Molecular Characteristics And Functional Analysis Of Two Homeobox Genes (KNOX) From Dendrocalamus Latiflorus

Bamboo is an important forest resource with fast growth, large biomass and other uniqueadvantages, of which development and utilization is increasing. Proteins encoded by homeoboxgene family are transcription factors playing important roles in biological morphogenesis,pattern formation, and cell fate decisions. To reveal the regulation mechanism of bamboofast-growing, Dendrocalamus latiflorus, an important species of bamboo with dual-purpose oftimber and shoots, was selected as material for experiment in this study. Homeobox genes wereisolated from the nodes of D. latiflorus, and their function analysis was carried out. This studywill not only be helpful to understand the genes’ function in the morphogenensis of nodes butalso provide theoretical basis for new varieties breeding with the regulation of fast-growingthrough genetic engineering. The main results were as follows:First, gene cloning and bioinformatics analysis. Two homeobox genes were isolated fromD. latiflorus using RT-PCR and RACE methods, and named as DlKNOX1and DlKNOX2respectively. The full length cDNA of DlKNOX1was1514bp encoding358amino acids, whileDlKNOX2was1452bp encoding330amino acids. Protein structure analysis showed thatDlKNOX1and DlKNOX2all contained four kinds of domain namely KNOX1area, KNOX2area, ELK area and HOMEOBOX area, which indicated that they were belonged to the KNOXprotein. Phylogenetic tree was constructed using MEGA4, and the analysis showed thatDlKNOX1and DlKNOX2were clustered together with monocotyledonous KNOX proteins,which coincided with the morphological classification.Second, tissue specific expression analysis. Tissue specific expression of KNOX geneswere analyzed with Real-time PCR. Results showed that DlKNOX1and DlKNOX2wereexpressed in all the tested tissues with the highest level in node. However, the expressionquantity of DlKNOX1in different tissues differed from that of DlKNOX2. The relativeexpression of DlKNOX1in node, stem and sheath was as much as136.8times,21.4times and 7.6times of that in root, respectively, while that of DlKNOX2in node, stem and sheath was asmuch as36.6times,14.5times and14.2times of that in root.Third, ectopic expression of DlKNOX1and DlKNOX2. Overexpression vectors ofDlKNOX1and DlKNOX2were constructed and transferred into Arabidopsis thalianarespectively. The transgenics were selected with antibiotics. The phenotypes of transgenicsshowed that there were a series of distinct changes from that of wild type, including:(1) Leafphenotype changes, manifested as wrinkled leaves, leaf spiral notch blades, small leaves, etc.(2) Plant phenotype changes, the performance of growth retardants, dwarf, loss of apicaldominance, shoot apical meristem missing, etc.(3) Flower pattern and flowering time changes,such as wrinkled corolla surface, irregularly shaped corolla, delayed flowering time, and so on;(4) Silique changes, some transgenics with two siliques on one node, some ones with siliqueson ipsilateral growth and some ones with siliques symmetric growth.Fourth,the prokaryotic expression of DlKNOX1and DlKNOX2. The sequences encodingmature proteins of DlKNOX1and DlKNOX2were used, and the prokaryotic expression vectorsof pMAL-c5X-DlKNOX1and pMAL-c5X-DlKNOX2were constructed respectively. Thevectors were transformed into Escherichia coli (ER2523) respectively and induced by IPTG.SDS-PAGE analysis showed that the best expression condition for the recombinant protein ofDlKNOX1was at28℃induced by0.3mmol·L-1IPTG for2h, while that for DlKNOX2wasat37℃induced by0.3mmol·L-1IPTG for2h. The recombinant protein of DlKNOX1was82kD (including MBP42.5kD and DlKNOX1mature protein39.5kD), and that of DlKNOX2was79.4kD (including MBP42.5kD and DlKNOX2mature protein36.9kD).Therecombinant proteins were purified with MBP label amylose resin column respectively.Western blotting analysis was carried out using anti-MBP monoclonal antibodies as probe. Theresults showed that there were positive reactions in the predicting locations which furtherconfirmed the recombinant proteins.Genetic engineering is one of the important means of modern breeding which would havea broad prospect of application in forest directional breeding. In this study, the proteinsencoded by homeobox genes KNOX of D. latiflorus are important transcription factors. Gene expression showed KNOX genes had regulatory effect on the development of bamboo nodes, itis helpful to reveal the molecular mechanism of bamboo fast development and new varietiesbreeding in the future.