Studies On The Mechanism Of The Accelerated Growth And Development In Transgenic Tobacco Overexpressing Wheat Ta-Ub2Gene

Protein, whose production and degradation are significant to organism, isconstitution of organism and plays an important role in many life activities. Proteincan be degraded by lysosomes and ubiquitin-26S proteasome system (UPS). Ubiquitin(Ub) is an evolutionarily conserved76-amino-acid polypeptide which is attached totarget proteins either for degradation by the multi-subunit26S proteasome orregulation of their function in a proteasome-independent manner. After a3-stepenzymatic (ubiquitin-activating enzymes-ubiquitin-conjugating enzyme-ubiquitinligases) conjugation cascade, target protein can be moderated by monoubiquitin orpoly-Ub chains of different styles, to regulate many cell activities such as signaltransduction, gene transcription and programmed cell death.Although previous studies have proved that UPS significantly contributes togrowth and development in plants, most of them focused on E3. The role of Ub inplant development remains elusive. The transgenic tobacco plants overexpressingTa-Ub2have been produced in our laboratory. It is observed that transgenic tobaccoplants exhibited a significant alternation compared to wild type tobaccos (WT).However, it is unclear of the main mechanism responsible for the alternation. Twotransgenic tobacco lines (T-2, T-11) were selected by genome PCR andsemi-quantitative RT-PCR, to study the effect of ubiquitin on plant growth anddevelopment and the physiological and molecular mechanism. The main results arelisted as follows:1. Transgenic tobaccos exhibited accelerated growth and early floweringcompared with WT. It was observed that transgenic tobaccos flowered about30daysearlier than WT. 2. Expression of developmental marker genes (NtMADS-4, NtMADS-11) wasdetected by real time quantitative reverse transcription PCR (qRT-PCR) to confirm thedifference in developmental stages. The results showed that expression of NtMADS-11were similar between transgenic tobaccos and WT of30days later, indicating thatoverexpression of Ta-Ub2accelerated the developmental pattern.3. The alternation of Ub proteins in wild type tobaccos during flowering weredetected by immunological blotting. The results showed that ubiquitin levels in apicaltissues altered obviously, suggesting that Ub is involved in development of plantapical tissues.4. The earlier bolting in transgenic tobacco plants was the typical phenotype andmay be responsible for the accelerated growth pattern. It is well-known that GA playsan important role in plant bolting. Total gibberellin (GA) content in apical leaves andstems were measured by high-performance liquid chromatography (HPLC). Total GAof transgenic tobaccos are higher than or equal to wild type tobaccos at specific stages.Considering that total GA contain GA precursor, active GA and inactive GA, activeGA contents were detected by enzyme linked immunosorbent assay (ELISA). Theresults showed that GA3and GA4of apical leaves and stems were higher in transgenictobaccos than in WT.5. Treatment with GA or GA biosynthesis inhibitors confirmed the contributionof GA to the altered development patterns of tobacco seedlings. Thus, an increase inGA may contribute to the accelerated growth and development in transgenic tobaccoplants overexpressing wheat Ta-Ub2.6. GA biosynthesis and metabolism are very complicated. Many key enzymes,including GA20-oxidases, GA3β-hydroxylase and GA2-oxidase, play significantroles in GA metabolism and regulation of active GA levels. Expressions of GA20ox,GA3ox and GA2ox at several key stages were detected by qRT-PCR. The resultsshowed that overexpression of Ta-Ub2affects expression of these GA metabolismgenes. Compared with wild type tobaccos of the same developmental stage,NtGA20ox1, NtGA20ox2, NtGA3ox1(GA biosynthesis genes) and NtGA2ox3(GAcatabolism genes) of transgenic tobaccos were upregulated. To sum up, overexpression of Ta-Ub2altered the developmental pattern oftransgenic tobacco plants. This alternation may be related to the variety of GAmetabolism. Overexpression of Ta-Ub2increase GA contents by regulating theexpression of GA metabolism genes, resulting in early bolting and flowering oftransgenic tobacco plants.