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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. 8  Published: 01 August 2024
 
Articles and Letters
Identification of DREB Gene Family in Phalaenopsis aphrodite and its Expression Analysis Under Abiotic Stresses
HU Zi-Ang, LUO Ping, XIN Jing-Jing, CUI Yong-Yi
2024 Vol. 32 (8): 1715-1728   DOI:10.3969/j.issn.1674-7968.2024.08.001 |  Full text (HTML) (1 KB)  |    PDF  (12437 KB)  ( 1 )
Abstract
The dehydration responsive element binding (DREB) gene family plays an important role in plant response to abiotic stresses such as low temperature, drought, and high salinity, and currently, there are few reports on the DREB gene family in Phalaenopsis spp. In this study, Phalaenopsis aphrodite was used as the reference genome for the identification. In addition, the subcellular localization, bioinformatics, and gene expression pattern of DREB family members under 3 abiotic stress conditions: low temperature stress, drought treatment, and salt stress, and abscisic acid treatment were studied and analysed. Using conserved structural domain analysis and BLAST comparison, 31 DREB family gene members were found from the P. aphrodite genome database. PaDREB transcription factors were divided into 6 subgroups, numbered A1 to A6, according to phylogenetic research. Conserved motif analysis revealed that all of PaDREB transcription factors had a AP2 conserved structural domain. The transcriptome expression pattern study of P. aphrodite revealed that different PaDREBs showed differential tissue expression and most of the genes were highly expressed in the roots. The 4 members of subgroup A1 were isolated and named PaDREB1A, PaDREB1B, PaDREB1C, and PaDREB1D, respectively, and subcellular localization was carried out on them, and the results showed that all of them were localized in the nucleus. Meanwhile, the results of expression analysis of the above 4 members showed that PaDREB1A mainly responded to drought and high salt and ABA treatments; PaDREB1B mainly responded to low temperature and high salt treatments; PaDREB1C mainly responded to drought and ABA treatments; and PaDREB1D mainly responded to low temperature, drought and ABA treatments. The above results suggested that the DREB family of Phalaenopsis had an important role in the response to abiotic stress pathway, and this study provides a theoretical basis for further research on the mechanism of the DREB family's response to abiotic stress in Phalaenopsis.
Functional Analysis of Rice (Oryza sativa) TFⅡB-related Factor OsBRF1
CHEN Xin-Hui, CHEN Guang-Na, NING He, CHEN Jia-Luo, WANG Si-Yi, ZHU Cheng, YANG Su
2024 Vol. 32 (8): 1729-1741   DOI:10.3969/j.issn.1674-7968.2024.08.002 |  Full text (HTML) (1 KB)  |    PDF  (8355 KB)  ( 0 )
Abstract
In most eukaryotes, transcription factor ⅡB (TFⅡB) is a general transcription factor of RNA polymerase Ⅱ in transcription of DNA into RNA. TFⅡB-related factor 1 (BRF1) serves as the general transcription factor for RNA polymerase Ⅲ, responsible for synthesizing tRNA and 5S rRNA. In Arabidopsis thaliana, AtBRF1 and AtBRF2 participate in gametogenesis in a functionally redundant manner. However, the function of BRF1 in rice (Oryza sativa), an important monocotyledonous model plant and staple crop, has not been reported yet. In this study, OsBRF1 gene promoter-driven β-glucuronidase (GUS) expression vector was constructed. The results of GUS staining showed that OsBRF1 exhibited high expression levels in root, leaf, leaf sheath, stigma, mature root, mature leaf, panicle, pollen, endosperm, and anther, but it was not expressed in the glume. The fusion expression vector of OsBRF1 and GFP was transfected into Nicotiana benthamiana leaves via Agrobacterium tumefaciens, and it was found that OsBRF1 protein predominantly localized in the nucleus. Unable to obtain homozygous frameshift mutant plants using CRISPR/Cas9 gene editing methods indicated that OsBRF1 gene was homozygous lethal. RNAi-mediated silencing led to a significant reduction in pollen activity and fertility (P<0.05), suggesting a potential role of OsBRF1 in regulating rice fertility by modulating pollen viability. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation assays demonstrated interaction between OsBRF1 and OsTBP1~3 (TBP: TATA-binding protein), indicating its potential role as a general transcription factor. This study provides basic material for further exploration of the gene function and regulatory mechanisms of OsBRF1.
Identification and Tissue Expression Pattern Analysis of KNOX Gene Family in Setaria italica
SHI Wei-Ping, ZHANG Xin, ZHAO Yuan, LIU Min, DAI Ke-li, ZHANG Ai-Ying, GUO Er-Hu, SHI Guan-Yan, GUO Jie
2024 Vol. 32 (8): 1742-1752   DOI:10.3969/j.issn.1674-7968.2024.08.003 |  Full text (HTML) (1 KB)  |    PDF  (8709 KB)  ( 1 )
Abstract
Knotted1-like homeobox (KNOX) is a transcription factor unique to plants, characterized by its homologous domain, and is widely involved in plant growth and development. To explore the potential function of the foxtail millet (Setaria italica) KONXs, bioinformatic methods were used to identify and analyze the SiKNOX gene family at the whole-genome level of foxtail millet, and the expression patterns of SiKNOXs in different tissues were also detected using RT-qPCR. The results showed that a total of 11 SiKNOXs were identified in the S. italica genome. Phylogenetic analysis classified them into 2 groups, classⅠ and classⅡ. ClassⅠ contained 5 SiKNOXs and classⅡ contained 6 SiKNOXs. Subcellular localization prediction revealed that all SiKNOX proteins were predicted to be nuclear. Chromosomal localization analysis showed that they were unevenly distributed among the 7 chromosomes of S. italica. Duplication analysis showed that there were 5 SiKNOXs forming 4 segmental duplication gene pairs. Gene structure analysis revealed that all 11 SiKNOXs consisted of 5~6 exons. Protein conserved domain analysis indicated 10 motifs in SiKNOXs. Motifs 2, 3, 5, and 6 corresponded to the KNOX1, KNOX2, ELK, and HD domains, respectively. The promoter region of the SiKNOXs harbored numerous cis-acting elements that were closely associated with various biological processes. Expression pattern analysis revealed that all SiKNOXs were detected in different tissues (including roots, stems, leaves, tassels, and seeds), and most of them showed tissue-specific expression. This study lays the foundation for further research on KNOX genes family in the growth and development of S. italica.
Cloning and Expression Analysis of CmWRKY7 Gene in Melon (Cucumis melo)
LI Jia-Qi, ZHANG Li-Chao, ZHAO Wang-Long, CHEN Chun-Lin, WANG Ji-Qing, LI Meng, XIAO Huai-Juan
2024 Vol. 32 (8): 1753-1765   DOI:10.3969/j.issn.1674-7968.2024.08.004 |  Full text (HTML) (1 KB)  |    PDF  (7405 KB)  ( 3 )
Abstract
In plants, WRKY transcription factors play a crucial role in responding to biotic and abiotic stresses. To explore the biological function of WRKY gene in defense against abiotic stress in melon (Cucumis melo), a candidate gene strongly induced by low temperature, CmWRKY7, was screened based on pre transcriptome sequencing. In this study, CmWRKY7 was cloned, and the molecular characteristics of its encoded protein were analyzed using bioinformatics methods. The expression patterns of CmWRKY7 under different stresses were detected by qRT-PCR, including low temperature, drought, salt, and ABA stress. Results showed that the total length of CmWRKY7 was 732 bp, encoding 243 amino acids, with a predicted molecular weight of 27.09 kD and a theoretical isoelectric point of 9.50. It was a hydrophilic protein with an aliphatic amino acid index of 64.20 and high thermal stability. The CmWRKY7 protein sequence contained 2 highly conserved domains: The WRKYGKK and the C2H2 zinc finger structural sequence (C-X4-C-X23-H-X-H). Moreover, the CmWRKY7 protein also contained 26 amino acid residues that might be phosphorylation modification sites, 7 protein kinase C phosphorylation sites, 4 N-myristoylation sites, and 1 tyrosine kinase phosphorylation site. The secondary structure of CmWRKY7 protein was dominated by irregular coils, accounting for 62.14%. Phylogenetic analysis showed that CmWRKY7 had the closest evolutionary relationship with the proteins of cucumber (C. sativus) and horned melon (C. metuliferus), gathering into one branch. Promoter prediction analysis showed that there were multiple hormone and stress-related cis acting elements on the CmWRKY7 promoter, and protein interaction prediction analysis showed that CmWRKY7 mostly interacted with stress-related proteins. Under abiotic stress, the CmWRKY7 gene participated in ABA signal transduction and actively responded to drought, high salt, and low temperature stresses. These results provided theoretical basis for further studying the biological function of CmWRKY7 and its application in melon breeding for abiotic stress resistance.
Cloning and Functional Validation of the ZjABR1 Gene in 'Laoqiu zao' (Ziziphus jujuba)
ZHANG Peng-Fei, QIU Li-Ping, CHAO Rui-Qiang, CUI Wen-Jing, LIANG Jin-Jun, WEN Peng-Fei
2024 Vol. 32 (8): 1766-1775   DOI:10.3969/j.issn.1674-7968.2024.08.005 |  Full text (HTML) (1 KB)  |    PDF  (7670 KB)  ( 0 )
Abstract
The AP2/ERF family is widely involved in processes such as plant growth and development, hormonal response, and can affect changes in fruit texture. In order to investigate the expression pattern and function of AP2/ERF family member ABR1 (abscisic acid repressor 1) in the ripening process of jujube (Ziziphus jujuba) fruit, this study used 'Laoqiu zao' (Ziziphus jujuba cv. 'Laoqiu zao') as the material, ZjABR1 gene (LOC107409847) was cloned and its bioinformatics, subcellular localization, and expression were analyzed. An overexpression vector was constructed for transient transformation. Yeast one hybrid experiment was performed. The study found that the CDS region of the ZjABR1 gene was 1 344 bp, encoded 447 amino acids, and contained an AP2 binding domain. The subcellular localization analysis revealed that the ZjABR1 protein was presented in nucleus. RT-qPCR analysis revealed that ZjABR1 was highly expressed in the jujube fruit fully ripe stage. Transient overexpression of ZjABR1 gene enhanced polygalacturonase (PG) activity and the expression level of ZjPG. Yeast one hybrid demonstrated that ZjABR1 could directly bind to the ZjPG promoter. This study provided a basis for research on the molecular mechanisms of jujube fruit ripening.
MAX1 Gene Cloning and Its Response to High Temperature Stress in Green Onions (Allium fistulosum)
ZHANG Yu-Chen, BAI Mi-Feng, XU Huan-Huan, XING Jia-Yi, ZHOU Xu-Yang, LIU Le-Cheng, WANG Yong-Qin
2024 Vol. 32 (8): 1776-1783   DOI:10.3969/j.issn.1674-7968.2024.08.006 |  Full text (HTML) (1 KB)  |    PDF  (8121 KB)  ( 0 )
Abstract
More axillary growth 1 (MAX1) is a key gene in the strigolactone synthesis pathway, which plays an important role in the tillering regulation of green onions (Allium fistulosum) and resistance to adversity stress. In this study, AfMAX1 gene (GenBank No. AfisC2G04224) was cloned from green onions, and bioinformatics analysis, subcellular localization, tissue expression patterns, and high-temperature stress response analysis were carried out. The results showed that the CDS of AfMAX1 was 1 545 bp long, encoded 514 amino acids, and had a relative molecular weight of 58.55 kD. AfMAX1 was a hydrophilic protein with no signal peptide and transmembrane domain. Protein sequence comparison analysis showed that MAX1 was conserved in multiple species. Subcellular localization results showed that AfMAX1 might be localized in the cytoplasm and nucleus of Nicotiana benthamiana. Expression pattern analysis showed that AfMAX1 had the highest expression level in the leaf sheath, followed by in the roots; The expression level of AfMAX1 significantly increased after 12 h of high temperature stress (P<0.01). The above results indicate that AfMAX1 could respond to high temperature stress, which might help enhance plant tillering regulation and heat tolerance. This study provides basic data for in-depth exploration of the molecular mechanisms of green onion plant type regulation and abiotic stress resistance.
NtCPS2 Gene Affects the Occurrence of Tobacco (Nicotiana tabacum) Glandular Hair by Regulating Gibberellin Synthesis
CAO Ke-Xin, YANG Hui-Juan, LIU Hua-Bing, WU Jian, CHEN Xiao-Xiang, XU Shi-Xiao
2024 Vol. 32 (8): 1784-1791   DOI:10.3969/j.issn.1674-7968.2024.08.007 |  Full text (HTML) (1 KB)  |    PDF  (2607 KB)  ( 0 )
Abstract
The growth and development of foliar glandular hairs in tobacco (Nicotiana tabacum) is related to the aroma quality of tobacco leaves. In order to investigate the relationship between Ent-copalyl diphosphate synthase 2 (NtCPS2) and the growth and development of glandular hair and the synthesis of glandular hair secretion in tobacco, the wild-type strain (WT), NtCPS2 homozygous editing strain (KD) and overexpression strain (OE) of '8306' were used as materials to observe the phenotype of glandular hair and the density of glandular hair. Content of cyperane substances was determined and the expression of key synthetic genes was analyzed. The results showed that compared with WT, the density of leaf secretory glandular hairs in KD strains increased by about 75%, and the secretion of leaf glandular hairs increased significantly (P<0.05), while the secretion of leaf glandular hairs in OE strains was significantly lower than that of WT strains (P<0.05), and decreased by about 50% compared with WT strains (P<0.05). The expression level of cyclin B2 (NtCycB2) gene in the negative regulation of glandular hair gene was significantly lower than that in WT and KD strain, and the expression level of NtCycB2 gene in OE and KD strains showed an opposite trend, and the expression level of NtCycB2 gene was opposite to that of glandular hair phenotype. The gibberellin content of KD tobacco plant was significantly higher than that of WT strain (P<0.05). The gibberellin content of OE strains was significantly lower than that of WT strains (P<0.05). The content of gibberellin in KD strains increased, the density of secretory glandular hairs increased, and the content of cyperane substances, the main glandular hair secretions in the foliar strain increased. The OE strain showed an opposite trend to the KD strain. The results suggested that the NtCPS2 gene might promote the increase of gonadal hair density and foliar glandular hair secretion by positively regulating gibberellin biosynthesis. This study provides a new research direction for the regulation of the synthesis of tobacco glandular hair and tobacco aroma compounds by NtCPS2 gene through gibberellin pathway.
Identification of EgrZFP-EARs of the C2H2 Zinc Finger Protein Gene Family in Eucalyptus grandis and Analysis of Their Expression Pattern Under Abiotic Stresses
YU Jian-Feng, HONG Jia-Du, ZHAO Shuang, LI Xiao-Mei, WEN Dan-Dan, CHENG Long-Jun
2024 Vol. 32 (8): 1792-1808   DOI:10.3969/j.issn.1674-7968.2024.08.008 |  Full text (HTML) (1 KB)  |    PDF  (6778 KB)  ( 0 )
Abstract
Eucalyptus spp. are widely planted in south of China as a fast-growing timber species. However, the resistance to abiotic stresses such as low temperature, drought, and salinity of them is very poor. Zinc finger protein (ZPF) transcription factor members containing the ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motif have been shown to be extensively involved in plant responses to abiotic stresses such as cold, high temperature, drought and salinity. Based on the genome-wide identification of Eucalyptus grandis C2H2-type ZFP, the EgrZFP-EAR genes with EAR motif was screened. The evolutionary relationships of EgrZFP-EAR, the types, numbers and distributions of EAR motifs in their proteins, chromosome localization and gene replication of them, as well as gene and protein sequence structure and the cis-elements on their promoters were analyzed. In addition, transcriptome data and qRT-PCR were used to analyze the expression patterns of EgrZFP-EAR in different tissues and 4 ℃, high temperature (42 ℃), drought, NaCl (300 mmol/L), ABA (100 μmol/L) and MeJA (100 μmol/L) treatments of E. grandis seedlings. The results showed that there were 118 C2H2-type ZFP in E. grandis, 57 of which were EgrZFP-EAR members, and 54 of these members encoded ZFP proteins containing Q-type sequence (QALGGH). They contained 3 types of EAR motifs: LxLxL, DLNx (x) p, and their overlap. The LxLxL type had the largest number of them, with a total of 98. Evolutionarily, genome-wide replication, tandem duplication and segment duplication all played roles in the expansion of these genes. Stress responsive elements such as DRE, MBS, LTR and STRE, and hormone responsive elements including ABRE and CGTCA-motif were distributed on the EgrZFP-EAR promoters. Tissue expression patterns were divided into 4 types: high expression in shoot tips and young leaves; high expression in phloem; high expression in mature xylem; and high expression in phloem, immature and mature xylem. The expression patterns of EgrZFP-EAR gene in leaves of E. grandis seedlings treated with 4 ℃ for different time (2, 6, 12, 24, 48 h) were classified into 3 types. Genes in the first type were induced persistently; the expression of genes in the second type ascended firstly and then descended; and in the third type, genes' expression was mainly inhibited. The selected 12 genes from different EgrZFP-EAR subgroup were also responsive differently to high temperature, drought, NaCl, ABA and MeJA treatments, implicating the complex function of EgrZFP-EAR gene under abiotic stresses. The results provide an foundation for further revealing the molecular mechanism of stress resistance, the exploitation of stress-resistant gene resources and molecular-assisted stress resistance breeding of Eucalyptus.
Genome-wide Identification of ZF-HD Family and Its Expression Pattern Analysis in Magnolia sinostellata
ZHU Sen-Yu, CHEN Xiao-Jun, MA Jing-Jing, REN Ming-Jie, WANG Jing-Jing, WU Chao, TIAN Feng, LIU Zhi-Gao, FAN Min-Liang, SHEN Ya-Mei
2024 Vol. 32 (8): 1809-1820   DOI:10.3969/j.issn.1674-7968.2024.08.009 |  Full text (HTML) (1 KB)  |    PDF  (7413 KB)  ( 0 )
Abstract
Stress from canopy shade is currently endangering a very small population of the indigenous Magnolia sinostellata found in Zhejiang province. A group of transcription factors known as zinc finger homeodomain (ZF-HD) is crucial for the plant development as well as the response to biotic and abiotic stress. In order to gain a better understanding of the mechanism underlying the MsZF-HD gene family under low-light stress, this study aimed to identify the members of the ZF-HD gene family and investigate their expression patterns based on transcriptome data. Twelve members of the MsZF-HD family were found using transcriptome data from M. sinostellata leaves and genomic data from M. biondii. The 12 MsZF-HDs located in the cell nucleus, and MsZF-HD2b also located in chloroplast. The ZF-HD_dimer structural domain was present in all members of the MsZF-HD gene family, which was revealed by phylogenetic analysis to be separated into 4 subfamilies. Additionally, the number and types of motifs were substantially conserved among the members of the same subfamily. According to GO analysis, members of the MsZF-HD family played a significant role in the expansion and evolution of M. sinostellata. Tissue-specific expression study revealed that most MsZF-HDs genes were substantially expressed in M. sinostellata leaf buds and leaves. MsZF-HD8 was highly expressed in both stems and leaves, while MsZF-HD13 and MsZF-HD14 were highly expressed in both flowers and stems, indicating that they might be involved in stem elongation and the regulation of floral development. Additionally, it was discovered that MsZF-HDs all responded favorably to low-light stress after studying the expression pattern of the MsZF-HDs gene under shade stress. Under low-light stress, the expression of MsZF-HD1a, MsZF-HD2b, MsZF-HD1b, MsZF-HD1c, MsZF-HD2a and MsZF-HD14 decreased. MsZF-HD13 gene expression was significantly upregulated after 21 and 28 d of low-light treatment (P<0.05). MsZF-HD11, MsZF-HD12 and MsZF-HD8 showed significant upregulation of gene expression after 7 d of low-light treatment (P<0.05), followed by a downward trend. The ZF-HD family members of M. sinostellata were identified in this study and it was discovered that there was a significant expression response to low-light stress in leaf, stem and other tissues. MsZF-HD13 and MsZF-HD8 were found to be key genes in the response to shading to coordinate reproductive growth, stem elongation and leaf senescence. This study offers a theoretical framework for examining M. sinostellata's shade response mechanism.
Transcriptomic Analysis of Big and Small Testis and Screening Genes Related to Reprodution in Blackette Black Cattle (Bos taurus)
WANG Xiu-Yuan, LIU Rui-Li, HAN Ming-Xuan, ZHANG Wen-Qi, CHEN Zhen-Peng, MEI Yan-Fang, MIAO Xiu-Ping, XIAO Chao-Zhu, Zhang Ren-Zheng, DONG Ya-Juan, BAI Xue-Jin
2024 Vol. 32 (8): 1821-1835   DOI:10.3969/j.issn.1674-7968.2024.08.010 |  Full text (HTML) (1 KB)  |    PDF  (10622 KB)  ( 1 )
Abstract
Testis size is significantly related to semen quality and count in mammals, and male fertility can be predicted by testicular size in adulthood. In order to explore key candidate genes affecting bull fertility in differently testicular sizes, peripheral blood and testis of Blackette black cattle (Bos taurus) of 12 months old were collected and divided big testis and small testis groups according to testicular weight, long diameter and short diameter for hormone determination, histological study of testis and transcriptomic sequencing in this study. The differential expression multiplier value |log2 (Fold change)|≥1 and P-value≤0.01 were used as the conditions for selecting the differentially expressed genes (DEGs) and were subjected to Gene Ontology (GO) and KEGG analysis. Clone, qPCR, Western blot and immunohistochemistry (IHC) methods were used to detect and locate for candidate gene. The results showed that there were more testicular leydig cells and the development of spermatogenic tubules was better in big group. The concentrations of testosterone and estradiol of reproductive hormones in big testis were significantly higher than those in small testis group (P<0.05). A total of 1 006 DEGs were obtained from the transcriptome results, 297 genes were up-regulated and 709 genes were down-regulated in small testis. Functional analysis showed that the DEGs was enriched in cAMP and mitogen-activated protein kinase (MAPK) signaling pathways and played a role in reproduction and development; 9 reproduction-related candidate genes were screened, such as dual specificity phosphatase 4 (DUSP4), heat shock 70 kD protein 1-like (HSPA1L) and mitogen activated protein kinase kinase kinase kinase 4 (MAP4K4). Further study on MAP4K4 gene and protein showed that the expression of MAP4K4 in big testis was significantly lower than that in small testis (P<0.05). IHC found that MAP4K4 protein was highly expressed in sperm cells and spermatocytes and was mainly expressed in cytoplasm. Inhibition of MAP4K4 expression might promote spermatocyte proliferation and facilitate spermatoblast survival. This work provides reference for further understanding of the relationship between testis size and reproductive function in cattle.
The Effect of Resveratrol on Sperm Capacitation In vitro and Early Embryo Development in Bovine (Bos taurus)
LI Xiao-Xia, CAO Ping-Hua, WANG Dou-Dou, QU Pei-Dong, XU Zhi-Qian, YU Xue-Li, LI Ying-Hua
2024 Vol. 32 (8): 1836-1843   DOI:10.3969/j.issn.1674-7968.2024.08.011 |  Full text (HTML) (1 KB)  |    PDF  (1420 KB)  ( 2 )
Abstract
An excess production of reactive oxygen species (ROS) during sperm capacitation in vitro will decrease sperm quality, and affect early embryonic development. Resveratrol is an effective anti-oxidant to protect sperm. In this study, the effect of resveratrol was evaluated on sperm capacitation in vitro and early embryo development in bovine (Bos taurus). The frozen-thawed semen from Holstein bulls was used in sperm capacitation medium supplementing with 0, 10, 50 100 and 500 μmol/L resveratrol, subsequently incubated by swim-up method for 1 h. This study was to evaluate the effects of resveratrol on bovine sperm progressive motility, acrosome integrity, mitochondrial activity, plasma membrane integrity, capacitation status and the capacity of early embryo development after in vitro fertilization. These results showed that compared with the control group (0 μmol/mL), the supplementation of 50, 100 μmol/L resveratrol in sperm capacitation medium could significantly increase sperm progressive motility, the percentage of sperm with acrosome integrity, high mitochondrial membrane potential and plasma membrane integrity in bovine after in vitro capacitation (P<0.05); and it significantly increased the percentage of the B pattern sperm. Both of chlortetracycline (CTC)fluorescent staining and coomassie staining could be used to assess acrosome integrity of bovine sperm, and there was no significant difference between the 2 methods (P>0.05). The results of in vitro fertilization showed that compared with the control group (0 μmol/mL), the supplementation of resveratrol at 100 μmol/L in the capacitation medium resulted in a higher cleavage rate and blastocyst rate (P<0.05). This result showed that adding 100 μmol /mL resveratrol to capacitation medium could significantly improve sperm function of bovine after in vitro capacitation, increase its capacitation rate, so that the cleavage and blastocyst rate were significantly increased after in vitro fertilization. This study could help to optimize in vitro fertilization procedures and accelerate the efficiency of in vitro embryo production.
The Expression of MST1 and LATS1 in the Formation, Maintenance and Degeneration of Corpus Luteum in Yak (Bos grunniens) and the Effect of HCG on Its Expression
ZHOU Ying-Cong, ZHANG Tong-Xiang, WANG Jing-Lei, ZHAO Ling, ZHANG Rui, YAO Ying, QIAN Wen-Jie, CUI Yan, YU Si-Jiu, FAN Jiang-Feng
2024 Vol. 32 (8): 1844-1856   DOI:10.3969/j.issn.1674-7968.2024.08.012 |  Full text (HTML) (1 KB)  |    PDF  (13934 KB)  ( 0 )
Abstract
Mammalian STe20-like kinase 1 (MST1) and large tumor suppressor protein 1 (LATS1) are key members of the core kinase cascade of the Hippo pathway, which participate in the reproductive process of female animals and play an important role. This study aimed to explore the relationship between MST1, LATS1, and the formation, maintenance, and degeneration of yak (Bos grunniens) corpus luteum (CL). In this study, the corpus luteum of female yaks was collected and divided into 4 groups according to the different stages of corpus luteum formation (corpus rubrum stage, lacunose luteum stage), maintenance (pregnancy luteal phase) and degeneration (degenerative luteal phase). qPCR and Western blot were used to detect the differences of MST1, LATS1 mRNA and protein expression in the formation, maintenance, and degeneration of the corpus luteum. Immunohistochemistry (IHC) was used to analyze the localization of MST1 and LATS1 in the formation, maintenance, and degeneration of the corpus luteum. In addition, granulosa cells (GC) of the yak ovary were isolated and cultured, and luteinization of granulosa cells was induced by adding human chorionic gonadotropin (HCG). qPCR, Western blot and IF methods were used to analyze the quantity and localization of MST1, LATS1 mRNA and protein before and after luteinization. The results showed that MST1, LATS1 mRNA and protein were expressed in the yak corpus luteum, and there were differences in the expression of MST1 at different developmental stages of corpus luteum. The expression of MST1 was the highest in the pregnancy luteal phase, while the expression of LATS1 was the most abundant in the lacunose luteum stage, and the expression of MST1 and LATS1 was the lowest in the degenerative luteal phase. The results of IHC showed that MST1 and LATS1 were widely distributed in yak granular luteal cells, and MST1 was characterized by expression in vascular endothelial cells. After luteinization of granulosa cells induced by 5 IU/mL HCG, the expression of MST1, LATS1 mRNA and protein increased significantly. By IF staining, it was found that MST1 and LATS1 proteins were mainly located in the cytoplasm of granulosa cells, and the position did not change before and after luteinization. The results showed that MST1 and LATS1-mediated Hippo signaling pathway participates in the formation, maintenance, degeneration and functional operation of corpus luteum in female yaks by controlling granulosa cell differentiation, luteal cell proliferation and apoptosis, and which was closely related to various reproductive hormones in the physiological process of corpus luteum. This study provides basic data for further exploring the mechanism of the Hippo signal pathway regulating the size of the corpus luteum in yaks and regulating the reproductive physiological process of mammals.
Effects of Knockdown with JAK2 gene Expression on Proliferation and Apoptosis of Porcine Mammary Epithelial Cells
LUO Jie, YANG Suan, MAO Tong-Hui, YANG Qi, ZHANG Yi-Yu
2024 Vol. 32 (8): 1857-1868   DOI:10.3969/j.issn.1674-7968.2024.08.013 |  Full text (HTML) (1 KB)  |    PDF  (2756 KB)  ( 2 )
Abstract
Janus kinase 2 (JAK2), a hormone receptor-coupled kinase, is involved in the proliferation, differentiation and apoptosis of cow (Bos taurus) mammary epithelial cells. However, the regulation mechanism of JAK2 on the proliferation or apoptosis of porcine (Sus scrofa) mammary epithelial cells is still unclear. To study the effect of RNA interference with JAK2 gene expression on the proliferation and apoptosis of porcine mammary epithelial cells, in this study, 4 pairs of shRNA (short hairpin RNA) interference sequences and 1 pair of negative control sequences designed and synthesized according to the JAK2 gene sequence were transfected into porcine mammary epithelial cells, and the vectors with the best interference effect were screened. After successfully interfering with JAK2, the mammary epithelial cells were subjected to quantitative proteomics by using TMT (tandem mass tags) technology. Proteome sequencing and screening of differential proteins were performed, and the mRNA expression levels of cell proliferation, apoptosis, cycle and related genes were detected by CCK-8 cell proliferation detection kit, flow cytometry and qRT-PCR, respectively. The results showed that 699 differentially expressed proteins were screened, including 309 up-regulated proteins and 390 down-regulated proteins. GO enrichment analysis showed that the differential proteins were mainly enriched in the peptide biosynthesis process and amide biosynthesis process, as well as enriched in the molecular functions such as carbohydrate derivatives binding and the structural composition of ribosomes; KEGG enrichment analysis showed that the differential proteins were mainly enriched in the ribosomes, the complement system, and the hepatitis C signaling pathway. After interfering with JAK2, the cell proliferation ability was shown to be reduced at different time nodes compared with the control group (P<0.05), with extremely significant reduction in the OD450 value at 72 h (P<0.01), and the apoptosis rate (24.0%±3.82%) was significantly lower than that of the shNC group (33.2%±2.55%)(P<0.05), and the blockage of the cell cycle in the G1/S phase was induced. IFIT1 (interferon-induced protein with tetratricopeptide repeats 1)、ISG15 (interferon-stimulated gene 15)、GRB2 (growth factor receptor-bound protein 2) and RACK1 (receptor for activated C kinase 1) gene expression levels were extremely significant lower than those in the control group after silencing JAK2 (P<0.01), and RSAD2 (radical S-adenosyl methionine domain containing 2) gene was extremely significant higher than that in the control group after silencing JAK2 (P<0.01). In conclusion, silencing JAK2 induced G1/S phase block of porcine mammary epithelial cell cycle, which inhibited cell proliferation and apoptosis, and B6E241 protein (encoded by the gene GRB2) could be served as a key protein related to mammary epithelial cell proliferation for future studies. This study provides basic information for further study of the regulation mechanism of mammary gland development and lactation in sows.
Detection of Genetic Structure of Conservation Population in Wannanhua Pig (Sus scrofa domesticus) Based on SNPs Chip
WANG Jing-Lin, WU Xu-Dong, HE Zhi-Bing, LIU Yang-Guang, WANG Xiao-Fen, DING Yue-Yun, ZHENG Xian-Rui, ZHANG Xiao-Dong, YIN Zong-Jun
2024 Vol. 32 (8): 1869-1877   DOI:10.3969/j.issn.1674-7968.2024.08.014 |  Full text (HTML) (1 KB)  |    PDF  (2031 KB)  ( 2 )
Abstract
The Wannanhua pig (Sus scrofa domesticus) is an indispensable valuable pig breed resource in Anhui province and even in China. Studying the genetic diversity, kinship relationships, and pedigree structure of the Wannanhua pig population is crucial for revealing the current population status. The SNP of 32 Wannanhua pigs was detected using Chinese Chip-1 BeadChip. PLINK software was used to calculate the observed heterozygosity (Ho), expected heterozygosity (He) and polymorphic information content (PIC), and to analyse the runs of homozygosity (ROH) of the Wannanhua pig population; Constructing G-matrix using GCTA software to analyze the genetic relationship of Wannanhua pig population. A colony evolutionary tree was built using MEGA X software to analyze the family structure of the Wannanhua pig population. The findings indicated that 42 662 SNP loci were identified, with 21 317 of them passing the quality control assessment; The polymorphism marker ratio (PN) was 0.500; The effective population size (Ne) was 3, the observed heterozygosity (Ho) was 0.372, the expected heterozygosity (He) was 0.348. The average relationship coefficient was 0.11. The G matrix results all indicated that most of the Wannanhua pigs were moderately related. There were 884 ROH fragments in the results, the average ROH length of (12.35±2.17) Mb and the individual ROH length of (341.19±139.58) Mb; The mean inbreeding coefficient based on ROH was 0.139±0.056, indicating that the inbreeding degree of the conserved population was not severe; The results of the cluster analysis showed that the Wannanhua pigs were derived from 5 bloodlines. In summary, the population of the Wannanhua pig conservancy herd had a low effective content, medium genetic diversity, medium degree of inbreeding, and might contain a small amount of foreign blood. This study provides scientific basis for the conservation and utilization of genetic resources of the southern Wannanhua pig.
Screening, Identification and Biological Characteristics Research of Digestive Enzyme Secreting Probiotics from Gibel Carp (Carassius auratus gibelio) CASⅤ
YU Peng, MA Jing-Jing, CHENG Yu, ZHANG Cheng-Shuo, WANG Kai, ZHOU Zheng-Hai
2024 Vol. 32 (8): 1878-1889   DOI:10.3969/j.issn.1674-7968.2024.08.015 |  Full text (HTML) (1 KB)  |    PDF  (3588 KB)  ( 2 )
Abstract
The low feed digestibility and absorption rate is an important reason for high feed cost and low profit of fish farming. Gibel carp (Carassius auratus gibelio) is one of the main current crucian carp breeding in China, the aim of this study was to screen safe and efficient probiotics which can secret digestive enzymes from healthy culture system of gibel carp CASⅤ, so as to improve feed digestion and absorption capacity of CASⅤ and reduce feed cost. Ten strains with protease, lipase and amylase activities were isolated from CASⅤ and pond water by spot inoculation method. Two highly efficient, non-hemolytic, sensitive to most antibiotics and non-pathogenic strains, ZKW7 and ZKF17, were obtained by further analysis of enzyme secretion ability and safety test (hemolysis, antibiotics susceptibility and intraperitoneal infection). Based on 16S rDNA sequence analysis, both strains had the highest similarity to Bacillus subtilis and B. halotolerans. The results of biological characteristics analysis showed that both strains had strong degrading activities for NH4+, NO2- and NO3-. The optimum growth conditions of ZKW7 were pH 8, salinity 15 and Fe2+ in culture medium, respectively. By contrast, the optimum growth conditions of ZKF17 were pH 7, salinity 10~30 and the presence of Mg2+. In addition, these 2 strains were resistant to acid, bile salt, intestinal fluid and succus gastricus to some extent. In the present study, 2 strains of safe and efficient enzyme-producing Bacillus sp. were obtained, which could be used as potential probiotics and would be conducive to the healthy farming of C. auratus gibelio var. CASⅤ.
Construction of Plant Elicitor Peptide Engineering Strain and Its Control Effect on Cotton (Gossypium hirsutum) Verticillium Wilt
YIN Ming-Shen, WU Guo-Han, LEI Sheng-Wei, WANG Yu, NIU Qiu-Hong
2024 Vol. 32 (8): 1890-1898   DOI:10.3969/j.issn.1674-7968.2024.08.016 |  Full text (HTML) (1 KB)  |    PDF  (6208 KB)  ( 1 )
Abstract
Cotton verticillium wilt has always been an important obstacle to the sustainable development of China's cotton (Gossypium hirsutum) industry. It has been shown that plant elicitor peptide (Pep) can promote immune responses in multiple plant families against many kinds of pathogens. The purpose of this study was to construct an engineering strain of plant immune-inducing peptide for cotton verticillium wilt, and explore the antibacterial effect of protein-inducing peptide after activating plant immunity, then screen the engineering strain with good control effect on cotton verticillium wilt. Firstly, the plant immune-inducing protein genes PEL1 (pectate lyase 1) and PevD1 (protein elicitor 1) in Verticillium dahliae, a pathogenic bacterium of cotton wilt disease, were cloned, and the recombinant plasmids pHT43-PEL1 and pHT43-PevD1 were constructed. After being verified by enzyme digestion and sequencing, the plasmid was introduced into Bacillus sp. T6, which was screened in the early study, to construct engineering strain. The heterologous expression of the immune-inducing peptide was verified by SDS-PAGE, and the activity of the pot experiment was verified, so as to further confirm the engineering strain with efficient control of cotton verticillium wilt. The results of SDS-PAGE showed that the 2 inducible peptides were successfully overexpressed in T6 strain, and the engineering strain V3-T with PEL1 overexpression and the engineering strain P18-T with PevD1 overexpression were obtained respectively. The engineering strain P18-T was injected into the leaves of cotton plants for 15 min, and the active oxygen content increased significantly (P<0.01). In the cotton pot experiment, the engineering strain P18-T acted on the susceptible cotton plants for 30 d. Compared with the control wild strain T6 group, the incidence of cotton verticillium wilt in the P18-T treatment group decreased significantly (P<0.05), and the control effect reached to 78.85% (P<0.05). In this study, an engineering strain P18-T with high efficiency against cotton verticillium wilt was obtained, which provides basic data for broadening the comprehensive prevention and control technology of cotton verticillium wilt.
Study of Recombinant Pasteurella multocida Toxin-mediated FGFRL1-induced Proliferation in Hela Cells
LIN Zhong-Sen, ZHAO Qin, YUAN Jian-Lin, WU Rui, YAN Qi-Gui, WEN Yi-Ping, HUANG Xiao-Bo, DU Sen-Yan, CAO San-Jie
2024 Vol. 32 (8): 1899-1908   DOI:10.3969/j.issn.1674-7968.2024.08.017 |  Full text (HTML) (1 KB)  |    PDF  (8958 KB)  ( 0 )
Abstract
Fibroblast growth factor receptor-like 1 (FGFRL1), as a member of the fibroblast growth factor receptor family, plays a role in the regulation of cell proliferation and differentiation. Pasteurella multocida toxin (PMT) is an important virulence factor produced by P. multocida (Pm), which belongs to mitogenic protein, and causes cell proliferation on a variety of cells, leading to tissue proliferation, occupancy, and causing impair growth and death of other normal tissue cells. However, the mechanism and the factor that PMT induce a pro-proliferative effect, are currently unclear. Previous CRISPR/Cas9 whole gene library screen revealed that FGFRL1 increased the survival rate of recombinant PMT protein (rPMT) treated Hela cells. In order to investigate whether PMT affects the proliferation of Hela cells through FGFRL1 in the PI3K-AKT pathway, in the present study, normal and FGFRL1-knockout Hela cells were treated with rPMT at 0, 0.01, 0.1, and 1.0 μg/ml for 48 h. The cell proliferation rate was detected by Cell Counting Kit-8 (CCK-8) assay, cell DNA replication by EdU-488 labeling assay, and the cell migration rate by scratch assay, The results showed that cell proliferation in the FGFRL1 knockout Hela cell was significantly decreased (P<0.05) when treated with rPMT at a concentration of 0.1 μg/mL for 48 h, while the rate of scratch recovery was decreased by nearly 70% compared with that of normal Hela cell (P<0.05). It indicated that the absence of FGFRL1 diminished the pro-proliferative effect of rPMT on Hela cell. Initial validation of the pathway was performed using qPCR to detect changes in the transcriptional levels of factors upstream of the phosphatidylinositol 3 kinase/serine-threonine protein kinase (PI3K-AKT) pathway in Hela cells after rPMT treatment, and phosphorylation levels of AKT, a key activator of the pathway, were detected by western blot.. The results showed that the upstream factors of PI3K-AKT pathway were extremely significantly elevated after rPMT treatment (P<0.05), and the levels of AKT and phosphorylated AKT in the FGFRL1 knockout Hela cell were significantly lower than those in the normal Hela cells (P<0.01), which indicated that the activation of the PI3K-AKT pathway was inhibited. Thus, it could be preliminarily confirmed that PMT might cause Hela cell proliferation by targeting the PI3K-AKT pathway through FGFRL1. This study enriches the theoretical basis of the PMT's proliferation-promoting effect, preliminarily explores the pathway activation mode of PMT-induced cell proliferation, and provides information for further revealing the PMT's proliferation-promoting effect.
Reviews and Progress
Research Progress on Genetic Improvement and Biosafety of Crop Phosphorus Efficiency
XU Ji-Ming, LU Hong, WU Zhong-Chang, MAO Chuan-Zao
2024 Vol. 32 (8): 1909-1922   DOI:10.3969/j.issn.1674-7968.2024.08.018 |  Full text (HTML) (1 KB)  |    PDF  (1628 KB)  ( 12 )
Abstract
Phosphorus (P), one of the essential nutrients, plays an irreplaceable role in plant growth and development. Elucidating the physiological and molecular biological regulatory mechanisms of phosphorus absorption and utilization in plants is the basis for the genetic improvement on the efficiency of crop phosphorus absorption and utilization. Due to the similar chemical properties of arsenate and phosphate, the absorption of pentavalent arsenic may increase simultaneously with the improvement of crop phosphorus absorption. Therefore, during the process of genetic improvement on crop phosphorus absorption efficiency, attention should also be focused on the content of arsenic elements in crops, especially in edible parts. This article reviews the research progresses on plant phosphate absorption and transport, phosphate signal regulation mechanisms, as well as the molecular mechanisms of coupled absorption of phosphorus and arsenic. The current studies on the genetic improvement of crop phosphorus efficiency were also summarized. This review provides reference for the genetic improvement on crop phosphate efficiency and related biosafety assessment.
Research Progress on Regulation of Endoplasmic Reticulum-Lipid Droplet-Mitochondrial Interactions in Animal Lipid Metabolism
WANG Ming-Yu, PANG Wei-Jun, CAI Rui
2024 Vol. 32 (8): 1923-1938   DOI:10.3969/j.issn.1674-7968.2024.08.019 |  Full text (HTML) (1 KB)  |    PDF  (2239 KB)  ( 6 )
Abstract
Lipid droplet (LD), endoplasmic reticulum (ER) and mitochondria are key organelles in animal cells, and their interaction plays an important role in regulating animal lipid metabolism. ER interacts with LD to promote LD outgrowth, growth, and lipid transfer, so that LD can be efficiently stored and distributed to maintain cellular energy balance and lipid homeostasis. In addition, the interaction between LD and mitochondria promotes the efficient uptake of free fatty acids (FFAs) by mitochondria, which helps to maintain cellular energy supply. This review focuses on LD, and summarize the interactions between LD and mitochondria, ER, peroxisomes, lysosomes, and other organelles, as well as the advances of the regulation of in animal lipid metabolism. This review provides a new way of investigating lipid metabolism from the perspective of organelles, as well as a new direction for the analysis of the mechanism of formation of livestock and poultry meat traits and treatment of metabolic diseases in human (Homo sapiens).
Research Progress and Development Trend of Gene Editing Technology in Pig (Sus scrofa) Molecular Breeding
ZHAO Ya-Ya, YUAN Li-Ming, HUA Jin-Lian
2024 Vol. 32 (8): 1939-1948   DOI:10.3969/j.issn.1674-7968.2024.08.020 |  Full text (HTML) (1 KB)  |    PDF  (1035 KB)  ( 10 )
Abstract
Gene editing technology provides a new solution for pig (Sus scrofa) molecular breeding. Gene editing technology can achieve accurate and efficient gene editing, providing a powerful tool for breeding pig. Gene editing can not only be used to quickly and accurately improve growth rate and disease resistance, thereby enhancing the feeding efficiency of pigs, but also optimize the quality, taste and even nutritional content of pork to meet the growing consumer demand for high-quality, healthy pork. However, there are still many technical challenges in the application of gene editing technology, such as off-target effects. It is necessary to increase technological innovation to solve these problems restricting the efficiency of pig gene editing breeding, and promote the wide application of gene editing technology in pig disease resistance breeding. This paper summarized the latest development of gene editing technology in pig molecular breeding, including technical progress, application status and challenges. This review provides reference for breeding of disease resistance in China.
Resources and Updated Technology
Development and Preliminary Application of LAMP Assay for Rapid Detection of Tobacco leaf curl Yunnan virus (TbLCYnV)
ZHAO Zheng-Ting, GE Xiao-Tong, ZHANG Jun-Lei, XIA Zhen-Yuan, LIU Di, LIU Ya-Ting, JIANG Ning
2024 Vol. 32 (8): 1949-1958   DOI:10.3969/j.issn.1674-7968.2024.08.021 |  Full text (HTML) (1 KB)  |    PDF  (7881 KB)  ( 0 )
Abstract
Tobacco leaf curl Yunnan virus (TbLCYnV) is a new virus of the Begomovirus discovered on tobacco (Nicotiana tabacum) in Baoshan, Yunnan, China. It is one of the important pathogens of tobacco leaf curl disease in Yunnan. In order to quickly detect the TbLCYnV, five sets of primers were designed and screened according to the conserved nucleotide sequence of the coat protein (CP) gene of TbLCYnV, and the loop mediated isothermal amplification (LAMP) system reaction temperature, time, betaine concentration, dNTP concentration, Mg2+ concentration, and concentration ratio of internal and external primers were optimized by single variable method, At the same time, parallel comparisons with traditional PCR were conducted to verify the specificity and sensitivity of LAMP. The practicality of the LAMP system was verified using field collected samples, and the optimal LAMP detection system for TbLCYnV was established. The results showed that the optimal parameters were as follows: Primer group was TbL-5, the reaction temperature was 60 ℃, and the final concentrations of Betaine, dNTPs, and Mg2+ were 1.0, 0.4, 4.0 mmol/L, respectively; The optimal concentration ratio of internal and external primers was 4∶1, and the optimal reaction time was 60 min. The test results show that the optimized LAMP can be directly judged by naked eyes after staining with SYBR GreenⅠ, with high specificity and 10 000 times higher sensitivity than that of conventional PCR. This study provides a convenient, efficient, and reliable method for the detection of TbLCYnV, which is of great significance for the early and rapid diagnosis and prevention and control of TbLCYnV.
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