Screening Of Anti-phage And Anti-caenorhabditis Elegans Genes By Library Construction

Honeysuckle(Lonicera japonica),Pinellia ternata and garlic(Allium sativum L.)are the most important Chinese herbal medicinal plants.These plants are famous for their medicinal value such as anti-bacterial and anti-viral.Bacteria and bacteriophages are important to maintain the balance of ecosystem,so they are found everwhere in nature like soil,water and air.Phage is simple due to its less number of genes,and can be used as well suited test system for molecular biology and genetic engineering.In our reseach,we used phage as a model organism to study interaction between plant and virus,such as screening of antiviral genes in plants and its mechanism of resistance.These genes can be manipulated in field crops to enhance resistance against variety of pathogens through genetic engineering technique in future studies.We screened and identified resistant genes from plants that have adverse effects on nematode,furthermore,these genes can be used for biological control of nematodes.For the isolation of temperate phage,which can infect host Escherichia coli BL21(DE-3),double layer plate method was used.Phage fragments were amplified,sequenced and NCBI blast was used to analyse the sequencing results.From NCBI blast,no homologous to these sequences was found.So these fragments are considerd as newly identified phages.In the present study we used same authentication method and successfully identified a Bacillus subtilis phage.The homology comparison results showed that the phage is B.subtilis phage Nf.In this study,we constructed and expressed the honeysuckle cDNA library in B.subtilis expression system,while for garlic,cDNA and genomic library was constructed and expressed in E.coli.Previously constructed P.ternata cDNA library was also used for this study.We randomly screened genes from these libraries against phage.Two screening methods,individual and group selection,were used.A series of experiments was conducted to explore and optimize the screening methods to improve screening efficiency,and finally a suitable and ideal screening method was found.In another experiment,Caenorhabditis elegans,sensitive to smell and taste,was used as model organism for the screening of garlic cDNA and genomic library.We used food preference assay and progeny assay to explore the influence of genes on nematodes.From the results,we analyzed that the genes from garlic library can influence the feeding choice of nematode,whereas these genes have not shown any effect on the egg production capacity of nematode.