The Genes Expression And Transgenic Study Of DELLA Family In Peanut

Peanut(Arachis hypogaea L.)is an important oil crop in China,and due to the remarkable economics benefits,planting area of peanut expanded rapidly.However,drought,high/low temperature stress,disease and insect pests are negatively affect the production of peanut.Studying the ability of abiotic and biotic stress resistance,understanding the mechanisms and governing these biological processes are important for peanut improvement and sustainable production of peanut.Gibberellin is a kind of important plant hormone,regulating plant growth and development in many aspects,including seed germination,stem elongation,flowering and fruit development.DELLA protein,a negative regulator of gibberellin signal transduction pathway,could inhibit the growth of plants if accumulated in a high level.However,the accumulation of DELLA protein could increase plant resistant to the adverse environment.In this study,The expression of AhDELLA genes and under different stress conditions was investigated.The AhDELLA genes and their promoters were cloned and analyzed though transgenic tobacco.These results provided useful information for the further studies on the roles of DELLA in peanut stress resistance.The main results of this study were summarized as follows:(1)Expression analysis of AhDELLA under adversity stresses.Peanut plant were treated with drought,low temperature,salt and pathogen of bacterial wilt disease.The expression of AhDELLA1、AhDELLA2、AhDELLA3 and AhDELLA4 were determined with fluorescence quantitative real time PCR(qRT-PCR).The results showed that four AhDELLAs responsed differentially to the above mentioned stresses.For example,48 h after 250 mM NaCl treatment,the expression of AhDELLA3 and AhDELLA4 were up-regulated significantly and were 251 and 943 folds of the control,respectively.Infection of bacterial wilt pathogen also strongly induced the expression of AhDELLAs.For example,after 48 h of infection the expression of AhDELLA2 was about 598 folds of the control,and the expression of AhDELLA3 was about 5554 folds of the control.These results suggested that AhDELLA may play important roles for peanut adaptation of the adverse environments.Compared to AhDELLA1 and AhDELLA2,the response of AhDELLA3 and AhDELLA4 were more obvious to stresses.(2)Promoter cloning of AhD2 pro and AhD4 pro.Based on the peanut genomic sequence primers were designed for promoter cloning.We obtained 1771 bp and 1446 bp promoter sequences for AhD2 pro and AhD4 pro,respectively.These two promoters were analyzedusing PLACE software for stress responsive cis-element prediction.The results showed that there were many stress responsive elements,such as drought responsive ACGTATERD1 and ABRERATCAL,pathogen responsive BIHD1OS、WBOX,and salt responsive DRE/CRT.(3)GUS staining of transgenic tobaccos harboring AhD2 pro and AhD4 pro promoter.Promoter GUS constructs were constructed and transformed to tobacco plant using Agrobacteria mediated method.The juvenile leaf,adult leaf,flower,immature seeds and germinating seeds were used for GUS staining to analyze the expression of the promoters.Dark blue color was detected in the young leaves indicating high expression of the promoters in young leaves.GUS was also expressed in the adult leaves in a relatively low level to compare with the yang leaves.In flower,GUS was expressed in stamen and gynoecia,especially in the stigma and anther was much higher than that in the petal,GUS staining was also detected in the germinating seeds in the hypocotyls and cotyledons.However,GUS staining was not detected in the region of root tip of the germinated seeds.These results showed that the expression of promoters in young tissues was higher than adult tissues.When the transgenic tobacco seedlings were treated with drought and salt stresses,an increased staining intensity of GUS was observed.(4)Heterologous expression of AhDELLAs in transgenic tobacco plants.Heterologous expression vectors of pCAMBIA2300-D1,pCAMBIA2300-D2,pCAMBIA2300-D3 and pCAMBIA2300-D4 were constructed and transformed to tobacco with Agrobacterium tumefaciens mediated method.The growth and development of the trangenic plants were observed.Results showed that the transgenic tobacco plants displayed yellow leaves and dwarfing,indicating AhDELLAs’ inhibitory roles to plant growth and senescence.(5)Prokaryotic expression of AhDELLA4.The gene of AhDELLA4 has been obtained already and was connected with prokaryotic expression vector pET28-a.The ligated products was transformed into Escherichia coli DE3(BL21),and screened the positive clones.Treated with 1mM IPTG of 2、4、6、12 h,the protein was induced expression.Constructing of the prokaryotic expression vector was laying the foundation for production of AhDELLA4 antibody.