In Silico Cloning and Sequence Analysis of Phospholipase Dα Gene from Peach Fruit

Phospholipase D （PLD, EC 3.1.4.4） plays an important role in adaptive response of postharvest fruit to environment. In this study, a novel cDNA of PLDα was isolated with the strategy of in silico cloning in combination with RT-PCR from peach （Prunus persica L. cv. Jiubao）. The obtained PLDα gene contained a complete open reading frame encoding a 92- kDa protein of 810 amino acid residues, which possessed the characteristic C2 domain and two catalytic HKD motifs. The alignment analysis of the deduced peach PLDa protein with other known PLDα family proteins indicated that peach PLDα was conserved and highly homologous with strawberry PLDα. Semi-quantitative RT-PCR and Northern blot analysis indicated PLDα mRNA in peach fruits could be induced by low temperature. This work provided a scientific basis for further investigating the mechanism of postharvest fruit adaptation to low temperature.

Feasibility Analysis of in silico Cloning of Functional Candidate Genes in Tea (Camellia sinensis)

[ Objective ] This study aimed to verify the feasibility of in silico cloning of functional candidate genes in tea. [ Method ] Theobroma cacao caffeine syn- thase gene BCS1 was used as a probe to search the established tea EST database using BLAST; 26 tea ESTs highly homologous to BCS1 were obtained, which were assembled using CAP （contig assembly program） of BioEdit software; subsequently, two EST configs harboring ORF were obtained, which were named TCSnewl and TCSnew2, respectively. Nucleotide sequences and deduced amino acid sequences of theses two genes were compared with those of cDNA of tea caffeine synthase gene TCS in the GenBank database that was cloned with experimental biological method. A phylogenetic tree was constructed for homalogous analysis of the deduced amino acid sequences of theses three genes. [ Result] in silico cloning of functional candidate genes in tea using a homologous gene of distantly related species as a probe is a feasible technical means. [ Conclusion] This study provided the basis for in silico cloning of other functional genes in tea.

Cloning and Characterization of a Pathogenesis-Related Protein Gene TaPR10 from Wheat Induced by Stripe Rust Pathogen

Pathogenesis-related proteins （PRs） play many important roles in plant defense response against pathogen attack. To better understand the molecular mechanism of PR genes involved in wheat adult plant resistance （APR） to stripe rust, based on a differentially expressed transcribed derived fragment （TDF）, a novel PR gene from wheat cv. Xingzi 9104 infected by the Puccinia striiformis Westend f. sp. tritici Erikss. pathotype CY32, which was highly similar to the maize ZmPRIO gene and designated as TaPRIO, was identified using in silico cloning and RT-PCR method. This novel TaPRIO gene was predicted to encode a 160-amino acid protein with a deduced molecular weight of 17.06 kDa and an isoelectronic point （pI） of 5.19. An amino acid sequence analysis of TaPR10 demonstrated the presence of a typical conserved domain of pathogenesis related protein Bet v I family. Multiple alignment analysis based on the amino acids encoded by 10 different PRIO genes from maize （Zea mays）, rice （Oryza sativa）, broomcorn （Sorghum bicolor）, and wheat （Triticum aestivum） indicated that PR proteins of class 10 was conserved among the 4 plant species with about 80% similarity. DNA sequence of TaPRIO suggested the presence of one 84-bp intron with the splicing sites of GT-AT bi-nucleotide sequence between 188 and 271 bp. Using a real-time quantitative RT-PCR （qRT-PCR）, expression profiles of TaPRIO revealed that at the adult-plant stage, TaPRIO transcript was up-regulated as early as 12 h post-inoculation （hpi）, with the occurrence of maximum induction at 24 hpi. At the seedling stage, TaPRIO was also slightly induced 18 hpi. However, the transcript amount was relatively lower than that of the adult-plant stage. Taken together, these results suggest that TaPRIO may participate in wheat defense response of APR to stripe rust.

Decoding bovine coronavirus immune targets:an epitope informatics approach

Bovine coronavirus(BCoV)poses a significant threat to the global cattle industry,causing both respiratory and gastrointestinal infections in cattle populations.This necessitates the development of efficacious vaccines.While several inactivated and live BCoV vaccines exist,they are predominantly limited to calves.The immunization of adult cattle is imperative for BCoV infection control,as it curtails viral transmission to calves and ameliorates the impact of enteric and respiratory ailments across all age groups within the herd.This study presents an in silico methodology for devising a multiepitope vaccine targeting BCoV.The spike glycoprotein(S)and nucleocapsid(N)proteins,which are integral elements of the BCoV structure,play pivotal roles in the viral infection cycle and immune response.We constructed a remarkably effective multiepitope vaccine candidate specifically designed to combat the BCoV population.Using immunoinformatics technology,B-cell and T-cell epitopes were predicted and linked together using linkers and adjuvants to efficiently trigger both cellular and humoral immune responses in cattle.The in silico construct was characterized,and assessment of its physicochemical properties revealed the formation of a stable vaccine construct.After 3D modeling of the vaccine construct,molecular docking revealed a stable interaction with the bovine receptor bTLR4.Moreover,the viability of the vaccine’s high expression and simple purification was demonstrated by codon optimization and in silico cloning expression into the pET28a(+)vector.By applying immunoinformatics approaches,researchers aim to better understand the immune response to bovine coronavirus,discover potential targets for intervention,and facilitate the development of diagnostic tools and vaccines to mitigate the impact of this virus on cattle health and the livestock industry.We anticipate that the design will be useful as a preventive treatment for BCoV sickness in cattle,opening the door for further laboratory studies.