The Application of Nicotiana benthamiana as a Transient Expression Host to Clone the Coding Sequences of Plant Genes

Coding sequences (CDS) are commonly used for transient gene expression, in yeast two-hybrid screening, to verify protein interactions and in prokaryotic gene expression studies. CDS are most commonly obtained using complementary DNA (cDNA) derived from messenger RNA (mRNA) extracted from plant tissues and generated by reverse transcription. However, some CDS are difficult to acquire through this process as they are expressed at extremely low levels or have specific spatial and/or temporal expression patterns in vivo. These challenges require the development of alternative CDS cloning technologies. In this study, we found that the genomic intron-containing gene coding sequences (gDNA) from Arabidopsis thaliana, Oryza sativa, Brassica napus, and Glycine max can be correctly transcribed and spliced into mRNA in Nicotiana benthamiana. In contrast, gDNAs from Triticum aestivum and Sorghum bicolor did not function correctly. In transient expression experiments, the target DNA sequence is driven by a constitutive promoter. Theoretically, a sufficient amount of mRNA can be extracted from the N. benthamiana leaves, making it conducive to the cloning of CDS target genes. Our data demonstrate that N. benthamiana can be used as an effective host for the cloning CDS of plant genes.

Pig pangenome graph reveals functional features of non‑reference sequences

Background The reliance on a solitary linear reference genome has imposed a significant constraint on our compre-hensive understanding of genetic variation in animals.This constraint is particularly pronounced for non-reference sequences(NRSs),which have not been extensively studied.Results In this study,we constructed a pig pangenome graph using 21 pig assemblies and identified 23,831 NRSs with a total length of 105 Mb.Our findings revealed that NRSs were more prevalent in breeds exhibiting greater genetic divergence from the reference genome.Furthermore,we observed that NRSs were rarely found within coding sequences,while NRS insertions were enriched in immune-related Gene Ontology terms.Notably,our investigation also unveiled a close association between novel genes and the immune capacity of pigs.We observed substantial differences in terms of frequencies of NRSs between Eastern and Western pigs,and the heat-resistant pigs exhibited a substantial number of NRS insertions in an 11.6 Mb interval on chromosome X.Additionally,we discovered a 665 bp insertion in the fourth intron of the TNFRSF19 gene that may be associated with the ability of heat tolerance in South-ern Chinese pigs.Conclusions Our findings demonstrate the potential of a graph genome approach to reveal important functional features of NRSs in pig populations.

Phylogenetic, phylogeographic and divergence time analysis of Anopheles subpictus species complex using ITS2 and COI sequences

Objective:To address the phylogenetic and phylogeographic relationship between different lineages of Anopheles(An.)subpictus species complex in most parts of the Asian continent by maximum utilization of Internal Transcriber Spacer 2(ITS2)and cytochrome C oxidase I(COI)sequences deposited at the GenBank.Methods:Seventy-five ITS2,210 COI and 26 concatenated sequences available in the NCBI database were used.Phylogenetic analysis was performed using Bayesian likelihood trees,whereas median-joining haplotype networks and time-scale divergence trees were generated for phylogeographic analysis.Genetic diversity indices and genetic differentiation were also calculated.Results:Two genetically divergent molecular forms of An.subpictus species complex corresponding to sibling species A and B are established.Species A evolved around 37-82 million years ago in Sri Lanka,India,and the Netherlands,and species B evolved around 22-79 million years ago in Sri Lanka,India,and Myanmar.Vietnam,Thailand,and Cambodia have two molecular forms:one is phylogenetically similar to species B.Other forms differ from species A and B and evolved recently in the above mentioned countries,Indonesia and the Philippines.Genetic subdivision among Sri Lanka,India,and the Netherlands is almost absent.A substantial genetic differentiation was obtained for some populations due to isolation by large geographical distances.Genetic diversity indices reveal the presence of a long-established stable mosquito population,at mutation-drift equilibrium,regardless of population fluctuations.Conclusions:An.subpictus species complex consists of more than two genetically divergent molecular forms.Species A is highly divergent from the rest.Sri Lanka and India contain only species A and B.

On power series statistical convergence and new uniform integrability of double sequences

In the present paper,we mostly focus on P_(p)^(2)-statistical convergence.We will look into the uniform integrability via the power series method and its characterizations for double sequences.Also,the notions of P_(p)^(2)-statistically Cauchy sequence,P_(p)^(2)-statistical boundedness and core for double sequences will be described in addition to these findings.

Secure Transmission of Compressed Medical Image Sequences on Communication Networks Using Motion Vector Watermarking

Medical imaging plays a key role within modern hospital management systems for diagnostic purposes.Compression methodologies are extensively employed to mitigate storage demands and enhance transmission speed,all while upholding image quality.Moreover,an increasing number of hospitals are embracing cloud computing for patient data storage,necessitating meticulous scrutiny of server security and privacy protocols.Nevertheless,considering the widespread availability of multimedia tools,the preservation of digital data integrity surpasses the significance of compression alone.In response to this concern,we propose a secure storage and transmission solution for compressed medical image sequences,such as ultrasound images,utilizing a motion vector watermarking scheme.The watermark is generated employing an error-correcting code known as Bose-Chaudhuri-Hocquenghem(BCH)and is subsequently embedded into the compressed sequence via block-based motion vectors.In the process of watermark embedding,motion vectors are selected based on their magnitude and phase angle.When embedding watermarks,no specific spatial area,such as a region of interest(ROI),is used in the images.The embedding of watermark bits is dependent on motion vectors.Although reversible watermarking allows the restoration of the original image sequences,we use the irreversible watermarking method.The reason for this is that the use of reversible watermarks may impede the claims of ownership and legal rights.The restoration of original data or images may call into question ownership or other legal claims.The peak signal-to-noise ratio(PSNR)and structural similarity index(SSIM)serve as metrics for evaluating the watermarked image quality.Across all images,the PSNR value exceeds 46 dB,and the SSIM value exceeds 0.92.Experimental results substantiate the efficacy of the proposed technique in preserving data integrity.

Molecular Phylogeny of the Lardizabalaceae Based on TrnL-F Sequences and Combined Chloroplast Data

The molecular phylogeny of the Lardizabalaceae is reconstructed based on chloroplast trn L_F sequences alone and combined trn L_F and rbc L sequences. The phylogenetic topologies agree well with Qin's and Takhtajan's tribal classification in both analyses. Decaisneae and Sinofranchetieae are basal clades in the phylogenetic trees and external to all other taxa in the family. Lardizabaleae consisting of Boquila and Lardizabala are well supported in both trn L_F (100%) analysis and trn L_F and rbc L combined analysis (99%). Tribe Akebieae are strongly supported by a bootstrap value of 100% in both trn L_F analysis and trn L_F and rbc L combined analysis. However, the new genus Archakebia is nested within the genus Akebia in the trn L_F trees. In the combined trees, Archakebia is sister to Akebia with high bootstrap support. The inter_relationships among three closely related genera Parvatia , Holboellia and Stauntonia are still problematic. P. brunoniana ssp. elliptica is sister to H. latifolia in both analyses with low bootstrap support. H. parviflora is nested within the Stauntonia and sister to S. cavalerieana . Therefore, these three genera of tribe Akebieae may not be monophylytic and their generic boundary and delimitation need to be further studied, by exploring more molecular data, together with more morphological characters.

ITS1 SEQUENCES OF NUCLEAR RIBOSOMAL DNA IN WILD RICES AND CULTIVATED RICES OF CHINA AND THEIR PHYLOGENETIC IMPLICATIONS

The first internal transcribed spacer(ITS1) of nuclear ribosomal DNA of three wild rice species and two subspecies of cultivated rice, which are distributed in China, was amplified using PCR technique and sequenced with automated fluorescent sequencing. The sequences of ITS1 ranged from 193 bp to 218 bp in size and G/C content varied from 69.3% to 72.7%. In pairwise comparisons among the five taxa, sequence site divergence ranged from 1.5% to 10.6%. Phylogenetic analysis of ITS1 sequences using Wagner parsimony generated a single well resolved tree, which revealed that Oryza rufipogon was much more closely related to cultivated rice species than to the other two wild species. Oryza granulata was less closely related to either cultivated rice species or the other two wild species, and might be a unique and isolated taxon in the genus Oryza. The phylogenetic relationships of the three wild rice species and two cultivated rice subspecies inferred from ITS1 sequences is highly concordant with those based on the molecular evidence from isozyme, chloroplast DNA (cpDNA), mitochondrial DNA(mtDNA) and nuclear DNA (nDNA) of the genus Oryza .

Characterization of Two Groups of Low_copy and Specific DNA Sequences Isolated from Chromosome 7B of Common Wheat

Recent work revealed that, in the genomes of polyploid wheat, there exists a class of low_copy and chromosome_specific sequences that are labile upon polyploid formation. This class of sequences was proposed to play a critical role in the stabilization and establishment of nascent plant polyploids as new species. To further study this issue, five wheat chromosome 7B_specific sequences, isolated from common wheat (Triticum aestivum L.) by chromosome microdissection, were characterized. The sequences were studied by genomic Southern hybridizations on a collection of polyploid wheats and their diploid progenitors. Four sequences hybridized to all polyploid species, but at the diploid level to only species closely related to the B_genome of polyploid wheat. This indicates that these sequences originated with the divergence of the diploid species, and was then vertically transmitted to polyploids. One sequence hybridized to all species at both the diploid and polyploid levels, suggesting its elimination after the polyploid wheat formation. The hybridization of this sequence to two synthetic polyploid wheats indicated that sequence elimination is a rapid event and probably related to methylation status of the sequence. Based on the above results, we suggest that selective changes of low_copy sequences occur rapidly after polyploid formation, which may contribute to the differentiation of chromosomes in newly formed allopolyploid wheats.

Isolation of Zizania latifolia Species-specific DNA Sequences and Their Utility in Identification of Z. latifolia DNA Introgressed into Rice

根据两个植物抗病基因N和RPS2中核酸结合位点 (NBS)和富亮氨酸重复区 (LRR)中的保守序列设计了一对特异引物 ,用PCR从具有水稻 (OryzasativaL .)改良所需要的许多优良性状的水稻近缘野生种菰 (Zizanialatifolia(Griseb .)Turcz.exStapf)的基因组DNA中扩增同源片段。PCR产物经克隆后 ,分别以菰和水稻的基因组DNA为探针 ,通过点杂交对所得克隆进行了分析。点杂交结果表明 ,在所分析的 6 0个克隆中有 2个克隆是菰专化的序列 ,即它们与水稻无杂交信号。基因组DNA的Southern杂交进一步证实了这 2个克隆的专化性。为了验证一些可能的“水稻_菰”渐渗杂交系是否确实含有源自供体菰的DNA ,以这 2个克隆为探针 ,与经EcoRⅠ酶切的 5个可能的渐渗杂交系进行了Southern杂交。结果表明 ,这 2个克隆均能检测出其中的一个系含有其同源序列。这一结果为曾经报道的经一种非常规有性杂交方法将菰DNA导入水稻提供了确凿的证据。

MINER RULE CONSIDERING FUZZINESS OF STRESS DAMAGE UNDER DIFFERENT LOAD SEQUENCES

Since the traditional Miner rule ignores the influence of the load sequence on the fatigue life, the fuzzy rules are used to analyze the fuzziness of the fatigue damage caused by the stress nearby the fatigue limit under different load sequences. The improved fuzzy Miner rule can reflect the influence of the load sequence on the fatigue life. Results of the example show that the prediction error can be reduced from 61.6% to 21.7%.

Classification and Identification of Marine Penicillium Species Based on ITS Sequences of rDNA

[ Objective ] The aim of this study is to identify 6 marine fungi species by analyzing ITS nucleotide sequence, which had been primarily identified as penicillium based on morphological characteristics. [ Method ] The ITS regions of these species were cloned by molecule biology method and phylogenetic analyzed using ClustalX1.83 software. [ Result] The ITS regions of these species were sequenced, and phylogenetic analysis between the yield sequences and the ITS sequences assessed in GenBank showed that the 6 strains all belonged to penicillium. [ Condusion] The present study suggests ITS sequence analysis could not be used as an only proof, but it is a very useful supplementary tool for the classification and identification of marine peniciUium combined with morphological characteristics.

Phylogenetic Relationships of Caragana(Fabaceae) by the Use of nrITS Sequences

[Objective] The aim was to explore the phylogenetic relationships of Cara- gana （Fabaceae） by the use of nrlTS sequences. [Method] Internal transcribed spac- er （ITS） sequences of nuclear ribosomal DNA （nrDNA） from 29 taxa of Caragana species and seven close relatives （all belong to Astralinae （Adens） Benth） were used for phylogenetic analysis. [Result] Length of the entire ITS region ranges from 611 to 614 bp in Caragana species. The aligned sequences nrlTS of Caragana are 655 bp, and 170 sites are variable, with 107 phylogenetically informative sites. The phylogenetically informative sites are 16.3% of the total aligned sequences. The ITS sequences data are useful to resolve some relationships at lower taxonomic levels within Caragana. The Caragana Fabr. is not a monophyletic group with very close connection with Calophaca tianschanica. The ITS data revealed that the species of Sect. tragacanthoides were dispersed in MP tree or ME tree. Although the morphol- ogy of C. ordosica is similar to C. tibetica, the ITS results revealed an unexpectedly close relativeship to C. roborovskyi. The ITS data also indicate C. davazamcii, C. korshinskii, C. intermedia, and C. microphylla are different species. [Conclusion] ITS sequences have an important reference value in exploring the relationships of Cara- gana.

SECURITY ANALYSIS BASED ON OVERSAMPLED CHAOTIC BINARY SEQUENCES

Some chaotic sequences generated from maps conjugated with tent map can be reconstructed precisely by short sequence prediction,since the problem of finite precision degrades the statistical properties of chaotic maps.This paper discusses the topological conjugation properties of the chaotic maps;analyzes in detail the reconstruction process of the chaotic sequences and the security of the oversampled chaotic map (OSCM) binary sequences.The result demonstrates that OSCM binary sequences can sustain the attack of the short sequence prediction,that is,they cant be reconstructed.Furthermore,all attack strategies aiming at the chaotic systems based on synchronization will fail,meaning that they are secure.

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.

Isolation of the Flanking Sequences Adjacent to Transgenic T-DNA in Brassica napus Genome by an Improved Inverse PCR Method

[Objective] The research aimed to isolate flanking sequences adjacent to the transgenic T-DNA in Brassica napus by an improved inverse PCR method.[Method] Using single clone of transgenic FS4 in Brassica napus as the research materials,total DNA was extracted from transgenic Brassica napus by using modified CTAB method.After enzyme digestion and purification,self-joining was made.Two circles of nested PCR and the sequence alignment were carried out.[Result] A fragement with the size of 4.0 kb was amplified ...

Microdissection of Haynaldia villosa Telosome 6VS and Cloning of Species-specific DNA Sequences

The material T240_6 derived from SC 2 young embryo of the combination CA9211/RW15 (6D/6V alien substitution) was telosomic substitution line of 6VS identified by GISH (genomic in situ hybridization) analysis. The 6VS was microdissected with a needle and transferred into a 0.5 mL Ep tube. In the 'single tube', all the subsequence steps were conducted. After two round of LA (Linker adaptor)_PCR amplification, the size of PCR bands ranged from 100 to 3 000 bp, with predominate bands 600-1 500 bp. The products were confirmed by Southern blotting analysis using Haynaldia villosa (L.) Schur. genomic DNA labeled with 32 P as probe. The PCR products were purified and ligated into clone vector-pGEM_T easy vector. Then, the plasmids were transformed into competence E. coli JM109 with cool CaCl 2. It was estimated that there were more than 17 000 white clones in the library. The size of insert fragments distributed from 100-1 500 bp, with average of 600 bp. Using H. villosa genomic DNA as probe, dot blotting results showed that 37% clones displayed strong and medium positive signals, and 63% clones had faint or no signals. It is demonstrated that there were about 37% repeat sequence clones and 67% single/unique sequence clones in the library. Eight H. villosa_specific clones were screened from the library, and two clones pHVMK22 and pHVMK134 were used for RFLP analysis and sequencing. Both of them were H. villosa specific clones. The pHVMK22 was a unique sequence clone, and the pHVMK134 was a repeat sequence clone. When the pHVMK22 was used as a probe for Southern hybridization, all the powdery mildew resistance materials showed a special band of 2 kb, while all the susceptible ones not. The pHVMK22 may be applied to detect the existence of Pm21.

Preliminary Study on the ITS Sequences of nrDNA from Rhodiola alsia

[Objective] The study aimed to analyze the ITS sequences of nrDNA from Rhodiola alisa and investigate the difference of evolution rate between nrDNA and trnS-trnG and rpl20-rps12 sequences of cpDNA(chloroplast DNA).[Method]Total DNA was extracted from silica-dried leaves of R.alsia by using modified CTAB method.With the extracted DNA sample as template,nrDNA ITS region was amplified,then purified and sequenced.In addition,the yielded ITS sequences were also compared with the known trnS-trnG and rpl20-rps12 sequences of cpDNA from R.alsia.[Result]The ITS sequence of nrDNA from R.alsia was 701 bp in length,of which 13 variable sites were found with a percentage of 1.85%.Of the 13 variable sites,8 were caused by point mutations,5 were the results of insertions or deletions.The(A+T)content and(G+C)content were 46.9% and 53.1%,respectively.The nucleotide diversity(π)was 0.004 27.[Conclusion]The ITS region of nrDNA from R.alsia was more conservative and evolved more slowly than the trnS-trnG and rpl20-rps12 sequences of its cpDNA.

Characteristic atom sequences of Nb-Mo alloys system in BCC structure and properties of disordered alloys

Comprehensively considering energy, volume and electronic structure of alloys, the ninth equation was determined as the interaction function of Nb-Mo alloys system in BCC structure on the basis of idea of systematic science of alloys, experimental lattice constants and heats of formation of disordered Nb（1-x）Mox alloys. The structural parameters and properties of Nb and Mo characteristic atoms sequences and corresponding characteristic crystals sequences were determined in Nb-Mo alloys system. The electronic structure and physical properties of disordered Nb（1-x）Mox alloys system were calculated according to concentration of characteristic atoms of disordered alloys. The change trend of physical properties is the same as that of electronic structure.

Auto-selection order of Markov chain for background sequences with chi-square test

Modeling non coding background sequences appropriately is important for the detection of regulatory elements from DNA sequences. Based on the chi square statistic test, some explanations about why to choose higher order Markov chain model and how to automatically select the proper order are given in this paper. The chi square test is first run on synthetic data sets to show that it can efficiently find the proper order of Markov chain. Using chi square test, distinct higher order context dependences inherent in ten sets of sequences of yeast S.cerevisiae from other literature have been found. So the Markov chain with higher order would be more suitable for modeling the non coding background sequences than an independent model.

Sequencing and Analysis of ITS Sequences of Bangia atropurpurea

[Object]To providing new data for phylogenesis of Bangia atropurpurea by the sequencing of internal transcribed spacer（ITS region）of rDNA.[Method]B.atropurpurea was collected from Niangziguan Spring in Shanxi,the DNA was extracted and the primers were designed for PCR amplification so as to obtain ITS gene sequence.[Result]The homology of ITS region between B.atropurpurea and Porphyra oligospermatangia was 75%,which was 79% between B.atropurpurea and P.yezoensis.[Conclusion]Compared with other genes,the ITS sequence had a greater evolution rate.The differences in classification and distribution resulted in the sequence diversity.