A method of identifying microorganisms with reduced adaptability to a particular environment, wherein (1) each microorganism is independent by insertional inactivation of a gene with a nucleic acid comprising a unique marker sequence. Mutating to provide a plurality of microorganisms, or clones of said microorganisms, such that each mutation has a different label sequence; (2) providing storage samples of each mutation produced by step (1), respectively, and providing storage nucleic acids each comprising a unique label sequence from each mutation; (3) introducing a plurality of mutations produced by step (1) into the specific environment such that the microorganisms capable of growing in the specific environment grow in the above environment; (4) recovering the microorganisms or selected portions thereof from the environment and separating nucleic acids from the recovered microorganisms; (5) comparing all label sequences in the nucleic acid isolated in step (4) with the unique label sequences of each of the stored mutations as in step (2); and (6) any label isolated in step (4). Screening for individual mutations that also do not have a sequence.

Identification of genes

The diverse color patterns of cichlid fishes play an important role in mate choice and speciation Here we develop the Nile tilapia (Oreochromis niloticus) as a model system for studying the developmental genetics of cichlid color patterns We identified 4 types of pigment cells: melanophores, xanthophores, iridophores and erythrophores, and characterized their first appearance in wild-type fish We mutated 25 genes involved in melanogenesis, pteridine metabolism, and the carotenoid absorption and cleavage pathways Among the 25 mutated genes, 13 genes had a phenotype in both the F0 and F2 generations None of F1 heterozygotes had phenotype By comparing the color pattern of our mutants with that of red tilapia (Oreochromis spp), a natural mutant produced during hybridization of tilapia species, we found that the pigmentation of the body and eye is controlled by different genes Previously studied genes like mitf, kita/kitlga, pmel, tyrb, hps4, gch2, csf1ra, pax7b, and bco2b were proved to be of great significance for color patterning in tilapia Our results suggested that tilapia, a fish with 4 types of pigment cells and a vertically barred wild-type color pattern, together with various natural and artificially induced color gene mutants, can serve as an excellent model system for study color patterning in vertebrates

Nile Tilapia: A Model for Studying Teleost Color Patterns

Body color is an important economic and ecological trait in aquatic animals, which influence their economic values and determine the animal ability to survive in the marine environment. Red-colored Plectropomus leopardus is in high demand owing to its bright color and potential value for artificial breeding. High-throughput transcriptome sequencing and LC-MS/MS approaches were used to identify transcript and metabolic differences between black-colored and red-colored P. leopardus. Compared with the black-colored group, 218 and 181 differentially expressed genes were up- and down- regulated, respectively, in the red-colored group. 425 and 56 significantly different metabolites were identified in LC-MS positive and LC-MS negative ion models, respectively, between two colored groups. Based on the integrative analysis, the red-colored group exhibited greater carotenoid uptake, transport, and accumulation activity potential than those in the black-colored group and may consume more arachidonic acid for body color formation. The black-colored group showed greater melanin synthesis activity compared with the red-colored group. These results substantially improve the understanding of molecular and metabolic mechanisms underlying body color formation in P. leopardus and the potential of body color for the environment adaptation.

/pdf/integrative-transcriptomics-and-metabolomics-analysis-of-2djrq8bebi.pdf

Integrative Transcriptomics and Metabolomics Analysis of Body Color Formation in the Leopard Coral Grouper (Plectropomus leopardus)

Integrated analysis of the role of miRNA-mRNA in determining different body colors of leopard coral grouper (Plectropomus leopardus)

Pristella maxillaris is known as the X-ray fish based on its translucent body. However, the morphological characteristics and the molecular regulatory mechanisms of these translucent bodies are still unknown. In this study, the following three phenotypes, a black-and-gray body color or wild-type (WT), a silvery-white body color defined as mutant I (MU1), and a fully transparent body with a visible visceral mass named as mutant II (MU2), were investigated to analyze their chromatophores and molecular mechanisms. The variety and distribution of pigment cells in the three phenotypes of P. maxillaris significantly differed by histological assessment. Three types of chromatophores (melanophores, iridophores, and xanthophores) were observed in the WT, whereas MU1 fish were deficient in melanophores, and MU2 fish lacked melanophores and iridophores. Transcriptome sequencing of the skin and peritoneal tissues of P. maxillaris identified a total of 166,089 unigenes. After comparing intergroup gene expression levels, more than 3,000 unigenes with significantly differential expression levels were identified among three strains. Functional annotation and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of the differentially expressed genes (DEGs) identified a number of candidates melanophores and iridophores genes that influence body color. Some DEGs that were identified using transcriptome analysis were confirmed by quantitative real-time PCR. This study serves as a global survey of the morphological characteristics and molecular mechanism of different body colors observed in P. maxillaris and thus provides a valuable theoretical foundation for the molecular regulation of the transparent phenotype.

/pdf/morphological-characteristics-and-comparative-transcriptome-3luwckzm00.pdf

Morphological Characteristics and Comparative Transcriptome Analysis of Three Different Phenotypes of Pristella maxillaris.

Self-healing polymers are categorized as smart materials that are capable of surface protection and prevention of structural failure. Polyurethane/polyurea, as one of the representative coatings, has also attracted attention for industrial applications. Compared with polyurethane, polyurea coating, with a similar formation process, provides higher tensile strength and requires shorter curing time. In this paper, extrinsic and intrinsic mechanisms are reviewed to address the efficiency of the self-healing process. Moreover, formulation optimization and strategic improvement to ensure self-healing within a shorter period of time with acceptable recovery of mechanical strength are also discussed. The choice and ratio of diisocyanates, as well as the choice of chain extender, are believed to have a crucial effect on the acceleration of the self-healing process and enhance self-healing efficiency during the preparation of polyurea coatings.

/pdf/mini-review-of-self-healing-mechanism-and-formulation-183ecf6y.pdf

Mini-Review of Self-Healing Mechanism and Formulation Optimization of Polyurea Coating

The highly conserved transcription factor Pax6 is involved in the development of the eyes, brain, and pancreas in vertebrates and invertebrates, whereas the additional expression pattern in other organs is still elusive. In this study, we cloned and characterized two pax6 homologs in blunt snout bream (Megalobrama amblycephala), named Mapax6a and Mapax6b. The protein alignment and phylogenetic tree showed that Mapax6a and Mapax6b were highly conserved compared with their counterparts in other species. Genomic information analysis revealed that the synteny conservation of Wilms tumor, Aniridia, genitourinary abnormalities, and mental retardation loci was also maintained in this species. By reverse transcription polymerase chain reaction, the expression of Mapax6a was later than that of Mapax6b which was found in the blastula stage, while the expression of Mapax6a started from the somite stage, and both of them persisted in a subsequent stage during the embryonic development. By RNA and protein detection, Mapax6a and Mapax6b were detected in the eye and brain as canonic patterns, and most importantly, they were also enriched in germ cells of the testis and ovary. Therefore, our findings validate the duplication of pax6 in fish, confirm the classical expression patterns in the brain and eye, and, for the first time, present a new acquisition of Mapax6a and Mapax6b in gonadal germ cells in particular. Therefore, our results enrich the expression pattern and evolutionary relationship of pax6 by suggesting that duplicated Mapax6 is involved in gametogenesis in Megalobrama amblycephala.

Gonadal, Not Maternal, Acquisition of Duplicated pax6 Orthologs in Megalobrama Amblycephala.

Generation of mutants with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is commonly carried out in fish species by co-injecting a mixture of Cas9 messenger RNA (mRNA) or protein and transcribed guide RNA (gRNA). However, the appropriate expression system to produce functional gRNAs in fish embryos and cells is rarely present. In this study, we employed a poly-transfer RNA (tRNA)-gRNA (PTG) system driven by cytomegalovirus (CMV) promoter to target the medaka ( Oryzias latipes ) endogenous gene tyrosinase ( tyr ) or paired box 6.1 ( pax6.1 ) and illustrated its function in a medaka cell line and embryos. The PTG system was combined with the CRISPR/Cas9 system under high levels of promoter to successfully induce gene editing in medaka. This is a valuable step forward in potential application of the CRISPR/Cas9 system in medaka and other teleosts. 目的 探究巨细胞病毒(CMV)启动子驱动转运RNA-导向RNA(tRNA-gRNA)的表达是否能够在青鳉细胞系及胚胎中产生功能性的gRNA并诱导基因的突变。 创新点 利用CMV启动子驱动tRNA-gRNA的表达在青鳉细胞系及胚胎中产生功能性的gRNA, 结合Cas9系统诱导了青鳉胚胎和细胞系中的基因突变, 为在鱼类胚胎和细胞系中进行基因编辑提供了一种可行的方案。 方法 将tRNA- tyr gRNA(酪氨酸酶基因, tyr )或 tyr gRNA与Cas9 mRNA注射到青鳉1胞期的胚胎中观察胚胎表型并通过聚合酶链式反应(PCR)和测序检测tRNA- tyr gRNA在青鳉胚胎中的功能。将pCS2-tRNA- tyr 和pCS2-tRNA-PT质粒分别与Cas9 mRNA注射到青鳉1胞期的胚胎中观察胚胎表型, 并通过PCR和测序检测CMV启动子驱动的tRNA-gRNA在胚胎中的功能。将pCMV-tRNA-PT-zCas9-puro以及1:1比例的pCVpf或pCS2-tRNA- tyr 与pCV-zCas9-puro的混合物分别转染SG3细胞, 通过PCR和测序检测CMV启动子驱动的tRNA-gRNA在青鳉细胞系中的功能。 结论 tRNA- tyr gRNA与 tyr gRNA一样能够引起青鳉胚胎中 tyr 基因的突变。CMV启动子驱动tRNA-gRNA的表达在青鳉细胞系及胚胎中能够产生功能性的gRNA, 结合Cas9系统诱导青鳉胚胎和细胞系中基因的突变。

Efficient gene editing in a medaka (Oryzias latipes) cell line and embryos by SpCas9/tRNA-gRNA

Generation of mutants with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is commonly carried out in fish species by co-injecting a mixture of Cas9 messenger RNA (mRNA) or protein and transcribed guide RNA (gRNA). However, the appropriate expression system to produce functional gRNAs in fish embryos and cells is rarely present. In this study, we employed a poly-transfer RNA (tRNA)-gRNA (PTG) system driven by cytomegalovirus (CMV) promoter to target the medaka (Oryzias latipes) endogenous gene tyrosinase (tyr) or paired box 6.1 (pax6.1) and illustrated its function in a medaka cell line and embryos. The PTG system was combined with the CRISPR/Cas9 system under high levels of promoter to successfully induce gene editing in medaka. This is a valuable step forward in potential application of the CRISPR/Cas9 system in medaka and other teleosts.

Junzhi Luo

Papers

Morphological Characteristics and Comparative Transcriptome Analysis of Three Different Phenotypes of Pristella maxillaris.

Mini-Review of Self-Healing Mechanism and Formulation Optimization of Polyurea Coating

Gonadal, Not Maternal, Acquisition of Duplicated pax6 Orthologs in Megalobrama Amblycephala.

Efficient gene editing in a medaka (Oryzias latipes) cell line and embryos by SpCas9/tRNA-gRNA

Efficient gene editing in a medaka (Oryzias latipes) cell line and embryos by SpCas9/tRNA-gRNA