To breed new varieties of medicinal plants with high resistance is the premise to ensure the production of high-quality medicinal materials. Molecular breeding using modern molecular biology and genetic technology can save time and effort and realize rapid and accurate breeding. Here we are trying to summarize the difference of breeding characteristics between medicinal plants and crops such as genetic background and breeding purpose. The strategy of molecular breeding of medicinal plants was summarized, and the four-phases breeding based on high-throughput sequencing and target gene mining was emphasized. We put forward the current molecular breeding of medicinal plants in the condition of four phases breeding is the optimal technological way of breeding, and gene editing breeding is the direction of medicinal plants breeding.
Breeding ; DNA Shuffling ; Gene Editing ; Plant Breeding ; Plants, Medicinal ; genetics

Genome shuffling methods were explored for Bacillus subtilis strain molecular breeding. Recycling protoplast fusion, recycling transformation and recycling universal transduction were used for genome shuffling in B. subtilis. Four strains with different nutrition-deficiency markers were used as initial strains. After five rounds protoplast fusion, transformation or transduction, the descendant with 4 markers had not been detected, and the rate of descendant with 3 markers were 4.53 x 10(-4), 1.64 x 10(-4), 4.47 x 10(-3), respectively. A computer program was made to simulate the recycling fusion process. Based on simulation result and comparing the genome shuffling result of B. subtilis in this experiment and that of Streptomyces coelicolor reported in references, effective genome shuffling needs a high recombination rate of at least between 10(-3) and 10(-2).
Bacillus subtilis ; classification ; genetics ; DNA Shuffling ; Genetic Techniques ; Genome, Bacterial ; genetics ; Protein Engineering ; Transformation, Bacterial

Cyclic amp receptor protein (CRP) is a global transcriptional factor in many prokaryotes, capable of governing nearly half of the total genes in Escherichia coli. Through the method of error-prone PCR or DNA shuffling, we can first obtain CRP mutant library and then get the expected cell phenotype with enhanced resistance. In this article, we reviewed the following desired phenotype: enhanced tolerance towards oxidative stress, improved osmotolerance, enhanced organic solvent (toluene) tolerance, improved acetate tolerance of E. coli fermentation and improved ethanol tolerance during bio-ethanol production. We then concluded that CRP can also be applied in other host cells to get desired phenotypes. Last, we predicted potential applications of mutant CRP transcriptional factor.
Cyclic AMP Receptor Protein ; biosynthesis ; DNA Shuffling ; Escherichia coli ; metabolism ; Fermentation ; Metabolic Engineering

On November 18, 2018, the Future Science Prize Awarding Ceremony was held in Beijing. In the area of life science, Professors Jiayang Li, Longping Yuan, and Qifa Zhang shared the prize for their pioneering contributions in producing high-yield, superior-quality rice through systematic study of molecular mechanisms associated with specific rice features and application of novel approaches in rice breeding. The Future Science Prize is also touted as "China's Nobel Prize", fully affirming their achievements in rice basic research and breeding.
Awards and Prizes ; China ; DNA Shuffling ; Genetic Engineering ; methods ; Oryza ; genetics ; growth & development ; Plant Breeding ; methods ; Research

'Zhizhang Guhong Chongcui' is a new cultivar of Prunus mume with cross-cultivar group characteristics. It has typical characteristics of cinnabar purple cultivar group and green calyx cultivar group. It has green calyx, white flower, and light purple xylem, but the mechanism remains unclear. In order to clarify the causes of its cross-cultivar group traits, the color phenotype, anthocyanin content and the expression levels of genes related to anthocyanin synthesis pathway of 'Zhizhang Guhong Chongcui', 'Yuxi Zhusha' and 'Yuxi Bian Lü'e' were determined. It was found that the red degree of petals, sepals and fresh xylem in branches was positively correlated with the total anthocyanin content. MYBɑ1, MYB1, and bHLH3 were the key transcription factor genes that affected the redness of the three cultivars of flowers and xylem. The transcription factors further promoted the high expression of structural genes F3'H, DFR, ANS and UFGT, thereby promoting the production of red traits. Combined with phenotype, anthocyanin content and qRT-PCR results, it was speculated that the white color of petals of 'Zhizhang Guhong Chongcui' were derived from the high expression of FLS, F3'5'H, LAR and ANR genes in other branches of cyanidin synthesis pathway, and the low expression of GST gene. The green color of sepals might be originated from the relatively low expression of F3'H, DFR and ANS genes. The red color of xylem might be derived from the high expression of ANS and UFGT genes. This study made a preliminary explanation for the characteristics of the cross-cultivar group of 'Zhizhang Guhong Chongcui', and provided a reference for molecular breeding of flower color and xylem color of Prunus mume.
Animals ; Anthocyanins ; DNA Shuffling ; Flowers/genetics* ; Porifera ; Prunus/genetics* ; Glutamine/analogs & derivatives* ; Plant Extracts

We have constructed a molecular linkage map of pepper (Capsicum spp.) in an interspecific F2 population of 107 plants with 320 RFLP, 136 AFLP, and 46 SSR markers. The resulting linkage map consists of 15 linkage groups covering 1,720 cM with an average map distance of 3.7 cM between framework markers. Most RFLP markers (80%) were pepper-derived clones and these markers were evenly distributed all over the genome. Genes for defense and biosynthesis of carotenoids and capsaicinoids were mapped on this linkage map. By using 30 primer combinations, AFLP markers were generated in the F2 population. For development of SSR markers in Capsicum, microsatellites were isolated from two small-insert genomic libraries and the GenBank database. This combined map provides a starting point for high-resolution QTL analysis, gene isolation, and molecular breeding.
Capsicum ; Carotenoids ; Chromosome Mapping* ; Clone Cells ; Databases, Nucleic Acid ; DNA Shuffling ; Genome* ; Genomic Library ; Microsatellite Repeats ; Polymorphism, Restriction Fragment Length

OBJECTIVETo develop directly molecular evolution of nitrite oxido-reductase using DNA-shuffling technique because nitrobacteria grow extremely slow and are unable to nitrify effectively inorganic nitrogen in wastewater treatment.

METHODSThe norB gene coding the ndtrite oxido-reductase in nitrobacteria was cloned and sequenced. Then, directed molecular evolution of nitrite oxido-reductase was developed by DNA-shuffling of 15 norB genes from different nitrobacteria.

RESULTSAfter DNA-shuffling with sexual PCR and staggered extension process PCR, the sequence was different from its parental DNA fragments and the homology ranged from 98% to 99%. The maximum nitrification rate of the modified bacterium of X16 by DNA-shuffling was up to 42.9 mg/L x d, which was almost 10 times higher than that of its parental bacteria. Furthermore, the modified bacterium had the same characteristics of its parental bacteria of E. coli and could grow rapidly in normal cultures.

CONCLUSIONDNA-shuffling was successfully used to engineer E. coli, which had norB gene and could degrade inorganic nitrogen effectively.

Cloning, Molecular ; DNA Shuffling ; Deltaproteobacteria ; enzymology ; genetics ; Directed Molecular Evolution ; Escherichia coli ; genetics ; Gammaproteobacteria ; enzymology ; genetics ; Nitrite Reductases ; chemistry ; genetics ; Nitrogen ; metabolism ; Phylogeny ; Sequence Analysis, DNA

Improvement of the productivity of industrial strains is an important field in micro-biology, because wild-type strains isolated from nature usually produce only a low level of antibiotics. Although random screening and simple rational screening are still effective without using genomic information, they are always time- and labor-consuming. With the broad application of recombinant DNA technology, protoplast fusion and X-omics, novel methods and strategies such as metabolic engineering, genome shuffling, system biology and system biotechnology, ribosome engineering, epigenetic modification are being exploited for the industry microbiology. In this review, we will focus on the progress of these novel methods and strategies for strain improvement in recent years.
Bacteria ; classification ; genetics ; metabolism ; Biotechnology ; trends ; DNA Shuffling ; methods ; DNA, Recombinant ; Gene Expression Regulation, Bacterial ; Genetic Engineering ; methods ; Industrial Microbiology ; methods ; Technology, Pharmaceutical

In the present study, the genome shuffling was used to improve lipase production of Penicillium expansum. A lipase producing mutant strain-Penicillium expansum FS8486 and a wild type of Aspergillus Tamarii FS-132 isolated from soil of a volcano in Xinjiang were used as the parental strains. After two rounds of genome shuffling, several elite daughter strains were screened. The lipase activity in one of the daughter strains was increased 317% over the starting strain FS8486. Comparisons of the morphology, RAPD (Random Amplification of Polymorphic DNA) polymorphism and the fatty acid compositions between the daughter and the parental strains suggested that the filial generation were generated by genome shuffling. In this study, the genome shuffling used successfully first time in eukaryotic microorganism and increases the production of the desired metabolite in short time, the study will be useful to spread the genome shuffling in eukaryotic microbial breeding.
Aspergillus ; genetics ; DNA Shuffling ; methods ; Genetic Enhancement ; methods ; Genome, Fungal ; genetics ; Lipase ; biosynthesis ; genetics ; Penicillium ; enzymology ; genetics ; Random Amplified Polymorphic DNA Technique

PURPOSE: The selective elimination of cancer stem cells (CSCs) in tumor patients is a crucial goal because CSCs cause drug refractory relapse. To improve the current conventional bispecific immune-engager platform, a 16133 bispecific natural killer (NK) cell engager (BiKE), consisting of scFvs binding FcγRIII (CD16) on NK cells and CD133 on carcinoma cells, was first synthesized and a modified interleukin (IL)-15 crosslinker capable of stimulating NK effector cells was introduced. MATERIALS AND METHODS: DNA shuffling and ligation techniques were used to assemble and synthesize the 1615133 trispecific NK cell engager (TriKE). The construct was tested for its specificity using flow cytometry, cytotoxic determinations using chromium release assays, and lytic degranulation. IL-15–mediated expansion was measured using flow-based proliferation assays. The level of interferon (IFN)-γ release was measured because of its importance in the anti-cancer response. RESULTS: 1615133 TriKE induced NK cell–mediated cytotoxicity and NK expansion far greater than that achieved with BiKE devoid of IL-15. The drug binding and induction of cytotoxic degranulation was CD133+ specific and the anti-cancer activity was improved by integrating the IL-15 cross linker. The NK cell–related cytokine release measured by IFN-γ detection was higher than that of BiKE. NK cytokine release studies showed that although the IFN-γ levels were elevated, they did not approach the levels achieved with IL-12/IL-18, indicating that release was not at the supraphysiologic level. CONCLUSION: 1615133 TriKE enhances the NK cell anti-cancer activity and provides a self-sustaining mechanism via IL-15 signaling. By improving the NK cell performance, the new TriKE represents a highly active drug against drug refractory relapse mediated by CSCs.
Antibody-Dependent Cell Cytotoxicity ; Chromium ; DNA Shuffling ; Flow Cytometry ; Humans ; Interferons ; Interleukin-15 ; Interleukins ; Killer Cells, Natural ; Ligation ; Neoplastic Stem Cells ; Recurrence ; Sensitivity and Specificity