Functional Analysis Of Vacuolar Processing Enzyme IbVPE2 In Sweet Potato

Vacuolar processing enzyme(VPE)is an aspartate specific cysteine endopeptidase existing in plant vacuoles.It belongs to the Peptidase-C13 family.VPE is similar to animal caspase-1 in structure and function.Therefore,VPE is also known as a caspase like protease in plants,which can mediate programmed cell death(PCD)in plants.VPE is responsible for the maturation and activation of vacuolar proteins in plants.It is also involved in the growth,development and death of many plant organs.Sweet potato(Ipomoea batatas(L.)Lam.)is an important food crop,energy and health crop next only to rice,corn and wheat.Previous studies in our laboratory showed that there were three vacuolar processing enzyme genes Ib VPE1,IbVPE2 and Ib VPE3 in sweet potato.On this basis,we used bioinformatics technology,tissue-specific and stress expression analysis,overexpression of Arabidopsis and sweet potato.The function of IbVPE2 in sweet potato growth and development and response to salt stress was analyzed.The function of VPE gene in sweet potato was further explored.The main results are as follows1.The VPE phylogenetic trees of 56 angiosperms were constructed by bioinformatics methods.It was found that IbVPE2 belongs to β-type VPE.Its tertiary structure is mainly composed of α-helix,irregular coil and a small amount of β-fold and corner,and does not contain transmembrane domain.2.Promoter analysis showed that the upstream promoter sequence of IbVPE2 gene contained not only common cis acting elements CAAT box and TATA box,but also cis acting elements related to light,metabolism,stress,hormone,injury response and tissue-specific expression,such as CAT-box,GATA-box,MYB1 AT,ABRE,EBOXBNNAPA,WRKY71 OS,ROOTMOTIFTAPOX1,etc.These results suggest that IbVPE2 may respond to stress and play an important role in plant growth and development.3.Subcellular localization showed that IbVPE2 protein was mainly located in vacuole.4.The results of tissue specificity and stress expression analysis showed that the expression of IbVPE2 was significantly different in different varieties of sweet potato and different tissues.The expression of IbVPE2 was relatively high in roots and mature leaves.These results indicate that the expression of IbVPE2 in sweet potato is tissue-specific.The expression of IbVPE2 was significantly up-regulated under salt stress and drought stress.After treatment with ABA,IAA and GA for 24 hours,the expression of IbVPE2 increased significantly and decreased with time.The expression of IbVPE2 increased significantly 48 h after ethephon treatment and decreased rapidly72 h after ethephon treatment.There was no significant difference in the expression of IbVPE2 between JA and SA groups.These results indicate that IbVPE2 can respond to salt stress,drought stress and some hormones related to growth and development.5.Phenotypic analysis showed that the phenotype of IbVPE2 overexpression Arabidopsis was different from that of wild type.The leaf size,leaf number,branch number,pod number and plant height of transgenic lines were significantly smaller than those of wild type.The bolting and flowering time of transgenic type was 1～2days earlier than that of wild type.These results suggest that the overexpression of IbVPE2 may be equivalent to stress in plants.Thus,the growth and development of Arabidopsis was accelerated.6.The results of salt stress response analysis showed that the activities of antioxidant enzymes(SOD,POD and CAT)of transgenic lines were lower than those of wild type under simulated salt stress.The content of MDA was higher than that of wild type,and the death of root tip cells was more serious than that of wild type.These results indicated that the overexpression of IbVPE2 reduced the tolerance of plants to salt stress,and thus accelerated the PCD process of plants.These results indicate that IbVPE2 plays an important role in plant growth and development and in response to drought,salt and hormone stresses.It is important to explore the function of IbVPE2 gene for further improving the function of VPE family members in sweet potato.