The Structure And Regulatory Function Of Intron In Inositol Phosphoryl Ceramide Synthase Gene From Botrytis Cinerea

It is a new areas to study the eukaryotic gene non-coding sequences structure andregulation mechanism in biological research today, the knowledge about plantpathogenic fungus intron-mediated regulate the gene expression is still not clear. TheBotrytis cinerea mutant with AbA resistance strain was already obtained in our lab,the mutant strain inositol phosphorylceramide (IPC) synthase gene (BcAUR1) lack ofintron, the amount of mRNA expression and the IPC synthetic enzyme vitality of themutant strain’s AUR1were measured by Real Time PCR and HPLC, respectively. It’salready confirmed that the intron in the BcAUR1inhibit the AUR1gene expression.Through the bioinformatics analysis of Botrytis cinerea AUR1gene intronsequences and the mode yeast intron sequences, we found Botrytis cinerea AUR1gene intron sequences is similar with the yeast，it has the conservative sequencecontains the5’splice sites,3’ splice sites and the branch sites, which can correctlysplice the intron.Taken cDNA of the Botrytis cinerea as a template, amplificated the3’UTRsequence of the BcAUR1gene, and then constructed into the pTFCM. The wild-typeBcAUR1gene and the BcAUR1gene without the sequence1located into the intronwere constructed into pTFCM-3’UTR, respectively. The3’UTR sequence was used asthe right arm of homologous recombination, AUR1gene5’sequence as the left arm.The two plasmids were transformed into Agrobacterium tumefaciens throughElectroporation, then transformed into Botrytis cinerea via Agrobacterium-mediatedtransformation.And gain the transformants be with a Hygromycin resistance knockedout the AUR1by the homologous recombination happened in the genome of Botrytiscinerea, the transformants positive control and the mutant with deletion of AUR1sequence1of Botrytis cinerea were selected by hygromycin resistance.The transformants’ relative mRNA expression were detected by the real-timequantitative, and the IPC synthetase activity were screened by the high performanceliquid determination. The results showed that the amount of the mRNA expression ofpositive control and the IPC synthetase activity is low than the wild-type Botrytis cinerea, these indicate that the hygromycin in the gene may be caused AUR1geneexpression down, IPC synthetase activity was decreased. The mutant with intronsequences1delection mRNA expression and the IPC synthetase activity was less thanpositive control, indicating the AUR1gene the sequence1of intron deletion also beable to down-regulate AUR1gene expression, to decrease the IPC synthetase activity.Taken together, we speculated that the sequences1of intron might be positiveregulatory element in the AUR1gene regulation mechanism.In the conditions of AbA absence culture, the growth of the positive controltransformant was slightly later than the wild type, transformant deleted intronsequence1growed seversely slowly, the spore germination and mycelial growthdelay of24h, no conidia was found in72h. These results indicated that thehygromycin gene in the transformant had a certain impacts on cell growth, and intronsequence1of AUR1gene deletion can also inhibit cell growth. Under the containingABA culture conditions, wild-type spores germination was delayed for12hs,72hs nosporulation; positive control transformant spore germination was delayed for24hs, thegermination and growth of the intron sequence1deletion transformant was notobserved at72hs later. These results showed that AbA can inhibit mutants’ sporegermination and the growth.The oxalic acid secretion volume of the positive control transformant and the intronsequences1deletion transformant strains compared with wild-type, decreased of33.7%,63.1%, respectively. It indicated that the hygromycin gene and the intronsequences deletion introduced oxalic acid secretion reduced, so that theirPathogenicity also weaker than the wild type. Containing AbA conditions, the oxalicacid secretion volume of the wild type transformant, the positive control transformant,deletion of intron sequences1transformant were decreased by45.5%,46.1%and58.3%, respectively, compared AbA absence. These showed that AbA can reduceoxalate secretion volume, the hygromycin gene and the absence of intron sequences1AbA leaded to oxalate secretion volume down and resistance was lower.The cellulase and xylanase activity of positive control and the absence of intronsequences1transformant strains were reduced with respect to wild-type,15.2%,48% and13.0%,59.7%, respectively. AbA decreased of28.3%,36.8%,46.4%and31.4%,31.2%and25.8%, respectively. The results showed that their virulence is weaker thanthe wild-type and AbA can reduce the activity of cellulase and xylanase, but alsoweaken the virulence.In summary, the hygromycin and intron sequences1deletion could down-regulatedthe IPC synthase gene expression and decreased the strains oxalic acid secretion,cellulase and xylanase activity were also reduced, resulted in decreased virulence,these suggested that intron sequences1was BcAUR1cis acting elements.