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2024 Vol. 32, No. 1  Published: 01 January 2024
Articles and Letters
Preliminary Study on ZmMYC7 Transcription Factor Regulating of Disease Resistance Related Functions in Maize (Zea mays)
WANG Yong-Gui, LI Wei, CUI Ting-Ru, ZHANG Fu-Yuan, YANG Wen-Hao, XU Dang-Fei, XING Ji-Hong, CAO Hong-Zhe, DONG Jin-Gao
2024, 32(1): 1-10  | doi: 10.3969/j.issn.1674-7968.2024.01.001 |  Full text (HTML) (1 KB)  | PDF   PDF  (4497 KB)  ( 79 )
Maize (Zea mays) is one of the most important food crops in the world. Maize diseases caused by pathogens directly affect maize production and lead to huge economic losses. Transcription factor MYC2 plays an important role in the field of plant disease resistance. ZmMYC7 (Zm00001d030028), ortholog of Arabidopsis thaliana AtMYC2, was previously obtained in the previous work. In order to explore the function of transcription factor ZmMYC7 in regulating maize disease resistance, the sensitivity of ZmMYC7 mutant to 4 maize fungal diseases was detected, and the effect of ZmMYC7 mutation on the expression level of pathogenesis-related protein gene ZmPRs was detected by qRT-PCR. The results showed that ZmMYC7 had the highest similarity (90%) with Sorghum bicolor SbMYC2. ZmMYC7 was highly expressed in leaves and anthers of maize. The expression of ZmMYC7 was up-regulated by UV irradiation and Fusarium graminearum infection. In ZmMYC7 mutants, the sensitivity of maize to leaf and stem diseases was significantly increased, and the expression level of disease resistance related gene ZmPRs was significantly down-regulated (P<0.01). The above results showed that ZmMYC7 played an important role in maize resistance to pathogen disease. The present study provides basic materials for further analysis of the molecular mechanism of ZmMYC7 regulating maize disease resistance.
Meta-analysis of QTL and Mining of Candidate Genes for Quality Traits in Wheat (Triticum aestivum)
LI Na, WANG Peng, KONG Bin-Xue, MA Jing-Fu, DOU Jia-Xin, CHEN Tao, ZHANG Pei-Pei, LIU Yuan, YANG De-Long
2024, 32(1): 11-25  | doi: 10.3969/j.issn.1674-7968.2024.01.002 |  Full text (HTML) (1 KB)  | PDF   PDF  (6208 KB)  ( 71 )
Wheat (Triticum aestivum) quality trait is a complex quantitative trait controlled by multiple genes. It is of great significance to explore the important genomic regions and candidate genes of wheat quality for molecular genetic improvement. In this study, the mapping integration and meta-analysis were conducted of 293 initial QTLs for quality traits from 31 QTL mapping studies in wheat, and 40 meta quantitative trait loci (MQTLs) were predicted which included 35 MQTLs for grain hardness (GH), 23 MQTLs for sedimentation value (SV) and 16 MQTLs for starch content (SC). The average confidence interval of these MQTLs was 3.87 cM, which was 3.82 times smaller than the average confidence interval of the initial QTL (14.80 cM), The average confidence interval of the 5 core MQTLs was reduced to 0.80 cM. A total of 839 candidate genes were obtained from the 40 MQTLs intervals by homology alignment and Ensembl Plants sequence information. Based on transcriptome date, 113 candidate genes with high expression in grain endosperm and aleurone layer were screened. The important genomic regions and candidate genes obtained in this study will provide a theoretical basis for the molecular genetic improvement of quality traits in wheat.
Identification of Soybean (Glycine max) GmBAS1 Gene and Its Effect on Soybean Plant Architecture Structure
CHEN Xiao-Rui, WANG Ying, QIU Li-Juan, CHEN Qing-Shan
2024, 32(1): 26-38  | doi: 10.3969/j.issn.1674-7968.2024.01.003 |  Full text (HTML) (1 KB)  | PDF   PDF  (8390 KB)  ( 28 )
Close planting is the main way to increase soybean (Glycine max) yield. The premise is to improve the soybean plant structure such as plant height, petiole angle, and internode length. Brassinolide (BR), as the sixth plant hormone, plays an important role in controlling the petiole angle of rice (Oryza sativa) and the plant structure of Arabidopsis thaliana. In soybean, most of the studies on ester genes are related to the flowering period, but the studies on plant architecture have not been reported. The cytochrome P450 734A1 (CYP734A1/BAS1) gene belongs to the brassinosteroid metabolism gene, which can control the size of the petiole angle of rice and improve the plant architecture of rice. In order to explore the influence of the CYP734A1/BAS1 gene on the soybean plant architecture. This study homologously cloned 2 homologous genes GmBAS1a and GmBAS1b of CYP734A1/BAS1 in soybean. Phylogenetic tree analysis showed that the homologous sequence of GmBAS1a (GenBank No. NC_038253.2) and GmBAS1b (GenBank No. NC_038241.2) were the closest related with that of Arabidopsis thaliana, and the evolution direction was different in monocots and dicots. Haplotype analysis showed that there were few natural mutations in the exon region of GmBAS1 gene, and the gene performed important functions in soybean; Promoter prediction showed that the promoter region of GmBAS1 gene had various cis-acting elements involved in light response and adversity stress, and could participate in multiple growth and development processes of soybean. Further, the BR mutants were obtained by Agrobacterium-mediated transformation of soybean cotyledon nodes into overexpression vectors. The plant height of soybean overexpressing GmBAS1a was reduced by 70%, the angle of petiole in different parts was reduced by 10%~50%, and the length of internode was reduced by 55%~82%, while soybean plants overexpressing GmBAS1b showed a more serious dwarf phenotype. These two phenotypes were consistent with the usual BR-deficient phenotypes. The results showed that overexpression of GmBAS1a and GmBAS1b could reduce soybean plant height and petiole angle, and promote soybean plant compactness, and GmBAS1b was more important in BR metabolism than GmBAS1a. GmBAS1b played a more important role and had a greater impact on plant architecture. The results provide a reference for exploring the role of BR in regulating soybean plant architecture.
Cloning, Subcellular Localization and Expression Analysis of the Transcription Factor Gene FtNAC16 in Fagopyrum tataricum
KE Jin, CHEN Qing-Fu, LI Hong-You
2024, 32(1): 39-49  | doi: 10.3969/j.issn.1674-7968.2024.01.004 |  Full text (HTML) (1 KB)  | PDF   PDF  (9944 KB)  ( 48 )
NAC (petunia NAM and Arabidopsis thaliana ATAF1, ATAF2, and CUC2) transcription factors play important regulatory roles in plant secondary wall thickening. In order to explore the function and molecular mechanism of NAC transcription factor in the formation of secondary wall of tartary buckwheat (Fagopyrum tataricum), reverse transcription PCR (RT-PCR) was used to clone a candidate NAC transcription factor previously identified by comparative transcriptome, named FtNAC16, which might be involved in regulating the synthesis of secondary wall of tartary buckwheat shell cells. Then the bioinformatics, subcellular localization and gene expression analysis of FtNAC16 were conducted. As results, the CDS length of FtNAC16 (GenBank No. OR723785) was 1 086 bp and it encoded 361 amino acids. Multiple sequence alignment and phylogenetic tree analysis indicated that FtNAC16 belonged to the NAC transcription factor family and was closely related to AtNST2 (NAC secondary wall thickening), which regulated secondary wall thickening in Arabidopsis thaliana. Subcellular localization showed that FtNAC16 was located in the nucleus. Gene expression analysis suggested that FtNAC16 was highly expressed in roots, stems, flowers and early developing grains with high cellulose content. In addition, the expression analysis of FtNAC16 in thick shell tartary buckwheat and thin shell tartary buckwheat at different developmental stages showed a similar expression pattern, with a trend of increasing and then decreasing, and the highest expression was observed at 7 d after pollination. However, the expression of FtNAC16 in thick shell tartary buckwheat was significantly higher than that in thin shell tartary buckwheat at early stage of development. Further expression analysis revealed that the expression of the downstream regulatory gene FtMYB103, cellulose synthase 4 (FtCESA4) and FtCESA8 involved in cellulose synthesis were highly positively correlated with FtNAC16 in the grains of thick shell tartary buckwheat and thin shell tartary buckwheat at different developmental stages. All these results suggested that FtNAC16 was a nucleo-specific NAC transcription factor, which may positively regulate the biosynthesis of secondary walls by positively regulating the expression of genes further related to the biosynthesis of cell secondary walls of tartary buckwheat in different tissue parts, especially in the fruit shell cells. It provides excellent genetic resources and theoretical basis for breeding new varieties of buckwheat with high yield, high quality and strong adaptability.
Expression Analysis of the StNF-YA8 Gene Associated with Dormancy Release in Potato (Solanum tuberosum)
WANG Xiao, WEI Han, LI Shi-Gui, WANG Kai-Tong, ZHAO Liang, ZHANG Ning, SI Huai-Jun
2024, 32(1): 50-59  | doi: 10.3969/j.issn.1674-7968.2024.01.005 |  Full text (HTML) (1 KB)  | PDF   PDF  (9792 KB)  ( 27 )
Nuclear factor-Y (NF-Y) is a class of transcription factors widely distributed in eukaryotes. It composed of NF-YA, NF-YB and NF-YC subunits. NF-YA is involved in regulating plant growth and development, and stress response. In this study, qRT-PCR method was used to analyze the expression pattern of 10 StNF-YA genes of potato (Solanum tuberosum) in the process of tuber dormancy release. The results showed that the StNF-YA8 gene was consistently up-regulated in expression during dormancy release in tubers. The StNF-YA8 gene was further cloned (GenBank No. XM_015309982.1), and its ORF was 591 bp, encoding 196 amino acids. The subcellular localization vector of StNF-YA8 protein was constructed, and the results showed that StNF-YA8 protein was localized in the nucleus and cell membrane. Tissue-specific analysis revealed that the StNF-YA8 gene was differentially expressed between different tissues. The results of bioinformatics analysis showed that the potato StNF-YA8 protein contained a conserved CBFB domain, and had the highest evolutionary relationship with wild potato (S. commersonii); StNF-YA8 protein closely interacted with StNF-YB3/5/10/14/17 and StNF-YC1/4/5/8/9 proteins; there were cis-acting elements involved in light response, circadian rhythm control and hormone response in the promoter sequence of this gene. The results can provide a basis for the molecular mechanism analysis of potato StNF-YA8 gene involved in regulating tuber dormancy release.
Cloning and Expression Pattern Analysis of BnaNAC14.1 Gene in Brassica napus
ZHANG Bing-Xin, ZHU Jin-Bo, LIU Cui, LI Qing-Yang, CUI Peng, LIU Hong-Bo
2024, 32(1): 60-69  | doi: 10.3969/j.issn.1674-7968.2024.01.006 |  Full text (HTML) (1 KB)  | PDF   PDF  (9320 KB)  ( 38 )
NAC transcription factors are widely involved in plant growth and development and abiotic stress response. In this study, the BnaNAC14.1 gene was cloned from the 'Zhongshuang 11' cDNA of Brassica napus by RT-PCR. Bioinformatics analysis showed that the coding sequence of BnaNAC14.1 gene was 1 908 bp, and encoded 635 amino acids, including the NAM conserved domain. The pCAMBIA1305.1-35S-sGFP-BnaNAC14.1 fusion expression vector was constructed and transformed into tobacco (Nicotiana tabacum) leaf cells by Agrobacterium tumefaciens-mediated transformation. The results of subcellular localization showed that BnaNAC14.1 localized in the nucleus; The recombinant vector pGBKT7-BD-BnaNAC14.1 was transformed into yeast (Saccharomyces cerevisiae) strain Y2HGold, and blue monoclonal colonies were observed on SD/-Trp+X-α-gal medium which indicated that the BnaNAC14.1 had transcriptional activation activity. The expression pattern demonstrated that the BnaNAC14.1 had relatively higher expression levels in flowers and development seeds than that in roots, stems and leaves. The results of qRT-PCR showed that BnaNAC14.1 gene was significantly up-regulated by salt, drought and exogenous hormones abscisic acid (ABA), and inhibited by indoleacetic acid (IAA) and methyl jasmonate (MeJA). The above results preliminarily identified that BnaNAC14.1 gene might be involved in abiotic stress and hormone response, and play an important role in seed development. This study provides theoretical support for further study on the BnaNAC14.1 biological function and rapeseed breeding with high quality and stress resistance.
Effect of Overexpression of F-box Gene BoFBX117 on Low-temperature Tolerance of Cabbage (Brassica oleracea var. capitata)
LUO Yu-Xia, XU Tao, DAI Qian, CHEN Yao, SONG Jiang-Hua
2024, 32(1): 70-79  | doi: 10.3969/j.issn.1674-7968.2024.01.007 |  Full text (HTML) (1 KB)  | PDF   PDF  (8396 KB)  ( 19 )
F-box gene plays an important role in plant abiotic stress response. In order to study the function of F-box gene BoFBX117 (GenBank No. XM_013731217.1) in cabbage (Brassica oleracea var. capitata) response to low-temperature stress, in this study, the BoFBX117 over-expression vector was constructed, and the over-expression of BoFBX117 cabbage (OE-BoFBX117) plants were obtained by Agrobacterium tumefaciens mediated method. After 24 h of low-temperature stress at 4, 0, -2 and -4 ℃, the phenotype of the over-expression of BoFBX117 cabbage showed that the degree of leaf yellowing wilting was less than that of non-transgenic cabbage (WT). After 7 d of recovery, it was found that the over-expression of BoFBX117 cabbage had stronger ability to return to normal growth. The results of qPCR showed that the expression of BoFBX117 in transgenic plants was significantly increased under low-temperature stress (P<0.01), which was 1.87~4.80 times that of WT plants. Under low-temperature stress at 0 ℃, the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) of transgenic plants were the highest, which were 80%, 16% and 99% higher than those of WT plants respectively. Under low-temperature stress, the content of malondialdehyde (MDA) continuously accumulated. At -4 ℃, the MDA content of transgenic plants was 0.62 μmol/g FW, 53.38% lower than WT. Under normal conditions and low-temperature stress, the proline and soluble sugar contents of transgenic plants were significantly higher than those of WT plants (P<0.05). The results showed that BoFBX117 might play a positive role in the response to low-temperature stress, and over-expression of BoFBX117 in cabbage could improve the low-temperature tolerance of cabbage. This study provides support for the breeding of low-temperature tolerance varieties of cabbage.
Identification of Resistance to Clubroot and Histological Microscopic Observation of Turnip (Brassica rapa ssp. rapifera) Germplasm
LEI Ting, LIU Xiao-Le, ZHOU Xiao-Qing, YANG Xin-Lei, HUI Mai-Xia
2024, 32(1): 80-90  | doi: 10.3969/j.issn.1674-7968.2024.01.008 |  Full text (HTML) (1 KB)  | PDF   PDF  (18176 KB)  ( 6 )
Clubroot is one of the main diseases of cruciferous crops. Screening the clubroot resistant (CR) materials of turnip (Brassica rapa ssp. rapifera), clarifying the distribution of CR genes, and analyzing the disease-resistance mechanism in turnip can provide a theoretical and practical basis for the breeding of turnip CR varieties. In this study, the resistance of 25 turnip materials to clubroot was identified by inoculating Plasraodiophora brassicae at the artificial seedling stage, the resistance genes were detected by molecular markers, and the infection process of Plasraodiophora brassicae and pathological changes of root were observed by Phloxine-B staining and paraffin section techniques. As a results, 4 materials (immune and highly resistant), among which WJ-9, WJ-21, and WJ-22 exhibited immunity, while WJ-14 exhibited high resistance. In addition, WJ-10, WJ-13, and WJ-18 exhibit disease tolerance; and 18 susceptible (susceptible and highly susceptible) materials were identified. The total number of CR genes in the tested materials was in descending order: Crr3>CRb>Crr1a=CrrA5>CRc>Crr1>CRa. WJ-22 was the material with the most resistance genes, containing a total of 6 resistance genes. There were differences in the infection process and pathological changes of Plasmodiophora brassicae in resistant and susceptible turnip roots. Compared with the susceptible material, the root hairs were infected later, the infection amount was less, and the cortex was not infected in the resistant materials. The root cells of the susceptible material would swell after Plasmodiophora brassicae invade, and the structure would be disordered. Swollen cells squeezed and destroyed root canals. The cell structure of the disease-resistant material was completed. In this study, 4 turnip resources with resistance to clubroot were identified, the CR genes carried by them were clarified, and the infection process and tissue structure differences between resistant and susceptible materials were explored. It provides a scientific basis for cultivating varieties with durable resistance to clubroot.
Identification and Expression Analysis of SAUR Gene Family in Fragaria vesca
LIU Fang, LIANG Guo-Ping, WANG Han, REN Jia-Xuan, CHEN Bai-Hong, MAO Juan
2024, 32(1): 91-106  | doi: 10.3969/j.issn.1674-7968.2024.01.009 |  Full text (HTML) (1 KB)  | PDF   PDF  (10991 KB)  ( 27 )
SAUR (small auxin up RNA) is a gene family that can respond quickly to auxin indole-3-acetic acid (IAA) treatment in the early stage, but how the gene family responds to auxin treatment in strawberries (Fragaria×ananassa) has not been reported. Therefore, this study used bioinformatics tools to analyze the evolutionary relationship, protein physical and chemical properties, gene structure and promoter elements of forest strawberry (F. vesca) SAUR gene family, as well as the changes of expression patterns of respond IAA. The results showed that 67 SAUR family members were identified in the forest strawberry genome. The amino acid number of these members varied from 83 to 286 aa. Except FvSAUR9, FvSAUR15, FvSAUR22, FvSAUR44, FvSAUR45 were hydrophobic, and the other members were hydrophilic. Subcellular localization prediction analysis showed that the expression of members of the gene family was mainly located in nucleus, mitochondria, chloroplast and cytoplasm. The results of phylogenetic tree showed that the SAUR gene members of Arabidopsis thaliana, forest strawberry and strawberry were divided into 10 subgroups. Among them, subgroup 5 had the highest number of members in F. vesca, with a total of 20 members. Additionally, subgroup 4 and subgroup 8 did not have any members. A total of 11 different types of elements were identified in the promoter sequences of forest strawberry SAUR gene members, included 5 types related to environmental stress and 6 types related to hormones, with the highest number of light responsive elements. The expression pattern of IAA in response of SAUR family members of forest strawberry was analyzed by qRT-PCR. The results showed that the expression levels of most members were significantly down-regulated after 2 h of IAA treatment (P<0.05), while 12 members were up-regulated after 12 h, and all members of the SAUR gene family members were up-regulated after 24 h of treatment. In addition, the results of collinearity analysis showed that there were 6 gene pairs in the SAUR gene family of forest strawberries with collinearity. These results provide a reference for strawberry SAUR gene family members in growth and development and hormone response, especially in response to IAA treatment.
Cloning, Bioinformatics Analysis of Transcription Factor NAC62 in Industrial Hemp (Cannabis sativa) and Its Response Analysis to Drought Stress
YANG Yu-Lei, ZHANG Han-Xue, WANG Shan-Shan, TANG Kai-Lei, DU Guang-Hui
2024, 32(1): 107-114  | doi: 10.3969/j.issn.1674-7968.2024.01.010 |  Full text (HTML) (1 KB)  | PDF   PDF  (3988 KB)  ( 53 )
Industrial hemp (Cannabis sativa) is an important economic crop, and its seed germination period is susceptible to drought stress, which ultimately has adverse effects on the growth and yield of hemp. NAC gene is a unique type of transcription factor in plants, which plays an essential role in plant response to abiotic stress. Based on the previous transcriptome data, a NAC gene from industrial hemp 'Yunma 1' was cloned by RT-PCR in this study, and named as CsNAC62 (GenBank No. XM_030652694). The results of sequence analysis found that the coding sequence of CsNAC62 gene contains 1 260 bp, encoding 419 amino acids, its protein molecular weight is 48.32 kD, and the isoelectric point is 8.24. The N-terminal of CsNAC62 contains a NAC domain composed of 150~160 amino acids. This domain is composed of 5 subdomains, which is a typical conservative domain of the NAM/NAC gene family. Phylogenetic analysis showed that CsNAC62 was closely related to the NAC3 protein of peach (Prunus persica) in all the tested species. The gene expression level of CsNAC62 in the germinated seeds of industrial hemp 'Yunma 1' gradually increased with the prolongation of the drought stress treatment (PEG-6000 simulation), and the expression level reached to the maximumon the 7 d of germinated seeds. In normal seed germination (control), the expression level of CsNAC62 increased slowly. These results indicated that the gene expression of CsNAC62 was induced by drought. This study preliminarily explored the expression mode of the CsNAC62 under PEG simulated drought stress, and provides a reference for further exploring its molecular mechanism of participating in the response to industrial cannabis drought stress.
Identification of the SNAC Gene Family in Eucalyptus grandis and Its Expression Analysis Under Abiotic Stresses
HONG Jia-Du, NI Xiao-Xiang, YU Jian-Feng, WU Meng-Jie, ZHAO Shuang, CHENG Long-Jun
2024, 32(1): 115-131  | doi: 10.3969/j.issn.1674-7968.2024.01.011 |  Full text (HTML) (1 KB)  | PDF   PDF  (9261 KB)  ( 28 )
Eucalyptus grandis has made important contribution to wood-related industries in South China, but they are sensitive to abiotic stresses, limiting the expansion of its cultivation and benefits improvement of plantation. NAC is a plant-specific transcription factor family, which is widely involved in plant growth, development, metabolism and stress response. And, the stress-related NACs were named SNAC. In this study, 166 NAC transcription factors identified in E. grandis genome and 74 SNAC in other plant species were used to identify EgrSNACs. And, EgrSNACs structure, coding protein sequence, chromosome localization, collinearity and gene duplication, cis-acting elements on promoters, and the tissue-specific expression patterns were analyzed. In addition, the seedlings of E. grandis were treated with low temperature, drought, high salt, abscisic acid (ABA) and methyl jasmonate (MeJA), and qPCR was used to analyze the expression of EgrSNACs under these treatments. The results showed that there were 22 EgrSNACs in E. grandis, belonging to ATAF, NAP, and AtNAC3 subfamily. Except for EgrSNAC20, the proteins encoded by these genes all contained 5 subdomains of the typical NAM domain, corresponding to Motif1~Motif5. The 22 EgrSNACs were distributed on 9 chromosomes, with 4 collinear gene pairs and 2 tandem repeat gene segments composed of 11 EgrSNACs. Cis-acting element of promoter analysis showed that multiple stress response elements were distributed on the EgrSNAC promoters. The expression pattern of tandem duplicated EgrSNACs were similar in leaf, stem, xylem and phloem. The qPCR analysis of different time treatments at 4 ℃ indicated that the expression of the 19 EgrSNACs were induced by low temperature, in addition to EgrSNAC1, EgrSNAC3, and EgrSNAC22; there were 19 EgrSNACs that responded to the drought, except EgrSNAC2, EgrSNAC5, EgrSNAC14 and EgrSNAC21 were suppressed, others were induced. Under high salt treatment, 19 EgrSNACs expression changes, and only EgrSNAC21 was suppressed. In addition, 14 EgrSNACs were induced under ABA (100 μmol/L) treatment. 13 EgrSNACs were induced and 4 were inhibited under MeJA (100 μmol/L) treatment . The results of this study revealed the relationship between EgrSNACs and their response of low temperature, drought, high salt, ABA, MeJA and other abiotic stress factors, providing a reference for further study of EgrSNACs in E. grandis.
Identification and Analysis of MYB Transcription Factors Related to Salt Stress in Pyrus betulaefolia based on Transcriptome Information
LI Hui, ZHANG Yu-Feng, LI Xiao-Gang, WANG Zhong-Hua, CHANG You-Hong, LIN Jing
2024, 32(1): 132-146  | doi: 10.3969/j.issn.1674-7968.2024.01.012 |  Full text (HTML) (1 KB)  | PDF   PDF  (15646 KB)  ( 16 )
Myeloblastosis (MYB) is one of the most common transcription factor families in plants, which widely takes part in various adversities responses including salt stress. So far, there is still no systematic comparative analysis on the expression pattern of MYB family among various ecotypes with different salt tolerance abilities from the same species. Based on the transcriptome sequencing (RNA-Seq) data from the salt-tolerant ecotype and common ecotype of Pyrus betulaefolia, the differentially expressed PbMYBs genes before and after salt stress were screened and annotated in this study. Then, their conserved domains were classified, subcellular localizations were predicted and the phylogenetic tree was constructed. TBtools was used to draw the heatmaps to analyze the differential expression pattern of PbMYBs genes in roots, stems and leaves from the salt-tolerant ecotype and common ecotype of P. betulaefolia. By using transcriptome analysis tools, 129 PbMYBs transcription factors were annotated and selected from the RNA-Seq data, which differentially expressed in 2 ecotypes of P. betulaefolia after salt stress. These genes can be divided into 3 categories (1R-MYB, R2R3-MYB and 3R-MYB) according to their structural characteristics.The number of PbMYBs genes distributed on chromosome 15 of P. betulaefolia was the largest. The phylogenetic trees of MYB families were constructed, which showed that the PbMYBs family of P. betulaefolia contained 3 major branches, and 23 evolutionary branches. The results of the subcellular localization demonstrated that 42 PbMYBs were located in the extracellular, and 87 PbMYBs were located in the nucleus. Analysis of the differential expression pattern of PbMYBs based on transcriptome data showed that PbMYBs genes in P. betulaefolia may be involved in the transcriptional regulation of different organs in response to salt stress. Additionally, the results of real-time quantitative PCR further verified that some PbMYBs transcription factors may play a role in stress regulation in the roots, stems or leaves of P. betulaefolia, respectively. During the process of salt stress, the transcriptional regulation of 129 PbMYBs members in response to stress were diversity in various organs. The results of real-time quantitative PCR further verified that different PbMYBs transcription factors were up-regulated or down-regulated to response to salt stress signals in the roots, stems or leaves of P. betulaefolia, respectively. The results provide the relative information for further research on gene structure and biological function of the MYB family in P. betulaefolia.
RNA Interference Efficiency Comparison of Bovine (Bos taurus) MSTN Gene Mediated by CRISPR-Cas13a and MicroRNA
LEI Jia-Ru, WANG Song, DI An-Qi, AN Chang-Suo, SUN Xue-Song, LIU Ming-Cheng, YUAN Hong-Min, YANG Lei, LI Guang-Peng
2024, 32(1): 147-157  | doi: 10.3969/j.issn.1674-7968.2024.01.013 |  Full text (HTML) (1 KB)  | PDF   PDF  (3614 KB)  ( 43 )
RNA interference (RNAi) is mainly mediated by small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA) and CRISPR/Cas13a. miRNA mediates RNAi through complementary pairing with target mRNA bases. CRISPR/Cas13a mediates RNAi through the combined action of CRISPR-derived RNA (crRNA) and Cas13a proteins. Comparison of CRISPR/Cas13a- and miRNA-mediated RNAi efficiency has not been reported. In this study, according to the mRNA homologous sequence of myostatin gene (MSTN) in Simmental cattle (Bos taurus) and mouse (Mus musculous), MSTN-crRNA-1/2/3 and MSTN-miRNA-1/2/3 were designed. In order to eliminate the fluctuations of CRISPR/Cas13a interference efficiency caused by unstable Cas13a protein expression, firstly, cattle muscle satellite cell lines (SCs) and mouse myoblast cell lines C2C12 with stable expression of Cas13a protein were prepared. Then, MSTN-crRNA-1/2/3 were transfected into cattle Cas13a-SCs and mouse Cas13a-C2C12, respectively. Meanwhile, MSTN-miRNA-1/2/3 were transfected into cattle SCs and mouse C2C12, respectively. The efficiency of CRISPR/Cas13a- and miRNA-mediated MSTN interference was detected by qRT-PCR after 48 h transfection. In addition, MTT assay and Cell Counting Kit-8 (CCK-8) assay were used to detect the effects of CRISPR/Cas13a- and miRNA-mediated MSTN knockdown on cell activity and proliferation. The results showed that the average and highest interference efficiency of MSTN-crRNA-1/2/3 in cattle Cas13a-SCs and mouse Cas13a-C2C12 were 45% and 54%, respectively. The average and highest interference efficiency of MSTN-miRNA-1/2/3 in cattle SCs and mouse C2C12 were 30% and 33%, respectively, indicating that CRISPR/Cas13a had higher interference efficiency than miRNA in cattle SCs and mouse C2C12. In addition, the cell viability and proliferation ability of MSTN-miRNA-1/2/3 in cattle SCs and mouse C2C12 decreased by 12% and 12.5% (P<0.05), respectively, while the cell viability and proliferation ability of MSTN-crRNA-1/2/3 had no effect. This study provides basic data for the research and application of gene editing in model animals and large livestocks.
Transcriptome Analysis Reveals Differences in Skeletal Muscle Gene Expression in Sheep (Ovis aries) of Different Genders
LOU Meng-Yu, LU Jia-Ni, DUAN Qin, YANG Wang-Xin, ZHENG Qi, ZHU Cui-Yun, ZHANG Si-Huan, LI Shuang, LING Ying-Hui
2024, 32(1): 158-167  | doi: 10.3969/j.issn.1674-7968.2024.01.014 |  Full text (HTML) (1 KB)  | PDF   PDF  (2183 KB)  ( 59 )
Sex is one of the main factors affecting skeletal muscle development and meat production performance in sheep (Ovis aries). This study investigated the differences in gene expression in sheep longest dorsal muscle of different sexes using transcriptome sequencing technology, aiming to reveal the molecular mechanism of the effect of sex on skeletal muscle development in sheep. A total of 14 587 genes were identified by transcriptome sequencing and analysis, among which 29 genes were significantly differentially expressed. 23 genes were significantly up-regulated and 6 were down-regulated in the ram group compared with the ewe group. The differential genes included xin actin binding repeat containing 1 (XIRP1), glutathione peroxidase 2 (GPX2), acyl-CoA synthetase medium chain family member 1 (ACSM1), fatty acid binding protein 4 (FABP4), testis specific serine kinase 6 (TSSK6), etc. The GO analysis showed that the differentially expressed genes were mainly enriched in ribonucleoside, actin-binding and cytoskeleton-binding categories, and the KEGG pathway analysis revealed that the differentially expressed genes were significantly enriched in 3 signaling pathways, namely PPAR signaling pathway, arachidonic acid metabolism and glutathione metabolism signaling pathway. Thus, differences in skeletal muscle development in sheep between the sexes may arise through the above pathways. Eight differentially expressed genes were randomly selected and their expression was verified by qPCR, and the results were generally consistent with the transcriptome sequencing results, which proved that the sequencing results were reliable. In conclusion, this study revealed the differences in skeletal muscle gene expression and related signaling pathways in sheep of different sexes, which provides reference for further understanding the mechanisms by which sex affects muscle development in sheep.
Effect of Pig (Sus scrofa) SELW Gene on the Differentiation of Skeletal Muscle Satellite Cells
HU Rong-Bin, WU Xing-Feng, PAN Zhi-Hong, LI Li, HE Yi-Yi, XU E
2024, 32(1): 168-179  | doi: 10.3969/j.issn.1674-7968.2024.01.015 |  Full text (HTML) (1 KB)  | PDF   PDF  (11278 KB)  ( 10 )
Selenoprotein W (SELW) is one of the important selenoproteins in skeletal muscle, which has important roles in cell differentiation and other biological functions. To investigate the effect of SELW gene on the differentiation of skeletal muscle satellite cells in pigs (Sus scrofa), three 7-day-old healthy white pigs were selected, and 7 tissue samples of heart, liver, spleen, lung, kidney, dorsal muscle and leg muscle were collected to extract RNA. qPCR was performed to detect the relative expression of SELW gene in different tissues. Bioinformatics analysis of porcine SELW proteins was performed using online software. The full-length sequence of the CDS region of porcine SELW gene (GenBank No. NM_213977) was cloned, a recombinant lentiviral vector overexpressing SELW gene was constructed and infected with skeletal muscle satellite cells, SELW stably expressed cell lines were obtained by puromycin screening, SELW protein expression sites was determined by subcellular localization, and SELW gene and its protein expression levels were detected by qPCR and Western blot. Cell samples were collected at 0 and 48 h of induction differentiation, and qPCR was performed to detect the expression of paired box 3 (PAX3), PAX7, myogenic factor 5 (MYF5), myogenic determinant (MYOD), and myocyte generating factor (MYOG) genes. The results showed that SELW genes were expressed in 7 different tissues of pigs, and the expression was extremely significant differenct in heart and dorsal muscle than in other tissues (P<0.01). The molecular formula of SELW protein was C421H677N109O119S3Se1, the relative molecular weight was 9 344.81 D, and the theoretical isoelectric point was 9.15, which was a stable protein. Genetic evolutionary analysis showed that pig SELW gene had the highest similarity with human (Homo sapiens), rhesus monkey (Macaca mulatta) and Sumatran orangutan (Pongo abelii). Subcellular localization results indicated that SELW proteins were localized in the nucleus and cytoplasm of cells. qPCR and Western blot results showed that the expression of SELW-OE group was very significant higher than that of SELW-NC group (P<0.01), indicating the successful construction of SELW gene stably expressing cell lines. qPCR results showed that the expression of PAX7 and MYF5 genes in SELW-OE group were extremely significantly upregulated at 0 h of induction differentiation, while MYOD gene expression was significantly down-regulated (P<0.05), and MYOG gene expression was extremely significantly down-regulated (P<0.01). The expression of PAX3 gene was significantly upregulated (P<0.05) and the expression of PAX7, MYF5, MYOD and MYOG genes were extremely significantly upregulated (P<0.01) after 48 h of induction of differentiation. It was shown that overexpression of SELW gene could promote the expression of genes related to skeletal muscle cell differentiation. It provides basic information for further study of the molecular regulation mechanism of SELW gene on muscle growth and development in pigs.
Analysis of plekhm2 Gene Structure and Differential Expression in Taihang Chickens (Gallus domesticus)
DING Hong, ZHANG Yin-Liang, ZHOU Rong-Yan, LI Yuan-Yuan, LIU Hua-Ge
2024, 32(1): 180-188  | doi: 10.3969/j.issn.1674-7968.2024.01.016 |  Full text (HTML) (1 KB)  | PDF   PDF  (4837 KB)  ( 37 )
Chicken salmonellosis is a highly infectious bacterial disease caused by Salmonella infection of chicken (Gallus domesticus). The pleckstrin homology and RUN domain containing M2 (plekhm2) gene acts as a target of the Salmonella effector protein SifA and plays a role in Salmonella infection by downregulating the recruitment of kinesin. The aim of this study was to analyze the sequence characteristics of the chicken plekhm2 gene and to investigate its tissue expression profile and the expression levels in the livers and spleens of Taihang chickens infected and uninfected with Salmonella. Differentially expressed genes were screened based on spleen transcriptome data from Salmonella-infected and uninfected chickens, online tools and software were used for bioinformatic analysis of plekhm2 gene, and qPCR was used to detect the expression levels of plekhm2 gene in different tissues of Taihang chickens, and a Taihang chicken Salmonella infection model was constructed to study the changes of its expression in the chicken liver and spleen. The results showed that the expression level of plekhm2 gene was elevated in the spleen-based transcriptome infected with Salmonella, which encoded 1 016 amino acids, with 2 motifs (RUN and PH), and the highest similarity of sequence with duck (Cairina moschata). Sequence comparison revealed a missense mutation site c.1013G>A at position 1 013 of the cDNA sequence of plekhm2 gene (p.Gly338Glu), A for Taihang chicken or G for Kashmir faverolla chicken; Tissue expression profiles showed that plekhm2 gene was expressed in 11 tissues of Taihang chickens, including heart, liver, spleen and lung. Compared with the uninfected group, the relative expression levels of plekhm2 gene in the liver (P<0.01) and spleen (P<0.05) of Taihang chickens were significantly higher after Salmonella infection. plekhm2 gene was expressed in several tissues of Taihang chickens, and Salmonella infection caused an increase in the expression level of plekhm2 gene. The present study provides a reference to reveal the function of plekhm2 gene in Salmonella infection.
Inhibitory Effect of PEX10 Gene Deletion on the Growth and Pathogenicity of Trichophyton mentagrophytes in Oryctolagus cuniculus
PAN Yao, XIAO Chen-Wen, LIU Yan, ZHANG Peng-Jun, YING Shu-Min, HUANG Ye-E, JI Quan-An, WEI Qiang, WANG Jing, WANG Jao-Yu, BAO Guo-Lian
2024, 32(1): 189-199  | doi: 10.3969/j.issn.1674-7968.2024.01.017 |  Full text (HTML) (1 KB)  | PDF   PDF  (9610 KB)  ( 11 )
Trichophyton mentagrophytes, also known as T. gypsioides, is severely hazardousness to rabbit (Oryctolagus cuniculus). Peroxisomal biogenesis factor 10 (PEX10) gene is closely related to the drug susceptibility of plant pathogenic fungi. However, the mechanism of whether PEX10 gene affects the growth and pathogenicity of animal pathogenic fungi such as T. mentagrophytes is still unclear. This study first constructed a PEX10 gene knockout vector and obtained a PEX10 gene knockout strain of T. mentagrophytes through Agrobacterium tumefaciens-mediated transformation (AtMT) and transformant screening. Phenotypic analysis such as growth characteristics showed that the growth rate of PEX10 gene knockout strains was significantly slowed down (P<0.01), and their sensitivity to berberine hydrochloride was significantly increased (P<0.001). The virulence and pathogenicity of the PEX10 knockout strain to rabbit skin was reduced. The results of this study provide reference for the study of PEX10 gene function and the prevention and treatment of T. mentagrophytes of rabbits.
Reviews and Progress
Research Progress on Follicle Selection and Atresia Regulatory Mechanism in Chicken (Gallus gallus)
ZHANG Wen-Hui, NIE Rui-Xue, LING Yao, LI Jun-Ying, ZHANG Bo, ZHANG Hao
2024, 32(1): 200-208  | doi: 10.3969/j.issn.1674-7968.2024.01.018 |  Full text (HTML) (1 KB)  | PDF   PDF  (3501 KB)  ( 57 )
The number and proportion of developmental follicles and atretic follicles in different stages of chicken ovary are related to egg production performance. A well-organized order of follicles is very important to improve egg production and maintain the continuity of egg production. In this paper, the physiological phenotypic changes during follicle selection and atresia in chickens were analyzed and the main regulatory mechanisms were summarized, and the roles of granulosa autophagy, apoptosis and inflammation in follicular atresia were discussed. The future research direction and suggestions are put forward, which has important reference value for analyzing the genetic mechanism of chicken egg laying traits.
Research Progress on Structural Characteristics and Preparation of Nanobodies
KONG Jing, ZHENG Nan, WANG Jia-Qi, ZHAO Sheng-Guo
2024, 32(1): 209-219  | doi: 10.3969/j.issn.1674-7968.2024.01.019 |  Full text (HTML) (1 KB)  | PDF   PDF  (2704 KB)  ( 200 )
Nanobodies are small molecules composed of only heavy chains with specific recognition of antigens, which have broad application prospects. In this paper, three different antibody subtypes in camels (Camelus) were introduced, and the similarities and differences between traditional antibodies and nanobodies in the types and positions of disulfide bonds, the composition of skeleton regions, and complementary determining regions were revealed. The characteristics of nanobodies, such as simple production, strong specificity, high stability, and high solubility, were discussed. The screening and expression methods of nanobodies were expounded, that is, phage display, yeast two-hybrid and other technologies can be used to screen and obtain specific nanobodies. Because of its simple structure, it can be expressed in Escherichia coli and yeast (Saccharomyces) and other organisms, to better study its function. This review provides a theoretical basis for the development and application of nanobodies in the future.
Resources and Updated Technology
Analysis of the Development Trends in Global Agricultural Animal Gene Editing Fundamental Research
ZHANG Meng-Meng, JIANG Ying, LI Ning
2024, 32(1): 220-233  | doi: 10.3969/j.issn.1674-7968.2024.01.020 |  Full text (HTML) (1 KB)  | PDF   PDF  (16149 KB)  ( 31 )
Gene editing technology is a precise modification technique that can manipulate DNA sequences in living organisms. In order to understand the global development trends in this field, particularly with regard to agricultural animal gene editing, the present study utilized the bibliometric methods to analyze research data from 2015 to 2022 in the field of gene editing technology. In this study, we systematically examined the main research institutions, teams, research frontiers, and current status of agricultural animal gene editing technology research. The results showed that China is in a leading position internationally in gene editing technology research at the literature level, as it is ranked second in the world in terms of the number of gene editing technology-related papers published between 2015 and 2022. Eight of the top 20 institutions being in China, accounting for 40% of the total in the world. However, compared with developed countries, such as the United States, China still lags behind in terms of core technology innovation, gene editing system development, optimization and Cas protein discoveries, all of which are mainly being conducted overseas. Although China ranks second in the world in terms of the number of papers published in top international academic journals, however, it is far behind the United States. In the Nature Biotechnology, China accounts for only one-fifth of the total number of papers, with 31 papers published. The number of research reports on gene editing technology in agricultural animals is relatively small, but it is increasing year by year. The application of gene editing technology in animal breeding has immense potential in the field of agricultural animal breeding, particularly in developing disease-resistant breeds. Finally, we look forward to the prospect of gene editing technology and the development trends of agricultural animal breeding field based on the current situation and national conditions, in order to provide us with a reference for using gene editing technology to solve major problems in agricultural animal breeding, especially disease resistance breeding.
Pharmacodynamics Study of Compound Huang-Long Cream in Promoting the Repair of Acute Soft Tissue Injury and Scald Injury
XIE Tong, ZHANG Pei-Hua, LIN Jia-Hao
2024, 32(1): 234-246  | doi: 10.3969/j.issn.1674-7968.2024.01.021 |  Full text (HTML) (1 KB)  | PDF   PDF  (28442 KB)  ( 6 )
Acute soft tissue injury and scald are common types of traumatic diseases in veterinary clinics, both characterized by local acute aseptic inflammatory response and tissue damage repair as the main pathological changes. In the field of veterinary medicine, there is not much research on drugs that promote tissue damage repair at present. Compound Huang-Long Cream is a Chinese herbal ointment for promoting tissue damage repair according to the empirical formula from Traditional Chinese Veterinary Medicine, which has a good clinical efficacy. This study aimed to conduct a preliminary exploration of the therapeutic effect of Compound Huang-Long Cream and provide a experimental basis for its development and clinical application. An acute soft tissue injury model in SD rats (Rattus norvegicus) and a scald model in C57BL/6J mice (Mus musculus) with high-temperature mental scald were established. The changes of symptomatic manifestations and the haematoxylin and eosin (HE) staining were used to evaluate the pharmacodynamics efficacy of Compound Huang-Long Cream in promoting the repair of acute soft tissue injury and scald injury. The results showed that in the acute soft tissue injury repair experiment, the syndrome score of rats with acute soft tissue injury decreased extremely significantly (P<0.01) and the swelling rate of hind limbs decreased extremely significantly (P<0.01) in the Compound Huang-Long Cream group compared with the model group at 72 h after molding. There was a significant difference (P<0.05) in the swelling rates in the Compound Huang-Long Cream group compared with the positive drug (Diclofenac Diethylamine Emulgel) group. In the scald injury repair experiment, the healing rate of scald focus in the Compound Huang-Long Cream group was (47.82±5.53)%, which was 1.4 times higher than that in the model group (P<0.05) on the day 7. The pathological section results showed that compared with the model group, hemorrhage and inflammation were reduced and collagen fibers were increased in both the soft tissue of rats and the skin of mice in the Compound Huang-Long Cream group. This study indicates that Compound Huang-Long Cream can significantly improve the symptoms of ecchymosis and edema, inhibit inflammation and promote the repair of damaged tissues in rats with acute soft tissue injury model. Compared with diclofenac diethylamine emulgel, it was better at eliminating edema. In terms of promoting scald repair, Compound Huang-Long Cream could accelerate the healing of scald wounds in scald model mice, promoted their wound repair, and inhibited inflammation. This study provides a preliminary experimental basis for the clinical application of Compound Huang-Long Cream.
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