Cloning and function analysis of DlWRKY9 gene in longan (Dimocarpus longan)

WRKY is one of the largest plant transcription factors (TFs) which is widely involved in plant growth, development, and responses to stresses. In the present study, a WRKY TF DlWRKY9 was cloned from longan (Dimocarpus longan). The coding sequence (CDS) of DlWRKY9 is 762 bp in length and encodes 253 amino acids. It has a typical WRKY domain and zinc finger structure which belongs to type IIa WRKY protein. The molecular weight of DlWRKY9 protein was 30.27kda and the theoretical isoelectric point (PI) was 5.24. It is an unstable hydrophilic protein. The secondary structure of DlWRKY9 protein consists of helical structure (17.39%), extended chain (8.70%) and other structures (turn and random coil) (73.91%). The amino acid sequence of DlWRKY9 protein had the highest similarity with DlWRKY9 (xp_006450293.1) of citrus Clementina. DlWRKY9 gene promoter elements contain light, abscisic acid, gibberellin, jasmonic acid and other response elements. The results of qRT-PCR showed that the relative expression level of DlWRKY9 gene in pericarp was higher, followed by young fruits and floral organs. Meanwhile, DlWRKY9 gene specifically down-regulated in the early stage of flower induction in ‘Sijimi’ (SJ) longan. The results of transient expression of Arabidopsis protoplasts showed that the fluorescence signal was mainly concentrated in the nucleus. Moreover, overexpression of DlWRKY9 in Arabidopsis promoted early flowering. These results provide useful information for revealing the biological roles of DlWRKY9 in longan and increase our understanding of the WRKY family in fruit trees

Cloning and function analysis of DlWRKY9 gene in longan (Dimocarpus longan)

WRKY is one of the largest plant transcription factors (TFs) which is widely involved in plant growth, development, and responses to stresses. In the present study, a WRKY TF DlWRKY9 was cloned from longan (Dimocarpus longan). The coding sequence (CDS) of DlWRKY9 is 762 bp in length and encodes 253 amino acids. It has a typical WRKY domain and zinc finger structure which belongs to type IIa WRKY protein. The molecular weight of DlWRKY9 protein was 30.27kda and the theoretical isoelectric point (PI) was 5.24. It is an unstable hydrophilic protein. The secondary structure of DlWRKY9 protein consists of helical structure (17.39%), extended chain (8.70%) and other structures (turn and random coil) (73.91%). The amino acid sequence of DlWRKY9 protein had the highest similarity with DlWRKY9 (xp_006450293.1) of citrus Clementina. DlWRKY9 gene promoter elements contain light, abscisic acid, gibberellin, jasmonic acid and other response elements. The results of qRT-PCR showed that the relative expression level of DlWRKY9 gene in pericarp was higher, followed by young fruits and floral organs. Meanwhile, DlWRKY9 gene specifically down-regulated in the early stage of flower induction in ‘Sijimi’ (SJ) longan. The results of transient expression of Arabidopsis protoplasts showed that the fluorescence signal was mainly concentrated in the nucleus. Moreover, overexpression of DlWRKY9 in Arabidopsis promoted early flowering. These results provide useful information for revealing the biological roles of DlWRKY9 in longan and increase our understanding of the WRKY family in fruit trees

Karyotype Analysis of Diploid and Spontaneously Occurring Tetraploid Blood Orange [Citrus sinensis (L.) Osbeck] Using Multicolor FISH With Repetitive DNA Sequences as Probes

Blood orange [Citrus sinensis (L.) Osbeck] has been increasingly appreciated by consumers worldwide owing to its brilliant red color, abundant anthocyanin and other health-promoting compounds. However, there is still relatively little known about its cytogenetic characteristics, probably because of the small size and similar morphology of metaphase chromosomes and the paucity of chromosomal landmarks. In our previous study, a naturally occurring tetraploid blood orange plant was obtained via seedling screening. Before this tetraploid germplasm can be manipulated into a citrus triploid seedless breeding program, it is of great importance to determine its chromosome characterization and composition. In the present study, an integrated karyotype of blood orange was constructed using sequential multicolor fluorescence in situ hybridization (FISH) with four satellite repeats, two ribosomal DNAs (rDNAs), a centromere-like repeat and an oligonucleotide of telomere repeat (TTTAGGG)3 as probes. Satellite repeats were preferentially located at the terminal regions of the chromosomes of blood orange. Individual somatic chromosome pairs of blood orange were unambiguously identified by repetitive DNA-based multicolor FISH. These probes proved to be effective chromosomal landmarks. The karyotype was formulated as 2n = 2x = 18 = 16m+2sm (1sat) with the karyotype asymmetry degree belonging to 2B. The chromosomal distribution pattern of these repetitive DNAs in this spontaneously occurring tetraploid was identical to that of the diploid, but the tetraploid carried twice the number of hybridization sites as the diploid, indicating a possible pathway involving the spontaneous duplication of chromosome sets in nucellar cells. Our work may facilitate the molecular cytogenetic study of blood orange and provide chromosomal characterization for the future utilization of this tetraploid germplasm in the service of seedless breeding programs

Comparative transcriptome analysis of molecular mechanisms underlying adventitious root developments in Huangshan Bitter tea (Camellia gymnogyna Chang) under red light quality

As the formation of adventitious roots (AR) is an important component of in vitro regeneration of tea plants, the propagation and preservation of Huangshan Bitter tea (Camellia gymnogyna Chang) cuttings have been hindered due to its lower rooting rate. As light is a crucial environmental factor that affects AR formation, this study aimed to investigate the special role of red light (RL) in the formation of AR in Huangshan Bitter tea plants, which has not been well understood. Huangshan Bitter tea plants were induced with white light (control, WL) and red light (660 nm, RL) qualities 36 days after induced treatment (DAI) to investigate dynamic AR formation and development, anatomical observation, hormones content change, and weighted gene co-expression network analysis (WGCNA) of the transcriptome. Results showed that RL promoted the rooting rate and root characteristics compared to WL. Anatomical observations demonstrated that root primordium was induced earlier by RL at the 4 DAI. RL positively affected IAA, ZT and GA3 content and negatively influenced ABA from the 4 to 16 DAI. RNA-seq and analysis of differential expression genes (DEGs) exhibited extensive variation in gene expression profiles between RL and WL. Meanwhile, the results of WGCNA and correlation analysis identified three highly correlated modules and hub genes mainly participated in 'response to hormone', 'cellular glucan metabolic progress', and 'response to auxin'. Furthermore, the proportion of transcription factors (TFs) such as ethylene response factor (ERF), myeloblastosis (MYB), basic helix-loop-helix (bHLH), and WRKYGQK (WRKY) were the top four in DEGs. These results suggested that the AR-promoting potential of red light was due to complex hormone interactions in tea plants by regulating the expression of related genes. This study provided an important reference to shorten breeding cycles and accelerate superiority in tea plant propagation and preservation

Global Optimization for Sum of Linear Ratios Problem Using New Pruning Technique

A global optimization algorithm is proposed for solving sum of general linear ratios problem (P) using new pruning technique. Firstly, an equivalent problem (P1) of the (P) is derived by exploiting the characteristics of linear constraints. Then, by utilizing linearization method the relaxation linear programming (RLP) of the (P1) can be constructed and the proposed algorithm is convergent to the global minimum of the (P) through the successive refinement of the linear relaxation of feasible region and solutions of a series of (RLP). Then, a new pruning technique is proposed, this technique offers a possibility to cut away a large part of the current investigated feasible region by the optimization algorithm, which can be utilized as an accelerating device for global optimization of problem (P). Finally, the numerical experiments are given to illustrate the feasibility of the proposed algorithm

Strength and deformation characteristics of compacted loess with different moisture content and compaction energy

The normal operation of superstructure in loess areas will be related closely to the strength and stability of compacted loess. Loess samples derived from two different engineering sites in China were compacted with different compaction energy, and the main factors which will affect the strength and deformation of compacted loess were analyzed. Different soil samples were prepared with varying moisture content of 11 %–19 %. As the moisture content increases, the compression deformation of compacted loess increases. The immersion influence coefficient suitable for compacted loess was calculated in this study. During soaking in water for 4 days, the California Bearing Ratio (CBR) decreases gradually. Especially, in the process of soak, the lower the initial moisture contents of sample, the more obvious the CBR degradation of sample. It is suggested that soil should be compacted with appropriate compaction energy and moisture content. Furthermore, the loess should be compacted under the condition that the moisture content is slightly greater than the optimal moisture content. When the current moisture content is about 1.02 times the optimal value, the compaction performance of loess is better. As disclosed in this research, the compacted loess in optimal compaction state has smaller compression deformation, better stability and weaker sensitivity to saturation

<i>NpPP2-B10</i>, an F-Box-Nictaba Gene, Promotes Plant Growth and Resistance to Black Shank Disease Incited by <i>Phytophthora nicotianae</i> in <i>Nicotiana tabacum</i>

Black shank, a devastating disease affecting tobacco production worldwide, is caused by Phytophthora nicotianae. However, few genes related to Phytophthora resistance have been reported in tobacco. Here, we identified NpPP2-B10, a gene strongly induced by P. nicotianae race 0, with a conserved F-box motif and Nictaba (tobacco lectin) domain, in the highly resistant tobacco species Nicotiana plumbaginifolia. NpPP2-B10 is a typical F-box-Nictaba gene. When it was transferred into the black shank-susceptible tobacco cultivar ‘Honghua Dajinyuan’, it was found to promote resistance to black shank disease. NpPP2-B10 was induced by salicylic acid, and some resistance-related genes (NtPR1, NtPR2, NtCHN50, and NtPAL) and resistance-related enzymes (catalase and peroxidase) were significantly upregulated in the overexpression lines after infection with P. nicotianae. Furthermore, we showed that NpPP2-B10 actively regulated the tobacco seed germination rate, growth rate, and plant height. The erythrocyte coagulation test of purified NpPP2-B10 protein showed that NpPP2-B10 had plant lectin activity, and the lectin content in the overexpression lines was significantly higher than that in the WT, which could lead to accelerated growth and improved resistance of tobacco. SKP1 is an adaptor protein of the E3 ubiquitin ligase SKP1, Cullin, F-box (SCF) complex. We demonstrated that NpPP2-B10 could interact with the NpSKP1-1A gene in vivo and in vitro through yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC), indicating that NpPP2-B10 likely participates in the plant immune response by mediating the ubiquitin protease pathway. In conclusion, our study provides some important insights concerning NpPP2-B10-mediated regulation of tobacco growth and resistance

Molecular cytogenetic analysis of genome-specific repetitive elements in Citrus clementina Hort. Ex Tan. and its taxonomic implications

Abstract Background Clementine mandarin (Citrus clementina Hort. ex Tan.) is one of the most famous and widely grown citrus cultivars worldwide. Variations in relation to the composition and distribution of repetitive DNA sequences that dominate greatly in eukaryote genomes are considered to be species-, genome-, or even chromosome-specific. Repetitive DNA-based fluorescence in situ hybridization (FISH) is a powerful tool for molecular cytogenetic study. However, to date few studies have involved in the repetitive elements and cytogenetic karyotype of Clementine. Results A graph-based similarity sequence read clustering methodology was performed to analyze the repetitive DNA families in the Clementine genome. The bioinformatics analysis showed that repetitive DNAs constitute 41.95% of the Clementine genome, and the majority of repetitive elements are retrotransposons and satellite DNAs. Sequential multicolor FISH using a probe mix that contained CL17, four satellite DNAs, two rDNAs and an oligonucleotide of (TTTAGGG)3 was performed with Clementine somatic metaphase chromosomes. An integrated karyotype of Clementine was established based on unequivocal and reproducible chromosome discriminations. The distribution patterns of these probes in several Citrus, Poncirus and Fortunella species were summarized through extensive FISH analyses. Polymorphism and heterozygosity were commonly observed in the three genera. Some asymmetrical FISH loci in Clementine were in agreement with its hybrid origin. Conclusions The composition and abundance of repetitive elements in the Clementine genome were reanalyzed. Multicolor FISH-based karyotyping provided direct visual proof of the heterozygous nature of Clementine chromosomes with conspicuous asymmetrical FISH hybridization signals. We detected some similar and variable distribution patterns of repetitive DNAs in Citrus, Poncirus, and Fortunella, which revealed notable conservation among these genera, as well as obvious polymorphism and heterozygosity, indicating the potential utility of these repetitive element markers for the study of taxonomic, phylogenetic and evolutionary relationships in the future