CRISPR/Cas9-Mediated Editing of AGAMOUS-like Genes Results in a Late-Bolting Phenotype in Chinese Cabbage (Brassica rapa ssp. pekinensis).

Due to the sudden change in temperature in spring, Chinese cabbage, a leafy vegetable cultivated for consumption, loses its commercial value due to the onset of bolting­the phenomenon of switching from vegetative to reproductive growth. In this study, we applied clustered regularly interspaced short palindromic repeats/(CRISPR)-associated system 9 (CRISPR/Cas9) technology to analyze AGAMOUS-like genes. We performed functional analysis of AGL19 and AGL24 genes related to bolting and flowering using CRISPR/Cas9-mediated Chinese cabbage transformation. Single-guide RNA (sgRNA) sequences were created with a low off-targeting probability to construct gene-editing vectors. Agrobacterium-mediated transformation was conducted, and tentative E0 AGL-edited lines were analyzed using molecular biotechnological methods. Two AGL19-edited lines with nucleotide sequence mutations in the target sequence of the AGL19 genes and four AGL24-edited lines with nucleotide sequence mutations in the target sequence of the AGL24 genes showed particularly late bolting compared to the inbred line 'CT001.' Generational progression using bud pollination obtained T-DNA-free E1 AGL-edited lines, which also showed late bolting. The loss of function of the AGL protein was caused by the occurrence of an indel mutation in the AGL19 and AGL24 genes, which results in an early stop codon. Furthermore, frameshift mutations led to structural changes and the introduction of an early stop codon in the AGL19 and AGL24 proteins. Our results indicate that CRISPR/Cas9-mediated editing of AGAMOUS-like genes results in a late-bolting phenotype and that CRISPR/Cas9 is a useful technology for analyzing gene function in Chinese cabbage (Brassica rapa ssp. pekinensis).

Identification and Characterization of PTE-2, a Stowaway-like MITE Activated in Transgenic Chinese Cabbage Lines.

Transposable elements (TEs) are DNA fragments that can be replicated or transposed within a genome. TEs make up a high proportion of the plant genome and contribute to genetic diversity and evolution, affecting genome structure or gene activity. Miniature inverted-repeat transposable elements (MITEs) are short, non-autonomous class II DNA transposable elements. MITEs have specific sequences, target site duplications (TSDs), and terminal inverted repeats(TIRs), which are characteristics of the classification of MITE families. In this study, a Stowaway-like MITE, PTE-2, was activated in transgenic Chinese cabbage lines. PTE-2 was revealed by in silico analysis as the putative activated element in transgenic Chinese cabbage lines. To verify the in silico analysis data, MITE insertion polymorphism (MIP) PCR was conducted and PTE-2 was confirmed to be activated in transgenic Chinese cabbage lines. The activation tendency of the copy elements of PTE-2 at different loci was also analyzed and only one more element was activated in the transgenic Chinese cabbage lines. Analyzing the sequence of MIP PCR products, the TSD sequence and TIR motif of PTE-2 were identified and matched to the characteristics of the Stowaway-like MITE family. In addition, the flanking region of PTE-2 was modified when PTE-2 was activated.

Function Analysis of the PR55/B Gene Related to Self-Incompatibility in Chinese Cabbage Using CRISPR/Cas9.

Chinese cabbage, a major crop in Korea, shows self-incompatibility (SI). SI is controlled by the type 2A serine/threonine protein phosphatases (PP2As). The PP2A gene is controlled by regulatory subunits that comprise a 36 kDa catalyst C subunit, a 65 kDa regulatory A subunit, and a variety of regulatory B subunits (50-70 kDa). Among them, the PP2A 55 kDa B regulatory subunit (PR55/B) gene located in the A05 chromosome has 13 exons spanning 2.9 kb, and two homologous genes, Bra018924 and Bra014296, were found to be present on the A06 and A08 chromosome, respectively. In this study, we performed a functional analysis of the PR55/B gene using clustered regularly interspaced short palindromic repeats/CRISPR-associated system 9 (CRISPR/Cas9)-mediated gene mutagenesis. CRISPR/Cas9 technology can be used to easily introduce mutations in the target gene. Tentative gene-edited lines were generated by the Agrobacterium-mediated transfer and were selected by PCR and Southern hybridization analysis. Furthermore, pods were confirmed to be formed in flower pollination (FP) as well as bud pollination (BP) in some gene-edited lines. Seed fertility of gene-edited lines indicated that the PR55/B gene plays a key role in SI. Finally, self-compatible T-DNA-free T2 gene-edited plants and edited sequences of target genes were secured. The self-compatible Chinese cabbage developed in this study is expected to contribute to Chinese cabbage breeding.

CRISPR/Cas9-Mediated Mutagenesis of BrLEAFY Delays the Bolting Time in Chinese Cabbage (Brassica rapa L. ssp. pekinensis).

Chinese cabbage has unintended bolting in early spring due to sudden climate change. In this study, late-bolting Chinese cabbage lines were developed via mutagenesis of the BrLEAFY (BrLFY) gene, a transcription factor that determines floral identity, using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system. Double-strand break of the target region via gene editing based on nonhomologous end joining (NHEJ) was applied to acquire useful traits in plants. Based on the 'CT001' pseudomolecule, a single guide RNA (sgRNA) was designed and the gene-editing vector was constructed. Agrobacterium-mediated transformation was used to generate a Chinese cabbage line in which the sequence of the BrLFY paralogs was edited. In particular, single base inserted mutations occurred in the BrLFY paralogs of the LFY-7 and LFY-13 lines, and one copy of T-DNA was inserted into the intergenic region. The selected LFY-edited lines displayed continuous vegetative growth and late bolting compared to the control inbred line, 'CT001'. Further, some LFY-edited lines showing late bolting were advanced to the next generation. The T-DNA-free E1LFY-edited lines bolted later than the inbred line, 'CT001'. Overall, CRISPR/Cas9-mediated mutagenesis of the BrLFY gene was found to delay the bolting time. Accordingly, CRISPR/Cas9 is considered an available method for the molecular breeding of crops.

DNA Methylation Level Changes in Transgenic Chinese Cabbage (Brassica rapa ssp. pekinensis) Plants and Their Effects on Corresponding Gene Expression Patterns.

Plant tissue culture is an in vitro technique used to manipulate cells, tissues, or organs, and plays an important role in genetic transformation. However, plants cultured in vitro often exhibit unintended genetic and epigenetic variations. Since it is important to secure the stability of endogenous and exogenous gene expressions in transgenic plants, it is preferable to avoid the occurrence of such variations. In this study, we focused on epigenetic variations, exclusively on methylation level changes of DNA, in transgenic Chinese cabbage (Brassica rapa ssp. pekinensis) plants. To detect these methylation level changes of DNA, bisulfite sequencing was performed and the obtained sequences were compared with the 'CT001' reference genome. Differentially methylated regions (DMRs) of DNA between the non-transgenic and transgenic lines were detected by bisulfite sequencing, and ten DMRs located in exonic regions were identified. The regions with methylation variations that were inherited and consistently maintained in the next generation lines were selected and validated. We also analyzed the relationship between methylation status and expression levels of transformant-conserved DMR (TCD) genes by quantitative reverse transcription-PCR. These results suggested that the changes in methylation levels of these DMRs might have been related to the plant transformation process, affecting subsequent gene expression. Our findings can be used in fundamental research on methylation variations in transgenic plants and suggest that these variations affect the expression of the associated genes.

Identification and Validation of Genetic Variations in Transgenic Chinese Cabbage Plants (Brassica rapa ssp. pekinensis) by Next-Generation Sequencing.

Transgenic plants are usually produced through tissue culture, which is an essential step in Agrobacterium-mediated plant transformation. However, genomic variations, termed somaclonal variations, have been detected in transgenic plants cultured in vitro. The occurrence of these variations should be as low as possible to secure the stability of transgenic crops. Determining the cause and mechanism of somaclonal variations in tissue culture-derived plants will help reduce the rate of variation and promote the stable expression of genes in transgenic plants. In order to determine the genetic variability in transgenic Chinese cabbage plants, we performed whole-genome resequencing and compared the sequencing data with the 'CT001' reference genome. The variation candidates that were expected to consistently occur in the transgenic lines were selected and validated. The single nucleotide polymorphism (SNP) and insertion and deletion (InDel) candidates were identified using the resequencing data and validated by reverse transcription (RT)-PCR analysis. The deduced amino acid sequences were used to determine whether the variations caused changes in the resulting polypeptide, and the annotations of the mutated genes were analyzed to predict the possible effects of the SNPs on gene function. In conclusion, we selected and validated the genetic variations identified in transgenic Chinese cabbage plants. Their genomes were expected to be affected by the process of Agrobacterium-mediated transformation. The findings of our study will provide a genetic basis for transgenic plant research.

[Effects of Self-directed Feedback Practice using Smartphone Videos on Basic Nursing Skills, Confidence in Performance and Learning Satisfaction].

PURPOSE: This study was done to verify effects of a self-directed feedback practice using smartphone videos on nursing students' basic nursing skills, confidence in performance and learning satisfaction. METHODS: In this study an experimental study with a post-test only control group design was used. Twenty-nine students were assigned to the experimental group and 29 to the control group. Experimental treatment was exchanging feedback on deficiencies through smartphone recorded videos of nursing practice process taken by peers during self-directed practice. RESULTS: Basic nursing skills scores were higher for all items in the experimental group compared to the control group, and differences were statistically significant ["Measuring vital signs" (t=-2.10, p=.039); "Wearing protective equipment when entering and exiting the quarantine room and the management of waste materials" (t=-4.74, p<.001) "Gavage tube feeding" (t=-2.70, p=.009)]. Confidence in performance was higher in the experimental group compared to the control group, but the differences were not statistically significant. However, after the complete practice, there was a statistically significant difference in overall performance confidence (t=-3.07. p=.003). Learning satisfaction was higher in the experimental group compared to the control group, but the difference was not statistically significant (t=-1.67, p=.100). CONCLUSION: Results of this study indicate that self-directed feedback practice using smartphone videos can improve basic nursing skills. The significance is that it can help nursing students gain confidence in their nursing skills for the future through improvement of basic nursing skills and performance of quality care, thus providing patients with safer care.

[Impact of Bowel Function, Anxiety and Depression on Quality of Life in Patients with Sphincter-preserving Resection for Rectal Cancer].

PURPOSE: This study was a descriptive survey research to identify the impact of bowel function, anxiety and depression on quality of life in patients with rectal cancer who had a sphincter-preserving resection. METHODS: Participants were 100 patients who had rectal cancer surgery at W hospital in Korea. Bowel function, anxiety & depression, and quality of life were measured using the BFI (Bowel Function Instrument), HADS (Hospital Anxiety-Depression Scale) and the FACT-C (Functional Assessment of Cancer Therapy-Colorectal). RESULTS: The mean scores were 39.81±5.16 for bowel function, 6.15±3.25 for anxiety, 7.24±3.13 for depression, and 72.50±13.27 for quality of life. There were significant negative correlations between quality of life and anxiety (r=-.59, p<.001) and between quality of life and depression (r=-.53, p<.001). But the correlation between quality of life and bowel function was significantly positive (r=.22, p=.025). The influence of the independent variables on the total quality of life was examined using multiple regression analysis. Anxiety (ß=-.38, p=.002), bowel function (ß=-.25, p=.028) and occupation (ß=.16, p=.048) were identified as factors affecting quality of life. The explanation power of this regression model was 44% and it was statistically significant (F=16.53, p<.001). CONCLUSION: The results of this study indicate that in order to improve the bowel function of patients after sphincter-preserving resection for rectal cancer, effective nursing interventions should be developed. As psychological problem such as anxiety and depression can relate to quality of life for these patients, nurses should work on improving the situation by providing continuous emotional nursing.

[Development and psychometric evaluation of a scale to measure health behaviors of adolescents].

PURPOSE: The purpose was to develop a preliminary scale to measure Korean adolescents' health behaviors through a qualitative approach, to evaluate the scale psychometrically, and to develop a final scale. METHODS: Participants were 61 adolescents for qualitative interviews and 1,687 adolescents for the psychometric evaluation. Procedure included content analysis of interviews to identify health behavior categories for Korean adolescents, pre-test to confirm that preliminary scale items were understandable, content validity by an expert panel, development of the web-based computer-assisted survey (CAS), and psychometric analysis to determine reliability and validity of the final scale. RESULTS: A final scale was developed for both paper-and-pencil and CAS. It consisted of 14 health behaviors (72 items), including stress and mental health (10), sleep habits (5), dietary habits (12), weight control (4), physical activity (4), hygiene habits (5), tobacco use (5), substance use (2), alcohol consumption (4), safety (4), sexual behavior (9), computer use (3), health screening (4), and posture (1). CONCLUSION: The scale's strong points are: 1) Two thirds of the final scale items are Likert scale items, enabling calculation of a health behavior score. 2) The scale is appropriate to Korean culture. 3) The scale focuses on concrete health behaviors, not abstract concepts.

[Development of a mentoring program to improve exercise and dietary habits of adolescents].

PURPOSE: The purpose of this study was to evaluate the effects of a mentoring program to improve the exercise and dietary habits of adolescents. METHODS: A non-equivalent control group, pretest-posttest design was used. The independent variable was a mentoring program for improvement of exercise and dietary habits of adolescents, in which the mentors were nursing students and the mentees were female middle school students. The dependent variables were weekly exercise frequency, weekly exercise time, perception of exercise benefit, frequency of vegetable intake, and dietary habits. The intervention was conducted by various methods such as group education, individual approach through the mentor-mentee relationship, and multimedia approaches. RESULTS: At follow-up, the perception of the exercise benefit was significantly greater in the intervention group than in the control group. The weekly exercise frequency and frequency of vegetable intake in the intervention group were significantly greater after the intervention than those before the intervention. CONCLUSION: This mentoring program is potentially of an effective health promotion program for adolescents and will enable nursing students who participate in the program as mentors to gain confidence in their professional capability.

Exercise self-efficacy, exercise benefits and barriers, and commitment to a plan for exercise among Korean women with osteoporosis and osteoarthritis.

This study compared perceived exercise self-efficacy, exercise benefits, exercise barriers, and commitment to a plan for exercise between Korean women with a diagnosis of either osteoporosis or osteoarthritis. The influence of exercise self-efficacy, exercise benefits and barriers on commitment to a plan for exercise was also assessed in each group. Participants in the study were 154 Korean women over 40 years of age who were recruited from hospitals, health centers or a nursing home. The two groups differed on commitment to a plan for exercise with commitment greater among women with osteoporosis. Exercise self-efficacy was the most influential variable on commitment to a plan for exercise accounting for 27% of the variance in commitment among osteoporosis patients and 53% of the variance among osteoarthritis patients. This study generated information relevant to tailoring exercise interventions to the differing needs and perceptions of Korean women with osteoporosis and osteoarthritis.

[Content analysis of the experience of preceptors in clinical education for senior student nurses].

PURPOSE: The purpose of this study was to identify experiences of nurses who served as preceptors in clinical education for senior student nurses in a college of medicine in Wonju city. METHOD: Data was collected from 20 preceptors instructing senior student nurses in 2001 using a self-completion questionnaire. To analyze data, content analysis was done using an analysis scheme developed by the investigators. RESULT: The analysis scheme consisted of 7 categories and 25 subcategories. 135 significant statements were analyzed and categorized. Preceptors indicated that they were role models, socialization facilitators and educators while instructing students in the clinical practicum. In performing the preceptors' role, preceptors reported that their most important change was self-enhancement,and positive experience was a constructive work atmosphere. The most important factor facilitating the preceptors' role performance was support from head nurses, and the most discouraging factor was work loads. CONCLUSION: This study suggests that interventions for encouragement and socialization of preceptors should be developed to promote clinical education for senior student nurses.