| Cassava(Manihot esculenta Crantz)is an important starch crop in tropical and subtropical regions,and is one of the most important economic crops in China.Cassava is happy to warm temperature,but sensitive to low temperature.Low temperature is the major abiotic stress to its growth and development,and its geographical limitation.Thus,to improve the cold tolerance of cassava by transgenic technology is significant.The annexins,are Ca2+-dependent phospholipid binding proteins,which widely distribute in eukaryotes.Plant annexins play important roles in response to environmental stresses.In this research,ten annexin genes have been isolated from cassava.The bioinformatic analyses,promoter activities as well as expression patterns of these genes were studied to analyze their function in response to abiotic stresses.It was found that the relative expression of MeAnn10 could be improved rapidly and efficiently under low temperature stress.Subsequently,the subcellular localization in cassava,the over-expression in Arabidopsis of MeAnn10 gene was studied.The main results as following:1,Ten annexin genes have been isolated from cassava South China 8(Manihot esculenta Crantz Var.SC8),and named as MeAnnl-10.There accession numbers are as follows:MeAnnl(KM975562),MeAnn2(KM975563),MeAnn3(KM975564),MeAnn4(KM975569),MeAnn5(KM975565),MeAnn6(KM975566),MeAnn7(KM975567),MeAnn8(KM975567),MeAnn9(KP171695),MeAnn10(KP171696).2,The evolution analysis showed that the MeAnns have a comparatively higher identity with AtAnns in Arabidopsis,OsAnn in Oryza sativa and PtrAnns in Populus spp.3,Chromosomal localization analysis showed that MeAnn genes were identified and distributed unevenly among 6 Manihot esculenta chromosomes;Gene structural analysis showed that MeAnn genes have 4-6 exons,and their length is far different.4,Protein conservative domain analysis showed that MeAnns have a typical annexins domain which consists of four repeats(Ⅰ to Ⅳ).In the Domain Ⅰ and Domain Ⅳ,there is a type Ⅱ Ca2+-binding site {K/GXGT-38-D/E} respectively.In addition,a potential phospholipid binding site and a potential heme-binding domain are also found in the Domain Ⅰ.In other three repeats,there are several other conserved sits important for annexin function,F-actin-binding site(IRI)in Domain Ⅲ and a GTP-binding site(DXXG)in the Domain Ⅳ.5,The 3D structure of MeAnns are similar with the AtAnnl in Arabidopsis.The annexin-domain is consist 5 a-helix,in which the A,B α-helix loops and D,E a-helix loops are close each other to form a type of Ca2+ binding site.In addition,MeAnn proteins have other XGD motifs for Ca2+ ion binding,which the sequences and positions are different among them.These results suggest that the ten MeAnns isolated from Cassava belong to members of the plant annexin family.6,Promoter analysis showed that MeAnns have many elements in response to stresses,such as:LTR,HSE,MBS,TC rich repeats,ABRE,ERE,TCA,TGA-element and GARE-motif etc..It speculate that the MeAnns might be involved in abiotic stress response,hormone signal response and regulation of photosynthesis.7,Expression analysis showed that the ten MeAnn genes revealed tissue specificity in cassava,among which MeAnn4 was preferentially expressed in cassava roots while MeAnn9 was mainly expressed in leaves.In addition,the expression of MeAnn genes was regulated by Ca2+ and phytohormone,among which MeAnn5 was much sensitive to Ca2+stress,while MeAnn10 was significantly increased by Ca2+ stress(157-fold;P<0.05).The expression levels of MeAnnl,MeAnn4,MeAnn5 and MeAnn10 were up-regulation under ABA,GA,IAA,SA and JA treatments.Also,the MeAnns were response to the stresses of salt,drought,low temperature,high temperature and oxidation stresses,among which MeAnn10 was significantly increased its expression under 300 mmol/L NaCl treatment(16-fold;P<0.05);The expression of MeAnn7 was increased under drought and high temperature and oxidative stresses;The expression of MeAnn6,MeAnn7 and MeAnn10 was significantly increased under low temperature stress,which was up to 23.8,101.2 and 24.4 folds respectively.8,The expression of MeAnn10 could be quickly and significantly increased,which was up to 11.7 folds,and maintained a high level under low temperature stress of 3 h.It speculates that MeAnn10 maybe play an important role in response to low temperature.Thus MeAnn10 was selected to further study on low temperature tolerance.The p1300-MeAnn10:GFP fusion expression vector was constructed and introduced into cassava.The stable transformed cassava friable embryogenic calli(FEC)were observed.The result indicated that MeAnn10 protein is localized in cytoplasm and nucleus of cassava cells.9,MeAnn10 was overexpressed and transformed into Arabidopsis.Phenotypic analysis showed that the transgenic plants were able to grow new leaves after 4℃treatment for 3 d,while the wild type plants were damaged and died.The physiological and biochemical analyses showed that the MeAnn10 transgenic Arabidopsis was stronger to cold tolerance than WT.Under natural conditions,the levels of chlorophyll,relative electrical conductivity,SOD,MDA,soluble sugar and Pro were similar in MeAnn10 transgenic and wild type Arabidopsis,while under low temperature stress,the levels of chlorophyll,SOD,soluble sugar and Pro were higher in MeAnn10 transgenic Arabidopsis than in wild type;however,the levels of relative electrical conductivity and MDA were higher in the wild type than in MeAnn10 transgenic Arabidopsis.The qRT-PCR results showed that MeAnn10 was significantly up-regulated in transgenic Arabidopsis under low temperature stress.The above results further proved that MeAnn10 could response to low temperature stress. |
