Identification and expression analysis of TCP family members in tobacco (Nicotiana tabacum L.) / 生物工程学报

TCP family as plant specific transcription factor, plays an important role in different aspects of plant development. In order to screen TCP family members in tobacco, the homologous sequences of tobacco and Arabidopsis TCP family were identified by genome-wide homologous alignment. The physicochemical properties, phylogenetic relationships and cis-acting elements were analyzed by bioinformatics. The homologous genes of AtTCP3/AtTCP4 were screened, and RT-qPCR was used to detect the changes of gene expression upon 20% PEG6000 treatment. The results show that tobacco contains 63 TCP family members. Their amino acid sequence length ranged from 89 aa to 596 aa, and their protein hydropathicity grand average of hydropathicity (GRAVY) ranged from -1.147 to 0.125. The isoelectric point (pI) ranges from 4.42 to 9.94, the number of introns is 0 to 3, and the subcellular location is all located in the nucleus. The results of conserved domain and phylogenetic relationship analysis showed that the tobacco TCP family can be divided into PCF, CIN and CYC/TB1 subfamilies, and each subfamily has a stable sequence. The results of cis-acting elements in gene promoter region showed that TCP family genes contain low docile acting elements (LTR) and a variety of stress and metabolic regulation related elements (MYB, MYC). Analysis of gene expression patterns showed that AtTCP3/AtTCP4 homologous genes (NtTCP6, NtTCP28, NtTCP30, NtTCP33, NtTCP42, NtTCP57, NtTCP63) accounted for 20% PEG6000 treatment significantly up-regulated/down-regulated expression, and NtTCP30 and NtTCP57 genes were selected as candidate genes in response to drought. The results of this study analyzed the TCP family in the tobacco genome and provided candidate genes for the study of drought-resistance gene function and variety breeding in tobacco.

Identification and expression analysis of citrate synthase 3 gene family members in apple / 生物工程学报

As one of the key enzymes in cell metabolism, the activity of citrate synthase 3 (CS3) regulates the substance and energy metabolism of organisms. The protein members of CS3 family were identified from the whole genome of apple, and bioinformatics analysis was performed and expression patterns were analyzed to provide a theoretical basis for studying the potential function of CS3 gene in apple. BLASTp was used to identify members of the apple CS3 family based on the GDR database, and the basic information of CS3 protein sequence, subcellular localization, domain composition, phylogenetic relationship and chromosome localization were analyzed by Pfam, SMART, MEGA5.0, clustalx.exe, ExPASy Proteomics Server, MEGAX, SOPMA, MEME, WoLF PSORT and other software. The tissue expression and inducible expression characteristics of 6 CS3 genes in apple were determined by acid content and real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). Apple CS3 gene family contains 6 members, and these CS3 proteins contain 473-608 amino acid residues, with isoelectric point distribution between 7.21 and 8.82. Subcellular localization results showed that CS3 protein was located in mitochondria and chloroplasts, respectively. Phylogenetic analysis divided them into 3 categories, and the number of genes in each subfamily was 2. Chromosome localization analysis showed that CS3 gene was distributed on different chromosomes of apple. The secondary structure of protein is mainly α-helix, followed by random curling, and the proportion of β-angle is the smallest. The 6 members were all expressed in different apple tissues. The overall expression trend from high to low was the highest relative expression content of MdCS3.4, followed by MdCS3.6, and the relative expression level of other members was in the order of MdCS3.3 > MdCS3.2 > MdCS3.1 > MdCS3.5. qRT-PCR results showed that MdCS3.1 and MdCS3.3 genes had the highest relative expression in the pulp of 'Chengji No. 1' with low acid content, and MdCS3.2 and MdCS3.3 genes in the pulp of 'Asda' with higher acid content had the highest relative expression. Therefore, in this study, the relative expression of CS3 gene in apple cultivars with different acid content in different apple varieties was detected, and its role in apple fruit acid synthesis was analyzed. The experimental results showed that the relative expression of CS3 gene in different apple varieties was different, which provided a reference for the subsequent study of the quality formation mechanism of apple.

Identification and expression analysis of cellulose synthase family genes in Aquilaria sinensis / 药学学报

Cellulose synthase (CesA), one of the key enzymes in the biosynthesis of cellulose in plants, plays an important role in plant growth and plant resistance. In this study, a total of 21 AsCesA genes from Aquilaria sinensis were systematically identified and the physico-chemical characteristics were analyzed based on genome database and bioinformatical methods. The phylogenetic tree was constructed and the gene location on chromosome, cis-acting elements in the 2 000 basepairs upstream regulatory regions and conservative motifs were analyzed. The AsCesA proteins were mainly located on the plasma membrane. The number of amino acids of the proteins ranged from 390 to 1 261. The isoelectric point distributed from 5.67 to 8.86. All of the 21 AsCesA proteins possessed the transmembrane domains, the number of which was from 6 to 8. The genes were classified into 3 groups according to the phylogenetic relationship. Obvious differences were observed in motif composition in genes from different groups. However, motif2, motif6, motif7 and motif10 were observed in all of AsCesA proteins. Analysis of cis-acting elements indicated that AsCesA genes family has cis-acting elements related to plant hormones, abiotic stresses, and biological processes. Seven AsCesA genes with differential expression were selected according to the calli transcriptome data induced by NaCl at different times and their expression levels under different abiotic stresses were analyzed by quantitative real-time PCR. The results indicated that salt, low temperature, drought, and heavy metal stresses could affect the expression level of AsCesA genes, and the abundance of AsCesA1, AsCesA3 and AsCesA20 showed a significant change, implying their potential important roles to the abiotic stresses. The accumulation pattern of cellulose content under different abiotic stresses was similar to the expression trend of AsCesA genes. Our results provide valuable insights into the role of cellulose synthase in A.sinensis in plant defense.

Cloning and catalytic analysis of Isatis indigotica chalcone isomerase in vitro / 中国中药杂志

Chalcone isomerase is a key rate-limiting enzyme in the biosynthesis of flavonoids in higher plants, which determines the production of flavonoids in plants. In this study, RNA was extracted from different parts of Isatis indigotica and reverse-transcribed into cDNA. Specific primers with enzyme restriction sites were designed, and a chalcone isomerase gene was cloned from I. indigotica, named IiCHI. IiCHI was 756 bp in length, containing a complete open reading frame and encoding 251 amino acids. Homology analysis showed that IiCHI was closely related to CHI protein of Arabidopsis thaliana and had typical active sites of chalcone isomerase. Phylogenetic tree analysis showed that IiCHI was classified into type Ⅰ CHI clade. Recombinant prokaryotic expression vector pET28a-IiCHI was constructed and purified to obtain IiCHI recombinant protein. In vitro enzymatic analysis showed that the IiCHI protein could convert naringenin chalcone into naringenin, but could not catalyze the production of liquiritigenin by isoliquiritigenin. The results of real-time quantitative polymerase chain reaction(qPCR) showed that the expression level of IiCHI in the aboveground parts was higher than that in the underground parts and the expression level was the highest in the flowers of the aboveground parts, followed by leaves and stems, and no expression was observed in the roots and rhizomes of the underground parts. This study has confirmed the function of chalcone isomerase in I. indigotica and provided references for the biosynthesis of flavonoid components.

Cloning and expression analysis of cytochrome P450 AsCYP71D1 from Aquilaria sinensis (Lour.) Gilg / 药学学报

Cytochrome P450 (CYP450) is a kind of superfamily oxidase containing heme, which is distributed in various aerobic organisms. They are widely involved in the biosynthesis of terpenoids, alkaloids, flavonoids, fatty acids, etc. In this study, the full-length cDNA sequence of a P450 was cloned by reverse transcription-PCR (RT-PCR) and rapid amplification of cDNA ends (RACE) technology, with the specific primers that designed according to the sequence of a transcript annotated as P450 from the Aquilaria sinensis (Lour.) Gilg transcriptome database. The tissue expression and subcellular localization were also studied. The full-length cDNA of the cloned P450 gene is 1 920 bp, with 88 bp 5′-untranslated region (UTR), 344 bp 3′-UTR and a 21 bp polyA tail, and 1 488 bp open reading frame (ORF), encoding 495 amino acids. Sequence alignment revealed that the protein belonged to CYP71D family of cytochrome P450 family, and named AsCYP71D1. Tissue expression analysis indicated that AsCYP71D1 was mainly expressed in stem. Further subcellular localization of onion epidermis showed that AsCYP71D1 was expressed in cytoplasm, nucleus and cell membrane. This study will provide a foundation for further research on its function in agarwood sesquiterpene biosynthesis.

Identification and Expression Analysis of AP2/ERF Family in Stress Responses of Pinellia ternata Based on Transcriptome Data / 中国实验方剂学杂志

ObjectiveTo identify the functions of the AP2/ERF family members in Pinellia ternata and promote the genetic improvement of P. ternata varieties. MethodWe identified and conducted a systematic bioinformatics analysis of the AP2/ERF family member genes in P. ternata based on the three generations of transcriptome data. Real-time polymerase Chain reaction　（Real-time） PCR was employed to determine the expression pattern of AP2/ERF genes in different tissues and under different stress conditions. ResultA total of eight full-length AP2/ERF family members were identified from the transcriptome data， which were classified into three sub-gene families： AP2， ERF， and DREB. The deduced AP2/ERF proteins in P. ternata had the length of 251-512 aa， the theoretical pI of 5.29-11.72， the instability index of 45.90-82.41， subcellular localization in the nucleus， and conserved domains and motifs. AP2/ERF genes were expressed in different tissues of P. ternata， with high expression levels in the leaf. The stress response experiments showed that PtERF1 mainly responded to NaCl stress. The expression of PtERF2 and PtERF4 was significantly up-regulated under low temperature and polyethylene glycol （PEG）-simulated stress. PtERF3 responded to both low temperature and NaCl stress. The expression of PtERF5 was induced by high temperature， low temperature， NaCl and PEG stress. The expression of PtERF7 was up-regulated under high temperature， while that of PtERF8 under low temperature. ConclusionThe AP2/ERF genes in P. ternata can respond to stress and have the potential functions of regulating photosynthesis and improving root stress resistance.

Cloning and Expression Patterns of Coumarate-3-hydroxylase Gene from Angelica sinensis and Its Correlation with Content of Ferulic Acid / 中国实验方剂学杂志

ObjectiveTo clone coumarate-3-hydroxylase gene （C3H） from Angelica sinensis， and analyze the correlation between its bioinformatics， expression patterns and content of ferulic acid， and to explore the functions of ASC3H. MethodReal-time polymerase chain reaction （Real-time PCR） was used to clone the full-length cDNA of ASC3H based on the transcriptome dataset of A. sinensis， and the bioinformatics analysis of the gene sequence was carried out. Real-time PCR and high performance liquid chromatography （HPLC） were used to determine relative expression of ASC3H and content of ferulic acid in different root tissues of A. sinensis （periderm， cortex and stele）. ResultThe open reading frame （ORF） of ASC3H （GenBank accession number： MN2550298） was 1 530 bp， encoding 509 amino acids， with a theoretical molecular weight of 57.86 kDa and an isoelectric point of 8.36. It was a hydrophilic protein that was located in the chloroplast with multiple phosphorylation sites and a transmembrane region， and contained a conserved domain CGYDWPKGYGPIINVW_P450 （383-399 aa） in cytochrome P450. Multiple amino acid sequence alignment analysis showed that ASC3H had high similarity with C3H from other plants， especially Ammi majus in Umbelliferae. The Real-time PCR revealed that ASC3H had different expressions in periderm， cortex and stele tissues of A. sinensis roots. It was found from HPLC that the cortex tissues had the highest content of ferulic acid， and the stele tissues had the lowest. ConclusionASC3H was successfully cloned from A. sinensis， and its sequence characteristics were understood more clearly， suggesting that ASC3H might be involved in the ferulic acid biosynthesis pathway of A. sinensis. This paper provided a basis for further studying the functions of the gene and exploring the biosynthesis and regulation mechanism of ferulic acid in A. sinensis， while laying the foundation for the genetic improvement of A. sinensis．

Cloning and expression analysis of JrGI gene in walnut / 生物工程学报

GI (GIGANTEA) is one of the output key genes for circadian clock in the plant. The JrGI gene was cloned and its expression in different tissues was analyzed to facilitate the functional research of JrGI. RT-PCR (reverse transcription-polymerase chain reaction) was used to clone JrGI gene in present study. This gene was then analyzed by bioinformatics, subcellular localization and gene expression. The coding sequence (CDS) full length of JrGI gene was 3 516 bp, encoding 1 171 amino acids with a molecular mass of 128.60 kDa and a theoretical isoelectric point of 6.13. It was a hydrophilic protein. Phylogenetic analysis showed that JrGI of 'Xinxin 2' was highly homologous to GI of Populus euphratica. The result of subcellular localization showed that JrGI protein was located in nucleus. The JrGI, JrCO and JrFT genes in female flower buds undifferentiated and early differentiated of 'Xinxin 2' were analyzed by RT-qPCR (real-time quantitative PCR). The results showed that the expression of JrGI, JrCO and JrFT genes were the highest on morphological differentiation, implying the temporal and special regulation of JrGI in the differential process of female flower buds of'Xinxin 2'. In addition, RT-qPCR analysis showed that JrGI gene was expressed in all tissues examined, whereas the expression level in leaves was the highest. It is suggested that JrGI gene plays a key role in the development of walnut leaves.

Cloning and expression analysis of superoxide dismutase SmMSD2 gene from Salvia miltiorrhiza / 药学学报

Superoxide dismutase (SOD) is a key enzyme that scavenge superoxide anion free radical (O2·-) in vivo, and plays an important role in plant growth and development and stress. In this study, according to the genome and transcriptome data of Salvia miltiorrhizae, 9 SOD genes were identified and the expression patterns of SOD family genes were further analyzed, including 5 Cu/Zn-SOD, 2 Fe-SOD and 2 Mn-SOD. On the basis of proteomic analysis, combined with transcriptome data, one full-length cDNA of Mn-SOD gene, namely SmMSD2 was cloned from Salvia miltiorrhizae. The results of amino acid sequence alignment and phylogenetic analysis showed that SmMSD2 protein belongs to the manganese superoxide dismutase (Mn-SOD) subfamily, and SmMSD2 protein shares high sequence identity with the Mn-SOD proteins of various plants that all contain a C-terminal conserved metal-binding domain "DVWEHAYY". The prokaryotic expression vector pMAL-c2X-SmMSD2 was constructed and transformed into E. coli BL21 expressing strain, and the target recombinant protein was successfully induced and its enzymatic properties were analyzed. Spatiotemporal expression analysis showed that SmMSD2 gene was expressed in all tissues, indicating that SmMSD2 gene was constitutively expressed at a stable level. Real-time quantitative PCR indicated that drought (15% PEG6000), abscisic acid (ABA) and indole-3-acetic acid (IAA) could induce the expression of SmMSD2 gene, suggesting that SmMSD2 may be involved in the response of Salvia miltiorrhizae to abiotic stress such as drought, as well as the signaling pathways of phytohormone ABA and IAA. These results lay the foundation for further elucidating the involvement of superoxide dismutase in the stress response and accumulation of active components of Salvia miltiorrhiza.

Genome-wide identification of the BmAKR gene family in the silkworm (Bombyx mori) and their expression analysis in diapause eggs and nondiapause eggs / 生物工程学报

The aldo-keto reductase super family (AKRs) has a wide range of substrate specificity. However, the systematic identification of insect AKR gene family members has not been reported. In this study, bioinformatics methods were used to predict the phylogenetic evolution, physical and chemical properties, chromosome location, conserved motifs, and gene structure of AKR family members in Bombyx mori (BmAKR). Transcriptome data or quantitative real time polymerase chain reaction (qRT-PCR) were used to analyze the expression level of BmAKR genes during different organizational periods and silkworm eggs in different developmental states. Moreover, Western blotting was used to detect the expression level of the BmAKR in silkworm eggs. The results showed that 11 BmAKR genes were identified. These genes were distributed on 4 chromosomes of the silkworm genome, all of which had the (α/β) 8-barrel motif, and their physical and chemical characteristics were relatively similar. Phylogenetic analysis showed that the BmAKR genes could be divided into 2 subgroups (AKR1 and AKR2). Transcriptome data analysis showed that the expression of BmAKR genes were quite different in different tissues and periods. Moreover, the expression analysis of BmAKR genes in silkworm eggs showed that some genes were expressed significantly higher in nondiapause eggs than in diapause eggs; but another gene, BmAKR1-1, was expressed significantly higher in diapause eggs than in nondiapause eggs. The detection of protein level found that the difference trend of BmAKR1-1 in diapause eggs and non-diapause eggs was consistent with the results of qRT-PCR. In conclusion, BmAKR1-1 was screened out as candidates through the identification and analysis of the BmAKR genes in silkworm, which may regulate silkworm egg development is worthy of further investigation.

Genome-wide identification of the banana GLR gene family and its expression analysis in response to low temperature and abscisic acid/methyl jasmonate / 生物工程学报

Glutamate receptor-like (GLR) is an important class of Ca2+ channel proteins, playing important roles in plant growth and development as well as in response to biotic and abiotic stresses. In this paper, we performed genome-wide identification of banana GLR gene family based on banana genomic data. Moreover, we analyzed the basic physicochemical properties, gene structure, conserved motifs, promoter cis-acting elements, evolutionary relationships, and used real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) to verify the expression patterns of some GLR family members under low temperature of 4 ℃ and different hormone treatments. The results showed that there were 19 MaGLR family members in Musa acuminata, 16 MbGLR family members in Musa balbisiana and 14 MiGLR family members in Musa itinerans. Most of the members were stable proteins and had signal peptides, all of them had 3-6 transmembrane structures. Prediction of subcellular localization indicated that all of them were localized on the plasma membrane and irregularly distributed on the chromosome. Phylogenetic analysis revealed that banana GLRs could be divided into 3 subclades. The results of promoter cis-acting elements and transcription factor binding site prediction showed that there were multiple hormone- and stress-related response elements and 18 TFBS in banana GLR. RT-qPCR analysis showed that MaGLR1.1 and MaGLR3.5 responded positively to low temperature stress and were significantly expressed in abscisic acid/methyl jasmonate treatments. In conclusion, the results of this study suggest that GLR, a highly conserved family of ion channels, may play an important role in the growth and development process and stress resistance of banana.

Cloning and expression analysis of wpHSP90 gene from Whitmania pigra at different temperatures / 中国中药杂志

HSP90 is a widely distributed molecular chaperone that participates in a variety of cellular processes and plays an important role in the meiosis of germ cells. However, its role in the gonadal development of hermaphroditic Whitmania pigra is not yet clear. To explore the effect of HSP90 on the germ cell development of Wh. Pigra, this study cloned the wpHSP90 gene, performed bioinformatics analysis, and measured its expression levels. The results showed that the cloned wpHSP90 was 2 592 bp in length, with an open reading frame(ORF) of 2 373 bp, encoding 790 amino acids. Prediction analysis revealed 85 phosphorylation modification sites on serine, threonine, and tyrosine residues of the wpHSP90 protein. Structural domain prediction and multiple sequence alignment results showed that wpHSP90 contained two conserved domains of HSP90 and exhibited the highest homology with Helobdella robusta, with a sequence similarity of 80.72%. RT-qPCR results showed that the relative expression level of wpHSP90 in the gonads of 5-month-old Wh. pigra was positively correlated with temperature within the range of 12 ℃ to 28 ℃. The expression level in the female gonads was significantly higher than in the male gonads and correlated with the trend of germ cell development in the ovaries and testes. In conclusion, wpHSP90 may be involved in regulating the development of germ cells, particularly oocytes, in Wh. pigra. This study provides a reference for further research on the gonadal development mechanism in Wh. pigra.

Screening and expression analysis of transcription factors involved in genuineness of Codonopsis pilosula in Shanxi / 中国中药杂志

This study aims to mine the transcription factors that affect the genuineness of Codonopsis pilosula in Shanxi based on the transcriptome data of C. pilosula samples collected from Shanxi and Gansu, and then analyze the gene expression patterns, which will provide a theoretical basis for the molecular assisted breeding of C. pilosula. Gene ontology(GO) functional annotation, conserved motif prediction, and gene expression pattern analysis were performed for the differential transcription factors predicted based on the transcriptome data of C. pilosula from different habitats. A total of 61 differentially expressed genes(DEGs) were screened out from the transcriptome data. Most of the DEGs belonged to AP2/ERF-ERF family, with the conserved motif of [2X]-[LG]-[3X]-T-[3X]-[AARAYDRAA]-[3X]-[RG]-[2X]-A-[2X]-[NFP]. Forty-three of the DEGs showed significantly higher gene expression in C. pilosula samples from Shanxi than in the samples from Gansu, including 11 genes in the AP2/ERF-ERF family, 5 genes in the NAC fa-mily, 1 gene in the bHLH family, and 2 genes in the RWP-RK family, while 18 transcription factors showed higher expression levels in the samples from Gansu. GO annotation predicted that most of the DEGs were enriched in GO terms related to transcriptional binding activity(103), metabolic process(26), and stress response(23). The expression of transcription factor genes, CpNAC92, CpNAC100, CpbHLH128, and CpRAP2-7 was higher in the samples from Shanxi and in the roots of C. pilosula. CpNAC92, CpbHLH128, and CpRAP2-7 responded to the low temperature, temperature difference, and iron stresses, while CpNAC100 only responded to low temperature and iron stresses. The screening and expression analysis of the specific transcription factors CpNAC92, CpNAC100, CpbHLH128, and CpRAP2-7 in C. pilosula in Shanxi laid a theoretical foundation for further research on the mechanism of genuineness formation of C. pilosula.

Cloning, expression analysis and enzyme activity verification of dihydroflavonol 4-reductase from Cistanche tubulosa (Schenk) Wight flower / 药学学报

Dihydroflavonol 4-reductase (DFR) plays an essential role in the biosynthesis of anthocyanin and regulation of plant flower color. Based on the transcriptome data of Cistanche tubulosa (Schenk) Wight, a full-length cDNA sequence of CtDFR gene was cloned by reverse transcription-polymerase chain reaction (RT-PCR). CtDFR contains an open reading frame (ORF) of 1 263 bp which encodes 420 amino acids with a predicted molecular weight of 47.5 kDa. The sequence analysis showed that CtDFR contains a nicotinamide adenine dinucleotide phosphate (NADPH) binding domain and a specific substrate binding domain. The expression analysis indicated that CtDFR was highly expressed in red and purple flowers, and the relative expression levels were 4.04 and 19.37 times higher than those of white flowers, respectively. The recombinant CtDFR protein was expressed in E.coli BL21 (DE3) using vector pET-28a-CtDFR and was purified. In vitro enzyme activity analysis, CtDFR could reduce three types of dihydroflavonols including dihydrokaempferol, dihydroquercetin, and dihydromyricetin to leucopelargonidin, leucocyanidin and leucodelphinidin. Subcellular localization analysis showed that CtDFR was mainly localized in the cytoplasm. These results demonstrate that CtDFR plays an important role in regulation of flower color in C. tubulosa and make a valuable contribution for the further investigation on the regulation mechanism of C. tubulosa (Schenk) Wight flower color.

Identification and expression analysis of flavonoid O-methyltransferases gene family in Artemisia argyi / 药学学报

italic>Artemisia argyi (A. argyi) is a Chinese herbal medicine in China. The main active components are volatile oils, flavonoids, and other compounds, which have various pharmacological activities. Methoxylated flavonoids are the main active ingredients in A. argyi. Flavonoid O-methyltransferase (FOMT) is a key enzyme in the O-methylation of flavonoids. In order to further understand the function and characteristics of FOMT proteins, this paper carried out the whole genome mining and identification of FOMT genes in A. argyi and performed phylogenetic, chromosomal localization, gene sequence characterization, subcellular localization prediction, protein structure, gene structure analysis, and expression pattern analysis. The results showed that a total of 83 FOMT genes were identified in the genome of A. argyi. The phylogenetic tree shows that FOMT genes are divided into two subgroups, CCoAOMT (caffeoyl CoA O-methyltransferase) subfamily (32 genes) and COMT (caffeic acid O-methyltransferase) subfamily (51 genes). Gene sequence analysis showed that the number of amino acids encoded by FOMT was 70-734 aa, the molecular weight was 25 296.55-34 241.3 Da, and the isoelectric point was 4.51-9.99. Compared with 32 members of the CCoAOMT subfamily, nearly 1/3 of the 51 members of the COMT subfamily were hydrophobic proteins and 2/3 were hydrophilic proteins. Subcellular localization prediction showed that more than 80% of CCoAOMT subfamily members were located in the cytoplasm, and 96% of COMT subfamily members were located in the chloroplast. COMT subfamily members have more motifs than CCoAOMT subfamily members. The N-terminal motifs of COMT subfamily proteins are relatively variable, while the C-terminal motifs are relatively conserved. Expression pattern analysis showed that CCoAOMT subfamily members were mainly expressed in roots, while COMT members were mainly expressed in leaves. Some FOMTs showed the tissue expression specificity by real-time quantitative PCR analysis, especially in leaves. In this study, we identified and analyzed the FOMT gene family in A. argyi, and provided a theoretical basis for further research on the function of FOMTs and the biosynthesis of methylated flavonoids in A. argyi.

Genome-wide identification of GSK gene family in Senna tora L. and expression analyses / 药学学报

Glycogen synthase kinase 3/SHAGGY-like kinase (GSK3) proteins play important roles in regulating plant growth, development, and stress response. In order to reveal the characteristics of GSK family members in the medicinal plant Senna tora L., in this study, we conducted the identification and expression analyses of GSKs in S. tora based on its whole genome data, combined with bioinformatics and gene expression research methods. The results showed that a total of nine S. tora GSK genes were identified, all of which contained the GSK characteristic kinase domains. All members were distributed on six chromosomes, the encoding amino acid length ranged from 465 to 943 aa, the protein molecular weight was from 33.57 to 88.83 kDa, and the average isoelectric point was 8.2. The StoSKs were divided into four evolutionary branches, and the StoSKs in the same evolutionary branch shared the same exon/intron structure and conserved motifs. The expansion of the StoSKs gene family was mainly due to segment duplication events, and there were 17, 11, 8 and 7 pairs of collinear genes with Glycine max, Medicago truncatula, Arabidopsis thaliana and Oryza sativa, respectively. The promoter regions of StoSKs mostly contained responses elements related to stress stimulation, growth and development, and hormone induction. Transcriptome data analysis showed that StoSKs were expressed in different tissues, with the highest expression level in roots. Quantitative real-time PCR (qRT-PCR) analysis indicated that StoSKs in different evolutionary branches displayed a synergistic expression pattern response to light, and most of StoSKs could rapidly respond to NaCl stress with significantly up-regulated expression. All the results provide a basis for further analysis of the biological functions of the GSKs gene family in S. tora.

Cloning, expression and activity analysises of chalcone synthase genes in Carthamus tinctorius / 中草药·英文版

OBJECTIVE@#Flavonoids are the bioactive compounds in safflower (Carthamus tinctorius), in which chalcone synthase (CHS) is the first limiting enzyme. However, it is unclear that which chalcone synthase genes (CHSs) are participated in flavonoids biosynthesis in C. tinctorius. In this study, the CHSs in the molecular characterization and enzyme activities were investigated.@*METHODS@#Putative chalcone biosynthase genes were screened by the full-length transcriptome sequences data in C. tinctorius. Chalcone biosynthase genes in C. tinctorius (CtCHSs) were cloned from cDNA of flowers of C. tinctorius. The cloned gene sequences were analyzed by bioinformatics, and their expression patterns were analyzed by real-time PCR (RT-PCR). The protein of CtCHS in the development of flowers was detected by polyclonal antibody Western blot. A recombinant vector of CtCHS was constructed. The CtCHS recombinant protein was induced and purified to detect the enzyme reaction (catalyzing the reaction of p-coumaryl-CoA and malonyl-CoA to produce naringin chalcone). The reaction product was detected by HPLC and LC-MS.@*RESULTS@#Two full-length CtCHS genes were successfully cloned from the flowers of safflower (CtCHS1 and CtCHS3), with gene lengths of 1525 bp and 1358 bp, respectively. RT-PCR analysis showed that both genes were highly expressed in the flowers, but the expression of CtCHS1 was higher than that of CtCHS3 at each developmental stage of the flowers. WB analysis showed that only CtCHS1 protein could be detected at each developmental stage of the flowers. HPLC and LC-MS analyses showed that CtCHS1 could catalyze the conversion of p-coumaryl-CoA and malonyl-CoA substrates to naringin chalcone.@*CONCLUSION@#CtCHS1 is involved in the biosynthesis of naringin chalcone in safflower.

Bioinformatics and expression analysis on MYB-related family in Angelica dahurica var. formosana / 中国中药杂志

In recent years, the MYB-related gene family has been found pivotal in plant growth and development. MYB-related gene family in Angelica dahurica var. formosana was systematically investigated based on "Chuanzhi No. 2" through transcriptome database search and bioinformatics and the temporal and spatial expression patterns were analyzed through real-time fluorescence-based quantitative polymerase chain reaction(PCR). The results showed that 122 MYB-related proteins family were identified, mainly including the unstable hydrophilic proteins with good thermal stability. Most of the proteins were located in nuclei. The majority of the proteins had the structures of random coil and α-helix. Five MYB-related proteins family of A. dahurica var. formosana had membrane-binding domains. The conserved domain analysis of MYB-related proteins family of A. dahurica var. formosana showed that the MYB domains of genes in five subgroups, similar to 2 R-, 3 R-, and 4 R-MYB proteins, contained three evenly distributed Trp(W) residues in the MYB repeat sequence. The phylogenetic analysis of MYB-related proteins family in A. dahurica var. formosana and Arabidopsis thaliana showed that the MYB-related members were unevenly distributed in five subgroups, and A. thaliana and A. dahurica var. formosana had almost the same number of genes in the CCA1-like subgroup. There were differences in the number, type, and distribution of motifs contained in 122 encoded proteins. Transcription factors with similar branches had similar domains and motifs. The expression pattern analysis showed that the transcription factors AdMYB53, AdMYB83, and AdMYB89 responded to hormones to varying degrees, and they were highly expressed in leaves and responded quickly in roots. This study lays a foundation for further investigating the function of MYB-related transcription factors of A. dahurica var. formosana and solving the corresponding biological problems such as bolting early.

Cloning and function analysis of chalcone isomerase gene and chalcone synthase gene in Lonicera macranthoides / 中国中药杂志

In order to explore the functions of genes of key rate-limiting enzymes chalcone isomerase(CHI) and chalcone synthase(CHS) in the biosynthesis of flavonoids in Lonicera macranthoides, this study screened and cloned the cDNA sequences of CHI and CHS genes from the transcriptome data of conventional variety and 'Xianglei' of L. macranthoides. Online bioinformatics analysis software was used to analyze the characteristics of the encoded proteins, and quantitative reverse-transcription polymerase chain reaction(qRT-PCR) to detect the expression of CHI and CHS in different parts of the varieties at different flowering stages. The content of luteo-loside was determined by high performance liquid chromatography(HPLC) and the correlation with the expression of the two genes was analyzed. The results showed that the CHI and CHS of the two varieties contained a 627 bp and 1170 bp open reading frame(ORF), respectively, and the CHI protein and CHS protein were stable, hydrophilic, and non-secretory. qRT-PCR results demonstrated that CHI and CHS of the two varieties were differentially expressed in stems and leaves at different flowering stages, particularly the key stages. Based on HPLC data, luteoloside content was in negative correlation with the relative expression of the genes. Thus, CHI and CHS might regulate the accumulation of flavonoids in L. macranthoides, and the specific functions should be further studied. This study cloned CHI and CHS in L. macranthoides and analyzed their expression for the first time, which laid a basis for investigating the molecular mechanism of the differences in flavonoids such as luteoloside in L. macranthoides and variety breeding.

Expression Analysis of miRNA Profiles in Apheresis Platelets during Storage / 中国实验血液学杂志

OBJECTIVE@#To study the expression profiles changes of miRNA in apheresis platelets after 1, 3 and 5 days of storage.@*METHODS@#The apheresis platelets were collected from 20 volunteer blood donors. After mixing fully, the platelets were stored in a shaker with (22±2) ℃ horizontal oscillation. The samples were taken on the 1st, 3rd and 5th day, and used to sequence for miRNAs by DNA nanoball (DNB) sequencing technology, which were named as C_1, C_3 and C_5, respectively. The expression level of platelets miRNA was standardized by transcripts per kilobase million (TPM) algorithm. MiRNAs with P-value < 0.001 and the expression difference of more than two times were considered as significant difference between two groups. The expression of miRNAs was verified by real-time fluorescence quantitative PCR (RT-qPCR).@*RESULTS@#By DNB sequencing, there were 688, 730, and 679 platelet miRNAs expressed in C_1, C_3 and C_5 group, respectively. Cluster analysis showed that the expression profile of miRNAs changed significantly. The expression level of the first 20 high abundance miRNAs was about 4/5 of the total amounts of expressed miRNAs in each group, which the top five miRNAs were miR-21-5p, miR-26a-5p, miR-199a-3p, miR-126-3p, and let-7f-5p. The correlation of high abundance platelet miRNAs among the three groups was high (R2=0.876, R2=0.979, R2=0.937, respectively) and the differences were not statistically significant (P>0.05). Compared with the differential expression of platelet miRNAs with more than 1 000 TPM in the C_3 and C_1 group, there were 6 differentially expressed miRNAs, including 3 up-regulated (miR-146a-5p, miR-379-5p, and miR-486-5p) and 3 down-regulated (miR-652-3p, miR-142-5p, and miR-7-5p). While in the C_5 and C_1 group, there were 4 differentially expressed miRNAs, including 2 up-regulated (miR-146a-5p and let-7b-5p) and 2 down-regulated (miR-30d-5p and miR-142-5p). Compared with the differentially expression of platelet miRNAs between 1-1 000 TPM in the C_3 and C_1 group, there were 133 differentially expressed miRNAs, in which 99 were up-regulated and 34 were down-regulated. While in the C_5 and C_1 group, there were 77 differentially expressed miRNAs, in which 31 were up-regulated and 46 were down-regulated. The six selected differentially expressed miRNAs verified by RT-qPCR were consistent with those of sequencing.@*CONCLUSION@#The expression profiles of platelets miRNAs change significantly among 1, 3, and 5 d of storage in vitro.