Cloning And Characterization Of Resistance Related Genes From Longan Embryogenic Cultures

Longan(Dimocarpus longan Lour.), which contained high nutritional value, is an important fruit in tropic and subtropic area. Longan is suspected to more than 20 kinds of diseases, with the global emphasized on environment and food safety day by day, the trendent of breeding is to create cultivars without relaying on chemical peticides. However, due to the long breeding cycle and lacked of genomic information, the development of longan resistance breeding was slow, and little was known about the molecure mechanism of longan resistance. Previously, we conducted the high-through put transcriptome sequencing in longan embryogenic cultures, there were 8.15% of unigenes were predicted to involved in plant-pathogen interaction, which made great foundation for quickly acquiring informations of resistance related genes in longan. In this paper, we aimed at using bioinformatic technology to mining information of resistance related genes including NBS-LRR type resistance genes, resistance related plant hormone siganal maker genes and broad spectrum resistance gene ferredoxins faimly genes in longan transcriptome,after aquired the full-length gene, we analyzed their expression profiles and used ectopic expression to indentify their physical functions. This study may help for isolating efficient resistancegenes(R genes) in longan, also for providing inforamtions for resistance breeding in other plant.1 Resistance genes analog cloningNBS-LRR genes are the most important part in R genes, based on the conserved domains can quickly acquired imforamation of R genes amnong species. According to motifs including P-loop, Kinase-2 and GLPL (AL), degenerate primes for isolating longan R genes were designed. After sequencing,46 sequencs from 55 postival single clones showed identity to R genes in other species, and not redundant between sequences. Named these sequences as NBS(X), and submitted to GenBank, the accession numbers were JQ900202-JQ900227, JX007920-JX007923, JN398315-JN398318 and JN398320-JN398329. After multi-alignment of the deduced amio acid sequences, motifs including P-loop, RNBS-A, Kinase-2, RNBS-B and GLPL (AL) were detected, among them,4 sequences were predicted to be TIR-NBS-LRR(TNL) type R genes according to Kinase-2 motifs, and 42 sequences were predicted to be CC-NBS-LRR(CNL) type R genes. Polygenetic tree analysis indicated that 46 sequences could be dived into TNL group and non-TNL group, and non-TNL group could be further dived into 4 subgroups. Motif analysis indicated that members in subgroup non-TNLl and non-TNL2 showed high identity to each other and not known R genes were be sorted into these subgroup, they might be longan specific R genes. In this experiment, we found diverse of R genes were expressing in logan embryogenic cultures, athrough these cultures were maintain on sterilized medium. Analog cloning is a useful tool in studing the diversity of R genes in longan cultures. However, due to the high evlution rate of R gene, the potaintial function of individ R genes were hardly deduced based on the information obation by analog cloning is limited between motifs, more information were need by full-length cloning and other sequencing technology.2 Mining R genes in longan embryogenic culturesUsing analog cloning method to isolated R genes is heavily depended on the conserved motifs, and hard to know the genomic and transcirptomic R genes’situation, and genomic and transcriptomic wide mining R genes based on high through-put sequencing database have been proved to be effective in many species. In this exprement, annotation key words searching and batch BLAST were proceeded to mine R genes in transcriptome database of longan embryogenic cultures.1406 from 68925 Unigenes were found related to R genes, among them,221 were found contained NB-ARC domain,40 were found contained TIR domain. After these,73 sequences which contained relative intactive NB-ARC domain were choosen for polygenetic tree analysis. Results indicated that, as R genes isolated by degenareted primers, these sequences also could be sorted in to 2 group and 4 subgroup. Among them,15 sequences were predicted to be TNL type R gene, and predicted non-TNL type R genes were 75.3% of the choosen R genes. Subgroups of non-TNL1 and non-TNL2 still showed relative long distance to known R genes. We also conducted the miRNA targets pretiction among R genes, well studied miRNA miR482 tent to target to R genes in subgroup non-TNL3, which shown relative higher conserved, meanwhile, miR5018 predicted to target non-TNL1, and this miRNA-target nod havn’t been reported in other species. In this experiment, we found using transcriptome database mining method can quickly obatain huge number of R genes, even those contain low conserved motifs and couldn’t be detected using degenerate primers. But this method heavily depended on the coverage of sequencing and the expression level of R genes. The transcriptome database minging could be more powerful once genomic information avaiurable.3 Full-length cloning of R genes in longanIn order to further understanding the both end of R genes and their function. The 13 full-length of R genes in different groups were amplifed, their GenBank asseccion number were JQ900226, KP266525-KP266536. Using BLASTp alignment to R genes in other species in NCBI GenBanK, longan R genes show most close relation with R genes from Citrus senensis, the identities were 66-79%. After conserved domain and Coil-Coil analysis, we found the all these 13 R genes were CNL type, these results confirmed the prediction by kinase2 motifs and polygentic tree analysis. As same as known R genes, all of these full-length R genes havn’t detected obvious signal peptites, but predicted to have divers subcelluar lotaion possidblity. Using qPCR to study the expression of full-length and miRNA targeted R genes, the expression level of R genes in leaves were significanltly higher than in callus, and most of the R genes showed up regulation after inoculated by pathogens. We also found different R genes showed different expression pattern in different horment treatment. These results indicated that, longan contain a large R gene family, and the evolution rate were high, they showed complicated gene regulation network after pathogen inoculation and treated by different plant hormones, the physic function of these R genes still needed to be confirm by transforamtion assaies.4 Resistance related plant hormone signal transduction path ways marker genes cloning and expression analysis in longanIn longan, little was knowed about the interaction between pathogen and plant, which heavily affected the isolation and charicterization of R genes. Marker genes in different hormone signal transduction path ways are useful tools for studing the interaction between plant and pathogens. In this study, SA signal pathway marker genes form Arabidopsis thaliana PR1(AT2G14610), PR-2(AT3G57260), JA and ET signal pathway marker gene ERF1(AT3G23240), JA signal pathway marker gene VSP2(AT5G24770), and ET signal pathway marker gene PDF1.2(AT5G44420) were choosen for mining analog genes in transcriptome database of longan embryogenic culture, and follow by full-length cloning. After these,9 full-length and 1.5’end sequences were obtained, the GenBank accession number were KP266536-KP66545. After BLAST and conserved domain analysis, all these 10 sequences shown high similar to their analog genes in A.thaliana. Expression analysis indictated that, all these genes can responsed to pathogen inoculation except VSP2-1, those PR1-1, PR2-1, ERF1-2, VSP2-2, PDF1.2-1 showed strickly and especially responsed to SA, JA and ET as predicted. Results indicated that using analog cloning method could be an efficient way to isolate the hormone maker gene in longan.5 Broad spectrum resistance gene ferredoxin faimly genes cloning, subcelluar location and expression analysis in longanFerredoxins are key proteins in photosynthetic electron transport chain, which have been proved to confer broad spectrum resistance to pathogen, also enhance plant resistancing to abiotic stress. Based on transctiptome database,5 full-length seqeuces of ferredoxin family were obtained, Genbank accession number were JF733784, JF957855, JF957856, JF957857 and JF957858. Polygenetic tree analysis indicted that, these sequences were contained 3 non-photosynthetic type Fd3, FdCl and FdC2 and 2 mitochondria type MFDX. Using Agrobacterium tumefaciens transformed Fd3, FdCl into tobacco, the subcelluar loction of them were confirmed in chloroplast as predicted. The expression level analysis during different embryogenic phase indicated that all these ferredoxin showed similar patterns, all abundantly up-regulated in heart emrbryo phase.6 Analysis of resistance and gene expression changes in Oncidium extopic expression of ferredoxinsTransformed logan Fd3, FdCl into Oncidium gower ramsey by A. tumefaciens, after screen positive cultures and obtained regeneration plantlets, we found overexpression of Fd3 enhanced the Onc. gower ramsey resistance to soft rot disease and promoted the transplant survival rate. Piriformospora indica is a model fungi for study the interaction between plant and symbiotic fungus. A miRNA high-throughput miRNA sequencing were conducted during symbiosis with Oncidium, miRNAs were found to involve in regulating auxin and development related genes to affect symbiotic process. qPCR expression level analysis indicated that, overexpression of Fd3 significantly up regulated genes including PHV, NTF, AGO and LAC during symbiosis with P.indica, but not in plants overexpression of FdCl.In conclusion, RGA cloning and transcriptome database mining were been conducted in this study, a large number of R genes’ segments and 13 full length sequences were obtained, the expression pattern analysis of these R genes in materials inoculated by pathgens and treated by different plant hormones were performed, results indicated that plants may globly regulate R genes to acquired SAR, and R genes predicted to targeted by miRNA were shown more significantly up regulation in materials inoculated by pathogens; for further characterizated the R genes in longan, the plant hormone signal path ways’ marker genes were mining and cloning,9 full length seeuqences and 1 5’end sequence were obtained, among them, PR1-1, PR2-1, ERF1-2, VSP2-2, PDF1.2-1 were found specially responsed to different hormone treatments; the mining and cloning of broad-spectrum resistance gene Fd family genes also be conducted based on longan transcriptome,5 full length sequences were obtained, they were included chloroplasmid type non-photosynthetic ferredoxin Fd3, FdCl and FdC2, meanwhile,2 mitochondrial ferredoxin MFDX were also acquired. Agrobacterium-mediated transformation were performed and introducted Fd3 and FdCl into PLBs of Oncidium, which significantly promoted the resistance to soft rot disease and transplant survival rate, meanwhile, over expression of Fd3 signicficantly modified the expression pattern of the miRNA target genes including PHV, NTF, AGO and LAC after inoculated by Piriformospora indica. This study would help further charecterization of efficient resistant related genes in longan, also provided technical base for molecular resistance breeding in longan and other plants.