Gene cloning is one of the most important and widely used technologies in molecular biology research. Generally, DNA fragment is cut with restriction enzyme, and then the product is ligated to a linearized vector with complementary sticky end or blunt end by DNA-ligase. This traditional DNA cloning method requires compatible enzyme recognition sites existing in both PCR fragment and targeted vector. Several ligase-free methods have been established to avoid the using of restriction enzyme. However, those methods are time-consuming, labor-intensive and expensive. To overcome these shortcomings, we developed an Exonuclease III based DNA cloning method that takes only 30 minutes with high cloning efficiency and significant economic advantage. Therefore, this method is suitable for large-scale gene cloning.
Cloning, Molecular ; methods ; DNA ; chemistry ; DNA Restriction Enzymes ; Exodeoxyribonucleases ; chemistry ; Genetic Vectors ; Polymerase Chain Reaction

Objective To prepare the standard molecular weight fragment mixtures. Methods Primers were designed to prepare clones which contained different sizes of standard molecular weight fragments. The template used for amplification of insert fragments was the pMD18-T vector. Bacteria culture and plasmid extraction were used to obtain abundant target fragment. Unlabeled DNA fragments were prepared by double digestion of the recombinant plasmids, and the fluorescent adaptor was prepared by annealing with two partial reverse complimentary DNA fragments. The unlabeled fragments and fluorescent adaptor were connected by DNA ligation reaction assisted with T4 DNA ligase. In this way, different sizes of standard molecular weight fragments were prepared. Standard molecular weight fragment mixture was finally prepared by mixing all the fragments together before purification. Results Ten standard molecular weight fragments of different sizes were prepared. The sizes of each fragment are 80bp, 124bp, 194bp, 224bp, 254bp, 304bp, 349bp, 399bp, 424bp and 454bp. The internal standard could accurately determine the size of PCR products amplified with the DNATyper15 kit. Conclusion Using this method, the standard molecular weight fragment mixture which meet the requirements of research and laboratory use was prepared, perfectly providing a new method for preparation of the DNA molecular weight standards. The peaks and the size of the prepared DNA internal lane standard are correct, which can be used to calculate the DNA fragments size in capillary electrophoresis.

Transcriptional regulation is one of the major regulations in plant adventious shoot regeneration, but the exact mechanism remains unclear. In our study, the RNA-seq technology based on the IlluminaHiSeq 2000 sequencing platform was used to identify differentially expressed transcription factor (TF) encoding genes during callus formation stage and adventious shoot regeneration stage between wild type and adventious shoot formation defective mutant be1-3 and during the transition from dedifferentiation to redifferentiation stage in wildtype WS. Results show that 155 TFs were differentially expressed between be1-3 mutant and wild type during callus formation, of which 97 genes were up-regulated, and 58 genes were down-regulated; and that 68 genes were differentially expressed during redifferentiation stage, with 40 genes up-regulated and 28 genes down-regulated; whereas at the transition stage from dedifferentiation to redifferention in WS wild type explants, a total of 231 differentially expressed TF genes were identified, including 160 up-regualted genes and 71 down-regulated genes. Among these TF genes, the adventious shoot related transcription factor 1 (ART1) gene encoding a MYB-related (v-myb avian myeloblastosis viral oncogene homolog) TF, was up-regulated 3 217 folds, and was the highest up-regulated gene during be1-3 callus formation. Over expression of the ART1 gene caused defects in callus formation and shoot regeneration and inhibited seedling growth, indicating that the ART1 gene is a negative regulator of callus formation and shoot regeneration. This work not only enriches our knowledge about the transcriptional regulation mechanism of adventious shoot regeneration, but also provides valuable information on candidate TF genes associated with adventious shoot regeneration for future research.
Arabidopsis ; growth & development ; Arabidopsis Proteins ; physiology ; Gene Expression Regulation, Plant ; Genes, Plant ; Plant Shoots ; growth & development ; RNA ; Regeneration ; Seedlings ; growth & development ; Transcription Factors ; physiology ; Up-Regulation

Objective This study aimed to explore clinical characteristics of four types of obesity based on metabolic classification. Methods Forty-eight obese patients were divided according to their clinical characteristics into 4 groups including metabolic healthy obesity (MHO), hypometabolic obesity (LMO), hypermetabolic obesity (HMO), and metabolic obesity with inflammation (IMO). 20 normal weight individuals were also recruited as a control group. Body fat, body weight, visceral index, and basal metabolism were measured by Omron body fat meter. Fat content and its distribution were measured by dual energy X-ray absorptiometry. All participating patients underwent various tests for 75 g oral glucose tolerance, blood glucose, insulin, C peptide. Lipid profile, thyroid function and sex hormones levels, and inflammation factors were also measured. Results (1)Patients in MHO group had higher body fat content, but had no metabolic disorder and inflammation. Their hormones levels were normal. (2) Lower metabolic rate and lower hormones levels were found in the patients in LMO group with increasing visceral fat. Trunk/subcutaneous fat mass was significantly higher than that in MHO group(1. 19 ± 0. 25 vs 0. 97 ± 0. 32, P<0. 05). There were abnormal lipid and glucose metabolism in LMO group. The insulin action index was significantly lower than that in MHO group(0. 006 6 ± 0. 002 7 vs 0. 012 1 ± 0. 009 5, P<0. 05). The area under the curve of glucoseconcentrationwassignificantlyhigherinLMOgroupthanthatinMHOgroup[(18.71±8.68vs12.70±4.63) mmol/L, P<0. 05]. (3)Heart rate and blood pressure were higher in HMO group. The heart rate was significantly increased compared with that in MHO group [(90. 50 ± 8. 24 vs 73. 20 ± 14. 11) beat/min, P<0. 05]. The waist circumference was significantly larger than that in MHO group [(111. 88 ± 10. 54 vs 98. 05 ± 15. 56) cm, P<0. 05]. (4) In IMO group, insulin action index was significantly lower than MHO group (0. 007 0 ± 0. 003 3 vs 0.0121±0.0095,P<0.05). ThetrunkfatmassanduricacidlevelsweresignificantlyhigherthanMHOgroup [(17236.38±4610.60vs15816.10±5453.42)gand(468.28±121.32vs376.84±97.14) μmol/L,bothP<0. 05]. Patients in IMO group had acanthosis nigricans, but their glucose level was relatively normal. Conclusion The metabolic-based obese diagnosis is essential for understanding the obesity etiology and providing individualized treatment.

An enantioselective method was developed for the separation and determination of three chiral hexabromocyclododecanes ( HBCDs ) including α-HBCD, β-HBCD, γ-HBCD in soil and earthworm by HPLC-ID-MS/MS. d18-HBCDs used as internal standards were added to the samples before extraction. HBCDs enantiomers were extracted from soil by accelerated solvent extraction ( ASE ) with n-hexane/DCM (1:1,V/V) at 100℃ and 10 MPa for 5 min, and further cleaned up using silica column. HBCDs enantiomers were extracted from earthworm by vortex turbulence with ethyl acetate. The extracts were orderly sulphonated by sulfuric acid, and purified by silica column. For all HBCDs enantiomers, good linearities were obtained in the concentration range of 0. 25-50 ng/mL. Limits of detection ( LOD) and limits of quantification ( LOQ) were 0. 00544-0. 00766 ng/g and 0. 0173-0. 0244 ng/g, respectively in soil. The recoveries of spiked samples at 0. 05 and 2. 5 ng/g levels were 80. 0%-95. 9% with relative standard deviations ( RSD, %) of 5. 7%-11. 9% in soil. Limits of detection (LOD) and limits of quantification (LOQ) were 0. 0103-0. 0148 ng/g and 0. 0328-0. 0471 ng/g, respectively in earthworm. The recoveries of spiked samples at 0. 1 and 5 ng/g levels were 78. 0% -94. 4% with relative standard deviations ( RSD, %) of 6. 1% -12. 2% in earthworm. This method can meet the requirements of determination of trace HBCDs in soil and earthworm.

Objective To construct a rapid genetic DNA extraction method, with nano magnetic beads, self-designed reagents system and extracting process. Method Part I: DNA extraction from old blood cotton swab sample with self-designed DNA extraction kit, then quantiifed by UV spectrophotometer. The method was further optimized on the preliminary results. Part II: All kinds of difficult DNA sample were tested with optimized kit, to detect the applicability of the kit. Result By improving the experimental condition, the extraction effects of different DNA sample is good, meanwhile, the extraction cost is relatively low.

The somatic embryogenesis in plant is a very complicated and highly ordered process, which is regulated by many internal and external factors. Among them, gene expression and regulation are key factors. Genes encoding regulatory proteins, for example PLANT GROWTH ACTIVATOR, LEAFY COTYLEDON, BABY BOOM, SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE and PICKLE, interact with each other and form a complicated regulatory network. Recent progress on this regulatory network was reviewed on the basis of our study on the PLANT GROWTH ACTIVATOR 37 gene. In addition, future research perspectives on plant somatic embryogenesis were discussed.
Arabidopsis Proteins ; genetics ; Gene Expression Regulation, Developmental ; Gene Expression Regulation, Plant ; Plant Development ; Plant Growth Regulators ; genetics ; Plant Somatic Embryogenesis Techniques ; Plants ; genetics ; Transcription Factors ; genetics

WRKY transcription factors, one of the largest families of transcriptional regulators in plants, involve in multiple life activities including plant growth and development as well as stress responses. However, little is known about the types and functions of WRKY transcription factors in Catharanthus roseus, an important medicinal plant. In this study, we identified 47 CrWRKY transcriptional factors from 26 009 proteins in Catharanthus roseus, and classified them into three distinct groups (G1, G2 and G3) according to the structure of WRKY domain and evolution of the protein family. The expression profiling showed that these CrWRKY genes expressed in a tissue/organ specific manner. The 47 CrWRKY genes were clustered into three types of expression patterns. The first type includes the CrWRKYs highly expressed in flowers and the protoplast treated with methy jasmonate (MeJA) or yeast extraction (YE). The second type contains the CrWRKYs highly expressed in stem and hairy root. The third type represents the CrWRKYs highly expressed in root, stem, leaf, seedling and the hairy root treated by MeJA. Real time quantitative PCR was employed to further identify the expression patterns of the 16 selected CrWRKY genes in various organs, the MeJA-treated protoplasts and hairy roots of Catharanthus roseus, and similar results were obtained. Notably, the expresion of more than 1/3 CrWRKY genes were regulated by MeJA or YE, indicating that these CrWRKYs are likely involed in the signalling webs which modulate the biosynthesis of terpenoid indole alkaloid and plant responses to various stresses. The present results provide a framework for functional identification of the CrWRKYs and understanding of the regulation network of terpenoid indole alkaloid biosynthesis in Catharanthus roseus.
Amino Acid Sequence ; Catharanthus ; genetics ; metabolism ; Gene Expression Regulation, Plant ; Genes, Plant ; Molecular Sequence Data ; Plant Proteins ; biosynthesis ; genetics ; Plants, Medicinal ; genetics ; metabolism ; Transcription Factors ; biosynthesis ; genetics

Most current research in the field of adventitious shoot formation is focused on the regulatory function of a single gene. However, a systematic transcriptomic analysis of the early adventitious shoot formation is still lacking. Here, we analyzed the transcriptome profiling of the early adventitious shoot formation in Arabidopsis by RNA-seq high throughput sequencing technology, and identified 2 457 differentially expressed genes. Detailed categorization revealed that these genes were mainly involved in hormone homeostasis or signal transduction, callus and lateral root formation, shoot apical meristem development and photosynthesis. Further pathway enrichment analysis showed that genes involved in phenylalanine metabolism and phenylpropanoid biosynthesis were significantly enriched. Moreover, exogenous phenylalanine could repress adventitious shoot formation, indicating that phenylalanine metabolism and phenylpropanoid biosynthesis might be important for adventitious shoot formation.
Arabidopsis ; genetics ; Gene Expression Profiling ; methods ; Gene Expression Regulation ; Genes, Plant ; Phenylalanine ; pharmacology ; Plant Shoots ; genetics ; growth & development

Objective To develop and validate a model based on deep learning for automatic diagnosis of early gastric cancer ( EGC) to improve detection and diagnosis of EGC. Methods A total of 5159 images ( including 1000 images of EGC and 4159 images of other benign lesions or normal patients) obtained from May 2014 to December 2016 were collected from endoscopic database in changhai Hospital. Then 4449 images were selected randomly for a deep convolutional neural network ( CNN ) training, of which 768 were diagnosed as EGC and 3681 diagnosed as other benign lesions or normal. The remaining 710 images were used to test the model by comparing with diagnostic results of four endoscopists. Results The deep learning model showed accuracy of 89. 4％ ( 635/710 ) , sensitivity of 88. 8％ ( 206/232 ) and specificity of 89. 7％ ( 429/478) for EGC. The mean time required for diagnosis was 0. 30 ± 0. 02 s. The performance of the model was superior to that of four endoscopists. Conclusion The model based on deep learning has high accuracy,sensitivity and specificity for detecting EGC,which could assist endoscopists in real-time diagnosis.