The proteome reveals the involvement of serine/threonine kinase in the recognition of self- incompatibility in almond.

The self-incompatibility recognition mechanism determines whether the gametophyte is successfully fertilized between pollen tube SCF (SKP1-CUL1-F-box-RBX1) protein and pistil S-RNase protein during fertilization is unclear. In this study, the pistils of two almond cultivars 'Wanfeng' and 'Nonpareil' were used as the experimental materials after self- and nonself/cross-pollination, and pistils from the stamen-removed flowers were used as controls. We used fluorescence microscopy to observe the development of pollen tubes after pollination and 4D-LFQ to detect the protein expression profiles of 'Wanfeng' and 'Nonpareil' pistils and in controls. The results showed that it took 24-36 h for the development of the pollen tube to 1/3 of the pistil, and a total of 7684 differentially accumulated proteins (DAPs) were identified in the pistil after pollinating for 36 h, of which 7022 were quantifiable. Bioinformatics analysis based on the function of DAPs, identified RNA polymerases (4 DAPs), autophagy (3 DAPs), oxidative phosphorylation (3 DAPs), and homologous recombination (2 DAPs) pathways associated with the self-incompatibility process. These results were confirmed by parallel reaction monitoring (PRM), protein interaction and bioinformatics analysis. Taken together, these results provide the involvement of serine/threonine kinase protein in the reaction of pollen tube recognition the nonself- and the self-S-RNase protein. SIGNIFICANCE: Gametophytic self-incompatibility (GSI) is controlled by the highly polymorphic S locus or S haplotype, with two linked self-incompatibility genes, one encoding the S-RNase protein of the pistil S-determinant and the other encoding the F-box/SLF/SFB (S haplotype-specific F-box protein) protein of the pollen S-determinant. The recognition mechanism between pollen tube SCF protein and pistil S-RNase protein is divided into nonself- and self-recognition hypothesis mechanisms. At present, two hypothetical mechanisms cannot explain the recognition between pollen and pistil well, so the mechanism of gametophytic self-incompatibility recognition is still not fully revealed. In this experiment, we investigated the molecular mechanism of pollen-pistil recognition in self-incompatibility using self- and nonself-pollinated pistils of almond cultivars 'Wanfeng' and 'Nonpareil'. Based on our results, we proposed a potential involvement of the MARK2 (serine/threonine kinase) protein in the reaction of pollen tube recognition of the nonself- and the self-S-RNase protein. It provides a new way to reveal how almond pollen tubes recognize the self and nonself S-RNase enzyme protein.

Case report: exome sequencing achieved a definite diagnosis in a Chinese family with muscle atrophy.

BACKGROUND: Due to large genetic and phenotypic heterogeneity, the conventional workup for Charcot-Marie-Tooth (CMT) diagnosis is often underpowered, leading to diagnostic delay or even lack of diagnosis. In the present study, we explored how bioinformatics analysis on whole-exome sequencing (WES) data can be used to diagnose patients with CMT disease efficiently. CASE PRESENTATION: The proband is a 29-year-old female presented with a severe amyotrophy and distal skeletal deformity that plagued her family for over 20 years since she was 5-year-old. No other aberrant symptoms were detected in her speaking, hearing, vision, and intelligence. Similar symptoms manifested in her younger brother, while her parents and her older brother showed normal. To uncover the genetic causes of this disease, we performed exome sequencing for the proband and her parents. Subsequent bioinformatics analysis on the KGGSeq platform and further Sanger sequencing identified a novel homozygous GDAP1 nonsense mutation (c.218C > G, p.Ser73*) that responsible for the family. This genetic finding then led to a quick diagnosis of CMT type 4A (CMT4A), confirmed by nerve conduction velocity and electromyography examination of the patients. CONCLUSIONS: The patients with severe muscle atrophy and distal skeletal deformity were caused by a novel homozygous nonsense mutation in GDAP1 (c.218C > G, p.Ser73*), and were diagnosed as CMT4A finally. This study expanded the mutation spectrum of CMT disease and demonstrated how affordable WES could be effectively employed for the clinical diagnosis of unexplained phenotypes.

Developmental transcriptome profiling uncovered carbon signaling genes associated with almond fruit drop.

Almond is one of the most featured nut crops owing to its high nutritional value. However, due to three different waves of flower and fruitlet drop, fruit drop is a major concern for growers. In this study, we carried out a time-course transcriptome analysis to investigate gene expression differences between normal and abnormal fruitlet development. By de novo assembly analysis, we identified 33,577 unigenes and provided their functional annotations. In total, we identified 7,469 differentially expressed genes and observed the most apparent difference between normal and abnormal fruits at 12 and 17 days after flowering. Their biological functions were enriched in carbon metabolism, carbon fixation in photosynthetic organisms and plant hormone signal transduction. RT-qPCR validated the expression pattern of 14 representative genes, including glycosyltransferase like family 2, MYB39, IAA13, gibberellin-regulated protein 11-like and POD44, which confirmed the reliability of our transcriptome data. This study provides an insight into the association between abnormal fruit development and carbohydrate signaling from the early developmental stages and could be served as useful information for understanding the regulatory mechanisms related to almond fruit drop.

Construction of a Competitive Endogenous RNA Network and Identification of Potential Regulatory Axis in Gastric Cancer.

Background: Increasing studies has found that long non-coding RNAs (lncRNAs) play critical roles in carcinogenesis, but the underlying mechanisms remain unclear. The aim of this study is to construct a competitive endogenous RNA (ceRNA) network and to identify potential regulatory axis in gastric cancer (GC). Methods: Differentially expressed (DE) mRNAs, miRNAs, and lncRNAs were obtained by analyzing the RNA expression profiles of stomach adenocarcinoma (STAD) retrieved from The Cancer Genome Atlas (TCGA) database. The lncRNA-miRNA-mRNA regulatory networks of GC were constructed by comprehensive bioinformatics methods including functional annotation, RNA-RNA interactomes prediction, correlation analysis, and survival analysis. The interactions and correlations among ceRNAs were validated by experiments on cancer tissues and cell lines. Results: A total of 41 lncRNAs, 9 miRNAs, and 10 mRNAs were identified and selected to establish the ceRNA regulatory network of GC. Several ceRNA regulatory axes, which consist of 18 lncRNAs, 4 miRNAs, and 6 mRNAs, were obtained from the network. A potential ADAMTS9-AS2/miR-372/CADM2 axis which perfectly conformed to the ceRNA theory was further analyzed. qRT-PCR showed that ADAMTS9-AS2 knockdown remarkably increased miR-372 expression but reduced CADM2 expression, whereas ADAMTS9-AS2 overexpression had the opposite effects. Dual luciferase reporter assay indicated that miR-372 could bound to the ADAMTS9-AS2 and the 3'UTR of CADM2. Conclusion: The constructed novel ceRNA network and the potential regulatory axes might provide a novel approach of the exploring the potential mechanisms of development in GC. The ADAMTS9-AS2/miR-372/CADM2 could act as a promising target for GC treatment.

Mutation analysis and pathogenicity identification of Mucopolysaccharidosis type IVA in 8 south China families.

BACKGROUND: Mucopolysaccharidosis type IVA (MPS IVA) is a rare autosomal recessive lysosomal storage disorder caused by GALNS gene mutation. The aim of our study is to detect pathogenic variants for patients suspected of MPS IVA and set the base for subsequent prenatal diagnosis and preimplantation genetic diagnosis. METHODS: In our study, 9 MPS IVA patients from south China families were investigated. Urine glycosaminoglycans (GAGS) screening was used as an initial method. For patients with abnormal result, all 14 exons and intron-exon junctions of the GALNS gene were sequenced after amplification from genomic DNA. The pathogenicity of novel mutations were analyzed with molecular genetics, bioinformatics and structure modeling in light of clinical manifestations and biochemical results. RESULTS: Among 12 mutations detected, direct sequencing found 3 novel mutations (c.686A>C, p.Y229S; c.1498G>T, p.G500C; c.278T>C, p.I93T). The pathogenicity of these novel mutations was illustrated by correlating clinical symptoms with pedigree analysis and bioinformatics analysis. CONCLUSION: The detection and variant analysis are essential for accurate diagnosis of MPS IVA patients. Our results enrich GALNS gene mutation spectrum of Chinese population. This information has important clinical value for molecular diagnosis and genetic counseling of patients with this disease.

CRISPR/Cas9-induced Targeted Mutagenesis and Gene Replacement to Generate Long-shelf Life Tomato Lines.

Quickly and precisely gain genetically enhanced breeding elites with value-adding performance traits is desired by the crop breeders all the time. The present of gene editing technologies, especially the CRISPR/Cas9 system with the capacities of efficiency, versatility and multiplexing provides a reasonable expectation towards breeding goals. For exploiting possible application to accelerate the speed of process at breeding by CRISPR/Cas9 technology, in this study, the Agrobacterium tumefaciens-mediated CRISPR/Cas9 system transformation method was used for obtaining tomato ALC gene mutagenesis and replacement, in absence and presence of the homologous repair template. The average mutation frequency (72.73%) and low replacement efficiency (7.69%) were achieved in T0 transgenic plants respectively. None of homozygous mutation was detected in T0 transgenic plants, but one plant carry the heterozygous genes (Cas9/*-ALC/alc) was stably transmitted to T1 generations for segregation and genotyping. Finally, the desired alc homozygous mutants without T-DNA insertion (*/*-alc/alc) in T1 generations were acquired and further confirmed by genotype and phenotype characterization, with highlight of excellent storage performance, thus the recessive homozygous breeding elites with the character of long-shelf life were generated. Our results support that CRISPR/Cas9-induced gene replacement via HDR provides a valuable method for breeding elite innovation in tomato.

Two novel mutations in the PPIB gene cause a rare pedigree of osteogenesis imperfecta type IX.

BACKGROUND: Osteogenesis imperfecta (OI) is a rare genetic skeletal disorder characterized by increased bone fragility and vulnerability to fractures. PPIB is identified as a candidate gene for OI-IX, here we detect two pathogenic mutations in PPIB and analyze the genotype-phenotype correlation in a Chinese family with OI. METHODS: Next-generation sequencing (NGS) was used to screen the whole exome of the parents of proband. Screening of variation frequency, evolutionary conservation comparisons, pathogenicity evaluation, and protein structure prediction were conducted to assess the pathogenicity of the novel mutations. Sanger sequencing was used to confirm the candidate variants. RTQ-PCR was used to analyze the PPIB gene expression. RESULTS: All mutant genes screened out by NGS were excluded except PPIB. Two novel heterozygous PPIB mutations (father, c.25A>G; mother, c.509G>A) were identified in relation to osteogenesis imperfecta type IX. Both mutations were predicted to be pathogenic by bioinformatics analysis and RTQ-PCR analysis revealed downregulated PPIB expression in the two carriers. CONCLUSION: We report a rare pedigree with an autosomal recessive osteogenesis imperfecta type IX (OI-IX) caused by two novel PPIB mutations identified for the first time in China. The current study expands our knowledge of PPIB mutations and their associated phenotypes, and provides new information on the genetic defects associated with this disease for clinical diagnosis.

Molecular diagnosis of a Chinese pedigree with α-mannosidosis and identification of a novel missense mutation.

α-Mannosidosis storage disease is a rare autosomal recessive disease that is caused by a deficiency of the lysosomal enzyme α-mannosidase. In this article, a proband in China was preliminarily diagnosed as having α-mannosidosis by clinical symptoms, imaging examination, and enzyme assay. Definitive diagnosis was performed by directly sequencing the MAN2B1 gDNA and cDNA of the peripheral blood leukocyte from the patient. Finally, denaturing high-performance liquid chromatography screening, conservative analysis, and protein secondary structure prediction were used to identify the novel mutation. The results showed that the patient has compound heterozygous mutations in the MAN2B1 gene, c.856G>A (p.E286K, novel) and c.788C>T (p.P263L). Her parents are heterozygote that carry one of these two mutations respectively. Pathogenicity identification of the novel mutation showed that the p.E286K mutation is a disease-causing mutation. Our work enriches the human MAN2B1 gene mutation database. As far as we know, this research is thus far the first gene diagnosis case of a Chinese patient with α-mannosidosis.

Ascorbic acid formation and profiling of genes expressed in its synthesis and recycling in apple leaves of different ages.

Ascorbic acid (AsA), as a unique antioxidant and enzyme cofactor, has multiple roles in plants. However, there is very limited information on the mechanism of AsA accumulation and controlling in leaves. In this study, we determined AsA accumulation levels, analyzed expression patterns of the genes involved in synthesizing via l-galactose pathway and recycling as well as enzyme activities in apple (Malus domestica Borkh) leaves with different age. AsA content was found to increase with leaf development, reaching the highest level in 20-day-old leaves. This level was maintained in mature leaves until the dropping in senescent leaves. Comparing with young and senescent leaves, mature leaves had higher capability for AsA synthesis with high expression levels and activity of l-galactose dehydrogenase and l-galactono-1,4-lactone dehydrogenase. The mRNA expression of genes involved in AsA synthesis also showed highest abundance in 20-day-old leaves, though GDP-mannose-3',5'-epimerase and l-galactose-1-phosphate phosphatase expression reached the highest levels before 20 days old. These results suggest that AsA accumulation in apple leaves mainly occurs during the transition phase from young to mature leaves with high rates of synthesis and recycling, and that l-galactose-1-phosphate phosphatase could play an important role in regulating AsA biosynthesis via the l-galactose pathway.

[Identification of a novel mutation of IDS gene from a Chinese pedigree with MPS II].

The purpose of this study was to understand the molecular genetic mechanism of mucopolysaccharidosis type II (MPS II) and to provide a prerequisite for future prenatal gene diagnosis. A preliminary diagnosis was made by qualitative detection of Urinary Glycosaminoglycans of the suspected MPS II proband. Then, mutation detection was performed on the proband and his family members with PCR and direct sequencing of PCR products. After the novel mutation of c.876 del 2 in IDS gene was detected, sequence analysis was performed on exon 6 of IDS gene of the 135 cases, which consisted of 120 randomly selected normal controls, and other 15 patients with MPS I, IV, and VI other than MPS II. Besides, the patho-genicity of the novel mutation was analyzed with the following 2 methods: conservative analysis of the sequence of muta-tion spots of different species and the direct test of the IDS enzyme activity of the patient and his relative family members. The result of uroscopy of the proband was strong positive (GAGs +++). There was a novel deletion mutation of c.876-877 del TC in the coding region of exon 6 of IDS gene, which was a hemizygous mutation. However, the mutation of his mother and sister was a heterozygous mutation. Detection of the exon 6 of IDS gene showed that the mutation was not found among normal controls and other patients with MPS I, IV, and VI other than MPS II. Homology comparison of amino acid sequences from different species showed that the phenylalanine (F) glutamine (Q) of the mutation site of c.876-877 del TC located in p.292-293 was highly conserved. The activity of IDS enzyme of the proband was only 2.3 nmol/4 h/mL, which was much lower than normal; but the activity of IDS enzyme of his father, mother and sister was 641.9 nmol/4 h/mL, 95.8 nmol/4h/mL and 103.2 nmol/4h/mL, respectively. These results illustrated that the deletion and frame-shift mutation of c.876-877 del TC detected was a novel pathologic mutation, which was the underlying cause of MPS II of this patient.