Regulon: An overview of plant abiotic stress transcriptional regulatory system and role in transgenic plants / Regulon: Uma visão geral do sistema regulador da transcrição do estresse abiótico em plantas e papel nas plantas transgênica

Abstract Population growth is increasing rapidly around the world, in these consequences we need to produce more foods to full fill the demand of increased population. The world is facing global warming due to urbanizations and industrialization and in this concerns plants exposed continuously to abiotic stresses which is a major cause of crop hammering every year. Abiotic stresses consist of Drought, Salt, Heat, Cold, Oxidative and Metal toxicity which damage the crop yield continuously. Drought and salinity stress severally affected in similar manner to plant and the leading cause of reduction in crop yield. Plants respond to various stimuli under abiotic or biotic stress condition and express certain genes either structural or regulatory genes which maintain the plant integrity. The regulatory genes primarily the transcription factors that exert their activity by binding to certain cis DNA elements and consequently either up regulated or down regulate to target expression. These transcription factors are known as masters regulators because its single transcript regulate more than one gene, in this context the regulon word is fascinating more in compass of transcription factors. Progress has been made to better understand about effect of regulons (AREB/ABF, DREB, MYB, and NAC) under abiotic stresses and a number of regulons reported for stress responsive and used as a better transgenic tool of Arabidopsis and Rice.

Resumo O crescimento populacional está aumentando rapidamente em todo o mundo, e para combater suas consequências precisamos produzir mais alimentos para suprir a demanda do aumento populacional. O mundo está enfrentando o aquecimento global devido à urbanização e industrialização e, nesse caso, plantas expostas continuamente a estresses abióticos, que é uma das principais causas do martelamento das safras todos os anos. Estresses abióticos consistem em seca, sal, calor, frio, oxidação e toxicidade de metais que prejudicam o rendimento da colheita continuamente. A seca e o estresse salino são afetados de maneira diversa pela planta e são a principal causa de redução da produtividade das culturas. As plantas respondem a vários estímulos sob condições de estresse abiótico ou biótico e expressam certos genes estruturais ou regulatórios que mantêm a integridade da planta. Os genes reguladores são principalmente os fatores de transcrição que exercem sua atividade ligando-se a certos elementos cis do DNA e, consequentemente, são regulados para cima ou para baixo para a expressão alvo. Esses fatores de transcrição são conhecidos como reguladores mestres porque sua única transcrição regula mais de um gene; nesse contexto, a palavra regulon é mais fascinante no âmbito dos fatores de transcrição. Progresso foi feito para entender melhor sobre o efeito dos regulons (AREB / ABF, DREB, MYB e NAC) sob estresses abióticos e uma série de regulons relatados como responsivos ao estresse e usados ​​como uma melhor ferramenta transgênica de Arabidopsis e Rice.

Expression analysis of transcription factors in sugarcane during cold stress / Análise de expressão de fatores de transcrição em cana-de-açúcar sob estresse por frio

Abstract Transcription factors (TF) are a wide class of genes in plants, and these can regulate the expression of other genes in response to various environmental stresses (biotic and abiotic). In the current study, transcription factor activity in sugarcane was examined during cold stress. Initially, RNA transcript reads of two sugarcane cultivars (ROC22 and GT08-1108) under cold stress were downloaded from SRA NCBI database. The reads were aligned into a reference genome and the differential expression analyses were performed with the R/Bioconductor edgeR package. Based on our analyses in the ROC22 cultivar, 963 TF genes were significantly upregulated under cold stress among a total of 5649 upregulated genes, while 293 TF genes were downregulated among a total of 3,289 downregulated genes. In the GT08-1108 cultivar, 974 TF genes were identified among 5,649 upregulated genes and 283 TF genes were found among 3,289 downregulated genes. Most transcription factors were annotated with GO categories related to protein binding, transcription factor binding, DNA-sequence-specific binding, transcription factor complex, transcription factor activity in RNA polymerase II, the activity of nucleic acid binding transcription factor, transcription corepressor activity, sequence-specific regulatory region, the activity of transcription factor of RNA polymerase II, transcription factor cofactor activity, transcription factor activity from plastid promoter, transcription factor activity from RNA polymerase I promoter, polymerase II and RNA polymerase III. The findings of above results will help to identify differentially expressed transcription factors during cold stress. It also provides a comprehensive analysis of the regulation of the transcription activity of many genes. Therefore, this study provides the molecular basis for improving cold tolerance in sugarcane and other economically important grasses.

Resumo Fatores de transcrição (FT) são uma ampla classe de genes em plantas e podem regular a expressão de outros genes em resposta a vários estresses ambientais (estresses bióticos e abióticos). No presente estudo, a atividade do fator de transcrição na cana-de-açúcar foi examinada durante o estresse pelo frio. Inicialmente, as leituras de transcrição de RNA de duas cultivares de cana-de-açúcar (ROC22 e GT08-1108) sob estresse frio foram baixadas do banco de dados SRA NCBI. As leituras foram alinhadas em um genoma de referência e as análises de expressão diferencial foram realizadas com o pacote R / Bioconductor edgeR. Com base em nossas análises no cultivar ROC22, 963 genes TF foram significativamente regulados positivamente sob estresse pelo frio entre um total de 5.649 genes regulados positivamente, enquanto 293 genes TF foram regulados negativamente entre um total de 3.289 genes regulados negativamente. No cultivar GT08-1108, 974 genes TF foram identificados entre 5.649 genes regulados positivamente e 283 genes TF foram encontrados entre 3.289 genes regulados negativamente. Os fatores de transcrição, em sua maioria, foram anotados com categorias GO relacionadas à ligação de proteína, ligação de fator de transcrição, ligação específica de sequência de DNA, complexo de fator de transcrição, atividade de fator de transcrição em RNA polimerase II, atividade de fator de transcrição de ligação de ácido nucleico, atividade de corepressor de transcrição, sequência específica da região reguladora, atividade do fator de transcrição da RNA polimerase II, atividade do cofator do fator de transcrição, atividade do fator de transcrição do promotor do plastídio, atividade do fator de transcrição do promotor da RNA polimerase I, polimerase II e RNA polimerase III. As descobertas dos resultados acima ajudarão a identificar fatores de transcrição expressos diferencialmente durante o estresse pelo frio. Ele também fornece uma análise abrangente da regulação da atividade de transcrição de muitos genes. Portanto, este estudo fornece base molecular para melhorar a tolerância ao frio em cana-de-açúcar e outras gramíneas economicamente importantes.

Mining and identification of members of MYB transcription factor family in Lonicera macranthoides / 中国中药杂志

As a large family of transcription factors, the MYB family plays a vital role in regulating flower development. We studied the MYB family members in Lonicera macranthoides for the first time and identified three sequences of 1R-MYB, 47 sequences of R2R3-MYB, two sequences of 3R-MYB, and one sequence of 4R-MYB from the transcriptome data. Further, their physicochemical properties, conserved domains, phylogenetic relationship, protein structure, functional information, and expression were analyzed. The results show that the 53 MYB transcription factors had different conserved motifs, physicochemical properties, structures, and functions in wild type and 'Xianglei' cultivar of L. macranthoides, indicating their conservation and diversity in evolution. The transcript level of LmMYB was significantly different between the wild type and 'Xianglei' cultivar as well as between flowers and leaves, and some genes were specifically expressed. Forty-three out of 53 LmMYB sequences were expressed in both flowers and leaves, and 9 of the LmMYB members showed significantly different transcript levels between the wild type and 'Xianglei' cultivar, which were up-regulated in the wild type. The results provide a theoretical basis for further studying the specific functional mechanism of the MYB family.

Comparative analysis of differentially expressed genes for biosynthesis of active ingredients in fruits of different cultivars of Lycium barbarum L. based on transcriptome sequencing / 生物工程学报

To explore the differentially expressed genes (DEGs) related to biosynthesis of active ingredients in wolfberry fruits of different varieties of Lycium barbarum L. and reveal the molecular mechanism of the differences of active ingredients, we utilized Illumina NovaSeq 6000 high-throughput sequencing technology to conduct transcriptome sequencing on the fruits of 'Ningqi No.1' and 'Ningqi No.7' during the green fruit stage, color turning stage and maturity stage. Subsequently, we compared the profiles of related gene expression in the fruits of the two varieties at different development stages. The results showed that a total of 811 818 178 clean reads were obtained, resulting in 121.76 Gb of valid data. There were 2 827, 2 552 and 2 311 DEGs obtained during the green fruit stage, color turning stage and maturity stage of 'Ningqi No. 1' and 'Ningqi No. 7', respectively, among which 2 153, 2 050 and 1 825 genes were annotated in six databases, including gene ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG) and clusters of orthologous groups of proteins (KOG). In GO database, 1 307, 865 and 624 DEGs of green fruit stage, color turning stage and maturity stage were found to be enriched in biological processes, cell components and molecular functions, respectively. In the KEGG database, the DEGs at three developmental stages were mainly concentrated in metabolic pathways, biosynthesis of secondary metabolites and plant-pathogen interaction. In KOG database, 1 775, 1 751 and 1 541 DEGs were annotated at three developmental stages, respectively. Searching the annotated genes against the PubMed database revealed 18, 26 and 24 DEGs related to the synthesis of active ingredients were mined at the green fruit stage, color turning stage and maturity stage, respectively. These genes are involved in carotenoid, flavonoid, terpenoid, alkaloid, vitamin metabolic pathways, etc. Seven DEGs were verified by RT-qPCR, which showed consistent results with transcriptome sequencing. This study provides preliminary evidences for the differences in the content of active ingredients in different Lycium barbarum L. varieties from the transcriptional level. These evidences may facilitate further exploring the key genes for active ingredients biosynthesis in Lycium barbarum L. and analyzing their expression regulation mechanism.

Identification and expression analysis of MADS-box gene family in Docynia delavayi (Franch.) Schneid / 生物工程学报

MADS-box gene family is a significant transcription factor family that plays a crucial role in regulating plant growth, development, signal transduction, and other processes. In order to study the characteristics of MADS-box gene family in Docynia delavayi (Franch.) Schneid. and its expression during different stages of seed germination, this study used seedlings at different stages of germination as materials and screened MADS-box transcription factors from the transcriptome database of D. delavayi using bioinformatics methods based on transcriptome sequencing. The physical and chemical properties, protein conservative motifs, phylogenetic evolution, and expression patterns of the MADS-box transcription factors were analyzed. Quantitative real-time PCR (qRT-PCR) was used to verify the expression of MADS-box gene family members during different stages of seed germination in D. delavayi. The results showed that 81 genes of MADS-box gene family were identified from the transcriptome data of D. delavayi, with the molecular weight distribution ranged of 6 211.34-173 512.77 Da and the theoretical isoelectric point ranged from 5.21 to 10.97. Phylogenetic analysis showed that the 81 genes could be divided into 15 subgroups, among which DdMADS27, DdMADS42, DdMADS45, DdMADS46, DdMADS53, DdMADS61, DdMADS76, DdMADS77 and DdMADS79 might be involved in the regulation of ovule development in D. delavayi. The combination of the transcriptome data and the qRT-PCR analysis results of D. delavayi seeds indicated that DdMADS25 and DdMADS42 might be involved in the regulation of seed development, and that DdMADS37 and DdMADS38 might have negative regulation effects on seed dormancy. Previous studies have reported that the MIKC* subgroup is mainly involved in regulating flower organ development. For the first time, we found that the transcription factors of the MIKC* subgroup exhibited a high expression level at the early stage of seed germination, so we speculated that the MIKC* subgroup played a regulatory role in the process of seed germination. To verify the accuracy of this speculation, we selected DdMADS60 and DdMADS75 from the MIKC* subgroup for qRT-PCR experiments, and the experimental results were consistent with the expression trend of transcriptome sequencing. This study provides a reference for further research on the biological function of D. delavayi MADS-box gene family from the perspective of molecular evolution.

Genome-wide identification of the banana GLR gene family and its expression analysis in response to low temperature and abscisic acid/methyl jasmonate / 生物工程学报

Glutamate receptor-like (GLR) is an important class of Ca2+ channel proteins, playing important roles in plant growth and development as well as in response to biotic and abiotic stresses. In this paper, we performed genome-wide identification of banana GLR gene family based on banana genomic data. Moreover, we analyzed the basic physicochemical properties, gene structure, conserved motifs, promoter cis-acting elements, evolutionary relationships, and used real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) to verify the expression patterns of some GLR family members under low temperature of 4 ℃ and different hormone treatments. The results showed that there were 19 MaGLR family members in Musa acuminata, 16 MbGLR family members in Musa balbisiana and 14 MiGLR family members in Musa itinerans. Most of the members were stable proteins and had signal peptides, all of them had 3-6 transmembrane structures. Prediction of subcellular localization indicated that all of them were localized on the plasma membrane and irregularly distributed on the chromosome. Phylogenetic analysis revealed that banana GLRs could be divided into 3 subclades. The results of promoter cis-acting elements and transcription factor binding site prediction showed that there were multiple hormone- and stress-related response elements and 18 TFBS in banana GLR. RT-qPCR analysis showed that MaGLR1.1 and MaGLR3.5 responded positively to low temperature stress and were significantly expressed in abscisic acid/methyl jasmonate treatments. In conclusion, the results of this study suggest that GLR, a highly conserved family of ion channels, may play an important role in the growth and development process and stress resistance of banana.

Identification and expression pattern analysis of RcACA gene family in castor under abiotic stresses / 生物工程学报

Auto-inhibited Ca2+-ATPase (ACA) is one of the Ca2+-ATPase subfamilies that plays an important role in maintaining Ca2+ concentration balance in plant cells. To explore the function and gene expression pattern of the RcACA gene family in castor, bioinformatics analysis was used to identify the members of the RcACA gene family in castor. The basic physical and chemical properties, subcellular location, protein secondary and tertiary structure, conserved domain, conserved motif, gene structure, chromosome location and collinear relationship, as well as the evolutionary characteristics and promoter cis-acting elements were predicted and analyzed. The expression pattern of the RcACA gene under abiotic stress was analyzed by expression (fragments per kilobase of exon model per million mapped fragments, FPKM) in castor transcriptome data. The results showed that 8 RcACA gene family members were identified in castor, acidic proteins located in the plasma membrane. In the secondary structure of all proteins, the α-helix and random coil is more; the RcACA genes were clustered into three categories, and the design of the genes in the same category was similar to the conserved motif. Both of them had four typical domains, RcACA3-RcACA8 had a Ca2+-ATPase N-terminal autoinhibitory domain. The RcACA gene is mostly located on the long arm of the chromosome and has 2 pairs of collinear relationships. There are more light response elements but fewer hormone-induced elements located upstream of the RcACA coding region. Interspecific clustering showed that the evolution of ACA genes among species was conservative. Tissue expression pattern analysis showed that RcACA genes showed apparent tissue expression specificity, and most of the genes showed the highest expression level in male flowers. Expression analysis under abiotic stress showed that RcACA2-RcACA8 were up-regulated under high salt and drought stress, and RcACA1 was up-regulated at 0-24 h under low-temperature stress, indicating that RcACA genes positively responded to abiotic stresses. The above results provide a theoretical basis for exploring the role of the RcACA gene in castor growth, development and stress response.

Functional analysis on sucrose transporters in sweet potato / 生物工程学报

Sweet potato is an important food crop that can also be used as an industrial raw material. Sucrose is the main form of long-distance carbohydrate transport in plants, and sucrose transporter (SUT) regulates the transmembrane transport and distribution of sucrose during plant growth and metabolism. Moreover, SUT plays a key role in phloem mediated source-to-sink sucrose transport and physiological activities, supplying sucrose for the sink tissues. In this study, the full-length cDNA sequences of IbSUT62788 and IbSUT81616 were obtained by rapid amplification of cDNA ends (RACE) cloning according to the transcripts of the two SUT coding genes which were differentially expressed in sweet potato storage roots with different starch properties. Phylogenetic analysis was performed to clarify the classification of IbSUT62788 and IbSUT81616. The subcellular localization of IbSUT62788 and IbSUT81616 was determined by transient expression in Nicotiana benthamiana. The function of IbSUT62788 and IbSUT81616 in sucrose and hexose absorption and transport was identified using yeast functional complementarity system. The expression pattern of IbSUT62788 and IbSUT81616 in sweet potato organs were analyzed by real-time fluorescence quantitative PCR (RT-qPCR). Arabidopsis plants heterologous expressing IbSUT62788 and IbSUT81616 genes were obtained using floral dip method. The differences in starch and sugar contents between transgenic and wild-type Arabidopsis were compared. The results showed IbSUT62788 and IbSUT81616 encoded SUT proteins with a length of 505 and 521 amino acids, respectively, and both proteins belonged to the SUT1 subfamily. IbSUT62788 and IbSUT81616 were located in the cell membrane and were able to transport sucrose, glucose and fructose in the yeast system. In addition, IbSUT62788 was also able to transport mannose. The expression of IbSUT62788 was higher in leaves, lateral branches and main stems, and the expression of IbSUT81616 was higher in lateral branches, stems and storage roots. After IbSUT62788 and IbSUT81616 were heterologously expressed in Arabidopsis, the plants grew normally, but the biomass increased. The heterologous expression of IbSUT62788 increased the soluble sugar content, leaf size and 1 000-seed weight of Arabidopsis plants. Heterologous expression of IbSUT81616 increased starch accumulation in leaves and root tips and 1 000-seed weight of seeds, but decreased soluble sugar content. The results obtained in this study showed that IbSUT62788 and IbSUT81616 might be important genes regulating sucrose and sugar content traits in sweet potato. They might carry out physiological functions on cell membrane, such as transmembrane transport of sucrose, sucrose into and out of sink tissue, as well as transport and unloading of sucrose into phloem. The changes in traits result from their heterologous expression in Arabidopsis indicates their potential in improving the yield of other plants or crops. The results obtained in this study provide important information for revealing the functions of IbSUT62788 and IbSUT81616 in starch and glucose metabolism and formation mechanism of important quality traits in sweet potato.

Soybean GmGolS2-2 improves drought resistance of transgenic tobacco / 生物工程学报

Galactinol synthase (GolS) genes play important roles in plant response to abiotic stress. In this research, the plant expression vector of soybean GmGolS2-2 gene was constructed and transformed into tobacco to study the drought tolerance of transgenic tobacco. A GmGolS2-2 gene with 975 bp coding sequence was cloned from soybean leaves by reverse transcription-polymerase chain reaction (RT-PCR). GmGolS2-2 was linked to the plant expression vector pRI101 by restriction enzyme sites Nde Ⅰ and EcoR Ⅰ, and transformed into tobacco by leaf disc method. Genomic DNA PCR and real-time PCR showed that three GmGolS2-2 transgenic tobacco plants were obtained. The growth status of GmGolS2-2 transgenic tobacco under drought stress was better than that of wild-type tobacco. After drought stress treatment, the electrolyte leakage and malondialdehyde content of transgenic tobacco were lower than those of wild-type tobacco, but the proline content and soluble sugar content were higher than those of wild-type tobacco. The results of real-time PCR showed that the heterologous expression of GmGolS2-2 increased the expression of stress-related genes NtERD10C and NtAQP1 in transgenic tobacco. The above results indicated that GmGolS2-2 improved drought resistance of transgenic tobacco.

Identification, expression and DNA variation analysis of high affinity nitrate transporter NRT2/3 gene family in Sorghum bicolor / 生物工程学报

Nitrate is the main form of inorganic nitrogen that crop absorbs, and nitrate transporter 2 (NRT2) is a high affinity transporter using nitrate as a specific substrate. When the available nitrate is limited, the high affinity transport systems are activated and play an important role in the process of nitrate absorption and transport. Most NRT2 cannot transport nitrates alone and require the assistance of a helper protein belonging to nitrate assimilation related family (NAR2) to complete the absorption or transport of nitrates. Crop nitrogen utilization efficiency is affected by environmental conditions, and there are differences between varieties, so it is of great significance to develop varieties with high nitrogen utilization efficiency. Sorghum bicolor has high stress tolerance and is more efficient in soil nitrogen uptake and utilization. The S. bicolor genome database was scanned to systematically analyze the gene structure, chromosomal localization, physicochemical properties, secondary structure and transmembrane domain, signal peptide and subcellular localization, promoter region cis-acting elements, phylogenetic evolution, single nucleotide polymorphism (SNP) recognition and annotation, and selection pressure of the gene family members. Through bioinformatics analysis, 5 NRT2 gene members (designated as SbNRT2-1a, SbNRT2-1b, SbNRT2-2, SbNRT2-3, and SbNRT2-4) and 2 NAR2 gene members (designated as SbNRT3-1 and SbNRT3-2) were identified, the number of which was less than that of foxtail millet. SbNRT2/3 were distributed on 3 chromosomes, and could be divided into four subfamilies. The genetic structure of the same subfamilies was highly similar. The average value of SbNRT2/3 hydrophilicity was positive, indicating that they were all hydrophobic proteins, whereas α-helix and random coil accounted for more than 70% of the total secondary structure. Subcellular localization occurred on plasma membrane, where SbNRT2 proteins did not contain signal peptides, but SbNRT3 proteins contained signal peptides. Further analysis revealed that the number of transmembrane domains of the SbNRT2s family members was greater than 10, while that of the SbNRT3s were 2. There was a close collinearity between NRT2/3s of S. bicolor and Zea mays. Protein domains analysis showed the presence of MFS_1 and NAR2 protein domains, which supported executing high affinity nitrate transport. Phylogenetic tree analysis showed that SbNRT2/3 were more closely related to those of Z. mays and Setaria italic. Analysis of gene promoter cis-acting elements indicated that the promoter region of SbNRT2/3 had several plant hormones and stress response elements, which might respond to growth and environmental cues. Gene expression heat map showed that SbNRT2-3 and SbNRT3-1 were induced by nitrate in the root and stem, respectively, and SbNRT2-4 and SbNRT2-3 were induced by low nitrogen in the root and stem. Non-synonymous SNP variants were found in SbNRT2-4 and SbNRT2-1a. Selection pressure analysis showed that the SbNRT2/3 were subject to purification and selection during evolution. The expression of SbNRT2/3 gene and the effect of aphid infection were consistent with the expression analysis results of genes in different tissues, and SbNRT2-1b and SbNRT3-1 were significantly expressed in the roots of aphid lines 5-27sug, and the expression levels of SbNRT2-3, SbNRT2-4 and SbNRT3-2 were significantly reduced in sorghum aphid infested leaves. Overall, genome-wide identification, expression and DNA variation analysis of NRT2/3 gene family of Sorghum bicolor provided a basis for elucidating the high efficiency of sorghum in nitrogen utilization.

The role of plant WRKY transcription factors against salt stress: a review / 生物工程学报

High salt content in soils severely hampers plant growth and crop yields. Many transcription factors in plants play important roles in responding to various stresses, but their molecular mechanisms remain unclear. WRKY transcription factors are one of the largest families of transcription factors in higher plants that are involved in and influence many aspects of plant growth and development. They play important roles in responding to salt stress. The regulation of gene expression by WRKY proteins is mainly achieved by binding to the DNA's specific cis-regulatory elements, the W-box elements (TTGACC). In recent years, there have been many studies revealing the roles and mechanisms of WRKY family members, from model plant Arabidopsis to agricultural crops. This paper reviews the latest research progress on WRKY transcription factors in response to salt stress and discusses the current challenges and future perspectives of WRKY transcription factor research.

Application of virus-induced gene silencing technology to investigate the phytochrome metabolism mechanism: a review / 生物工程学报

Color is an important indicator for evaluating the ornamental traits of horticultural plants, and plant pigments is a key factor affecting the color phenotype of plants. Plant pigments and their metabolites play important roles in color formation of ornamental organs, regulation of plant growth and development, and response to adversity stress. It has therefore became a hot topic in the field of plant research. Virus-induced gene silencing (VIGS) is a vital genomics tool that specifically reduces host endogenous gene expression utilizing plant homology-dependent defense mechanisms. In addition, VIGS enables characterization of gene function by rapidly inducing the gene-silencing phenotypes in plants. It provides an efficient and feasible alternative for verifying gene function in plant species lacking genetic transformation systems. This paper reviews the current status of the application of VIGS technology in the biosynthesis, degradation and regulatory mechanisms of plant pigments. Moreover, this review discusses the potential and future prospects of VIGS technology in exploring the regulatory mechanisms of plant pigments, with the aim to further our understandings of the metabolic processes and regulatory mechanisms of different plant pigments as well as improving plant color traits.

Genome-wide identification of SUN gene family in Fragaria vesca and stresses-response analysis / 生物工程学报

SUN gene is a group of key genes regulating plant growth and development. Here, SUN gene families of strawberry were identified from the genome of the diploid Fragaria vesca, and their physicochemical properties, genes structure, evolution and genes expression were also analyzed. Our results showed that there were thirty-one FvSUN genes in F. vesca and the FvSUNs encoded proteins were classified into seven groups, and the members in the same group showed high similarity in gene structures and conservative motifs. The electronic subcellular localization of FvSUNs was mainly in the nucleus. Collinearity analysis showed that the members of FvSUN gene family were mainly expanded by segmental duplication in F. vesca, and Arabidopsis and F. vesca shared twenty-three pairs of orthologous SUN genes. According to the expression pattern in different tissues shown by the transcriptome data of F. vesca, the FvSUNs gene can be divided into three types: (1) expressed in nearly all tissues, (2) hardly expressed in any tissues, and (3) expressed in special tissues. The gene expression pattern of FvSUNs was further verified by quantitative real-time polymerase chain reaction (qRT-PCR). Additionally, the seedlings of F. vesca were treated by different abiotic stresses, and the expression level of 31 FvSUNs genes were assayed by qRT-PCR. The expression of most of the tested genes was induced by cold, high salt or drought stress. Our studies may facilitate revealing the biological function and molecular mechanism of SUN genes in strawberry.

Cloning and functional analysis of flavanone 3-hydroxylase gene in Rhododendron hybridum Hort / 生物工程学报

Flavanone 3-hydroxylase (F3H) is a key enzyme in the synthesis of phycocyanidins. In this experiment, the petals of red Rhododendron hybridum Hort. at different developmental stages were used as experimental materials. The R. hybridum flavanone 3-hydroxylase (RhF3H) gene was cloned using reverse transcription PCR (RT-PCR) and rapid-amplification of cDNA ends (RACE) techniques, and bioinformatics analyses were performed. Petal RhF3H gene expression at different developmental stages were analyzed by using quantitative real-time polymerase chain reaction (qRT-PCR). A pET-28a-RhF3H prokaryotic expression vector was constructed for the preparation and purification of RhF3H protein. A pCAMBIA1302-RhF3H overexpression vector was constructed for genetic transformation in Arabidopsis thaliana by Agrobacterium-mediated method. The results showed that the R. hybridum Hort. RhF3H gene is 1 245 bp long, with an open reading frame of 1 092 bp, encoding 363 amino acids. It contains a Fe2+ binding motif and a 2-ketoglutarate binding motif of the dioxygenase superfamily. Phylogenetic analysis showed that the R. hybridum RhF3H protein is most closely related to the Vaccinium corymbosum F3H protein. qRT-PCR analysis showed that the expression level of the red R. hybridum RhF3H gene tended to increase and then decrease in the petals at different developmental stages, with the highest expression at middle opening stage. The results of the prokaryotic expression showed that the size of the induced protein of the constructed prokaryotic expression vector pET-28a-RhF3H was about 40 kDa, which was similar to the theoretical value. Transgenic RhF3H Arabidopsis thaliana plants were successfully obtained, and PCR identification and β-glucuronidase (GUS) staining demonstrated that the RhF3H gene was integrated into the genome of A. thaliana plants. qRT-PCR, total flavonoid and anthocyanin contentanalysis showed that RhF3H was significantly higher expressed in the transgenic A. thaliana relative to that of the wild type, and its total flavonoid and anthocyanin content were significantly increased. This study provides a theoretical basis for investigating the function of RhF3H gene, as well as for studying the molecular mechanism of flower color in R. simsiib Planch.

Cloning and expression analysis of JrGI gene in walnut / 生物工程学报

GI (GIGANTEA) is one of the output key genes for circadian clock in the plant. The JrGI gene was cloned and its expression in different tissues was analyzed to facilitate the functional research of JrGI. RT-PCR (reverse transcription-polymerase chain reaction) was used to clone JrGI gene in present study. This gene was then analyzed by bioinformatics, subcellular localization and gene expression. The coding sequence (CDS) full length of JrGI gene was 3 516 bp, encoding 1 171 amino acids with a molecular mass of 128.60 kDa and a theoretical isoelectric point of 6.13. It was a hydrophilic protein. Phylogenetic analysis showed that JrGI of 'Xinxin 2' was highly homologous to GI of Populus euphratica. The result of subcellular localization showed that JrGI protein was located in nucleus. The JrGI, JrCO and JrFT genes in female flower buds undifferentiated and early differentiated of 'Xinxin 2' were analyzed by RT-qPCR (real-time quantitative PCR). The results showed that the expression of JrGI, JrCO and JrFT genes were the highest on morphological differentiation, implying the temporal and special regulation of JrGI in the differential process of female flower buds of'Xinxin 2'. In addition, RT-qPCR analysis showed that JrGI gene was expressed in all tissues examined, whereas the expression level in leaves was the highest. It is suggested that JrGI gene plays a key role in the development of walnut leaves.

Identification of CjSPL gene family in Ziyang Xiangcheng rootstock and expression pattern analysis / 生物工程学报

Squamosa promoter binding protein-like (SPL) family is a group of important transcription factors involved in the regulation of plant growth and development and the response to environmental stress, but there are few studies in perennial fruit trees such as citrus. In this study, Ziyang Xiangcheng (Citrus junos Sib.ex Tanaka), an important rootstock of Citrus, was used as the material for analysis. Based on plantTFDB transcription factor database and sweet orange genome database, 15 SPL family members were genome-widely identified and cloned from Ziyang Xiangcheng, and named CjSPL1-CjSPL15. Sequence analysis showed that the open reading frame (ORF) length of CjSPLs ranged from 393 bp to 2 865 bp, encoding 130-954 amino acids. Phylogenetic tree divided 15 CjSPLs into 9 subfamilies. Gene structure and conserved domain analysis predicted 20 different conserved motifs and SBP basic domains. Analysis of cis-acting promoter elements predicted 20 different promoter elements, including those related to plant growth and development, abiotic stress and secondary metabolites. The expression patterns of CjSPLs under drought, salt and low temperature stresses were analyzed by real-time fluorescence quantitative PCR (qRT-PCR), and many CjSPLs were significantly up-regulated after stress treatment. This study provides a reference for further study on the function of SPL family transcription factors in citrus and other fruit trees.

Effect of ACC oxidase gene AhACOs on salt tolerance of peanut / 生物工程学报

ACC oxidase (ACO) is one of the key enzymes that catalyze the synthesis of ethylene. Ethylene is involved in salt stress response in plants, and salt stress seriously affects the yield of peanut. In this study, AhACO genes were cloned and their functions were investigated with the aim to explore the biological function of AhACOs in salt stress response, and to provide genetic resources for the breeding of salt-tolerant varieties of peanut. AhACO1 and AhACO2 were amplified from the cDNA of salt-tolerant peanut mutant M29, respectively, and cloned into the plant expression vector pCAMBIA super1300. The recombinant plasmid was transformed into Huayu22 by pollen tube injection mediated by Agrobacterium tumefaciens. After harvest, the small slice cotyledon was separated from the kernel, and the positive seeds were screened by PCR. The expression of AhACO genes was analyzed by qRT-PCR, and the ethylene release was detected by capillary column gas chromatography. Transgenic seeds were sowed and then irrigated with NaCl solution, and the phenotypic changes of 21-day-seedings were recorded. The results showed that the growth of transgenic plants were better than that of the control group Huayu 22 upon salt stress, and the relative content of chlorophyll SPAD value and net photosynthetic rate (Pn) of transgenic peanuts were higher than those of the control group. In addition, the ethylene production of AhACO1 and AhACO2 transgenic plants were 2.79 and 1.87 times higher than that of control peanut, respectively. These results showed that AhACO1 and AhACO2 could significantly improve the salt stress tolerance of transgenic peanut.

Identification and expression analysis of NHX gene family in Chinese cabbage / 生物工程学报

Na+/H+ antiporter (NHX) gene subfamily plays an important role in plant response to salt stress. In this study, we identified the NHX gene family members of Chinese cabbage and analyzed the expression patterns of BrNHXs gene in response to abiotic stresses such as high temperature, low temperature, drought and salt stress. The results showed that there were 9 members of the NHX gene family in Chinese cabbage, which were distributed on 6 chromosomes respectively. The number of amino acids was 513-1 154 aa, the relative molecular weight was 56 804.22-127 856.66 kDa, the isoelectric point was 5.35-7.68. Members of BrNHX gene family mainly existed in vacuoles, the gene structure is complete, and the number of exons is 11-22. The secondary structures of the proteins encoded by the NHX gene family in Chinese cabbage had alpha helix, beta turn and random coil, and the alpha helix occurred more frequently. Quantitative real-time PCR (qRT-PCR) analysis showed that the gene family members had different responses to high temperature, low temperature, drought and salt stress, and their expression levels differed significantly in different time periods. BrNHX02 and BrNHX09 had the most significant responses to these four stresses, and their expression levels were significantly up-regulated at 72 h after treatments, which could be used as candidate genes to further verify their functions.

Genome-wide identification and characterization of the WOX gene family in Brassica juncea / 生物工程学报

The WUSCHEL related-homeobox (WOX) family is one of the plant-specific transcription factor families, playing important roles in plant growth and development. In this study, 51 WOX gene family members were identified from the genome data of Brassica juncea by searching and screening with HUMMER, Smart and other software. Their protein molecular weight, amino acids numbers, and isoelectric point were analyzed by using Expasy online software. Furthermore, bioinformatics software was used to systematically analyze the evolutionary relationship, conservative region, and gene structure of the WOX gene family. The mustard WOX gene family was divided into three subfamilies: ancient clade, intermediate clade, and WUS clade/modern clade. Structural analysis showed that the type, organization form and gene structure of the conservative domain of WOX transcription factor family members in the same subfamily were highly consistent, while there was a certain diversity among different subfamilies. 51 WOX genes are distributed unevenly on 18 chromosomes of mustard. Most of the promoters of these genes contain cis acting elements related to light, hormone and abiotic stress. Using transcriptome data and real-time fluorescence quantitative PCR (qRT-PCR) analysis, it was found that the expression of mustard WOX gene was spatio-temporal specific, among which BjuWOX25, BjuWOX33, and BjuWOX49 might play an important role in the development of silique, and BjuWOX10, BjuWOX32, and BjuWOX11, BjuWOX23 respectively might play an important role in the response to drought and high temperature stresses. The above results may facilitate the functional study of mustard WOX gene family.

The pattern of alternative splicing and DNA methylation alteration and their interaction in linseed (Linum usitatissimum L) response to repeated drought stresses

BACKGROUND: Drought stress has significantly hampered agricultural productivity worldwide and can also result in modifications to DNA methylation levels. However, the dynamics of DNA methylation and its association with the changes in gene transcription and alternative splicing (AS) under drought stress are unknown in linseed, which is frequently cultivated in arid and semiarid regions. RESULTS: We analysed AS events and DNA methylation patterns in drought-tolerant (Z141) and drought-sensitive (NY-17) linseed under drought stress (DS) and repeated drought stress (RD) treatments. We found that the number of intron-retention (IR) and alternative 3' splice site (Alt3'SS) events were significantly higher in Z141 and NY-17 under drought stress. We found that the linseed response to the DS treatment was mainly regulated by transcription, while the response to the RD treatment was coregulated by transcription and AS. Whole genome-wide DNA methylation analysis revealed that drought stress caused an increase in the overall methylation level of linseed. Although we did not observe any correlation between differentially methylated genes (DMGs) and differentially spliced genes (DSGs) in this study, we found that the DSGs whose gene body region was hypermethylated in Z141 and hypomethylated in NY-17 were enriched in abiotic stress response Gene Ontology (GO) terms. This finding implies that gene body methylation plays an important role in AS regulation in some specific genes. CONCLUSION: Our study is the first comprehensive genome-wide analysis of the relationship between linseed methylation changes and AS under drought and repeated drought stress. Our study revealed different interaction patterns between differentially expressed genes (DEGs) and DSGs under DS and RD treatments and differences between methylation and AS regulation in drought-tolerant and drought-sensitive linseed varieties. The findings will probably be of interest in the future. Our results provide interesting insights into the association between gene expression, AS, and DNA methylation in linseed under drought stress. Differences in these associations may account for the differences in linseed drought tolerance.