analytical tools such as ExPASy, ProScale and MEGA. The expression characteristics of TtTret1-like and TtTret1 at different development stages and different high temperatures (38, 42, 46 and 50 ℃) were analyzed by qRT-PCR. The trehalose content of T. truncatus at different high temperatures was determined by microanalysis. RNA interference (RNAi) technique was used to silence TtTret1-like and TtTret1, and to explore their functions in coping with high temperature.【Result】 Bioinformatics analysis showed that the full-length CDS of TtTret1-like and TtTret1 was 1 389 and 1 569 bp, respectively, encoding 463 and 523 amino acids. The predicted protein molecular weights were 50 189.03 and 57 358.10 Da, and the isoelectric points were 8.87 and 8.70, respectively. TtTret1-like and TtTret1 are both basic amino acids and hydrophobic amino acids, belonging to unstable proteins. The secondary structure is a spiral and coiled structure. Both trehalose transporters have conserved domains and 12 transmembrane domains of major facilitator superfamily (MFS). The amino acid sequence similarity and phylogenetic analysis showed that TtTret1-like and TtTret1 were highly consistent with the trehalose transporter sequences of other arachnids, especially with T. urticae. The expression level of TtTret1-like was higher in egg, larva and adult stages. TtTret1 was highly expressed in larva stage. With the increase of treatment temperature, the expression level of TtTret1-like increased, and reached the highest level at 50 ℃. However, with the increase of treatment temperature, the expression level of TtTret1 increased first and then decreased, reaching the maximum at 42 ℃. The trehalose content in T. truncatus increased significantly after high temperature stress. After silencing TtTret1-like and TtTret1 for 48 h, the trehalose content in T. truncatus increased, but the trehalose content in hemolymph decreased. The survival rates of T. truncatus were 11% and 46.67% after treated at 50 ℃ for 2 h and then recovered for 96 h, which were significantly lower than those of the control group.【Conclusion】 It is hypothesized that TtTret1-like and TtTret1 in T. truncatus play an important role in their responses to high temperature stress.

Effect and Mechanism of Phosphate-Solubilizing Bacterial on Activating of Low-Grade Phosphate Rock Powder in Red Paddy Soil

SONG YaRong, CHANG DanNa, ZHOU GuoPeng, GAO SongJuan, DUAN TingYu, CAO WeiDong

Scientia Agricultura Sinica. 2024, 57(6): 1102-1116. doi:10.3864/j.issn.0578-1752.2024.06.007

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【Objective】 The biological utilization of phosphorus (P) in low grade phosphate rock powder (PRP) is of great significance. The study explore the effect and mechanism of activating low-grade PRP by phosphate-solubilizing bacterial (PSB) which can provide basis for improving the fertilization effect of PRP on red soil paddy soil. 【Method】 PRP with different particle sizes (0.18, 0.10 and 0.05 mm) was added to the red paddy soil collected in Hunan Province. Three treatments were set up: inoculated with Acinetobacter calcoaceticus (P1), Acinetobacter pittii (P2) and no inoculation with PBS (P0) as control. Bottles were cultured in a dark incubator at 25 ℃ for 60 days, during which different forms of P and pH were dynamically monitored. On the 60th day, the activity of organic acid, acid and alkaline phosphatase (ACP, ALP) and alkaline phosphatase gene (phoD) were determined to study the activation effect and mechanism of PSB on PRP with different particle sizes. 【Result】 Both strains of PSB could activate insoluble P, but the activation effect was not significantly different. During the whole culture period, the average available P content of three particle sizes of PRP inoculated with PSB was 13.4-14.7 mg·kg^-1, which was higher than that of P0 treatment and increased by 31.1%-53.1%; The average content of available P increased by 53.1% and 47.5% after inoculation with P1 and P2 bacteria (P<0.05), respectively. The average content of Resin-Pi and NaHCO₃-Pi were 13.9-16.6 mg·kg^-1 and 14.9-16.5 mg·kg^-1, respectively, which were higher than those without inoculation, and increased by 36.4%-78.5% and 13.7%-25.0%, respectively; the increase of average content of Resin-Pi in 0.18 mm PRP was the most obvious, which was 78.5% and 49.5%, respectively (P<0.05). Compared with P0 treatment, the increase of active P in 0.18 mm PRP treatment was the most obvious, ranging from 28.4% to 46.7%, and the decrease of stable P was 2.1%-8.0%. Compared with the P0 treatment, inoculation of PSB significantly reduced pH value by 0.18-0.35 units (P<0.05) and increased acetic acid and propionic acid content by 5.2%-13.7% and 45.9%-127.5% (P<0.05), respectively. ALP content and phoD abundance under P1 treatment increased by 6.5%-13.4% and 24.0%-98.6% (P<0.05), respectively, and ACP in P2 treatment increased by 12.8%-17.2% (P<0.05), which indicated that P1 mainly secreted ALP, while P2 mainly secreted ACP. The results of correlation analysis showed that the two PSB strains dissolved insoluble Conc.HCl-Po, Conc.HCl-Pi and NaOH-Pi by secreting acetic acid and propionic acid, dissolved insoluble Conc.HCl-Po by secreting ACP and ALP, and converted into these insoluble P to Resin-Pi and NaHCO₃-Pi which promoted the turnover of P pool. The structural equation model showed that the addition of small particle size PRP and the inoculation of PSB could directly increase the soil available P content, but the inoculation of PSB had a greater effect on available P. 【Conclusion】 Inoculation of phosphate- solubilizing bacterial can promote the activation of insoluble phosphorus in phosphate rock powder with the biggest increasement of available phosphorus for particle size of 0.05 mm, and biggest increasement of active phosphorus proportion for particle size of 0.18 mm. The two phosphate-solubilizing bacteria A. calcoaceticus and A. pittii mainly secreted organic acids and phosphatases, such as acetic acid and propionic acid, activated insoluble phosphorus, increased active phosphorus content, and improved the application effect of phosphate rock powder in red paddy soil.

Assessment of Application Efficacy for Agro-Forestry Absorbent Polymers and Their Environmental Risks

WANG XiaoBin, YAN Xiang, LI XiuYing, SUN ZhaoKai, TU Cheng

Scientia Agricultura Sinica. 2024, 57(6): 1117-1136. doi:10.3864/j.issn.0578-1752.2024.06.008

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Since the performing of Seventh Five-Year (1986-1990) National Scientific and Technological Projects in China, when the national dryland farming research projects started, the super-absorbent polymers for agriculture and forestry (SAP-AF), as one of the technical products for drought-resistance and water-retention in agriculture and forestry, have been given concerned. A Chinese agricultural standard of Agro-forestry absorbent polymer (NY 886) was issued in 2004, and then revised three times in 2010, 2016 and 2022, respectively. However, so far there is still no relevant standard or regulation on experiment and assessment for the SAP-AF application efficacy, thus greatly affecting its promotion and application. Based on the literature review on the relevant research and application status of the SAP-AF for more than 30 years (1990-2023), this paper focused on the research about the effects of the SAP-AF application on soil water retention, crop water use, and crop yield, as well as environmental impact. The results showed as follows. (1) The SAP-AF products could enhance the ability to soil water storage and retention (especially for sandy soils), and be beneficial to protecting crop seedlings against drought, water-saving, and yield-increasing, as well as water use efficiency (WUE)-improving in dry-farming areas. (2) The assessment indicators (mainly including soil water storage, crop water consumption, crop yield, and WUE) were proposed to be applicable to evaluate the experiments for the SAP-AF product application efficacy. (3) According to the phenomenon that the SAP-AF application for crops in some regions could not always show a significant effect on yield increase, or sometimes had a negative return, it was proposed that SAP-AF application should formulate corresponding technical regulations, and determine the appropriate product types, and their application methods and dosage for crops. (4) The environmental safety of the SAP-AF products (mainly in the form of polyacrylamide or polyacrylic acid (PAM or PAA)-based materials) mainly involved the biodegradability of the PAM or PAA-based materials and the biological toxicity induced by the residual monomers (acrylamide or acrylic acid (AM or AA)) in the products. The residual AM or AA monomer content detected in the PAM or PAA-related products on the market were partly at the risk of exceeding the allowable limits (accounting for about 22%-100% of the total, referred to as the standard limits for some similar products). The review also indicated that some natural polymer materials such as such as starch grafted based polymer materials would be the replaycement of the SAP-AF in the future. The results of this study provided the reference for the formulation of evaluation standards or regulations for SAP-AF product application efficacy. It was suggested that relevant departments should strengthen the detection of residual monomer and its limit requirements for PAM or PAA-based SAP-AF products, and quantify the biodegradability index of products, in order to provide protection for the environmental safety of agricultural and forestry water protection agent products. It was suggested to strengthen the detection of residual AM or AA monomers, and research on the environmental safety threshold of residual monomers for such PAM or PAA-based SAP-AF products; but also need to further explore the relationship between the product biodegradability indicators in the soils and the safety of soil ecological environment, in order to ensure the product environmental safety.

Water and Carbon Footprint and Layout Optimization of Major Grain Crops in the Northwest China

WANG ChuFan, NIU Jun

Scientia Agricultura Sinica. 2024, 57(6): 1137-1152. doi:10.3864/j.issn.0578-1752.2024.06.009

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【Objective】 This paper assessed the carbon footprint and water footprint of major food crops in the northwest region, and established a multi-objective planting structure optimization model based on the carbon footprint and water footprint to adjust the spatial distribution of food crops in each province, so as to provide a theoretical basis for reducing carbon emissions and enhancing water resource management. 【Method】 Based on the planting area, yield, and agricultural inputs of wheat, maize, and rice in the northwest region, the carbon footprint was evaluated using Life Cycle Analysis (LCA); Based on evapotranspiration and effective precipitation, the water footprint was evaluated by Penman-Monteith formula; Multi objective optimization of the layout of three major grain crops in the five northwest provinces was performed based on entropy weight method. 【Result】 The data showed that the carbon footprint and water footprint of wheat, maize and rice in northwest China from 1999 to 2020 showed significant differences in different provinces. In the northwest region, the carbon emissions from the production of three grain crops, wheat, maize and rice showed a distribution pattern of higher levels in the eastern and western regions and lower levels in the central region; the range of carbon footprint per unit production (PCF) was 0.36-0.63, 0.33-0.56, 0.57-0.97 t CO₂-eq·t^-1, respectively; the carbon footprint per unit area (FCF) was (2.46±0.77), (3.21±0.49) and (5.57±0.91) t CO₂-eq·hm^-2, respectively. From 2010 to 2018, the total green water content of wheat, maize and rice in the northwest region showed a steady upward trend, with regions with higher average green water content distributed in Shaanxi, Gansu, and Ningxia. The total amount of blue water showed an upward trend from 2010 to 2015, and a downward trend from 2015 to 2018. The regions with higher average total blue water were distributed in Gansu, Ningxia, and Xinjiang. Among the three major grain crops in northwest China, maize consumes the least blue water footprint, with an average blue water footprint of 0.45 m³·kg^-1; the blue water footprint consumption of rice was the largest, and the average production blue water footprint was 0.77 m³·kg^-1. The optimization of food crop cultivation structure was performed, based on carbon and water footprints, using the cultivated area of different crops in each province as decision variables, and setting optimization scenarios focusing on reducing carbon emissions (ecological benefits) and increasing green water use (water resource benefits) according to different weights. In Scenario 1, the total carbon emissions decreased by 1.9% and the total green water increased by 5.0%; In scenario 2, the total carbon emissions decreased by 11.8%, while in scenario 3, the total utilization of green water increased by 6.7%. 【Conclusion】 There were significant spatial and temporal differences in carbon emissions and total water volume of the three major food crops in the Northwest Region. In terms of carbon footprint, the average FCF of the three major grain crops in the region shows an increasing trend, and the PCF shows a decreasing trend. In terms of water footprint, the green water footprint of the three major food crops in the region was higher than the national average, with maize having the largest green water footprint and rice having the smallest green water footprint. Under the premise of ensuring food crop security, the planting area of wheat has increased by 6.7%, while the planting area of maize and rice has decreased by 5.8% and 8.0%, respectively. The economic, resource, and ecological benefits have all been improved to a certain extent. In summary, multi-objective optimization could improve the utilization of green water resources, reduce carbon emissions, and alleviate environmental pressure.

Research on Leaf Nutritional Diagnosis of Liaoning 1 Walnut in Taihang Mountain Area of Hebei Province

AN XiuHong, SUN Yan, WANG Fang, FENG QiKe, WANG Ning, LI JinJin, ZHANG JunPei, WANG HongXia

Scientia Agricultura Sinica. 2024, 57(6): 1153-1166. doi:10.3864/j.issn.0578-1752.2024.06.010

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【Objective】 The mineral element content in leaves of Liaoning 1 walnut in Taihang Mountain area of Hebei Province were determined and the nutritional status of leaves were diagnosed, so as to provide the basis for scientific fertilization for Liaoning 1. 【Method】 The content of mineral elements in Liaoning 1 leaves at different developmental stages were measured to determine the sampling time of leaf nutritional diagnosis. Component nutritional diagnosis (CND) method was used to establish the standard values of element contents for the nutritional diagnosis of Liaoning 1. The diagnosis and recommendation integrated system (L-DRIS), CND, and deviation from optimum percentage (DOP) methods were used to diagnose the leaf nutritional status in low-yielding orchards to provide fertilization strategies. 【Result】 The suitable period for leaf nutritional diagnosis of Liaoning 1 in Taihang Mountains of Hebei province from August to September. Correlation analysis showed that the mineral elements in Liaoning 1 leaves from Taihang Mountain area mainly had synergistic effects, and there was strong antagonistic interaction between K and Mg. Based on the CND analysis, the yield of 3.64 t∙hm^-2 was selected as the yield cutoff value of high-yielding orchards. The optimal values of mineral element content determined by high-yielding orchards were N (13.04-17.04) g∙kg^-1, P (0.67-1.23) g∙kg^-1, K (5.94-10.64) g∙kg^-1, Ca (15.64-22.16) g∙kg^-1, Mg (3.80-6.62) g∙kg^-1, Cu (5.14-8.48) mg∙kg^-1, Fe (437.38-794.58) mg∙kg^-1, Mn (126.94-172.02) mg∙kg^-1, and Zn (14.59-34.25) mg∙kg^-1. There were differences in the abundance and deficiency of mineral elements in Liaoning 1 leaves of low-yielding orchards obtained through L-DRIS, CND, and DOP methods, and the diagnosis results of L-DRIS and CND methods were more consistent. The fertilization order of low-yielding orchards in Taihang Mountain of Hebei province was K>P>N>Zn>Mn>Ca>Fe>Mg>Cu. 【Conclusion】 It was suggested that K should be the main fertilizer, combined with N and P, while excessive applications of Mg and Cu should be avoided in low-yielding orchards.

Effects of Different Ratios of Green-Blue Light on Basil Growth and Its Energy Use Efficiency

YANG Yang, JIA MengHan, CHEN Can, ZHANG YiHan, TONG YuXin

Scientia Agricultura Sinica. 2024, 57(6): 1167-1179. doi:10.3864/j.issn.0578-1752.2024.06.011

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【Objective】 This study aimed to investigate the effects of different ratios of green-blue light (G/B) with constant red light (R, 660 nm) on the growth, stomatal characteristics, photosynthetic capacity, and energy utilization efficiency of basil plants (Ocimum basilicum Linn.). The ultimate goal of this study was to provide the technical support for efficient and energy-saving production of basil plants in plant factory. 【Method】 Basil seedlings were subjected to seven different light quality treatments of RB (R:G:B=8:0:8, set as control), RG₁B₇(R:G:B=8:1:7), RG₁B₃(R:G:B=8:2:6), RG₁B₁(R:G:B=8:4:4), RG₃B₁(R:G:B=8:6:2), RG₇B₁(R:G:B=8:7:1), and RG (R:G:B=8:8:0) for a duration of 19 days after transplanting. The total light intensity was 160 μmol∙m^-2∙s^-1, with red light accounting for 50% of the total light intensity. The photo/dark period was 16/8 hours per day. The air temperature during the light/dark period was set at 25 ℃/23 ℃, and the relative humidity was set at 65%. Additionally, the CO₂ concentration during the light period was 1 000 μmol∙mol^-1. The photosynthesis parameters of basil plants were determined after 13 days of experimental treatment, while the growth parameters, stomatal characteristics, and energy use efficiency of basil plants were determined after 19 days of experimental treatment. 【Result】 Except for treatment RG₁B₃, the addition of green light significantly enhanced height, leaf area, and dry and fresh weight of basil plants. The shoot dry and fresh weights of basil plants under different G/B treatments showed an increase of 19%-51% and 26%-64%, respectively, compared with those under the control. However, no significant differences in leaf area and dry/fresh weights were observed among the G/B treatments, except those under treatment RG. The treatment with low G/B (RG₁B₇) only caused a reduction in the stomatal density on the abaxial surface of basil lower leaves. As G/B increased, the stomatal density decreased on the adaxial surface of the lower leaves and both surfaces of the upper leaves. In general, the leaf nitrogen content decreased with an increase in the G/B. This decrease in leaf nitrogen content and stomatal density resulted in a decline in CO₂ assimilation capacity and light utilization capacity of basil plants. The reduction in these capacities generally increased with the increasing G/B. Due to the lower light-emitting efficiency of green LEDs compared with blue light, the total power consumption increased with an increase in the G/B. Only the treatment RG₁B₇significantly improved electric use efficiency by 25% than that under the control. With the exception of RG₁B₃, all other G/B treatments improved light utilization efficiency by 30%-57% than that under the control, and no significant differences were found among different G/B treatments, except for RG. RG₃B₁ and RG improved water use efficiency of both upper and lower basil leaves, with RG₃B₁ showing a 58% and 74% increase, and RG showing a 67% and 90% increase, respectively compared with that under the control. 【Conclusion】 By comprehensively analyzing, basil plants growth and energy utilization efficiency, the RG₁B₇ could be considered as a more suitable light quality combination for basil plants production in plant factories. The findings of this study could serve as a technical support for understanding the impact of different G/B ratios on basil growth, photosynthetic characteristics, and energy utilization efficiency. This research provided the valuable insights for developing optimized light formulations that enable efficient and energy-saving production of basil plants in plant factory environments.

Analysis of the Effect of Dielectric Barrier Discharge Cold Plasma on Phenolic Metabolism of Stored Paddy Rice Under High Temperature Stress

HOU Shuai, ZHANG YiJia, ZHOU DanDan, MA FeiYang, WANG DaPeng, ZHAO SiQi, DING Chao, LIU Qiang

Scientia Agricultura Sinica. 2024, 57(6): 1180-1190. doi:10.3864/j.issn.0578-1752.2024.06.012

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【Objective】 The aim of his study was to explore the impact of dielectric barrier discharge cold plasma (DBD-CP) treatment on the phenolic metabolism of stored rice under high-temperature stress.【Method】 Under simulated summer high- temperature conditions (35 ℃), fresh high-moisture rice (Japonica rice, 16.0% moisture content on a wet basis) was used as the raw material, the effects of DBD-CP treatment on quality parameters such as L^*, a^*, b^*, yellowness index (YI), reactive oxygen species, and malondialdehyde (MDA) in stored rice were analyzed. Additionally, the study aims to analyze the changes in total phenols, total flavonoids, and antioxidant activities during the storage process. The investigation would involve multiple validations, including ABTS⁺ and DPPH radical scavenging activities, ferric ion reducing power, and the activity changes of key rate-limiting enzymes in phenylpropane metabolism. The ultimate goal was to explore the impact of DBD-CP treatment on the phenolic metabolism of stored rice. 【Result】 After DBD-CP treatment, the color stability of rice during storage significantly improved. The yellowing index of the treatment group decreased significantly by 12.6% after 60 days of storage. After 20 days of storage, the levels of superoxide anion, hydrogen peroxide, and MDA decreased by 14.8%, 41.6%, and 21.6%, respectively, and the deterioration rate of fresh high-moisture rice was significantly inhibited. Additionally, during the high-temperature stress storage period, the total phenol and total flavonoid content in the treatment group increased simultaneously (P<0.05), with maximum increases of 1.23 and 1.34 times, respectively. The antioxidant properties and iron ion reduction capacity of the corresponding samples were also improved (P<0.05). Combined with the analysis of the activity of key limiting enzymes in phenylpropane metabolism, the activities of phenylalanine ammonia-lyase (PAL), cinnamic acid-4-hydroxylase (C4H), and chalcone synthase (CHS) were significantly enhanced, with increases ranging from 1.71 to 2.28 times. Further correlation analysis confirmed the close association (P<0.05) between the content of phenolic substances in high-moisture rice under high-temperature stress and the activities of PAL, C4H, and CHS enzymes, and a significant negative correlation (P<0.05) with internal reactive oxygen species and MDA content. 【Conclusion】 Dielectric barrier discharge cold plasma treatment could promote the synthesis rate of phenolic compounds within the grains of high-moisture rice by activating phenylpropane metabolism. This treatment enhanced the tissue's antioxidant properties, alleviated membrane lipid peroxidation, and reduced the accumulation of free radicals. As a result, it delayed the quality deterioration of rice under high-temperature storage conditions.

Study on the Role of FoxO1 in Regulating the Proliferation, Apoptosis and Differentiation of Bovine Skeletal Muscle Cells

JIANG Chao, ZHANG JiuPan, SONG YaPing, SONG XiaoYu, WU Hao, WEI DaWei

Scientia Agricultura Sinica. 2024, 57(6): 1191-1203. doi:10.3864/j.issn.0578-1752.2024.06.013

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【Objective】 Skeletal muscle is an important component of animal body, and its growth and development directly affect the meat yield of livestock and poultry. As an important transcription regulator, fork head box protein O1 (FoxO1) is closely related to the growth and development of skeletal muscle. Exploring the effects of overexpression of FoxO1 gene on the proliferation, apoptosis and differentiation of bovine skeletal muscle cells could provide the theoretical basis for genetic improvement of beef. 【Method】 The tissue expression profile of FoxO1 was constructed by real-time fluorescence quantitative PCR (qPCR). To test the cell differentiation performance of bovine skeletal muscle cells which were isolated by enzyme digestion, the myotubes formation was observed and the expressions of differentiation marker genes were detected by qPCR. The subcellular localization of bovine skeletal muscle cells was carried out by immunofluorescence technique. In order to improve the expression of FoxO1 in bovine skeletal muscle cells, bovine FoxO1 overexpression adenovirus were designed and packaged. The effect of FoxO1 overexpression on the relative proliferation rate of cells was detected by EdU staining. Flow cytometry was used to detect the effect of FoxO1 overexpression on cell cycle distribution. The effects of FoxO1 overexpression on the expression levels of genes related to proliferation, apoptosis and differentiation of bovine skeletal muscle cells were investigated by qPCR.【Result】 The results of tissue expression spectrum showed that FoxO1 was expressed in many tissues, with the highest expression in the back fat of adult cattle and the lowest expression in the longissimus dorsi muscle tissue, while the expression of FoxO1 in the longissimus dorsi muscle tissue of calves was significantly higher than that of adult cattle (P<0.01). The results of subcellular localization showed that FoxO1 was expressed in the nucleus and cytoplasm of bovine skeletal muscle cells, and the fluorescence intensity in the nucleus was higher than that in the cytoplasm. The FoxO1 overexpression vector was successfully constructed, and the packaging and propagation of FoxO1 recombinant adenovirus were completed. After infecting bovine skeletal muscle cells, the FoxO1 expression level was significantly improved (P<0.01). EdU detection showed that overexpression of FoxO1 significantly reduced the cell proliferation rate (P<0.01), and flow cytometry showed that overexpression of FoxO1 significantly increased the number of cells in G1 phase and decreased the number of cells in S phase and G2 phase, inhibited the transformation of cells in G1/S phase and reduced the formation of cells in G2 phase. Further detection by qPCR found that the proliferation-related genes of PCNA, CDK1, CDK2, CCNA2, CCNB1, CCND1 and CCNE2 were significantly down-regulated (P<0.01), the apoptosis-related genes BAD and BAX were significantly up-regulated, and the apoptosis-inhibiting gene BCL2 was significantly down-regulated (P<0.05). After FoxO1 was overexpressed, the myotube formation of bovine skeletal muscle cells decreased, and the expression levels of MYOD, MYOG, MYF5, MYF6 and MYHC related to skeletal muscle differentiation were significantly decreased by qPCR detection (P<0.05). 【Conclusion】 FoxO1 was expressed in different tissues of cattle, and it was a ubiquitous transcription regulator. There were differences in expression at different stages of the growth and development of longissimus dorsi muscle, which played a role in stage regulation. FoxO1 played a transcriptional regulatory role in both the nucleus and cytoplasm, especially in the nucleus. Overexpression of FoxO1 might inhibit the proliferation and differentiation of bovine skeletal muscle cells by inhibiting the expression of genes related to cell proliferation and muscle cell differentiation, and might promote the apoptosis of bovine skeletal muscle cells by up-regulating the expression of apoptosis-promoting genes and down-regulating the expression of apoptosis- inhibiting genes.

Screening of Mycobacterium Avium Subsp. Paratuberculosis Immunogenic Proteins and Its Evaluation of Immunological Effect

CHEN FanRuo, ZHANG JiaJun, LU Ping, CUI Ning, CUI YingYing, CUI ZiYin, DANG GuangHui, LIU SiGuo

Scientia Agricultura Sinica. 2024, 57(6): 1204-1214. doi:10.3864/j.issn.0578-1752.2024.06.014

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【Background】 Paratuberculosis (PTB) is a chronic, wasting infectious disease caused by Mycobacterium avium subsp. paratuberculosis (MAP) in ruminants. PTB causes huge economic losses to the livestock industry and poses a serious threat to public health safety. Since the current clinical methods for the detection and control of PTB are inadequate, and the PTB vaccine used is ineffective and interferes with the diagnosis of bovine tuberculosis, there is a need for developing a vaccine with strong immunogenicity, good safety, and excellent protection for the prevention and control of PTB. 【Objective】 The immunogenic protein of MAP was screened and its immunoprotective effect was evaluated, so as to provide the data support for the prevention and control of PTB. 【Method】 Five recombinant plasmids were constructed based on six genes of MAP: p22, map1272c, map3531c, map3783, map3701c, and map3527. The five recombinant proteins were combined with MONTANIDE ISA 61 VG adjuvant to immunize mouse by subcutaneous injection, and the best immunogen was screened by IFN-γ ELISPOT assay. The best immunogen was then mixed with the reported 66NC fusion protein. Mouse were immunized by subcutaneous multi-point injection. At 3 weeks after the second immunization, mice were immunized with 1×10⁸ CFU of the MAP K-10 strain intraperitoneally. The immunogenicity and immunoprotective effect of the candidate subunit vaccine were comprehensively evaluated by IFN-γ ELISPOT assay, monitoring antibody titers and serum cytokines, as well as detecting weight changes, liver pathological and histopathological observations and charge count differences of infected mouse. 【Result】 Five recombinant proteins, such as 58F, 62F, 69F, 46F, and 52F, were expressed based on the genes p22, map1272c, map3531c, map3783 and map3701c. 58F produced the highest level of IFN-γ after immunization and was the most promising candidate immunogen. The fusion protein combination 66NC+58F induced persistent high titers of IgG, IgM, IgG₁ and IgG_2a, and also induced specific release of IFN-γ, TNF-α, and IL-17A. In the evaluation of protective effects, the fusion protein combination 66NC+58F resisted the weight loss caused by MAP infection, significantly reduced pathological damage in the liver, and decreased MAP colonization in the liver. 【Conclusion】 The fusion protein combination 66NC+58F induced Th1 and Th17-type immune responses in mouse, provided immune protection against MAP infection and was an important candidate subunit vaccine for PTB.

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