Cloning And Salt-resistant Function Analysis In Tobacco Of LpNAC6 And LpNAC20 Genes From Lilium Pumilum

Lilium pumilum is a kind of perennial herb in northern China.It has a wide range of adaptability,extremely cold resistant,drought tolerant and salt tolerant.It is an important parent of lily resistant breeding.The NAC transcription factor family is one of the largest family of transcription factors in plants,which is induced by a variety of biotic and abiotic stresses.Many studies have confirmed that NAC transcription factors are involved in plant development,growth regulation,and stress response.In this study,the total RNA was extracted from the bulb of Lilium pumilum,and the cDNA was reverse transcribed from total RNA.The LpNAC6 and LpNAC20 genes were cloned from the bulb of Lilium pumilum,Bioinformatics and gene expression patterns were analyzed for these two genes.Transformation of tobacco by plant expression vector to obtain transgenic tobacco lines.The transgenic tobacco was used to identify the overexpression of LpNAC6 and LpNAC20 genes against salt stress,and revealed the function of the NAC gene of Lilium pumilum under salt stress.The specific research results are as follows:1.LpNAC6 and LpNAC20 genes from Lilium pumilum were successfully cloned by homologous cloning.The LpNAC6 gene is 909 bp in length and encodes 302 amino acids,and the LpNAC20 gene is 1986 bp in length and encodes 661 amino acids.Conservative structures and analysis showed that both the LpNAC6 and LpNAC20 genes are NAC transcription factors.Fluorescence quantitative PCR was used to analyze the expression of LpNAC6 and LpANAC20 genes under various abiotic stresses(high salt,drought,cold and ABA).It was found that LpNAC6 and LpNAC20 were sensitive to high salt and drought stress.2.The intermediate vectors pMD18-T-LpNAC6 and pM D18-T-LpNAC20 were successfully constructed.After double digestion,the plant expression vector pBI121-LpNAC6-GFP and pBI121-LpNAC20-GFP were constructed by ligation with the plant expression vector pBI121-GFP.The subcellular localization analysis of LpNAC6 and LpNAC20 genes in onion epidermal cells was performed by gene gun method.The results showed that both LpNAC6 and LpNAC20 genes were expressed in the nucleus.The LpNAC6 and LpNAC20 genes were successfully integrated into the tobacco genome and expressed by Agrobacterium-mediated method.According to the phenotypic changes of LpNAC6 and LpNAC20 tobacco TO plants,LpNAC6-2,Lp NAC6-3,LpNAC6-5 and LpNAC20-4,LpNAC20-6 and LpNAC20-9 strains were selected.Their seeds were sown on 1/2 MS medium with a pressure of 50 mg/L kanamycin to obtain transgenic T1 plants.3.The germination rate and seedling growth of LpNAC6 and LpNAC20 genes and wild type tobacco seeds were observed under salt stress.The results showed that the transgenic LpNAC6 and LpNAC20 strains showed some tolerance under salt treatment,and the seed germination rate and relative root length were greater than those of wild type tobacco.After the mature tobacco plants(5-6 leaves)were treated with 300mmol/L NaC1 for 15d,the wild type tobacco showed yellowing and wilting and growth retardation.The overexpressing LpNAC6 and LpNAC20 genes tobacco still grew normally,and the contents of chlorophyll,proline,soluble protein and SOD,POD and CAT of transgenic plants were higher than those of wild type plants.It was demonstrated that overexpression of LpNAC6 and LpNAC20 genes in tobacco can enhance tolerance to salt stress.