【Objective】 In order to lay a basis for the further functional research and application of MdLIM genes, this study were carried out to analyze the bioinformatics (e.g promoter action element, conserved domain, gene clustering, gene structure, chromosome localization) and expression of the LIM gene family in apple. 【Method】Based on the apple genome database GDR and PLAZA, the members of LIM gene were identified. The MdLIM amino acid sequence prediction, subcellular localization prediction, LIM domain analysis, and phylogenetic tree the gene structure were completed by ExPASy Proteomics Server, Cell-PLoc, CD-Search Tool, MEGA7, and GSDS, respectively. In addition, the expression pattern of MdLIM genes in different tissues and in peels with different degree of fruit russeting in samples was analyzed by real-time qRT-PCR.【Result】A total of eleven MdLIM genes were identified from apple genome. These MdLIM proteins contained 96-222 amino acid residues with isoelectric points ranging from 6.14 to 9.01. The results of subcellular localization showed that the apple LIM proteins were distributed in the nucleus. Analysis of promoter showed these 11 MdLIM genes contained cis-acting elements related to hormone responses, environmental adaptability and adversity induction. Conserved domains showed that ten MdLIM proteins had double LIM domains except MdLIM8. According to the phylogeny relationship, MdLIM genes were divided into four categories. The expression patterns of the 11 MdLIM genes in flowers, leaves, fruit peels and stems were determined by real-time RT-PCR, and the results showed that their diverse and specific expression could be detected in all of the four tissues, suggesting that they might play different roles in different tissues. 【Conclusion】Eleven MdLIM genes were identified from the whole genome of apple, and they could be divided into four groups, and distributed on 7 chromosomes with diverse and specific tissues expression patterns.
【Objective】The study was carried out to explore the whole genome characteristics and expression patterns of NLP transcription factors in apple, and further understand its structural characteristics and mechanism.【Method】Based on the local BLAST database and Pfam database, the members of NLP transcription factor family in the whole genome of apple were identified by using two query strategies with blastp and hmmsearch. Through strict filtration and confirmation, the results were used for further analysis, and analysis mainly divides into three parts, including NLP proteins analysis, analysis of NLP genes and NLP expression analysis in apple. Programs or softwares, such as ProtParam, Clustal Omega, MEGA7, MEME 5.0.2, SOPMA, Phyre 2, WoLF PSORT and STRING, were used for protein analysis, online tools included MG2C, GSDS2.0, PlantCARE, psRNATarget, were used for gene analysis, and the expression of MdNLP gene was quantitatively detected by qRT-PCR.【Result】6 NLP members were identified from apple protein databases, which were classified into three categories by phylogenetic analysis: I, II and III. The protein secondary structure was dominated by random coil, followed by alpha-helix, and the smallest proportion was beta-turn. The prediction of subcellular localization was located in the nucleus, which was consistent with the characteristics of transcription factors. Chromosome localization showed that five genes (except MDP0000584547) were located on four chromosomes. Promoter analysis revealed a large number of cis-acting elements related to hormone and stress response, suggesting that MdNLP genes might be involved in the regulation of hormone and stress signals. In addition, a nitrogen-responsive GCN4 element was also identified, further indicating that such transcription factors were closely related to nitrogen. By quantitative detection, the pattern which NLP family had high expression in stem and leaf of apple was revealed. And the expression analysis results also confirmed that MdNLP genes responded to nitrogen starvation and drought stress, and so on.【Conclusion】Through the apple genome analysis, 6 NLP transcription factors were found; the analysis of structure and conserved domain of protein for 6 gene suggested that there had a very high similarity and conservation between them, at the same time, they were different in a way; the associated protein network of NLP family in Arabidopsis thaliana were used for MdNLPs, MDP0000132856, which had the highest homology with AtNLP7, might also have complicated features and functions.
【Objective】Columnar growth in apple (Malus × domestica Borkh.) is a special type of dwarf mutation. Due to short internodes and lateral branches, a limited canopy and minimal pruning, columnar apple trees are well adapted to high density plantings. Based on the fine mapping of Co, the genes in the localization interval were screened, which laid a foundation for elucidating the molecular mechanism of columnar apple formation and breeding new varieties of columnar apples.【Method】Based on the latest apple genome data and transcriptome information, the buds, stem tips and leaves of columnar apple Wujia, Runtai No.1 and standard apple Fuji and Huashuo were used as test materials, and the genes between the fine mapping Co gene in interval from 27.66 Mb to 29.05 Mb were annotated and predicted. The coding sequence of the target gene was selected to detect primer specificity by RT-PCR, the real-time quantitative PCR was used to analyze the expression characteristics of target genes in different tissues and organs, and differential genes were screened out as candidate genes.【Result】The results showed that there were 67 genes between 27.66 Mb and 29.05 Mb in chromosome 10, 12 of which were non-coding RNAs (ncRNA), and the rest were genes with encoding proteins. According to the columnar and standard apple RNA-seq, there were 25 genes with more than 1 fold difference, 13 of which were up-regulated, and 12 genes were down-regulated in columnar apples. Among the 14 predicted genes, there were significant differences in the relative expression of the four genes MD10G1184100, MD10G1185400, MD10G1185600 and MD10G1190500 between shoot tips and lateral shoot tips in columnar and standard apples. The relative expression levels of MD10G1184100 and MD10G1185600 in the tip of terminal bud of two columnar apples were significantly higher than those of both standard apples. The relative expression of MD10G1185400 and MD10G1190500 genes in the tip of lateral bud of two columnar apples was significantly higher than that of two standard apples, while the expression of MD10G1184100 gene in two columnar apples was significantly lower than that of standard apples. The gene expression patterns of different tissues or organs of four candidate genes were analyzed. The result showed that the expression of MD10G1184100 gene in the roots of columnar apples was significantly higher than that in other tissues. The MD10G1185400 and D10G1185600 genes were significantly expressed in lateral tips in columnar apples, while MD10G1190500 gene was prominently expressed in the terminal bud of columnar apples.【Conclusion】4 genes with significant differences in columnar and standard apple could be regarded as Co candidate genes, which laid a foundation for gene cloning and functional verification and apple tree-oriented genetic improvement.
【Objective】U6 promoter is an important element for the transcription of sgRNA in the CRISPR/Cas9 genome editing system. There may be species-specific factors in U6 promoter, and the activity of U6 promoter would be altered when its length changed. So far, in apple (Malus×domestica), the transcriptional characterization of U6 promoters has not been reported. Therefore, selecting an apple U6 promoter with high transcriptional activity and suitable fragment size would provide a basis for optimizing apple CRISPR/Cas9 gene editing technology system. 【Method】 DNAMAN, promoter cis element online predicting website PLACE and plant CARE were used to do the comparative analysis of apple U6 promoters; U6 promoters were cloned and constructed into firefly luciferase vector; the apple callus and tobacco (Nicotiana benthamiana) leaves were transformed via Agrobacterium-mediated transient transformation; the transcriptional activity of U6 promoter was determined according to the luciferase activity. 【Result】 There were six alternative U6 promoters in apple genome (E-value<3e -40), which were located on chr 6, chr 7, chr 9, chr 10, chr 15 and chr 17, respectively. 27 bp snRNA at 5′ end and its upstream 1 500 bp were selected as candidate U6 promoter. Sequence analysis results showed that the upstream sequence element (USE) and TATA-like elements were contained in six U6 promoters of apple, the same as Arabidopsis. After transient transformation, the luciferase activity assay showed that, the U6 promoter on chromosome 10 had the highest transcriptional activity. Among all U6 promoters which were shortened at 5′ end (1 500 bp, 959 bp, 275 bp, and 116 bp) on chromosome 10, the 275 bp one had the highest transcriptional activity. In addition, compared with the Arabidopsis U6 promoter, in apple callus, the transcriptional activity of the apple U6 promoter was higher. 【Conclusion】Six U6 promoters were cloned from the apple genome, and a U6 promoter with high transcriptional activity and suitable sequence fragment was obtained.
【Objective】Ethylene response factor (ERF) , a plant-specific transcription factor, is involved in the growth and development of root formation, hypocotyl elongation, fruit ripening, and organ senescence. It also plays a vital role in regulating responses of plant biological and abiotic stress, as well as fruit qualities. In this study, we cloned the apple ethylene response factor MdERF72. Subsequently, a series of expression analysis and functional identification of transgenic apple calli were performed to study its role in abiotic stress responses. These results provided a theoretical basis for exploring the functions of MdERF72 in plant growth and development.【Method】Using Orin apple calli (Malus calli stica Borkh.) as the test material, the MdERF72 was cloned from apple fruits by RT-PCR assay. Bioinformatics methods were used to analyze its amino acid sequence, physicochemical properties genetic relationship, and spatial structure. MEGA5.0 was used to construct the phylogenetic tree for analyzing the homology of its protein sequence with ERF-B2 subfamily in Arabidopsis. The real-time fluorescence PCR (qRT-PCR) assays were performed to analyse the expression of MdERF72 in different organs and tissues of apple, as well as in apple fruits during different developmental stages. Meanwhile, the expression of MdERF72 in Gala apple tissue culture seedlings treated with ACC, NaCl and low-temperature was detected by qRT-PCR assay. We also constructed its overexpression vector and obtained stable overexpression apple calli through Agrobacterium-mediated genetic transformation. The fresh weight, malondialdehyde content, electrical conductivity, hydrogen peroxide content and superoxide anion content of the wild type and transgenic apple calli were detected after NaCl and low temperature treatment. 【Result】 MdERF72 was located on chromosome 13 in apple genome, which had an AP2/ERF domain, unique to ERF family. Phylogenetic tree analyses indicated that the apple MdERF72 exhibited the highest sequence similarity to Arabidopsis AtERF72, and belonged to the B2 subfamily of ERF family. Analysis of amino acid physicochemical properties indicated that MdERF72 encodes 253 amino acids, and its protein molecular weight was predicted as 27.61 kD, the isoelectric point (pI) was 5.10. In addition, the pro-hydrophobic prediction showed that the hydrophobic portion of MdERF72 was larger than the hydrophilic portion, indicating that it belonged to a hydrophobic protein. Phosphorylation site analysis revealed that MdERF72 had only one threonine phosphorylation site, suggesting that the protein might be regulated by phosphorylation. The results revealed that the MdERF72 promoter sequence contains cis-acting elements associated with jasmonic acid (JA), auxin and drought signals. qRT-PCR analysis showed that MdERF72 was a positive regulatory transcription factor of ethylene, which was expressed in all tissues of apple. Its expression in fruits and stems was relatively high, and gradually increased with the fruit ripening. The expression of MdERF72 in apple tissue culture seedlings was significantly induced by high salt and low temperature. Under the treatment of high salt and low temperature stresses, the MdERF72- overexpressing apple calli had stronger growth potential than the wild type control, and the conductivity, malondialdehyde, hydrogen peroxide and superoxide anion content were lower than the wild type control, indicating that MdERF72 increased the resistance to salt and low temperature stresses.【Conclusion】MdERF72 played an important role in the regulation of high salt and low temperature stresses. Overexpression of MdERF72 could increase the resistance of apple calli to high salt and low temperature stresses.
