| MicroRNAs(miRNAs)are a class of approximately 20-22nt non-coding RNAs that play a key role in many biological processes in animals and plants.Although many miRNAs have been identified in maize,there are few studies on the function of miRNAs during maize ear development.Identification of miRNAs involved in maize ear development,especially in the early stage of reproductive development and grain filling,will lay a theoretical foundation for our study on the physiological basis of maize ear development.In this study,Small RNA sequencing was used to identify the known miRNAs and novel miRNAs related to the development of maize young ears before and after pollination.The target genes of novel miRNAs predicted by software were verified by RLM-RACE and qRT-PCR.The main results are as follows:(1)Analysis of high-throughput sequencing results of four small RNA libraries of maize inbred lines Xu 178 and Ye 478,Xul78 before pollination(xu178BP),Xu 17810days after pollination(xu178 10DAP),Ye478 before pollination(ye479BP)and Ye47810 days after pollination(ye478 10DAP)produced 21,070,902,21532706,21,070,902 and 21532706 clean readings,respectively.In xu178BP,xu178 10DAP,ye479BP and ye478 10DAP,there were 16,056,493;14,624,016;14,472,955 and 17,176,565 readings,respectively,which matched the maize genome,accounting for 76.2%;67.92%;74.68%and 68.02%of the total reads,respectively.(2)The known miRNAs in 178 and Ye478 were identified.A total of 44 known miRNAs from 16 maize miRNA families were identified in Xu178,and 33 miRNAs from 12 maize miRNA families were differentially expressed in Ye478.Among them,22 miRNA members of Xu 178 10DAP were significantly up-regulated,22 members were down-regulated significantly;25 members of Ye478 10DAP were significantly up-regulated,and 8 members were significantly down-regulated.Among the known minas identified jointly in Xu178 and Ye478,16 members were significantly up-regulated in both Xu178 and Ye478,4 members were down-regulated in both Xu178 and Ye478,and 2 members were down-regulated in Xu178 but up-regulated in Ye478.expression.(3)The novel miRNAs in 178 and Ye478 were identified.A total of 32 new post-pollination development-related miRNAs were identified,named miRx and miRy,respectively.Among the 32 novel miRNAs,18 of them were significantly differentially expressed in Xu178 10DAP,and 16 up-regulated and 2 down-regulated;14 of them were significantly differentially expressed in Ye478,8 up-regulated and 6 down-regulated;4 of them were up-regulated in both Xu178 and Ye478,and one was down-regulated in both Xu 178 and Ye478.In addition,one miRNA was up-regulated in Xu 178 but down-regulated in Ye478.(4)Upon bioinformatics analysis,The target gene of zma-miRx2 was predicted to be GRMZM2G461230,the target gene of zma-miRy23 was GRMZM2G129288,and the target gene of zma-miRy28 was GRMZM2G418749.The 5’ RACE method was used to verify the cleavage site of the target gene GRMZM2G12230 mediated by zma-miRy23 and target gene GRMZM2G12928 mediated by zma-miRn2.The results of real-time PCR showed that zma-miRx2 was inversely proportional to the expression abundance of its target genes GRMZM2G461230,and zma-miRy28 was inversely proportional to the expression abundance of its target gene GRMZM2G418749.The results showed that the negative regulation of miRNA on its target gene may be one of the physiological bases for the development regulation of maize young ears. |
