Showing papers on "Complementary DNA published in 2023"

Gene cloning, expression pattern, and response to dietary total lipids and phospholipids of hormone-sensitive lipase (HSL) in the Oriental river prawn Macrobrachium nipponense De Haan, 1849 (Decapoda: Caridea: Palaemonidae)

Abstract: Hormone-sensitive lipase (HSL) is an important regulator of cellular lipid homeostasis and catalyzes the hydrolysis of stored triacylglycerol. We identified and cloned for the first time the full-length cDNA sequence of HSL of the prawn Macrobrachium nipponense De Haan, 1849 [De Haan, 1833–1850] from a hepatopancreas cDNA library. The complete HSL sequence is 3,575 bp and encoded a 785 amino acid peptide with the catalytic core (GXSXG) containing a serine residue. The phylogenetic tree revealed that the gene of HSL of M. nipponense is closely related with that of Penaeus vanmameiBoone, 1931. The tissue distribution showed that the mRNA expression level of HSL in the hepatopancreas was significantly higher than that in other tissues (P < 0.05). Furthermore, the HSL expression in hepatopancreas was upregulated with the increase of dietary lipids but partially inhibited when the ratio of phospholipids was increased in the lipid mixture. These results demonstrate that HSL is involved in the lipid metabolism of M. nipponense and highlights the importance of phospholipids in lipid metabolism.

A Cold-Inducible RNA Binding Protein Gene from Acanthopagrus schlegelii: Molecular Characterization, Expression, and Association with Apoptosis to Low-Temperature Stress

Abstract: In this study, the complete cDNA sequence (1552 bp) of the cold-inducible RNA binding protein gene (cirbp gene) was successfully cloned from the liver in Acanthopagrus schlegelii (initial weight: 15.0 ± 2.3 g). Results showed that Ascirbp (cirbp gene from A. schlegelii) gene has 24 phosphorylation sites, no signal peptide, and no transmembrane helix structure. AsCIRBP, with a molecular weight of 18.84 ku and an isoelectric point of 9.04 was a stable protein that encodes 182 amino acids. Subcellular localization analysis of this protein showed that it was located in the nucleus. Sequence alignment results showed that the AsCIRBP amino acid sequences of various fishes including black porgy were highly conserved, especially the RNA recognition motif (RRM). Those results of real-time quantitative PCR (qRT-PCR) demonstrated that Ascirbp gene was specifically expressed in the liver tissue of black porgy and its expression was significantly increased under cold stress or cold acclimation. The RNA interference experiment results showed that Ascirbp-dsRNA could suppress the expression of Ascirbp gene in the liver of black porgy through intraperitoneal injection. After silencing the expression of Ascirbp gene, RNAi groups were more severely damaged in the structure of the liver tissue and more prone to apoptosis under cold stress than control groups. The results of the study on the linkage between Ascirbp gene expression and mitochondrial apoptosis pathways showed that changes in the expression of the Ascirbp gene had a significant effect on the expression of key genes of apoptosis. The most striking result from silencing the expression of the Ascirbp gene was that expressions of the bcl-2 and apaf-1 gene in the liver of black porgy decreased significantly, which can block the normal apoptotic process. After the disruption of the normal apoptotic process, the expressions of p53, bax, cyto-c, caspase-9, caspase-3, diablo, and caspase-1 gene were significantly affected. These results suggest that Ascirbp gene can inhibit apoptosis and protect tissue structure in the liver tissue of black porgy at low temperatures.

An efficient electrochemical biosensor for the detection of heavy metal lead in food based on magnetic separation strategy and Y-DNA structure.

Abstract: Herein, an electrochemical biosensor was developed based on a magnetic separation strategy for the sensitive detection of the heavy metal Pb2+. The specific binding of Pb2+ and the aptamer (Apt) is used to trigger the release of the complementary chain (cDNA) on the magnetic bead system. The cDNA completes base complementary pairing with hairpins HP1 and HP2 at the electrode to form a Y-DNA structure. Then, the Y-DNA runs continuously with the assistance of the signal tag methylene blue (MB) and the current signal increases. However, in the absence of Pb2+, cDNA cannot be released and the Y-DNA structure cannot be formed on the electrode, resulting in a relatively low current signal. Under the optimal experimental conditions, the reduced peak current difference (ΔI) showed a good linear relationship with lg CPb2+ between 0.1 and 1000 nM, with a detection limit of 5.9 pM. In addition, the stability, reproducibility and detection capability of the sensors were investigated with satisfactory results.

Extraction of Total DNA and RNA from Marine Filter Samples and Generation of a Universal cDNA as Universal Template for Marker Gene Studies.

Abstract: Microbial communities play an important role in marine ecosystem processes. Although the number of studies targeting marker genes such as the 16S rRNA gene has increased during the last years, the vast majority of marine diversity are rather unexplored. Moreover, most studies focused on the entire microbial community and thus do not assess the active fraction of the microbial community. Here, we describe a detailed protocol for the simultaneous extraction of DNA and RNA from marine water samples and the generation of cDNA from the isolated RNA that can be used as a universal template in various marker gene studies.

Newly identified cytochrome P450 3A genes of tree shrews and pigs are expressed and encode functional enzymes.

Abstract: Novel cytochrome P450 3A5 (CYP3A5) cDNA in tree shrews (which are non-rodent primate-like species) and pig CYP3A227 cDNA were identified, along with known pig CYP3A22, CYP3A29, and CYP3A46 cDNAs. All five cDNAs contained open reading frames encoding a polypeptide of 503 amino acids that shared high sequence identity (72-78 %) with human CYP3A4 and were more closely related to human CYP3As than rat CYP3As by phylogenetic analysis. CYP3A5 was the only CYP3A in the tree shrew genome, but pig CYP3A genes formed a CYP3A gene cluster in the genomic region corresponding to that of human CYP3A genes. Tree shrew CYP3A5 mRNA was predominantly expressed in liver and small intestine, among the tissues analyzed, whereas pig CYP3A227 mRNA was most abundantly expressed in jejunum, followed by liver. Metabolic assays established that tree shrew CYP3A5 and pig CYP3A proteins heterologously expressed in Escherichia coli metabolized typical human CYP3A4 substrates nifedipine and midazolam. These results suggest that novel tree shrew CYP3A5 and pig CYP3A227 were functional enzymes able to metabolize human CYP3A4 substrates in liver and small intestine, similar to human CYP3A4, although pig CYP3A227 mRNA was minimally expressed in all tissues analyzed.

Host defence peptide LEAP2 contributes to antimicrobial activity in a mustache toad (Leptobrachium liui)

Abstract: The liver-expressed antimicrobial peptide 2 (LEAP2) is essential in host immunity against harmful pathogens and is only known to act as an extracellular modulator to regulate embryonic development in amphibians. However, there is a dearth of information on the antimicrobial function of amphibian LEAP2. Hence, a LEAP2 homologue from Leptobrachium liui was identified, characterized, and chemically synthesized, and its antibacterial activities and mechanisms were determined.In this study, LEAP2 gene (Ll-LEAP2) cDNA was cloned and sequenced from the Chong'an Moustache Toad (Leptobrachium liui). The predicted amino acid sequence of Ll-LEAP2 comprises a signal peptide, a mature peptide, and a prodomain. From sequence analysis, it was revealed that Ll-LEAP2 belongs to the cluster of amphibian LEAP2 and displays high similarity to the Tropical Clawed Frog (Xenopus tropicalis)'s LEAP2. Our study revealed that LEAP2 protein was found in different tissues, with the highest concentration in the kidney and liver of L. liui; and Ll-LEAP2 mRNA transcripts were expressed in various tissues with the kidney having the highest mRNA expression level. As a result of Aeromonas hydrophila infection, Ll-LEAP2 underwent a noticeable up-regulation in the skin while it was down-regulated in the intestines. The chemically synthesized Ll-LEAP2 mature peptide was selective in its antimicrobial activity against several in vitro bacteria including both gram-positive and negative bacteria. Additionally, Ll-LEAP2 can kill specific bacteria by disrupting bacterial membrane and hydrolyzing bacterial gDNA.This study is the first report on the antibacterial activity and mechanism of amphibian LEAP2. With more to uncover, the immunomodulatory functions and wound-healing activities of Ll-LEAP2 holds great potential for future research.

Nanopore Direct RNA Sequencing of Monosome- and Polysome-Bound RNA.

Abstract: Polysome fractionation makes use of density gradients and ultracentrifugation to separate transcripts based on their specific number of bound ribosomes, and can be combined with downstream analysis such as cDNA-seq (commonly known as RNA-seq), microarray analysis, RT-qPCR, or Northern blotting. Here, we describe the application of Nanopore direct RNA sequencing to quantify monosome- and polysome-bound full-length transcripts after polysome fractionation, RNA cleanup, and size selection, using the yeast glucose stress response as an example use case.

Cloning, Characterization, and Expression Prolife Analysis of Tyrosinase Genes: Insights into Black Shell Formation in Cyclina sinensis

Abstract: Shell colors that exhibit a positive relationship with excellent traits can be employed as marker colors for breeding new varieties of bivalves. The clam Cyclina sinensis is an economically important marine bivalve that has three main colors: black, white, and purple. In the present study, we cloned and analyzed the full-length cDNA of the tyrosinase gene (TYR), which is the key gene for melanin synthesis, to explore the formation mechanism of black shells. The full-length cDNA of TYR was cloned by RACE-PCR, and the results showed that the full-length cDNA of TYR was 2993 bp, including a 2262 bp open reading frame encoding 753 amino acids. The results of functional domain prediction showed that deduced TYR protein contains a typical functional domain of tyrosinase, the common central domain. Phylogenetic analysis was conducted using neighbor-joining and maximum likelihood methods, and the results indicated that the evolutionary positions of C. sinensis and Meretrix meretrix were closest. The qRT‒PCR results showed that TYR mRNA was highly expressed in the outer edge of the mantle, which suggested that TYR was involved in the synthesis of melanin in the mantle of C. sinensis and might play an important regulatory role in the formation of melanin in black clam shells.

Primary Structure and Conformation of a Tetrodotoxin-Binding Protein in the Hemolymph of Non-Toxic Shore Crab Hemigrapsus sanguineus

Abstract: Tetrodotoxin (TTX)-binding proteins are present in toxic TTX-bearing animals, such as pufferfish and gastropods. These may prevent autotoxicity. However, TTX-binding proteins are also found in the nontoxic marine shore crab, Hemigrapsus sanguineus. Here, we isolated the TTX-binding protein, HSTBP (Hemigrapsus sanguineus TTX-binding protein), from the hemolymph of H. sanguineus and elucidated its primary structure using cDNA cloning. HSTBP, a 400 kDa acidic glycoprotein by gel filtration high-performance liquid chromatography, comprises 3 subunits, 88 kDa (subunit-1), 65 kDa (subunit-2), and 26 kDa (subunit-3) via sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reduced conditions. The open reading frame of the cDNA comprises 5049 base pairs encoding 1683 amino acid residues, and the mature protein contains 1650 amino acid residues from Arg34 to Ser1683. The three subunits are arranged in tandem in the following order: subunit-3 (Arg34-Gln261), subunit-1 (Asp262-Phe1138), and subunit-2 (Val1139-Ser1683). A BLAST homology search showed weak similarity of HSTBP to clotting proteins of crustaceans (29–40%). SMART analysis revealed a von Willebrand factor (vWF)-type (⇒delete hyphen) D domain at Phe1387-Gly1544. We confirmed that the recombinant protein of HSTBP subunit-2 containing the vWF-type (⇒delete hyphen) D domain bound to TTX at a molecular ratio of 1:1.

Molecular Cloning of Toll-like Receptor 2 and 4 (SpTLR2, 4) and Expression of TLR-Related Genes from Schizothorax prenanti after Poly (I:C) Stimulation

Abstract: Toll-like receptor (TLR) signaling is conserved between fish and mammals, except for TLR4, which is absent in most fish. In the present study, we aimed to evaluate whether TLR4 is expressed in Schizothorax prenanti (SpTLR4). The SpTLR2 and SpTLR4 were cloned and identified, and their tissue distribution was examined. The cDNA encoding SpTLR4 and SpTLR2 complete coding sequences (CDS) were identified and cloned. Additionally, we examined the expression levels of seven SpTLRs (SpTLR2, 3, 4, 18, 22-1, 22-2, and 22-3), as well as SpMyD88 and SpIRF3 in the liver, head kidney, hindgut, and spleen of S. prenanti, after intraperitoneal injection of polyinosinic-polycytidylic acid (poly (I:C)). The SpTLR2 and SpTLR4 shared amino acid sequence identity of 42.15–96.21% and 36.21–93.58%, respectively, with sequences from other vertebrates. SpTLR2 and SpTLR4 were expressed in all S. prenanti tissues examined, particularly in immune-related tissues. Poly (I:C) significantly upregulated most of the genes evaluated in the four immune organs compared with the PBS-control (p < 0.05); expression of these different genes was tissue-specific. Our findings demonstrate that TLR2 and TLR4 are expressed in S. prenanti and that poly (I:C) affects the expression of nine TLR-related genes, which are potentially involved in S. prenanti antiviral immunity or mediating pathological processes with differential kinetics. This will contribute to a better understanding of the roles of these TLR-related genes in antiviral immunity.

A visualized automatic particle counting strategy for single‐cell level telomerase activity quantification

Abstract: The accurate evaluation of telomerase activity, a typical cancer biomarker, is vital for early cancer screening. In this study, we developed a dark-field microscopy (DFM) visual single-particle detection scheme to detect telomerase activity based on automatic counting gold nanoparticles (AuNPs). This method started with attaching the telomerase substrate (TS) primer to the magnetic beads (MBs) through streptavidin-biotin interaction. In the presence of telomerase and dNTPs, the TS primer was expanded with (TTAGGG)n repeat units to form the telomerase extension product (MBs-telomerase extension product), which could be hybridized with the complementary DNA (cDNA) modified with AuNPs through Au-S bonds (AuNPs-SH-cDNA). After magnetic separation and DNA double-strand unwinding, AuNPs were collected from the supernatant, and the telomerase activity was quantitatively measured by visually counting bright spots based on DFM. This strategy achieved a limit of detection as low as 1 HeLa cell and distinguished telomerase activity among different cell lines, thus verifying its excellent sensitivity and specificity. Further, two common telomerase inhibitors (BIBR1532 and curcumin) were screened with the consistent IC50 values with other methods, respectively. It is worth mentioning that this strategy can clearly identify bladder cancer among various urinary diseases. Consequently, the visualized automatic particle counting strategy is potential as a powerful tool in early and noninvasive diagnosis of bladder cancer.

Data from Tissue-Wide Expression Profiling Using cDNA Subtraction and Microarrays to Identify Tumor-Specific Genes

Abstract: <div>Abstract<p>With the objective of discovering novel putative intervention sites for anticancer therapy, we compared transcriptional profiles of breast cancer, lung squamous cell cancer (LSCC), lung adenocarcinoma (LAC), and renal cell cancer (RCC). Each of these tumor types still needs improvement in medical treatment. Our intention was to search for genes not only highly expressed in the majority of patient samples but which also exhibit very low or even absence of expression in a comprehensive panel of 16 critical (vital) normal tissues. To achieve this goal, we combined two powerful technologies, PCR-based cDNA subtraction and cDNA microarrays. Seven subtractive libraries consisting of ∼9250 clones were established and enriched for tumor-specific transcripts. These clones, together with ∼1750 additional tumor-relevant genes, were used for cDNA microarray preparation. Hybridizations were performed using a pool of 16 critical normal tissues as a reference in all experiments. In total, we analyzed 20 samples of breast cancer, 11 of LSCC, 11 of LAC, and 8 of RCC. To select for genes with low or even no expression in normal tissues, expression profiles of 22 different normal tissues were additionally analyzed. Importantly, this tissue-wide expression profiling allowed us to eliminate genes, which exhibit also high expression in normal tissues. Similarly, expression signatures of genes, which are derived from infiltrating cells of the immune system, were eliminated as well. Cluster analysis resulted in the identification of 527 expressed sequence tags specifically up-regulated in these tumors. Gene-wise hierarchical clustering of these clones clearly separated the different tumor types with RCC exhibiting the most homogenous and LAC the most diverse expression profile. In addition to already known tumor-associated genes, the majority of identified genes have not yet been brought into context with tumorigenesis such as genes involved in bone matrix mineralization (<i>OSN</i>, <i>OPN</i>, and <i>OSF-2</i>) in lung, breast, and kidney cancer or genes controlling Ca²⁺ homeostasis (<i>RCN1</i>,<i>CALCA</i>, S100 protein family). EGLN3, which recently has been shown to be involved in regulation of hypoxia-inducible factor, was found to be highly up-regulated in all RCCs and in half of the LSCCs analyzed. Furthermore, 42 genes, the expression level of which correlated with the overall survival of breast cancer patients, were identified. The gene dendogram clearly separates two groups of genes, those up-regulated such as cyclin B1, <i>TGF-β3</i>, <i>B-Myb</i>, <i>Erg2</i>, <i>VCAM-1</i>, and <i>CD44</i> and those down-regulated such as <i>MIG-6</i>, <i>Esp15</i>, and <i>CAK</i> in patients with short survival time.</p></div>

miR-330 targeting BCO2 is involved in carotenoid metabolism to regulate skin pigmentation in rainbow trout (Oncorhynchus mykiss)

Abstract: Abstract Background MicroRNAs (miRNAs) play a critical role in regulating skin pigmentation. As a key economic trait, skin color directly affects the market value of rainbow trout ( Oncorhynchus mykiss ), however, the regulatory mechanism of most miRNAs in fish skin color is still unclear. Results In this study, the full-length cDNA sequence of β-carotene oxygenase 2 ( BCO2 , a key regulator of carotenoid metabolism) from the rainbow trout was obtained using rapid-amplification of cDNA ends (RACE) technology, and qRT-PCR was used to investigate the differential expression of miR-330 and BCO2 in 14 developmental stages and 13 tissues between wild-type rainbow trout (WTrt) and yellow mutant rainbow trout (YMrt). Additionally, the function of miR-330 was verified by overexpression and silencing in vitro and in vivo. The results showed that the complete cDNA sequence of BCO2 was 2057 bp with a 1707 bp ORF, encoding a 568 amino acid protein having a molecular weight of 64.07 kD. Sequence alignment revealed that higher conservation of BCO2 protein amongst fishes than amongst other vertebrates, which was further confirmed by phylogenetic analysis. The analysis of spatial and temporal expression patterns suggested that BCO2 and miR-330 were abundantly expressed from fertilized-stage to multi-cell as well as in the dorsal and ventral skin of WTrt and YMrt, and their expression patterns were opposite in most of the same periods and tissues. In vitro, luciferase reporter assay confirmed that BCO2 was a direct target of miR-330, and transfection of miR-330 mimics into rainbow trout liver cells resulted in a decrease in the expression of BCO2 ; conversely, miR-330 inhibitor had the opposite effect to the miR-330 mimics. In vivo, miR-330 agomir significantly decreased BCO2 expression in dorsal skin, tail fin, and liver. Furthermore, overexpression of miR-330 could suppress cell proliferation and induce apoptosis. Conclusion Our results showed that miR-330 is involved in the regulation of skin pigmentation in rainbow trout by targeting BCO2 and shows its promise as a potential molecular target to assist the selection of rainbow trout with better skin color patterns.

Construction of Full-Length Infectious cDNA Clones of Citrus Mosaic Virus RNA1 and RNA2 and Infection of Citrus Seedlings by <i>Agrobacterium</i>-Mediated Vacuum-Infiltration

Abstract: The development of full-length infectious cDNA clones for plant RNA viruses is important for studying their molecular biological characteristics, functional genomics, pathogenesis, and vectorization applications. Citrus mosaic virus (CiMV), a member of the genus Sadwavirus, is of economic importance to the citrus industry and comprises a bipartite, positive-sense, single-stranded RNA genome encapsidated in icosahedral virions. In the present study, full-length cDNA clones of CiMV RNA1 and RNA2 were constructed based on a ternary yeast-Escherichia coli-Agrobacterium tumefaciens shuttle vector, pTY, using transformation-associated recombination (TAR) strategy. Infectivity of cDNA clones of CiMV RNA1 and RNA2 was examined in multiple citrus varieties via Agrobacterium-mediated vacuum-infiltration (AVI) through symptom observation, RT-PCR, and virion detection with an electron microscope. Furthermore, the genome-sized RT-PCR fragments of RNA1 and RNA2 were obtained from symptomatic Jinchengyou (Citrus grandis) plants infected by the cloned virus (CiMV211). In addition, CiMV211 produced typical symptoms of wild-type CiMV in cowpea (Vigna angularis) plants inoculated by Agrobacterium-mediated injection. This is the first report of infectious cDNA clones of CiMV, which may lay the foundation for research on the pathogenesis and vectorization of the virus.

Reverse transcription-quantitative PCR (RT-qPCR) without the need for prior removal of DNA

Abstract: Abstract The procedure illustrated in this paper represents a new method for transcriptome analysis by PCR (Polymerase Chain Reaction), which circumvents the need for elimination of potential DNA contamination. Compared to the existing methodologies, our method is more precise, simpler and more reproducible because it preserves the RNA’s integrity, does not require materials and/or reagents that are used for elimination of DNA and it also reduces the number of samples that should be set up as negative controls. This novel procedure involves the use of a specifically modified primer during reverse transcription step, which contains mismatched bases, thus producing cDNA molecules that differ from genomic DNA. By using the same modified primer in PCR amplification, only cDNA template is amplified since genomic DNA template is partially heterologous to the primer. In this way, amplification by PCR is unaffected by any potential DNA contamination since it is specific only for the cDNA template. Furthermore, it accurately reflects the initial RNA concentration of the sample, which is prone to changes due to various physical or enzymatic treatments commonly used by the current methodologies for DNA elimination. The method is particularly suitable for quantification of highly repetitive DNA transcripts, such as satellite DNA.

Figure S5 from Single-Cell Transcriptomics of Pancreatic Cancer Precursors Demonstrates Epithelial and Microenvironmental Heterogeneity as an Early Event in Neoplastic Progression

Abstract: <p>Tapestation traces following (A) cDNA amplification of single cell and (B) library construction demonstrating sufficient quality and yield of material. Vertical lines demonstrate regions utilized to calculate cDNA and library yield through area under the curve.</p>

Novel strategy for in-vitro validation of Babesia orientalis heat shock proteins chaperone activity and thermostability

Abstract: Abstract Background: Babesia orientalis, an intracellular protozoan, which infects red blood cells and causes water buffalo babesiosis. The genome of B. orientalis has been reported and various genes have been accurately annotated, including heat shock proteins (HSP). Three B. orientalis HSPs (HSP90, HSP70 and HSP20) have been previously identified as potential antigenic targets. Here, a new validation strategy for the chaperone activities and cell protection characteristics of the three B. orientalis HSPs was developed in vitro. Methods: BoHSP20, BoHSP70 and BoHSP90B were amplified from cDNA, followed by cloning them into the pEGFP-N1 vector and transfecting the vector plasmid separately into 293T and Hela mammalian cells. Their expression and localization were determined by fluorescence microscopy. The biological functions and protein stability were testified through an analysis of the fluorescence intensity duration. Their role in the protection of cell viability from heat-shock treatments was examined by MTT assay. Results: Fusion proteins pEGFP-N1-BoHSP20, pEGFP-N1-BoHSP70, and pEGFP-N1-BoHSP90B (pBoHSPs: pBoHSP20; pBoHSP70 and pBoHSP90B) were identified as 47 kDa/97 kDa/118 kDa with a 27 kDa GFP tag, respectively. Prolonged fluorescent protein half-time was observed specifically in pBoHSPs under heat shock treatment at 55 ℃, and BoHSP20 showed relatively better thermotolerance than BoHSP70 and BoHSP90B. Significant difference was found between pBoHSPs and controls in the cell survival curve after 2 h of 45 ℃ heat shock. Conclusion: Significant biological properties of heat stress-associated genes of B. orientalis were identified in eukaryote by a new strategy. Fusion proteins pBoHSP20, pBoHSP70 and pBoHSP90B showed good chaperone activity and thermo-stability in this study, implying that BoHSPs played a key role in protecting B. orientalis against heat-stress environment during parasite life cycle. In a word, the in-vitro model explored in this study provides a new way to investigate the biological functions of B. orientalis proteins during the host -parasite interaction.

Cloning the full-length CDNA of actin gene and analysing alliinase gene expression in tillering onion

Abstract: Tillering onion is a herbaceous plant belonging to the Liliaceae family. We cloned the cDNAs of the actin gene (AcACT, GenBank: MF919598) of tillering onion using rapid amplification of the cDNA ends. The full-length cDNA of AcACT was 1,357 bp long with an open reading frame of 1,131 bp encoding 376 amino acids. The amino acid sequence of AcACT shared > 96% similarity with the amino acid sequences of other ACTs and was found (by means of phylogenetic tree analysis) to be closely related to those of Ananas comosus and Papaver somniferum. AcACT expressions showed no significant differences (p > 0.01) in two cultivars L-SH and L-SY over three growth periods and under suitable conditions, low temperature, and short-day conditions. In addition, AcACT was used as an internal reference gene to analyse the expression of the alliinase gene (AcALL). AcALL expression trends in the roots, stems and leaves were consistent with those of diallyl disulphide and diallyl trisulphide. Thus, AcACT is highly conserved and can be used as a suitable internal reference gene when analysing gene expression in tillering onion.