Research On The Application Of Oncidium MiRNA In Growth And Development And Resistance To Soft Rot

Oncidium hybridum is a genus of the genus Orchidaceae.It is regarded as one of the“five beautiful people”of cut flowers in the flower arrangement industry and has high economic value.Although Oncidium has a wide range of adaptability compared with other tropical orchids,its outputs reach peaks at seasons with high temperature and high humidity,and the Oncidium plant is very susceptible to various pathogens,thus hindering the development of Oncidium industry.The soft rot caused by Erwinia spp has brought devastating damage to the cultivation industry of Oncidium.Therefore,it is an important direction for studying the disease resistance in the industrialization of Oncidium.It has been shown that miRNA is involved in plant growth and disease resistance through negative regulation of target genes.However,no research has been done in Oncidium.To understand of the application of miRNA in the growth and development and resistance to soft rot of Oncidium,in this study,’Onc.Gower Ramsey’ Oncidium was used as research material;based on the Oncidium transcriptome and miRNA database,26 precursor miRNAs(pre-miRNAs),16 miRNAs,their candidate target genes were screened out;then their expression patterns of different tissues and different susceptible periods of soft rot pathogen in Oncidium were detected by qPCR;transient transformation of p1301-miR319 and miRNA over-expression in Oncidium pseudobulb were csarried out.The main findings are as follows:1 miRNA Precursor bioinformatics analysis based on the Oncidium transcriptome databaseBased on the Oncidium transcriptome database,26 miRNA precursor sequences that may be associated with disease resistance were screened out.Bioinformatics analysis showed that 26 miRNA precursors were distributed into 14 families.Among the 14 families,the miR398,miR535,and miR2950 families contain two loci,the miR162,miR167,and miR396 families contain three loci,and the miR168 family contains four loci.The 26 miRNA precursor sequences ranged in length from 259(miR2950-unigene0006151)to 1016 nt(miR319-unigene 0000840),and their GC contents ranged from 29.0 to 40.0%.The prediction of Mfold secondary structure shows that the minimum folding free energy of 26 miRNA precursors of Oncidium is between-32.04～-120.98 kal/mol,which can form a typical and stable hairpin structure.In addition,16 mature sequences distributed into 14 families were screened from the transcriptome database and miRNA library,and multi-sequence alignments of 26 miRNA precursors and 16 miRNA matures bodies were carried out.Compared with pre-miRNAs,there were 2～3 base differences in overlap region in miR319,miR162 and miR167 families,only 8 bases overlap of miR396-unigene0019032 and miR396 family,and complete overlap region among the remaining miRNA family sequences and their corresponding pre-miRNAs were detected,suggesting that the miRNA matures may originate from this overlap region.2 Analysis of expression patterns of 26 miRNA precursors in different organs and different susceptible diseases periods of OncidiumReal-time quantitative PCR(qRT-PCR)results showed that pre-miRNAs such as miR159-unigene0037857,miR167-unigene 0011236,miR167-unigene0002619,miR169-unigene0022341,miR172-unigene006514,miR319-unigene0000840,miR396-unigene0019032,miR398-unigene0009996,miR2950-unigene0006151 and miR2950-unigene0006422 accumulated in leaf may be helpful for the morphogenesis of their leaves.miR162-unigene0003615,miR162-unigene0013441,miRl 66-unigene0011870 and miR168-unigene 0009958 were highly expressed in roots and leaves,which may be beneficial to the development of their roots and leaves.Meanwhile,the abundances of miR171-unigene0045985,miR396-unigene0011179 and miR396-unigene0011180 in roots and pseudobulbs may be beneficial to the development of roots and pseudobulbs.In pseudobulbs infected by soft rot pathogen,the expression levels of miR159-unigene0037857,miR167-unigene0011236,miR396-unigene0011179,miR396-unigene 0011180,miR396-unigene0019032,miR845-unigene0012489 showed a trend of decreased and reached the lowest level after 12 h infection.The expression of miR162-unigene0040566,miR166-unigene0011870,miR168-unigene0009958,miR171-unigene0045985 increased in pseudobulbs infected by soft rot pathogen after 4 h,and then down-regulated.The expression of miR162-unigene0003615,miR167-unigene0002619,miR168-unigene0047942,miR169-unigene 0022341,miR845-unigene0012489 showed the tendency of first decreased and then increased in the process of pathogen infection,and reached the minimum after 8 h.The above results showed that different members of the 14 families of the pre-miRNAs of Oncidium are highly conserved and specific in their evolutionary processes,and may be widely involved in the growth and development of different tissues and in response to soft rot pathogen infection in Oncidium.3 Analysis of expression patterns of 16 miRNAs and their candidate target genes in different tissues and susceptible diseases of Oncidium26 precursors screened from the transcriptome database can be extracted 16 miRNA matures,bioinformatics were used to predict and annotate their candidate target,qRT-PCR analysis was applied to clarify the expression patterns of miRNAs and their candidate targets in different tissues and pseudobulbs infected by soft rot.miRNA reads analysis based on Oncidium miRNA database suggested that different members of miRNAs might have different functions in normal plants and different stages of Oncidium susceptibility.psRNATarget prediction showed that there are 852 candidate targets for 16 miRNAs in Oncidium.Among them,71 targets may relate to disease resistance,including serine/threonine-protein,F-box protein gene,zinc finger protein,RGA disease resistance protein.Based on the reads of miRNAs and the candidate targets RPKM values,miRNAs and their candidate targets were found to be differential expression in healthy plants and different susceptible periods of Oncidium.qPCR analysis showed that miRl59a,miR167b,miR168a,miR169a,miR171a,and miR172a were abundantly expressed in the pseudobulbs and leaves of Oncidium,and negativly correlated with the expression of the corresponding candidate targets,indicating that they are involved in the development and morphogenesis of pseudobulbs and leaves.In the pseudobulbs infected by soft rot pathogen,miR159a,miR168a,miR169a,miR171a,and miR172a were highly expressed at 8 h,while the expression of the corresponding candidate targets was opposite from 0 to 8 h,its speculated that the above miRNAs can respond to the infection process of soft rot pathogen in the medium term through negative regulation candidate targets;however,miR167b highly-expressed at 0 h and negative regulated the candidate targets expression at 0-8 h,speculated that its down-regulation may participate in the infection process of soft rot pathogen.The above research shows that miRNAs may be widely involved in the development of different tissues of Oncidium by mediating the cleavage of candidate targets and response of the infected process of soft rot pathogens.4 Study on the application of the transient expression of transforming p1301-miR319 vector to Oncidium pseudobulbsIn this experiment,the p1301-miR319 vector established in the laboratory was transferred into the pseudobulbs of Oncidium by Agrobacterium-mediated method.After total culture for 3 days,the total DNA was extracted for PCR detection of GUS and hygromycin.The experimental results show that both GUS and hygromycin gene fragments can be amplified by PCR.qPCR results showed that the expression level of Oncidium miR319 was significantly different between transformation and wild type.In the transformed plants and wild-type plants infected bysoft rot pathogens,it was found that the wild-type plants were more susceptible to that of the transformation of Oncidium.In summary,it can be speculated that the transformed plants can respond to the treatment of Oncidium against soft rot by regulating the expression of miR319.5 Study on the application of miRNA over-expression in Oncidium pseudobulbs infected by soft rot diseaseIn this experiment,the agomirna of miR166a,miR172a and miR398a(over-expression)was used to treated the Oncidium,and the expression of miRNAs were significantly up-regulated after miRNA over-expression.In order to further study the regulation of miRNA over-expression in Oncidium infection soft rot,the plants were first treated with miRNA over-expression,then infected with soft rot pathogens,and wild type plants were used as mock.The results showed that theOncidium plants transfected with miRNA over-expression with soft rot pathogens were significantly less susceptible than wild type,and the expression of miRNAs in Oncidium was higher than that in the mock group.Therefore,it can be speculated that miRNA over-expression can respond to the anti-soft rot disease process of Oncidium by regulating the expression of miRNA.