Isolation And Fuctional Analysis Of Lr14-3-3Gene From Lilium Regale Wilson

Crop plants are constantly threatened with a variety of stresses during growth and development, such as drought, salinity, high temperature, fungal and bacterial pathogens and so on. To survive under unfavorable conditions, plants have evolved a variety of mechanisms to perceive stress signal transduction, as well as the induction of specific stress-related genes and synthesis of proteins with defense roles. The14-3-3s are highly conserved regulatory proteins encoded by mutiple gene family in eukaryotes. They are known to function as regulators of a wide range of target proteins and operate in stress responses via regulating complex environmental signaling pathways and networks.14-3-3can bind to the key enzyme which involve in energy metabolism and regulating their metabolic processes, for example, by regulating the activities of key enzymes in carbon and nitrogen metabolism, nitrate reductase, sucrose phosphate synthase, trehalose-6-phosphate synthase. glycerol3phosphate dehydrogenase and glutamine synthetase enzymes,14-3-3s are involved in plant carbon and nitrogen metabolic pathways.14-3-3genes also control transmembrane transport of substances through binding to the H+-ATPase of plasma membrane. In this study, we cloned a14-3-3gene expression from the root of Lilium regale Wilson with the primers designed by Fusarium oxysporutn suppression subtractive hybridization cDNA library EST sequences encoding14-3-3(GenBank number JZ390973) and made fully bioinformatics analysis for understanding the gene. Subsequently, the quantitative reverse transcription-PCR (QRT-PCR) analysis was used to detect the expression characteristics of Lr14-3-3. Furthermore, in order to verify the function of Lr14-3-3, the constitutive plant expression vector of Lr14-3-3was constructed and Lr14-3-3transgenic T1tobacco(Nicotiana tabacum L. cv Xanthi) lines were obtained. The results of the present research are as follows:1. The full length cDNA of14-3-3(Lr14-3-3) was cloned from Lilium regale Wilson by RACE and fully bioinformation was made to understand the gene and its coded protein properties. The full-length cDNA of Lr14-3-3was1067bp in length with an ORF (open reading frame) of780bp. The ORF of Lr14-3-3encoded a predicted polypeptide of259amino acids. Additionally, the Lr14-3-3was clustered into the non-epsilon group and should be the14-3-3nu.2. The Lr14-3-3was expressed at relatively low level in root, stem, and leaf of L. regale under normal development, however, under the stress-related signaling molecules treatment of salicylic acid, jasmonic acid, ethylene and H2O2in root of L. regale, the Lr14-3-3was significantly induced by a stress-related signaling molecule, ethylene. But it was down-regulated by the signaling molecules SA and JA, and the H2O2treatment nearly did not affect the expression level of Lr14-3-3. Moreover, in an incompatible interaction between L. regale and Fusarium oxysporum as well as in a compatible interaction between susceptible Lilium Oriental Hybrid’Siberia’and F. oxysporum, Lr14-3-3was significantly up-regulated in L. regale from2hpi to12hpi and still up-regulated in slow speed with the maximum expression at12hpi. However, the transcription level of Lr14-3-3in’Siberia’was dramatically down-regulated after2hpi with the lowest transcription level at12hpi. Altogether, the expression level of Lr14-3-3in L. regale was evidently higher than that in ’Siberia’ during F. oxysporum infection.3. pCAMBIA2300S-Lr14-3-3was constructed for stable tansformation of tobacco plants. Then Lr14-3-3transgenic plants were generated through agrobacterium tumefaciens-mediated transformation and obtained39transgenic plants. Subsequently, the seedlings of Lr14-3-3transgenic T1tobacco lines were acquired. The following quantitative reverse transcription-PCR (QRT-PCR) analysis demonstrated that the Lr14-3-3gene was steadily expressed in the T1transgenic tobacco lines, and the disease resistance-related genes including N gene, osmotin, MYC, and NADPH oxidase gene were up-regulated in the transgenic lines compared with WT. What is more, the antioxidant enzymes, superoxide dismutase (SOD), glutathione S-transferase (GST), and ascorbate peroxidase (APX), showed significantly higher activities in the transgenic lines than that in WT. Antifungal activity of the crude protein extracts of the5Lr14-3-3transgenic tobacco lines showed different levels of resistance against Botrosphaeria dothidea, Phomopsis sp. and F. oxysporum. Subsequently, the4select Lr14-3-3transgenic tobacco lines displayed enhanced resistance of against the3fungal in vivo inoculation assay. Under the abiotic stresses (drought. NaCl), the4Lr14-3-3transgenic lines showed better growth momentum than WT, and the MDA (malondialdehyde) contents in transgenic lines were lower than that in WT, which showed that the overexpression of Lr14-3-3can significantly reduce the membrane lipid oxidation.Undoubtedly can we conclude from the results, as a stress-related gene, Lr14-3-3could not only be induced by ET signal molecule, but also could regulate the expression of disease resistance-related genes. The overexpression of Lr14-3-3in tobacco significantly increased the resistance to the3kinds of pathogenic fungi, and reduced tobacco membrane lipid oxidation under drought and NaCl stresses, so it can be more resist to drought, NaCl stresses. Therefore, the functional studies of14-3-3in L. regale can understand the molecular mechanisms of disease-resistance in L. regale in depth, moreover, the studies can lay the material and theoretical foundation to cultivate stress-resistant plants using genetic engineering techniques.