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Molecular Cloning And Expression Analysis Of MiR397 Target Gene LeLACmiR397 In Tomato

Posted on:2009-08-20Degree:MasterType:Thesis
Country:ChinaCandidate:M L PangFull Text:PDF
GTID:2120360248953121Subject:Developmental Biology
Abstract/Summary:PDF Full Text Request
LACs(EC 1.10.3.2)/Laccases are glycoproteins with polyphenol oxidase acitivity depending on copper. They ubiquitously exist in plants, fungi, insects, bacteria and other organisms with crucial roles in lignin synthesis, ion absorption and stress responses. It has been documented that several LAC genes were regulated by microRNAs in post–transcrip tional level.In this study, homology BLAST was conducted in GenBank for LAC ESTs. Resultly, 15 tomato ESTs highly similar with LAC genes were obtained, and spliced into a LAC fragment containing a recognition site of miR397. Then, its full length cDNA was cloned with RT-PCR and 5'-and 3'-RACEs. it was named as LeLACmiR397 because of its miR397 recognition site, and registered as EU503151 in GenBank.Sequence alignment showed that LeLACmiR397 protein was highly similar with AtLAC7 and PsLAC. In addition, a Cu-oxidase domain existed in the deduced LeLACmiR397 protein. To check the temporal and spatial expression of LeLACmiR397, semi-quantitative RT-PCR was performed. It was found that LeLACmiR397 specifically expressed in roots, flowers, ripe fruit and callus, but not in leaves and stems.Because miR397 is highly conserved in many plants, miR397a gene of Arabidopsis was integrated into tomato genome with agrobacterium-mediated transformation. The transformants produced high level of miR397 and were used to check the miR397-mediated posttranscriptional regulation to LeLACmiR397. It was found that posttranscriptional gene silence occurred in transgenic tomatos overexpressing miR397a, suggesting the expression of LeLACmiR397 was regulated by miR397 in posttranscriptional level.LeLACmiR397 gene encodes a polyphenol oxidase, so polyphenol oxidase activity was assayed in transgenic and nontransgenic tomatos. The result showed that transgenic tomato exhibited lower enzyme activity than nontransgenic control. In addition, high salinity induced high enzyme activity for SOD, PPO and POD both in nontransgenic and transgenic tomatos, however, the activities of those enzymes were lower in transgenic tomato at all time points tested than in nontransgenic control. It seems that the posttranscriptional regulation of miR397 to LeLACmiR397 is negatively related with tomato resistance to high salinity.
Keywords/Search Tags:tomato, laccase, miR397a, posttranscriptional regulation, stress resistance
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