Cloning And Function Analysis Of Peanut Root-specific Promoters

Cultivated peanut(Arachishypogaea L.)is an important oil crops and cash crops.The yield and quality of peanut were severely affected by damage caused by subterranean pests-white grubs.A series of cry8-type genes with insecticidal activity to white grubs were obtained in our laboratory.If peanut root specific promoter could be cloned to drive the exogenous insecticidal gene highly expressed in peanut root grubs,this will provide an new green control method.In this study,root-specific promoters of peanut were cloned based on the gene digital expression profile established by our lab in order to obtain the promoters which are highly expressed during the growing season.This will provide important cis-acting elements for the cultivation of insect-resistant germplasmof peanut.The main results are as follows:1.The promoter region of the 5'-methylthioadenosine / S-adenosyl-homocysteine nucleosidase gene(1486bp)were cloned,named AhMtan promoter.The bioinformatics analysis showed that this promoter contains 12 ROOTMOTIFTAPOX1 elements,2OSEROOTNODULE elements,both of which are associated with the root-specific expression.According to the distribution of root expression cis-acting elements,3 expression vectors with 5'-deletion were constructed,and transferred into tobacco.GUS staining results of seedlings with the kanamycin-resistance showed that roots oftobacco transformed with vector pAhMtan1247,pAhMtan523,and pAh Mtan235 were stained blue,no blue spots in leaves.This demonstratd that AhMtan promoter is a root-specific promoter,and the minimum core region(235 bp promoter fragment)was able to guide root specific expression of the reporter gene.2.The promoter region of the Gibberellin 20 oxidase gene(2111bp)were cloned,named AhGAox promoter.The bioinformatics analysis showed that this promoter contains 15 ROOTMOTIFTAPOX1 elements,1OSE2 ROOTNODULE elements,2OSE2 ROOTNODULE elements,both of which are associated with the root-specific expression.According to the distribution of root expression cis-acting elements,3 expression vectors with 5'-deletion were constructed,and transferred into tobacco.GUS staining results of seedlings with the kanamycin-resistance showed that roots of tobacco transformed with vecto pAh GAox1805,pAh GAox518 were stained blue,no blue spots in leaves.This demonstratd that Ah Mtan promoter is a root-specific promoter,and the minimum core region(518 bp promoter fragment)was able to guide root specific expression of the reporter gene.3.The promoter region of the NBS-LRR type disease resistance gene(1966bp)were cloned,named AhNDrp promoter.The bioinformatics analysis showed that this promoter contains 10 ROOTMOTIFTAPOX1 elements,2OSE1 ROOTNODULE elements,2OSE2 ROOTNODULE elements,both of which are associated with the root-specific expression.According to the distribution of root expression cis-acting elements,3 expression vectors with 5'-deletion were constructed,and transferred into tobacco.GUS staining results of seedlings with the kanamycin-resistance showed that roots of tobacco transformed with vector pAhNDrp1360 was stained blue,no blue spots in leaves.This demonstratd that Ah Mtan promoter is a root-specific promoter,and the minimum core region(1360 bp promoter fragment)was able to guide root specific expression of the reporter gene.4.Constructed the natural resistance-associated macrophage peomoter,and transferred into tobacco.GUS staining results of seedlings with the kanamycin-resistance showed that roots of tobacco transformed with vector 1021 and 920 were stained blue,no blue spots in leaves.This demonstratd that the minimum core region(920 bp promoter fragment)was able to guide root specific expression of the reporter gene.5.The Synthetic root specific promoter SRSP was cloned,and found that the promoter specifically expressed in roots,no blue spots in leaves.Observed the transverse and longitudinal of the tobacco root,GUS gene was highest expressed in the phloem,dispersed in the cortex,there were no expressing in epidermis,xylem and pith almost.