Cloning and Expression of Conserved Sequences of cry Gene in E.coli Strain Rosetta(DE3)

[Objective] The aim was to clone the conserved sequences of cry gene and express them in Rosetta （DE3）. [Method] Specific primers were designed according to NCBI database information and the conserved sequences of cry gene were amplified by PCR from Bt transgenic cotton. Then recombinant plasmids were constructed and expressed in E. coil strain Rosetta （DE3）. Finally, the effects of different concentrations and inducing time of IPTG on the expression level of protein were investigated. [Result] Two conserved sequences （304 and 853 bp respectively） of cry gene were amplified. The result of SDS-PAGE confirmed that the recombinant plasmids pGEX-4t-I-304 and pGEX-4t-1-853 could express fusion proteins by IPTG induction and the molecular weight of protein products was 39 and 62.4 kDa respectively, which was in accordance with predicted result. The optimal protein ex- pression conditions were confirmed as induction with 0.15 mmol/L IPTG for 7 h. [Conclusion] This study prepared the ground for the further detection of Bt transgenic crops.

Infinite Sequence of Conservation Laws and Analytic Solutions for a Generalized Variable-Coefficient Fifth-Order Korteweg-de Vries Equation in Fluids

In this paper, an infinite sequence of conservation laws for a generalized variable-coefficient fifth-order Korteweg-de Vries equation in fluids are constructed based on the Backlund transformation. Hirota bilinear form and symbolic computation are applied to obtain three kinds of solutions. Variable coefficients can affect the conserved density, associated flux, and appearance of the characteristic lines. Effects of the wave number on the soliton structures are also discussed and types of soliton structures, e.g., the double-periodic soliton, parallel soliton and soliton complexes, are presented.

Comparative Analysis of Mitochondrial Control Region Sequence from Three Flatfish Species(Pleuronectidae)

The 5’-end of the mitochondrial control region sequences of three flatfishes (Pleuronectiformes: Pleuronectidae) were amplified and sequenced. These sequences were compared with those of other three Pleuronectids species retrieved from GenBank. A phylogenetic tree was constructed based on the partial control region sequences. The results of phyloge- netic analysis are consistent with those of conventional systematics. Compared to previous studies, the structure of the 5’-end of mitochondrial control region was analyzed. The terminal associated sequence motif and its complementary motif were i- dentified at the 5’-end of the sequences. A conserved sequence block, named as CM5’d, was identified in the 5’-end of con- trol region sequences in all Pleuronectids. Another central conserved sequence block, named as CSB-F, was detected in the central conserved blocks.

STAG-CNS： An Order-Aware Conserved Noncoding Sequences Discovery Tool for Arbitrary Numbers of Species

One method for identifying noncoding regulatory regions of a genome is to quantify rates of divergence between related species, as functional sequence will generally diverge more slowly. Most approaches to identifying these conserved noncoding sequences （CNSs） based on alignment have had relatively large minimum sequence lengths （≥15 bp） compared with the average length of known transcription factor binding sites. To circumvent this constraint, STAG-CNS that can simultaneously integrate the data from the promoters of conserved orthologous genes in three or more species was developed. Using the data from up to six grass species made it possible to identify conserved sequences as short as 9 bp with false discovery rate ≤0.05. These CNSs exhibit greater overlap with open chromatin regions identified using DNase I hypersensitivity assays, and are enriched in the promoters of genes involved in transcriptional regulation. STAG-CNS was further employed to characterize loss of conserved noncoding sequences associated with retained duplicate genes from the ancient maize polyploidy. Genes with fewer retained CNSs show lower overall expression, although this bias is more apparent in samples of complex organ systems containing many cell types, suggesting that CNS loss may correspond to a reduced number of expression contexts rather than lower expression levels across the entire ancestral expression domain.

Researches on Sequence of Plant Cystatin:Phytocystatin

Plant cystatins or phytocystatins are cysteine proteinase inhibitors exist widely in different plant species. Because they can kill insects by inhibiting the digestive function of the cysteine proteinase in gut, they are believed to play an important role in plant’s defense against pests. Phytocystatins contain the conserved QXVXG motif and show some features on their sequence different to animal cystatins. After sequencing the protein directly and the cDNA clone, a large number of plant cystatins have been characterized. A multialignment with BLAST software and a detail analysis of 38 phytocystatins show that phytocystatins possess a specific conserved amino acid sequence [LRVI]-[AGT]-[RQKE]-[FY]- [AS]-[VI]-X-[EGHDQV]-[HYFQ]-N different to the conserved sequence demonstrated by Margis in 1998. This conserved sequence can be enough to detect with exclusivity phytocystatin sequences on protein data banks. A classification of these phytocystatins is performed and they can be divided into 3 groups according to their features on amino acid sequence, and the group-I can be still divided into 3 subgroups based on the feature of their amino acid and genomic sequence. By the CLUSTALX software, the most conserved nucleotide sequences of phytocystatins were found, which could be used to design the degenerate premiers to search new phytocystatins with PCR reaction.

Conserved noncoding sequences correlate with distant gene contacts in Arabidopsis and Brassica

Physical contact between genes distant on chromosomes is a potentially important way for genes to coordinate their expressions.To investigate the potential importance of distant contacts,we performed high-throughput chromatin conformation capture(Hi-C)experiments on leaf nuclei isolated from Brassica rapa and Brassica oleracea.We then combined our results with published Hi-C data from Arabidopsis thaliana.We found that distant genes come into physical contact and do so preferentially between the proximal promoter of one gene and the downstream region of another gene.Genes with higher numbers of conserved noncoding sequences(CNSs)nearby were more likely to have contact with distant genes.With more CNSs came higher numbers of transcription factor binding sites and more histone modifications associated with the activity.In addition,for the genes we studied,distant contacting genes with CNSs were more likely to be transcriptionally coordinated.These observations suggest that CNSs may enrich active histone modifications and recruit transcription factors,correlating with distant contacts to ensure coordinated expression.This study advances our knowledge of gene contacts and provides insights into the relationship between CNSs and distant gene contacts in plants.

Systematic annotation of conservation states provides insights into regulatory regions in rice

Plant genomes contain a large fraction of noncoding sequences.The discovery and annotation of conserved noncoding sequences(CNSs)in plants is an ongoing challenge.Here we report the application of comparative genomics to systematically identify CNSs in 50 well-annotated Gramineae genomes using rice(Oryza sativa)as the reference.We conduct multiple-way whole-genome alignments to the rice genome.The rice genome is annotated as 20 conservation states(CSs)at single-nucleotide resolution using a multivariate hidden Markov model(Cons HMM)based on the multiple-genome alignments.Different states show distinct enrichments for various genomic features,and the conservation scores of CSs are highly correlated with the level of associated chromatin accessibility.We find that at least 33.5%of the rice genome is highly under selection,with more than 70%of the sequence lying outside of coding regions.A catalog of 855,366 regulatory CNSs is generated,and they significantly overlapped with putative active regulatory elements such as promoters,enhancers,and transcription factor binding sites.Collectively,our study provides a resource for elucidating functional noncoding regions of the rice genome and an evolutionary aspect of regulatory sequences in higher plants.