Cloning And Functional Analysis Of Fatty Acid- And Retinoid-binding Protein Family Genes In Aphelenchoides Besseyi

The rice white tip nematode(RWTN)Aphelenchoides besseyi is a migratory plant parasitic nematode that infects the aboveground parts of plants,endangers a variety of plants.Fatty acid-and retinoid-binding(FAR)proteins are nematode-specific proteins that are involved in many important biological processes.FAR proteins have potential roles in nutrient acquisition,manipulation of the host tissues,and inhibition of host defence reactions.Therefore,FAR proteins have attracted increasing attention in recent years as a promising target for controlling parasitic nematodes.RNA interference(RNAi)is a powerful tool for gene functional analysis of plant-parasitic nematodes.RNAi methods involving dsRNA solution soaking and in planta methods are commonly applied in the study of gene function in plant-parasitic nematodes.However,certain problems restrict the application of these methods.Therefore,a more reasonable,convenient and effective RNAi method must be established for different plant-parasitic nematodes according to their biological characteristics.In this study,the transcriptome database of two mixed-stage RWTN populations were used to analyze FAR protein family genes in RWTN,some transcript sequences related to fatty acid and retinol binding protein genes(far)were obtained.Seven new Ab-far genes were identified based on the rapid amplification of cDNA ends(RACE),the structure and characteristics of seven Ab-far genes were analyzed by using the bioinformatic method;the prokaryotic expression of seven Ab-far genes were conducted and the fluorescence spectra of ligand binding were used to detect the fatty acid and retinol binding activity to Ab-FAR proteins;the tissue localization and expression characteristics of seven Ab-far genes were conducted with in situ hybridization and qRT-PCR.The effect of RNAi on the reproduction of RWTN with Ab-far-2 and Ab-far-4 dsRNA solution soaking for different soaking time was determined.In addition,a new fungi-mediated method for RNAi induced in RWTN feeding on dsRNA-producing fungi to study the Ab-far-1 gene function of RWTN is described.The function of Ab-far-1 in the development and pathogenesis of RWTN was studied with fungi-mediated RNAi.The main results of this study are listed as following:1.Seven new Ab-far genes(Ab-far-2~Ab-far-8)were identified in the study.The full-length cDNA sequences of Ab-far-2~Ab-far-8 were 609 bp,847 bp,1109 bp,622 bp,1137 bp,749 bp and 609 bp,respectively,including 519 bp,501 bp,507 bp,519 bp,555 bp,537 bp and 567 bp of open reading frames that encodes 172,166,168,172,184,178 and 188 amino acids,respectively.Ab-FAR-2,Ab-FAR-5 and Ab-FAR-7 had a transmembrane domain,seven Ab-FAR proteins had a conserved casein kinase II phosphorylation sites except for Ab-FAR-7 and all had a protein kinase C phosphorylation site.All the Ab-FAR proteins had 1-3 glycosylation sites except for Ab-FAR-2 and Ab-FAR-5 and had a signal peptide.2.Ab-FAR-3,Ab-FAR-4,Ab-FAR-6~Ab-FAR-8 all could bind fatty acid and retinol.Ab-far-2~Ab-far-8 genes had two copies,two copies,one copy,two copies,two copies and one copy,respectively.The localisation of the seven Ab-far genes was different,the Ab-far-2 mRNA was located in genital system of males,the localisation of Ab-far-3~Ab-far-5 were extensive and the Ab-far-3 and Ab-far-5 mRNAs were also present in the nerve ring,the Ab-far-6 and Ab-far-8 mRNAs were located in oesophageal glands,Ab-far-7 mRNA was located in genital system of females.Seven Ab-far genes were present in all developmental stages of RWTN,and the expression levels of Ab-far-2,Ab-far-5,Ab-far-7 and Ab-far-8 were highest in males,Ab-far-3 and Ab-far-6 were highest in eggs and Ab-far-4 were highest in females.3.The RNAi results showed that the silence effect of Ab-far-2 and Ab-far-4 increased with treatment time within a certain time with Ab-far-2 and Ab-far-4 treated in vitro RNAi soaking RWTN.The silence effect of Ab-far-2 and Ab-far-4 were highest after Ab-far-2 and Ab-far-4 treated with RNAi for 24 h and 36 h,respectively.When Ab-far-2 and Ab-far-4 were treated with RNAi for 36 h,the reproduction number of RWTN decreased significantly(p<0.05)than than other treatments.Therefore,the Ab-far-2 and Ab-far-4 has important roles in the reproduction of RWTN.4.The Ab-far-1 of RWTN was used as the target gene to construct the fungi RNAi vector pBS2-Ab-far-1 expressing hairpin Ab-far-1 dsRNA.Then the Ab-far-1 dsRNA transgenic Botrytis cinerea(ARTB1)was generated using Agrobacterium-mediated transformation technology.The Ab-far-1 expression of RWTN feeding on ARTB1 was efficiently silenced,and the reproduction number and pathogenicity of RWTN on Arabidopsis thaliana were significantly inhibited(p<0.05).The Ab-far-1 expression levels of RWTN extracted from A.thaliana continued to be significantly inhibited.Thus,the RNAi induced in RWTNs feeding on ARTB1 had the characteristics of persistence and inheritance.In conclusion,seven new Ab-far genes(Ab-far-2~Ab-far-8)were identified and it is the first to identify multiple genes in the plant parasitic nematodes.The expression pattern and developmental expression pattern were diversity and two Ab-far mRNA were present in nerve ring of RWTN and it is the first to identify far genes in nervous system of nematodes.These findings may also contribute to the identification of new target genes and development of new methods to control RWTN.Meanwhile,a new method of fungi-mediated RNAi was established for fungivorous plant-parasitic nematodes and was verified as effective and applicable to the study of nematode gene function.It is first to study the gene function of plant parasitic nematodes with fungi-mediated RNAi and would provide a new strategy and new method for RNAi of the functional gene in parasitic nematode parasitic nematodes,these findings may provide foundation and established the foundation for the wide application of this method.