Cloning And Characterization Of Zma-miR397a And Its Target Gene ZmLAC In Zea Mays

Abiotic stresses adversely affect plant growth,development and productivity.miRNAs constitute a group of endogenous,single stranded,non-coding regulatory RNAs of about 20-25 nt in length.They negatively regulate various developmental and physiological processes in plants.miR397 a is a conserved miRNA which has potential functions in plant growth and development.Laccase(LAC)genes are target genes of mi R397 a which are involved in lignification.Little is known on the role they play in abiotic stress response.In a co-stress(drought,salt and alkali)Zea mays Zheng58 miRNA library constructed in our laboratory,Zma-miR397 a was down-regulated seven-fold whilst the ZmLAC was up-regulated two-fold as verified by qRT-PCR.In the current research,expression analysis of Zma-miR397 a and ZmLAC under various abiotic stresses,hormone treatments and in different maize tissues was investigated using qRT-PCR.The results showed that Zma-mi R397 a and ZmLAC were differentially expressed under drought,salt,alkali,low temperature,K+ deficiency,abscisic acid(ABA),salicylic acid(SA)treatments in shoots and roots of maize seedlings which suggests that these genes might play a role in the maize abiotic stress and hormone response.Zma-miR397 a and ZmLAC expression was observed in various maize plant tissues such as leaf,stem,root,silk,tassel and immature seed.Meanwhile,higher transcript levels were observed in the root,tassel,silk and seeds which suggests that these genes might play a role during maize reproduction.A 143 bp Zma-pre-miR397 a fragment flanked with 226 bp upstream and 288 bp downstream was amplified by PCR.The resultant sequence was analyzed by blastn and showed 100% complementarity with the B73 Zma-pre-miR397 a sequence from miRBase.The conserved region of ZmLAC was PCR amplified and sequenced.5? RACE and 3? RACE PCR were used to amplify the 5? end and 3? end respectively.The resultant ZmLAC open reading frame(ORF)was 1749 bp and the amino acid sequence was 582 aa.Features such as highly conserved copper ligands,N-glycosylation sites,proline-rich region etc.and phylogenetic analysis indicates that ZmLAC belongs to the laccase gene family.A plant expression vector(35s: ZmLAC)was constructed and used to transform tobacco plants via the Agrobacterium-mediated transformation method.Transgenic tobacco plants were screened using PCR and semi-quantitative RT-PCR.Four transgenic lines showed higher ZmLAC expression and were used for subsequent physiological analysis.The transgenic tobacco plants showed improved salt tolerance and ABA response.The results suggested that ZmLAC might play a role in salt tolerance and ABA regulation in plants.