The Effect Of Thermotherapy On MicroRNAs And ASGV-derived Small Interfering RNAs(vsiRNAs) In Vitro Pyrus Pyrifolia Tissues

Apple stem grooving virus(ASGV) infected universally pear trees planted in main producing areas of china, which has a serious effect on yield and quality. Currently, the most effective measures to prevent viral diseases are to obtain virus-free seeldings. Thermotherapy in combination with meristem tip cultures are the best way. However, the molecular mechanism of thermotherapy elimination virus in meritstm tip is unclear. In this study, pyrus pyrofolia meristem tip mixed samples with equal amount of total RNA for 1 and 5 days treated at 37℃ were designated as T37 sample, meanwhile equal amounts of total RNA for 1 and 5 days treated at 24℃ were designated as T24 sample of control. The two mixed samples were used for small RNA high-throughput sequencing. On the one hand, the study aims to reveal the effect of thermotherapy on the mi RNAs and the roles of mi RNA-mediated m RNA target genes expression level,on the other hand, to reveal the effect of thermotherapy on decreasing apple stem grooving virus(ASGV) in the tips of vitro pear plants under analysing ASGV-derived vsi RNA characterics in vitro pyrus pyrofolia plants. The results obtained are as follows:1. ASGV-J2 isolates full-length genomic sequence amplification and analysis: The complete genomic sequence of ASGV-J2 is 6497nt(excluding Ploy A tail). The complete genomic sequence of ASGV-J2 has similarity of 79.6% to 82.6% with 17 ASGV isolates reported at nt level, which has the highest similarity 82.6% with P-209 and T-47 apple hosts from Japan and China. ASGV-J2 has two open reading frames(ORF1 and ORF2), in which coat protein(CP) coded by ORF1 has a similarity of 94.1-98.7% at amino acid level with other chinese isolates, and has the highest similarity with apple isolate YTG. Meanwhile, the phylogenetic tress analsis of full-length genomic nt and cp aa sequences demonstrated that different ASGV isolates have the host selectivity.The results revealed ASGV-J2 isolates molecular characterics, which provides sequence information for further exploring the effect of thermotherapy on ASGV-derived vsi RNAs.2. Characterization of known mi RNAs in pyrus pyrofolia: 149 conserved mi RNA and 141 novel mi RNA were identified, among them 32 novel mi RNA by the presence of mature mi RNAs and corresponding complementary mi RNA*s strand were also detected. Meanwhile, 7 conserved mi RNA and 77 novel mi RNA were significant differentially expressed by comparative analysis of 24℃ and 37℃ libraries from in vitro pear shoots. Target genes for differentially expressed known and novel mi RNAs were predicted and annotated their function, further gene ontology(GO) revealing that high-ranking mi RNA target genes are involved in the metabolic process, response to stress, and the signaling, which indicated that these mi RNAs in responsive to thermotherapy have extensive function on gene regulation networks.3. The effect of thermotherapy on spatial expression patterns of potential mi RNA-mediated target genes in in vitro P. pyrofollia shoots: The spatial expression patterns of the mi RNAs and target genes were characterized in tip and base tissuess by sequencing analysis and q RT-RCR. The results revealed mi RNA/m RNA in pear have different expression profiling in the shoot meristem tip and base tissuess, which are in accordance with high throughput sequencing results, indicating that mi RNAs/m RNAs inducible to thermotherapy have the characteristics of the tissues-specific expression. In addition, thermotherapy decrease viral titer in shoot meristem tip, meanwhile negative regulated mi RNA-mediated target genes related to resistance disease defense and hormone signal transduction pathway were up-regulated in tip of P. Pyrofolia shoots under thermotherapy.4. The effect of thermotherapy on ASGV-derived vsi RNAs: the number of ASGV-derived vsi RNA reads are 7495 and 7949, respectively under 24 ℃ and 37 ℃ condition. The majority of the virus-derived small RNAs(vsi RNAs) are 21 nt, followed by 22 nt; Analysis of the 5′-terminal nt of 21-24 nt vsi RNAs revealed a clear bias with U. The vsi RNA profiles showed it almost cover the complete ASGV genome, with slight higher percentage of s RNAs derived from T37 than T24(7949 vs 7495 reads). The polarity distribution of sequenced 21 nt vsi RNAs from virus-infected pear from T37 than T24(4928 vs 4592 reads). Vsi RNAs distribution has no obvious differences in ASGV with genome from T37 than T24, revealing no silencing hotspots are generated in response to thermotherapy.The vsi RNA expression profiles from ORF1, Rd Rp, Helicase region and negative chain ORF1, cp area of ASGV genome were analysed by q RT-PCR at 24℃ and 37 ℃ tip tissuess of “Jinshui No. 2” shoots. The results demonstrate that thermotherapy has no significant effect on vsi RNA expression, whic are consistent with vsi RNAs distribution on ASGV genome sequencing. However, vsi RNA expression from negtive chain cp gene are higher under 37℃ than in 24℃. The results reveals the thermotherapy has no directly effect on in vitro pear ASGV-derived vsi RNA, speculated that thermotherapy decrease ASGV cp titer in exception of virus gene silencing, and maybe participation in the other regulated pathways.