Tissue-specific Expression Pattern Analysis Research Articles

Metabolomics integrated with transcriptomics provides insights into the phenylpropanoids biosynthesis pathway in Lilium davidii var. unicolor and L. lancifolium Thunb.

Lilium spp. is a world-famous bulbous flower with outstanding ornamental, edible, and medicinal value. Evaluating the taste of edible lilies and identifying important active substances and genes are necessary for germplasm improvement, new variety breeding, and industrial application. To better understand the phenylpropanoids and regalosides biosynthesis, L. davidii var. unicolor and L. lancifolium Thunb. bulbs were used for transcriptome and metabolite analysis. Results showed that the phenols and flavonoid contents in JT were lower than in LT, while the saponins and alkaloid contents in JT were higher than in LT. A total of 20,520 differentially expressed genes (DEGs) and 383 differential metabolites were searched. Integrated transcriptomics and metabolomics analysis showed that phenylpropanoid biosynthesis and flavonoid biosynthesis were differentially altered. Ninety-nine unigenes encoding ten phenolic acids and two flavonoids were identified as candidate genes involved in phenylpropanoid and flavonoid biosynthesis. WGCNA analysis showed 76 phenylpropanoid and flavonoid biosynthesis-related unigenes were verified as likely to be involved in phenylpropanoid metabolism and regalosides accumulation. Among them, 15 genes were used for qRT-PCR, and four genes were utilized for tissue-specific expression pattern analysis. Down-regulation of LdPAL2 and LdC4H1 in bulbs of L. davidii var. unicolor via virus induced gene silence (VIGS) reduced the contents of p-coumaric acid and cinnamic acid. These results contribute to understanding phenylpropanoid metabolism and identifying potential functional genes for improving the regalosides and flavonoids content in Lilium bulbs.

Identification, Evolution and Expression Analysis of GRF Family Reveals Their Involvement in Shoot Growth and Abiotic Stress Response in Moso Bamboo

Growth-regulating factors (GRFs) play an important role in regulating plant organ development, acting primarily as positive regulators of cell proliferation. However, research on the evolutionary history and expression patterns of the moso bamboo GRF family has been limited. In this study, a total of 24 GRFs have been identified in the Moso bamboo genome, and they have been categorized into four subfamilies. Estimation of the divergence time of paralogous gene pairs provided evidence supporting the significant contribution of recent whole-genome duplication events in the expansion of the GRF gene family. Sliding window analysis revealed that coding regions of a few PheGRFs, including the WRC and QLQ domains, may have undergone positive selection, possibly due to the redundant functions of paralogous genes. Coexpression network analysis further revealed the regulatory role of miR396 and various lncRNAs in controlling PheGRF expression. Based on the analysis of tissue-specific expression patterns using transcriptome sequencing, qRT-PCR results, and in situ hybridization, it was observed that most GRFs, particularly PheGRF6a and PheGRF9b, exhibited high levels of accumulation in the moso bamboo shoot. This suggests that the involvement of most PheGRF genes may be crucial for the growth and development of the bamboo shoot. A yeast two-hybrid screening revealed interactions between PheGRF9b and several proteins associated with plant growth and development, including PH02Gene11097.t1 (GIF3), PH02Gene37618.t (Phytochrome B), and PH02Gene01921.t3 (WD40). Based on transcriptome expression analysis, it was observed that a substantial number of PheGRFs exhibited significant variations under cold or drought stress treatments, and most of these genes were found to be downregulated, suggesting their role as abiotic stress-responsive genes. Our findings offer new insights into the GRF family of moso bamboo and provide some experimental evidence to support further gene functional validation research of PheGRF.

WGCNA and transcriptome profiling reveal hub genes for key development stage seed size/oil content between wild and cultivated soybean

BackgroundSoybean is one of the most important oil crops in the world. The domestication of wild soybean has resulted in significant changes in the seed oil content and seed size of cultivated soybeans. To better understand the molecular mechanisms of seed formation and oil content accumulation, WDD01514 (E1), ZYD00463 (E2), and two extreme progenies (E23 and E171) derived from RILs were used for weighted gene coexpression network analysis (WGCNA) combined with transcriptome analysis.ResultsIn this study, both seed weight and oil content in E1 and E171 were significantly higher than those in E2 and E23, and 20 DAF and 30 DAF may be key stages of soybean seed oil content accumulation and weight increase. Pathways such as “Photosynthesis”, “Carbon metabolism”, and “Fatty acid metabolism”, were involved in oil content accumulation and grain formation between wild and cultivated soybeans at 20 and 30 DAF according to RNA-seq analysis. A total of 121 oil content accumulation and 189 seed formation candidate genes were screened from differentially expressed genes. WGCNA identified six modules related to seed oil content and seed weight, and 76 candidate genes were screened from modules and network. Among them, 16 genes were used for qRT-PCR and tissue specific expression pattern analysis, and their expression-levels in 33-wild and 23-cultivated soybean varieties were subjected to correlation analysis; some key genes were verified as likely to be involved in oil content accumulation and grain formation.ConclusionsOverall, these results contribute to an understanding of seed lipid metabolism and seed size during seed development, and identify potential functional genes for improving soybean yield and seed oil quantity.

Identification of metabolizing enzyme genes associated with xenobiotics and odorants in the predatory stink bug Arma custos based on transcriptome analysis

The predatory stink bug, Arma custos, is a highly effective beneficial predator of crop pests. The lack of gene information related to xenobiotic detoxification and odorant degrading enzymes in the predator stink bugs to date has limited our ability for more in-depth studies of biological control. Hence, we conducted de novo assembly of the A. custos transcriptome from guts, antennae, and other tiussue samples of 5th instar larvae using Illumina sequencing technology. A total of 91, 50 and 23 genes of cytochrome P450 monooxygenases (CYPs), carboxyl/choline esterases (CCEs) and glutathione S-transferases (GSTs) genes were identified, respectively. Gene expansions of CYP3 and CYP4 clans and the hormone and pheromone processing CCE class were found in A. custos. Analysis of tissue-specific expression patterns showed that 37 CYPs, 14 CCEs and 8 GSTs were enriched in guts, and 6 CYPs, 5 CCEs and 2 GSTs were up-regulated in antennae, suggesting their potential roles on xenobiotics detoxification and ordorant degradation. Gene information data presented here could be useful for a deeper understanding of the ecology, physiology and behavior of this beneficial species and could be helpful to improve their bio-control efficiency.

Genomic characterization of the NAC transcription factors, directed at understanding their functions involved in endocarp lignification of iron walnut (Juglans sigillata Dode).

The NAC (NAM, ATAF1/2, and CUC2) transcription factors (TF), one of the largest plant-specific gene families, play important roles in the regulation of plant growth and development, stress response and disease resistance. In particular, several NAC TFs have been identified as master regulators of secondary cell wall (SCW) biosynthesis. Iron walnut (Juglans sigillata Dode), an economically important nut and oilseed tree, has been widely planted in the southwest China. The thick and high lignified shell derived endocarp tissues, however, brings troubles in processing processes of products in industry. It is indispensable to dissect the molecular mechanism of thick endocarp formation for further genetic improvement of iron walnut. In the present study, based on genome reference of iron walnut, 117 NAC genes, in total, were identified and characterized in silico, which involves only computational analysis to provide insight into gene function and regulation. We found that the amino acids encoded by these NAC genes varied from 103 to 1,264 in length, and conserved motif numbers ranged from 2 to 10. The JsiNAC genes were unevenly distributed across the genome of 16 chromosomes, and 96 of these genes were identified as segmental duplication genes. Furthermore, 117 JsiNAC genes were divided into 14 subfamilies (A-N) according to the phylogenetic tree based on NAC family members of Arabidopsis thaliana and common walnut (Juglans regia). Furthermore, tissue-specific expression pattern analysis demonstrated that a majority of NAC genes were constitutively expressed in five different tissues (bud, root, fruit, endocarp, and stem xylem), while a total of 19 genes were specifically expressed in endocarp, and most of them also showed high and specific expression levels in the middle and late stages during iron walnut endocarp development. Our result provided a new insight into the gene structure and function of JsiNACs in iron walnut, and identified key candidate JsiNAC genes involved in endocarp development, probably providing mechanistic insight into shell thickness formation across nut species.

Single-Molecule Real-Time Sequencing of Full-Length Transcriptome and Identification of Genes Related to Male Development in Cannabis sativa.

Female Cannabis sativa plants have important therapeutic properties. The sex ratio of the dioecious cannabis is approximately 1:1. Cultivating homozygous female plants by inducing female plants to produce male flowers is of great practical significance. However, the mechanism underlying cannabis male development remains unclear. In this study, single-molecule real-time (SMRT) sequencing was performed using a mixed sample of female and induced male flowers from the ZYZM1 cannabis variety. A total of 15,241 consensus reads were identified, and 13,657 transcripts were annotated across seven public databases. A total of 48 lncRNAs with an average length of 986.54 bp were identified. In total, 8202 transcripts were annotated as transcription factors, the most common of which were bHLH transcription factors. Moreover, tissue-specific expression pattern analysis showed that 13 MADS transcription factors were highly expressed in male flowers. Furthermore, 232 reads of novel genes were predicted and enriched in lipid metabolism, and qRT-PCR results showed that CER1 may be involved in the development of cannabis male flowers. In addition, 1170 AS events were detected, and two AS events were further validated. Taken together, these results may improve our understanding of the complexity of full-length cannabis transcripts and provide a basis for understanding the molecular mechanism of cannabis male development.

PHD-finger family genes in wheat (Triticum aestivum L.): Evolutionary conservatism, functional diversification, and active expression in abiotic stress.

Plant homeodomain (PHD) transcription factors (TFs) are a class of proteins with conserved Cys4-His-Cys3 domains that play important roles in plant growth and development and in response to abiotic stresses. Although characterization of PHDs has been performed in plants, little is known about their function in wheat (Triticum aestivum L.), especially under stress conditions. In the present study, 244 TaPHDs were identified in wheat using comparative genomics. We renamed them TaPHD1-244 based on their chromosomal distribution, and almost all PHD proteins were predicted to be located in the nucleus. According to the unrooted neighbor-joining phylogenetic tree, gene structure, and motif analyses, PHD genes were divided into four clades. A total of 149 TaPHD genes were assigned to arise from duplication events. Furthermore, 230 gene pairs came from wheat itself, and 119, 186, 168, 7, 2, and 6 gene pairs came from six other species (Hordeum vulgareto, Zea mays, Oryza sativa, Arabidopsis thaliana, Brassica rapa, and Gossypium raimondii, respectively). A total of 548 interacting protein branches were identified to be involved in the protein interaction network. Tissue-specific expression pattern analysis showed that TaPHDs were highly expressed in the stigma and ovary during flowering, suggesting that the TaPHD gene plays an active role in the reproductive growth of wheat. In addition, the qRT-PCR results further confirmed that these TaPHD genes are involved in the abiotic stress response of wheat. In conclusion, our study provides a theoretical basis for deciphering the molecular functions of TaPHDs, particularly in response to abiotic stress.

Genome-wide identification and characterization of AINTEGUMENTA-LIKE (AIL) family genes in apple (Malus domestica Borkh.).

AINTEGUMENTA-LIKE (AIL) genes play a key role in various growth and developmental processes in plants. Thus far, the genome-wide identification of AIL genes has been reported for some plant species. However, genome-wide identification of AIL genes has not been conducted in apple (Malus domestica Borkh.). The current study focused on a comprehensive analysis of the AIL genes in the apple genome (i.e., MdAIL genes). In total, 27 MdAIL genes in the apple genome were identified and then divided into four groups according to phylogenetic analysis. The chromosomal locations, gene and protein structures, and physicochemical characteristics of MdAIL genes were analyzed. Synteny analysis revealed that segmental duplication events played a major role in the expansion of the AIL gene family in apple. The analysis of cis-regulatory elements in MdAIL promoter regions indicated that most of the MdAIL genes are involved in embryo development and seed germination. Moreover, the analysis of tissue-specific expression patterns and transcript levels in adventitious bud regeneration indicated that MdAIL genes play an extensive regulatory role in apple growth and development, especially in the regulation of germination and adventitious bud regeneration from in vitro leaves of apple. In conclusion, this is the first genome-wide analysis of the AIL genes in apple. The current results may help in better understanding the evolution and function of MdAIL genes and thus facilitate further research on plant growth and development.

Silencing of Cytochrome P450 in Spodoptera frugiperda (Lepidoptera: Noctuidae) by RNA Interference Enhances Susceptibility to Chlorantraniliprole.

Fall armyworm, Spodoptera frugiperda (Smith), has caused significant losses for crop production in China. The fall armyworm is mainly controlled by the chemical insecticides, whereas the frequent application of insecticides would result in the resistance development. Insect cytochrome P450 monooxygenases play an essential part in the detoxification of insecticides. In this study, five P450 genes were selected to determine the role in response to insecticides by RNA interference (RNAi). Developmental expression pattern analysis revealed that S. frugiperda CYP321A8, CYP321A9, and CYP321B1 were highest in second-instar larvae among developmental stages, with 2.04-, 3.39-, and 8.58-fold compared with eggs, whereas CYP337B5 and CYP6AE44 were highest in adult stage, with 16.3- and 10.6-fold in comparison of eggs, respectively. Tissue-specific expression pattern analysis exhibited that CYP321A8, CYP321B1, and CYP6AE44 were highest in the midguts, with 3.56-, 3.33-, and 3.04-fold compared with heads, whereas CYP321A9 and CYP337B5 were highest in wings, with 3.07- and 3.36-fold compared with heads, respectively. RNAi was also conducted to explore detoxification effects of the five P450 genes on chlorantraniliprole. The second-instar larvae became more sensitive to chlorantraniliprole with a higher mortality rate than the control, after silencing CYP321A8, CYP321A9, and CYP321B1, respectively. These findings strongly supported our viewpoint that CYP321A8, CYP321A9, and CYP321B1 may play a critical role in insecticide detoxification. It will provide a basis for further study on regulation of P450 genes and the management of S. frugiperda.

Genome-wide identification of growth-regulating factors in moso bamboo (Phyllostachys edulis): in silico and experimental analyses.

Growth-regulating factor (GRF), a small plant-specific transcription factor (TF) family, is extensively involved in the regulation of growth and developmental processes. However, the GRF family has not been comprehensively studied in moso bamboo (Phyllostachys edulis), a typical non-timber forest member. Here, 18 GRF genes were identified and characterized from the moso bamboo genome, and they clustered into three subfamilies (A, B and C). PeGRF genes were analyzed to determine their gene structures, conserved motifs and promoter. The non-synonymous/synonymous substitution ratios of paralogous and orthologous were less than 1, indicating that the GRF family mainly experienced purifying selection during evolution. According to the analysis of tissue-specific expression patterns, the participation of moso bamboo GRFs might be required during the formation and development of these five tissues. Moreover, PeGRF proteins might be involved in the regulation of plant development in biological processes. The qRT-PCR analysis demonstrated that PeGRF genes played essential roles in combating hormonal stresses and they might be involved in hormone regulation. PeGRF11, a nuclear localized protein as assessed by a subcellular localization assay, could interact with PeGIF3 in yeast and in planta according to yeast two-hybridization and bimolecular fluorescence complementation assays (BiFC) assays. But PeGRF11, as a TF, had no transcriptional activity in yeast. These results provide useful information for future functional research on the GRF genes in moso bamboo.

Genome-wide characterization of SPL family in Medicago truncatula reveals the novel roles of miR156/SPL module in spiky pod development

BackgroundSQUAMOSA Promoter Binding Protein-Likes (SPLs) proteins are plant-specific transcription factors that play many crucial roles in plant growth and development. However, there is little information about SPL family in the model legume Medicago truncatula.ResultsIn this study, a total of 23 MtSPL genes were identified in M. truncatula genome, in which 17 of the MtSPLs contained the putative MtmiR156 binding site at the coding or 3′ UTR regions. Tissue-specific expression pattern analysis showed that most MtmiR156-targeted MtSPLs were highly expressed in seed and pod. The observation of MtmiR156B-overexpressing plants reveals that MtmiR156/MtSPL modules are not only involved in the development of leaves and branches, but also in the seed pod development, especially the formation of spine on pod.ConclusionThe spines on pods are developed in many plant species, which allow pods to adhere to the animals, and then be transported on the outside. This study sheds light on the new function of SPL family in seed dispersal by controlling the formation of spiky pod, and provides insights on understanding evolutionary divergence of the members of SPL gene family among plant species.

Identification and RNAi-based function analysis of chitinase family genes in diamondback moth, Plutella xylostella.

Insect chitinases play a vital part in chitin degradation in exoskeletons and gut linings during the molting process, and therefore are considered potential targets for new insecticide designs or RNA interference (RNAi)-based pest management. Systematic functional analysis of chitinase genes has already been conducted in several insect pests, but not Plutella xylostella. In this study, 13 full-length chitinase transcripts were obtained in P. xylostella. Developmental and tissue-specific expression pattern analysis revealed that seven chitinase transcripts were periodically expressed during molting stage and mainly expressed in the integument or midgut, including PxCht3, PxCht5, PxCht6-2, PxCht7, PxCht8, PxCht10 and PxCht-h. RNAi-mediated knockdown of these specific expressed genes revealed that PxCht5 and PxCht10 were essential in larval molting, pupation and eclosion, and PxCht7 was indispensable only in eclosion. No significant effects were observed on insect survival or normal development when the rest chitinase transcripts were suppressed by RNAi. Our results indicated the function of P. xylostella chitinase family genes during the molting process, and may provide potential targets for RNAi-based management of P. xylostella. © 2018 Society of Chemical Industry.

NCAPG is differentially expressed during longissimus muscle development and is associated with growth traits in Chinese Qinchuan beef cattle.

Based on RNA-seq analysis, we recently found that the bovine NCAPG (non-SMC condensin I complex, subunit G) gene is differentially expressed during development of the longissimus muscle. In the present study, we validated this result and, using quantitative real-time PCR analysis, identified two adjacent genes, LCORL and DCAF16, that are more abundant in fetal muscle tissue; further analysis of tissue-specific expression patterns indicated high abundance of NCAPG in muscle. Since no polymorphisms were detected in a previous study of Qinchuan cattle, we extended our investigation to examine the occurrence of single-nucleotide polymorphisms (SNPs) in the NCAPG gene. Three SNPs, i.e., one located in the intron region (g47747: T > G), a synonymous mutation (g52535: A > G) and a missense mutation (g53208: T > G) that leads to a change in the amino acid of interest (pIle442Met), were detected in a population of Qinchuan beef cattle (n = 300). Association analysis showed that these SNPs were significantly associated with the growth traits of Qinchuan beef cattle. Our results indicate that the bovine NCAPG gene may be involved in the development of the longissimus muscle. These polymorphisms in the NCAPG gene may be useful for marker-assisted selection of optimal body size in Qinchuan beef cattle.

Major latex protein-like protein 43 (MLP43) functions as a positive regulator during abscisic acid responses and confers drought tolerance in Arabidopsis thaliana.

Drought stress is one of the disadvantageous environmental conditions for plant growth and reproduction. Given the importance of abscisic acid (ABA) to plant growth and abiotic stress responses, identification of novel components involved in ABA signalling transduction is critical. In this study, we screened numerous Arabidopsis thaliana mutants by seed germination assay and identified a mutant mlp43 (major latex protein-like 43) with decreased ABA sensitivity in seed germination. The mlp43 mutant was sensitive to drought stress while the MLP43-overexpressed transgenic plants were drought tolerant. The tissue-specific expression pattern analysis showed that MLP43 was predominantly expressed in cotyledons, primary roots and apical meristems, and a subcellular localization study indicated that MLP43 was localized in the nucleus and cytoplasm. Physiological and biochemical analyses indicated that MLP43 functioned as a positive regulator in ABA- and drought-stress responses in Arabidopsis through regulating water loss efficiency, electrolyte leakage, ROS levels, and as well as ABA-responsive gene expression. Moreover, metabolite profiling analysis indicated that MLP43 could modulate the production of primary metabolites under drought stress conditions. Reconstitution of ABA signalling components in Arabidopsis protoplasts indicated that MLP43 was involved in ABA signalling transduction and acted upstream of SnRK2s by directly interacting with SnRK2.6 and ABF1 in a yeast two-hybrid assay. Moreover, ABA and drought stress down-regulated MLP43 expression as a negative feedback loop regulation to the performance of MLP43 in ABA and drought stress responses. Therefore, this study provided new insights for interpretation of physiological and molecular mechanisms of Arabidopsis MLP43 mediating ABA signalling transduction and drought stress responses.

Molecular cloning and sequence analysis of lipoxygenase (AsLOX1) gene in Aquilaria sinensis (Lour.) Gilg and its expression responding to MeJA and crush wounding treatment

Lipoxygenase (LOX) not only acts as a key enzyme in the octadecanoid pathway and jasmonate (JA) synthesis, but also plays a significant role in plant growth and development, especially plant defense. The LOX gene, however, remains unclear in Aquilaria sinensis, a “wounding-induced-type” medicinal plant with great economic value. Based on the transcriptome data, a full-length cDNA sequence of LOX gene (termed as AsLOX1) was cloned for A. sinensis by RT-PCR and RACE cloning strategy. The sequence analysis showed that the AsLOX1 was 3168bp in length (GenBank accession number: KJ868174), and contained a complete open frame (ORF) of 2754bp. The deduced protein is composed of 917 amino acids, with a predicted molecular weight of 103.55kDa and an isoelectric point of 6.70. The amino acid sequence of AsLOX1 was determined to have nearly 80% sequence identity with the LOX genes of several other species by multiple alignments, suggesting that LOX belongs to a kind of conservative proteins. The deduced AsLOX1 protein was predicted to possess PLAT_LH2 and the lipoxygenase superfamily domain. The phylogenetic tree analysis reveals that AsLOX1 is more closely related to Theobroma cacao LOX than to those of other plants. According to the tissue-specific expression pattern analysis, the LOX gene in A. sinensis tissues is mainly expressed in roots and stems, the main agarwood formation parts. In both the methyl jasmonate (MeJA) and crush wounding treatment, the expression of AsLOX1 was observed to be significantly induced in A. sinensis calli, which was relatively high at 1h, reached the peak at 6h, and then declined rapidly to the normal level. These results demonstrate that AsLOX1 gene is an early damage-responsive gene, and has its expression modulated in response to MeJA stress and wounding, indicating the role of AsLOX1 in defense responses to wounding-induced agarwood formation in A. sinensis.

Chitinase-like gene family in the brown planthopper, Nilaparvata lugens.

Chitinases are important enzymes required for chitin degradation and reconstruction in insects. Based on a bioinformatics investigation, we identified 12 genes encoding putative chitinase-like proteins, including 10 chitinases (Cht), one imaginal disc growth factor (IDGF) and one endo-β-N-acetylglucosaminidase (ENGase) in the genome of the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae). These 12 genes were clustered into nine different groups, with 11 in glycoside hydrolase family 18 groups (groups I-VIII) and one in the ENGase group. Developmental and tissue-specific expression pattern analysis revealed that the transcript levels of eight genes peaked periodically during moulting and were mainly expressed in the integument, except NlCht2, NlCht4, NlIDGF and NlENGase. NlCht2, NlIDGF and NlENGase were expressed at all stages with slight periodical changes and mainly expressed in the female reproductive organs in adults, whereas NlCht4 was highly expressed only at the adult stage in the male reproductive organs. Lethal phenotypes were observed in insects challenged by double-stranded RNAs for NlCht1, NlCht5, NlCht7, NlCht9 and NlCht10 during moulting, suggesting their significant roles in old cuticle degradation. NlCht1 was the most sensitive gene, inducing 50% mortality even at 0.01 ng per insect. Our results illustrate the structural and functional differences of chitinase-like family genes and provide potential targets for RNA interference-based rice planthopper management.

Isolation, structural analysis, and expression characteristics of the maize (Zea mays L.) hexokinase gene family.

Hexokinases (HXKs, EC 2.7.1.1) play important roles in metabolism, glucose (Glc) signaling, and phosphorylation of Glc and fructose and are ubiquitous in all organisms. Despite their physiological importance, the maize HXK (ZmHXK) genes have not been analyzed systematically. We isolated and characterized nine members of the ZmHXK gene family which were distributed on 3 of the 10 maize chromosomes. A multiple sequence alignment and motif analysis revealed that the maize ZmHXK proteins share three conserved domains. Phylogenetic analysis revealed that the ZmHXK family can be divided into four subfamilies. We identified putative cis-elements in the ZmHXK promoter sequences potentially involved in phytohormone and abiotic stress responses, sugar repression, light and circadian rhythm regulation, Ca(2+) responses, seed development and germination, and CO2-responsive transcriptional activation. To study the functions of maize HXK isoforms, we characterized the expression of the ZmHXK5 and ZmHXK6 genes, which are evolutionarily related to the OsHXK5 and OsHXK6 genes from rice. Analysis of tissue-specific expression patterns using quantitative real time-PCR showed that ZmHXK5 was highly expressed in tassels, while ZmHXK6 was expressed in both tassels and leaves. ZmHXK5 and ZmHXK6 expression levels were upregulated by phytohormones and by abiotic stress.

A Sperm–Plasma β-N-Acetyl-D-Hexosaminidase Interacting with a Chitinolytic β-N-Acetyl-D-Hexosaminidase in Insect Molting Fluid

Insects require molting fluids to shed the old cuticle during molting. β-N-acetyl-D-hexosaminidase, known as Hex1, together with various chitinases, is responsible for degrading the chitin component of the old cuticle. This study showed that another β-N-acetyl-D-hexosaminidase, termed OfHex3, interacted with Hex1 and functioned in the molting fluid, although the homolog of OfHex3 was known as a sperm–plasma enzyme functioning in egg–sperm recognition. OfHex3 is an enzyme cloned from the insect Asian corn borer, Ostrinia furnacalis, which is one of the most destructive pests of maize. The enzymatic activity analysis indicated that OfHex3 was able to degrade chitooligosaccharides, but at a lower rate than that of OfHex1. Because OfHex3 did not have substrate inhibition, we deduced that the presence of OfHex3 might help OfHex1 relieve substrate inhibition during chitin degradation during molting. The expression patterns of OfHex3 during O. furnacalis development were studied by real-time PCR as well as western blot. The results showed that both gene transcription and protein translation levels of OfHex3 were up-regulated during larval–larval molting. The tissue-specific expression pattern analysis indicated that OfHex3 was mostly localized in the fat body and testis. All these data further supported that Hex3 was involved in molting as well as in fertilization. This study may help to understand the complexity of cuticle degradation during insect molting, and may provide a possible target for pest control.

De novo sequencing and analysis of the American ginseng root transcriptome using a GS FLX Titanium platform to discover putative genes involved in ginsenoside biosynthesis

BackgroundAmerican ginseng (Panax quinquefolius L.) is one of the most widely used herbal remedies in the world. Its major bioactive constituents are the triterpene saponins known as ginsenosides. However, little is known about ginsenoside biosynthesis in American ginseng, especially the late steps of the pathway.ResultsIn this study, a one-quarter 454 sequencing run produced 209,747 high-quality reads with an average sequence length of 427 bases. De novo assembly generated 31,088 unique sequences containing 16,592 contigs and 14,496 singletons. About 93.1% of the high-quality reads were assembled into contigs with an average 8-fold coverage. A total of 21,684 (69.8%) unique sequences were annotated by a BLAST similarity search against four public sequence databases, and 4,097 of the unique sequences were assigned to specific metabolic pathways by the Kyoto Encyclopedia of Genes and Genomes. Based on the bioinformatic analysis described above, we found all of the known enzymes involved in ginsenoside backbone synthesis, starting from acetyl-CoA via the isoprenoid pathway. Additionally, a total of 150 cytochrome P450 (CYP450) and 235 glycosyltransferase unique sequences were found in the 454 cDNA library, some of which encode enzymes responsible for the conversion of the ginsenoside backbone into the various ginsenosides. Finally, one CYP450 and four UDP-glycosyltransferases were selected as the candidates most likely to be involved in ginsenoside biosynthesis through a methyl jasmonate (MeJA) inducibility experiment and tissue-specific expression pattern analysis based on a real-time PCR assay.ConclusionsWe demonstrated, with the assistance of the MeJA inducibility experiment and tissue-specific expression pattern analysis, that transcriptome analysis based on 454 pyrosequencing is a powerful tool for determining the genes encoding enzymes responsible for the biosynthesis of secondary metabolites in non-model plants. Additionally, the expressed sequence tags (ESTs) and unique sequences from this study provide an important resource for the scientific community that is interested in the molecular genetics and functional genomics of American ginseng.