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Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(5): 591-754.  
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Young Scientist Forum
Research progresses on target of rapamycin kinase in plants
Wenzhen CHEN,Jiaqi LIU,Hao DU
Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(5): 591-606.   https://doi.org/10.3785/j.issn.1008-9209.2023.05.101
Abstract( 242 )   HTML( 8 )   HTML (   PDF(1889KB)( 216 )

In plants, target of rapamycin (TOR) functions as a pivotal signaling and metabolic hub, integrating nutrient availability, energy status, and environmental cues through phosphorylation. This regulatory mechanism plays a crucial role in governing plant growth, development, and environmental adaptation. In this paper, we provide a comprehensive review of the discovery and characterization of TOR in plants. We summarize previous and recent studies on the signaling pathway of plant TOR, highlighting the identification of upstream effect factors and downstream substrates. Additionally, we discuss the diverse roles of TOR in plant embryogenesis, meristem formation, nutrient utilization, flowering, senescence, and responses to both abiotic and biotic stresses. Furthermore, we explore the potential research prospects for TOR kinase and its application in agriculture.

Function and mechanism of cohesin REC8 during meiosis
Jingling DAI,Chao YU
Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(5): 607-617.   https://doi.org/10.3785/j.issn.1008-9209.2022.08.121
Abstract( 253 )   HTML( 4 )   HTML (   PDF(2028KB)( 147 )

Cohesin is a functionally and evolutionarily conserved multi-subunit protein complex that is required for sister chromatid cohesion and chromatin loop structure in both mitosis and meiosis. The meiotic cell-cycle consisting of one DNA replication and two successive rounds of chromosome segregation completes the segregation of homologous chromosomes and sister chromatids. Cohesin is crucial for faithful and proper segregations. There is a group of distinctive cohesin subunits that are only expressed in meiotic cells. The study of meiosis-specific cohesin is of great significance for understanding chromosome architecture and dynamics in meiosis. REC8 is a typical meiosis-specific cohesin subunit that plays essential roles in sister chromatid cohesion and meiotic chromosome events. Here, we review the function and mechanism of meiotic cohesin REC8 based on the current study and hypothesize that phosphorylation modification and microRNAs (miRNAs) could be the subsequent research directions of REC8.

Research advances on pathogenic nucleic acid detection technology based on CRIPSR/Cas system
Hongzhao LI,Hao WANG,Rui YIN,Min YUE,Yan LI
Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(5): 618-632.   https://doi.org/10.3785/j.issn.1008-9209.2022.08.051
Abstract( 152 )   HTML( 16 )   HTML (   PDF(4481KB)( 89 )

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) (CRISPR/Cas) system, an ancient bacterial and archaeal immune system, has rapidly developed into a popular gene-editing tool, which largely promotes the development of several biology-related fields. By combining the CRISPR/Cas systems with the isothermal amplification techniques, the novel and effective detection methods with high sensitivity and independence of equipment have been established, such as DNA endonuclease-targeted CRISPR trans-reporter (DETECTR) and specific high-sensitivity enzymatic reporter unlocking (SHERLOCK). These new technologies not only improve the performance of the CRISPR/Cas system in different situations, but also inspire its application potential in the on-site detection. In this review, we summarized the nucleic acid detection methods developed on the three widely-used CRISPR/Cas systems (CRISPR/Cas9, CRISPR/Cas12a, and CRISPR/Cas13), and elucidated their biological significance and the principles of action. We also reviewed the recent studies on the applications of CRISPR/Cas systems in pathogen detection, and analyzed the characteristics and possible defects of different detection systems in practical applications. This review aims to provide more constructive advice on developing adaptable and efficient CRISPR/Cas-based detection methods for different pathogens in various practical scenarios.

Special Topic: Major Bacterial and Viral Diseases in Crops
Research progress on the regulation of vascular lignification on defense against bacterial wilt of plants
Chenying LI,Ran WANG,Yan LIANG
Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(5): 633-643.   https://doi.org/10.3785/j.issn.1008-9209.2023.06.171
Abstract( 241 )   HTML( 8 )   HTML (   PDF(2188KB)( 211 )

Bacterial wilt, a typical vascular disease caused by Ralstonia solanacearum, is one of the most devastating diseases and dramatically reduces crop yield and quality. The most effective strategy for controlling wilt disease is breeding disease-resistant varieties, which requires understanding the molecular mechanisms underlying plant immune responses against R. solanacearum. However, more and more evidence suggests that vascular immune responses are cell type specific. After sensing of R. solanacearum, the cell wall lignification of vascular tissues plays a vital role in restricting the spread of R. solanacearum. Lignin biosynthesis pathway genes are strictly controlled at the transcriptional, translational, and spatial-temporal specific expression aspects. Here, we summarized the current understanding of the recognition and signal transductionupon R. solanacearum infection and the research progress of pathogen-induced vascular lignification on regulating resistance to R. solanacearum, including the expression of lignin biosynthesis genes, the transport and polymerization of monolignols, and the generation of different types of lignin. We hope that this review will provide a theoretical basis for breeding bacterial wilt disease-resistant cultivars by modifying vascular lignification.

Research progress on the molecular basis of plant-Ralstonia solanacearum recognition
Zhiliang XIAO,Aiguo YANG,Meixiang ZHANG
Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(5): 644-650.   https://doi.org/10.3785/j.issn.1008-9209.2023.06.161
Abstract( 252 )   HTML( 6 )   HTML (   PDF(832KB)( 203 )

Ralstonia solanacearum causes bacterial wilt disease in multiple crops, which severely threatens the global crop safety production. This pathogen exhibits high genetic diversity and evolves rapidly, and there is a lack of effective disease-resistant varieties in production, which brings great challenges for effective disease control. Identifying receptor proteins in plants that recognize associated molecular patterns or effectors of R. solanacearum and elucidating their molecular recognition mechanisms can provide clues to understand the mechanisms of plant-pathogen interaction, and lay a basis for the development of broad-spectrum disease resistance in plants. This paper reviewed the recent progress on the molecular basis of plant-R. solanacearum recognition. We mainly focused on the identification and functional analysis of membrane and intracellular receptors that recognize R. solanacearum in plants, as well as the mechanism behind receptor recognition of the associated molecular patterns or effectors from R. solanacearum. Besides, we provide research prospects for the exploration and utilization of disease-resistant resources against R. solanacearum in the future.

Pathogenicity and avirulence mechanism of Ralstonia solanacearum type Ⅲ effectors
Peipei QI,Xiao YU,Bo LI
Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(5): 651-661.   https://doi.org/10.3785/j.issn.1008-9209.2023.06.011
Abstract( 249 )   HTML( 3 )   HTML (   PDF(1680KB)( 114 )

Ralstonia solanacearum is a very harmful plant pathogenic bacterium, and the plant bacterial wilt caused by it seriously affects the healthy production of tomato and potato crops. It has broad host varieties and can acquire new virulence through horizontal gene transfer and gene recombination to extend the host range. The pathogenic mechanism of R. solanacearum is complex, type Ⅲ secretion system (T3SS) is the key pathogenic factor, and the type Ⅲ effectors (T3Es) secreted by it play important roles in the pathogenic processand inhibit innate immune response of hosts at different levels. Moreover, plant hosts can recognize R. solanacearum effectors and activate effector-triggered immunity (ETI) to achieve disease resistance. In this review, the virulence and avirulence mechanisms of R. solanacearum T3Es were discussed and summarized, providing insights for further understanding the pathogenesis of R. solanacearum and the mechanisms of plant resistance to bacterial wilt.

Uridine diphosphate-glucose 4-epimerase encoding gene galE affects the pathogenicity and carbon metabolism of Ralstonia pseudosolanacearum
Hong ZHANG,Zhijian LIN,Jindong ZHU,Zhaomiao LIN,Guoliang LI,Yongqing XU,Zhonghua LIU,Yongxiang QIU,Sixin QIU
Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(5): 662-676.   https://doi.org/10.3785/j.issn.1008-9209.2023.09.191
Abstract( 100 )   HTML( 1 )   HTML (   PDF(6149KB)( 42 )

In order to clarify the pathogenic function of uridine diphosphate (UDP)-glucose 4-epimerase encoding gene galE on Ralstonia pseudosolanacearum, the association between galE and the pathogenic-related phenotypes, and the gene deletion effect on physiological and biochemical metabolism of R. pseudosolanacearum were explored by constructing galE gene knock-out strain ΔgalE and its complementary strain CΔgalE. The results showed that the deletion of galE gene significantly decreased the pathogenicity of R. pseudosolanacearum on sweet potatoes and affected pathogenic-related phenotypes. The colony fluidity, swimming mobility, exopolysaccharide content and biofilm formation of ΔgalE reduced compared to those of wild-type SPRS911 and CΔgalE. In the galactose metabolism pathway, after deletion of galE gene, the expression levels of galU, pgm, and glk genes involved in the metabolism between uridine diphosphate glucose (UDPG) and glucose decreased, and D-glucose 6-phosphate accumulated. The expression levels of UDP-galactose (UDP-Gal) metabolism related genes dgoK, dgoAa, dgoAb, malZ and galM increased. The deletion of galE gene also enhanced the assimilation of malic acid in R. pseudosolanacearum. These results indicate that the galE gene has significant effects on the pathogenicity and the carbon metabolism of R. pseudosolanacearum.

Preparation and application of polyclonal antibody against a cysteine-rich protein encoded by Chinese wheat mosaic virus
Yuanxing DAI,Liuming GUO,Jing HE,Zhengrong SHEN,Yanfei GENG,Mingfang Lü,Zhengjie YUAN,Jing LI,Hengmu ZHANG
Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(5): 677-686.   https://doi.org/10.3785/j.issn.1008-9209.2023.02.011
Abstract( 92 )   HTML( 7 )   HTML (   PDF(3859KB)( 88 )

Chinese wheat mosaic virus (CWMV) is one of the most important pathogens causing mosaic disease in wheat and has threatened the yield and quality of wheat for a long time. Cysteine-rich protein (CRP) of CWMV plays important and complex roles in viral infection. To further study CRP functions and CWMV infection mechanisms, the CRP coding region was amplified by reverse transcription-polymerase chain reaction (RT-PCR) from leaves of CWMV-infected wheat and cloned into the prokaryotic expression vector pET-32a. The recombinant plasmid pET-CRP was transformed into Escherichia coli BL21 (DE3) for inducible expression. The recombinant CRP was purified by nickel-column affinity chromatography and used as an antigen to immunize New Zealand white rabbits for polyclonal antibody preparation. A series of immunological assays, including Western blot, indirect enzyme-linked immunosorbent assay (ELISA) and dot ELISA, showed that the purified CRP antibody had high specificity, and its titer was as high as 1∶4 096 000, which was four times higher than that of the unpurified antibody. The antibody could recognize 0.5 ng antigen, showing its high sensitivity. In addition, the purified CRP antibody could specifically and sensitively recognize native CRP even at a 1∶120 000 dilution. In conclusion, the polyclonal antibody can be not only used for precise diagnosis of the CWMV-infected plant samples from fields, but also applied to detect CRP expressed transiently in plants, which lays a foundation for subsequent detection, quantification and subcellular localization of CRP.

Whole genome sequencing and analysis of isolates of tomato leaf curl New Delhi virus collected from watermelon
Yuxin CHENG,Hongxia HU,Yanqing LIANG,Yajuan QIAN
Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(5): 687-695.   https://doi.org/10.3785/j.issn.1008-9209.2023.05.122
Abstract( 98 )   HTML( 2 )   HTML (   PDF(3557KB)( 98 )

Watermelon diseased leaves exhibiting leaf curl and yellowing symptoms were collected from Shanghai. To clarify the pathogenic type, small RNA high-throughput sequencing of the diseased leaves was performed, and the diseased samples were confirmed to be infected by tomato leaf curl New Delhi virus (ToLCNDV) through sequence splicing and dot-enzyme linked immunosorbent assay (Dot-ELISA). The DNA was further enriched by rolling circle replication (RCR) and was amplified by polymerase chain reaction (PCR) using back-to-back primers to obtain the full-length DNA-A and DNA-B sequences of two ToLCNDV watermelon isolates, which were tentatively named as ToLCNDV SH-WM1 and ToLCNDV SH-WM2. Phylogenetic analysis using MEGA 11.0 and SDT v1.2 softwares showed that ToLCNDV SH-WM1 was closely related to ToLCNDV SH-WM2 and other reported ToLCNDV isolates from China, with a total nucleotide sequence similarity of 98.99%-99.70%. Whole genome polymorphism analysis and population variation analysis showed that the single nucleotide variation rate of ToLCNDV isolates from China was less than 3%, with a total of 46 synonymous mutation sites and 29 non-synonymous mutation sites. There were no insertion or deletion mutations, and the amino acid sequence of AC4 protein was the most conservative among all encoded proteins. This study reveals that the ToLCNDV isolates from China have low intraspecific genetic variation.

Identification of genes in response to cucurbit aphid-borne yellows virus infection in melon
Siyu YANG,Zihui GONG,Zhongyuan HU,Mingfang ZHANG,Jinghua YANG
Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(5): 696-707.   https://doi.org/10.3785/j.issn.1008-9209.2023.05.121
Abstract( 112 )   HTML( 3 )   HTML (   PDF(4586KB)( 118 )

Cucurbit aphid-borne yellows virus (CABYV) is one of the most important viruses infecting cucurbits, such as melon (Cucumis melo), and severely affects the yield and quality of crops. Identification of CABYV-responsive genes can provide target genes for breeding of melon resistant to the viral disease. In this study, we used the CABYV infectious cloning vector to inoculate the melon XZM, and the disease identification and transcriptome analysis in the melon XZM after CABYV inoculation were performed at 0 dpi (days post inoculation), 5 dpi, 10 dpi, 15 dpi and 20 dpi. The results indicated that melon leaves showed typical disease symptoms of leaf chlorosis, yellowing and leaf thickening at 20 dpi. A total of 1 654 differentially expressed genes (DEGs) in response to CABYV infection were identified by transcriptome sequencing analysis, including 677 up-regulated genes and 977 down-regulated genes. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that the responsive genes were mainly enriched in plant-pathogen interaction, photosynthesis, starch and sucrose metabolism, glyoxylate and dicarboxylate metabolism, etc., pathways and processes. Co-expression and interaction analysis of DEGs revealed that RIN4, a key gene in pathogen defense responses, may negatively regulate responses to CABYV infection in melon. This study demonstrates the possible molecular mechanism of responses to CABYV infection and provides a basis for breading melon against CABYV.

Horticultural sciences
Bioinformatics and expression analyses of heat shock protein 90 gene family in Rhododendron ovatum
Fengyan LI,Fangqian QU,Fangmeng ZHAO,Qi WANG,Hong ZHOU,Liangsheng ZHANG,Yiping XIA,Xiuyun WANG
Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(5): 708-718.   https://doi.org/10.3785/j.issn.1008-9209.2022.08.012
Abstract( 134 )   HTML( 8 )   HTML (   PDF(3229KB)( 183 )

To explore the roles of heat shock protein 90 (Hsp90) in plant growth development and response to high temperature stress, this study identified the Hsp90 family through bioinformatics methods in the whole genome of Rhododendron ovatum,which is a highly ornamental and widely adaptive species of Rhododendron. Gene structure, cis-acting elements, evolution, and expression pattern of the Hsp90 family were subsequently analyzed. The results showed that 11 members of the Hsp90 family were identified in R. ovatum, which were located on five chromosomes. Cis-acting element analysis showed that all the 11 members of the Hsp90 family were involved in the processes of plant hormone metabolism and stress responses. The phylogenetic tree of Hsp90 family in Arabidopsis thaliana, Solanum lycopersicum, Camellia sinensis, R. simsii, R. delavayi, R. henanense, and R. ovatum was constructed, within four main clades. Evolutionary analysis showed that the Hsp90 family has undergone purification selection during the differentiation of Rhododendron species. Moreover, the expression patterns of Hsp90 family in different tissues and under high temperature treatments indicated that Hsp90 family plays roles in flower development and the response of plants to high temperature stress. This study provides a foundation for further study of the functions of Hsp90 genes in Rhododendron.

Animal sciences & veterinary medicines
Effects of fermented soybean residues on the growth performance, serum indexes, and digestive performance of Xianju chickens
Lai JIANG,Lei YAO,Chunchun YUAN,Wenxin YE,Jinghui FAN,Lichun QIAN
Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(5): 719-728.   https://doi.org/10.3785/j.issn.1008-9209.2022.08.091
Abstract( 110 )   HTML( 4 )   HTML (   PDF(948KB)( 119 )

This study aims to use microbial fermentation technology to improve the utilization of soybean residues and to develop a new type of fermented feed with superior quality and competitive price. In this experiment, fermented soybean residues were used to feed 1 day-of-age Xianju chickens, lasting for 42 d. Five groups were set up, including the control group T1, fed with the basal diets; the antibiotic group T2, fed with the basal diets supplemented with 40 mg/kg methylene salicylic acid bacitracin; the treatment groups T3, T4, and T5, fed with the basal diets in which 2%, 4%, and 6% soybean meal were replaced by fermented soybean residues, respectively. The results were shown as follows. 1) Compared with the control group T1, the 42 day-of-age body mass and average daily gain of Xianju chickens were significantly higher in each group, and the feed to gain ratio was significantly lower in T4 group (P<0.05). 2) Compared with the control group, the albumin content was extremely significantly higher in T4 group (P<0.01), and the superoxide dismutase activity was extremely significantly higher in T2 and T5 groups (P<0.01), while the malondialdehyde content was extremely significantly lower in T2 group (P<0.01). 3) Compared with the control group, the apparent digestibility of crude protein and crude fiber was significantly increased in T4 group (P<0.05) and the apparent digestibility of crude protein was extremely significantly increased in T5 group (P<0.01); the duodenal amylase activity in T4 group (P<0.05), the duodenal protease activities in T4 and T5 groups (P<0.01), and the chymotrypsin activities in T2, T3, and T5 groups (P<0.05) were significantly improved. In conclusion, replacing 4% soybean meal in the basal diets with an equal amount of fermented soybean residues has the best feeding effect, which could significantly promote the growth performance, apparent digestibility of crude protein and crude fiber, and digestive enzyme activities in vivo of Xianju chickens, improve their serum indexes, and has the potential to replace antibiotics.

Combined analysis of embryonic gonadal development differences of mulard duck and muscovy duck using long non-coding RNAs and mRNAs
Li LI,Linli ZHANG,Qingwu XIN,Zhongwei MIAO,Zhiming ZHU,Junzhi QIU,Xiaona HAO,Qinlou HUANG,Nenzhu ZHENG
Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(5): 729-743.   https://doi.org/10.3785/j.issn.1008-9209.2022.07.071
Abstract( 88 )   HTML( 2 )   HTML (   PDF(3442KB)( 95 )

The purpose of this study was to screen key mRNAs and long non-coding RNAs (lncRNAs) that affect gonadal development in duck embryos and to explain scientifically gonadal development defects in the mulard duck. Three male embryonic gonadal tissues of the mulard duck (BF1, BF2, BF3) and the muscovy duck (F1, F2, F3) were collected to extract RNA and perform high-throughput sequencing, and differential genes and lncRNAs were screened to predict target genes and perform functional annotations. Finally, the sequencing data were verified by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). The results showed that a total of 1 109 differentially expressed genes were screened from the gonadal tissues of the mulard duck and the muscovy duck. Compared with the muscovy duck, 857 genes were up-regulated and 252 genes were down-regulated in the mulard duck. Among them, the aldo-keto reductase family 1 member D1 gene (AKR1D1), 17β-hydroxysteroid dehydrogenase 3 gene (17β-HSD3), and cholesterol side-chain cleavage enzyme gene (P450scc) may be related to gonadal differentiation and development in the mulard duck. Meanwhile, 733 significantly differentially expressed lncRNAs were obtained. Compared with the muscovy duck, a total of 660 lncRNAs were significantly up-regulated and 73 lncRNAs were significantly down-regulated in the mulard duck. Target gene prediction analysis showed that a total of 136 down-regulated lncRNAs and 893 up-regulated lncRNAs may be involved in differential gene expression and had potential regulatory relationships, among which, TCONS_00246198 targeted 17β-HSD3, and TCONS_00229529 targeted tetraspanin-2 gene (TSPAN2), suggesting that the above lncRNAs may participate in duck embryonic gonadal development by targeting key genes. The qRT-PCR results showed that the expression levels of differential genes and lncRNAs were consistent with the expression trends in transcriptome sequencing, indicating that the data obtained by high-throughput sequencing are relatively reliable. RNA binding protein immunoprecipitation (RIP) assay results revealed that compared with IgG, the enrichment level of TCONS_00246198 reached 71.51 times. The above results indicate that TCONS_00246198 interacts directly or indirectly with the 17β-HSD3 protein, which means that they may have a targeting relationship. In summary, this study obtains a batch of key mRNAs and lncRNAs that may affect duck embryonic gonadal development, and it is speculated that the differential lncRNAs can regulate the expression of differential genes. This study provides a scientific basis for understanding the differences in duck embryonic gonadal development and the mechanisms of avian gonadal development.

Preparation of polyclonal antibodies and subcellular localization of non-structural protein 3 encoded by feline coronavirus
Ziyi WANG,Zi’an JIN,Chenhe LU,Zhi QIAO,Shengwen WANG,Yan YAN,Jiyong ZHOU,Xiaojuan ZHENG
Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(5): 744-754.   https://doi.org/10.3785/j.issn.1008-9209.2022.08.011
Abstract( 83 )   HTML( 5 )   HTML (   PDF(11572KB)( 60 )

The non-structural protein 3 (Nsp3) of coronavirus, a component of the replication and transcription complex, is one of the potentially important antiviral targets. In this study, the transmembrane region, signal peptide, and epitope of Nsp3 were predicted, and then the region with better antigenicity (50-550 amino acids) of Nsp3 protein in a representative strain (WSU 79-1683) of type Ⅱ feline coronavirus (FCoV) was amplified by polymerase chain reaction. Subsequently, it was subcloned into pCOLD-TF prokaryotic expression vector. Under the low-temperature condition, the recombinant fusion protein His-Nsp3 with a molecular weight of about 130 kDa was successfully induced by isopropylthio-β-D-galactoside. The targeted recombinant protein His-Nsp3 was purified using a non-denaturing nickel affinity column, and the purified protein was used as an antigen to immunize BALB/c mice for preparing Nsp3 polyclonal antiserum. Western blotting (WB) and indirect immunofluorescence assay (IFA) results showed that Nsp3 polyclonal antiserum could specifically recognize Nsp3 protein in FCoV-infected cells. The subcellular localization of Nsp3 protein in FCoV-infected cells was studied by double-labeling IFA combined with laser confocal microscopy. The results showed that Nsp3 protein aggregated in FCoV-infected cells and co-localized with the endoplasmic reticulum. The specific antibody preparation and subcellular localization study of Nsp3 protein provided an important basis for further analysis of the biological function of Nsp3 protein.

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