Transcriptome analysis of flower colour reveals the correlation between SNP and differential expression genes in Phalaenopsis

Ding Yu,Wang Ma-Yin,Yang Ding-Hai,Hao Dai-Cheng,Li Wei-Shi,Ling Peng,Xie Shang-Qian 한국유전학회 2023 Genes & Genomics Vol.45 No.12

Background Phalaenopsis is an important ornamental plant that has great economic value in the world flower market as one of the most popular flower resources. Objective To investigate the flower colour formation of Phalaenopsis at the transcription level, the genes involved in flower color formation were identified from RNA-seq in this study. Methods In this study, white and purple petals of Phalaenopsis were collected and analyzed to obtained (1) differential expression genes (DEGs) between white and purple flower color and (2) the association between single nucleotide polymorphisms (SNP) mutations and DEGs at the transcriptome level. Results The results indicated that a total of 1,175 DEGs were identified, and 718 and 457 of them were up- and down-regulated genes, respectively. Gene Ontology and pathway enrichment showed that the biosynthesis of the secondary metabolites pathway was key to color formation, and the expression of 12 crucial genes (C4H, CCoAOMT, F3’H, UA3’5’GT, PAL, 4CL, CCR, CAD, CALDH, bglx, SGTase, and E1.11.17) that are involved in the regulation of flower color in Phalaenopsis. Conclusion This study reported the association between the SNP mutations and DEGs for color formation at RNA level, and provides a new insight to further investigate the gene expression and its relationship with genetic variants from RNA-seq data in other species.

Relationship among porcine lncRNA TCONS_00010987, miR-323, and leptin receptor based on dual luciferase reporter gene assays and expression patterns

Ding, Yueyun,Qian, Li,Wang, Li,Wu, Chaodong,Li, DengTao,Zhang, Xiaodong,Yin, Zongjun,Wang, Yuanlang,Zhang, Wei,Wu, Xudong,Ding, Jian,Yang, Min,Zhang, Liang,Shang, Jinnan,Wang, Chonglong,Gao, Yafei Asian Australasian Association of Animal Productio 2020 Animal Bioscience Vol.33 No.2

Objective: Considering the physiological and clinical importance of leptin receptor (LEPR) in regulating obesity and the fact that porcine LEPR expression is not known to be controlled by lncRNAs and miRNAs, we aim to characterize this gene as a potential target of SSC-miR-323 and the lncRNA TCONS_00010987. Methods: Bioinformatics analyses revealed that lncRNA TCONS_00010987 and LEPR have SSC-miR-323-binding sites and that LEPR might be a target of lncRNA TCONS_00010987 based on cis prediction. Wild-type and mutant TCONS_00010987-target sequence fragments and wild-type and mutant LEPR 3'-UTR fragments were generated and cloned into pmiRRB-REPORT^TM-Control vectors to construct respective recombinant plasmids. HEK293T cells were co-transfected with the SSC-miR-323 mimics or a negative control with constructs harboring the corresponding binding sites and relative luciferase activities were determined. Tissue expression patterns of lncRNA TCONS_00010987, SSC-miR-323, and LEPR in Anqing six-end-white (AQ, the obese breed) and Large White (LW, the lean breed) pigs were detected by real-time quantitative polymerase chain reaction; backfat expression of LEPR protein was detected by western blotting. Results: Target gene fragments were successfully cloned, and the four recombinant vectors were constructed. Compared to the negative control, SSC-miR-323 mimics significantly inhibited luciferase activity from the wild-type TCONS_00010987-target sequence and wild-type LEPR-3'-UTR (p<0.01 for both) but not from the mutant TCONS_00010987-target sequence and mutant LEPR-3'-UTR (p>0.05 for both). Backfat expression levels of TCONS_00010987 and LEPR in AQ pigs were significantly higher than those in LW pigs (p<0.01), whereas levels of SSC-miR-323 in AQ pigs were significantly lower than those in LW pigs (p<0.05). LEPR protein levels in the backfat tissues of AQ pigs were markedly higher than those in LW pigs (p<0.01). Conclusion: LEPR is a potential target of SSC-miR-323, and TCONS_00010987 might act as a sponge for SSC-miR-323 to regulate LEPR expression.

Expression of Smoothened Protein in Colon Cancer and its Prognostic Value for Postoperative Liver Metastasis

Ding, Yin-Lu,Wang, Qi-San,Zhao, Wei-Min,Xiang, Lei Asian Pacific Journal of Cancer Prevention 2012 Asian Pacific journal of cancer prevention Vol.13 No.8

Backgrouds: The hedgehog (Hh) signaling pathway is composed of patched (PTCH) and smoothened (SMO), two transmembrane proteins, and downstream glioma-associated oncogene homolog (Gli) transcription factors. Hh signaling plays a pathological role in the occurrence and development of various cancers. Methods: To investigate the expression of SMO protein in colon cancer and its association with clinicopathological parameters and postoperative liver metastasis, immunohistochemistry was performed with paraffin-embedded specimens of 96 cases. Relationships between SMO protein expression and clinicopathological parameters, postoperative liver metastasis were analyzed. Results: IHC examination showed that SMO protein expression was significantly increased in colon cancer tissues compared to normal colon tissues (P = 0.042), positively related to lymph node metastases (P = 0.018) and higher T stages (P = 0.026). Postoperative live metastasis-free survival was significantly longer in the low SMO expression group than in those with high SMO expression ($48.7{\pm}8.02$ months vs $28.0{\pm}6.86$ months, P=0.036). Multivariate analysis showed SMO expression level to be an independent prognostic factor for postoperative live metastasis-free survival (95% confidence interval [CI] =1.46-2.82, P = 0.008). Conclusions: Our results suggest that in patients with colon cancer, the SMO expression level is an independent biomarker for postoperative liver metastasis, and SMO might play an important role in colon cancer progression.

Polymorphism, Expression of Natural Resistance-associated Macrophage Protein 1 Encoding Gene (NRAMP1) and Its Association with Immune Traits in Pigs

Ding, Xiaoling,Zhang, Xiaodong,Yang, Yong,Ding, Yueyun,Xue, Weiwei,Meng, Yun,Zhu, Weihua,Yin, Zongjun Asian Australasian Association of Animal Productio 2014 Animal Bioscience Vol.27 No.8

Natural resistance-associated macrophage protein 1 encoding gene (NRAMP1) plays an important role in immune response against intracellular pathogens. To evaluate the effects of NRAMP1 gene on immune capacity in pigs, tissue expression of NRAMP1 mRNA was observed by real time quantitative polymerase chain reaction (PCR), and the results revealed NRAMP1 expressed widely in nine tissues. One single nucleotide polymorphism (SNP) (ENSSSCG00000025058: g.130 C>T) in exon1 and one SNP (ENSSSCG00000025058: g.657 A>G) in intron1 region of porcine NRAMP1 gene were demonstrated by DNA sequencing and PCR-RFLP analysis. A further analysis of SNP genotypes associated with immune traits including contain of white blood cell (WBC), granulocyte, lymphocyte, monocyte (MO), rate of cytotoxin in monocyte (MC) and $CD4^-CD8^+$ T lymphocyte subpopulations in blood was carried out in four pig populations including Large White and three Chinese indigenous breeds (Wannan Black, Huai pig and Wei pig). The results showed that the SNP (ENSSSCG00000025058: g.130 C>T) was significantly associated with level of WBC % (p = 0.031), MO% (p = 0.024), MC% (p = 0.013) and $CD4^-CD8^+$ T lymphocyte (p = 0.023). The other SNP (ENSSSCG00000025058: g.657 A>G) was significantly associated with the level of MO% (p = 0.012), MC% (p = 0.019) and $CD4^-CD8^+$ T lymphocyte (p = 0.037). These results indicate that the NRAMP1 gene can be regarded as a molecular marker for genetic selection of disease susceptibility in pig breeding.

Relationship between porcine miR-20a and its putative target low-density lipoprotein receptor based on dual luciferase reporter gene assays

Yueyun Ding,Shujiao Zhu,Chaodong Wu,Li Qian,DengTao Li,Li Wang,Yuanlang Wang,Wei Zhang,Min Yang,Jian Ding,Xudong Wu,Xiao-Dong Zhang,Yafei Gao,Zongjun Yin 아세아·태평양축산학회 2019 Animal Bioscience Vol.32 No.7

Objective: Mutations in low-density lipoprotein receptor (LDLR), which encodes a critical protein for cholesterol homeostasis and lipid metabolism in mammals, are involved in cardiometabolic diseases, such as familial hypercholesterolemia in pigs. Whereas microRNAs (miRNAs) can control LDLR regulation, their involvement in circulating cholesterol and lipid levels with respect to cardiometabolic diseases in pigs is unclear. We aimed to identify and analyze LDLR as a potential target gene of SSC-miR-20a. Methods: Bioinformatic analysis predicted that porcine LDLR is a target of SSC-miR-20a. Wild-type and mutant LDLR 3′-untranslated region (UTR) fragments were generated by polymerase chain reaction (PCR) and cloned into the pGL3-Control vector to construct pGL3 Control LDLR wild-3′-UTR and pGL3 Control LDLR mutant-3′-UTR recombinant plasmids, respectively. An miR-20a expression plasmid was constructed by inserting the porcine pre-miR-20a-coding sequence between the HindIII and BamHI sites in pMR-mCherry, and constructs were confirmed by sequencing. HEK293T cells were co-transfected with the miR-20a expression or pMR-mCherry control plasmids and constructs harboring the corresponding 3′-UTR, and relative luciferase activity was determined. The relative expression levels of miR-20a and LDLR mRNA and their correlation in terms of expression levels in porcine liver tissue were analyzed using reverse-transcription quantitative PCR. Results: Gel electrophoresis and sequencing showed that target gene fragments were successfully cloned, and the three recombinant vectors were successfully constructed. Compared to pMR-mCherry, the miR-20a expression vector significantly inhibited wild-type LDLR-3′-UTR-driven (p<0.01), but not mutant LDLR-3′-UTR-driven (p>0.05), luciferase reporter activity. Further, miR-20a and LDLR were expressed at relatively high levels in porcine liver tissues. Pearson correlation analysis revealed that porcine liver miR-20a and LDLR levels were significantly negatively correlated (r = –0.656, p<0.05). Conclusion: LDLR is a potential target of miR-20a, which might directly bind the LDLR 3′-UTR to post-transcriptionally inhibit expression. These results have implications in understanding the pathogenesis and progression of porcine cardiovascular diseases.

Nonionic Amphiphilic Surfactant Conjuncted Polyethyleneimine as a New and Highly Efficient Non-viral Gene Carrier

Yin, Dongfeng,Chu, Cang,Ding, Xueying,Gao, Jing,Zou, Hao,Gao, Shen The Polymer Society of Korea 2009 Macromolecular Research Vol.17 No.1

In order to enhance the gene delivery efficiency and decrease the cytotoxicity of polyplexes, we synthesized Solutol-g-PEI by conjugating polyethyleneimine (PEI) to Solutol (polyoxyethylene (10) stearate), and evaluated its efficiency as a possible nonviral gene carrier candidate. Structural analysis of synthesized polymer was performed by using $^1H$-NMR. Gel retardation assay, particle sizes and zeta potential measurement confirmed that the new gene carrier formed a compact complex with plasmid DNA. The complexes were smaller than 150 nm, which implicated its potential for intracellular delivery. It showed lower cytotoxicity in three different cell lines (Hela, MCF-7, and HepG2) than PEI 25 kDa. pGL3-lus was used as a reporter gene, and the transfection efficiency was in vitro measured in Hela cells. Solutol-g-PEI showed much higher transfection efficiency than unmodified PEI 25 kDa.

Methodology of Mapping Quantitative Trait Loci for Binary Traits in a Half-sib Design Using Maximum Likelihood

Yin, Zongjun,Zhang, Qin,Zhang, Jigang,Ding, Xiangdong,Wang, Chunkao Asian Australasian Association of Animal Productio 2005 Animal Bioscience Vol.18 No.12

Maximum likelihood methodology was applied to analyze the efficiency and statistical power of interval mapping by using a threshold model. The factors that affect QTL detection efficiency (e.g. QTL effect, heritability and incidence of categories) were simulated in our study. Daughter design with multiple families was applied, and the size of segregating population is 500. The results showed that the threshold model has a great advantage in parameters estimation and power of QTL mapping, and has nice efficiency and accuracy for discrete traits. In addition, the accuracy and power of QTL mapping depended on the effect of putative quantitative trait loci, the value of heritability and incidence directly. With the increase of QTL effect, heritability and incidence of categories, the accuracy and power of QTL mapping improved correspondingly.

S100A4 Gene is Crucial for Methionine-Choline-Deficient Diet-Induced Non-Alcoholic Fatty Liver Disease in Mice

Yin-Hua Zhang,De-Ping Ding,De-Qiang Ma,Juan Li,Lin-Li Chen,Kang-Jian Ao,You-You Tian 연세대학교의과대학 2018 Yonsei medical journal Vol.59 No.9

Purpose: To explore the influence of S100 calcium binding protein A4 (S100A4) knockout (KO) on methionine-choline-deficient(MCD) diet-induced non-alcoholic fatty liver disease (NAFLD) in mice. Materials and Methods: S100A4 KO mice (n=20) and their wild-type (WT) counterparts (n=20) were randomly divided into KO/MCD, Ko/methionine-choline-sufficient (MCS), WT/MCD, and WT/MCS groups. After 8 weeks of feeding, blood lipid and liverfunction-related indexes were measured. HE, Oil Red O, and Masson stainings were used to observe the changes of liver histopathology. Additionally, expressions of S100A4 and proinflammatory and profibrogenic cytokines were detected by qRT-PCR andWestern blot, while hepatocyte apoptosis was revealed by TUNEL staining. Results: Serum levels of aminotransferase, aspartate aminotransferase, triglyceride, and total cholesterol in mice were increasedafter 8-week MCD feeding, and hepatocytes performed varying balloon-like changes with increased inflammatory cell infiltrationand collagen fibers; however, these effects were improved in mice of KO/MCD group. Meanwhile, total NAFLD activity scoresand fibrosis were lower compared to WT+MCD group. Compared to WT/MCS group, S100A4 expression in liver tissue of WT/MCD group was enhanced. The expression of proinflammatory (TNF-α, IL-1β, IL-6) and profibrogenic cytokines (TGF-β1, COL1A1,α-SMA) in MCD-induced NAFLD mice were increased, as well as apoptotic index (AI). For MCD group, the expressions ofproinflammatory and profibrogenic cytokines and AI in KO mice were lower than those of WT mice. Conclusion: S100A4 was detected to be upregulated in NAFLD, while S100A4 KO alleviated liver fibrosis and inflammation, inaddition to inhibiting hepatocyte apoptosis.

Variants on ESR1 and their Association with Prostate Cancer Risk: A Meta-analysis

Ding, Xiang,Cui, Feng-Mei,Xu, Song-Tao,Pu, Jin-Xian,Huang, Yu-Hua,Zhang, Jiang-Lei,Wei, Xue-Dong,Hou, Jian-Quan,Yan, Chun-Yin Asian Pacific Journal of Cancer Prevention 2012 Asian Pacific journal of cancer prevention Vol.13 No.8

Background: Epidemiological studies evaluating the association of two variants rs9340799 and rs2234693 on estrogen receptor 1 (ESR1) with prostate risk have generated inconsistent results. Methods: A meta-analysis was here conducted to systematically evaluate the relationship of these two variants with prostate cancer susceptibility. Results: For rs9340799, heterozygosity of T/C carriers showed a significant increased prostate cancer risk with a pooled odds ratio (OR) of 1.34 (95% CI = 1.06-1.69) while homozygote C/C carriers showed an increased but not statistically significant association with prostate cancer risk (pooled OR = 1.29, 95% CI = 0.94-1.79). Compared to the homozygous TT carriers, the allele C carriers showed a 31% increased risk for prostate cancer (pooled OR = 1.31, 95% CI = 1.06-1.63). No significant association between the rs2234693 and prostate cancer risk was found with the pooled OR of 1.15 (95% CI = 0.97-1.39, T/C and C/C vs. T/T) under the dominant genetic model. Compared to the homozygote T/T carriers, the heterozygous T/C carriers did not show any significantly different risk of prostate cancer (pooled OR = 1.13, 95% CI = 0.94-1.36) and the homozygous C/C carriers also did not show a significant change for prostate cancer risk compared to the wide-type T/T carriers (pooled OR = 1.26, 95% CI = 0.98-1.62). Conclusion: These data suggested that variant rs9340799, but not rs2234693, on ESR1 confers an elevated risk of prostate cancer.

Transcriptome analysis of male and female mature gonads of Japanese scallop Patinopecten yessonsis

Jun Ding,Dan Yang,Chao Yin,Yaqing Chang,Yan Dou,Zhenlin Hao 한국유전학회 2016 Genes & Genomics Vol.38 No.11

The Japanese scallop Patinopecten yessoensis is an important commercial culture shellfish in China and Japan. In addition to its commercial interest, it has also attracted much attention because of its value in studying sex determination and differentiation mechanisms. Herein, two transcriptome libraries from male and female gonads at maturing stage were constructed. The two libraries were paired-end sequenced using Illumina sequencing techniques. A total of 81,986,898 cleaned reads were obtained, and assembled into 29,897 unigenes by using Trinity software and removing redundancy. Compared with the public Swiss-Prot and NR databases by BlastX, 9354 unigenes were significantly matched to known unique proteins. As determined by GO annotation and KEGG pathway mapping, functional annotation of the unigenes uncovered diverse biological functions and processes. After comparing the two transcriptome libraries, 8652 upregulated and 2973 down-regulated genes were identified in the male gonads. According to annotation information, at least 30 genes related to sex determination and differentiation, such as Dmrt1, Sox9, fem1 and vasa, were identified and characterized. The expression patterns of the random eight genes were then validated by quantitative real-time PCR (qRT-PCR) suggesting the high reliability of RNA-Seq results. The study provides an archive for further studies of molecular mechanisms of bivalve sex determination and differentiation.