Selection and Verification of Reference Genes for qRT-PCR Analysis in <i>Iris domestica</i> under Drought

Iris domestica is a plant of the Iridaceae family and is drought-tolerant, but its drought-resistance mechanism is not yet clear. Analysing the gene expression changes of I. domestica by qRT-PCR is an important mean to understand its drought resistance characteristics. Nevertheless, a lack of reference genes greatly hinders investigation and research on the adaptation of I. domestica to drought at the molecular and genetic levels. In this study, we assessed the expression stability of 11 candidate gene in I. domestica under drought stress conditions and different tissues using geNorm, NormFinder, BestKeeper and RefFinder tools. The results showed that EF1β was the most stable reference genes under drought stress and in different tissues. To validate further the stability of the identified reference genes, the expression patterns of VP gene in I. domestica was analysed. These results will be conducive to more accurate quantification of gene expression levels in I. domestica.     

Cloning and Characterization of <i>EuGID1</i> in <i>Eucommia ulmoides</i> Oliver

Gibberellic acid controlled the key developmental processes of the life cycle of landing plants, and regulated the growth and development of plants. In this study, a novel gibberellin receptor gene EuGID1 was obtained from Eucommia ulmoides Oliver. The cDNA of EuGID1 was 1556 bp, and the open reading frame was 1029 bp, which encoded 343 amino acids. EuGID1 had the homology sequence with the hormone-sensitive lipase family. Amino acid sequence alignment confirmed EuGID1 protein had the highest homology with the GID1 protein of Manihot esculenta. EuGID1 was located in the nucleus and cell membrane and had expression in four plant organs. Overexpression of EuGID1 in transgenic Arabidopsis plants promoted plant elongation and increased siliques yield.     

Genome-Wide Identification,Evolution and Expression Analyses of <i>GA2ox</i> Gene Family in <i>Brassica napus</i> L.

Gibberellin 2-oxidases (GA2ox) are important enzymes that maintain the balance of bioactive GAs in plants. GA2ox genes have been identified and characterized in many plants, but these genes were not investigated in Brassica napus. Here, we identified 31 GA2ox genes in B. napus and 15 of these BnaGA2ox genes were distributed in the A and C subgenomes. Subcellular localization predictions suggested that all BnaGA2ox proteins were localized in the cytoplasm, and gene structure analysis showed that the BnaGA2ox genes contained 2–4 exons. Phylogenetic analysis indicated that BnGA2ox family proteins in monocotyledons and dicotyledons can be divided into four groups, including two C₁₉-GA2ox and two C₂₀-GA2ox clades. Group 4 is a C₂₀-GA2ox Class discovered recently. Most BnaGA2ox genes had a syntenic relationship with AtGA2ox genes. BnaGA2ox genes in the C subgenome had experienced stronger selection pressure than genes in the A subgenome. BnaGA2ox genes were highly expressed in specific tissues such as those involved in growth and development, and most of them were mainly involved in abiotic responses, regulation of phytohormones and growth and development. Our study provided a valuable evolutionary analysis of GA2ox genes in monocotyledons and dicotyledons, as well as an insight into the biological functions of GA2ox family genes in B. napus.     

Genome-Wide Analysis of the <i>KANADI</i> Gene Family and Its Expression Patterns under Different Nitrogen Concentrations Treatments in <i>Populus trichocarpa</i>

KANADI (KAN) is a plant-specific gene that controlled the polarity development of lateral organs. It mainly acted on the abaxial characteristics of plants to make the lateral organs asymmetrical. However, it had been less identified in woody plants. In this study, the members of the KAN gene family in Populus trichocarpa were identified and analyzed using the bioinformatics method. The results showed that a total of 8 KAN family members were screened out, and each member contained the unique GARP domain and conserved region of the family proteins. Phylogenetic analysis and their gene structures revealed that all KAN genes from P. trichocarpa, Arabidopsis thaliana, and Nicotiana benthamiana could be divided into four subgroups, while the eight genes in P. trichocarpa were classified into three subgroups, respectively. The analysis of tissue-specific expression indicated that PtKAN1 was highly expressed in young leaves, PtKAN6 was highly expressed in young leaves and mature leaves, PtKAN2, PtKAN5, and PtKAN7 were highly expressed in nodes and internodes, PtKAN8 was highly expressed in roots, and PtKAN3 and PtKAN4 showed low expression levels in all tissues. Among them, PtKAN2 and PtKAN6, and PtKAN4 and PtKAN5 might have functional redundancy. Under high nitrogen concentrations, PtKAN2 and PtKAN8 were highly expressed in mature stems and leaves, respectively, while PtKAN4, PtKAN5, and PtKAN7 were highly expressed in roots. This study laid a theoretical foundation for further study of the KAN gene-mediated nitrogen effect on root development.     

Molecular cloning and characterization of an F-box family gene <Emphasis Type="Italic">CarF</Emphasis>-<Emphasis Type="Italic">box1</Emphasis> from chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.)

F-box protein family has been found to play important roles in plant development and abiotic stress responses via the ubiquitin pathway. In this study, an F-box gene CarF-box1 (for Cicer arietinum F-box gene 1, Genbank accession no. GU247510) was isolated based on a cDNA library constructed with chickpea seedling leaves treated by polyethylene glycol. CarF-box1 encoded a putative protein with 345 amino acids and contained no intron within genomic DNA sequence. CarF-box1 is a KFB-type F-box protein, having a conserved F-box domain in the N-terminus and a Kelch repeat domain in the C-terminus. CarF-box1 was localized in the nucleus. CarF-box1 exhibited organ-specific expression and showed different expression patterns during seed development and germination processes, especially strongly expressed in the blooming flowers. In the leaves, CarF-box1 could be significantly induced by drought stress and slightly induced by IAA treatment, while in the roots, CarF-box1 could be strongly induced by drought, salinity and methyl jasmonate stresses. Our results suggest that CarF-box1 encodes an F-box protein and may be involved in various plant developmental processes and abiotic stress responses.     

Genome Wide Characterization of <i>CBL-CIPK</i> Family Genes and Their Responsive Expression in <i>Rosa chinensis</i>

Calcium (Ca²⁺) plays a pivotal role in various signal transduction pathways. Calcineurin B-like proteins (CBLs) are a unique group of Ca²⁺ sensors that decode Ca²⁺ signals by activating the plant specific protein kinase known as the CBL-interacting protein kinase (CIPK). In plants, the CBL-CIPK signaling network regulates multiple signals in response to different extracellular cues including abiotic stress. However, the genome wide annotation and expression patterns of CBLs and CIPKs in woody cutting flower plants are still unclear. In this study, a total number of 7 CBLs (RcCBLs) and 17 CIPKs (RcCIPKs) genes, divided into four and five subfamilies, respectively, were identified from the rose genome. All RcCBLs possess a classic elongation factor-hand (EF-hand) domain, while all RcCIPKs possess both the classic kinase and NAF domains. Most RcCBLs were predicted to be plasma membrane localized, whereas most RcCIPKs were predicted to be cytoplasmic localized. Synteny analysis showed that one RcCBL gene pair and five RcCIPK gene pairs have gone through whole genome duplication events. Promoter cis-element prediction assays indicated that RcCBLs and RcCIPKs could function in different abiotic stress responses in rose plants. Further quantitative real-time PCR analysis demonstrated that RcCBLs and RcCIPKs were expressed in different organs with overlapped but distinct patterns in response to various abiotic stresses. The findings in this work will provide fundamental information and gene resources for further functional research on RcCBLs and RcCIPKs.     

Overexpression of <i>IbSINA5</i> Increases Cold Tolerance through a CBF SINA-COR Mediated Module in Sweet Potato

Seven in absentia (SINA) family proteins play a central role in plant growth, development and resistance to abiotic stress. However, their biological function in plant response to cold stress is still largely unknown. In this work, a seven in absentia gene IbSINA5 was isolated from sweet potato. Quantitative real-time polymerase chain reaction (qRT-PCR) analyses demonstrated that IbSINA5 was ubiquitously expressed in various tissues and organs of sweet potato, with a predominant expression in fibrous roots, and was remarkably induced by cold, drought and salt stresses. Subcellular localization assays revealed that IbSINA5-GFP fusion protein was mainly localized in cytoplasm and nucleus. Overexpression of IbSINA5 in sweet potato led to dramatically improved resistance to cold stress in transgenic plants, which was associated with the up-regulated expression of IbCOR (cold-regulated) genes, increased proline production, and decreased malondialdehyde (MDA) and H₂O₂ accumulation in the leaves of transgenic plants. Furthermore, transient expression of IbCBF3, a C-repeat binding factor (CBF) gene, in the leaf protoplasts of wild type sweet potato plants up-regulated the expression of both IbSINA5 and IbCOR genes. Our results suggest that IbSINA5 could function as a positive regulator in the cold signaling pathway through a CBF-SINA-COR mediated module in sweet potato, and have a great potential to be used as a candidate gene for the future breeding of new plant species with improved cold resistance.     

Genome-Wide Identification and Expression Profiling Suggest that Invertase Genes Function in Silique Development and the Response to <i>Sclerotinia sclerotiorum</i> in <i>Brassica napus</i>

Invertase (INV), a key enzyme in sucrose metabolism, irreversibly catalyzes the hydrolysis of sucrose to glucose and fructose, thus playing important roles in plant growth, development, and biotic and abiotic stress responses. In this study, we identified 27 members of the BnaINV family in Brassica napus. We constructed a phylogenetic tree of the family and predicted the gene structures, conserved motifs, cis-acting elements in promoters, physicochemical properties of encoded proteins, and chromosomal distribution of the BnaINVs. We also analyzed the expression of the BnaINVs in different tissues and developmental stages in the B. napus cultivar Zhongshuang 11 using qRT-PCR. In addition, we analyzed RNA-sequencing data to explore the expression patterns of the BnaINVs in four cultivars with different harvest indices and in plants inoculated with the pathogenic fungus Sclerotinia sclerotiorum. We used WGCNA (weighted coexpression network analysis) to uncover BnaINVregulatory networks. Finally, we explored the expression patterns of several BnaINV genes in cultivars with long (Zhongshuang 4) and short (Ningyou 12) siliques. Our results suggest that BnaINVs play important roles in the growth and development of rapeseed siliques and the defense response against pathogens. Our findings could facilitate the breeding of high-yielding B. napus cultivars with strong disease resistance.     

Bioinformatics and Functional Analysis of High Oleic Acid-Related Gene Gm<i>SAM22</i> in Soybean [<i>Glycine max</i> (L.) Merr.]

High yield, high quality, stable yield, adaptability to growth period, and modern mechanization are the basic requirements for crops in the 21st century. Soybean oleic acid is a natural unsaturated fatty acid with strong antioxidant properties and stability. Known as a safe fatty acid, it has the ability to successfully prevent cardiovascular and cerebrovascular disorders. Improving the fatty acid composition of soybean seeds, can not only speed up the breeding process of high-quality high-oil and high-oleic soybeans, but also have important significance in human health, and provide the possibility for the development of soybean oil as a new energy source. Hence, the aim of this study was to analyze the high oleic acid elated gene GmSAM22 in soybean. In this research the soybean oleic acid-related gene GmSAM22 was screened out by Genome-wide association analysis, a 662 bp fragment was acquired by specific PCR amplification, and the pMD18T cloning vector was linked by the use of a seamless cloning technique. Bioinformatics analysis of the signal peptide prediction, subcellular localization, protein hydrophobicity, transmembrane region analysis, a phosphorylation site, protein secondary and tertiary structure and protein interaction analysis of the protein encoded by the SAM22 gene was carried out. The plasmid of the gene editing vector is pBK041. The overexpression vector was transformed from pCAMBIA3301 as the base vector, and overexpression vector were designed. Positive plants were obtained by genetic transformation by the pollen tube channel method. Fluorescence quantitative PCR was performed on the T₂ generation plants to detect the relative expression levels in different tissues. Southern Blot was used to detect the presence of hybridization signal. Screening genes BAR, 35S, and NOS in plants were identified by conventional PCR. 10 seeds with high and low oleic acid content were chosen for quantitative PCR identification, and finally, the concentration and morphology of soybean fatty acids were identified by near-far infrared spectroscopy. On 10 seeds with an upper and lower oleic acid content, a quantitative fluorescence analysis was done. In Southern blot hybridization, the SAM22 gene was integrated into the recipient soybean plant in hands of a sole copy. Fluorescence quantitative PCR appeared that the average relative expression of the SAM22 gene in roots, stems, leaves, and seeds was 1.70, 1.67, 3.83, and 4.41, respectively. Positive expression seeds had a 4.77% increase in oleic acid content. The level of oleic acid in the altered seeds was reduced by 4.13% when compared to CK, and it was discovered that the GmSAM22 gene could be a regulatory and secondary gene that promotes the conversion of stearic acid to oleic acid in soybean. There has not been a discussion of gene cloning or functional verification. The cloning and genetic transformation of the soybean SAM22 gene can effectively increase the content of oleic acid, which lays a foundation for the study of soybean with high oleic acid.     

Molecular mechanisms underlying Grateloupia imbricata (Rhodophyta) carposporogenesis induced by methyl jasmonate

When applied in vitro, methyl jasmonate is sensed by the red seaweed Grateloupia imbricate, substantially and visually affecting its carposporogenesis. However, although there is some understanding of the morphological changes induced by methyl jasmonate in vitro, little is known about the genes that are involved in red seaweed carposporogenesis and how their protein products act. For the work reported herein, the expression of genes in red seaweed that encode enzymes involved in the synthesis of methyl jasmonate (jasmonic acid carboxyl methyl transferase and a putative methyl transferase) was monitored. Additionally the genes involved in oxidation (cytochrome P450 and WD40), jasmonate synthesis, signal transduction, and regulation of reactive oxygen species (MYB), and reproduction (ornithine decarboxylase) were monitored. To determine when or if the aforementioned genes were expressed during cystocarp development, fertilized and fertile thalli were exposed to methyl jasmonate and gene expression was measured after 24 and 48 h. The results showed that methyl jasmonate promoted differential gene expression in fertilized thalli by 24 h and upregulated expression of the ornithine decarboxylase gene only by 48 h in fertile thalli (0.75 ± 003 copies · μL^?1 at 24 h vs. 1.11 ± 0.04 copies · μL^?1 at 48 h). We conclude that Ornithine decarboxylase expression involves methyl jasmonate signaling as well as development and maturation of cystocarps.     

Development of a New Cold-Tolerant Maize (<i>Zea mays</i> L.) Germplasm Using the <i>ICE1</i> Gene from <i>Arabidopsis thaliana</i>

To develop cold-tolerant maize germplasms and identify the activation of INDUCER OF CRT/DRE-BINDING FACTOR EXPRESSION (ICE1) expression in response to cold stress, RT-PCR was used to amplify the complete open reading frame sequence of the ICE1 gene and construct the plant expression vector pCAMBIA3301-ICE1-Bar. Immature maize embryos and calli were transformed with the recombinant vector using Agrobacterium tumefaciens-mediated transformations. From the regenerated plantlets, three T1 lines were screened and identified by PCR. A Southern blot analysis showed that a single copy of the ICE1 gene was integrated into the maize (Zea mays L.) genomes of the three T1 generations. Under low temperature-stress conditions (4°C), the relative conductivity levels decreased by 27.51%–31.44%, the proline concentrations increased by 12.50%–17.50%, the malondialdehyde concentrations decreased by 16.78%–18.37%, and the peroxidase activities increased by 19.60%–22.89% in the T1 lines compared with those of the control. A real-time quantitative PCR analysis showed that the ICE1 gene was ectopically expressed in the roots, stems, and leaves of the T1 lines. ICE1 positively regulates the expression of the CBF genes in response to cold stress. Thus, this study showed the successful transformation of maize with the ICE1 gene, resulting in the generation of a new maize germplasm that had increased tolerance to cold stress.     

Identification of Resistance to Pathogenesis Related Protein <i>GmPR1L</i> in Tobacco <i>Botrytis cinerea</i> Infection

Soybean (Glycine max (Linn.) Merr.) annual leguminous crop is cultivated all over the world. The occurrence of diseases has a great impact on the yield and quality of soybean. In this study, based on the RNA-seq of soybean variety M18, a complete CDS (Coding sequence) GmPR1L of the pathogenesis-related protein 1 family was obtained, which has the ability to resist fungal diseases. The overexpression vector and interference expression vector were transferred into tobacco NC89, and the resistance of transgenic tobacco (Nicotiana tabacum L.) to Botrytis cinerea infection was identified. The results show that: Compared with the control, the activities of related defense enzymes SOD (Superoxide dismutase), POD (Peroxidase), PAL (L-phenylalanine ammonia-lyase) and PPO (Polyphenol oxidase) in the over-expressed transgenic tobacco OEA1 and OEA2 increased to different degrees, and increased significantly at different infection time points. The activities of defense enzymes in the interfering strains IEA1 and IEA2 were significantly lower than those in the control strains. The results of resistance level identification showed that the disease spot rate of OEA1 was significantly lower than that of the control line, and the disease spot rate of OEA2 was significantly lower than that of the control line. The plaque rate of the interfering expression line IEA1-IEA2 was significantly higher than that of the control line. It is preliminarily believed that the process related protein GmPR1L can improve the resistance of tobacco to B. cinerea.