Genome-wide analysis of the MADS-box gene family in Lonicera japonica and a proposed floral organ identity model.

BACKGROUND: Lonicera japonica Thunb. is widely used in traditional Chinese medicine. Medicinal L. japonica mainly consists of dried flower buds and partially opened flowers, thus flowers are an important quality indicator. MADS-box genes encode transcription factors that regulate flower development. However, little is known about these genes in L. japonica. RESULTS: In this study, 48 MADS-box genes were identified in L. japonica, including 20 Type-I genes (8 Mα, 2 Mß, and 10 Mγ) and 28 Type-II genes (26 MIKCc and 2 MIKC*). The Type-I and Type-II genes differed significantly in gene structure, conserved domains, protein structure, chromosomal distribution, phylogenesis, and expression pattern. Type-I genes had a simpler gene structure, lacked the K domain, had low protein structure conservation, were tandemly distributed on the chromosomes, had more frequent lineage-specific duplications, and were expressed at low levels. In contrast, Type-II genes had a more complex gene structure; contained conserved M, I, K, and C domains; had highly conserved protein structure; and were expressed at high levels throughout the flowering period. Eleven floral homeotic MADS-box genes that are orthologous to the proposed Arabidopsis ABCDE model of floral organ identity determination, were identified in L. japonica. By integrating expression pattern and protein interaction data for these genes, we developed a possible model for floral organ identity determination. CONCLUSION: This study genome-widely identified and characterized the MADS-box gene family in L. japonica. Eleven floral homeotic MADS-box genes were identified and a possible model for floral organ identity determination was also developed. This study contributes to our understanding of the MADS-box gene family and its possible involvement in floral organ development in L. japonica.

The non-specific lipid transfer protein McLTPII.9 of Mentha canadensis is involved in peltate glandular trichome density and volatile compound metabolism.

Mentha canadensis L. is an important spice crop and medicinal herb with high economic value. The plant is covered with peltate glandular trichomes, which are responsible for the biosynthesis and secretion of volatile oils. Plant non-specific lipid transfer proteins (nsLTPs) belong to a complex multigenic family involved in various plant physiological processes. Here, we cloned and identified a non-specific lipid transfer protein gene (McLTPII.9) from M. canadensis, which may positively regulate peltate glandular trichome density and monoterpene metabolism. McLTPII.9 was expressed in most M. canadensis tissues. The GUS signal driven by the McLTPII.9 promoter in transgenic Nicotiana tabacum was observed in stems, leaves, and roots; it was also expressed in trichomes. McLTPII.9 was associated with the plasma membrane. Overexpression of McLTPII.9 in peppermint (Mentha piperita. L) significantly increased the peltate glandular trichome density and total volatile compound content compared with wild-type peppermint; it also altered the volatile oil composition. In McLTPII.9-overexpressing (OE) peppermint, the expression levels of several monoterpenoid synthase genes and glandular trichome development-related transcription factors-such as limonene synthase (LS), limonene-3-hydroxylase (L3OH), geranyl diphosphate synthase (GPPS), HD-ZIP3, and MIXTA-exhibited varying degrees of alteration. McLTPII.9 overexpression resulted in both a change in expression of genes for terpenoid biosynthetic pathways which corresponded with an altered terpenoid profile in OE plants. In addition, peltate glandular trichome density was altered in the OE plants as well as the expression of genes for transcription factors that were shown to be involved in trichome development in plants.

Systematic Discovery of a New Catalogue of Tyrosine-Type Integrases in Bacterial Genomic Islands.

Site-specific recombinases (integrases) can mediate the horizontal transfer of genomic islands. The ability to integrate large DNA sequences into target sites is very important for genetic engineering in prokaryotic and eukaryotic cells. Here, we characterized an unprecedented catalogue of 530 tyrosine-type integrases by examining genes potentially encoding tyrosine integrases in bacterial genomic islands. The phylogeny of putative tyrosine integrases revealed that these integrases form an evolutionary clade that is distinct from those already known and are affiliated with novel integrase groups. We systematically searched for candidate integrase genes, and their integration activities were validated in a bacterial model. We verified the integration functions of six representative novel integrases by using a two-plasmid integration system consisting of a donor plasmid carrying the integrase gene and attP site and a recipient plasmid harboring an attB site in recA-deficient Escherichia coli. Further quantitative reverse transcription-PCR (qRT-PCR) assays validated that the six selected integrases can be expressed with their native promoters in E. coli. The attP region reductions showed that the extent of attP sites of integrases is approximately 200 bp for integration capacity. In addition, mutational analysis showed that the conserved tyrosine at the C terminus is essential for catalysis, confirming that these candidate proteins belong to the tyrosine-type recombinase superfamily, i.e., tyrosine integrases. This study revealed that the novel integrases from bacterial genomic islands have site-specific recombination functions, which is of physiological significance for their genomic islands in bacterial chromosomes. More importantly, our discovery expands the toolbox for genetic engineering, especially for efficient integration activity. IMPORTANCE Site-specific recombinases or integrases have high specificity for DNA large fragment integration, which is urgently needed for gene editing. However, known integrases are not sufficient for meeting multiple integrations. In this work, we discovered an array of integrases through bioinformatics analysis in bacterial genomes. Phylogeny and functional assays revealed that these new integrases belong to tyrosine-type integrases and have the ability to conduct site-specific recombination. Moreover, attP region extent and catalysis site analysis were characterized. Our study provides the methodology for discovery of novel integrases and increases the capacity of weapon pool for genetic engineering in bacteria.

Luteolin Alleviates Oxidative Stress in Chronic Obstructive Pulmonary Disease Induced by Cigarette Smoke via Modulation of the TRPV1 and CYP2A13/NRF2 Signaling Pathways.

The current study aims to investigate the therapeutic potential of luteolin (Lut), a naturally occurring flavonoid found in various medicinal plants, for treating chronic obstructive pulmonary disease (COPD) through both in vitro and in vivo studies. The results demonstrated that Lut increased body weight, reduced lung tissue swelling and lung damage indices, mitigated systemic oxidative stress levels, and decreased alveolar fusion in cigarette smoke (CS)- and lipopolysaccharide (LPS)-induced COPD mice. Additionally, Lut was observed to downregulate the expression of the TRPV1 and CYP2A13 proteins while upregulating SIRT6 and NRF2 protein expression in CS + LPS-induced COPD mice and cigarette smoke extract (CSE)-treated A549 cells. The concentrations of total reactive oxygen species (ROS) and mitochondrial ROS in A549 cells induced by CSE significantly increased. Moreover, CSE caused a notable elevation of intracellular Ca2+ levels in A549 cells. Importantly, Lut exhibited inhibitory effects on the inward flow of Ca2+ and attenuated the overproduction of mitochondrial and intracellular ROS in A549 cells treated with CSE. In conclusion, Lut demonstrated a protective role in alleviating oxidative stress and inflammation in CS + LPS-induced COPD mice and CSE-treated A549 cells by regulating TRPV1/SIRT6 and CYP2A13/NRF2 signaling pathways.

Genetic control of RNA splicing and its distinct role in complex trait variation.

Most genetic variants identified from genome-wide association studies (GWAS) in humans are noncoding, indicating their role in gene regulation. Previous studies have shown considerable links of GWAS signals to expression quantitative trait loci (eQTLs) but the links to other genetic regulatory mechanisms, such as splicing QTLs (sQTLs), are underexplored. Here, we introduce an sQTL mapping method, testing for heterogeneity between isoform-eQTL effects (THISTLE), with improved power over competing methods. Applying THISTLE together with a complementary sQTL mapping strategy to brain transcriptomic (n = 2,865) and genotype data, we identified 12,794 genes with cis-sQTLs at P < 5 × 10-8, approximately 61% of which were distinct from eQTLs. Integrating the sQTL data into GWAS for 12 brain-related complex traits (including diseases), we identified 244 genes associated with the traits through cis-sQTLs, approximately 61% of which could not be discovered using the corresponding eQTL data. Our study demonstrates the distinct role of most sQTLs in the genetic regulation of transcription and complex trait variation.

Isolation, Characterization of Bamboo Leaf Flavonoids by Size Exclusion Chromatography and Their Antioxidant Properties.

The utilization of bamboo industry exhibits varied but still needs to be improved. Bamboo leaf flavonoid (BLF) is an important resource of bamboo which has become a research focus. However, the isolation and purification techniques of four flavonoid carbon glycosides (orientin, isoorientin, vitexin, and isovitexin) from BLF were still confronted with difficulties due to their complex and similar structures, which obstructed the development of bamboo utilization. In this article, a purification technology of four flavonoid carbon glycosides from BLF by Sephadex LH-20 was improved. The results were evaluated by HPLC and pharmacological activity. Specifically, the eluent, flow rate, and loading amount were investigated, respectively. According to the results, the eluent would dominate the isolation effect among three factors. High concentration of isoorientin and four flavonoid carbon glycosides would be obtained under the optimized condition (The eluent was 70 % methanol, the loading amount was 1.5 g, and the flow rate was 0.5 mL/min). Meanwhile, the link between flavonoid carbon glycosides content and their antioxidant activity in vitro was also revealed. Overall, the results suggested that BLF may serve as potential functional food additives and medicine.

Comparative transcriptome analysis revealed candidate genes involved in fruiting body development and sporulation in Ganoderma lucidum.

Ganoderma lucidum is an edible mushroom highly regarded in the traditional Chinese medicine. To better understand the molecular mechanisms underlying fruiting body development in G. lucidum, transcriptome analysis based on RNA sequencing was carried out on different developmental stages: mycelium (G1); primordium (G2); young fruiting body (G3); mature fruiting body (G4); fruiting body in post-sporulation stage (G5). In total, 26,137 unigenes with an average length of 1078 bp were de novo assembled. Functional annotation of transcriptomes matched 72.49% of the unigenes to known proteins available in at least one database. Differentially expressed genes (DEGs) were identified between the evaluated stages: 3135 DEGs in G1 versus G2; 120 in G2 versus G3; 3919 in G3 versus G4; and 1012 in G4 versus G5. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs identified in G1 versus G2 revealed that, in addition to global and overview maps, enriched pathways were related to amino acid metabolism and carbohydrate metabolism. In contrast, DEGs identified in G2 versus G3 were mainly assigned to the category of metabolism of amino acids and their derivatives, comprising mostly upregulated unigenes. In addition, highly expressed unigenes associated with the transition between different developmental stages were identified, including those encoding hydrophobins, cytochrome P450s, extracellular proteases, and several transcription factors. Meanwhile, highly expressed unigenes related to meiosis such as DMC1, MSH4, HOP1, and Mek1 were also analyzed. Our study provides important insights into the molecular mechanisms underlying fruiting body development and sporulation in G. lucidum.

Characterisation of the Mentha canadensis R2R3-MYB transcription factor gene McMIXTA and its involvement in peltate glandular trichome development.

BACKGROUND: Mentha canadensis L. has important economic value for the production of essential oils, which are synthesised, secreted and stored in peltate glandular trichomes. As a typical multicellular secretory trichome, glandular trichomes are important biological factories for the synthesis of some specialised metabolites. However, little is known about the molecular mechanism of glandular trichome development in M. canadensis. RESULTS: In this study, the R2R3-MYB transcription factor gene McMIXTA was isolated to investigate its function in glandular trichome development. Bioinformatics analysis indicated that McMIXTA belonged to the subgroup 9 R2R3-MYB, with a R2R3 DNA-binding domain and conserved subgroup 9 motifs. A subcellular localisation assay indicated that McMIXTA was localised in the nucleus. Transactivation analysis indicated that McMIXTA was a positive regulator, with transactivation regions located between positions N253 and N307. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that McMIXTA formed a complex with McHD-Zip3, a trichome development-related HD-ZIP IV transcription factor. Overexpression of McMIXTA in Mentha × piperita L. caused an increase in peltate glandular trichomes density of approximately 25% on the leaf abaxial surface. CONCLUSIONS: Our results demonstrated that the subgroup 9 R2R3-MYB transcription factor McMIXTA has a positive effect on regulating peltate glandular trichome development and the MIXTA/HD-ZIP IV complexes might be conserved regulators for glandular trichome initiation. These results provide useful information for revealing the regulatory mechanism of multicellular glandular trichome development.

Effect of light quality on the growth and main chemical composition of Bletilla striata.

This study aimed to investigate the effects of light quality on the morphological traits, photosynthetic pigment content, protective enzyme (superoxide dismutase, peroxidase, and catalase) activity, and bioactive compound (BSP, total phenol, and militarine) content in Bletilla striata. Plants of B. striata were grown under light filtered through three differently colored films. The treatments were red film (RF), yellow film (YF), and blue film (BF), and an uncovered treatment was included as a control (CK). Compared with the B. striata plants in the RF, YF, and CK treatment groups, those receiving BF treatment showed significantly promoted growth of the aerial parts. Meanwhile, the total phenol and militarine contents in B. striata tubers were increased without affecting the accumulation of B. striata polysaccharides. These results show that growing B. striata plants under blue film could be a useful technique to improve quality and production. This technique is conducive to achieving large-scale sustainable production of high-quality plant materials.

Characterization of the Glehnia littoralis Non-specific Phospholipase C Gene GlNPC3 and Its Involvement in the Salt Stress Response.

Glehnia littoralis is a medicinal halophyte that inhabits sandy beaches and has high ecological and commercial value. However, the molecular mechanism of salt adaptation in G. littoralis remains largely unknown. Here, we cloned and identified a non-specific phospholipase C gene (GlNPC3) from G. littoralis, which conferred lipid-mediated signaling during the salt stress response. The expression of GlNPC3 was induced continuously by salt treatment. Overexpression of GlNPC3 in Arabidopsis thaliana increased salt tolerance compared to wild-type (WT) plants. GlNPC3-overexpressing plants had longer roots and higher fresh and dry masses under the salt treatment. The GlNPC3 expression pattern revealed that the gene was expressed in most G. littoralis tissues, particularly in roots. The subcellular localization of GlNPC3 was mainly at the plasma membrane, and partially at the tonoplast. GlNPC3 hydrolyzed common membrane phospholipids, such as phosphotidylserine (PS), phosphoethanolamine (PE), and phosphocholine (PC). In vitro enzymatic assay showed salt-induced total non-specific phospholipase C (NPC) activation in A. thaliana GlNPC3-overexpressing plants. Plant lipid profiling showed a significant change in the membrane-lipid composition of A. thaliana GlNPC3-overexpressing plants compared to WT after the salt treatment. Furthermore, downregulation of GlNPC3 expression by virus-induced gene silencing in G. littoralis reduced the expression levels of some stress-related genes, such as SnRK2, P5SC5, TPC1, and SOS1. Together, these results indicated that GlNPC3 and GlNPC3-mediated membrane lipid change played a positive role in the response of G. littoralis to a saline environment.

A generalized linear mixed model association tool for biobank-scale data.

Compared with linear mixed model-based genome-wide association (GWA) methods, generalized linear mixed model (GLMM)-based methods have better statistical properties when applied to binary traits but are computationally much slower. In the present study, leveraging efficient sparse matrix-based algorithms, we developed a GLMM-based GWA tool, fastGWA-GLMM, that is severalfold to orders of magnitude faster than the state-of-the-art tools when applied to the UK Biobank (UKB) data and scalable to cohorts with millions of individuals. We show by simulation that the fastGWA-GLMM test statistics of both common and rare variants are well calibrated under the null, even for traits with extreme case-control ratios. We applied fastGWA-GLMM to the UKB data of 456,348 individuals, 11,842,647 variants and 2,989 binary traits (full summary statistics available at http://fastgwa.info/ukbimpbin ), and identified 259 rare variants associated with 75 traits, demonstrating the use of imputed genotype data in a large cohort to discover rare variants for binary complex traits.

Transcriptome-Wide Identification and Characterization of the JAZ Gene Family in Mentha canadensis L.

Jasmonate ZIM-domain (JAZ) proteins are the crucial transcriptional repressors in the jasmonic acid (JA) signaling process, and they play pervasive roles in plant development, defense, and plant specialized metabolism. Although numerous JAZ gene families have been discovered across several plants, our knowledge about the JAZ gene family remains limited in the economically and medicinally important Chinese herb Mentha canadensis L. Here, seven non-redundant JAZ genes named McJAZ1-McJAZ7 were identified from our reported M. canadensis transcriptome data. Structural, amino acid composition, and phylogenetic analysis showed that seven McJAZ proteins contained the typical zinc-finger inflorescence meristem (ZIM) domain and JA-associated (Jas) domain as conserved as those in other plants, and they were clustered into four groups (A-D) and distributed into five subgroups (A1, A2, B1, B2, and D). Quantitative real-time PCR (qRT-PCR) analysis showed that seven McJAZ genes displayed differential expression patterns in M. canadensis tissues, and preferentially expressed in flowers. Furthermore, the McJAZ genes expression was differentially induced after Methyl jasmonate (MeJA) treatment, and their transcripts were variable and up- or down-regulated under abscisic acid (ABA), drought, and salt treatments. Subcellular localization analysis revealed that McJAZ proteins are localized in the nucleus or cytoplasm. Yeast two-hybrid (Y2H) assays demonstrated that McJAZ1-5 interacted with McCOI1a, a homolog of Arabidopsis JA receptor AtCOI1, in a coronatine-dependent manner, and most of McJAZ proteins could also form homo- or heterodimers. This present study provides valuable basis for functional analysis and exploitation of the potential candidate McJAZ genes for developing efficient strategies for genetic improvement of M. canadensis.

Transcriptome Analysis of Light-Regulated Monoterpenes Biosynthesis in Leaves of Mentha canadensis L.

Light is a key environmental aspect that regulates secondary metabolic synthesis. The essential oil produced in mint (Mentha canadensis L.) leaves is used widely in the aromatics industry and in medicine. Under low-light treatment, significant reductions in peltate glandular trichome densities were observed. GC-MS analysis showed dramatically reduced essential oil and menthol contents. Light affected the peltate glandular trichomes' development and essential oil yield production. However, the underlying mechanisms of this regulation were elusive. To identify the critical genes during light-regulated changes in oil content, following a 24 h darkness treatment and a 24 h recovery light treatment, leaves were collected for transcriptome analysis. A total of 95,579 unigenes were obtained, with an average length of 754 bp. About 56.58% of the unigenes were annotated using four public protein databases: 10,977 differentially expressed genes (DEGs) were found to be involved in the light signaling pathway and monoterpene synthesis pathway. Most of the TPs showed a similar expression pattern: downregulation after darkness treatment and upregulation after the return of light. In addition, the genes involved in the light signal transduction pathway were analyzed. A series of responsive transcription factors (TFs) were identified and could be used in metabolic engineering as an effective strategy for increasing essential oil yields.

Genome-Wide Analysis of Terpene Synthase Gene Family in Mentha longifolia and Catalytic Activity Analysis of a Single Terpene Synthase.

Terpenoids are a wide variety of natural products and terpene synthase (TPS) plays a key role in the biosynthesis of terpenoids. Mentha plants are rich in essential oils, whose main components are terpenoids, and their biosynthetic pathways have been basically elucidated. However, there is a lack of systematic identification and study of TPS in Mentha plants. In this work, we genome-widely identified and analyzed the TPS gene family in Mentha longifolia, a model plant for functional genomic research in the genus Mentha. A total of 63 TPS genes were identified in the M. longifolia genome sequence assembly, which could be divided into six subfamilies. The TPS-b subfamily had the largest number of genes, which might be related to the abundant monoterpenoids in Mentha plants. The TPS-e subfamily had 18 members and showed a significant species-specific expansion compared with other sequenced Lamiaceae plant species. The 63 TPS genes could be mapped to nine scaffolds of the M. longifolia genome sequence assembly and the distribution of these genes is uneven. Tandem duplicates and fragment duplicates contributed greatly to the increase in the number of TPS genes in M. longifolia. The conserved motifs (RR(X)8W, NSE/DTE, RXR, and DDXXD) were analyzed in M. longifolia TPSs, and significant differentiation was found between different subfamilies. Adaptive evolution analysis showed that M. longifolia TPSs were subjected to purifying selection after the species-specific expansion, and some amino acid residues under positive selection were identified. Furthermore, we also cloned and analyzed the catalytic activity of a single terpene synthase, MlongTPS29, which belongs to the TPS-b subfamily. MlongTPS29 could encode a limonene synthase and catalyze the biosynthesis of limonene, an important precursor of essential oils from the genus Mentha. This study provides useful information for the biosynthesis of terpenoids in the genus Mentha.

A GPAT1 Mutation in Arabidopsis Enhances Plant Height but Impairs Seed Oil Biosynthesis.

Glycerol-3-phosphate acyltransferases (GPATs) play an important role in glycerolipid biosynthesis, and are mainly involved in oil production, flower development, and stress response. However, their roles in regulating plant height remain unreported. Here, we report that Arabidopsis GPAT1 is involved in the regulation of plant height. GUS assay and qRT-PCR analysis in Arabidopsis showed that GPAT1 is highly expressed in flowers, siliques, and seeds. A loss of function mutation in GPAT1 was shown to decrease seed yield but increase plant height through enhanced cell length. Transcriptomic and qRT-PCR data revealed that the expression levels of genes related to gibberellin (GA) biosynthesis and signaling, as well as those of cell wall organization and biogenesis, were significantly upregulated. These led to cell length elongation, and thus, an increase in plant height. Together, our data suggest that knockout of GPAT1 impairs glycerolipid metabolism in Arabidopsis, leading to reduced seed yield, but promotes the biosynthesis of GA, which ultimately enhances plant height. This study provides new evidence on the interplay between lipid and hormone metabolism in the regulation of plant height.

Correction: Chen et al. Genome-Wide Analysis of Terpene Synthase Gene Family in Mentha longifolia and Catalytic Activity Analysis of a Single Terpene Synthase. Genes 2021, 12, 518.

The authors have requested that the following changes be made to their paper [...].

De novo transcriptomic analysis of light-induced flavonoid pathway, transcription factors in the flower buds of Lonicera japonica.

KEY MESSAGE: Transcriptomic analysis of the relationship between gene expression patterns and flavonoid contents in the flower buds of Lonicera japonica under light-induced conditions, especially the flavonoid pathway genes and transcription factors. ABSTRACT: Flos Lonicerae Japonicae (FLJ), the flower buds of Lonicera japonica Thunb., has been used to treat some human diseases including severe respiratory syndromes and hand-foot-and-mouth diseases owing to its putative antibacterial, and antiviral effects. Luteoloside is a flavonoid that is used by the Chinese Pharmacopoeia to evaluate the quality of FLJ. Light is an important environmental factor that affects flavonoid biosynthesis in the flower buds of L. japonica. However, how light triggers increases in flavonoid production remains unclear. To enhance our understanding of the mechanism involved in light-regulated flavonoid biosynthesis, we sequenced the transcriptomes of L. japonica exposed to three different light conditions: 100% light intensity (CK), 50% light intensity (LI50), and 25% light intensity (LI25) using an Illumina HiSeq 4000 System. A total of 77,297 unigenes with an average length of 809 bp were obtained. Among them, 43,334 unigenes (56.06%) could be matched to at least one biomolecular database. Additionally, 4188, 1545 and 1023 differentially expressed genes (DEGs) were identified by comparative transcriptomics LI25-vs-CK, LI50-vs-CK, and LI25-vs-LI50, respectively. Of note, genes known to be involved in flavonoid biosynthesis, such as 4-coumarate coenzyme A ligase (4CL), and chalcone synthase (CHS) were up-regulated. In addition, a total of 1649 transcription factors (TFs) were identified and divided into 58 TF families; 98 TFs exhibited highly dynamic changes in response to light intensity. Quantitative real-time PCR (qRT-PCR) was used to test the expression profiles of the RNA sequencing (RNA-Seq) data. This study offers insight into how transcriptional expression pattern is influenced by light in the flower buds of L. japonica, and will enhance the understanding of molecular mechanisms of flavonoid biosynthesis in response to light in L. japonica.

Selection and Validation of Reference Genes for Normalisation of Gene Expression in Glehnia littoralis.

Glehnia littoralis is an important medicinal halophyte-the dried root of which is used as Chinese herbal medicine. However, the use, selection and stability of reference genes are rarely verified in studies of G. littoralis, which hampers investigation of its salt tolerance and metabolism. In this study, we selected 13 candidate reference genes from the transcriptome data of G. littoralis-serine/threonine-protein phosphatase PP2A (PP2A), polyubiquitin 10 (UBQ10), actin (ACT), elongation factor 1-α (EF1-α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), α-tubulin (α-TUB), ß-tubulin (ß-TUB), polypyrimidine tract-binding protein 1 (PTBP1), expressed protein 1 (EXP1), expressed protein 2 (EXP2), TIP41-like (TIP41), SAND family (SAND), and cyclophilin 2 (CYP2), and used qRT-PCR to analyse their expression levels in roots of G. littoralis treated with NaCl, polyethylene glycol (PEG), abscisic acid (ABA), and methyl jasmonate (MeJA), as well as in various organs of G. littoralis. The ΔCt, geNorm, NormFinder, and BestKeeper algorithms were used to assess the expression stability of the candidate reference genes and the results were then used to generate a comprehensive rank list with the RankAggreg R package. The most stable reference genes for normalisation were EXP1 and PP2A in response to NaCl, EXP2 and PP2A in response to ABA, CYP2 and α-TUB in response to MeJA, and ACT and EXP1 in the PEG and the organ subsets. GAPDH, ß-TUB, and UBQ10 exhibited low stability and so were unsuitable for normalisation. This study is the first systematic analysis of candidate reference genes in G. littoralis and will facilitate further investigation of normalisation of gene expression in G. littoralis.

Screening of an Alkaline CMCase-Producing Strain and the Optimization of its Fermentation Condition.

OBJECTIVE: Alkaline Carboxymethyl Cellulase (CMCase) is an attractive enzyme for the textile, laundry, pulp, and paper industries; however, commercial preparations with sufficient activity at alkaline conditions are scarce. METHODS: High CMCase-producing bacterial isolate, SX9-4, was screened out from soil bacteria, which was identified as Flavobacterium sp. on the basis of 16S rDNA sequencing. RESULTS: The optimum pH and temperature for CMCase reaction were 8.0 and 55°C, respectively. Alkaline CMCase was stable over wide pH (3.0-10.6) and temperature (25-55°C) ranges. Enzyme activity was significantly inhibited by the bivalent cations Mn2+ and Cu2+, and was activated by Fe2+. To improve the alkaline CMCase production of SX9-4, fermentation parameters were selected through onefactor- at-a-time and further carried out by response surface methodologies based on a central composite design. CONCLUSION: High CMCase production (57.18 U/mL) was achieved under the optimal conditions: 10.53 g/L carboxymethylcellulose sodium, 7.74 g/L glucose, 13.71 g/L peptone, and 5.27 g/L ammonium oxalate.