Functional Study Of Wheat TaFBA1Gene In Plant Growth And Development

UPS (ubiquitin-26S proteasome system) is now known to be the most important protein degradation pathways and it is mainly carried out through the actions of three enzymes:E1(the ubiquitin activating enzyme), E2(the ubiquitin conjugating enzyme) and E3(the ubiquitin ligase). Under the effect of these three enzymes, target proteins are moderated by ubiquitin. Then tagged proteins are degradated by the proteasome or participate in the regulation of some signal pathway, so regulating many cell activities, such as signal transduction, gene transcription and programmed cell death.F-box protein is a member of F-box domain containing protein family. As an important group of E3ubiquitin ligase enzyme, F-box protein can format ubiquitin ligase E3complex (SCF complex) with SKP1and Cde53(Cullin) and selectively bind target protein for degradation through ubiquitin protein degradation pathway.An F-box protein gene TaFBA1was cloned and transformed to the model plant tobacco, gaining transgenic tobacco overexpressing wheat F-box protein TaFBA1in our laboratory previously. Preliminary observation found that transgenic tobacco plants exhibited a significant alternation compared to wild type tobaccos (WT). However, the main mechanism responsible for the alternation is unclear. Two transgenic tobacco lines (T3, T8) were selected by genome PCR and quantitative RT-PCR. Through a series of physiological index statistics and experimental research, we want to study the function of F-box protein TaFBA1on plant growth and development and analysed variation mechanism from plant physiological and molecular levels. The main results are listed as follows:(1) Phenotypic observation and result statistics showed that the overexpressing TaFBA1transgenic line T3and T8showed obvious growth retardation and later flowering compared to wild type. Flowering time of the transgenic tobacco was delayed by about10days. It showed that the gene TaFBA1had a great influence on plant growth and development.(2) In order to further study the effects of overexpressiong TaFBA1on different development status of overexpression tobacco plants, we made systematic analysis of phenotypic observation and data statistics from seed germination, seedling formation to the end of the blooms. The results indicated that the germination rate of transgenic tobacco plants was higher than the wild type, and the trend continued until seedling formation. However, the growth of transgenic tobacco plants slowed down gradually after30days sown, and had similar phenotype to WT at60days. Then the growth of transgenic plants was slower than the wild type after60days. Slower growth rate and delayed blossom were the most significant characters of the transgenic tobacco.(3) Through the observation of the pod, we found that overexpressing TaFBA1affect the growth of the seeds and the formation of pod. The pod shape of transgenic plants was long-narrow and unfull; whereas the pod of wild type was full and round. Further data statistics of thousand seed weight indicated that the grain of transgenic seeds was less than wild type seeds.(4) Gibberellin (GA) plays an important role in processes of plant bolting and blossom, so active GA contents were detected by enzyme linked immunosorbent assay (ELISA). The results showed that the GA3and GA4of apical leaves were raising gradullly at30-,60-,90-d in WT, just downregulated at120-d; while the contents in transgenic plants were remained or decreased, just with an increase at120days. The reason for increased GA content in transgenic plants at late stage might be wild type tobacco has begun to enter the period of blossom while transgenic plants also in vigorous growth period.(5) We detected the reaction of transgenic plants when applied with exogenous GA and GA synthesis inhibitor PAC. Transgenic tobacco showed higher germination rate than wild type when applied PAC exogenously at seed germination stage, indicating less sensitivity to PAC. The application of the exogenous GA3or PAC on60-days plants changed the phenotype both of transgenic and wild plants. When applied GA3, dwarf traits of transgenic tobacco was improved, performing the similar phenotype to wild type tobacco. When applied PAC exogenously, transgenic and wild type plants were both dwarf shape, but the inhibition effect of PAC on transgenic tobacco was smaller than the wild type, showing less sensitivity to PAC of transgenic tobacco. Taken together, a decrease in GA may contribute to the restrained growth and development in transgenic tobacco plants overexpressing wheat TaFBAl.(6) GA biosynthesis and metabolism are very complicated. Many key enzymes, including GA20-oxidases, GAβ-hydroxylase and GA2-oxidase, play key roles in GA metabolism and regulation of active GA levels. Expressions of GA20ox, GA3ox and GA2ox at several key stages were detected using qRT-PCR. The results showed that overexpression of TaFBA1affected expression of these GA metabolism genes and different metabolic gene expression trends were various. Overexpression of TaFBAl up-regulated the expression of GA20oxl gene at30,60,90d, but down regulated its expression at120-day. While another gibberellin biosynthesis gene, the expression of GA3ox1was down regulated at30-day, rose subsequently and presented obvious up-regulation at90-day, but went down again at120-d, showing no difference between wild type. As gibberellin catabolism enzyme, GA2ox3showed contrary expression trend to GA20oxl.Based on the above results, overexpression of TaFBAl changed the growth process of transgenic tobacco. This may be related to the alterant GA metabolism. Overexpression of TaFBAl may regulate the expression of genes which involved in GA metabolism, thus changes the tobacco active GA levels, resulting in transgenic tobacco dwarf phenotype and delayed blossom.