Xiaoliheidou SSH-cDNA Library Construction And Expression Analysis Of Important Genes Under Heterodera Glycines Infection

Soybean Cyst Nematode，Heterodera glycines Ichinohe，is the most destructivepathogen in soybean production and accounts for substantial economic losses in worldwide.Resistant cultivars are the most primary methods for control SCN. Strategies of geneticengineering have been an effective breeding measure for plant resistance diseases due to thecharacteristics with short cycle，high effecincy，long resistance and broad resistant spectrum.Finding and identifying important genes are the essential for genetic engineering breeding andsoybean genetics improvement. China is the original area and abundance of soybeangermplasm sources. Exploring and utilizing landraces and caltivars to expand expressionprofiles for resistant disease genes，and isolation and cloing novel resistance genes havebecome more and more important in soybean research.In the present study，Xiaoloheidou （Glycine max），No ZDD1412in NationalGermplasm Repository， resistant to SCN3races was used as material to constructsuppression subtractive hybridazation cDNA libraries. Clone the target genes after screeningand analysis the differentially expressed genes in the libraries. Study Xiaoliheidou geneprofiles under soybean cyst nematode infection to explore the genes involved insoybean-nematode incompatible interaction and resistance mechanism at molecular level. Themain results are as followed:1. Duplex PCR for identifying the H. glycinesTwo pairs primers in the duplex PCR were used for identifying soybean cyst nematode.The first pair primers were universal primer D3A and D3B to amplify ribosome28S gene ofnematode；the second ones were specific primer GlyF1and universal primer rDNA2toamplify the partial ITS2and28S gene. The size of PCR fragments were181bp and345bp bygel electrophoresis detection. The result confirmed that the PCR products was the ITS regionof soybean cyst nematode by sequenced and analyzed by BLASTn. 2. Construction of SSH-cDNA librarySMARTerTMPCR cDNA synthesis and suppression subtractive hybridization （SSH）technique were combined in this study. RNA from Xiaoliheidou roots tips were extracted at12h，24h，36h，48h，72h post inoculation and non-inoculation，respectively. The inoculationgroup was used as Tester and non-inoculation group as Driver to construct forward andreverse libraries. After analyzing the characteristics of libraries，the result showed that the titerof library was4×108pfu/ml，the size of insert fragments between200-1000bp，recombinationfrequency of recombinants more than90%。The recombinants with lysates were propagatedand stored.3. Screening and biological functional analysis of resistant-nematode relative genesTotal of548positive clones with the size more than100bp were randomly picked outand hybridized with Tester-cDNA and Driver-cDNA labeled by DIG. There were362positivecDNA were screened as differential expressed genes and sequenced.280ESTs were obtainedand clustered by CAP3software.166unique ESTs were identified and analyzed with Blastxand Blastn by comparing sequences in the GenBank. All the unigenes showed high homologywith the function known genes or proteins. The function-known ESTs were annotated intofunctional categories including signal recognition and transduction，energy and materialmetabolism， stress responses， transcriptional regulation， protein synthesis and/ormodification，transport functions，cellular architecture. Discreet gene tag clusters primarilyincluding catalase，ubiquitin，chitinase，lipoxygenase，aquaporin，ripening related protein，metallothionein， plasma membrane intrinsic protein， cytochrome P450，glyceraldehyde-3-phosphate dehydrogenase were abundant in the SCN-infected roots. It wasspeculated that those genes played an important function in the incompatible interactionbetween Xiaoli black bean and H. glycines.4. Detection expression of resistant-nematode relative genes by Real Time PCRAccording to the analyzed results in above study，20genes playing important roles inresistance to nematode were picked out for further detection. The expression of a total of15genes were designed with specific primers，and detected by Real Time PCR after soybean cystnematode inoculation at12h，24h，36h，48h，72h. The trends of genes expression profiles were elevated in SCN-infected roots compared to uninfected roots including glutathioneS-transferase，syringolide-induced protein，glucose-6-phosphate-dehydrogenase，nodulin-26，S-adenosylethionine synthetase，isoliquiritigenin2’-O-methyltransferase-like，cinnamoyl coA reductase-like protein，ring-H2finger protein，glutathione peroxidase. Theexpression of genes like cyclin-dependant kinases and serine hydroxymethyltransferase1wereinduced to decrease after infection. Genes encoding lipoxygenase，phenylacetaldehydereductase，MYB transcription factor，histone showed to be up-regulated or down-regulated atdifferent time point. There was an expression transition for several genes at72h. Thisindicates that some genes were up-or down-regulated with sustained level during earlyinfection at all five time points，whereas others were induced expressed with intervals.5. Glutathione peroxidase cDNA Cloning and expression under H. glycines InfectionA gene named GmGPX1encoding glutathione peroxidase was cloned and sequencedfrom soybean roots infested H. glycines by Reverse Transcription PCR，which is a crucialenzyme in plant cells regulating reactive oxygen species（ROX）. The cDNA length of clonedgene was693bp，flanked by a5’-untranslated region of7bp and a3’-untranslated region of185bp，containing six exons and five introns. Genomic DNA fragment was located at G. maxchromosome5. The open reading frame of cDNA which encodes a polypeptide of166aminoacid residues and protein molecular weight was18375.8Da，theoretical isoelectric point6.59.The deduced amino acid sequence showed about99%and64%homology with G. maxputative PHGPX （XP₀03532707.1）and Arabidopsis thaliana GPX （NP₅64813.1），respectively. The expression profile of the GmGPX1in Xiaoliheidou under H. glycinesinfection，which generates oxidative stress，was analyzed. Real time PCR analysis revealedthat the GmGPX1mRNA levels was increased stabilized from1.3to1.47times afterexposure to H. glycines from12h to48h，and reduced to1.07times at72h comparing withnon-inoculation control. These results suggested that the GmGPX1gene was induced by H.glycines at early stage in soybean roots and played an important role in removing oxidativedamage.6. GmHs1pro-1cDNA Cloning and expression under H. glycines InfectionGmHs1pro-1was cloned and sequenced by Reverse Transcription PCR. The cDNA length of cloned gene was1477bp，flanked by a5’-untranslated region of27bp and a3’-untranslatedregion of185bp，containing six exons and five introns. Genomic DNA fragment was locatedat G. max chromosome2. The open reading frame of cDNA which encoded438amino acidresidues. Real time PCR analysis revealed that the GmHs1pro-1mRNA levels was increasedmore than10times after exposure to H. glycines at12h when compared with non-inoculationcontrol. These results suggested that the induced GmHs1pro-1by H. glycines at early stage insoybean roots involved in interation between soybean and H. Glycines.