Genome-wide identification of B-box zinc finger (BBX) gene family in Medicago sativa and their roles in abiotic stress responses.

BACKGROUND: B-box (BBX) family is a class of zinc finger transcription factors (TFs) that play essential roles in regulating plant growth, development, as well as abiotic stress. However, no systematic analysis of BBX genes has yet been conducted in alfalfa (Medica go sativa L.), and their functions have not been elucidated up to now. RESULTS: In this study, 28 MsBBX genes were identified from the alfalfa genome, which were clustered into 4 subfamilies according to an evolutionary tree of BBX proteins. Exon-intron structure and conserved motif analysis reflected the evolutionary conservation of MsBBXs in alfalfa. Collinearity analysis showed that segmental duplication promoted the expansion of the MsBBX family. Analysis of cis-regulatory elements suggested that the MsBBX genes possessed many growth/development-, light-, phytohormone-, and abiotic stress-related elements. MsBBX genes were differentially expressed in leaves, flowers, pre-elongated stems, elongated stems, roots and nodules, and most MsBBXs were remarkably induced by drought, salt and various plant growth regulators (ABA, JA, and SA). Further functional verification demonstrated that overexpressing of the MsBBX11 gene clearly promoted salt tolerance in transgenic Arabidopsis by regulating growth and physiological processes of seedlings. CONCLUSIONS: This research provides insights into further functional research and regulatory mechanisms of MsBBX family genes under abiotic stress of alfalfa.

Rapidly mining candidate cotton drought resistance genes based on key indicators of drought resistance.

BACKGROUND: Focusing on key indicators of drought resistance is highly important for quickly mining candidate genes related to drought resistance in cotton. RESULTS: In the present study, drought resistance was identified in drought resistance-related RIL populations during the flowering and boll stages, and multiple traits were evaluated; these traits included three key indicators: plant height (PH), single boll weight (SBW) and transpiration rate (Tr). Based on these three key indicators, three groups of extreme mixing pools were constructed for BSA-seq. Based on the mapping interval of each trait, a total of 6.27 Mb QTL intervals were selected on chromosomes A13 (3.2 Mb), A10 (2.45 Mb) and A07 (0.62 Mb) as the focus of this study. Based on the annotation information and qRT‒PCR analysis, three key genes that may be involved in the drought stress response of cotton were screened: GhF6'H1, Gh3AT1 and GhPER55. qRT‒PCR analysis of parental and extreme germplasm materials revealed that the expression of these genes changed significantly under drought stress. Cotton VIGS experiments verified the important impact of key genes on cotton drought resistance. CONCLUSIONS: This study focused on the key indicators of drought resistance, laying the foundation for the rapid mining of drought-resistant candidate genes in cotton and providing genetic resources for directed molecular breeding of drought resistance in cotton.

Genome-wide identification of Aux/IAA gene family in white clover (Trifolium repens L.) and functional verification of TrIAA18 under different abiotic stress.

BACKGROUND: White clover (Trifolium repens L.) is an excellent leguminous cool-season forage with a high protein content and strong nitrogen-fixing ability. Despite these advantages, its growth and development are markedly sensitive to environmental factors. Indole-3-acetic acid (IAA) is the major growth hormone in plants, regulating plant growth, development, and response to adversity. Nevertheless, the specific regulatory functions of Aux/IAA genes in response to abiotic stresses in white clover remain largely unexplored. RESULTS: In this study, we identified 47 Aux/IAA genes in the white clover genome, which were categorized into five groups based on phylogenetic analysis. The TrIAAs promoter region co-existed with different cis-regulatory elements involved in developmental and hormonal regulation, and stress responses, which may be closely related to their diverse regulatory roles. Collinearity analysis showed that the amplification of the TrIAA gene family was mainly carried out by segmental duplication. White clover Aux/IAA genes showed different expression patterns in different tissues and under different stress treatments. In addition, we performed a yeast two-hybrid analysis to investigate the interaction between white clover Aux/IAA and ARF proteins. Heterologous expression indicated that TrIAA18 could enhance stress tolerance in both yeast and transgenic Arabidopsis thaliana. CONCLUSION: These findings provide new scientific insights into the molecular mechanisms of growth hormone signaling in white clover and its functional characteristics in response to environmental stress.

Functional Study on the Key Gene LaLBD37 Related to the Lily Bulblets Formation.

Oriental hybrid lilies, known for their vibrant colors, diverse flower shapes, and long blooming seasons, require annual bulb propagation in horticultural production. This necessity can lead to higher production costs and limit their use in landscaping. The LA hybrid lily 'Aladdin' has shown strong self-reproduction capabilities in optimal cultivation environments, producing numerous high-quality underground stem bulblets. This makes it a valuable model for studying bulblet formation in lilies under natural conditions. Through transcriptome data analysis of different developmental stages of 'Aladdin' bulblets, the LaLBD37 gene, linked to bulblet formation, was identified. Bioinformatics analysis, subcellular localization studies, and transcriptional activation activity tests were conducted to understand the characteristics of LaLBD37. By introducing the LaLBD37 gene into 'Sorbonne' aseptic seedlings via Agrobacterium-mediated transformation, resistant plants were obtained. Positive plants were identified through various methods such as GUS activity detection, PCR, and fluorescence quantitative PCR. Phenotypic changes in positive plants were observed, and various physiological indicators were measured to confirm the role of LaLBD37 in bulblet formation, including soluble sugar content, starch content, sucrose synthase activity, and endogenous hormone levels. The findings suggest that the LaLBD37 gene plays a significant role in promoting the development of lily bulblets, offering insights for enhancing the reproductive capacity of Oriental hybrid lilies and exploring the molecular mechanisms involved in lily bulb regeneration.

Identification and Functional Studies on the Role of PlSPL14 in Herbaceous Peony Stem Development.

Stem strength plays a crucial role in the growth and development of plants, as well as in their flowering and fruiting. It not only impacts the lodging resistance of crops, but also influences the ornamental value of ornamental plants. Stem development is closely linked to stem strength; however, the roles of the SPL transcription factors in the stem development of herbaceous peony (Paeonia lactiflora Pall.) are not yet fully elucidated. In this study, we obtained and cloned the full-length sequence of PlSPL14, encoding 1085 amino acids. Quantitative real-time PCR (qRT-PCR) analysis revealed that the expression level of PlSPL14 gradually increased with the stem development of P. lactiflora and was significantly expressed in vascular bundles. Subsequently, utilizing the techniques of virus-induced gene silencing (VIGS) and heterologous overexpression in tobacco (Nicotiana tabacum L.), it was determined that PlSPL14-silenced P. lactiflora had a thinner xylem thickness, a decreased stem diameter, and weakened stem strength, while PlSPL14-overexpressing tobacco resulted in a thicker xylem thickness, an increased stem diameter, and enhanced stem strength. Further screening of the interacting proteins of PlSPL14 using a yeast two-hybrid (Y2H) assay revealed an interactive relationship between PlSPL14 and PlSLR1 protein, which acts as a negative regulator of gibberellin (GA). Additionally, the expression level of PlSLR1 gradually decreased during the stem development of P. lactiflora. The above results suggest that PlSPL14 may play a positive regulatory role in stem development and act in the xylem, making it a potential candidate gene for enhancing stem straightness in plants.

Grape SnRK2.7 Positively Regulates Drought Tolerance in Transgenic Arabidopsis.

In this study, we obtained and cloned VvSnRK2.7 by screening transcriptomic data to investigate the function of the grape sucrose non-fermenting kinase 2 (SnRK2) gene under stress conditions. A yeast two-hybrid (Y2H) assay was used to further screen for interaction proteins of VvSnRK2.7. Ultimately, VvSnRK2.7 was heterologously expressed in Arabidopsis thaliana, and the relative conductivity, MDA content, antioxidant enzyme activity, and sugar content of the transgenic plants were determined under drought treatment. In addition, the expression levels of VvSnRK2.7 in Arabidopsis were analyzed. The results showed that the VvSnRK2.7-EGFP fusion protein was mainly located in the cell membrane and nucleus of tobacco leaves. In addition, the VvSnRK2.7 protein had an interactive relationship with the VvbZIP protein during the Y2H assay. The expression levels of VvSnRK2.7 and the antioxidant enzyme activities and sugar contents of the transgenic lines were higher than those of the wild type under drought treatment. Moreover, the relative conductivity and MDA content were lower than those of the wild type. The results indicate that VvSnRK2.7 may activate the enzyme activity of the antioxidant enzyme system, maintain normal cellular physiological metabolism, stabilize the berry sugar metabolism pathway under drought stress, and promote sugar accumulation to improve plant resistance.

[Cloning and functional verification of farnesyl pyrophosphate synthase gene(AvFPS) from Aconitum vilmorinianum].

Aconitum vilmorinianum is an authentic and superior medicinal herbal in Yunnan, which is rich in yunaconitine and other diterpene alkaloids. Diterpene alkaloids are its main active components. Farnesyl pyrophosphate synthase(FPS) is a key enzyme in the terpene biosynthetic pathway and plays an important role in diterpene alkaloid biosynthesis. Functional studies of FPS help to reveal the molecular mechanism of diterpene alkaloid biosynthesis. In this study, one FPS gene(AvFPS) was selected based on the transcriptome data of A. vilmorinianum. Its full-length sequence was cloned, and bioinformatic analysis, functional verification, and gene expression analysis were performed. The open reading frame(ORF) of AvFPS was 1 056 bp, encoding 351 amino acids. Its molecular weight was 41 kDa. AvFPS had two typical conserved functional domains of isopentenyl transferase, " DDIMD" and " DDYXD". The recombinant protein of AvFPS was expressed in Escherichia coli, and purified recombinant protein was used for in vitro enzymatic reaction. The results revealed that AvFPS was able to catalyze the synthesis of farnesyl pyrophosphate(FPP). The results of qRT-PCR analysis showed that AvFPS was expressed in the roots, stems, leaves, and flowers of A. vilmorinianum, with the highest expression level in the roots. The expression level of AvFPS was significantly up-regulated by MeJA induction. This study clarified the catalytic function of AvFPS, revealed the expression pattern of AvFPS in different tissue, as well as at different time induced by MeJA, and provided a reference for a deeper understanding of the function of FPS in the biosynthesis of diterpenoid components.

S100A9 plays a key role in Clostridium perfringens beta2 toxin-induced inflammatory damage in porcine IPEC-J2 intestinal epithelial cells.

BACKGROUND: As an important regulator of autoimmune responses and inflammation, S100A9 may serve as a therapeutic target in inflammatory diseases. However, the role of S100A9 in Clostridium perfringens type C infectious diarrhea is poorly studied. The aim of our study was to screen downstream target genes regulated by S100A9 in Clostridium perfringens beta2 (CPB2) toxin-induced IPEC-J2 cell injury. We constructed IPEC-J2 cells with S100A9 knockdown and a CPB2-induced cell injury model, screened downstream genes regulated by S100A9 using RNA-Seq technique, and performed functional enrichment analysis. The function of S100A9 was verified using molecular biology techniques. RESULTS: We identified 316 differentially expressed genes (DEGs), of which 221 were upregulated and 95 were downregulated. Functional enrichment analysis revealed that the DEGs were significantly enriched in cilium movement, negative regulation of cell differentiation, immune response, protein digestion and absorption, and complement and coagulation cascades. The key genes of immune response were TNF, CCL1, CCR7, CSF2, and CXCL9. When CPB2 toxin-induced IPEC-J2 cells overexpressed S100A9, Bax expression increased, Bcl-2 expression and mitochondrial membrane potential decreased, and SOD activity was inhibited. CONCLUSION: In conclusion, S100A9 was involved in CPB2-induced inflammatory response in IPEC-J2 cells by regulating the expression of downstream target genes, namely, TNF, CCL1, CCR7, CSF2, and CXCL9; promoting apoptosis; and aggravating oxidative cell damage. This study laid the foundation for further study on the regulatory mechanism underlying piglet diarrhea.

Study on the role of calcium channel protein TRPV4 in the inflammatory pathway of type 2 diabetic adipose tissue based on gene databases.

BACKGROUND: Chronic inflammation of adipose tissue may be one of the key factors contributing to the development of insulin resistance in T2DM adipose tissue. Transient receptor potential vanilloid type 4 (TRPV4) can be involved in a variety of cellular inflammatory responses. In this study, we evaluated the role of TRPV4 channelin in the T2DM adipose tissue inflammatory pathway. METHODS: Based on the gene expression profiling data of the public database, bioinformatics methods were used to screen the target gene population of the TRPV4 channel protein involved in the regulation of T2DM fat cells. A mature adipocyte model was constructed to verify the expression level of target genes and to evaluate the regulatory effect of TRPV4 channel inhibition on target genes of inflammation-related pathways. RESULTS: In shTRPV4 adipocytes, 144 genes with downregulation expression were screened, a PPI network was constructed and a core module containing 15 genes was screened out, and the core genes were mainly enriched in the Toll-like receptor signaling pathway through enrichment analysis. Constructing a mature adipocyte model found that the TRPV4 inhibitor HC067047 inhibited the effect of upregulation of the expression level of the relevant gene in the signaling pathway. CONCLUSIONS: Our findings suggest that the expression of highly expressed pro-inflammatory cytokines and chemokines in T2DM adipose tissue decreases after inhibiting the expression of TRPV4 in adipocytes, suggesting that TRPV4 may become a potential drug target for the treatment of T2DM.

Regulatory mechanism of the miR172e-LbrAP2 module during the vegetative growth phase transition in Lilium.

MAIN CONCLUSION: It was proved for the first time that the miR172e-LbrAP2 module regulated the vegetative growth phase transition in Lilium, which provided a new approach to shorten the juvenile stage of Lilium, improved the reproduction rate, and reduced the propagation cost of Lilium commercial bulbs. Lilium is an ornamental bulb plant that takes at least 3 years to cultivate into commercial seed bulbs under natural conditions. The aim of this study was to shorten the Lilium expansion cycle. In this study, the growth cycle of lily tubers induced by low temperature of 15 °C was significantly shorter than that of tubers grown at a conventional temperature. Quantitative real-time PCR analysis showed that the expression patterns of miR172e and LbrAP2 were negatively correlated. GUS histochemical staining confirmed that miR172e and LbrAP2 in tobacco leaves interacted with each other after co-transformation. The shear sites of miR172e and its target gene, LbrAP2, upon binding, were identified by RLM 5' RACE analysis. In addition, miR172e and LbrAP2 showed opposite expression patterns after the transformation of Arabidopsis. miR172e overexpression accelerated the transition from juvenile to adult plants, whereas LbrAP2 overexpression inhibited this process, thus indicating that miR172e negatively regulated the target gene LbrAP2. Upregulation of the transcription factor LbrAP2 delayed the phase transition of plants, whereas miR172 inhibited the transcriptional translation of LbrAP2, thereby accelerating the phase transition. Low-temperature treatment of Lilium bulbs can shorten Lilium development, which provides a new approach to accelerating Lilium commercial bulb breeding and reducing breeding costs.

Integrated metabolite profiling and transcriptome analysis reveal candidate genes involved in the formation of yellow Nelumbo nucifera.

As a worldwide major ornamental flower and a edible plant, lotus (Nelumbo nucifera) is also used as medicine and tea beverage. Here, transcriptome and metabolites of yellow (MLQS) and white (YGB) lotus cultivars during five key flower coloration stages were profiled. 2014 differentially expressed genes were detected with 11 carotenoids in lotus were identified for the first time. Then, regulatory networks between and within functional modules was reconstructed, and the correlation between module-metabolites and gene-metabolites was conducted within 3 core modules. 18 candidate genes related to the formation of yellow flower were screened out and a gene regulatory model for the flower color difference between MLQS and YGB were speculated as follows: The substrate competition between F3'H and F3'5'H and substrate specificity of FLS, together with differential expression of CCD4a and CCD4b were contribute to the differences in flavonoids and carotenoids. Besides, UGT73C2, UGT91C1-2 and SGTase, and regulation of UGTs by transcription factors PLATZ, MADS, NAC031, and MYB308 may also play a role in the upstream regulation. The following verification results indicated that functional differences existed in the coding sequences of NnCCD4b and promoters of NnCCD4a of MLQS and YGB. In all, this study preliminarily reveals the mechanism of yellow flower coloration in lotus and provides new ideas for the study of complex ornamental characters of other plants.

The Expression of the StNRAMP2 Gene Determined the Accumulation of Cadmium in Different Tissues of Potato.

Cadmium (Cd) is a toxic metal that threatens human health when enriched in crops. NRAMPs are a family of natural macrophage proteins reported to play a key role in Cd transport in plants. In order to explore the gene regulation mechanism of potato under Cd stress and the role of NRAMPs family in it, this study analyzed the gene expression differences of two different Cd accumulation levels in potato after 7 days of 50 mg/kg Cd stress and screened out the key genes that may play a major role in the differential accumulation of Cd in different varieties. Additionally, StNRAMP2 was selected for verification. Further verification showed that the StNRAMP2 gene plays an important role in the accumulation of Cd in potato. Interestingly, silencing StNRAMP2 increased Cd accumulation in tubers but significantly decreased Cd accumulation in other sites, suggesting a critical role of StNRAMP2 in Cd uptake and transport in potatoes. To further confirm this conclusion, we performed heterologous expression experiments in which overexpression of StNRAMP2 gene in tomato resulted in a threefold increase in Cd content, which further confirmed the important role of StNRAMP2 in the process of Cd accumulation compared with wild-type plants. In addition, we found that the addition of Cd to the soil increased the activity of the plant antioxidant enzyme system, and silencing StNRAMP2 partially reversed this effect. This suggests that the StNRAMP2 gene plays an important role in plant stress tolerance, and future studies could further explore the role of this gene in other environmental stresses. In conclusion, the results of this study improve the understanding of the mechanism of Cd accumulation in potato and provide experimental basis for remediation of Cd pollution.

[Cloning and functional verification of PhAEP gene, a key enzyme for biosynthesis of heterophyllin A in Pseudostellaria heterophylla].

This paper aimed to study the role of asparagine endopeptidase(AEP) gene in the biosynthesis mechanism of cyclic peptide compounds in Pseudostellaria heterophylla. The transcriptome database of P. heterophylla was systematically mined and screened, and an AEP gene, tentatively named PhAEP, was successfully cloned. The heterologous function verification by Nicotiana benthamiana showed that the expression of the gene played a role in the biosynthesis of heterophyllin A in P. heterophylla. Bioinformatics analysis showed that the cDNA of PhAEP was 1 488 bp in length, encoding 495 amino acids with a molecular weight of 54.72 kDa. The phylogenetic tree showed that the amino acid sequence encoded by PhAEP was highly similar to that of Butelase-1 in Clitoria ternatea, reaching 80%. The sequence homology and cyclase active site analysis revealed that the PhAEP enzyme may specifically hydrolyse the C-terminal Asn/Asp(Asx) site of the core peptide in the HA linear precursor peptide of P. heterophylla, thereby participating in the ring formation of the linear precursor peptide. The results of real-time quantitative polymerase chain reaction(RT-qPCR) showed that the expression level of PhAEP was the highest in fruits, followed by in roots, and the lowest in leaves. The heterophyllin A of P. heterophylla was detected in N. benthamiana that co-expressed PrePhHA and PhAEP genes instantaneously. In this study, the PhAEP gene, a key enzyme in the biosynthesis of heterophyllin A in P. heterophylla, has been successfully cloned, which lays a foundation for further analysis of the molecular mechanism of PhAEP enzyme in the biosynthesis of heterophyllin A in P. heterophylla and has important significance for the study of synthetic biology of cyclic peptide compounds in P. heterophylla.

Genome-wide identification of MAPK gene family members in Fagopyrum tataricum and their expression during development and stress responses.

BACKGROUND: Mitogen-activated protein kinases (MAPKs) plays essential roles in the development, hormone regulation and abiotic stress response of plants. Nevertheless, a comprehensive study on MAPK family members has thus far not been performed in Tartary buckwheat. RESULTS: Here, we identified 16 FtMAPKs in the Fagopyrum tataricum genome. Phylogenetic analysis showed that the FtMAPK family members could be classified into Groups A, B, C and D, in which A, B and C members contain a Thr-Glu-Tyr (TEY) signature motif and Group D members contain a Thr-Asp-Tyr (TDY) signature motif. Promoter cis-acting elements showed that most ProFtMAPks contain light response elements, hormone response elements and abiotic stress response elements, and several ProFtMAPks have MYB-binding sites, which may be involved in the regulation of flavonoid biosynthesis-related enzyme gene expression. Synteny analysis indicated that FtMAPKs have a variety of biological functions. Protein interaction prediction suggested that MAPKs can interact with proteins involved in development and stress resistance. Correlation analysis further confirmed that most of the FtMAPK genes and transcription factors involved in the stress response have the same expression pattern. The transient transformation of FtMAPK1 significantly increased the antioxidant enzymes activity in Tartary buckwheat leaves. In addition, we also found that FtMAPK1 can respond to salt stress by up-regulating the transcription abundance of downstream genes. CONCLUSIONS: A total of 16 MAPKs were identified in Tartary buckwheat, and the members of the MAPK family containing the TDY motif were found to have expanded. The same subfamily members have relatively conserved gene structures and similar protein motifs. Tissue-specific expression indicated that the expression of all FtMAPK genes varied widely in the roots, stems, leaves and flowers. Most FtMAPKs can regulate the expression of other transcription factors and participate in the abiotic stress response. Our findings comprehensively revealed the FtMAPK gene family and laid a theoretical foundation for the functional characterization of FtMAPKs.

Genome-wide analysis of the DREB family genes and functional identification of the involvement of BrDREB2B in abiotic stress in wucai (Brassica campestris L.).

Dehydration responsive element binding protein (DREB) is a significant transcription factor class known to be implicated in abiotic stresses. In this study, we systematically conducted a genome-wide identification and expression analysis of the DREB gene family, including gene structures, evolutionary relationships, chromosome distribution, conserved domains, and expression patterns. A total of 65 DREB family gene members were identified in Chinese cabbage (Brassica rapa L.) and were classified into five subgroups based on phylogenetic analysis. Through analysis of the conserved domains of BrDREB family genes, only one exon existed in the gene structure. Through the analysis of cis-acting elements, these genes were mainly involved in hormone regulation and adversity stress. In order to identify the function of BrDREB2B, overexpressed transgenic Arabidopsis was constructed. After different stress treatments, the germination rate, root growth, survival rate, and various plant physiological indicators were measured. The results showed that transgenic Arabidopsis thaliana plants overexpressing BrDREB2B exhibited enhanced tolerance to salt, heat and drought stresses. Taken together, our results are the first to report the BrDREB2B gene response to drought and heat stresses in Chinese cabbage and provide a basis for further studies to determine the function of BrDREBs in response to abiotic stresses.

GhAAO2 was observed responding to NaHCO3 stress in cotton compared to AAO family genes.

BACKGROUND: Abscisic acid (ABA) is an important stress hormone, the changes of abscisic acid content can alter plant tolerance to stress, abscisic acid is crucial for studying plant responses to abiotic stress. The abscisic acid aldehyde oxidase (AAO) plays a vital role in the final step in the synthesis of abscisic acid, therefore, understanding the function of AAO gene family is of great significance for plants to response to abiotic stresses. RESULT: In this study, 6, 8, 4 and 4 AAO genes were identified in four cotton species. According to the structural characteristics of genes and the traits of phylogenetic tree, we divided the AAO gene family into 4 clades. Gene structure analysis showed that the AAO gene family was relatively conservative. The analysis of cis-elements showed that most AAO genes contained cis-elements related to light response and plant hormones. Tissue specificity analysis under NaHCO3 stress showed that GhAAO2 gene was differentially expressed in both roots and leaves. After GhAAO2 gene silencing, the degree of wilting of seedlings was lighter than that of the control group, indicating that GhAAO2 could respond to NaHCO3 stress. CONCLUSIONS: In this study, the AAO gene family was analyzed by bioinformatics, the response of GhAAO gene to various abiotic stresses was preliminarily verified, and the function of the specifically expressed gene GhAAO2 was further verified. These findings provide valuable information for the study of potential candidate genes related to plant growth and stress.

Genome-Wide Identification and Analysis of bZIP Gene Family and Resistance of TaABI5 (TabZIP96) under Freezing Stress in Wheat (Triticum aestivum).

The basic leucine zipper (bZIP) regulates plant growth and responds to stress as a key transcription factor of the Abscisic acid (ABA) signaling pathway. In this study, TabZIP genes were identified in wheat and the gene structure, physicochemical properties, cis-acting elements, and gene collinearity were analyzed. RNA-Seq and qRT-PCR analysis showed that ABA and abiotic stress induced most TabZIP genes expression. The ectopic expression of TaABI5 up-regulated the expression of several cold-responsive genes in Arabidopsis. Physiological indexes of seedlings of different lines under freezing stress showed that TaABI5 enhanced the freezing tolerance of plants. Subcellular localization showed that TaABI5 is localized in the nucleus. Furthermore, TaABI5 physically interacted with cold-resistant transcription factor TaICE1 in yeast two-hybrid system. In conclusion, this study identified and analyzed members of the TabZIP gene family in wheat. It proved for the first time that the gene TaABI5 affected the cold tolerance of transgenic plants and was convenient for us to understand the cold resistance molecular mechanism of TaABI5. These results will provide a new inspiration for further study on improving plant abiotic stress resistance.

Sequence Analysis and Functional Verification of the Effects of Three Key Structural Genes, PdTHC2'GT, PdCHS and PdCHI, on the Isosalipurposide Synthesis Pathway in Paeonia delavayi var. lutea.

Isosalipurposide (ISP) is the most important yellow pigment in tree peony. In ISP biosynthesis, CHS catalyzes 1-molecule coumaroyl-CoA and 3-molecule malonyl-CoA to form 2',4',6',4-tetrahyroxychalcone (THC), and THC generates a stable ISP in the vacuole under the action of chalcone2'-glucosyltransferases (THC2'GT). In tree peony, the details of the THC2'GT gene have not yet been reported. In this study, the candidate THC2'GT gene (PdTHC2'GT) in Paeonia delavayi var. lutea was screened. At the same time, we selected the upstream CHS gene (PdCHS) and the competitive CHI gene (PdCHI) to study the biosynthesis pathway of ISP. We successfully cloned three genes and sequenced them; subcellular localization showed that the three genes were located in the nucleus and cytoplasm. The overexpression of PdTHC2'GT in tobacco caused the accumulation of ISP in tobacco petals, which indicated that PdTHC2'GT was the key structural gene in the synthesis of ISP. After the overexpression of PdCHS and PdCHI in tobacco, the accumulation of anthocyanins in tobacco petals increased to different degrees, showing the role of PdCHS and PdCHI in anthocyanin accumulation. The analysis of NtCHS and NtCHI of transgenic tobacco lines by qRT-PCR showed that the THC2'GT gene could increase the expression of CHS. THC2'GT and CHI were found to be competitive; hence, the overexpression of THC2'GT could lead to a decrease in CHI expression. The CHS gene and CHI gene could increase the expression of each other. In conclusion, we verified the key structural gene PdTHC2'GT and studied the operation of the genes in its upstream and competitive pathway, providing a new perspective for the biosynthesis of ISP and new candidate genes for the directional breeding of tree peony.

Flavonoid 3'-hydroxylase of Camellia nitidissima Chi. promotes the synthesis of polyphenols better than flavonoids.

Camellia nitidissima Chi. is an ornamental plant of the genus Camellia L. Its flowers contain a lot of flavonoids and polyphenols. Flavonoid 3'-hydroxylase (F3'H) plays an important role in the synthesis of flavonoids, polyphenols and anthocyanins. We used PCR amplification, quantitative PCR, High-performance liquid chromatography, subcellular localization, and agrobacterium-mediated leaf disk method to study the the function of CnF3'H. The full length of CnF3'H was 1859 bp (GenBank code: HQ290518.1), with an open reading frame of 1577 bp, and encoded 518 amino acid. A phylogenetic tree analysis showed that CnF3'H was closely related to Camellia sinensis L. and C. sinensis cultivar Zhonghuang. CnF3'H was expressed in flowers, leaves, fruits, sepals, petals and stamens of C. nitidissima, and during the flowering process the expression level in flower decreased initially and then increased. CnF3'H expression was significantly positive correlated with polyphenol contents in C. nitidissima. A CnF3'H-EGFP expression vector was constructed to do the subcellular localization, we found that CnF3'H was obviously localized in the nuclear envelope and cytomembrane. In transgenic tobacco flowers, the total polyphenol content and various polyphenol constituents were significantly increased with high CnF3'H expression level, while total flavonoid contents and some flavonol constituents were increased slightly. These findings suggest that CnF3'H promotes the synthesis of polyphenols better than flavonoids.

The validation of the role of several genes related to Bombyx mori nucleopolyhedrovirus infection in vivo.

Bombyx mori nucleopolyhedrovirus (BmNPV) is one of primary silkworm pathogens and causes a serious damage of cocoon losses every year. Recent years, many works have been done to clarify the silkworm anti-BmNPV mechanism, and a significant progress has been made in screening and studying of genes and proteins related to BmNPV infection, but several of them lacked the proofs in vivo. In this study, to further validate the function of seven newly reported genes in vivo, including BmAtlatin-n, Bmferritin-heavy chain (BmFerHCH), Bmthymosin (BmTHY), Bmseroin1, Bmseroin2, Bmnuclear hormone receptors 96 (BmNHR96), and BmE3 ubiquitin-protein ligase SINA-like 10 (BmSINAL10), the response of them in the midgut, fat body, and hemolymph of differentially resistant strains (resistant strain YeA and susceptible strain YeB) at 48 h following BmNPV infection were analyzed. The results showed that the relative stable or upregulated expression level of BmAtlatin-n, BmTHY, Bmseroin1, and Bmseroin2 in YeA resistant strain following BmNPV infection further indicated their antiviral role in vivo, compared with susceptible YeB strain. Moreover, the significant downregulation of BmFerHCH, BmNHR96, and BmSINAL10 in both strains following BmNPV infection revealed their role in benefiting virus infection, as well as the upregulation of BmFerHCH in YeB midgut and BmSINAL10 in YeB hemolymph. These data could be used to complementary the proofs of the function of these genes in response to BmNPV infection.