Minjin Han

Body Shape and Coloration of Silkworm Larvae Are Influenced by a Novel Cuticular Protein

Body shape and color patterns of insect larvae are fundamental traits for survival. Typically, transcription factors or members of signaling pathways... The genetic basis of body shape and coloration patterns on caterpillars is often assumed to be regulated separately, but it is possible that common molecules affect both types of trait simultaneously. Here we examine the genetic basis of a spontaneous cuticle defect in silkworm, where larvae exhibit a bamboo-like body shape and decreased pigmentation. We performed linkage mapping and mutation screening to determine the gene product that affects body shape and coloration simultaneously. In these mutant larvae we identified a null mutation in BmorCPH24, a gene encoding a cuticular protein with low complexity sequence. Spatiotemporal expression analyses showed that BmorCPH24 is expressed in the larval epidermis postecdysis. RNAi-mediated knockdown and CRISPR/Cas9-mediated knockout of BmorCPH24 produced the abnormal body shape and the inhibited pigment typical of the mutant phenotype. In addition, our results showed that BmorCPH24 may be involved in the synthesis of endocuticle and its disruption-induced apoptosis of epidermal cells that accompanied the reduced expression of R&R-type larval cuticle protein genes and pigmentation gene Wnt1. Strikingly, BmorCPH24, a fast-evolving gene, has evolved a new function responsible for the assembly of silkworm larval cuticle and has evolved to be an indispensable factor maintaining the larval body shape and its coloration pattern. This is the first study to identify a molecule whose pleiotropic function affects the development of body shape and color patterns in insect larvae.

Comparative analysis of the integument transcriptomes of the black dilute mutant and the wild-type silkworm Bombyx mori

The insect cuticle is a critical protective shell that is composed predominantly of chitin and various cuticular proteins and pigments. Indeed, insects often change their surface pigment patterns in response to selective pressures, such as threats from predators, sexual selection and environmental changes. However, the molecular mechanisms underlying the construction of the epidermis and its pigmentation patterns are not fully understood. Among Lepidoptera, the silkworm is a favorable model for color pattern research. The black dilute (bd) mutant of silkworm is the result of a spontaneous mutation; the larval body color is notably melanized. We performed integument transcriptome sequencing of the wild-type strain Dazao and the mutant strains +/bd and bd/bd. In these experiments, during an early stage of the fourth molt, a stage at which approximately 51% of genes were expressed genome wide (RPKM ≥1) in each strain. A total of 254 novel transcripts were characterized using Cuffcompare and BLAST analyses. Comparison of the transcriptome data revealed 28 differentially expressed genes (DEGs) that may contribute to bd larval melanism, including 15 cuticular protein genes that were remarkably highly expressed in the bd/bd mutant. We suggest that these significantly up-regulated cuticular proteins may promote melanism in silkworm larvae.

Identification of MBF2 family genes in Bombyx mori and their expression in different tissues and stages and in response to Bacillus bombysepticus infection and starvation

The Multiprotein bridge factor 2 (MBF2) gene was first identified as a co‐activator involved in BmFTZ‐F1‐mediated activation of the Fushi tarazu gene. Herein, nine homologous genes of MBF2 gene are identified. Evolutionary analysis showed that this gene family is insect‐specific and that the family members are closely related to response to pathogens (REPAT) genes. Tissue distribution analysis revealed that these genes could be expressed in a tissue‐specific manner. Developmental profiles analysis showed that the MBF2 gene family members were highly expressed in the different stages. Analysis of the expression patterns of nine MBF2 family genes showed that Bacillus bombysepticus treatment induced the up‐regulation of several MBF2 family genes, including MBF2‐4, ‐7, ‐9, ‐8. Furthermore, we found the MBF2 family genes were modulated by starvation and the expression of these genes recovered upon re‐feeding, except for MBF2‐5, ‐9. These findings suggested roles for these proteins in insect defense against pathogens and nutrient metabolism, which has an important guiding significance for designing pest control strategies.

Microarray analysis of New Green Cocoon associated genes in silkworm, Bombyx mori

Green cocoons in silkworm, Bombyx mori, are caused by flavonoids accumulation in the silk proteins, fibroin and sericin. Despite the economic value of natural green cocoon and medical value of flavonoids, there is limited understanding of the molecular mechanism regulating flavonoids uptake in silkworm, which is tightly associated with the trait of green cocoon. The purpose of this study is to perform a comprehensive analysis to understand the molecular mechanisms of flavonoids uptake in silkworm based on microarray analyses. The study subject was the New Green Cocoon from the silkworm strains, G200 and N100, a new spontaneous dominant green cocoon trait identified in the 2000s. The genes regulating this trait are independent of other green cocoon genes previously reported. Genome‐wide gene expression was compared between the New Green Cocoon producing silkworm strains, G200 and N100, and the control sample, which is the white cocoon producing strain 872B. Among these strains, N100 and 872B are near‐isogenic lines. The results showed that 130 genes have consistently changing expression patterns in the green cocoon strains when compared with the white cocoon strain. Among these, we focused on the genes related to flavonoids metabolism and absorption, such as sugar transporter genes and UDP‐glucosyltransferase genes. Based on our findings, we propose the potential mechanisms for flavonoids absorption and metabolism in silkworm. Our results imply that silkworm might be used as an underlying model for flavonoids in pharmaceutical research.

Variation of lifespan in multiple strains, and effects of dietary restriction and BmFoxO on lifespan in silkworm, Bombyx mori

Established animal models have accelerated our understanding of the mechanisms involved in lifespan determination. However, more experimental animals are required to clarify the complex mechanisms behind the phenomena of aging and lifespan. In this study, we reported the variation of lifespan in nine distinct silkworm strains. Lifespan correlated significantly with BmFoxO gene expression in the representative silkworm strains tested (Xiafang, Dazao-N, and N4). In general, the female silkworm was longer lived than the male of the same strain. Dietary restriction extended the silkworm lifespan compared with that of silkworms fed ad libitum. The expression of BmFoxO was significantly elevated in the dietary restriction group on day 3 of the 4th instar and day 3 of the 5th instar, suggesting that BmFoxO contributes to dietary restriction-mediated lifespan extension. The RNA interference and overexpression of the BmFoxO gene significantly shortened and extended the silkworm adulthood, respectively. In conclusion, our findings suggest that the silkworm might serve as a promising experimental animal to explore the complex biological mechanisms of lifespan determination.

A serine protease homologue Bombyx mori scarface induces a short and fat body shape in silkworm: Bmscarface for silkworm tubby mutant

Body shape is one of the most prominent and basic characteristics of any organism. In insects, abundant variations in body shape can be observed both within and amongst species. However, the molecular mechanism underlying body shape fine‐tuning is very complex and has been largely unknown until now. In the silkworm Bombyx mori, the tubby (tub) mutant has an abnormal short fat body shape and the abdomen of tub larvae expands to form a fusiform body shape. Morphological investigation revealed that the body length was shorter and the body width was wider than that of the Dazao strain. Thus, this mutant is a good model for studying the molecular mechanisms of body shape fine‐tuning. Using positional cloning, we identified a gene encoding the serine protease homologue, B. mori scarface (Bmscarface), which is associated with the tub phenotype. Sequence analysis revealed a specific 312‐bp deletion from an exon of Bmscarface in the tub strain. In addition, recombination was not observed between the tub and Bmscarface loci. Moreover, RNA interference of Bmscarface resulted in the tub‐like phenotype. These results indicate that Bmscarface is responsible for the tub mutant phenotype. This is the first study to report that mutation of a serine protease homologue can induce an abnormal body shape in insects.

Enhancer activity of Helitron in sericin-1 gene promoter from Bombyx mori.

Sericin is a kind of water‐soluble protein expressed specifically in the middle silk gland of Bombyx mori. When the sericin‐1 gene promoter was cloned and a transgenic vector was constructed to express a foreign protein, a specific Helitron, Bmhel‐8, was identified in the sericin‐1 gene promoter sequence in some genotypes of Bombyx mori and Bombyx mandarina. Given that the Bmhel‐8 Helitron transposon was present only in some genotypes, it could be the source of allelic variation in the sericin‐1 promoter. The length of the sericin‐1 promoter sequence is approximately 1063 or 643 bp. The larger size of the sequence or allele is ascribed to the presence of Bmhel‐8. Silkworm genotypes can be homozygous for either the shorter or larger promoter sequence or heterozygous, containing both alleles. Bmhel‐8 in the sericin‐1 promoter exhibits enhancer activity, as demonstrated by a dual‐luciferase reporter system in BmE cell lines. Furthermore, Bmhel‐8 displays enhancer activity in a sericin‐1 promoter‐driven gene expression system but does not regulate the tissue‐specific expression of sericin‐1.

Cuticular protein defective Bamboo mutant of Bombyx mori is sensitive to environmental stresses

Insect cuticle acts as a primary protective barrier against environment stresses that may directly impact the insect body. Here, we report the mechanical defense function of a structural cuticular protein, BmorCPH24, to environmental stresses using a silkworm Bamboo (Bo) mutant with this gene mutation. Ultraviolet (UV) irradiation and topical application of an acetone insecticide were used as environmental stresses to determine the differences in susceptibility between Bo and wild-type larvae. UV irradiation resulted in a sunburn phenotype in the Bo strains earlier than the wild-type indicating the sensitivity of Bo. Higher malondialdehyde (MDA) content and a lower survival ratio were also observed in the Bo strains. Treatment with deltamethrin revealed that Bo larvae were more sensitive to insecticides than the wild-type. Furthermore, cuticle analysis by microsection revealed thinner cuticle and a significant decrease in the endocuticle layer (∼64.0%) in Bo. These results suggest that BmorCPH24 mutation can lead to deficiency in resources required to construct the cuticle in Bo resulting in thin cuticle and reduced resistance to UV and insecticides. These results provide us new insight into the role of structural cuticular proteins in insect cuticle against environment stresses.

Genome-wide identification and analysis of elongase of very long chain fatty acid genes in the silkworm, Bombyx mori

Very long chain fatty acids (VLCFAs), such as sphingolipids, are components of cellular lipids, which are essential for cell proliferation. Mutations in the genes that encode proteins participating in VLCFA biosynthesis may cause inherited diseases, such as macular degeneration. Elongases of very long chain fatty acid (ELOVL) are enzymes that are involved in the biosynthesis of VLCFAs. Here, a total of 13 ELOVL genes, distributed across three chromosomes, were identified in the silkworm genome; all the ELOVL members contain a distinct ELO domain and a conserved HXXHH motif. Phylogenetic reconstruction was performed to analyze the evolutionary relationships among different species and to predict gene functions. The 13 ELOVL genes were assigned to the ELOVL3/6, ELOVL1/7, and ELOVL4 clades. Microarray and semiquantitative PCR analyses indicated that these genes are differentially expressed among various tissues, in turn suggesting functional divergence in the growth and development of each tissue. Further investigation showed that the expression level of the BGIBMGA000424 gene is significantly negatively correlated with the cocoon-shell weight among different silkworm strains. Taken together, the present study is the first comprehensive analysis of ELOVL genes in silkworm, and the results may serve as a foundation for further analysis of the physiological functions of ELOVL genes in silkworm.

p27 inhibits CDK6/CCND1 complex formation resulting in cell cycle arrest and inhibition of cell proliferation

ABSTRACT p27 plays critical roles in cell proliferation, differentiation, and apoptosis, which have been well studied in mammals and Drosophila. However, the mechanisms underlying p27 regulation of the cell cycle have not been thoroughly researched. In this study, Genevestigator, Kaplan–Meier Plotter, and the Human Protein Atlas databases were used to analyze the expression of p27, cell division protein kinase 6 (CDK6), and cyclin D1 (CCND1), as well as its prognostic value in different tumor tissues and corresponding normal tissues. Quantitative PCR and immunohistochemistry were used to detect the expression of p27, CDK6, and CCND1 in the tissues of cancer patients. The effects of p27, CDK6, and CCND1 on the proliferation of lung cancer cells were examined by the MTT assay, and flow cytometry was used to investigate the mechanism by which p27 affected cell proliferation. Immunofluorescence, co-immunoprecipitation, and Western blotting were used to determine if p27 interacted with CDK and CCND1 to regulate the cell cycle. The results showed that p27, CDK6, and CCND1 played different roles in tumorigenesis and development, which are in accordance with CDK6 and CCND1 in affecting the cell cycle and cell proliferation. p27 regulated the cell cycle and inhibited cell proliferation by affecting formation of the cell cycle-dependent complex CDK6/CCND1, but did not directly affect the expression of CDK6 and CCND1. Moreover, CCND1 did not regulate the cell cycle alone, but rather, functioned together with CDK6. This study provides insights into the effects of p27 on tumor formation and development, and the underlying regulatory mechanisms.