Cloning And Genetic Transformation Of NtNAC3153 And NtNAC4729 Gene From Narcissus Tazetta Var.chinensis 'Yunxiang'

Chinese Narcissus(Narcissus tazetta var.Chinensis)is a variant of the French narcissus.It has a cultivation history above one thousand years in our country.Being known as 'Lingbo fairies',Chinese daffodils have great ornamental value and are very popular among people because the elegant color,beautiful flower stance and thick aroma of the flowers.Chinese daffodils are mainly distributed in coastal areas like Fujian,Zhejiang and Shanghai,etc.There are daffodils such as 'Zhangzhou daffo-dils' from Fujian,'Zhoushan daffodils' from Zhejiang and 'Chongming daffodils' from Shanghai,etc.Among all kinds of the daffodils,'Zhangzh-ou daffodils',which has been sold overseas,is the most famous.It is also the representive flower of Fujian province.However,the Chinese daffodil-s always have serious problems such as the single main cultivars,slow breeding process,serious viruses accumulation and other issues.Chinese narcissus is triploid that is highly sterile and can not grow seeds after flowering,so only through asexual reproduction method can it multiply descendants.After a long period of asexual reproduction,varieties degenerated,pathogens accumulated and resistance ability declined,the quality of Chinese daffodils has been declined.Because the lack of diploid parental resources,the traditional hybrid breeding methods are limited,thus increasing the difficulty of new varieties cultivation.Technology research efforts are also not enough,seriously hampered the further development of Chinese daffodils industry.In recent years,due to the degradation of the main cultivars,the cultivation of new varieties has been update slowly,the cost of land and labor has been increased and the income has been reduced.The enthusiasm of narcissus farmers has been generally reduced,and the cultivated area has been shrinking.The traditio-nal superior agricultural industry has suffered severe challenges.Therefo-re,to improve the resistance of Chinese daffodils,so that it can adapt to the lack of water and high salt beach,to expand the daffodils cultivation area,reduce production costs,is of great essential to enhance the competit-iveness of the industry.In this study,molecular biology method was used to study the resistance system of NAC transcription factors in 'Yunxiang'narcissus.The contents and results are mainly as follows:1.Seven NAC genes were selected from the transcriptome database of 'Yunxiang' narcissus.The expression of these seven NtNAC genes are analyzed through qPCR.Two special expressed NtNAC gene were obtained as target genes for further study.The study provides reference about gene information and screening methods in the study of resistance related genes in 'Yunxiang'.2.Based on the sequence information in the transcriptome database,the specific primers of two NtNAC genes were designed.Two stress resistance related genes(NtNAC3153 and NtNAC4729)were successfully cloned from 'Yunxiang' narcissus by RT-PCR.Physical and chemical properties and conserved domain analysis showed that NtNAC3153 and NtNAC4729 are both hydrophilic proteins with no transmembrane structure and have a conserved domain at the N-terminus.Phylogenetic tree analysis showed that NtNAC3153 and NtNAC4729 have the closest genetic relationship with OsNAC from rice,which is also a monocotyledonous plant.The cloning and bioinformatics analysis of NtNAC3153 and NtNAC4729 provide valuable genetic materials and information for further resistance research of 'Yunxiang' narcissus.3.The plant expression vectors pBI121-NtNAC3153 and pMDC140-NtNAC4729 were successfully constructed.The verification of the correct insertion were carried out using PCR,enzyme digest and sequencing.4.Leaf discs were used as explants,the recombinant plasmids of pBI121-NtNAC3153 and pMDC-NtNAC4729 were transferred into tobacco through Agrobacterium tumefaciens-mediated method.NtNAC3153 and NtNAC4729 transgenic tobacco plants were obtained.Because of this work,we can continue to study the function of NAC gene in 'Yunxiang' narcissus adversity reaction through genetic engineering method.It also provide technical support for the resistent variety improvement and germplasm resources innnovation of narcissus.