Identification And Differential Expression Analysis Of MiRNA In Strawberry Fruit Induced With Botrytis Cinerea

MicroRNA(miRNA)is a class of non-coding single strand RNAs,which play an important role in plant growth and resistance to various kinds of responses.At present,the research of plant miRNA has become more and more in-depth.However,the reports of miRNA’s response to fungi are still not widely reported,especially strawberry miRNA.In this study,the octoploid ’Yanli’ strawberry fruits were used as experimental materials,the small RNAs in strawberry fruits treated by Botrytis cinerea(B.cinerea)were sequenced to identify miRNAs and target genes.Differentially expressed miRNAs were screened out.The function of target genes was predicted and analyzed in order to explore the response of strawberry miRNA to B.cinerea.The main results are as follows:1.The strawberry red fruits with 48 h(T1),72 h(T2),96 h(T3)and 120 h(T4)treated by B.cinerea,strawberry red fruits treated by clear water(CK1)and B.cinerea(CK2)were used as test materials,six small RNA libraries were constructed,and then we found that the most abundant class of sRNAs was 21 nt.In six libraries,141 conserved miRNA belonging to58 miRNA families and 35 novel miRNAs were identified.A total of 506 and 287 target genes were predicted for the 176 miRNAs in the conserved and novel miRNAs,respectively.The types of these target genes mainly were SPL,NAC,HL-Zip,TMV,LRR,F-box/kelch,MYB and some other disease resistance related genes.2.Using high throughput sequencing,the differential expression of miRNA was analyzed,results showed that: compared with CK1,most of the conserved miRNAs and novel miRNAs were up-regulated at the early infected stages(T1,T2)and then down-regulated in the late infected stages(T3,T4).Using the qRT-PCR technology,the expression levels of six differentially expressed miRNAs(miR166a,miR167,miR5290 a,fan-18,fan-20,fan-24)were detected.It showed that the results of two expression analysis methods were basically the same.3.With ’Yanli’ strawberry fruits as test materials,the target gene PIRL of strawberry miR5290 a was successfully cloned by RT-PCR technology.The coding region of strawberry was 1554 bp and encoded 517 amino acids.The differential expression of PIRL gene in different processing times of B.cinerea was analyzed by qRT-PCR technology.It was found that the expression level of PIRL gene was down regulated,and the expression trend in T2 and T3 phase was opposite to miR5290 a.It was speculated that miR5290 a negatively regulates the expression of target gene PIRL.4.Using plant pRI101-AN as the vector,and then using Xbal I/Smal I and Kpn I/EcoR I restriction enzymes,the interference vector containing PIRL specific fragments was obtained by adding intron sequences and two reverse complementary PIRL specific fragments.Using Rapid PCR site-directed mutagenesis technology,we got the mPIRL gene coding sequence,whose target site of miR5290 a was eliminated,but the amino acid sequence was unchanged.On this basis,using plant pRI101-AN as the vector,and then using Nde I/Kpn I restriction enzymes,the over expression vector containing mPIRL coding region was obtained.5.Three transgenic ’Ruegen’ strawberry lines with overexpressed mPIRL genes were obtained by Agrobacterium mediated genetic transformation.The expression levels of three transgenic lines were detected.The results showed that the expression of mPIRL in three strawberry lines was much higher than that in the control.It can be concluded that three strains were all transgenic plants.6.Injecting the bacterial solution containing mPIRL overexpression vector and PIRL interference vector into the ‘Yanli’ strawberry white fruits,and processing them in the red fruit stage with B.cinerea,the result showed that compared with CK treated with clear water,the phenotypic susceptibility of mPIRL over expressed fruits were lighter,while PIRL interfered with heavy phenotypic susceptibility.It is conjecture that PIRL participates in the response mechanism of strawberry against B.cinerea.