Preliminary Study On Rice LncRNA(MSTRG.100908.3) And Its Mediated RNA Network

The pollen fertility of Photoperiod-thermo-sensitive genic male sterility(PTGMS)rice can be obviously converted under the influence of photoperiod and temperature.It has been reported that long non-coding RNA(lnc RNA)plays an important regulatory role in its fertility conversion.lnc RNAs are regulatory non-coding RNA transcript with a length of more than 200 nt,which participate in regulating various biological processes in plants through various mechanisms.One of the mechanisms is that lnc RNA binds and isolates specific micro RNA(miRNA)through target simulation to play a role as a competing endogenous RNA(competing endogenous RNAs,ce RNA),which indirectly regulates the expression of target genes.At present,many lnc RNAs that can play as ce RNAs are predicted in a variety of higher plants,but only a few have been experimentally verified.Moreover,there is no detailed report about the study on the mechanism of lnc RNA as ce RNA to regulate the fertility conversion of PTGMS rice.In the previous studies of our laboratory,RNA-sequencing was performed on the young panicles at the critical period of fertility conversion in the PTGMS line rice(Wuxiang S)WXS,and some significantly differentially expressed lnc RNAs were screened and further analyzed to predict these lnc RNAs mediated ce RNA network.In order to further understand the molecular mechanism and regulatory network of lnc RNA in the fertility conversion of WXS,in this study,we selected one group of ce RNA networks: MSTRG.100908.3-miR160a-ARF10/ARF22.And we mainly studied on three main aspects: analyzed the correlation of the expression of the three in the ce RNA network,verified of non-coding characteristics of MSTRG.100908.3,and verifized regulatory relationshipthe among the three.The main findings are as follow:1.We analyzed the expression of lnc RNA-MSTRG.100908.3,osa-miR160 a,and ARF10/22 in three developmental stages(P2,P3,P4)of WXS young panicles by real-time quantitative PCR.Its result showed that the expression level of lnc RNA in fertile young panicles(WXS-F)is significantly higher than that in sterile young panicles(WXS-S).In addition,in three different developmental stages of WXS-F young panicles,the expression of MSTRG.100908.3 and ARF10/ARF22 were positively correlated,while the expression of osa-miR160 a with MSTRG.100908.3 and ARF10/22 were negatively correlated.2.We obtained the full-length sequence 2852 bp of MSTRG.100908.3 by rapid amplification of cDNA ends(RACE).And we analyzed its coding potential by ORF Finder,pfam,CPC2,the results showed that the longest ORF coding product is less than100 amino acids.In addition,the ORF had no potential for protein coding through compared with known protein databases.Therefore,these results verified that MSTRG.100908.3 is a long non-coding RNA.3.We divided the MSTRG.100908.3 sequence into two segments,and designed designed internal primers with reverse complementary nucleotides.Finally,we successfully obtained the sequence of MSTRG.100908.3 by overlapping extension PCR.Then we constructed the sequencing sequence of MSTRG.100908.3 and the osa-miR160 a precursor sequence into plant overexpression vectors respectively;and constructed the CDS sequence of ARF10 and ARF22 into plant expression luciferase reporter vectors.Finally,we co-transfected the protoplasts of rice WXS with the recombinant luciferase reporter vector and the overexpression vector.The experimental results showed that miR160 a could down-regulate the expression of ARF10/ARF22,and MSTRG.100908.3 could be used as a ce RNA to bind oas-miR160 a,thereby indirectly up-regulate the expression of ARF10/ARF22.