Preliminary Study On Function And Regulation Of LATERAL SUPPRESSOR From Two Plants Of Nervilia Genus

Branches are complex developmental traits which plants rely on for adaption to the environment and are the result of development and expansion of the spermatophyte apical buds and axillary buds,which determine the architecture of the plant.The type of plants is often the key to the impact on crop biomass and seed yield.By studying plant branch related genes and using biological genetic engineering techniques to change the plant morphology to obtain more ideal plant type,we can make full use of natural resources and increase production,and improve the current situation of endangered species declining year by year.Herba Nervilia Plicatae is a dry whole plant or leaf which comes from Nervilia fordii?Hance?Schiltr.from Nervilia Genus of Orchidaceae family.Clinically,it is mainly used for tuberculosis,hemoptysis,infantile malnutrition,children pneumonia and other diseases.Because of its natural reproductive characteristics,low propagation efficiency,and commercially overexploitation,the wild plant resources have became reduced.The medicinal effect and chemical composition of its congeneric plant,Nervilia aragoana Gaud.,are very similar to that of Nervilia fordii which is often used as Herba Nervilia Plicatae in folk.Different from the one leaf per bulb of the N.fordii,one plant with more than one bulb or leaf appeared commonly in N.aragoana.Tomato LATERAL SUPPRESSOR?LS?was the first discovered and the most deeply understood branching gene.At present,its homologue genes have been found in Arabidopsis,rice,chrysanthemum and other plants that can regulate the formation of plant branches.Previous study of our research group obtained the branching gene NfLS from N.fordii through data mining from the transcriptome database and cloned from the plant;and the NaLS was also obtained from N.aragoana by homologous cloning.Therefore,in the current study,we preliminarily compared the functions of two LS genes and observed whether there were difference in the effects of lateral suppressor?LS?from two species of Nervilia Genus on the growth and development of the model plant Arabidopsis thaliana.The results will deepen the understanding of the regulation mechanism of branching development,help adopting appropriate measures in the production practice to increase yields,alleviate the currernt status of the endangered resources of the Herba Nervilia Plicatae.The main research contents and results are as follows:?1?Subcellular localization observation of two LS genesAgrobacterium tumefaciens carrying pRI101-EGFP,pRI101-NaLS-EGFP or pRI101-NfLS-EGFP were injected into hypodermis of tobacco leaves,and transient expression of the proteins was observed by confocal laser scanning microscopy.A large amount of pRI101-EGFP,pRI101-NaLS-EGFP or pRI101-NfLS-EGFP plasmids were transformed into tobacco protoplasts by PEG mediated transformation.The subcellular localization of NaLS and NfLS genes was also observed by confocal laser scanning microscopy.Results: EGFP gene was highly expressed in the nucleus,cell membrane and cytoplasm of tobacco hypodermic leaf;NaLS,NfLS were distributed in the nucleus of tobacco hypodermic leaf;both of them are mainly located in the nucleus.It shows that both of the LS expressed in the nucleus,possessing the general characteristics of transcription factors for certain biological functions.?2?Transcriptional activation activity and DNA binding analysis of two LS proteinsArabidopsis LAS sequence?Genebank:NC₀03070.9?was queried and download from NCBI database,and the CDS coding region was obtained by designing primers based on the downloaded LAS sequence.Specific primers were designed for integration of CDS of NaLS,NfLS and LAS genes and the restriction sites into multiple cloning sites of pGBKT7 and pGADT7 separately,and the recombinant yeast expression vectors pGBKT7-NaLS/NfLS/LAS and pGADT7-NaLS/NfLS/LAS were constructed by one-step cloning method.Specific primers were designed according to the distinctive motif?AAAACTGAAAGGGAGA?of GRAS family transcription factors and the restriction site of the vector pHIS2.The two primers were denatured and renatured,and then constructed into the yeast vector pHIS2 by one-step cloning method to construct a recombinant plasmid pHIS2-SCL.The yeast recombinant expression vectors pGBKT7-NaLS/NfLS/LAS were transferred into yeast for expression and analysis of ?-galactosidase activity.The yeast recombinant expression vector pHIS2-SCL and pGADT7-NaLS/NfLS/LAS were co-transformed into yeast for expression to analyze the combination with NaLS/NfLS/LAS proteins and DNA.Results: Both NaLS and NfLS protein showed no transcriptional activation activity,LAS has transcriptional activation activity in the yeast system.NaLS,NfLS and LAS have no DNA binding activity and can not bind to gene-specific regulatory DNA sequence?AAAACTGAAAGGGAGA?.?3?Genetic transformation and expression analysis of two LS genes in tabacco and Arabidopsis thalianaTobacco leaf dishes were dipped in culture medium containing Agrobacterium tumefaciens carrying pRI101-EGFP,pRI101-NaLS-EGFP or pRI101-NfLS-EGFP to obtain regenerative tobacco overexpressing the correspondent genes.Gene expression levels of the plants were analyzed using qRT-PCR and the phenotypes were investigated.At the same time,Arabidopsis thaliana was transfected by the floral dip method and the positive plants were screened on resistant medium and validated by gene specific PCR.The phenotypes of the positive plants were compared,and the LS gene expression levels in different parts?rosettes,cauline leaves,stems,siliques,roots?of transgenic Arabidopsis were analyzed by qRT-PCR.The effects of LS on the development of shoots were analyzed in genetic transformed model plants.Results: Tobacco plants overpressing the genes were successfully obtained,and the expression levels were analyzed for every single tobacco plant.Compared to wild type plants,the gene transformation showed a certain impact on the physiological growth of tobacco.Arabidopsis thaliana overexpressing the target genes were also successfully generated.Through its phenotypic analysis and statistics showed that there were significant differences in the number of total leaves,the number of first and second branches of rosette leaves,the height of plant and other branch-related traits between the NaLS and NfLS overexpressors.qRT-PCR analysis showed that the expression levels of NfLS in different parts were in the followed order: rosette > stem >cauline leaves > roots > siliques,which was nearly the same as that in Nervilia fordii,with higher leaf expression than in rhizome or corm.