Comparison of cell lineage development and regulation during early embryonic development in cattle and mice
Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(6): 765-775.
Low reproductive efficiency of high-yielding dairy cows is a worldwide challenge, among which the high mortality rate of early embryos is one of the main reasons. Scientists have gained insightful knowledge into early embryonic development in mice while very little work has been performed in cattle. To better understand the early embryonic development in cattle, we compared early embryonic development in cattle and mice from the perspectives of embryo morphology, transcription factors and signaling pathways. It was found that after fertilization, degradation of maternal factors, zygotic genome activation, construction of cell polarity and asymmetric division occurred at different periods in the early embryos of cattle and mice. Eventually, embryos develop to blastocysts with three germ layers (trophectoderm, epiblast and primitive endoderm). Furthermore, multiple transcription factors and signaling pathways form complicated networks to regulate cell lineage differentiation during early embryonic development in cattle and mice. In summary, similar biological events occur one after another at the early embryonic development in cattle and mice, but the regulation of cell lineage differentiation is different, suggesting the limitations of using the early embryo of mouse as a research model. The study in this field plays a crucial role in improving reproductive efficiency of dairy cows and promoting the genetic improvement in cattle.
Bioinformatics and expression analysis of heat shock protein genes in
Trametes gibbosa Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(6): 776-786.
To investigate the function and structure of the heat shock protein (HSP) family in
Trametes gibbosa, a cDNA library was constructed by collecting mycelial samples at different time under the sawdust treatment. All the HSP genes in this strain were screened by analyzing their transcriptome data; subsequently, bioinformatics analysis was performed for all the HSPs. Gene cloning and sequence structure analysis were performed for the HSP100 family, and the expression levels of the HSP100 genes were verified under the sawdust treatment by real-time fluorescent quantitative polymerase chain reaction (qRT-PCR). The results were as follows: A total of 32 HSP genes were screened and divided into five subclasses in T. gibbosa. Among the 32 HSPs, there were two HSP100, two HSP90, seven HSP70, one HSP60 and twenty small HSPs (sHSPs). In growth regulation, they had important functions, such as protein posttranslational modification, protein folding, and chaperonin. In T. gibbosa, HSPs were hydrophobic proteins with distinct physicochemical properties for different subclasses. The HSP100 family consist of an N-terminus, nucleotide-binding domain 1 (NBD1), NBD2, and the linker between the two NBDs. The NBDs had highly conserved Walker A and Walker B motifs and arginine finger residues. The qRT-PCR amplification results showed that there was obvious upregulation expression of HSP100 gene in T. gibbosa under the sawdust treatment. In summary, the classification of the HSP family in T. gibbosa is diverse and complex. Under stress conditions, the HSP100 family plays an important role in protein depolymerization, and its sequence and structure are relatively conserved. The above results can provide a theoretical basis for the study of T. gibbosa under stress.
Differential transcriptome analysis of different resistant citrus varieties infected with wild-type and toxin synthesis-impaired strains causing Alternaria brown spot
Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(6): 787-801.
To investigate the differences in the response of citrus varieties with different resistances to
Alternaria citri toxin (ACT) during interaction with the Alternaria alternata pathotype tangerine causing Alternaria brown spot, we inoculated the wild-type toxin-producing strain Z7 and the ACT synthesis-impaired mutant strain ΔΔ ACTT6 onto the leaves of resistant variety Clementine ( Citrus clementina Hort. ex Tan. cv. Clementine) and the susceptible variety Dancy ( Citrus reticulata Blanco cv. Dancy), and analyzed the gene expressions at 24 h and 48 h after inoculation using transcriptome sequencing technology. By comparing the corresponding differences in transcription between the two citrus varieties against the toxin-producing and non-toxin-producing strains, we found that the genes involved in the pathogen-associated molecular patterns (PAMPs)-triggered immunity (PTI) and effector-triggered immunity (ETI) pathways, pathogenesis-related proteins, WRKY transcription factors, and the secondary metabolic pathway were induced by the toxin-producing strain in both the varieties. At the later stage of inoculation (48 h), the toxin-producing strain mainly activated macromolecular degradation processes such as lipid and protein degradation in the susceptible citrus variety, indicating that its cellular integrity was disrupted due to toxin production, whereas the resistant citrus variety was mainly enriched in biological processes such as detoxification and jasmonic acid metabolism. Some multidrug and toxic compound extrusion protein (MATE) genes were specifically activated in the resistant citrus variety, which implies their ability to resist fungal toxins. In conclusion, the molecular regulatory mechanisms and key functional genes involved in the response of citrus varieties to ACT are preliminarily clarified, which provide a theoretical basis for the molecular breeding of citruses against Alternaria brown spot.
Analysis of main functional components in different parts of
Citrus× aurantium L.Changshanhuyou fruits Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(6): 802-812.
In this study, the whole fruit of
Citrus×aurantium L. Changshanhuyou was divided into five parts, namely, the flavedo, albedo, segment membrane, juice sacs and seeds, from which polyphenols and pectin polysaccharides were extracted sequentially. Ultra-high performance liquid chromatography-tandem mass spectrometry was used to determine 30 kinds of citrus polyphenols, and the compositions and molecular weights of pectin polysaccharides were compared to comprehensively analyze the compositions of the main functional components in different parts of Citrus×aurantium L. Changshanhuyou fruits. The results showed that flavonoids in various parts mainly existed in flavanones, and the contents of neohesperidin and neoeriocitrin in the albedo were the highest, reaching 13 700.72 μg/g and 6 270.24 μg/g (by dry mass and the same as below), respectively. The contents of cinnamic acids in various parts were higher than that of benzoic acids, and the contents of cinnamic acids in the flavedo were the highest. The yield of pectin polysaccharides extracted from albedo by amylase-assisted ethylenediamine tetraacetic acid solution was the highest (27.46%). The yield of pectin polysaccharides from the flavedo by the extraction of NaOH solution was the highest (11.30%), and the molecular weights ( M W) of pectin polysaccharides extracted by the two extraction methods in the flavedo were up to 6.888×10 5 g/mol and 2.343×10 5 g/mol, respectively. The flavedo, albedo and segment membrane of Citrus×aurantium L. Changshanhuyou can be used as the main processing sources of flavonoids and pectin, and their rich contents of neoeriocitrins provide new development direction for processing byproducts.
Optimization of extraction process and activity of protoilludane sesqui-terpene aryl esters from
Floccularia luteovirens Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(6): 813-824.
In this study, the effects of different methods on the extraction of protoilludane sesquiterpene aryl esters from
Floccularia luteovirens were studied. Using the ultrasonic-assisted extraction method, the extraction process conditions were optimized by Box-Behnken response surface methodology based on a single-factor experiment. The in vitro antioxidant activity of protoilludane sesquiterpene aryl esters was determined, and the antibacterial activities against three common pathogenic bacteria were evaluated via the inhibition zone method. The results showed that ethyl acetate was the most suitable solvent for the extraction of protoilludane sesquiterpene aryl esters. The optimal extraction conditions for accessing protoilludane sesquiterpene aryl esters from F. luteovirens were as follows: solid-liquid ratio of 1∶33 (mass to volume), ultrasonic time of 33 min, and ultrasonic power of 166 W. Under these conditions, the extraction content of protoilludane sesquiterpene aryl esters was 10.107 μmol/g. In addition, the protoilludane sesquiterpene aryl esters had strong scavenging effects against 2, 2-diphenyl-1-picrylhydrazyl free radicals and hydroxyl free radicals, and had different antibacterial effects on Escherichia coli, Staphylococcus aureus, and Bacillus cereus. This study provides data support and a theoretical basis for the further development of F. luteovirens related products.
Analysis of differential metabolites between ‘Zijuan’ and ‘Yingshuang’ tea flowers based on widely targeted metabolomics combined with high performance liquid chromatography
Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(6): 825-839.
To explore the differences in flavor between flowers of ‘Zijuan’ and ‘Yingshuang’ tea varieties, the abundance of non-volatile metabolites in two light fermented tea flowers was tested, and the metabolites identified via widely targeted metabolomic technology based on ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The evaluation factors were evaluated by tea sensory evaluation methods and the contents of taste components, such as catechins and flavonoids, were detected by high performance liquid chromatography and colorimetric method. The results showed that ‘Zijuan’ tea flowers tasted sweet and fresh, and a little bitter and slightly astringent, while ‘Yingshuang’ tea flowers tasted sweet, slightly bitter and a little astringent. There were 219 kinds of significantly differential metabolites, including phenolic acids (56 kinds), flavonoids (46 kinds), lipids (26 kinds), tannins (19 kinds), amino acids and their derivatives (17 kinds) and others between the two varieties. Furthermore, metabolic pathway annotations revealed that amino acid-related metabolic pathways and flavone and flavonol metabolism pathways were the main differential metabolic pathways between ‘Zijuan’ and ‘Yingshuang’ tea flowers. In addition, the total contents of flavonoids and anthocyanins and the contents of catechins and some alkaloids between ‘Zijuan’ and ‘Yingshuang’ tea flowers were significantly different (
P＜0.05), while the differences in the soluble sugar contents were not significant. The above results preliminarily showed that the flavonoids made the taste of tea flowers had a certain degree of bitterness and astringency, while compared with those in ‘Yingshuang’, the increase of contents of some amino acids and their derivatives in ‘Zijuan’ tea flowers was the main reason for its fresh taste.
Effects of straw burning on pathogens and antibiotic resistance genes in paddy fields with bacterial leaf blight
Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(6): 840-852.
Bacterial leaf blight in rice is a significant disease, and understanding the effects of straw burning on pathogens and antibiotic resistance genes (ARGs) in paddy fields is crucial. Soil and rice stubble samples were collected from bacterial leaf blight outbreak areas in Ningbo City of Zhejiang Province, and these samples were subjected to high-throughput metagenomic sequencing to investigate the effects of straw burning. The results indicated that the relative abundance of pathogens in the rice stubble was significantly higher than that in the soil. After straw burning, except for the significant increase in available potassium content, there were no significant changes in the other physicochemical properties of the soil. Overall, there were no significant differences in pathogen community diversity after straw burning, but straw burning led to a sharp decrease in the relative abundance of
Xanthomonas oryzae in the rice stubble. The relative abundances of ARGs in the soil and rice stubble increased after straw burning, but there were no significant differences compared with those before straw burning ( P＞0.05), while the relative abundance of mobile genetic elements (MGEs) in the rice stubble decreased significantly after straw burning ( P＜0.05). The proportion of positively correlated links in the coexisting networks of pathogens-ARGs decreased after straw burning. Mantel analysis revealed that the water content ( r=0.642, p＜0.01), pH value ( r=0.582, p＜0.05), total organic carbon content ( r=0.325, p＜0.05), total nitrogen content ( r=0.570, p＜0.01), and available phosphorus content ( r=0.311, p＜0.05) were closely related to the soil ARG subtypes; the dissolved organic carbon (DOC) content ( r=0.275, p＜0.05) and available potassium content ( r=0.312, p＜0.05) were correlated with the ARG subtypes, and the DOC content ( r=0.201, p＜0.05) significantly affected the pathogen community structure in the rice stubble. In summary, straw burning can reduce the relative abundance of X. oryzae in the rice stubble, which suggests that it may be a viable option for preventing and controlling rice bacterial leaf blight; moreover, a reduction in the positive correlation ratio of pathogens-ARGs and in the relative abundance of MGEs after straw burning may inhibit the formation of potentially resistant pathogens. However, given the complexity of soil biological compound pollution, the effects of straw burning on pathogens and ARGs in paddy fields need to be further explored by long-term location experiments.
Study on safe utilization technology model of high acidity and mild cadmium-contaminated paddy fields: a case of the cadmium-contaminated pilot area in Yongkang City of Zhejiang Province
Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(6): 853-862.
In the highly acidic soil pilot area contaminated by cadmium (Cd) in Yongkang City of Zhejiang Province, we screened low-Cd-accumulating rice cultivars and soil passivators in the field and performed the combined effect tests for two consecutive years to explore the safe utilization technology model suitable for local Cd-polluted paddy fields. The results showed that ‘Xiushui 519’ (XS519), ‘Zhenuo 106’ (ZN106), ‘Zhongzheyou 1’ (ZZY1H) and ‘Zhongjia 8’ (ZJ8H) could be recommended as low-Cd-accumulating rice cultivars suitable for local planting. Among them, ‘Xiushui 519’ had the lowest and most stable Cd accumulation ability, and the average bioconcentration factors (BCFs) of Cd in the rice grains in the two years were 0.090 and 0.159, respectively; in the screening tests of passivators, the improvement effect of lime (L) on soil pH was significant. Lime, iron-modified biochar (FeC) and calcium-magnesia phosphate fertilizer (CaMgP) could reduce soil available Cd contents to a certain extent (passivation rates were 14.8%, 7.1% and 6.9%, respectively). In addition, 1 800 kg/hm
2 iron-modified biochar and 2 400 kg/hm 2 soil conditioner had better effects on reducing the Cd content in the rice grains of ‘Zhongjia 8’, which decreased by 32.2% and 29.0%, respectively, after application. According to the results of the combination tests of the low-Cd-accumulating cultivars and passivators, both the application of 2 250 kg/hm 2 iron-modified biochar and 2 250 kg/hm 2 soil conditioner increased obviously the soil pH values at the tillering stage and filling stage, the soil conditioner of which had a better improvement effect. The Cd contents in the rice grains of ‘Xiushui 519’ and ‘Zhenuo 106’ were extremely significant correlations with the soil pH values (5.19-5.61) at the mature stage. Within the range of soil pH values involved in the test, except for the combination of ‘Xiushui 519’ and the 1 500 kg/hm 2 soil conditioner, the Cd content in the rice grains increased with increasing application amount of passivators. Therefore, planting ‘Xiushui 519’ (a low-Cd cultivar) directly is the best choice for the safe utilization of Cd-contaminated paddy fields in this area, which is both economical and convenient.
Effects of aboveground parts of
Tetrastigma hemsleyanum on the intestinal microflora, immune function and growth performance of broilers Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(6): 863-872.
The effects of aboveground parts of
Tetrastigma hemsleyanum, T. hemsleyanum leaves (THL), on the growth performance, immune function and intestinal microflora of broilers were evaluated. A total of 240 broilers were randomly divided into four groups: the control group (fed with a basal diet) and low dose group, medium dose group and high dose group (fed with a basal diet supplemented with 1%, 3% and 5% THL powder, and denoted as THL-L, THL-M and THL-H, respectively). The body masses of broilers at 21 and 42 days of age in each group was measured, and the immune organ indexes and the contents of immunoglobulin A (IgA), IgG and IgM in serum and the relative abundance of intestinal microflora in cecum contents of broilers at 42 days of age were determined. The results showed that compared with the control group, the supplementation of THL powder significantly enhanced the average daily feed intake ( P＜0.05) and average daily gain ( P＜0.05) of broilers at 42 days of age, and the promoting effect of the THL-M group was the best. At the same time, compared with the control group, the thymus index and the bursa of fabricius index of broilers in THL-L and THL-M groups were significantly increased ( P＜0.05). The contents of IgA and IgM in serum of broilers in THL-L, THL-M and THL-H groups were significantly higher than those in the control group ( P＜0.05). However, IgG content in THL-M and THL-H groups was significantly higher than that in the control group ( P＜0.05). The relative abundances of Lachnospiraceae and Clostridiaceae were significantly increased by the supplementation of appropriate amount of THL powder (3%) ( P＜0.05). This study indicated that T. hemsleyanum leaf powder could significantly improve the immune function, the intestinal microflora composition and the growth performance of broilers, and the most significant effect was found with supplementation of 3% THL powder.
Comparisons on soluble expression and immunoreactivity of African swine fever virus CD2v protein expressed by different prokaryotic expression vectors
Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(6): 873-880.
The aim of this study was to systematically investigate the differences in the soluble expression level of the African swine fever virus (ASFV) CD2v protein by different prokaryotic expression vectors, and the immunoreactivities of the inclusion body and soluble CD2v proteins were compared using clinical anti-ASFV antibody-positive sera. Five prokaryotic expression vectors, namely, pCold-TF, pET28a, pMAL-C6T, pGEX-4T-1 and pET32a, were utilized to express the CD2v protein without the signal peptide and transmembrane region, respectively. The inclusion body CD2v protein expressed by the pET28a vector and the soluble CD2v protein expressed by the pCold-TF vector were purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography, and the immunoreactivity of the purified proteins was detected by indirect enzyme-linked immunosorbent assay (ELISA). The results showed that the CD2v protein expressed by the pCold-TF vector was soluble mainly, while the CD2v protein expressed by the pMAL-C6T vector was insoluble (inclusion body) and soluble, and the CD2v protein expressed by the other vectors was mainly as inclusion body. The indirect ELISA results for clinical anti-ASFV antibody-positive sera showed that the immunoreactivity of soluble protein was significantly better than that of the inclusion body protein (
P＜0.05). The trigger factor (TF) tag of pCold-TF promoted the soluble expression of the CD2v protein, and the immunoreactivity of the expressed protein was greater than that of the inclusion body protein. This study lays the foundation for further immunogenicity research on the CD2v protein and provides a candidate strategy for the soluble expression of other important antigens.
Fritillaria thunbergii appearance quality based on machine vision and machine learning technology Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(6): 881-892.
In order to classify the appearance quality level of
Fritillaria thunbergii, the F. thunbergii dataset was constructed with the DigiEye system followed by an image annotation tool. Several statistical learning and object detection algorithms were selected to train and test the F. thunbergii dataset. The results showed that the model trained by the YOLO-X of YOLO (you only look once) series had relatively better performance. In addition, to optimize YOLO-X, according to the unique features of F. thunbergii dataset, a dilated convolution structure was embedded into the end of the backbone feature extraction network of YOLO-X as it could improve the model sensitivity to the dimension feature. The mean average precision (mAP) of the improved model was raised to 99.01%; the average precision (AP) for superfine, level one, level two, moth-eaten, mildewed, and broken F. thunbergii were raised to 99.97%, 98.33%, 98.47%, 98.71%, 99.73%, and 98.85%, respectively; and the weighted harmonic mean of precision and recall ( F 1) were raised to 0.99, 0.92, 0.94, 0.97, 0.99, and 0.97, respectively. The tune-up in this study enhanced the detection performance of the model without increasing the number of parameters, computational complexity, or major changes to the original model. This study provides a scientific basis for the subsequent construction of F. thunbergii detection platform.