Viscosity of Ionic Liquids: Theories and Models.

Ionic liquids (ILs) offer a wide range of promising applications due to their unique and designable properties compared to conventional solvents. Further development and application of ILs require correlating/predicting their pressure-viscosity-temperature behavior. In this review, we firstly introduce methods for calculation of thermodynamic inputs of viscosity models. Next, we introduce theories, theoretical and semi-empirical models coupling various theories with EoSs or activity coefficient models, and empirical and phenomenological models for viscosity of pure ILs and IL-related mixtures. Our modelling description is followed immediately by model application and performance. Then, we propose simple predictive equations for viscosity of IL-related mixtures and systematically compare performances of the above-mentioned theories and models. In concluding remarks, we recommend robust predictive models for viscosity at atmospheric pressure as well as proper and consistent theories and models for P-η-T behavior. The work that still remains to be done to obtain the desired theories and models for viscosity of ILs and IL-related mixtures is also presented. The present review is structured from pure ILs to IL-related mixtures and aims to summarize and quantitatively discuss the recent advances in theoretical and empirical modelling of viscosity of ILs and IL-related mixtures.

Hydrolysis mechanism of the cyclohexaborate anion: Unraveling the intricacies.

B 6 O 7 OH 6 2 - is a highly polymerized borate anion of three six-membered rings. Limited research on the B 6 O 7 OH 6 2 - hydrolysis mechanism under neutral solution conditions exists. Calculations based on density functional theory show that B 6 O 7 OH 6 2 - undergoes five steps of hydrolysis to form H3BO3 and B OH 4 - . At the same time, there are a small number of borate ions with different degrees of polymerization during the hydrolysis process, such as triborate, tetraborate, and pentaborate anions. The structure of the borate anion and the coordination environment of the bridging oxygen atoms control the hydrolysis process. Finally, this work explains that in existing experimental studies, the reason for the low B 6 O 7 OH 6 2 - content in solution environments with low total boron concentrations is that it depolymerizes into other types of borate ions and clarifies the borate species. The conversion relationship provides a basis for identifying the possibility of various borate ions existing in the solution. This work also provides a certain degree of theoretical support for the cause of the "dilution to salt" phenomenon.

Hydrogen sulfide antagonizes cytokinin to change root system architecture through persulfidation of CKX2 in Arabidopsis.

Hydrogen sulfide (H2S) is an endogenous gaseous signaling molecule, which has been shown to play an important role in plant growth and development by coupling with various phytohormones. However, the relationship between H2S and cytokinin (CTK) and the mechanisms by which H2S and CTK affect root growth remain poorly understood. Endogenous CTK was analyzed by UHPLC-ESI-MS/MS. Persulfidation of cytokinin oxidase/dehydrogenases (CKXs) was analyzed by mass spectrometry (MS). ckx2/CKX2wild-type (WT), OE CKX2 and ckx2/CKX2Cys(C)62alanine(A) transgenic lines were isolated with the ckx2 background. H2S is linked to CTK content by CKX2, which regulates root system architecture (RSA). Persulfidation at cysteine (Cys)62 residue of CKX2 enhances CKX2 activity, resulting in reduced CTK content. We utilized 35S-LCD/oasa1 transgenic lines to investigate the effect of endogenous H2S on RSA, indicating that H2S reduces the gravitropic set-point angle (GSA), shortens root hairs, and increases the number of lateral roots (LRs). The persulfidation of CKX2Cys62 changes the elongation of cells on the upper and lower flanks of LR elongation zone, confirming that Cys62 of CKX2 is the specificity target of H2S to regulate RSA in vivo. In conclusion, this study demonstrated that H2S negatively regulates CTK content and affects RSA by persulfidation of CKX2Cys62 in Arabidopsis thaliana.

Evolutionary cues of gene fusion and fission in plants.

KEY MESSAGE: Here, we systematically analyzed the potential fusion and fission events of neighboring genes in Arabidopsis genome and analyzed the influence on the protein targeting.

Serine Hydroxymethyltransferase 2 Deficiency in the Hematopoietic System Disrupts Erythropoiesis and Induces Anemia in Murine Models.

Serine and folate metabolism play critical roles in erythroid development in both embryonic and adult mice; however, the precise roles of these metabolic pathways in erythropoiesis and the pathophysiology of anemia remain inadequately characterized in the literature. To delineate the contributions of serine and folate metabolism to erythroid differentiation, we focused on serine hydroxymethyltransferase 2 (SHMT2), a key regulatory enzyme within these metabolic pathways. Using gene-editing techniques, we created fetal and adult mouse models with targeted deletion of Shmt2 in the hematopoietic system. Our findings demonstrated that the deletion of Shmt2 within the hematopoietic system led to the distinctive anemia phenotype in both fetal and adult mice. Detailed progression analysis of anemia revealed that Shmt2 deletion exerts stage-specific effects on the development and maturation of erythroid cells. Specifically, Shmt2 deficiency promoted erythroid differentiation in the R2 (CD71+ Ter119-) cell population residing in the bone marrow while concurrently inhibiting the proliferation and erythroid differentiation of the R3 (CD71+ Ter119+) cell population. This disruption resulted in developmental arrest at the R3 stage, significantly contributing to the anemia phenotype observed in the models. This study elucidates the critical role of Shmt2 in erythroid development within the hematopoietic system, highlighting the underlying mechanisms of erythroid developmental arrest associated with Shmt2 loss.

Persulfidation maintains cytosolic G6PDs activity through changing tetrameric structure and competing cysteine sulfur oxidation under salt stress in Arabidopsis and tomato.

Glucose-6-phosphate dehydrogenases (G6PDs) are essential regulators of cellular redox. Hydrogen sulfide (H2 S) is a small gasotransmitter that improves plant adaptation to stress; however, its role in regulating G6PD oligomerization to resist oxidative stress remains unknown in plants. Persulfidation of cytosolic G6PDs was analyzed by mass spectrometry (MS). The structural change model of AtG6PD6 homooligomer was built by chemical cross-linking coupled with mass spectrometry (CXMS). We isolated AtG6PD6C159A and SlG6PDCC155A transgenic lines to confirm the in vivo function of persulfidated sites with the g6pd5,6 background. Persulfidation occurs at Arabidopsis G6PD6 Cystine (Cys)159 and tomato G6PDC Cys155, leading to alterations of spatial distance between lysine (K)491-K475 from 42.0 Å to 10.3 Å within the G6PD tetramer. The structural alteration occurs in the structural NADP+ binding domain, which governs the stability of G6PD homooligomer. Persulfidation enhances G6PD oligomerization, thereby increasing substrate affinity. Under high salt stress, cytosolic G6PDs activity was inhibited due to oxidative modifications. Persulfidation protects these specific sites and prevents oxidative damage. In summary, H2 S-mediated persulfidation promotes cytosolic G6PD activity by altering homotetrameric structure. The cytosolic G6PD adaptive regulation with two kinds of protein modifications at the atomic and molecular levels is critical for the cellular stress response.

Hydrogen sulfide improves salt tolerance through persulfidation of PMA1 in Arabidopsis.

KEY MESSAGE: A new interaction was found between PMA1 and GRF4. H2S promotes the interaction through persulfidated Cys446 of PMA1. H2S activates PMA1 to maintain K+/Na+ homeostasis through persulfidation under salt stress. Plasma membrane H+-ATPase (PMA) is a transmembrane transporter responsible for pumping protons, and its contribution to salt resistance is indispensable in plants. Hydrogen sulfide (H2S), a small signaling gas molecule, plays the important roles in facilitating adaptation of plants to salt stress. However, how H2S regulates PMA activity remains largely unclear. Here, we show a possible original mechanism for H2S to regulate PMA activity. PMA1, a predominant member in the PMA family of Arabidopsis, has a non-conservative persulfidated cysteine (Cys) residue (Cys446), which is exposed on the surface of PMA1 and located in cation transporter/ATPase domain. A new interaction of PMA1 and GENERAL REGULATORY FACTOR 4 (GRF4, belongs to the 14-3-3 protein family) was found by chemical crosslinking coupled with mass spectrometry (CXMS) in vivo. H2S-mediated persulfidation promoted the binding of PMA1 to GRF4. Further studies showed that H2S enhanced instantaneous H+ efflux and maintained K+/Na+ homeostasis under salt stress. In light of these findings, we suggest that H2S promotes the binding of PMA1 to GRF4 through persulfidation, and then activating PMA, thus improving the salt tolerance of Arabidopsis.

An efficient strategy for large-scale preparation of low polarity gingerols directly from ginger crude extract by high-speed countercurrent chromatography with different rotation mode.

This study presents an efficient strategy for large-scale preparation of low polarity gingerols directly from ginger crude extract by high-speed countercurrent chromatography with different rotation mode. The ultrasonic-assisted extraction conditions were optimized by response surface methodology and the results showed the major low polarity gingerols could be well enriched under the optimized extraction conditions. Then the crude extract without any pretreatment was directly separated by high-speed countercurrent chromatography with different rotation mode using n-hexane/ethyl acetate/methanol/water (6:4:6:4, v/v/v/v) as the solvent system. In about 400 min, five major gingerols including 150 mg of [6]-gingerol, 50 mg of [8]-gingerol, 20 mg of [6]-shogaol, 43 mg of [6]-dehydrogingerdione, and 40 mg of [10]-gingerol were obtained from 1.2 g of crude extract in a single run with repeated injection. Their structures were identified by 1 H-NMR spectroscopy.

Correction to: Evaluation of salinity resistance and combining ability analysis in the seedlings of mulberry hybrids (Morus alba L.).

[This corrects the article DOI: 10.1007/s12298-023-01304-w.].

Evaluation of salinity resistance and combining ability analysis in the seedlings of mulberry hybrids (Morus alba L.).

Soil salinization has become one of the major abiotic stresses influencing food security and maintenance of sustainable eco-environment. Highly salt-tolerant germplasm in mulberry, an important perennial woody plant, could restore the ecology and increase the agricultural income. Studies on the salt tolerance of mulberry are limited. Therefore, the aim of this study was to estimate the genetic variation and develop a reliable and effective evaluation of salt tolerance in 14 F1 mulberry hybrids that were directionally constructed using nine genotypes, including two females and seven males. A salt stress test was performed using 0.3%, 0.6%, and 0.9% (w/v) NaCl to investigate four morphological indexes of the growth rate: the shoot height (SHR), leaf number (LNR), leaf area (LAR), and the total weight of the whole plant after defoliation (BI) in the seedlings of the 14 combinations. The most suitable concentration for evaluating salt tolerance was identified as 0.9% NaCl based on the changes in the salt tolerance coefficient (STC). Comprehensive evaluation (D) values were obtained using principal components and membership functions based on four morphological indexes and their STCs, grouped into three principal component indexes cumulatively contributing to approximately 88.90% of the total variance. Two highly salt-tolerant, three moderately salt-tolerant, five salt-sensitive, and four highly salt-sensitive genotypes were screened. Anshen × Xinghainei and Anshen × Xinghaiwai had the highest D values. The analyses of combining ability further showed that the variances for LNR, LAR, and BI were elevated significantly with the increasing NaCl concentrations. Anshen × Xinghainei from two superior parents (female: Anshen, male: Xinghainei) with relatively higher general combing abilities for SHR, LAR, and BI was the best hybrid combination under high salinity stress, and presented the best specific combining ability for BI. Of all the traits tested, LAR and BI were greatly affected by additive effects and might be the two most reliable indexes. These traits show higher correlation with the salt tolerance of mulberry germplasm at the seedling stage. These results may enrich the mulberry resources by breeding and screening for elite germplasms with high salt tolerance. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01304-w.

Hydrogen Sulfide and Reactive Oxygen Species, Antioxidant Defense, Abiotic Stress Tolerance Mechanisms in Plants.

Various stress conditions, such as drought, salt, heavy metals, and extreme temperatures, have severe deleterious effects on plant growth and directly lead to a decline in yield and quality [...].

Exogenous salicylic acid regulates cell wall polysaccharides synthesis and pectin methylation to reduce Cd accumulation of tomato.

Cadmium (Cd) is harmful to plant growth and can be easily transferred from soil to plants. Plant cell wall plays important role in preventing Cd from entering cells. Salicylic acid (SA) mediated defense response increases plant resistance to heavy metals. In this study, all tomato seedlings were pre-treated with 100 µM SA for 3 d, then seedlings were used to analyze the role of SA in regulating plant cell wall resistance to Cd stress. The results showed that exogenous SA significantly reduced Cd accumulation in tomato plants and changed Cd distribution. By analyzing the cell wall composition, it was found cellulose, hemicellulose, pectin, and lignin were induced by SA. Interestingly, the content of Cd in pectin decreased by SA pretreatment, however it was increased in cellulose. Gene expression analysis showed SA up-regulated the expression level of lignin and cellulose synthase genes, but down-regulated the expression of pectin methylesterase related genes. In addition, SA down-regulated the activity of pectin methylesterase. These results indicated that SA pretreatment up-regulated cell wall polysaccharide synthesis and related gene expression to thicken the cell wall and block Cd from passing through. Furthermore, SA decreased pectin methylesterase activity and content to reduce cell wall Cd accumulation and change the Cd partition ratio.

Comparative Analysis of Herbaceous and Woody Cell Wall Digestibility by Pathogenic Fungi.

Fungal pathogens have evolved combinations of plant cell-wall-degrading enzymes (PCWDEs) to deconstruct host plant cell walls (PCWs). An understanding of this process is hoped to create a basis for improving plant biomass conversion efficiency into sustainable biofuels and bioproducts. Here, an approach integrating enzyme activity assay, biomass pretreatment, field emission scanning electron microscopy (FESEM), and genomic analysis of PCWDEs were applied to examine digestibility or degradability of selected woody and herbaceous biomass by pathogenic fungi. Preferred hydrolysis of apple tree branch, rapeseed straw, or wheat straw were observed by the apple-tree-specific pathogen Valsa mali, the rapeseed pathogen Sclerotinia sclerotiorum, and the wheat pathogen Rhizoctonia cerealis, respectively. Delignification by peracetic acid (PAA) pretreatment increased PCW digestibility, and the increase was generally more profound with non-host than host PCW substrates. Hemicellulase pretreatment slightly reduced or had no effect on hemicellulose content in the PCW substrates tested; however, the pretreatment significantly changed hydrolytic preferences of the selected pathogens, indicating a role of hemicellulose branching in PCW digestibility. Cellulose organization appears to also impact digestibility of host PCWs, as reflected by differences in cellulose microfibril organization in woody and herbaceous PCWs and variation in cellulose-binding domain organization in cellulases of pathogenic fungi, which is known to influence enzyme access to cellulose. Taken together, this study highlighted the importance of chemical structure of both hemicelluloses and cellulose in host PCW digestibility by fungal pathogens.

Hydrogen sulfide promotes hypocotyl elongation via increasing cellulose content and changing the arrangement of cellulose fibrils in alfalfa.

Hydrogen sulfide (H2S) is known to have positive physiological functions in plant growth, but limited data are available on its influence on cell walls. Here, we demonstrate a novel mechanism by which H2S regulates the biosynthesis and deposition of cell wall cellulose in alfalfa (Medicago sativa). Treatment with NaHS was found to increase the length of epidermal cells in the hypocotyl, and transcriptome analysis indicated that it caused the differential expression of numerous of cell wall-related genes. These differentially expressed genes were directly associated with the biosynthesis of cellulose and hemicellulose, and with the degradation of pectin. Analysis of cell wall composition showed that NaHS treatment increased the contents of cellulose and hemicellulose, but decreased the pectin content. Atomic force microscopy revealed that treatment with NaHS decreased the diameter of cellulose fibrils, altered the arrangement of the fibrillar bundles, and increased the spacing between the bundles. The dynamics of cellulose synthase complexes (CSCs) were closely related to cellulose synthesis, and NaHS increased the rate of mobility of the particles. Overall, our results suggest that the H2S signal enhances the plasticity of the cell wall by regulating the deposition of cellulose fibrils and by decreasing the pectin content. The resulting increases in cellulose and hemicellulose contents lead to cell wall expansion and cell elongation.

Hydrogen Sulfide Disturbs Actin Polymerization via S-Sulfhydration Resulting in Stunted Root Hair Growth.

Hydrogen sulfide (H2S) is an important signaling molecule in plants. Our previous report suggested that H2S signaling affects the actin cytoskeleton and root hair growth. However, the underlying mechanisms of its effects are not understood. S-Sulfhydration of proteins is regulated directly by H2S, which converts the thiol groups of cysteine (Cys) residues to persulfides and alters protein function. In this work, we studied the effects of S-sulfhydration on actin dynamics in Arabidopsis (Arabidopsis thaliana). We generated transgenic plants overexpressing the H2S biosynthesis-related genes l-CYSTEINE DESULFHYDRASE (LCD) and d-CYSTEINE DESULFHYDRASE in the O-acetylserine(thiol)lyase isoform a1 (oasa1) mutant and Columbia-0 backgrounds. The H2S content increased significantly in overexpressing LCD/oasa1 plants. The density of filamentous actin (F-actin) bundles and the F-actin/globular actin ratio decreased in overexpressing LCD/oasa1 plants. S-Sulfhydration also was enhanced in overexpressing LCD/oasa1 plants. An analysis of actin dynamics suggested that S-sulfhydration inhibited actin polymerization. We also found that ACTIN2 (ACT2) was S-sulfhydrated at Cys-287. Cys-287 is adjacent to the D-loop, which acts as a central region for hydrophobic and electrostatic interactions and stabilizes F-actin filaments. Overaccumulation of H2S caused the depolymerization of F-actin bundles and inhibited root hair growth. Introduction of ACT2 carrying a Cys-287-to-Ser mutation into an act2-1 mutant partially suppressed H2S-dependent inhibition of root hair growth. We conclude that H2S regulates actin dynamics and affects root hair growth.

Loss of cytosolic glucose-6-phosphate dehydrogenase increases the susceptibility of Arabidopsis thaliana to root-knot nematode infection.

Background and Aims: Root knot nematodes (RKNs, Meloidogyne spp.) are microscopic roundworms with a wide host range causing great economic losses worldwide. Understanding how metabolic pathways function within the plant upon RKN infection will provide insight into the molecular aspects of plant-RKN interactions. Glucose-6-phosphate dehydrogenase (G6PDH), the key regulatory enzyme of the oxidative pentose phosphate pathway (OPPP), is involved in plant responses to abiotic stresses and pathogenesis. In this study, the roles of Arabidopsis cytosolic G6PDH in plant-RKN interactions were investigated. Methods: Enzyme assays and western blotting were used to characterize changes in total G6PDH activity and protein abundance in wild-type Arabidopsis in response to RKN infection. The susceptibility of wild-type plants and the double mutant g6pd5/6 to RKNs was analysed and the expression of genes associated with the basal defence response was tested after RKN infection using quantitative reverse transcription PCR. Key Results: RKN infection caused a marked increase in total G6PDH activity and protein abundance in wild-type Arabidopsis roots. However, the transcript levels of G6PDH genes except G6PD6 were not significantly induced following RKN infection, suggesting that the increase in G6PDH activity may occur at the post-transcriptional level. The double mutant g6pd5/6 with loss-of-function of the two cytosolic isoforms G6PD5 and G6PD6 displayed enhanced susceptibility to RKNs. Moreover, reactive oxygen species (ROS) production and gene expression involved in the defence response including jasmonic acid and salicylic acid pathways were suppressed in the g6pd5/6 mutant at the early stage of RKN infection when compared to the wild-type plants. Conclusions: The results demonstrated that the G6PDH-mediated OPPP plays an important role in the plant-RKN interaction. In addition, a new aspect of G6PDH activity involving NADPH production by the OPPP in plant basal defence against RKNs is defined, which may be involved in ROS signalling.

Integrated Exploitation of the Structural Diversity Space of Chemotherapy Drugs to Selectively Inhibit HER2 T798M Mutant in Lung Cancer.

An acquired T798M gatekeeper mutation in human epidermal growth factor receptor 2 (HER2) kinase can cause drug resistance to anti-HER2 chemotherapy drugs in lung cancer. Previously, the reversible pan-kinase inhibitor staurosporine has been found to selectively inhibit the HER2 T798M mutant over wild-type kinase, suggesting that the staurosporine scaffold is potentially to develop mutant-selective inhibitors. Here, we systematically evaluated the chemical space of staurosporine scaffold-based compounds in response to HER2 T798M mutation at structural, energetic and molecular levels by using an integrated analysis strategy. With this strategy, we were able to identify several novel wild-type sparing inhibitors with high or moderate selectivity, which are comparable to or even better than that of the parent compound staurosporine. Molecular modeling and structural analysis revealed that noncovalent contacts can form between the side chain of mutated residue Met798 and selective inhibitor ligands, which may improve the favorable interaction energy between the kinase and inhibitor and reduce the unfavorable desolvation penalty upon the kinase-inhibitor binding.

Paired box 5 is a frequently methylated lung cancer tumour suppressor gene interfering ß-catenin signalling and GADD45G expression.

Recent studies suggest that paired box 5 (PAX5) is down-regulated in multiple tumours through its promoter methylation. However, the role of PAX5 in non-small cell lung cancer (NSCLC) pathogenesis remains unclear. The aim of this study is to examine PAX5 expression, its methylation status, biological functions and related molecular mechanism in NSCLC. We found that PAX5 was widely expressed in normal adult tissues but silenced or down-regulated in 88% (7/8) of NSCLC cell lines. PAX5 expression level was significantly lower in NSCLC than that in adjacent non-cancerous tissues (P = 0.0201). PAX5 down-regulation was closely associated with its promoter hypermethylation status and PAX5 expression could be restored by demethylation treatment. Frequent PAX5 promoter methylation in primary tumours (70%) was correlated with lung tumour histological types (P = 0.006). Ectopic expression of PAX5 in silenced lung cancer cell lines (A549 and H1975) inhibited their colony formation and cell viability, arrested cell cycle at G2 phase and suppressed cell migration/invasion as well as tumorigenicity in nude mice. Restoration of PAX5 expression resulted in the down-regulation of ß-catenin and up-regulation of tissue inhibitors of metalloproteinase 2, GADD45G in lung tumour cells. In summary, PAX5 was found to be an epigenetically inactivated tumour suppressor that inhibits NSCLC cell proliferation and metastasis, through down-regulating the ß-catenin pathway and up-regulating GADD45G expression.

Arabidopsis type I proton-pumping pyrophosphatase expresses strongly in phloem, where it is required for pyrophosphate metabolism and photosynthate partitioning.

Phloem loading is a critical process in plant physiology. The potential of regulating the translocation of photoassimilates from source to sink tissues represents an opportunity to increase crop yield. Pyrophosphate homeostasis is crucial for normal phloem function in apoplasmic loaders. The involvement of Arabidopsis (Arabidopsis thaliana) type I proton-pumping pyrophosphatase (AVP1) in phloem loading was analyzed at genetic, histochemical, and physiological levels. A transcriptional AVP1 promoter::GUS fusion revealed phloem activity in source leaves. Ubiquitous AVP1 overexpression (35S::AVP1 cassette) enhanced shoot biomass, photoassimilate production and transport, rhizosphere acidification, and expression of sugar-induced root ion transporter genes (POTASSIUM TRANSPORTER2 [KUP2], NITRATE TRANSPORTER2.1 [NRT2.1], NRT2.4, and PHOSPHATE TRANSPORTER1.4 [PHT1.4]). Phloem-specific AVP1 overexpression (Commelina Yellow Mottle Virus promoter [pCOYMV]::AVP1) elicited similar phenotypes. By contrast, phloem-specific AVP1 knockdown (pCoYMV::RNAiAVP1) resulted in stunted seedlings in sucrose-deprived medium. We also present a promoter mutant avp1-2 (SALK046492) with a 70% reduction of expression that did not show severe growth impairment. Interestingly, AVP1 protein in this mutant is prominent in the phloem. Moreover, expression of an Escherichia coli-soluble pyrophosphatase in the phloem (pCoYMV::pyrophosphatase) of avp1-2 plants resulted in severe dwarf phenotype and abnormal leaf morphology. We conclude that the Proton-Pumping Pyrophosphatase AVP1 localized at the plasma membrane of the sieve element-companion cell complexes functions as a synthase, and that this activity is critical for the maintenance of pyrophosphate homeostasis required for phloem function.

The clustered regularly interspaced short palindromic repeats/associated proteins system for the induction of gene mutations and phenotypic changes in Bombyx mori.

To probe the general phenomena of gene mutations, Bombyx mori, the lepidopterous model organism, was chosen as the experimental model. To easily detect phenotypic variations, the piggyBac system was utilized to introduce two marker genes into the silkworm, and 23.4% transposition efficiency aided in easily breeding a new strain for the entire experiment. Then, the clustered regularly interspaced short palindromic repeats/an associated protein (Cas9) system was utilized. The results showed that the Cas9 system can induce efficient gene mutations and the base changes could be detected since the G0 individuals in B. mori; and that the mutation rates on different target sites were diverse. Next, the gRNA2-targeted site that generated higher mutation rate was chosen, and the experimental results were enumerated. First, the mutation proportion in G1 generation was 30.1%, and some gene mutations were not inherited from the G0 generation; second, occasionally, base substitutions did not lead to variation in the amino-acid sequence, which decreased the efficiency of phenotypic changes compared with that of genotypic changes. These results laid the foundation for better use of the Cas9 system in silkworm gene editing.