Evaluation and selection of reliable reference genes for gene expression under abiotic stress in cotton (Gossypium hirsutum L.)

Reference genes are critical for normalization of the gene expression level of target genes. The widely used housekeeping genes may change their expression levels at different tissue under different treatment or stress conditions. Therefore, systematical evaluation on the housekeeping genes is required for gene expression analysis. Up to date, no work was performed to evaluate the housekeeping genes in cotton under stress treatment. In this study, we chose 10 housekeeping genes to systematically assess their expression levels at two different tissues (leaves and roots) under two different abiotic stresses (salt and drought) with three different concentrations. Our results show that there is no best reference gene for all tissues at all stress conditions. The reliable reference gene should be selected based on a specific condition. For example, under salt stress, UBQ7, GAPDH and EF1A8 are better reference genes in leaves; TUA10, UBQ7, CYP1, GAPDH and EF1A8 were better in roots. Under drought stress, UBQ7, EF1A8, TUA10, and GAPDH showed less variety of expression level in leaves and roots. Thus, it is better to identify reliable reference genes first before performing any gene expression analysis. However, using a combination of housekeeping genes as reference gene may provide a new strategy for normalization of gene expression. In this study, we found that combination of four housekeeping genes worked well as reference genes under all the stress conditions.     

Cloning and expression analysis of novel Aux/IAA family genes in Gossypium hirsutum

The associations between polymorphisms of prostate stem cell antigen (PSCA-rs2294008C > T and -rs2976392G > A) and gastric cancer (GC) risk for Eastern Asians have been commonly studied, but the results were conflicting. The aim of the present study was to further assess the associations by the method of meta-analysis. The databases of Medline, Embase and CNKI (up to May 25th, 2011) were retrieved to identify eligible case-control studies. Odds ratio (OR) and 95% confidence interval (95%CI) were used to present the strength of the associations. In total, eight case-control studies in seven articles with 16792 individuals (9738 cases of GC and 7054 controls) were included in this meta-analysis. Through quantitative analyses, we found that T allele of rs2294008C > T and A allele of rs2976392G > A were significantly associated with increased GC risk [rs2294008C > T: OR (95%CI) = 1.31 (1.22-1.42), P_z-test < 0.001, P_{heterogeneity} = 0.166 for TT vs. C carriers; rs2976392G > A: OR (95%CI) = 1.36(1.24-1.50), P_z-test = 0.015, P_{heterogeneity} = 0.111 for AA vs. G carriers]. The results of subgroup analyses (according to histopathology, countries and sources of controls) indicated that T allele of rs2294008C > T and A allele rs2976392G > A were associated with increased risk of both intestinal- and diffuse-type GC, and associated with increased risk of GC for Chinese, Japanese, Koreans, PCC and HCC/PHCC. Furthermore, T allele of rs2294008C > T was also associated with increased risk of cardia and non-cardia GC, and associated with increased risk of GC for males and females. Besides those, this meta-analysis also indicated that the interactions between T allele of rs2294008C > T and A allele of rs2976392G > A was associated with increased risk of GC (A-T vs. G-T: OR = 1.16, 95%CI = 1.06-1.27, P_z-test = 0.001, P_{heterogeneity} = 0.835). Although modest limitations and potential bias cannot be eliminated, this meta-analysis suggests that PSCA -rs2294008C > T and -rs2976392G > A are potential factors of GC development for Eastern Asians, and future work may incorporate these findings and evaluate these variants as potential markers for screening and early diagnosis of GC.     

Cloning and expression analysis of ten genes associated with picrosides biosynthesis in Picrorhiza kurrooa

Picrorhiza kurrooa Royle ex Benth. is an economically important medicinal plant known to yield picrosides which have high medicinal value. Picroside I and picroside II are major picrosides associated with various bioactivities. The present work analyzed the expression of various genes of the picrosides biosynthesis pathway in different tissues of the plant in relation to the picrosides content. Eight full-length cDNA sequences namely, 1-deoxy-d-xylulose-5-phosphate synthase (2.317 kb), 1-deoxy-d-xylulose-5-phosphate reductoisomerase (1.767 kb), 4-diphosphocytidyl-2-C-methyl-d-erythritol kinase (1.674 kb), 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (1.701 kb), acetyl-CoA acetyltransferase (1.545 kb), 3-hydroxy-3-methylglutaryl coenzyme A reductase (2.241 kb), isopentenyl pyrophosphate isomerase (987 bp) and geranyl diphosphate synthase (1.434 kb), were cloned to full-length followed by expression analysis of ten genes vis-à-vis picrosides content analysis. There is maximum accumulation of picrosides in leaf tissue followed by the rhizome and root, and a similar pattern of expression was found in all the ten genes. The genes responded to the modulators of the picrosides biosynthesis. Picrosides accumulation was enhanced by application of hydrogen peroxide and abscisic acid, whereas methyl jasmonate and salicylic acid treatment decreased the content.     

Expression pattern of Aux/IAA genes in the iaa3/shy2-1D mutant of Arabidopsis thaliana (L.)

A semi-dominant mutant suppressor of hy2 (shy2-1D) of Arabidopsis thaliana, originally isolated as a photomorphogenesis mutant, shows altered auxin responses. Recent molecular cloning revealed that the SHY2 gene is identical to the IAA3 gene, a member of the primary auxin-response genes designated the Aux/IAA gene family. Because Aux/IAA proteins are reported to interact with auxin response factors, we investigated the pattern of expression of early auxin genes in the iaa3/shy2-1D mutant. RNA hybridization analysis showed that levels of mRNA accumulation of the early genes were reduced dramatically in the iaa3/shy2-1D mutants, although auxin still enhanced gene expression in the iaa3/shy2-1D mutant. Histochemical analysis using a fusion gene of the auxin responsive domain (AuxRD) and the GUS gene showed no IAA-inducible GUS expression in the root elongation zone of the iaa3/shy2-1D mutant. On the other hand, ectopic GUS expression occurred in the hypocotyl, cotyledon, petiole and root vascular tissues in the absence of auxin. These results suggest that IAA3/SHY2 functions both negatively and positively on early auxin gene expression.     

Mutation and expression analysis of the IDH1, IDH2, DNMT3A, and MYD88 genes in colorectal cancer

Colorectal cancer (CRC) is one of the leading causes of death around the world. Its genetic mechanism was intensively investigated in the past decades with findings of a number of canonical oncogenes and tumor-suppressor genes such as APC, KRAS, and TP53. Recent genome-wide association and sequencing studies have identified a series of promising oncogenes including IDH1, IDH2, DNMT3A, and MYD88 in hematologic malignancies. However, whether these genes are involved in CRC remains unknown. In this study, we screened the hotspot mutations of these four genes in 305 CRC samples from Han Chinese by direct sequencing. mRNA expression levels of these genes were quantified by quantitative real-time PCR (RT-qPCR) in paired cancerous and paracancerous tissues. Association analyses between mRNA expression levels and different cancerous stages were performed. Except for one patient harboring IDH1 mutation p.I99M, we identified no previously reported hotspot mutations in colorectal cancer tissues. mRNA expression levels of IDH1, DNMT3A, and MYD88, but not IDH2, were significantly decreased in the cancerous tissues comparing with the paired paracancerous normal tissues. Taken together, the hotspot mutations of IDH1, IDH2, DNMT3A, and MYD88 gene were absent in CRC. Aberrant mRNA expression of IDH1, DNMT3A, and MYD88 gene might be actively involved in the development of CRC.     

Drosophila Cand1 regulates Cullin3-dependent E3 ligases by affecting the neddylation of Cullin3 and by controlling the stability of Cullin3 and adaptor protein

Cullin-RING ubiquitin ligases (CRLs), which comprise the largest class of E3 ligases, regulate diverse cellular processes by targeting numerous proteins. Conjugation of the ubiquitin-like protein Nedd8 with Cullin activates CRLs. Cullin-associated and neddylation-dissociated 1 (Cand1) is known to negatively regulate CRL activity by sequestering unneddylated Cullin1 (Cul1) in biochemical studies. However, genetic studies of Arabidopsis have shown that Cand1 is required for optimal CRL activity. To elucidate the regulation of CRLs by Cand1, we analyzed a Cand1 mutant in Drosophila. Loss of Cand1 causes accumulation of neddylated Cullin3 (Cul3) and stabilizes the Cul3 adaptor protein HIB. In addition, the Cand1 mutation stimulates protein degradation of Cubitus interruptus (Ci), suggesting that Cul3-RING ligase activity is enhanced by the loss of Cand1. However, the loss of Cand1 fails to repress the accumulation of Ci in Nedd8^AN015 or CSN5^null mutant clones. Although Cand1 is able to bind both Cul1 and Cul3, mutation of Cand1 suppresses only the accumulation of Cul3 induced by the dAPP-BP1 mutation defective in the neddylation pathway, and this effect is attenuated by inhibition of proteasome function. Furthermore, overexpression of Cand1 stabilizes the Cul3 protein when the neddylation pathway is partially suppressed. These data indicate that Cand1 stabilizes unneddylated Cul3 by preventing proteasomal degradation. Here, we propose that binding of Cand1 to unneddylated Cul3 causes a shift in the equilibrium away from the neddylation of Cul3 that is required for the degradation of substrate by CRLs, and protects unneddylated Cul3 from proteasomal degradation. Cand1 regulates Cul3-mediated E3 ligase activity not only by acting on the neddylation of Cul3, but also by controlling the stability of the adaptor protein and unneddylated Cul3.     

Isolation and expression analysis of proline metabolism-related genes in Chrysanthemum lavandulifolium

Proline plays a significant role in plant resistance to abiotic stresses, and its level is determined by a combination of synthesis, catabolism and transport. The primary proteins involved are Δ¹-pyrroline-5-carboxylate synthetase (P5CS), proline dehydrogenase (PDH) and proline transporter (ProT). To utilise proline metabolism to improve the stress resistance of Chrysanthemum × morifolium, we isolated two P5CS-homologous genes (ClP5CS1 and ClP5CS2), one PDH gene (ClPDH) and four ProT-homologous genes (ClProT1-4) (GenBANK accession numbers: KF743136–KF743142) from Chrysanthemum lavandulifolium, which is closely related to chrysanthemums and exhibits strong resistance to stresses. Expression analysis of these genes in different organs and under various stresses indicated that ClP5CSs showed substantial constitutive expression, while ClPDH was only strongly expressed in the capitulum and was inhibited under most stresses. The expression patterns of four ClProT genes presented characteristics of organ specificity and disparity under stresses. Above all, the expression of ClProT2 was restricted to above-ground organs, especially strong in the capitulum and could be obviously induced by various stress conditions. Promoters of ClPDH and ClProTs contained many cis-acting regulatory elements involved in stress responses and plant growth and development. High levels of free proline were found in flower buds, the capitulum under the non-stress condition and later periods of stress conditions except cold treatment. Interestingly, organ specificity and disparity also exist in the level of free proline under different stress conditions. Our study indicates that ClProTs play significant roles in proline accumulation and stress responses, and that ClProT2 could be used to genetically modify the stress resistance of chrysanthemums. In addition, proline metabolism might be closely related to plant flowering and floral development.     

Cloning,characterization, and heterologous expression of a novel glucosyltransferase gene from sophorolipid-producing Candida bombicola

Candida bombicola is well-studied for the production of a biosurfactant, the sophorolipids. In this paper, the cloning of a glucosyltransferase gene using polymerase-chain-reaction (PCR) technique is described. Degenerative primer-pairs were first designed based on the highly conserved amino-acid sequences of several selected yeast glucosyltransferases. Using these primers, an amplified sequence (amplicon) of 700 base-pair from C. bombicola was obtained and subsequently sequenced. Based on the sequence of this amplicon, additional target-specific PCR primers were designed for use in subsequent rounds of 3′- and 5′-extension using DNA walking technique to eventually obtain a C. bombicola genomic sequence containing an open-reading-frame putatively identified as a glucosyltransferase (gtf-1). The gene was subcloned in Saccharomyces cerevisiae for expression and functional characterization. Quantitative RT-PCR confirmed the expression of gtf-1 in the recombinant S. cerevisiae. In vitro assay with the sonicated cells of the recombinant yeast confirms the presence of glucosylation activity on sterol and hydroxy fatty acid substrates. This study reports for the first time the cloning and characterization of a broad-specificity lipid glucosylation gene from C. bombicola, and the functional activity of its gene product.     

Cloning and functional expression in E. coli of a polyphenol oxidase transcript from Coreopsis grandiflora involved in aurone formation

Polyphenol oxidases are involved in aurone biosynthesis but the gene responsible for 4-deoxyaurone formation in Asteraceae was so far unknown. Three novel full-length cDNA sequences were isolated from Coreopsis grandiflora with sizes of 1.80 kb (cgAUS1) and 1.85 kb (cgAUS2a, 2b), encoding for proteins of 68–69 kDa, respectively. cgAUS1 is preferably expressed in young petals indicating a specific role in pigment formation. The 58.9 kDa AUS1 holoproenzyme, was recombinantly expressed in E. coli and purified to homogeneity. The enzyme shows only diphenolase activity, catalyzing the conversion of chalcones to aurones and was characterized by SDS–PAGE and shot-gun type nanoUHPLC–ESI-MS/MS.