Dissertation > Agricultural Sciences > Gardening > Vegetable gardening > Solanaceous > Tomatoes ( tomatoes )

Isolation and Functional Analysis of Tomato ICE1a

Author FengHaiLong
Tutor MengQingWei
School Shandong Agricultural University
Course Botany
Keywords Tomato SlICE1a Transcription factor Abiotic stress Expression pattern Transgenic tobacco Function analysis
CLC S641.2
Type PhD thesis
Year 2013
Downloads 39
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In the natural environment, plants are unavoidable subjected to the environmentalstresses, such as drought, high salinity and low temperature. Plants adapt to these stressesthrough the physiological and biochemical changes, including the expression of stressresponsively functional and regulatory genes, which reprogram the biological activities andestablish a new metabolism balance.Low temperature is an important environmental factor limiting the geographicaldistribution and growing season of plants. Cold stress often affects plant growth and cropproductivity, which causes significant crop losses. Cold stress limits the normal growth anddevelopment of plants directly by the inhibition of metabolic reactions and indirectly throughcold-induced osmotic and oxidative stresses. Temperate plants are capable of developingchilling tolerance when they are exposed to low non-freezing temperatures, a process knownas cold acclimation, which is associated with biochemical and physiological changes. ICE(inducer of CBF expression) is a key transcription factor in cold signaling pathway as well asa master regulator of cold acclimation. It regulates the expression of CBF/DREB1by bindingto MYC recognition elements and activate COR (cold-regulated) genes in turn.So far, researches about ICEs are mainly focus on the LT-tolerant plants, such asArabidopsis and wheat, and the information of ICE in tomato is relatively limited and hasemerged only recently. Tomato is an important vegetable around the world, while it ischilling-sensitive and incapable of cold acclimation. Thus, the research of chilling tolerancemechanism is an important theoretical and practical value. A series of studies have beenconducted on the isolation, sequence and expression analysis, function identification ofSlICE1a. The main results are as follows:(1) We isolated a novel MYC-type bHLH transcription factor, designated SlICE1a(JX625139), from tomato. It consists of a1596bp open reading frame (ORF) that encodes apolypeptide of531amino acids with a predicted molecular weight of58.3kDa. SlICE1acontains all conserved bHLH domain, S-rich region and ACT-domain, and a putative SUMO conjugation motif.(2) To observe the subcellular localization of SlICE1a, an expression cassette wasconstruct by fusing SlICE1a with the GFP reporter gene, and expressed transiently in onionepidermal cells. For transcriptional activation analysis, different coding regions of SlICE1awere constructed with the sequence of GAL4DNA-binding domain, respectively. The fusionconstructs were introduced into yeast strain Y2HGold. It is confirmed that SlICE1a localizesto the nucleus and harbors transcription-activating activity in the N-terminal.(3) RT-qPCR analysis showed that the expression of SlICE1a was stronger in leaves thanin other tissues. SlICE1a transcript was slightly up-regulated by cold, high salt, osmotic andoxidative stresses, and ABA can also induce the expression of SlICE1a. These results indicatethat SlICE1a may be involved in the adaptive responses to abiotic stresses.(4) The expression vector pBI-SlICE1a was constructed and transformed into tobaccoplant (Nicotiana benthamiana) with Agrobacterium tumefaciens-mediated leaf disc method.Transformants were evaluated by RT-qPCR after the filtering with kanamycin. Base on thevarious advanced levels of SlICE1a over-expression in transgenic lines, L8, L10and L26(T3)were chosen for further experiments. There were no obvious morphological differencesbetween WT and transgenic plants during all lifecycles under normal growth conditions.(5) SlICE1a conferred transgenic tobacco enhanced tolerance against cold, osmotic,oxidative and high salinity. Over-expression of SlICE1a in tobacco enhanced the induction ofCBF/DREB1and their target gene, consequently elevated the levels of proline, soluble sugarsand LEA proteins, which play important roles in stabilizing membranes of plant cells and inalleviating osmotic and oxidative injuries induced by many abiotic stresses.(6) SlICE1a could bind to two MYC DNA fragment in SlCBF1and SlCBF3promoter,indicating that tomato has complete ICE-CBF cold response pathways. Since both SlICE1aand SlICE1can transactive the expression of cold-responsive genes and regulate coldtolerance, suggesting that some downstream factors may be involved in chilling-sensitivity oftomato.These results broaden our knowledge about ICE genes in stress tolerance and provide a new clue for the application of genetic engineering in crop improvement.

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