The Analysis Of Function On AmRad23 Genes In Amygdalus Mira Koehne

Amygdahis mira Koehne,known as "Koehne"(Tibetan),belongs to Rosaceae Prunes persica.As Chinese endemic peach species,it exhibits a high resistance to drought,barren and disease.It is one of the life span hit several thousand years and belonged to the secondary protected plant in China.A.mira could be used as fruit,flower and medicine.It has enormous ecological and economic values.In this research,a radiation sensitivity protein-23(Rad23)gene,AmRad23d,was cloned from A.mira in Tibet,it's expression patterns were investigated in drought stress.Bioinformatics analysis indicated that the structural characteristics of AmRad23d.In addition,we studied the subcellular localization of the AmRad23d,the Plant transient expression vector was transiently expressed in onion epidermal cells.To study the recombinant fusion protein expressed and optimized,the recombinant prokaryotic expression vector was constructed.In addition,the plant overexpression vector was constructed to study heterologous expression in Arabidopsis thaliana.The main results are as follows:1 AmRad23d,was cloned with cDNA library from three months old leaves of Amygdalus mira(Koehne)in Tibet by RT-PCR.its expression patterns in leaves and roots were analyzed under drought stress.The results of qPCR showed that AmRad23d gene was increased under drought stress,in addition the expression of AmRad23d gene was mainly expressed in leaves.2 Using bioinformatics methods to analyze the structural characteristics of AmRad23d protein which cloned from Amira.The sequence of open reading frame(ORF)of AmRad23d was 1158 bp,with the encoding protein molecular weight and theoretical isoelectric point(pI)is 42.3 kDa and 4.69 respectively.Amino acid sequence analysis showed that the protein belonged to hydrophilic unstable proteins and no signal peptide.The significant similarity of the AmRad23d protein amino acid sequence and the relevant one of Amygdalus persica was found.The UBQ,UBA and STI1 function structure domains of the protein were highly conservative.The AmRad23d is located in the nucleus.3 The recombinant prokaryotic expression vector pET-15b-AmRad23d was constructed,and the expression conditions of pET-15b-AmRad23d protein were optimized.The recombinant fusion protein was well expressed by inducing its gene with 0.1 mmol/L IPTG at 30? when the induction initiated at OD600?0.5 and lasted 8 h.The recombinant fusion protein AmRad23d(25 mg)with high purity was obtained after purification and dialysis.4 The plant over-expression vector pC1390-AmRad23d was constructed.Arabidopsis transgenic plants were prepared.The T3 transgenic plants of OE2,OE3,0E8 were obtained,Overexpression AmRad23d gene in Arabidopsis thaliana have higher survival rates.The results indicated that AmRad23d gene increased the tolerance of Arabidopsis thaliana to stress.When exposed to drought stress,The H2O2 content,O2-content of the wild-type Arabidopsis were significantly higher than those of the transgenic plants.At the same time,we further studied the expression levels of stress related genes RD29B?KIN2?COR15?COR47 and KIN1 in transgenic Arabidopsis thaliana and wild-type Arabidopsis.The expression of stress related genes were up-regulate at different time points.In the exogenous application of ABA,the expression of AmRad23d gene in Arabidopsis thaliana was significantly induced.The transgenic lines germination rate and root length was significantly higher than the wild type,the result suggest that AmRad23d increased the tolerance of Arabidopsis thaliana to exogenous ABA.At the same time,the expression levels of ABA-related genes ABA2,ABI1 and DREB2A were significantly reduced.