玉米Zma-miR397a及其靶基因ZmLAC的克隆與表達(dá)分析.pdf

1、Abiotic stresses adversely affect plant growth, development and productivity. miRNAs constitute a group of endogenous, single stranded, non-coding regulatory RNAs of about 20-25nt in length. They negatively regulate vari

2、ous developmental and physiological processes in plants. miR397a is a conserved miRNA which has potential functions in plant growth and development. Laccase (LAC) genes are target genes of miR397a which are involved in l

3、ignification. 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-miR397a was down-regulated seven

4、-fold whilst the ZmLAC was up-regulated two-fold as verified by qRT-PCR. In the current research, expression analysis of Zma-miR397a and ZmLAC under various abiotic stresses, hormone treatments and in different maize tis

5、sues was investigated using qRT-PCR. The results showed that Zma-miR397a and ZmLAC were differentially expressed under drought, salt, alkali, low temperature, K+deficiency, abscisic acid (ABA), salicylic acid (SA) treatm

6、ents 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-miR397a and ZmLAC expression was observed in various maize plant tissues

7、 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

8、. A 143bp Zma-pre-miR397a fragment flanked with 226bp upstream and 288bp downstream was amplified by PCR. The resultant sequence was analyzed by blastn and showed 100%complementarity with the B73 Zma-pre-miR397a sequence

9、 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 1749bp and the a

10、mino acid sequence was 582aa. 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 e

11、xpression 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

12、 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