Effect Of Phytoplasma Invasion On MicroRNA Expression Profiles And Metabolome Of Mulberry

Mulberry yellow dwarf is one of the most serious infectious diseases caused byphytoplasma in mulberry. Inability to culture phytoplasmas in vitro has hindered theirbiological characterization at the molecular level. Plant microRNAs recognized as importantregulatory factors in the regulation of gene expression have a wide range of biologicalfunctions and play important roles in plant defense system. Metabolites are the final productsin cell regulation process, and their quantity changes are considered to be the final response ofthe biological system to genetic or environmental changes. In this study, the effects ofphytoplasma infection on the miRNA expression profiles and metabolome of mulberry wereinvestigated. The information obtained in this study will provide a basis for elucidating themolecular mechanism that operates under phytoplasma infection comprehensively andsystematically and for the research on the functional genomics of mulberry. Furthermore, itwill provide reference for the research on prevention and cure of phytoplasma diseases.Results presented in this thesis can be summarized as follows:(1) To examine the phytoplasma-infection-responsive miRNAs in mulberry, two smallRNA libraries were constructed from phytoplasma-infected and healthy leaves, and then weresubjected to Solexa deep sequencing. When our libraries were compared to known miRNAsfrom other plant species,146conserved miRNA families including166miRNA memberswere identified. Among the62miRNAs which were found to be differential expression inresponse to phytoplasma infection,37miRNAs were significantly decreased and25miRNAswere up-regulated in the infected leaves compared with those in the healthy ones.Furthermore,23novel miRNAs belonged to20miRNA families were also identified.Compared with the miRNAs in healthy leaves, there were7novel miRNAs which expressionswere significantly decreased and6miRNAs which expressions were up-regulated in theinfected leaf. There was a very strong correlation between PCR data and read frequencies,demonstrating that our sequencing profiles are quantitative and reliable.(2) To understand the functions of phytoplasma-responsive miRNAs, the targets of thephytoplasma-responsive miRNAs were predicted. The target genes of the differentialexpression conserved and new miRNAs were predicted and classified into15categoriesaccording to KEGG functional annotations and the pathways regulated, respectively. Most ofthe target genes are known with the roles in the control of genes related to metabolic processes, development and signaling. All of the results obtained showed the complexity ofthe regulatory network of the mulberry in response to phytoplasma infection, and based on theresults the mechanisms involved in the response of mulberry to phytoplasma infection werediscussed.(3) Based on the achievement of the transcriptomic information of mulberry, the precursorgenes of miR393a and miR393b of mulberry were cloned using PCR. At the same time, theprecursor gene of miR482of mulberry was cloned from the cDNA and genome libraries,respectively. All the precursor genes cloned were inserted into the expression vector pBI121,respectively, and their plant expression vectors were constructed successfully. This will begood help in revealing the functions and regulatory mechanisms of these miRNAs involved inthe response to phytoplasma-infection.(4) The effect of phytoplasma infection on the metabolic profilings of the leaves andphloem saps of mulberry were analyzed using GC/MS. Among the153metabolites identifiedin the infected and healthy leaf samples, there are18metabolites which were unique to theinfected samples, whereas there are only five metabolites which were unique to the healthysamples. Among the130metabolites shared by the two samples,65metabolites havesignificant concentration changes between the two samples.13metabolites were significantlyincreased in the infected leaves, whereas52metabolites were significantly decreased. A totalof147metabolites could be identified from mulberry phloem saps. Among all the metabolitesidentified, there are18metabolites peculiar to the infected phloem saps, in contrast, there are15metabolites detected only in the healthy phloem saps. Among the114metabolites commonto the two samples, there are56metabolites which concentration were changed significantly.A total of25metabolites were increased whereas31metabolites were reduced in the infectedsaps, when compared with those of healthy ones. PCA of all metabolites showed separationnot only between leaf and sap samples but also infected and healthy samples. The firstcomponent separated leaf samples from sap samples and the second component separatedinfected samples from healthy samples. Based on the results above and from the analyses ofthe physiological and biochemical indexes related to the metabolites changed, the molecularmechanisms implicated in the changes of the metabolites were discussed.