Change On The Allelopathy And Rhizosphere Soil Microbial Diversity By Regulating OsMYB57 Expression On Rice

The allelochemicals of the donor plant,the plant rhizosphere microorganisms,and the bio-interactions between allelochemicals and microorganisms are research topics of plant allelopathy.Most of the allelochemicals are biosynthesized from plant secondary metabolic pathways.MYB transcription factors are documented that effectively regulate the expression of related genes involved in the plant secondary metabolic pathways.Therefore,it is reasonable to construct the artificial expression vectors infused with the hybrid MYB transcription factors,and then using the rice as donor plant to transform and express these MYB contained vectors,which is able to further reveal the bio-function of these transcription factors in vivo.In this study,the regulatory effect of rice allelopathic potential by transcription factor OsMYB57(LOC_Os02g40530)was investigated.The transcriptional enhancer(VP64),transcriptional repressor(EAR),and rootspecific promoter(RSP)fusion with OsMYB57 were used to construct recombinant expression vectors and respectively transformed into wild-type rice Kitake(KK)to obtain positive transgenic rice lines,which were taken to compare the allelopathic potential between three different transgenic rice and their wild type(WT).The results were summarized as follows:(1)The study here analyzed the homology of the amino acid sequences of OsMYB57 and the previously reported MYB transcription factors with capacity on regulation of phenylpropanoid pathway.A separate branch is generated which shows the target OsMYB57 has low homology with the remaining MYB transcription factors.(2)Western blot was used to detect the expression level of OsMYB57 in the three transgenic rice lines and the WT.The results showed that the protein expression level of OsMYB57 in the VP64-and RSP-mediated transgenic rice was higher than that in the WT of KK,and it was lower in the EAR-mediated transgenic rice line than the KK.On this basis,the allelopathic potential of VP64-,EAR-,and RSP-hybrid transgenic rice were evaluated by using indoor relay seeding on ager co-culture with barnyard grass,and also in the soil medium.The results showed that the transgenic rice with increased OsMYB57 expression by VP64 has higher inhibitory rates on barnyardgrass than that of WT.In addition,combining the above two experimental results,three lines of each hybrid transcription factor transgenic rice types were selected as representatives for follow-up experiments.(3)The expression level of the genes involving in phenolic acids and terpenoids biosynthesis were detected in the three transgenic rice lines and the WT by using qPCR.The results showed that the gene expression level of O-methyltransferase(OMT)and cinnamoyl coenzyme A(CoA)were upregulated in OsMYB57-enhanced rice,when it was compared with that of the WT,whilst expression of the two genes was down-regulated in the EAR transgenic rice in comparison with KK.The reverse was showed in the gene expression of coenzyme A ligase(COL)from transgenic rice lines and WT.The results suggested that the OsMYB57 play a regulatory roles in gene expression of OMT,CoA,and COL from rice phenylpropanoid pathway.Meanwhile,the concentrations of benzeneacetic acid and p-Coumaric acid were higher in the VP64-OsMYB57 transgenic rice than that in the WT.(4)High-throughput sequencing and quantitative PCR were used to determine the population and diversity of rhizosphere soil microorganisms in three transgenic rice lines and KK.The results showed that the expression enhancement of OsMYB57 led to increase the amount of microbes in the rhizosphere of rice.Comparison of difference on the rhizosphere soil microbial diversity from the transgenic rice lines and the WT showed that increasing the expression of OsMYB57 increased the relative abundance of varietal bacteria such as Rhizobiales,Anaerolineales,Rhodospirillales,Ktetoobacterales,Sphingomonadales,and Burkholderiales in rice rhizosphere soil.The same tendency was also found in the relative abundance of Chaetomium,Penicillium,and Fusarium,which belong to fungi.(5)Additionally,some microorganisms from the rhizosphere soil of rice were further isolated and purified.The allelopathic inhibition potential of these microbial fermentation broths on the barnygrass was studied.The results showed that the fermentation broth of Bacillus,Pseudomonas,and Streptomyces suppressed the barnyardgrass growth with higher inhibitory rate,and the relative abundance of these microorganisms in the rhizospheric soil of the increased expression of OsMYB5 7 transgenic rice was higher than that of other rice,indicating that the high expression of OsMYB57 promotes the gathering of these microorganisms with allelpathic potential.(6)Exogenous phenolic acid induction on selected microorganisms revealed that the p-hydroxybenzoic acid,cinnamic acid and ferulic acid,and combination of these three phenolic acids facilitated the growth of Bacillus subtilis,Streptomyces and Pseudomonas spp.,which indicated that these microorganisms were able to interact with phenolic acids.In summary,transcription factor of OsMYB57 functions in the regulation of the allelopathic potential from rice by regulating the gene in rice phenylpropanoid pathway.During this process,the microbial diversity in rice rhizosphere soil also changed,and it was closely correlative to the rhizosphere biological processes of allelochemicals.The allelochemicals secreted by rice roots promoted the gathering of these microorganisms,and the joint-interactions between allelochemicas and particular microorganisms dominates the allelopathic effect on target barnyardgrass.