Objective: to construct human neuromuscular disease-related gene site-specific mutant cell line by Cas9 mutation system. Methods: according to the principle of CRISPR/Cas9 target design, the exon region of CXCR4 gene sequence was found in the National Center for Biotechnology Information (NCBI) of the United States. Two sgRNAs were designed. Lenticrisprv2 was used as the vector to construct the lenticrisprv2-sgrna recombinant plasmid, which was transformed into the sensitive stbl3 strain. The monoclonal sequencing was selected to verify and expand the culture of the plasmid, then it was transferred to 293T cells for packaging to a slow virus. The virus was collected and infected with 4T1 cells. The monoclonal cells were isolated and cultured by puromycin screening and limited dilution method. The genomic DNA of the selected monoclonal cells was extracted and the DNA fragment near the knockout site was amplified by PCR and sequenced. Results: one cell line had 6 deletion mutations, including DYSF mutation site of neuromuscular disease gene and HEK293T cell model knocked out by DYSF mutation site of neuromuscular disease gene. Conclusion: the recombinant plasmid targeting CXCR4 gene was obtained by CRISPR/Cas9 system, and the human neuromuscular disease-related gene site-specific mutant cell line was successfully constructed.

Objective: To construct prokaryotic expression vector of Amuc_1100 gene, the ELP (ELSTIN-LIKE PROTEIN) is used as purification label to express fusion with the target protein and detect the biological activity of recombinant protein at cell level and animal level. Methods: the recombinant plasmid pET28a-Amuc_1100-ELP is designed and synthesized, and transformed into e. coli competent cell bl21 (de3) to induce expression. The recombinant egg was isolated and purified by the reversible phase change cycling (ITC) characteristic of ELP10 white. At the cellular level, PBMCs cells from mice were extracted to detect the expression level of IL-10. At the animal level, obese mice induced by gastric gavage with high fat detected the improvement effect of the protein on mouse metabolism. Results: The recombinant protein with the expected molecular weight of 60 kDa was expressed by E.coli expression system. After circulating through ITC, the recombinant protein with high purity was obtained. The protein increased the expression level of IL-10 in PBMCs cells, and significantly improved the obesity and metabolic disorders induced by high-fat diet in mice. Conclusion: The recombinant protein expression plasmid pET28a-Amuc_1100-ELP was successfully constructed. After expression and purification, the recombinant protein expression plasmid has high purity and has 15 biologically active recombinant protein. This study laid a foundation for the industrial production and clinical application of Amuc_1100.

To understand the function of Sly-miR393 in tomato, the precursor sequences and potential target genes of Sly-miR393 were identificated from tomato genome database by computational homology search method. The Sly-miR393 gene was amplified from the genomic DNA by PCR and cloned into plant expression vector pLP35s-100. Sly-miR393 guided-cleavage to putative target  transcripts was validated u sing 5RACE RT-PCR. In this study, our results indicated that the precursor sequence of Sly-miR393 contains the complete hairpin  structure. TIR1/AFB auxin receptor genes contain recognition sites with high complementarities to Sly-miR393 sequence. In tomato, Sly-miR393 directs the cleavage of SlTIR1，SlTIR1-like1 and SlAFB mRNA, then auxin receptor homologous was validated to be as target of Sly-miR393. The pLP35s-pre-SlymiR393 vector containing Sly-miR393 gene was successfully constructed, which would provide significant evidence for further study of Sly-miR393 function in auxin signaling pathway in tomato.

Low phosphorus in soil has become an important limit factor affecting the yield and quality of soybean. The excavation and utilization of high phosphorus efficient related genes is an important prerequisite for the analysis of high pho sphorus mechanism and the improvement of genetic breeding. In this study, the high- and low-efficiency soybean germplasms were used to analyze the root transcriptome data under two different phosphorus conditions through the weight gene co-expression network method.The results showed that there were 15305 high-expressed related genes obtained and were divided into 20 modules, and four of them showed different expressions between these two varieties under two phosphorus treatments. Further analysis results of the Melightcyan module revealed that 268 genes were found in this module, and 13 genes of them were up-regulated with low-phosphorus induction and involved in multiple metabolic pathways. Moreover, the related genes in this module which participate in the phospholipid metabolism pathways showed the most highest expression levels. Finally, combined with the previous reports, six kinds of related genes with high efficient utilization of soybean phosphorus were screened out, which could provide 

In this work we comprehensively compared the transcriptome profiles of ripe fruits (RFs) and unripe fruits (UFs) and identified the genes related to flavonoid biosynthesis in Solanum nigrum Linn. (S. nigrum). Firstly, 118198 unigenes with a N50 length of 1339 bp were de novo assembled. In addition, 527 genes that were differential expression in RFs and UFs have been identified, including 329 down-regulated genes and 198 up-regulated genes. Especially, 8 differential expression genes (DEGs) were identified to be involved in flavonoid biosynthesis. Coincidentally, most of the 8 flavonoid-related genes were highly expressed in RFs. Finally, we found that the differential expression of three genes that were related to the synthesis of dihydroflavonol-4-reductase (DFR), flavonoid 3’, 5’-hydroxylase (F35H) and anthocyanin synthase (ANS) was the main reason for differences in flavonoid biosynthesis between RFs and UFs of S. nigrum. This study provides some supporting information for the flavonoid accumulation in RFs. Moreover, this study can improve our understanding of the molecular mechanisms of flavonoid biosynthesis in S. nigrum.