CRISPR/Cas系统作为抗菌药的现状及展望

抗生素长期滥用导致了人体内菌群失调及细菌耐药性的产生,因此需要寻找新型、靶向抗菌方法来治疗耐药细菌的感染。近年来,CRISPR/Cas系统的深入研究为设计特异性靶向耐药基因,定向清除耐药细菌的药物提供了新的思路。在此介绍了CRISPR/Cas系统作为新型抗菌方法,通过靶向切割抗性质粒或细菌基因组以实现对耐药基因或病原菌的特异性清除,并对CRISPR抗菌药的不同类型核酸酶的选择,以及CRISPR递送系统的运载工具进行了评价。     

Plant microRNAs in molecular breeding

MicroRNAs are small, endogenous, non-coding RNAs found in plants, animals, and in some viruses, which negatively regulate the expression of genes by promoting the degradation of target mRNAs or by translation inhibition. Ever since the discovery of miRNAs, its biology, mechanisms, and functions were extensively studied in the past two decades. Plant and animal miRNAs both regulate target mRNAs, but they differ in scope of complementarity to their target mRNA. Plant microRNAs are known to play essential roles in a wide array of plant development. To date, there are many studies giving evidence that the regulation of miRNA levels can reprogram plant responses to abiotic (physical environment) and biotic stresses (pathogen and herbivore). Most of these studies were first carried out in the model plant Arabidopsis thaliana. Recently, the trend of miRNA research is furthering its role in crop breeding and its evolutionary origin. In this review, we presented the dynamic biogenesis of microRNAs, the diverse functions of miRNAs in plants, and experimental designs used in studying microRNAs in plants, and most importantly, we presented the applications of microRNA-based technology to improve the resistance of crops in abiotic and biotic stresses.     

Gene loss/retention and evolutionary pattern of ascorbic acid biosynthesis and recycling genes in <Emphasis Type="Italic">Brassica rapa</Emphasis> following whole genome triplication

Ascorbic acid (AsA) is an inevitable antioxidant found abundantly in higher plants. Despite the importance of AsA in plants, how AsA biosynthesis (ABGs; d-mannose/l-galactose pathway) and AsA recycling genes (ARGs) evolved through polyploidization has not been addressed to date. Here, we evaluated the impacts of whole genome triplication (WGT) on ABGs and ARGs in Chinese cabbage (Brassica rapa ssp. pekinensis), which diverged from Arabidopsis thaliana before the WGT event. Twenty-three ABGs coded in 13 loci representing nine different enzyme classes and 29 ARGs coded in 19 loci representing five different enzyme classes were identified in the B. rapa genome by whole-genome screening through comparative genomic analyses. Five of these loci maintained three gene copies, 10 loci maintained two gene copies and the majority of the loci (n = 17) maintained single gene copies. Segmental (62 %) and tandem duplication (6 %), and fragment (21 %) and large-scale recombination (10 %) events accelerated the diversification of ABGs and ARGs. Thirteen of the 52 (25 %) identified genes experienced intron losses and two (4 %) experienced intron gains implying that intron losses outnumbered intron gains. The expansion and the retention of ABGs and ARGs were similar to the whole genome gene expansion and retention (P > 0.05). These findings provide new insights into the structural characteristics and evolutionary trends of ABGs and ARGs. In addition, our data could become a useful resource to further the functional characterization of these genes.     

Genotyping-by-sequencing map permits identification of clubroot resistance QTLs and revision of the reference genome assembly in cabbage (Brassica oleracea L.)

Clubroot is a devastating disease caused by Plasmodiophora brassicae and results in severe losses of yield and quality in Brassica crops. Many clubroot resistance genes and markers are available in Brassica rapa but less is known in Brassica oleracea. Here, we applied the genotyping-by-sequencing (GBS) technique to construct a high-resolution genetic map and identify clubroot resistance (CR) genes. A total of 43,821 SNPs were identified using GBS data for two parental lines, one resistant and one susceptible lines to clubroot, and 18,187 of them showed >5× coverage in the GBS data. Among those, 4,103 were credibly genotyped for all 78 F₂ individual plants. These markers were clustered into nine linkage groups spanning 879.9 cM with an average interval of 1.15 cM. Quantitative trait loci (QTLs) survey based on three rounds of clubroot resistance tests using F_{2 : 3} progenies revealed two and single major QTLs for Race 2 and Race 9 of P. brassicae, respectively. The QTLs show similar locations to the previously reported CR loci for Race 4 in B. oleracea but are in different positions from any of the CR loci found in B. rapa. We utilized two reference genome sequences in this study. The high-resolution genetic map developed herein allowed us to reposition 37 and 2 misanchored scaffolds in the 02–12 and TO1000DH genome sequences, respectively. Our data also support additional positioning of two unanchored 3.3 Mb scaffolds into the 02–12 genome sequence.     

Orange color is associated with <Emphasis Type="Italic">CYC</Emphasis>-<Emphasis Type="Italic">B</Emphasis> expression in tomato fleshy fruit

Carotenes are plant secondary metabolites that are important for human health. Additionally, carotenes influence fruit color, which is a major trait for breeding. We compared the expression and sequences of genes related to color phenotypes in tomato inbred lines that produce different colors of fleshy fruit. Up-regulation of CYC-B expression and higher amount of β-carotene content in fruit ripening stage and nucleotide variations in the 5′ region of the gene were detected in orange fruited inbred lines compared to the other lines. Our results indicated that there is a close relationship between the expression pattern of the CYC-B gene and the orange color of fleshy fruit. We identified 4 SNPs in the promoter region of CYC-B genes associated with the orange fruit color. Moreover, the segregation ratio and color phenotypes in an F2 generation further indicated that one of the detected SNPs were associated with the orange color in the tested inbred lines. Our study provides valuable information to breeders for marker-assisted selection to produce desirable tomato varieties with health benefits by varying carotenoid levels.     

A novel amino-benzosuberone derivative is a picomolar inhibitor of mammalian aminopeptidase N/CD13

A new class of low molecular weight, highly potent and selective non peptidic inhibitors of aminopeptidase N (APN/CD13) is described. We report the synthesis and in vitro evaluation of racemic substituted analogues of 7-amino-benzocyclohepten-6-one 1a. We investigated various substitutions on the aromatic ring with phenyl and halogen groups. In vitro kinetic studies revealed that these compounds are among the most effective APN/CD13 inhibitors found so far. Hydrophobic substituents placed at position 1 or 4 on the cycloheptenone 1a led to the potent compounds 1c-h,b'-c',f',h' with K(i) in the nanomolar range. The key finding of the present work was the observed additive effect of 1,4-disubstitutions which led to the discovery of the picomolar inhibitor 1d' (K(i)=60 pM). The designed inhibitors retain the selectivity of our lead structure 1a towards selected members of the aminopeptidase family, combined with an impressive increase in inhibitory potency and a conserved stability.