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Monthly, Started in1993
ISSN 1674-7968  CN 11-3342/S
Administrated by: Ministry of Education of People's Republic of China
Sponsored by: China Agricultural University and Chinese Society of Agricultural
                          Biotechnology
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WU Wei-Hua
Associate editor-in-chief:CHEN Hua-Lan   LI Kui   LI Yi   LIN Min   PENG Yu-Fa
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农业生物技术学报
2024 Vol. 32, No. 4  Published: 01 April 2024
 
Articles and Letters
Cloning of ZlALDH2C4 Related to Ferulic Acid Synthesis and Its Response to Swelling Development of Zizania latifolia Stem
LIU Jing-Xi, XIA Shu-Qian, QI Xiao-Qing, QIAN Zhi-Yuan, MA Ying-Ran, TANG Jin-Tian, XIA Wen-Qiang, ZHANG Ya-Fen, YE Zi-Hong, CUI Hai-Feng
2024 Vol. 32 (4): 739-750   DOI:10.3969/j.issn.1674-7968.2024.04.001 |  Full text (HTML) (1 KB)  |    PDF  (11453 KB)  ( 37 )
Abstract
Ferulic acid, an important metabolic component in monolignol synthesis catalyzed by aldehyde dehydrogenase family 2 member C4 (ALDH2C4), was involved in the basic defense of plant cells in response to pathogen infection, and played an important regulatory role in the formation of secondary wall and lignification differentiation in plant cells. In this research, the ALDH2C4-related gene of ZlALDH2C4 (GenBank No. OQ552555) was cloned from Zizania latifolia plant with the full-length cDNA sequence of 1 527 bp, containing a ALDH2C4 domain of PLN02766. The phylogenetic tree analysis showed that ZlALDH2C4 was closely related to OsALDH2C4, a homolog protein of Oryza sativa ssp. japonica. Based on qPCR analysis, it was showed that the expression of ZlALDH2C4 was significantly up-regulated after infected by U. esculenta in stem of Z. latifolia, which had higher expression in tissues with higher degree of lignification in plants of Z. latifolia. Based on the submicroscopic observation of the different parts cells in swollen stem of normal Jiaobai and the analysis of spatial and temporal expression of ZlALDH2C4 during the swelling development of stem, it was found that the expression of ZlALDH2C4 was basically positively correlated with the changes of lignification in stem. The lower expression in the initiation and the early development stage of swollen stem was related to the lower degree of lignification, while the enhancement of lignification in swollen stem cells at the late development stage was accompanied by a significant increasing expression of ZlALDH2C4. Among the different parts of the swollen stem, the ZlALDH2C4 gene expression in the lower part with thicker secondary wall was significantly higher than that in the upper part with thinner secondary wall. The above results indicated that ZlALDH2C4 was closely related to the regulation of secondary wall formation in stem cells, and could respond to the infection induction of U. esculenta, and participate in the regulation of lignification differentiation of stem cell in Z. latifolia. This study provides a theoretical reference for the research on the regulatory mechanism of stem swelling and development induced by infection of U. esculenta.
Transcriptome Analysis of the Candidate Key Genes Regulating Post-harvest Chilling Injury and Firmness in Peach (Prunus persica)
ZHANG Zhi-Gang, CHEN Yu-Feng, WANG Chang-Jiang, XU Jin, JIANG Yan-Wei, ZHENG Ya-Qin, LIU Zhen-Ning, CHEN Shou-Bing, LIU Yi-Hua, XU Meng
2024 Vol. 32 (4): 751-761   DOI:10.3969/j.issn.1674-7968.2024.04.002 |  Full text (HTML) (1 KB)  |    PDF  (8076 KB)  ( 14 )
Abstract
Cold-sensitive fruit stored at low temperatures after harvest is vulnerable to fruit chilling injury, and low-temperature conditioning (LTC) treatment has notable effect in alleviating chilling injury of cold-sensitive fruit such as peach (Prunus persica), but slight fruit softening occurs in the initial stage of storage. In this study, weighted gene co-expression network analysis (WGCNA) was used to identify gene modules that were highly correlated with chilling injury and firmness phenotypes. The analysis revealed that WRKY transcription factor PpWRKY43 and P. persica auxin response factor 1 (PpARF1) might be involved in postharvest chilling injury regulation by regulating cold-regulated 413 inner membrane protein 2 (PpCOR413-2) and the lipoxygenase 5 (PpLOX5), and NAC transcription factor PpNAC5 might be involved in postharvest softness of peach fruit by regulating P. persica pectate lyase 2 (PpePL2). The results of this study provides basic data for postharvest preservation and reduction of peach fruit injury.
Genome-wide Analysis of the Expansin Gene Family in Grape (Vitis vinifera) and Functional Study of VvEXPA27
ZHANG Jian-Peng, SHANG Xiao-Li
2024 Vol. 32 (4): 762-774   DOI:10.3969/j.issn.1674-7968.2024.04.003 |  Full text (HTML) (1 KB)  |    PDF  (8402 KB)  ( 17 )
Abstract
The size of berry is one of the important factors determining the appearance quality and yield of grapes (Vitis vinifera), and cell expansion is crucial for determining the final size of berry. Expansin (EXP) is a cell wall relaxation protein, which promotes cell expansion by breaking the noncovalent bond between cellulose microfibers and related matrix polysaccharides in the cell wall. This study used the 'Pinot Noir PN4002' genome as a reference and used HMMER to identify the whole genome of grape expansin family members based on Pfam database. After eliminating redundant sequences, a total of 31 members were screened. Two candidate genes, VvEXPA06 and VvEXPA27, were identified through bioinformatics analysis. The RT-qPCR results showed that VvEXPA06 was only upregulated at 20 d after flowering, while VvEXPA27 was upregulated at both 20 and 48 d after flowering. Subsequently, the VvEXPA27 were subjected to heterologous expression in Arabidopsis thaliana and their transgenic plants were functionally validated. Compared with the vector control (VC), the cell area of the third leaf, silique length, and rosette diameter of T3-generation homozygous transgenic A. thaliana overexpressing (OE) the VvEXPA27 gene showed a significant increase. The results showed that VvEXPA27 played a positive role in cell expansion. This study provides a theoretical basis for analyzing the molecular mechanism of berry cell expansion and genetic resources for molecular breeding.
Cloning of Cold Tolerance-related Genes RsMYBS3 and RsRCI2B of Rhododendron sphaeroblastum var. wumengense and Their Expression Analysis
PENG Wan-Ming, TAN Yi, ZHANG Xiao-Li, LUO Liang, LI Xin-Yi, HUANG Hai-Quan, HUANG Mei-Juan
2024 Vol. 32 (4): 775-784   DOI:10.3969/j.issn.1674-7968.2024.04.004 |  Full text (HTML) (1 KB)  |    PDF  (2368 KB)  ( 18 )
Abstract
Rhododendron sphaeroblastum var. wumengense is a high-altitude and cold-tolerant plant. MYBS3 and rare cold inducible 2 (RCI2B) genes were found to play a significant role in enhancing cold stress tolerance in plants based on the pre-screening from R. sphaeroblastum transcriptome. In this study, the key transcription factors RsMYBS3 (GenBank No. OR178946) and RsRCI2B (GenBank No. OR178945) genes that regulated cold tolerance of R. sphaeroblastum were cloned, whose cDNA lengths were 795 and 165 bp, encoding 264 and 54 aa, respectively. Bioinformatics analysis showed that RsMYBS3 was a hydrophilic unstable protein with a typical SANT conserved structural domain of the SANT superfamilies; RsRCI2B was a hydrophobic stable protein with a highly conservative structural domain typical PMP3. The results of gene homology comparison showed that both RsMYBS3 and RsRCI2B proteins were highly homologous to MYBS3 and RCI2B proteins in red horse psyllium (Rhododendron vialii). The similarities were 93.73% and 94.44%, respectively. Phylogenetic analysis showed that RsMYBS3 and RsRCI2B were clustered in a clade with RvMYBS3 and RvRCI2B from R. vialii, which suggested that 4 genes were orthologous. qPCR analysis showed that both genes were significantly expressed in R. sphaeroblastum under the low-temperature stress, but had different expression patterns under different stress temperatures and times. The RsMYBS3 gene had the highest expression level at -6 ℃ treatment on 1 d, and then gradually decreased on 4 d with the prolonging of stress time, and expression gradually up-regulated on 7 d. The expression level gradually increased and reached a peak from 1 d to 4 d, and gradually decreased on 7 d under the -12 ℃ treatment. While the expression level of RsRCI2B gene had gradually increased from 1 d to the 7 day at -6 ℃ treatment, and at -12 ℃ treatment, except for the slight decrease expression on 1 d, the expression level gradually increased and reached a peak from 4 d to 7 d as the stress time prolonged. The above mentioned results suggested that both RsMYBS3 and RsRCI2B genes might be involved in the cold-tolerance-response physiological process of R. sphaeroblastum. This study provides some certain basic data and theoretical basis for exploring their cold tolerance molecular mechanism in the future.
Expression, Distribution and Function Analysis of STAB1 in the Placenta of Yak (Bos grunniens) Early and Middle Pregnancy Phase
WEI Qian-Qian, ZHANG Quan-Wei, ZHANG Bo-Hao, BAI Xu, ZHAO Xing-Xu, ZHANG Yong
2024 Vol. 32 (4): 785-794   DOI:10.3969/j.issn.1674-7968.2024.04.005 |  Full text (HTML) (1 KB)  |    PDF  (10990 KB)  ( 6 )
Abstract
The development of mammalian placenta is crucial for regulation of intrauterine environment and pregnant maintenance. Dysplasia or abnormality placenta could cause a variety of pregnant complications. Stabilin-1 (STAB1) is involved in regulating the development of several organs in animals, but its function and regulatory mechanisms are not clearly understood in yak (Bos grunniens) placenta. This study aimed to investigate the distribution, expression pattern and potential function of STAB1 in yak placenta during early and middle pregnancy. Yak placental tissues were collected at early and middle pregnancy. The distribution and expression pattern of STAB1 in yak placental tissues during early and middle pregnancy were observed using hematoxylin-eosin (HE) staining, immunohistochemistry (IHC) staining, immunofluorescence (IF) staining, qRT-PCR and Western blot. Meanwhile, the interacting proteins and potential functions of STAB1 were predicted based on data-independent acquisition (DIA) proteomics data. The results showed that STAB1 protein was mainly localized in the cytoplasm of yak placenta trophoblast giant cells and uninucleate trophoblast cells. Compared to early pregnancy placenta, the expression of STAB1 gene and its protein levels were significantly down-regulated in yak middle pregnancy (P<0.05). Bioinformatics analysis revealed that STAB1 might regulate cell adhesion, calcium binding and plasma membrane composition and other pathways involved in the dynamic development of yak's placenta during the early and middle pregnancy. In conclusion, STAB1 might be involved in placental development and pregnancy maintenance in early and middle pregnancy through regulating the function of placental trophoblast cells in yaks. This study provides a reference for further investigation of the function and mechanism of STAB1 in yak placental growth and development.
Construction of Porcine (Sus scrofa) SCD1 and SCD5 Double Knockout Cell Lines and Their Effects on Fat Deposition
FANG Qian-Hai, GUO Shuai, REN Hong-Yan, BAI Wen-Zhe, GAO Si, LIU Wen-Wen, CHEN Hong-Bo, ZHANG Li-Ping
2024 Vol. 32 (4): 795-806   DOI:10.3969/j.issn.1674-7968.2024.04.006 |  Full text (HTML) (1 KB)  |    PDF  (9170 KB)  ( 3 )
Abstract
Fat deposition and fatty acid composition have a decisive impact on pork quality. Stearoyl CoA desaturase (SCD) plays an important regulatory role in fatty acid composition and metabolism. SCD1 and SCD5 are the only 2 subtypes of the SCD family in various vertebrates currently, including humans (Homo sapiens). In order to clarify the effects of SCD1 and SCD5 on the types and composition of fatty acids in pigs (Sus scrofa) and their mechanisms, this study utilized CRISPR/Cas9 technology mediated by a combination of single and double single guide RNA (sgRNA) to perform targeted knockout of SCD1 and SCD5 genes. Homozygous deletion monoclone cell lines were screened, and the fatty acid content was detected and analyzed, their effects on fat deposition and lipid metabolism were examined. The results showed that the efficiency of SCD1 and SCD5 double genome editing exceeded 70%; Moreover, the deletion of both SCD1 and SCD5 genes reduced the relative content of palmitoleic acid (C16∶1) and oleic acid (C18∶1) in the rate limiting reaction products mediated by them; Moreover, the content of polyunsaturated fatty acids (PUFAs) in cells increased significantly. The absence of SCD1 and SCD5 also affects the content of fatty acids such as erucic acid (C18∶2n6), docosapentaenoic acid (DPA), docosahexenoic acid (DHA), eicosapentaenoic acid (EPA) and linoleic acid; The overall absence of SCD1 and SCD5 also significantly reduced the content of triglycerides, affecting the expression of factors related to fatty acid and lipid metabolism, such as fatty acid synthase (FASN) and acyl-CoA synthatase long-chain family member 3 (ACSL3). The results indicate that SCD might play an important regulatory role in the composition, metabolism, and deposition of fatty acids. This study provides a research model and reference for the improvement of pork quality and the study of obesity diseases.
Genome-wide eQTL Reveals Novel Candidate Loci for Meat Quality Traits on Chromosome 11 in Pigs (Sus scrofa)
ZHENG Yun-Di, RAN Xue-Qin, NIU-Xi, HUANG Shi-Hui, LI Sheng, WANG Jia-Fu
2024 Vol. 32 (4): 807-819   DOI:10.3969/j.issn.1674-7968.2024.04.007 |  Full text (HTML) (1 KB)  |    PDF  (2565 KB)  ( 21 )
Abstract
Genome-wide association study (GWAS) has detected a considerable amount of genetic variants associated with pork meat quality traits, however, the regulatory mechanism of numerous variants are generally unknown. Genetic variants mainly exert effects on complex traits by regulating gene expression levels. Thus, mapping expression quantitative trait locus (eQTL) has identified variants associated with gene expression levels, and will contribute to interpretate the association between variant loci and traits. In order to identify genetic variants/genes correlative with meat traits, this study used 19 hybrid Xiang pigs (Sus scrofa)(Large White pig ♂ × Congjiang Xiang pig ♀) of F2 generation as tested materials, whole genome sequencing (WGS) and high-throughput RNA sequencing (RNA-seq) data of the pork longissimus muscle tissues were integrated for eQTL analysis. The results showed a total of 1 332 cis-eQTL (PFDR<0.01) and 18 078 trans-eQTL (PFDR<1E-12) on chromosome 11. Subsequently, annotation and filtering were conducted on the eQTL-associated single nucleotide polymorphism (eSNP) which identified by cis-eQTL associations, and PCR-restriction fragment length polymorphism (RFLP) and Sanger sequencing were used to verify the candidate eSNP. An eSNP event rs319855910 c.-46C>G located in mitochondrial intermediate peptidase gene (MIPEP) 5'UTR was ultimately identified, and rs319855910 significantly associated with MIPEP gene expression (PFDR=8.81E-03). Statistical analysis combined with genotype and RNA-seq showed that rs319855910 genotypes significantly affected MIPEP gene expression (P<0.05), and it was observed that significant down-regulation (P<0.05) of MIPEP gene expression when the rs319855910 genotype was mutated from homozygote (CC) to homozygous mutant (GG). Furthermore, the transcription factor binding analysis revealed that rs319855910 was located in the region of transcription factor binding sequences, which could change the binding type or number of 5'UTR sequence with transcription factors. The above results suggested that rs319855910 was a novel candidate locus associated with pork meat quality traits, which could affect gene expression and regulate pork meat quality traits by changing the binding of transcription factors. This study provides basic data for pig breeding and genetic improvement.
DLD, NDUFA9 and Their Crotonylation Promote Browning of Primary Subcutaneous White Adipocytes in Piglets (Sus scrofa)
LIU Yue-Xia, LIU Zun-Hai, LIANG Jun-Tong, CHEN Qi, CHEN Xin-Hao, ZUO Yun-Fei, SUN Chao
2024 Vol. 32 (4): 820-832   DOI:10.3969/j.issn.1674-7968.2024.04.008 |  Full text (HTML) (1 KB)  |    PDF  (5401 KB)  ( 8 )
Abstract
Dihydrolipoyl dehydrogenase (Dld) and nicotinamide adenine dinucleotide (NADH) dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9 (Ndufa9) gene were found to be related to energy metabolism and mitochondrial function. It was suggested that Dld and Ndufa9 may be intrinsically linked to white adipocytes browning. This study aimed to investigate the effect of Dld, Ndufa9 and their crotonylation modifications on the browning of primary subcutaneous white adipocytes in piglets (Sus scrofa). To investigate this intrinsic linkage, this study was conducted with primary subcutaneous white adipocytes from piglets, and induced their differentiation into mature adipocytes. Interference and overexpression treatment of Dld and Ndufa9, detection of expression of lipid metabolism genes using real-time quantitative PCR (qPCR) and Western blot. The results revealed that lipogenesis-related genes fatty acid binding protein 4 (Fabp4) and fatty acid synthase (Fasn) were significantly lower (P<0.05) and lipolysis-related genes adipose triglyceride lipase (Atgl) and hormone sensitivelipase (Hsl), browning of white fat-related genes peroxisome proliferator-activated receptor gamma coactivator 1 alpha (Pgc-) and PR domain containing 16 (Prdm16) and thermogenesis-related genes uncoupling protein 2 (Ucp2), bone morphogenetic proteins 4 (Bmp4), bone morphogenetic proteins 7 (Bmp7) and cell death-inducing DNA fragmentation factorα-like effector A (Cidea) were significantly up-regulated (P<0.05) after overexpression of Dld and Ndufa9 compared with the control group, which indicated that Dld and Ndufa9 promoted browning of subcutaneous white adipocytes in piglets. Next, mature adipocytes were treated with vorinostat (SAHA) and sodium crotonate (NaCr) to give the cells a crotonylation-modified environment. It was found that crotonylation modification of Dld and Ndufa9 similarly promoted browning of subcutaneous white adipocytes in piglets. And through co-immunoprecipitation (Co-IP), it was further discovered that DLD combined with NDUFA9. These results suggested that DLD, NDUFA9 and their crotonylation modifications promoted primary subcutaneous browning of white adipocytes in piglets. Browning of piglet subcutaneous white adipocytes was critical to lipid metabolism in pigs. Therefore, it also indicated that Dld and Ndufa9 were key factors in regulating pig lipid metabolism. The results of this study provide an experimental basis and rationale for further studies on pork quality improvement.
SNP Detection of ARHGAP24 Gene and Correlation Analysis of Growth Traits in Xiangsu Hybrid Pigs (Sus scrofa)
JIANG Chuan-Mei, RUAN Yong, LI Ji-Feng, XIAO Mei-Mei, XU Jia-Li, HUANG Jia-Jin, XU Hou-Qiang
2024 Vol. 32 (4): 833-842   DOI:10.3969/j.issn.1674-7968.2024.04.009 |  Full text (HTML) (1 KB)  |    PDF  (2233 KB)  ( 6 )
Abstract
RhoGTPase activating protein 24 (ARHGAP24) gene is closely related to actin remodeling, cell polarity control and cell migration, and this study aims to explore the relationship between ARHGAP24 gene polymorphisms and the growth traits of Xiangsu hybrid pigs Sus scrofa, so as to provide a reference for the molecular marker selection of Xiangsu hybrid pigs. In this study, the blood DNA of 164 Xiangsu hybrid pigs under the same feeding conditions was collected, and the SNP sites of ARHGAP24 genes were determined by PCR amplification and sequencing. The general linear model in SPSS 25.0 software was used to analyze the association between the SNP site of the ARHGAP24 gene and the growth traits of Xiangsu hybrid pigs. A total of 5 SNP sites were detected in 9 exons of Xiangsu hybrid pigs, located in exon 1 and exon 8, respectively.g.735024 A>G nonsynonymous mutations caused valine at position 455 to change to aspartic acid on mRNA, which had no obvious effect on the structure of mRNA, but leaded to the α-helix and irregular coil of the protein secondary structure, and the decrease of β-corner and extension chain. After the nonsynonymous mutation of the g.735242 A>C site, the 528th position valine changed to serine, which produced obvious changes in the mRNA structure, while the proportion of α-helix and extended chain in the secondary structure of the protein decreased slightly, and the proportion of β-turn angle and irregular curl increased slightly. The analysis of association results showed that the C>T locus of g.735313 had significant effect on chest circumference (P<0.01) and abdominal circumference (P<0.05). Linkage imbalance analysis of 5 SNPs sites detected 5 haplotypes and 15 doubles. The g.735313 C>T loci in the ARHGAP24 gene of Xiangsu hybrid pigs were significantly associated with the chest and abdominal circumference of Xiangsu hybrid pigs, and were moderately polymorphic and in Hardy-Weinberg equilibrium, which could be used as candidate sites for molecular breeding of genetic markers of Xiangsu hybrid pigs in the later stage.
Cloning of UePkc1 in Ustilago esculenta and Its Expression Analysis in Hypha Growth and the Infected Zizania latifolia
QI Xiao-Qing, ZHOU Ya-Ping, LIU Jing-Xi, ZHENG Yu-Jie, SHANG Yuan-Yuan, TANG Jin-Tian, XIA Wen-Qiang, ZHANG Ya-Fen, YE Zi-Hong, CUI Hai-Feng
2024 Vol. 32 (4): 843-858   DOI:10.3969/j.issn.1674-7968.2024.04.010 |  Full text (HTML) (1 KB)  |    PDF  (24578 KB)  ( 5 )
Abstract
The development of swollen stem was induced by the infection of Ustilago esculenta in the stem of Zizania latifolia. The mycelium growth of U. esculenta and its response to environmental factors played an important role in regulating the development of swollen stem in Z. latifolia. Protein kinase C1 (Pkc1) was an important regulator of fungi which regulated the cell polar growth and responsed to stimulus feedback. In this research, the UePkc1 gene (GenBank No. OQ572333) was cloned with the cDNA length of 3 573 bp, which encoded 1 190 amino acids and had the STKc_PKC conserved domain of PKC protein. After treatment with 3 types cell wall interferers (Congo red, fluorescent brightener and sodium dodecyl sulfate) and 2 different temperatures (15 ℃ and 35 ℃) during culture of U. esculenta in vitro, the swelling and shortening of basidiospore cells were observed by treatments with cell wall interfering agents and high temperature. The fusion mycelium of U. esculenta was shortened in the treatments with cell wall interfering agents, while no fusion mycelium was found in the 2 treatments with different temperatures. qPCR analysis showed that the expression of UePkc1 in U. esculenta was significantly increased after treatment with cell wall interferers for 48 h, and the expression of UePkc1 in U. esculenta treated with low temperature for 72 h was also significantly increased, while it was significantly decreased after treatment with high temperature. These results suggested that UePkc1 gene was involved in the maintenance of cell wall integrity and responsed to ambient temperature regulation in U. esculenta. Besides, there were significant differences in the expression of UePkc1 among the development stems in Z. latifolia between different swelling phenotypes. The expression of UePkc1 was the highest in the development stem of 10 cm in Grey-Jiaobai, while it was the highest at the early stage of stem in Normal-Jiaobai. Combined with the changes of growth and distribution of U. esculenta during stem development of Grey-Jiaobai and normal Jiaobai, It was preliminarily clarified that the mycelial growth of U.esculanta in the stem of plants was regulated by the expression of UePkc1, which might be positively correlated with the increasing of mycelial quantity related to the swollen of the stem. This study provides technical reference and theoretical basis for the research on the interaction mechanism of inducing the swollen and development of the stem in Z. latifolia infected by U. esculenta.
Antibacterial Effect of Bacillus velezensis YFB3-1 on Phytophthora capsici and Its Antibacterial Substances
MU Xiao-Qing, LU Yao-Xiong, DAI Liang-Ying, HUANG Guo-Lin, CHEN Wei, ZHANG Ju-Ye, WEI Chen-Xi
2024 Vol. 32 (4): 859-872   DOI:10.3969/j.issn.1674-7968.2024.04.011 |  Full text (HTML) (1 KB)  |    PDF  (11209 KB)  ( 4 )
Abstract
Pepper phytophthora blight is a soil-borne disease caused by Phytophthora capsic, which causes devastating losses of pepper (Capsicum annuum) and seriously threatens the sustainable development of pepper industry in China. In order to obtain a safe, efficient and effective biocontrol agent for the prevention of pepper phytophthora blight, a strain of Bacillus velezensis YFB3-1 isolated and identified from the intestinal bacteria of earthworms (Eisenia foetida) was selected as a biocontrol strain, and Phytophthora capsici was used as the target strain. The antibacterial effects of different types of Bacillus on P. capsici were compared by plate confrontation method, and the antibacterial effects of YFB3-1 on different soil-borne pathogenic fungi of pepper were compared, and the main antibacterial secondary metabolites and biocontrol mechanisms were analyzed. The antibacterial test showed that B. velezensis YFB3-1 had the best inhibitory effect on P. capsici, and the inhibition rate was 85.21%, which was 19.8% higher than that of B. subtilis GCK5-1. It had good antibacterial effect on different soil-borne pathogenic fungi of pepper, and had the characteristics of broad-spectrum resistance to soil-borne pathogenic fungi of pepper. Microscopic examination showed that B. velezensis YFB3-1 could lead to abnormal mycelial growth, multi-branching or disordered branches, resulting in distortion and enlargement of mycelial cells to varying degrees, and even cell wall rupture. The results of liquid chromatography tandem mass spectrometry (LC-MS) targeted metabolomics showed that the main lipopeptide compounds in the fermentation broth of B. velezensis YFB3-1 were surfactin and fengycin, which reached the highest content at 3 d of fermentation, which were 1.19×10-3 and 1.14×10-3 mg/mL, respectively.This study provides an excellent biocontrol strain for the green prevention and control of pepper blight, and provides a reference for further excavation and utilization of secondary metabolites of B. velezensis.
Analysis of Biofilm Formation Ability and Influencing Factors of Yak (Bos grunniens) Pathogenic Escherichia coli
GAO Xiang, LI Hui-Ping, LANG Xiao, HE Zi-Wen, WANG Jing-Hong, WANG Xia, NING Jian-Gang
2024 Vol. 32 (4): 873-881   DOI:10.3969/j.issn.1674-7968.2024.04.012 |  Full text (HTML) (1 KB)  |    PDF  (2103 KB)  ( 8 )
Abstract
Biofilms exist widely in the natural environment. As one of the important anti-stress mechanisms of bacteria, they have strong resistance, pathogenicity and immune escape functions. The aim of this study was to establish a biofilm-forming model strain of yak (Bos grunniens) pathogenic Escherichia coli (YPEC), and to explore the effects of various boundary factors on the biofilm formation of YPEC. In this study, the isolates strains of YPEC Tianzhu-1, Tianzhu-2, Tianzhu-3, Qinghai-2, Qinghai-3 and Qinghai-4 were selected as the research object. Microplate quantative method was used to identify the biofilm formation ability of YPEC, and strains with strong biofilm formation were selected as the dominant strains, and then the influencing factors of biofilm formation amount were analyzed. The results showed that different isolates of YPEC had different biofilm forming abilities. Tianzhu-1, Tianzhu-2, and Tianzhu-3 strains were all strong biofilm formers, while Qinghai-3 and Qinghai-4 strains were medium biofilm formers, and Qinghai-2 strain were weak biofilm formers. Among them, Tianzhu-3 strain had the strongest ability to form biofilms (P<0.05). The optimal amount of biofilm formation of Tianzhu-3 strains was achieved when culture time was 24 h, temperature was 37 ℃, pH 7.5, initial bacterial solution concentration was 1.3×109 CFU/mL and 10% bacterial liquid ratio. The addition of 20 μg/mL DNA and 2% sucrose in LB medium could promote the formation of biofilm, while the addition of different concentrations of NaCl, glucose, and lactose all had inhibitory effects on the formation of biofilms. The above results provide data support and theoretical basis for YPEC prevention and control from the perspective of biofilm.
Reviews and Progress
Molecular Composition and Mechanism of Action of PANoptosis
WANG Yi-Ru, WANG Yuan, CAO Ling-Xia, YANG Jie, WANG Shu-Min
2024 Vol. 32 (4): 882-891   DOI:10.3969/j.issn.1674-7968.2024.04.013 |  Full text (HTML) (1 KB)  |    PDF  (3606 KB)  ( 15 )
Abstract
Programmed cell death (PCD) is a line of the innate immune defense system, and plays an important role in the growth development and immune regulation of the organism. As a new PCD pathway, PANoptosis is regulated by the multi-protein complex PANoptosome, which can simultaneously regulate the three key modes of programmed cell death including pyroptosis, apoptosis and necrosis, thereby defending against invasion of various pathogens. Consequently, studying the mechanism of PANoptosis is of great significance for deepening the understanding of cell death patterns, suggesting the occurrence of inflammation and diseases. In this paper, the concept, mechanism of action, molecular composition and assembly method of PANoptosis were reviewed, then a view was proposed that the formation of PANoptosome was a sign of PANoptosis in cells. This review provides new ideas for the treatment of inflammatory diseases.
Research Progress on the Effect of IL-33/ST2 Signaling Pathway on Stem Cell Function
XU Wen-Jie, HUA Jin-Lian
2024 Vol. 32 (4): 892-902   DOI:10.3969/j.issn.1674-7968.2024.04.014 |  Full text (HTML) (1 KB)  |    PDF  (3322 KB)  ( 10 )
Abstract
Interleucin 33 (IL-33) is a cytokine with both pro-inflammatory and anti-inflammatory functions, and its signaling is mediated through its specific receptor suppressor of tumorigenicity 2 (ST2). Activation of the IL-33/ST2 signaling pathway regulates NF-κb, TNFα, and Wnt signaling pathways depending on the cell type, thereby triggering inflammatory response and affecting the stemness and differentiation of stem cells. In this paper, the basic structure and composition of the IL-33/ST2 signaling pathway were reviewed, and the recent progress in stem cell function research was summarized. This review provides new ideas for the treatment of inflammatory and immune diseases by elucidating the mechanism of action of stem cells.
Research Progress on Functionalized Ferritin Nanoparticle Vaccine
ZHANG Rong, WEI Yan-Ming, RU Yi
2024 Vol. 32 (4): 903-910   DOI:10.3969/j.issn.1674-7968.2024.04.015 |  Full text (HTML) (1 KB)  |    PDF  (918 KB)  ( 18 )
Abstract
Among the multitude of nanocarriers, protein nanoparticles are hot topic in vaccine nanobiotechnology due to their biocompatibility, and flexibility of design. Compared with isolated protein subunits, protein nanoparticle vaccines are more readily taken up by antigen-presenting dendritic cells. Ferritin is a ubiquitous iron storage and detoxification protein that protects cells from iron-induced oxidative damage. Ferritin have remarkable chemical and thermal stability, reversible assembly and disassembly processes, and ability for engineering to display antigens. Ferritin is not only a useful nanoreactor and nanocarrier, but also an effective platform for vaccine development platform. This review summarized structure-function properties, self-assembly, novel bioengineering strategies, production and purification of ferritin nanoparticles, and functionalization of ferritin. Application progress and foreground to the field of vaccine development of ferritin were also discussed. This review provides theoretical support for nanobiotechnology in vaccine development.
Resources and Updated Technology
Application of Double Resonator Piezoelectric Cytometry in Salt Tolerance Evaluation of Rice (Oryza sativa) Varieties
YANG Hou-Peng, ZHOU Tie-An, PAN Wei-Song, XIAO Ying-Hui
2024 Vol. 32 (4): 911-925   DOI:10.3969/j.issn.1674-7968.2024.04.016 |  Full text (HTML) (1 KB)  |    PDF  (7301 KB)  ( 6 )
Abstract
The selective breeding of rice (Oryza sativa) varieties and the testing of stress resistance (salt tolerance) are usually conducted on the tested plants, which is not only laborious, time-consuming and destructive, but also susceptible to environmental factors, which greatly limits the efficiency and reliability of selective breeding. The purpose of this study was to establish a new method for evaluating salt tolerance of rice varieties based on double resonant piezoelectric cytometry (DRPC) and cell mechanics testing, using live rice cells as the characterization object. Firstly, 'Haidao 86' and 'Nipponbare' rice varieties with known strong salt tolerance and salt-sensitive characteristics respectively were selected to prepare their adherent cell culture systems, and then DRPC was used to measure the responses and variations of cells generated surface stress (ΔS) and cell viscoelasticity index (CVI) to different concentrations of NaCl stress. The results showed that the ΔS of 'Nipponbare' cells decreased first and then increased under the conditions of 20 and 40 mmol/L NaCl, and decreased monotonously under the conditions of 60~120 mmol/L NaCl; at the same time, the cells became softer and then harder under the conditions of 20 and 40 mmol/L NaCl, and became softer monotonously under the conditions of 60~120 mmol/L NaCl, and reached the limit of resisting salt stress under the condition of 40 mmol/L NaCl. The ΔS of 'Haidao 86' cells decreased first and then increased under the condition of 20 mmol/L NaCl, increased first and then decreased under the conditions of 40 and 60 mmol/L NaCl, fluctuated up and down under the conditions of 80 and 100 mmol/L NaCl, and increased sharply under the condition of 120 mmol/L NaCl. At the same time, the cells became harder under the conditions of 20, 80 and 100 mmol/L NaCl, and became harder first and then softer under the conditions of 40, 60 and 120 mmol/L NaCl, and reached the limit of resisting salt stress under the condition of 100 mmol/L NaCl. By a comprehensive comparison of the changes in cells generated stress and CVI produced by the cells of the 2 varieties under different concentrations of NaCl stress, it could be confirmed that the salt tolerance of 'Haidao86' was higher than that of 'Nipponbare', which was consistent with the result that the salt tolerance of 'Haidao86' was higher than that of Nipponbare' reported at the level of field plants. Therefore, this study not only confirms the accuracy and reliability of the DRPC for the evaluation of salt tolerance of rice varieties, but also provides an important reference for the wide application of this new method to efficiently evaluate crop stress resistance, which is of great significance for crop breeding for abiotic stress resistance.
Screening and Stability Evaluation on qRT-PCR Reference Genes of Cremastra appendiculata
ZHANG Yu-Jin, JI Jun, XIAO Xin, ZHANG Jing-Yi, WANG Li-Qin, GAO Yan-Yan, TIAN Yu-Hang, ZHANG Ming-Sheng
2024 Vol. 32 (4): 926-938   DOI:10.3969/j.issn.1674-7968.2024.04.017 |  Full text (HTML) (1 KB)  |    PDF  (2677 KB)  ( 7 )
Abstract
As a rare medicinal plant of orchid family, Cremastra appendiculata has been widely concerned for its high medicinal value, but its resources are depleted by severe reproductive barriers and predatory human extraction, using modern biomolecular technology to solve the reproduction difficulties of C. appendiculata species is one of the effective ways of resource conservation. In this study, different organs (root, stem and leaf) and seeds at different stages (the symbiotic and non-symbiotic germination) of C. appendiculata were used as research materials, qRT-PCR was used to detect the expression of 7 commonly used household genes of β-actin (ACT), translation elongation factor 1 beta (ef-), cyclophilin (CYP), ribosomal protein (RPL), ribosomal protein S8 (RPS), ubiquitin C (UBC), α-tubulin (TUB). The stability of candidate genes was comprehensively evaluated by geNorm, NormFinder and BestKeeper software, and finally the reference genes suitable for qRT-PCR of C. appendiculata were selected. The stability of the reference genes was verified by the target genes GA 3-beta dioxygenase (GA3ox) and lectin protein coding gene (Lectin10). The result showed that 2 reference genes could be introduced into different organs and treatments of C. appendiculata; The expression of RPL and ef- in different organs was relatively consistent, with good stability, and suitable as reference genes; The expression of UBC and TUB were the most stable in the symbiotic germinated seeds at different germination stages; RPL and ef- were the most stable reference genes in the non-symbiotic germination seeds at different germination stages. When the relatively stable UBC and TUB genes were used as internal reference genes, the target genes GA3ox and Lectin10 showed similar expression trends in the seed of C. appendiculata at different symbiotic germination stages, while the poorly stable ACT genes could not effectively correct the expression analysis of target genes, and the data showed extremely obvious deviations. This study provides correction and standardization genes for the expression analysis of related genes in C. appendiculata under different research conditions, which would effectively improve the accuracy and reliability of subsequent studies.
Establishment and Application of Real-time Fluorescent LAMP Rapid Detection of Fusarium solani of Panax notoginseng Root Rot
CHE Xiao-Li, LIANG Ting-Ting, QU Yuan, LIU Di-Qiu
2024 Vol. 32 (4): 939-948   DOI:10.3969/j.issn.1674-7968.2024.04.018 |  Full text (HTML) (1 KB)  |    PDF  (5978 KB)  ( 6 )
Abstract
Root rot is a serious disease of Panax notoginseng in China, and Fusarium solani is the main fungal pathogen causing root rot of P. notoginseng. In this study, a loop mediated isothermal amplification (LAMP) system was established to rapidly detect F. solani from P. notoginseng. Firstly, the D-amino acid oxidase gene (DAO) was screened by PCR as a specific gene locus for detection of F. solani, and the specific primer pairs of LAMP reaction were designed and the reaction conditions were optimized. The optimum reaction temperature was 65 ℃; the optimum concentration of MgSO4 was 6 mmol/L; the optimum concentration of dNTPs was 0.6 mmol/L; the amplification reaction was more efficient in the presence of cyclic primers. Sensitivity of LAMP to F. solani was tested, and the template concentration could be as low as 0.009 pg/μL. At last, the on-site rapid detection technology of F. solani was established based on the combination of DNA extraction-free and calcein coloration. This study provides technical support for the rapid diagnosis of F. solani of root rot in P. notoginseng cultivation and seedling trade.
Construction of Sesuvium portulacastrum VIGS System Based on PDS Gene Family
MA-Wei, WANG Xi-Hao, ZHANG Xue-Yan
2024 Vol. 32 (4): 949-960   DOI:10.3969/j.issn.1674-7968.2024.04.019 |  Full text (HTML) (1 KB)  |    PDF  (10954 KB)  ( 5 )
Abstract
Sesuvium portulacastrum is a succulent saline plant, which is extremely tolerant to high salt stress. Virus-induced gene silencing (VIGS) is an important research tool to characterise the gene function of plants. In order to establish an efficient VIGS system in the important salt-tolerant plant S. portulacastrum, phytoene desaturase (PDS) gene was selected as a marker gene using S. portulacastrum of Sanya as the material. According to the transcriptome data, 5 PDS family members of S. portulacastrum were identified by local Blast and other biosignature means, and the common sequences of the 5 members were obtained by PCR, with a length of 431 bp.Meanwhile, SpTRV2-PDS silencing expression vector was constructed, which was transformed by Agrobacterium tumefaciens GV3101 and then infiltrated into the plants by injection. The phenotype was observed, and the changes of carotenoid content and PDS gene expression were measured. The results showed that after infection, the plants showed slow growth and gradual yellowing of leaves, and the leaves showed albino phenomenon after 7 d, and the carotenoid content and the relative expression of PDS in the experimental group continued to exist, indicating that the VIGS system of S. portulacastrum was successfully constructed, indicating that the VIGS system could effectively silence the expression of PDS gene in S. portulacastrum. This study provides a technical means for the identification of the function of salt tolerance genes in S. portulacastrum.
Detection of Acute Hepatopancreatic Necrosis Disease in Pacific White Shrimp (Penaeus vannamei) by Recombinase Polymerase Amplification
JIN Dong-Sheng, LOU Yi-Rong, WANG Yao-Hua, DUAN Li-Jun, LI Qi-Ang, CHEN Chen, YAN Mao-Cang, CHEN Jiong, ZHOU Qian-Jin
2024 Vol. 32 (4): 961-973   DOI:10.3969/j.issn.1674-7968.2024.04.020 |  Full text (HTML) (1 KB)  |    PDF  (4429 KB)  ( 11 )
Abstract
Acute hepatopancreatic necrosis disease (AHPND) is a newly emerged bacterial disease in shrimps (Penaeidae), causing significant economic losses in the global shrimp farming industry. AHPND is primarily caused by Vibrio spp. carrying virulent pirA and pirB. In this study, specific primers and probes were designed and screened for pirA and pirB, and a recombinase polymerase amplification (RPA) method was developed. This RPA reaction ran in 39 ℃ for 20 min. The results revealed successful screening of primer/probe combinations for both pirA and pirB genes. Through a series of tests using the RPA method, these primer/probe combinations demonstrated specific detection of the target virulence genes, without amplification of genomic DNA from 13 microorganisms, such as Vibrio alginolyticus, V. parahaemolyticus, and Enterocytozoon hepatomegaly. The detection limit of the RPA method was 0.01 and 0.1 fg/μL of recombinant plasmid pirAB-pMD19, when the primers and probes targeting pirA or pirB were used. The primer concentrations were optimized at 0.4 µmol/L each, while the optimal concentration for the probe was 0.16 µmol/L. The RPA method was used to detect suspicious tissues of Penaeus vannamei using optimized sets of primers and probe. The results showed that the RPA method could accurately detect pirA and pirB from shrimp tissues, which was consistent with the PCR method recommended by the World Organization for Animal Health (WOAH). In summary, this PRA methods targeting pirA and pirB respectively could rapid and accurately detect ANPND-causing bacteria with easy operation and short time-consumption, making it a promising supplementary tool for routine monitoring and rapid detection of AHPND.
Construction and Analysis of Biological Characteristics of Gallibacterium anatis tolC Traceless Knockout Strain
MA Li-Jing, SHI Zi-Cong, WANG Xin-Wei, CHEN Lu, PENG Ze-Yu, LI Dong-Liang, YANG Xia
2024 Vol. 32 (4): 974-982   DOI:10.3969/j.issn.1674-7968.2024.04.021 |  Full text (HTML) (1 KB)  |    PDF  (2522 KB)  ( 8 )
Abstract
TolC family protein (TolC), an outer membrane channel protein, is widespread in Gram-negative bacteria, which mediates extracellular transport of various substances and plays important roles in the bacterial biological functions. The identification of the functions of TolC in the virulence of Gallibacterium anatis would play crucial roles for prevention and control of this disease. In this study, traceless deletion mutant of tolCtolC) was constructed by natural transformation using a clinical isolate Yu-PDS-RZ-1-SLG (WT) and the biological characteristics of the mutant strain was investigated. Compared with WT strain, the results of ΔtolC showed that bacteria colonies and zone of hemolysis were smaller, the logarithmic growth phase was delayed 2 h with slower growth rate and weakened biofilm formation ability (P<0.001). In addition, the survival rates under high temperature, oxidative stress and osmotic pressure circumstance were significantly reduced (P<0.01). The sensitivity to some antibiotics in the planktonic and membrane states increased: in the planktonic state, the sensitivity of ΔtolC to SDS, fosfomycin, and doxycycline increased by 32, 8, and 4 times, respectively; in the membrane state, the sensitivity of ΔtolC to SDS, fosfomycin, and doxycycline increased by 16, 8, and 8 times, respectively; while the sensitivity to neomycin sulfate and kanamycin remained unchanged. The adhesion ability of ΔtolC to human (Homo sapiens) cervical cancer cell line (Hela) reduced. The construction and analysis of biological characteristics of tolC traceless knockout strain ΔtolC provides basic data for the study of the biological function, pathogenic mechanism, and drug resistance mechanism of TolC protein in G. anatis.
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