Functional Analysis Of The Tkn4 Gene During Tomato Growth And Development

KNOXs encode homeodomain-containing transcription factors. Since the first KNOX was cloned, scientists have been isolated a large number of homologous genes from Arabidopsis, rice, barley and tomato. The expression and function of these genes have been studied a lot. Recent researches demonstrated that KNOX plays important roles in the meristem maintenance, leaf and petal formation, and the hormones homeostasis in the meristem. Tkn4 belongs to the KNOX transcription factor family,but the function of Tkn4 gene has been reported little in tomato. Therefore, Cloning and functional analysis of Tkn4 gene will further illuminate the role of the KNOXs in the regulation of plant growth and fruit development, so this study has some theoretical significance.The main research results and conclusions are as follows:① According to the information of tomato database, Tkn4 gene and promoter were cloned. The bioinformatics analysis showed that Tkn4 belonged to the TALE family of the KNOX. Tkn4 gene contains these conserved domains: Helix-loop-helix, Amphipathic helix, GSE, ELK and HD. There are hormone, meristem and stress responsing elements in the promoter of Tkn4.② By performing RT-PCR, the transcript level of Tkn4 was separately found in stem, root, bud, flower and fruit but hardly visible in the leaf. Moreover, expression of Tkn4 is induced by a wide spectrum of plant hormone including NAA, GA3, KT, ABA, SA, MeJA and ethylene treatments. It is significant responsed to the GA3 and NAA treatments. The GUS gene which is driven by the promoter of Tkn4 expressed in root, stem, leaf, sepal, stamen and fruit in tomato, but strongly expressed in vascular tissue, pollen and pollen tube. By tissue staining, the expression pattern of GUS gene was consistent with the quantitative PCR results. The in situ subcellular localization and yeast one-hybrid experiments demonstrated that Tkn4 has transcriptional activation and is targeted to the nucleus in plant cell.③ There are two phenotypes of the transgenic plants overexpressing Tkn4. The transgenic plants overexpressing Tkn4 exhibit extremely dwarf phenotype and the leaves are curled and deeply lobed. Over-expression of Tkn4 increases chlorophyll content of leaves. Anatomic analysis of the leaves find that typical anatomical structure of leaves in transgenic lines changed and there was no obvious cell palisade tissues. In leaf and shoot, the formation of xylem and phloem tissues in transgenic lines is strongly repressed compared to WT plants. Compared with the wild type plants, the chlorophyll content of the fruits were increased and the rate of fruit set was improved in the transgenic plants. The transgenic seedings overexpressing Tkn4 were sensitive to the GA3 and IAA; RNA-seq data showed that Tkn4 gene play a role in auxin, gibberellin and cytokinin signal transduction; it also involved in the differentiation of the meristem and vascular tissuses; overexpression Tkn4 gene changed the genes expression patterns, which are related with the developmental of flowers.In conclusion, our results demonstrate that Tkn4 plays important roles in regulating the biosynthesis, signal transduction of diverse plant hormones, the development(including formation and differentiation) of meristems and vasculature in tomato. In addition, the formation of tomato flower tissues is also regulated by Tkn4 via modulating the expression of flowering related genes. In this study, we have confirmed that KNOX genes involved in the formation and development process of meristem tissue. Furthermore, we had confirmed a new function of Tkn4 gene which is related to the development of flower organ. These results provide a new pathway to study the formation of the floral organs.