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2024 Vol. 32, No. 5  Published: 01 May 2024
Articles and Letters
Responses of SiPHYA and SiPHYB Gene to Light Quality, Photoperiod and Abiotic Stresses in Foxtail Millet (Setaria italica)
DU Xiao-Fen, SHENG Meng-Meng, WANG Zhi-Lan, HAN Kang-Ni, LI Yan-Fang, CHENG Kai, LI Yu-Xin, LIAN Shi-Chao, WANG Jun
2024, 32(5): 983-994  | doi: 10.3969/j.issn.1674-7968.2024.05.001 |  Full text (HTML) (1 KB)  | PDF   PDF  (5562 KB)  ( 41 )
Phytochromes are an red/far-red receptors that regulate seed germination, plant height, flowering,and shade avoidance response in plants. To explore the response patterns of photochrome genes (SiPHYA and SiPHYB) to light quality, photoperiod and abiotic stress in foxtail millet, The cDNA sequences of SiPHYA (Seita.9G113600) and SiPHYB (Seita.9G427800) were cloned by RT-PCR, and the results showed that the full length of SiPHYA coding region was 3 396 bp, encoding 1 131 amino acids, and SiPHYB was 3 507 bp, encoding 1 169 amino acids. Phylogenetic trees of multi-species were constructed by MEGA-X, which indicated that SiPHYA and SiPHYB had a closer evolutionary relationship to ZmPHYA and ZmPHYB, respectively. Transcription factors binding to the promoters of the photochrome genes were predicted via the online website PlantRegMap, the results showed that 109 transcription factor from 29 families could bind to the promoter region of SiPHYA, and 93 transcription factor from 27 families could bind to the promoter region of SiPHYB. Furthermore, the expression patterns in different tissues and organs, light quality treatments, photoperiod treatments and stress treatments were analysed using qRT-PCR. qRT-PCR results showed that SiPHYA and SiPHYB were expressed in roots, stems, leaves and young panicles, with the highest expression levels in the flag leaves. The response patterns of SiPHYA and SiPHYB were diverse under different light quality treatment between 'Henggu12' and 'Changnong35', of which, SiPHYA had the highest expression level under darkness in 'Henggu12', while SiPHYA had the highest expression level under far-red light in 'Changnong35', and SiPHYB had the highest expression level under white light, and lowest expression under red light in both 'Henggu12' and 'Changnong35'. Under long-day, SiPHYA showed similar circadian rhythm changes in both 'Henggu12' and 'Changnong35', while SiPHYB showed rhythmic changes in 'Changnong35', but no rhythmic changes in 'Henggu12'. Under short-day, SiPHYA and SiPHYB showed no circadian rhythm changes in both 'Henggu12' and 'Changnong35'. Both SiPHYA and SiPHYB were responsive to high salt, high temperature, low temperature and drought stresses, but the response patterns were diverse. This study provides clues for furher exploring the mechanisms of SiPHYA and SiPHYB in the development and abiotic stresses, and provides a basis for their application in the genetic improvement of foxtai millet.
Screening of Heat Tolerance Genes in Tomato (Solanum lycopersicum) Based on BSA-seq and Plant Hormone Combined Analysis
JIANG Yue-Chang, DUAN Jin-Hu, LI Yi-Feng, MAO Xiu-Jie, WANG Shuai, ZHANG Ning
2024, 32(5): 995-1007  | doi: 10.3969/j.issn.1674-7968.2024.05.002 |  Full text (HTML) (1 KB)  | PDF   PDF  (7007 KB)  ( 15 )
High temperatures affect the physiology and hormone metabolism of tomatoes (Solanum lycopersicum). Localization of genes involved in heat tolerance in tomato can provide theoretical and technical support for subsequent gene cloning and functional analysis. In this study, electrical conductivity was used as the identification index, and 2 tomato strains at seedling stage were identified by high temperature and heat resistance, and the F2 generation isolated population formed by heat-resistant strain CH and heat-sensitive strain DH and their F1 generation were used as materials to detect plant hormones in parents CH and DH. The F2 generation isolated population was resequenced by bulked sergeant analysis (BSA), and candidate genes related to tomato heat resistance were screened through the combined analysis. The results showed that 4 metabolic pathways related to abiotic stress were obtained by phytohormone differential metabolite screening and KEGG annotation, namely glycine, serine and threonine metabolic pathways, amino acid biosynthetic pathway, tryptophan metabolic pathway and aminoacyl-tRNA biosynthetic pathway; 6.32 Mb size candidate interval was obtained on chromosome 3 by BSA-seq sequencing. which included 752 candidate genes. Combined with the differential metabolic pathways, 5 candidate genes associated with heat tolerance in tomato were finally screened within the candidate regional metabolic pathways, namely: D-glycerate 3-kinase (DG3K), Hydroxypyruvate reductase (HYDR), prolyl-tRNA synthetase (PARS2) and ribulose phosphate 3-epimerase (RPE), aldehyde dehydrogenase (ALDH). The expression of the candidate genes was analysed by qPCR, the expression of candidate genes DG3K, PARS2, and RPE increased extremely significant after 3 hours of high temperature stress (P<0.01), it was hypothesized that these 3 candidate genes might play an important role in the regulation of heat tolerance in tomato. This study has important implications for molecular assisted selection of heat-resistant tomato lines.
Genome-wide Identification of Tomato (Solanum lycopersicum) β-BAM Gene Family and Its Expression Analysis Under Salt Stress and Exogenous Plant Growth Regulators
LI Xian-Guo, CHEN Zhao-Long, WANG Ze-Peng, DAI Qi, YU Qing-Hui, LI Ning
2024, 32(5): 1008-1019  | doi: 10.3969/j.issn.1674-7968.2024.05.003 |  Full text (HTML) (1 KB)  | PDF   PDF  (8339 KB)  ( 19 )
β-amylase (BAM) is widely involved in the regulation of various biological processes in plants and responds to various external stimuli such as hormone and abiotic stress. In this study, 8 SlBAM members in tomato (Solanum lycopersicum) were identified through bioinformatics analysis, and their physical and chemical properties, gene structure, phylogenetic evolution and collinearity were analyzed. The expression pattern of SlBAM gene under salt stress and fruit development was analyzed by qPCR. The results showed that SlBAM possessed typical Glyco_hydro_14 domain, and most of them were acidic and hydrophilic proteins. Phylogenetic analysis showed that SlBAM could be divided into 6 groups, and the same groups had similar gene structure and motif distribution. The composition of cis-acting elements indicated that the expression of most SlBAM may be related to plant hormones, abiotic stress and light response. The results of collinearity analysis showed that tomato had certain homology with Arabidopsis thaliana BAM gene family, and was more closely related to potato (Solanum tuberosum). qRT-PCR analysis showed that SlBAM were mainly expressed in leaves, but almost not expressed in roots and stems. Under salt stress, the expressions of SlBAM2, SlBAM4 and SlBAM6 were up-regulated (P<0.05), while the expressions of SlBAM1 and SlBAM3 were down-regulated (P<0.05). When the concentration of 2,4-epibrassinolide (EBR) solution was 0.1 mol/L, the expression of all genes in green ripening stage was significantly up-regulated (P<0.05). Under the influence of 1-naphthaleneacetic acid (NAA), all the genes except SlBAM6 were up-regulated and only SlBAM6 was down-regulated. These results provide a reference for further elucidations of the functions of the BAM gene family in tomato in response to abiotic stress and fruit development.
Identification and Expression Analysis of Cellulose Synthase/Cellulose Synthase-like (Cesa/Csl) Superfamily Genes in Grape (Vitis vinifera)
HU Ling-Ling, XU Tao, YANG Jie, GONG Li-Li, YANG Yu-Xin, YANG Zhong-Yi, WU Yue-Yan
2024, 32(5): 1020-1036  | doi: 10.3969/j.issn.1674-7968.2024.05.004 |  Full text (HTML) (1 KB)  | PDF   PDF  (8064 KB)  ( 24 )
Fruit firmness is one of the important indicators of the quality of fresh grapes (Vitis vinifera), which greatly affects the commercial and market value of grapes. The structure and composition of cell walls are important factors leading to differences in grape fruit hardness. The cellulose synthase/cellulose synthase like (CesA/Csl) superfamily is a key enzyme involved in the synthesis of cellulose and hemicellulose in plant cell walls, which has a potential impact on fruit hardness. However, the grape CesA/Csl superfamily has not been systematically identified. In this study, to investigate the expression patterns of the grape CesA/Csl superfamily in different hard and soft meat grape varieties, bioinformatics methods were used to identify the genes of the grape CesA/Csl superfamily at the whole genome level. A phylogenetic evolution tree was constructed and the physicochemical properties, gene structure, and variety expression characteristics of the genes were analyzed. The results showed that there were 67 members of the grape CesA/Csl family, which were unevenly distributed on 15 chromosomes; Phylogenetic tree analysis showed that grape CesA/Csl members were divided into 6 subgroups, which were relatively conservative in evolution; The proteins encoded by members of this family were mainly distributed on the plasma membrane, with an amino acid length of 128~1 406 aa; The transcriptome analysis results showed that there were significantly differentially expressed CesA/Csl genes in the mature fruits of hard and soft meat grapes, which might be a key gene leading to differences in fruit hardness. In addition, there were significant differences in the expression of VvCesA4 and VvCslB5 at different developmental stages of the same variety, which might regulate the hardness changes during fruit growth and development. Through qRT-PCR and correlation analysis of fruit hardness among different varieties, it was found that VvCesA4 may negatively regulate fruit hardness, while VvCesA2 and VvCesA6 might positively regulate fruit firmness. Based on the above results, it could be found that the CesA/Csl superfamily plays different regulatory roles in grape hardness differences. This study provide theoretical basis for further exploring the mechanism by which the CesA/Csl superfamily regulates grape fruit firmness.
Functional Analysis of OfMYB12 Gene Involved in Flavonoid Synthesis in Osmanthus fragrans
ZHANG Hui-Lian, WANG Yi-Guang, ZHONG Shi-Wei, DENG Jin-Ping, FANG Qiu, ZHAO Hong-Bo
2024, 32(5): 1037-1048  | doi: 10.3969/j.issn.1674-7968.2024.05.005 |  Full text (HTML) (1 KB)  | PDF   PDF  (12251 KB)  ( 13 )
MYB transcription factor is widely involved in plant growth and development, and plays an important role in regulating plant secondary metabolism. The OfMYB12 gene, which was related to flavonoid synthesis and screened by RNA-Seq technology in previous study of our research group, was further cloned from the flower buds of Osmanthus fragrans 'Yanhonggui'. The ORF length of OfMYB12 gene (GenBank No. WMD01176) was 1 026 bp and encoded 341 amino acids. The subcellular localization results indicated that this gene was located in the nucleus. Multiple sequence alignment and phylogenetic tree indicated that the OfMYB12 gene belonged to a typical R2R3 transcription factor and had the closest relationship with anthocyanin activator AtMYB56 in Arabidopsis thaliana. The analysis of tissue-specific results showed that OfMYB12 gene was expressed in different tissues of O. fragrans and had the highest expression level in petals. Heterologous overexpression of OfMYB12 in Petunia hybrida 'Mitchell' resulted in a significantly higher total flavonoid content in the transgenic petals compared to the control group (P<0.05), and the expression levels of related genes (PhCHS (P. hybrida chalcone synthase), PhF3H (P. hybrida flavanone 3-hydroxylase), PhFLS (P. hybrida flavonol synthase)) in the flavonoid synthesis pathway in transgenic petals were significantly increased. Based on the above results, it is speculated that the OfMYB12 gene might be involved in the regulation of flavonoid secondary metabolism biosynthesis. The results of this study provides a theoretical basis for exploring the molecular mechanism of flavonoid synthesis in Osmanthus and improving the quality of Osmanthus.
Study on Cross-compatibility Among the Three Subgenera of Rhododendron subgen. Tsutsusi, subgen. Azaleastrum and subgen. Pentanthera
GAO Jia-Jia, XIAO Zheng, DONG Bin, ZHAO Hong-Bo
2024, 32(5): 1049-1060  | doi: 10.3969/j.issn.1674-7968.2024.05.006 |  Full text (HTML) (1 KB)  | PDF   PDF  (21311 KB)  ( 16 )
Rhododendron resources are abundant in China and have a long history of cultivation. Hybridization breeding technology can greatly enrich Rhododendron resources. Rhododendron subgen. Tsutsusi, subgen. Azaleastrum and subgen. Pentanthera belong to the same evolutionary branch of hairy Rhododendron. In order to explore the compatibility of hybridization and the stages of hybridization barriers among 3 genus, Rhododendron simsii, R. pulchrum and 8 cultivars of subgen. Tsutsusi, R. ovatum of subgen. Azaleastrum, R. molle and 2 cultivars of subgen. Pentanthera were studied. The cross compatibility between subgenera was analyzed from the aspects of style length ratio, pollen germination, pollen tube growth, ovary and embryo development. The results showed that there was obvious prezygotic unidirectional discompatibility among subgen. Tsutsusi, subgen. Azaleastrum and subgen. Pentanthera. All pollen combinations could germinate in large quantities in stigma. The pollen tubes of subgen. Tsutsusi × subgen. Pentanthera, subgen. Azaleastrum × subgen. Pentanthera, and subgen. Azaleastrum × subgen. Tsutsusi reached the base of the style in large numbers and were able to set fruit. Reciprocal cross pollen tubes mostly stop growing in the middle of the style, a few reach the base of the style, and could not set fruit. The embryo development of subgen. Azaleastrum × subgen. Pentanthera and subgen. Azaleastrum × subgen. Tsutsusi were slower than that of subgen. Tsutsusi × subgen. Pentanthera, and the capsule dehiscent time was also late, most embryos of mature seeds in each hybridization direction were incomplete. The results of compatibility experiments showed that the cross barriers appeared in pollen tube growth, double fertilization and embryo development. The present study explored the law of cross compatibility and could provide reference for cross breeding of Rhododendron subgenera.
Cloning and Functional Analysis of CtWRI1 Gene from Safflower (Carthamus tinctorius)
LI Jia-Qi, REN Ying-Ying, LIU Sha, LIU Hong-Ye, TIAN Yin-Shuai
2024, 32(5): 1061-1070  | doi: 10.3969/j.issn.1674-7968.2024.05.007 |  Full text (HTML) (1 KB)  | PDF   PDF  (7057 KB)  ( 21 )
Safflower (Carthamus tinctorius) is an economic crop with the medicinal and oil values, and analyzing the molecular mechanism of safflower seed oil biosynthesis will be benefiting for the genetic breeding of safflower. WRINKLED1 (WRI1) gene plays an important regulatory role in plant lipid biosynthesis. In order to analyze the molecular mechanism of oil accumulation in safflower seeds, the CDS sequence (GenBank No. OP785157) of safflower CtWRI1 gene was cloned from 'Yuhonghua No. 3' combined with the early transcriptomic data and homologous sequence method, and its bioinformatics and expression patterns were analyzed. The function of CtWRI1 gene was identified through tobacco (Nicotiana tabacum) genetic transformation method. The results showed that the total CDS length of CtWRI1 was 1 128 bp, encoding 375 amino acids. The CtWRI1 protein was predicted containing 2 conserved AP2/ERF domains. CtWRI1 protein sequence was closely related to sunflower (Helianthus annuus) and lettuce (Lactuca sativa) by the evolutionary tree analysis. The expression level of CtWRI1 was high during seed development and reached the highest level in 10-days embryo. The analysis results of CtWRI1 transgenic tobacco showed that CtWRI1 could induce the expression of genes involved in fatty acid biosynthesis, and the oil content of transgenic tobacco seeds was significantly increased. This study lays the foundation for further study of the molecular mechanism of oil biosynthesis in safflower seeds.
Analysis of Copy Number Variation of PPP3CA Gene and Its Association with Growth Traits in Goats (Capra hircus)
BAI Yang-Yang, DU Shu-Zeng, LI Xin, ZHU Hai-Jing, QU Lei, GUO Zhen-Gang, YANG Qing, LAN Xian-Yong
2024, 32(5): 1071-1080  | doi: 10.3969/j.issn.1674-7968.2024.05.008 |  Full text (HTML) (1 KB)  | PDF   PDF  (2571 KB)  ( 17 )
Protein phosphatase 3 catalytic subunit alpha (PPP3CA) plays an important role in animal growth and development. This study aims to explore PPP3CA gene expression in different tissues and the genetic effects of copy number variation on the growth traits of different breeds of goats (Capra hircus). First, a total of 11 Shaanbei white cashmere goats were chosen to identify the expression levels of PPP3CA mRNA in the heart, liver, spleen, lung, kidney, muscle, brain, ovary, testis tissues. Then, copy number variation (CNV) of PPP3CA gene was detected in 306 Shaanbei white cashmere goats and 110 Guizhou Heima goats by qPCR. The associations between PPP3CA gene CNV loci and growth traits in goats were analyzed using ANOVA. In addition, the linkage disequilibrium block in these regions and the potential transcription factors that could bind to the 3 CNVs were analyzed by using bioinformatics. The results showed that PPP3CA mRNA was highly expressed in the lung, followed by the spleen, fallopian tube, testis, heart, liver, kidney, muscle and brain. Three CNVs were detected in PPP3CA gene. In the Shaanbei white cashmere goats population, CNV1 and CNV2 significantly correlated with goat chest circumference (P<0.05); CNV3 was significantly correlated with goat chest width, chest depth and chest circumference (P<0.05). In the Guizhou Heima goat population, there was no significant relationship between 3 CNVs and goat growth traits. The linkage disequilibrium (LD) haplotype block analysis showed that there was no LD block for PPP3CA gene, but these CNVs might bind to SPT23, SIP4, and YOX1 transcription factors. In summary, these findings indicated that the CNV1, CNV2, and CNV3 loci of the PPP3CA gene could be used as molecular markers for the selection of growth traits in Shaanbei white cashmere goats.
Identification and Analysis of Interacting Proteins Regulating IFNβ Expression in PPARγ Activated and Inhibited States
MA Zhuo-Yuan, YANG Bin, CHEN Yun-Long, FAN Gang, WU Xiao-Long, WANG Xi-Yin, HUA Jin-Lian, WANG Yan, ZHANG Yong-Qiang, ZHANG Shi-Qiang
2024, 32(5): 1081-1092  | doi: 10.3969/j.issn.1674-7968.2024.05.009 |  Full text (HTML) (1 KB)  | PDF   PDF  (7486 KB)  ( 20 )
Peroxisome proliferator activated receptor γ (PPARγ) is specifically overexpressed in alveolar macrophages and has metabolic and immunoregulatory roles. It is not clear which interacting proteins of PPARγ are involved in the regulation of typeⅠinterferon production induced by innate immunity. In this study, wild boar (Sus scrofa) lung cell line (WSL) with stable overexpression of PPARγ-3×Flag was constructed using lentiviral vector system. Taking this cell strain as a model, fluorescence quantitative PCR assay revealed that the addition of the PPARγ agonist rosiglitazone significantly reduced the level of interferon β (IFNβ) expression induced by stimulating with the dsDNA mimic Poly (dA:dT); In contrast, addition of the PPARγ inhibitor T0070907 significantly increased the level of IFNβ expression induced by Poly (dA:dT) stimulation. Further, the PPARγ-3×Flag protein complex regulating the above process was precipitated by immunomagnetic beads of anti-FLAG protein. The PPARγ-3×Flag protein complexes enriched in cytoplasmic proteins and nuclear proteins were digested into peptides and analyzed by liquid chromatography and tandem mass spectrometry (LC-MS/MS), respectively. The results showed that there were 101 cytoplasmic proteins and 95 nucleoproteins interacting specifically with PPARγ when the PPARγ agonist rosiglitazone was added. Under the condition of adding PPARγ inhibitor T0070907, there were 24 cytoplasmic proteins and 14 nuclear proteins that specifically interacted with PPARγ. GO and KEGG analysis showed that specific PPARγ interactions of cytoplasmic proteins and nuclear proteins were related to translation regulation. This study provides a new clue to further study the mechanism of regulating of IFNβ expression by PPARγ.
Analysis of Genetic Diversity and Population Structure Changes of Dapulian pigs (Sus scrofa) Based on Genomic Information
ZHANG Xin, ZHANG Ting, WANG Huai-Zhong, YANG Qing, GAO Shi-Chao, WANG Yan-Ping, WANG Ji-Ying
2024, 32(5): 1093-1101  | doi: 10.3969/j.issn.1674-7968.2024.05.010 |  Full text (HTML) (1 KB)  | PDF   PDF  (3605 KB)  ( 12 )
Due to pedigree errors, unbalanced breeding and hybrid individuals introgression, population coefficient of inbreeding may increase too fast and the genetic relationship between individuals may be unclear in the process of local pig (Sus scrofa) conservation. In order to analyze the genetic diversity and population structure of the Dapulian conserved population, and monitor the genomic information changes of the conserved population during the 5 years from 2017 to 2022, in this study, 110 Dapulian pigs were genotyped using high-density SNP array, and the genetic diversity, inbreeding coefficient, phylogenetic tree and the relationship with the imported pigs of Dapulian pigs of 2017 and 2022 were comparatively analyzed based on genome-wide SNP markers. The results showed that: 1) Compared with those of 2017, the samples of 2022 had higher observe heterozygosity (HO) (0.356 vs. 0.292), expected heterozygosity (HE) (0.342 vs. 0.285) and genetic distance (0.270 vs. 0.233), smaller identity by descent (IBD) proportion (0.108 vs. 0.127) and genomic inbreeding coefficients (FROH) based on run of homozygosity (ROH) (0.108 vs. 0.163), similar phylogenetic tree but increased balance between families, indicating the samples of 2022 had higher genetic diversity and improved population structure than those of 2017. 2) The samples of 2022 had higher HO and HE ratio (1.041) than those of 2017 (1.025), and the sample of 2022 were more influenced by the imported pig breeds than 2017 as shown by principal component analysis (PCA) among Dapulian pigs and 5 imported breeds. These results demonstrated that Dapulian pigs had been impacted by other pig breeds, and the impact on the conserved population of 2022 was greater than on that of 2017. This study analyzed the change trend of Dapulian pigs population. It provides scientific reference basis for further conservation and utilization of Dapulian pigs.
Cloning of Gonadotropin-inhibitory Hormone Receptor Gene NPPFR1 in Rabbit (Oryctolagus cuniculus) and Its Expression Analysis in HPT Axis
SANG Lei, GAO Cheng-Fang, SUN Shi-Kun, WANG Jin-Xiang, CHEN Dong-Jin, XIE Xi-Ping
2024, 32(5): 1102-1110  | doi: 10.3969/j.issn.1674-7968.2024.05.011 |  Full text (HTML) (1 KB)  | PDF   PDF  (2304 KB)  ( 7 )
Neuropeptide FF receptor 1 (NPFFR1) is a primary receptor to the gonadotropin-inhibitory hormone (GnIH), which plays an obvious effect on the controls of animal reproductive physiology. To investigate the function of NPFFR1, the NPFFR1 gene (GeneBank No. OP791886) was cloned from the Minxinan Black rabbit (Oryctolagus cuniculus) by rapid amplification of cDNA ends (RACE) method, and the bioinformatics and spatiotemporal expression profiles were analyzed. Moreover, the male rabbits were intraperitoneally injected with 0, 0.5, 5, or 50 μg GnIH-related peptides, and the NPFFR1 mRNA level in the hypothalamic-pituitary-testicular (HPT) axis was detected by qPCR. The results of bioinformatics analysis showed that the full CDS sequence of rabbit NPFFR1 gene was 3 712 bp, encoded 432 amino acids, its protein was a 7-transmembrane polypeptide distributed in the plasma membrane, which was composed of α-helix, random curl, and β-fold, and highly homologous with Ochotona curzoniae and Ochotona princeps. qPCR analysis showed that NPFFR1 expressed in all tissues of the HPT axis, and the expression level in the hypothalamus was significantly higher than that in the pituitary gland and testis (P<0.05). After being born, the hypothalamic mRNA levels of NPFFR1 gene in 90-day-old rabbits were lower than those in 11-, 30-, 60- or 150-day-old rabbits (P<0.05), the pituitary mRNA level of NPFFR1 gene in 30- or 120-day-old rabbits were lower than those in 11- or 90-day-old rabbits (P<0.05), and the testicular mRNA levels of NPFFR1 gene in 30-day-old rabbits were lower than those in 11- or 90-day-old rabbits (P<0.05). The hypothalamic mRNA levels of NPFFR1 increased in a dose-dependent manner after the rabbits administered with GnIH using a dose from 5 to 50 μg, and the testicular NPPFR1 expression levels in the 50 μg GnIH-injected group were higher than those in the 5 μg group (P<0.05); there were no significant differences in the mRNA levels of pituitary NPFFR1 among the 4 dose-groups. This study provides a theoretical support and reference in research on the function of rabbit NPFFR1.
LncRNA-TUG1 Affects 22Rv1 Cell Proliferation and Migration by Modulating miR-31-5p/YWHAE Axis
ZHAO Jia-Fu, LI Yong, ZHOU Ming-Shuai, WEN Xiao-Yan, LU Qing-Mei, LIU Bin
2024, 32(5): 1111-1120  | doi: 10.3969/j.issn.1674-7968.2024.05.012 |  Full text (HTML) (1 KB)  | PDF   PDF  (4483 KB)  ( 6 )
Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activating protein ε (YWHAE) was highly expressed in prostate cancer, and miR-31-5p could target and regulate the expression of YWHAE to affect the proliferation of prostate cancer cells. However, it was not clear which lncRNAs were involved in the targeted regulation of miR-31-5p against YWHAE. In this study, 3 LncRNAs with long non-coding RNA tauronic upregulation gene (LncRNA-TUG1), LINC02604 and PDCD6IP-DT were screened to target and regulate the expression of miR-31-5p based on the results of previous studies and bioanalysis software. The expression of 3 LncRNAs in prostate cancer LNCaP, PC3, 22Rv1 and normal prostate stromal immortem cells (WPMY-1) was detected by RT-qPCR, and the optimal LncRNAs regulating the miR-31-5p/YWHAE axis were screened. The effects of interfering LncRNA-TUG1 and overexpression of miR-31-5p on the expression of LncRNA-TUG1, miR-31-5p and YWHAE in 22Rv1 cells were detected by RT-qPCR. Meanwhile, the effects of co-transfection of sh-TUG1 and miR-31-5p mimics on the proliferation and migration of 22Rv1 cells were detected by CCK-8 and cell scratch assay. Western blot was used to detect the expression of proliferating cell nuclear antigen (PCNA), B cell lymphoma-2 (BCL-2), Bcl-2 associated X protein (BAX) and cysteine proteinase-3 (Caspase-3). The results showed that LncRNA-TUG1, LINC02604 and PDCD6IP-DT were the 3 LncRNAs regulating the miR-31-5p/YWHAE axis, and compared with normal cells, LncRNA-TUG1 was highly expressed in all PCa cell lines, especially in 22Rv1 cells. Taking LncRNA-TUG1 as the research object, after transfecting sh-TUG1 and miR-31-5p mimics into 22Rv1 cells, respectively, qPCR results showed that the expression of miR-31-5p in the miR-31-5p mimics group was significantly up-regulated compared with the NC mimics group (P<0.01). The expression of LncRNA-TUG1 and YWHAE genes was significantly down-regulated (P<0.05). Compared with the sh-NC group, there was no significant change in the expression of miR-31-5p in the sh-TUG1 transfection group, and the expression of LncRNA-TUG1 and YWHAE were significantly down-regulated (P<0.05). CCK-8 and cell scratch experiments showed that co-transfection of sh-TUG1 and miR-31-5p mimics could significantly inhibit cell proliferation and migration. In addition, Western blot results showed that co-transfection of sh-TUG1 and miR-31-5p mimics could significantly inhibit the expression of PCNA and BCL-2/BAX protein, and promoted the expression of caspase-3 protein.These results indicated that LncRNA-TUG1 could regulate the expression of PCNA, BCL-2, BAX and caspase-3 proteins by regulating miR-31-5p/YWHAE, and further affected the proliferation of 22Rv1 cells. This study provides theoretical basis and experimental ideas for the study of targeted drugs for prostate cancer.
Identification and Expression Analysis of Siderophore Synthetic Gene Cluster of Setosphaeria turcica
YUE Hao-Feng, ZHAO Tian-Yi, LI Hai-Xiao, YAN Qiu-Jing, XU Ya-Di, MENG Ya-Nan, CAO Zhi-Yan, LIU Ning, DONG Jin-Gao
2024, 32(5): 1121-1131  | doi: 10.3969/j.issn.1674-7968.2024.05.013 |  Full text (HTML) (1 KB)  | PDF   PDF  (5342 KB)  ( 15 )
Iron is an essential element for microbial growth and plays a crucial role in processes such as oxygen metabolism, electron transfer, DNA and RNA synthesis. Microbes primarily rely on siderophores to absorb iron ions from their environment. Siderophores, as low molecular weight iron chelators secreted by microbes, assist in transporting iron ions within the organism. Fungi primarily use non-ribosomal peptide synthetase-encoded genes as the core of secondary metabolic biosynthetic gene clusters to synthesize siderophores . This study aimed to clarify the siderophore function and biosynthetic process of Setosphaeria turcica. The type of siderophores synthesized by S. turcica was identified using CAS (chrome azurol sulphonate), and its biosynthetic gene cluster was predicted using the AntiSMASH website. Transcriptional data from different developmental stages of S. turcica spores and qRT-PCR analysis of the expression levels of key genes in the siderophore biosynthetic gene cluster during pathogen infection were utilized. The results showed that S. turcica produces hydroxamate-type siderophores. One biosynthetic gene cluster responsible for hydroxamate-type siderophore production was identified in the genome. This cluster consisted of 12 genes with the core gene StNPS6 encoding non-ribosomal peptide synthetase. During spore germination and infection process of S. turcica, several genes in the cluster exhibited significant upregulation. The core gene StNPS6 had an expression level 5.52 times higher at 6 d post-infection compared to the early infection stage, and the auxiliary biosynthesis gene StrhbE showed a 24.12-fold increase in expression at 9 d post-infection. Additionally, the transport-related gene Stpmd1 increased significantly by 19.95-fold at 9 d post-infection. At the same time, it was observed that both iron-rich and iron-deficient conditions had a certain inhibitory effect on the growth of the pathogen. Moreover, under iron-rich conditions, melanin synthesis in the pathogen was hindered. The differences in iron content in the medium led to significant changes in the expression levels of multiple genes within the gene cluster, especially the transport-related protein gene Stpmd1, which showed a 73.28-fold increase in expression under iron-rich conditions. This study provides a foundation for understanding the potential role of siderophores in S.turcica.
Cloning and Functional Analysis of Transcription Factor Gene StbHLH10 in Setosphaeria turcica
HAN Dan, ZHANG Xin-Yu, WANG Xiao-Min, XIAO Yi-Ming, PEI Zhi-Yong, Gu Shou-Qin, LIU Yu-Wei, GONG Xiao-Dong
2024, 32(5): 1132-1140  | doi: 10.3969/j.issn.1674-7968.2024.05.014 |  Full text (HTML) (1 KB)  | PDF   PDF  (3159 KB)  ( 13 )
As a conserved transcription factor family, basic Helix-Loop-Helix (bHLH) is widely involved in the development and infection of plant pathogenic fungi. StbHLH10 is a member of bHLH transcription factor family in Setosphaeria turcica. In order to understand the function of this gene, the full-length CDS of StbHLH10 (GenBank No. XP_008030228.1) was cloned from S. turcica, and the structural characteristics of its protein sequence were analyzed. The results showed that the CDS of the StbHLH10 was 1 659 bp in length, encoding 552 amino acids. The protein sequence of StbHLH10 contained a HLH conserved domain at residues 335~441 aa, which formed a spatial structure of protein by 2 α-helices and a loop ring. The expression pattern analysis showed that the expression level of StbHLH10 gene increased with the prolongation of the time that the pathogen infected maize leaves. By constructing its regulatory network, it was found that the StHLH10 may be involved in the development and infection of S. turcica by regulating the expression of 36 downstream target genes. Prokaryotic expression analysis showed that the StbHLH10-His fusion protein was highly expressed in Escherichia coli at 28 ℃ for 6 h, and the molecular weight was 78.5 kD, which was consistent with the theoretical value. The soluble purified protein of StbHLH10 was obtained by nickel column affinity chromatography. This study preliminarily explores the function of StbHLH10 transcription factor, and provides reference for further research on the mechanism of StbHLH10.
Analysis of Mating Type Composition and Distribution of Setosphaeria turcica in Northern Maize Producting Areas of China
WANG Chao-Hong, ZHANG Lu, GUO Zheng-Yu, YAN Xiao-Guang, LIU Xin-Fang, LIU Ning, CAO Zhi-Yan, DONG Jin-Gao
2024, 32(5): 1141-1149  | doi: 10.3969/j.issn.1674-7968.2024.05.015 |  Full text (HTML) (1 KB)  | PDF   PDF  (1512 KB)  ( 8 )
Setosphaeria turcica can harm the leaves, leaf sheaths and bracts of maize (Zea mays), resulting in the decrease of maize yield and quality, thus causing serious economic losses. Northern corn leaf blight has become one of the most important fungal diseases on maize in China. There were 3 mating types (MAT) in S. turcica, namely A mating type containing only StMAT1-1 gene, a mating type containing only StMAT1-2 gene, and Aa mating type containing both genes simultaneously. In China, there were significant differences in the composition and distribution of mating types of S. turcica in China between years and regions, leading to frequent genetic variations of the pathogens. To clarify the composition and distribution of mating types of the pathogen in different years of maize production areas in northern China, in this study, mating type identification specific primers of S. turcica were used to determine the mating type of 365 isolates collected from 10 provinces (autonomous regions) of Shaanxi, Shanxi, Henan, Hebei, Gansu, Heilongjiang, Ningxia, Inner Mongolia, Liaoning and Jilin in China from 2018 to 2022. The results showed that a total of 61 isolates of type A, 224 isolates of type a, and 80 isolates of type Aa were detected from 365 isolates of S. turcica, with isolation frequencies of 16.71%, 61.37%, and 21.92%, respectively, indicating that a mating type was the main mating type in North China. There were little differences in the frequency of mating types in S. turcica populations between different years. The isolation frequencies of mating type a in 2018, 2019, 2020, 2021, and 2022 was 61.73%, 72.50%, 55.74%, 58.65%, and 63.29%, respectively. There were differences in the composition of mating types of S. turcica in different regions. No A mating type isolate was isolated in Heilongjiang Province, while the distribution of A, a, and Aa mating types, were found in the remaining 9 provinces (autonomous regions). However, there were differences in the distribution frequency of different mating type isolates among regions. The frequency of a mating type isolates was the highest in Heilongjiang Province, at 87.50%, and the frequency of A mating type isolates was the highest in Shaanxi Province, at 26.67%, the frequency of Aa mating type isolates was the highest in Inner Mongolia and Jilin provinces, both at 33.33%, indicating that there was diversity in mating types among S. turcica populations. This study further clarified the composition and distribution of mating types of S. turcica in northern China, which provides reference for the analysis of pathogen variation trends and effective control.
Reviews and Progress
Single-cell RNA Sequencing Technology and Its Application in Animal Bone Research
LIU Zhe, TIAN Xue-Kai, HE Yu-Lin, YANG Pei-Yu, YU Tai-Yong
2024, 32(5): 1150-1168  | doi: 10.3969/j.issn.1674-7968.2024.05.016 |  Full text (HTML) (1 KB)  | PDF   PDF  (1671 KB)  ( 66 )
The skeleton is an important part of the animal body, playing a vital role in providing biomechanical support, maintaining movement, hematopoiesis, and calcium homeostasis. The application of bulk RNA sequencing (bulk RNA-seq) technology in skeletal research has provided important references for revealing the development of bone cells, but it still has limitations in revealing the heterogeneity of bone cells. In recent years, the emerging single-cell RNA sequencing (scRNA-seq) technology has greatly improved the efficiency of cell capture and survival rate, allowing researchers to interpret biological information from the perspective of individual cells in a population of cells, providing a new research strategy for studying the heterogeneity of bone cells and the relationship between cell populations. This article reviewed the progress of single-cell RNA sequencing technology and its application in animal bone research, with the aim of providing reference for future studies.
Research Progress on the Effect of Endoplasmic Reticulum Stress on Skeletal Muscle Cells
LIU Ying, SHI Jian-Kai, GUO Xiao-Rong, LIN Xiao, ZHOU Rong
2024, 32(5): 1169-1177  | doi: 10.3969/j.issn.1674-7968.2024.05.017 |  Full text (HTML) (1 KB)  | PDF   PDF  (2549 KB)  ( 23 )
Skeletal muscle has very important roles in both human body and livestock, related to physical function and meat production of livestock. External stimulation or the internal environment changes could make the endoplasmic reticulum (ER) to start a series of protective mechanisms, that is, ER stress. ER stress response has a two-way effect. Proper ER stress can alleviate the cell's inadaptability to living conditions through autophagy, unfolded protein reaction and other behaviors, but the continuous stress of ER caused by adverse factors will affect the normal function of ER and lead to apoptosis. This paper mainly reviewed the researches about the effects of ER stress on lipid metabolism, autophagy, apoptosis, differentiation and other physiological processes and mechanisms of skeletal muscle cells, aiming to provide a theoretical basis for studying the effect of ER stress on skeletal muscle cells.
Molecular Mechanism of Synonymous Codons Usage Patterns Control Ribosome Scanning Rhythms
GAO Ming-Yang, WU Yu-Hu, YANG Xuan-Ye, WANG Jin-Qian, HU Xin-Yan, ZHOU Jian-Hua
2024, 32(5): 1178-1187  | doi: 10.3969/j.issn.1674-7968.2024.05.018 |  Full text (HTML) (1 KB)  | PDF   PDF  (2420 KB)  ( 17 )
In translation process, the correct folding of the native polypeptide chain is regarded as the cornerstone for the biological activity of proteins. However, the correct spatial conformation of the native polypeptide chain is generally performed by the co-translation folding mechanism. The biogenetic effects of synonymous codon usage patterns on translation play important roles in ribosomal scanning mRNA. More and more studies have shown that although synonymous codon usage variations fail to impair amino acid sequence of protein, the preference of synonymous usage pattern perform a profound effect on the folding dynamic for the native polypeptide chain, especially on the regulation of the ribosomal scanning speeds. The purpose of this review is to explain the important role of synonymous codon usage patterns in the translation process and the mechanism of their influence on translation dynamics, and to identify their important role in the correct folding of proteins into their natural conformations. Synonymous codons are also associated with many diseases, this paper provides ideas for the research of protein synthesis and diseases caused by dysrhythmicity of ribosomes.
Resources and Updated Technology
Construction of Fluorescently Labeled Strains of Nucleus and Mitochondria for Ustilago esculenta
TANG Jin-Tian, YANG Fu-Rong, ZHANG Lei-Lei, LI Yu-Kang, ZHANG Ya-Fen, FU Hui-Lan, XIA Wen-Qiang, CUI Hai-Feng, YE Zi-Hong
2024, 32(5): 1188-1197  | doi: 10.3969/j.issn.1674-7968.2024.05.019 |  Full text (HTML) (1 KB)  | PDF   PDF  (4406 KB)  ( 15 )
Stem swollen of Zizania latifolia was associated with the successful infection of Ustilago esculenta, which depended on the dimorphic transition of U. esculenta. The dynamic changes of the nucleus and mitochondria during the process of dimorphic transition remain unknown. This study will focus on the coding gene for histone H1 and the coding sequence for the mitochondrial targeting signal (MTS) in the nucleus, respectively. After connecting them with the GFP and mCherry fluorescent protein genes, respectively, fluorescent labeling vectors for the nucleus and mitochondria was constructed. Wild-type strains UeT14 and UeT55 of U. esculenta as experimental materials to genetic transformation by Agrobacterium tumefaciens-mediated transformation (ATMT), and the recombinant strains that stably expressed red and green fluorescent proteins in nucleus or mitochondria were obtained: UeT55-H1-GFP, UeT14-H1-mCherry, UeT55-MTS-GFP and UeT14-MTS-mCherry. In UeT55-H1-GFP and UeT14-H1-mCherry strains, bright and round fluorescent spots could be seen in the center of the spores, which were co-located with the blue fluorescence formed by DAPI. In UeT55-MTS-GFP and UeT14-MTS-mCherry strains, small dot green or red fluorescence could be seen in the spores, and the green fluorescence was co-located with the red fluorescence formed by MitoTracker. In this study, the stable expression of fluorescent protein in nucleus or mitochondria was obtained, which provides an important reference and material for studying the molecular mechanism of growth, development, fusion and infection of U. esculenta.
Establishment and Application of SYBR GreenⅠReal-time Fluorescent Quantitative PCR Method for Penicillium sclerotiorum
SONG Jia-Yi, ZHANG Hai-Xia, QI Shi, ZHANG Ji-Li, CHEN Dong-Hao, WEI Jian-Yu, LI Ping, CHEN Ze-Peng, YAN Jian
2024, 32(5): 1198-1205  | doi: 10.3969/j.issn.1674-7968.2024.05.020 |  Full text (HTML) (1 KB)  | PDF   PDF  (5083 KB)  ( 19 )
Penicillium sclerotiorum is an endophytic fungus in the genus Penicillium with high biological activity that produces secondary metabolites with certain biological control potentials and has been widely used in medicine, agriculture, and other fields. This study utilized P. sclerotiorum obtained from Portulaca oleracea, and developed primers for its specific conserved sequence. The target segment was inserted into the pBM73-T vector by molecular cloning to create a recombinant plasmid as a positive control. A real-time PCR method for P. sclerotiorum was developed using the SYBR GreenⅠdye. PCR amplification revealed that only P. sclerotiorum exhibited bands, and there were no melting curves or amplification curves for P. chrysogenum and P. polonicum. The findings demonstrated a good linear relationship between the cycling threshold (Ct) and the template concentration between 2.35×103 and 2.35×1010 copies/μL in the standard curve created using recombinant plasmid as the standard. The obtained standard curve was y=-3.211x+39.42 with a correlation coefficient R2=0.999 3. The findings from the soil detection showed that the method's ability to accurately count endophytic P. sclerotiorum. The method established in this study had good specificity, reproducibility and practicability, which could be used to investigate the colonization of P. sclerotiorum in plants and soil and quantify the number in different tissues of plants. This study provides some data and theoretical support for the study of the promotion and resistance mechanism of P. sclerotiorum.
Therapeutic Effect of Recombinant Attenuated Listeria monocytogenes Expressing E7 Protein in a Mouse (Mus musculus) Model of Cervical Cancer
PAN Xiao-Xin, CUI Ming-Zhu, LUO Ya-Ru, SUN Jing, CHENG Chang-Yong, WANG Jing, SONG Hou-Hui
2024, 32(5): 1206-1215  | doi: 10.3969/j.issn.1674-7968.2024.05.021 |  Full text (HTML) (1 KB)  | PDF   PDF  (4005 KB)  ( 7 )
Listeria monocytogenes is a high-quality vaccine candidate live vector that can migrate between cells and stimulate the body to promote the proliferation of antigen-specific CD4+ and CD8+ T cells to produce adaptive cellular immunity. This research group found that attenuated Listeria monocytogenes (LAM) had significantly lower hemolytic and proliferative ability than wild-type Listeria. At the same time, LAM could normally secrete Listeriolysin O (LLO) protein. In order to study the safety of LAM and its delivery and immune effect on the cervical cancer specific antigen E7 protein, this study tested the virulence of LAM in a mouse (Mus musculus) model, and to evaluate the role of attenuated Listeria monocytogenes-E7 (LAM-E7) in the immunotherapy and prevention of cervical cancer in mice. The results showed that the virulence of LAM to mice was 1/8709 of that of wild-type Listeria, and LAM could be eliminated from the organs of mice within a week. In this study, E7 gene was integrated into LAM genome by homologous recombination method. PCR identification results showed that LAM-E7 strain was successfully constructed, and Western blot results showed that E7 protein could be fused with LLO and expressed and released in the recombinant strain. The analysis of the growth ability and hemolytic activity of LAM-E7 in vitro showed that the fusion had no significant effect on the growth ability and hemolytic ability of LAM, indicating that the fusion of E7 protein would not affect the safety of LAM. In the cervical cancer treatment trial, the average tumor volume of the LAM-E7 group (161.1202 mm3) was notably smaller than that of the LAM group (349.4625 mm3) and the PBS group (512.4149 mm3)(P<0.01), indicating a significant inhibitory effected on tumor growth. The immunoprevention test showed that the average tumor volume of the LAM-E7 group (3.86 mm3) was significantly smaller than that of the LAM group (172.67 mm3) and the PBS group (57.38 mm3)(P<0.001), demonstrating the preventive effect of LAM-E7 on cervical cancer. LAM-E7 has demonstrated potent therapeutic and preventive properties on cervical cancer tumors in mice, which offers a new perspective for using attenuated Listeria as a vaccine vector in the treatment of cervical cancer. This study also provides valuable data that supports the advancement of therapeutic vaccines for cervical cancer in the future.
Preparation of Ferritin Nanoparticles Displaying the Dominant Neutralizing Epitopes of Foot-and-mouth disease virus and Their Immunogenicity Evaluation in Mouse (Mus musculus) Models
LU Bing-Zhou, RU Yi, HAO Rong-Zeng, LI Ya-Jun, YANG Yang, YANG Rui, MAO Yu-Han, JIANG Cheng-Hui, TAO Shi-Yu, ZHENG Hai-Xue, YU Si-Jiu, CUI Yan
2024, 32(5): 1216-1226  | doi: 10.3969/j.issn.1674-7968.2024.05.022 |  Full text (HTML) (1 KB)  | PDF   PDF  (4021 KB)  ( 15 )
Nanoparticles (NPs) can display multiple copies of antigen on the surface and simulate the structural morphology of viruses, which is more conducive to uptake by immune cells and promote antigen presentation, thus enhancing protective efficacy. This study aimed to generate ferritin nanoparticles harboring a dominant neutralizing epitope of Foot-and-mouth disease virus (FMDV) by using the Escherichia coli expression system, and to evaluate its immunogenicity. The recombinant expression plasmid pET28a-FBT was constructed based on the Helicobacter pylori ferritin, the dominant neutralizing epitope sequence of FMDV was inserted between the 33th and 34th amino acids of ferritin, and the T cell epitope sequence was inserted at the N-terminus of ferritin. The recombinant expression plasmid pET28a-Fer expressing Fer protein was used as control. The recombinant plasmid pET28a-Fer and pET28a-FBT were transformed into Escherichia coli competent cells, the target proteins were obtained by induced expression and size exclusion chromatography purification, and identified using SDS-PAGE, Western blot, particle size analysis, and transmission electron microscopy, then evaluated immunogenicity by immunizing BALB/c mice (Mus muculus). The results showed that the recombinant protein Fer and FBT were successfully prepared by using prokaryotic expression system. The recombinant protein FBT could be recognized specifically by FMDV positive serum. After 14 d of immunization with FBT, serum antibodies were produced and continued to rise, and the neutralizing antibody titer could reach up to 1∶128 on 35 d. At the same time, the proliferation level of spleen lymphocytes and the secretion level of interleukin 2 (IL-2), IL-4, IL-10, and interferon-γ (IFN-γ) in FBT immunized mice were significantly higher than the control (P<0.05). This study prepared ferritin nanoparticles displaying FMDV neutralizing epitopes with good immunogenicity, providing technical support for the development of a novel foot-and-mouth disease engineered vaccine.
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