MeFtsZ2 Gene Affects The Plastid Division And Starch Formation In Cassava

Cassava(Manihot esculenta Crantz)is a species of the Euphorbiaceae family.It has higher starch accumulation capacity and can be used as food,industrial raw materials and feed.The amyloplasts as the organelle that synthesize and store starch,and is developed from the proplastid.Previous studies indicated that Fts Z plays an essential role in plastid division process in potato,rice,and Arabidopsis.In order to better understand the biological function of Fts Z in root crops,we have conducted research MeFtsZ2 gene in cassava.The laboratory has cloned three MeFtsZ genes from the cassava genome in the early stage,named MeFtsZ1(XP＿021608385),MeFtsZ2(XM＿021766529.1)and MeFtsZ3(XM＿021765141.1).We have gotten transgenic plants.This article mainly studies the function of the plastid division protein MeFtsZ2 in cassava.The main research results are as follows:1.The number of amino acids,molecular weight,theoretical p I,grand average of hydropathicity,instability index,and aliphatic index of cassava MeFtsZ protein are highly similar.The subcellular location indicates that most of the cassava Fts Z members were located on cytoplasm,and a few were located on chloroplast.The results showed that MeFtsZ genes in the cassava genome were distributed on different chromosomes.MeFtsZ1 was located on chromosome 3;MeFtsZ2 was located on chromosome 9;MeFtsZ3 was located on chromosome 8.Predicting the conserved motifs of cassava MeFtsZ protein,analysis of MeFtsZ1,MeFtsZ2 and MeFtsZ3,there are 3 identical motifs,which are distributed within 500 amino acids.And MeFtsZ2 and MeFtsZ3 are arranged in the same way.The phylogenetic tree analysis showed that MeFtsZ2 and MeFtsZ3 were closely related in cassava.Analysis of the promoter of MeFtsZ gene family showed that there are multiple light-responsive elements,such as GA-motif,TCCC-motif,Box4,et al.,and also contain multiple hormone-responsive elements,such as abscisic acid response elements and gibberellin response elements,Jasmonic acid response element,et al.2.The expression of MeFtsZ gene in different organs.In cassava,MeFtsZ gene expression is the highest in leaves,followed by stems,and the lowest expression in roots.MeFtsZ1 and MeFtsZ3 are highly expressed in leaves,while MeFtsZ2 gene expression is slightly lower in leaves.We transformed the transient expression of GFP-MeFtsZ1,GFP-MeFtsZ2,GFP-MeFtsZ3 plasmid in E.coli.After induction,Lac Z can drive the expression of MeFtsZ1,MeFtsZ2 and MeFtsZ3 genes.The results showed that the E.coli treated with IPTG showed a bead shape,indicating that the division of E.coli was affected after the MeFtsZ protein was expressed in E.coli.3.Functional analysis of MeFtsZ2 gene in cassava.Transgenic plants were obtained by overexpression and interference of MeFtsZ2 gene.The phenotype of MeFtsZ2 transgenic cassava in the field.Compared with wild type,The results showed that all of plants were generally smaller and lower.And root weight and root thickness were significantly lower.Observed the phenotype of chloroplasts and conduct biostatistical analysis on the size and the number of chloroplast.The results showed that the number and size of chloroplast,compared with wild type,the chloroplasts of RNAi were significantly fewer and larger.And observed the phenotype of amyloplasts and conduct biostatistical analysis on the size and the number of amyloplast.The results showed that the amyloplasts of RNAi were significantly fewer and larger.Compared with wild type,the starch granule size of RNAi-5 were larger.From the results,MeFtsZ2 gene has a great influence on the starch granule size,so it is speculated that MeFtsZ2 gene performed function in amyloid division.4.Observing the localization of MeFtsZ2 protein in protoplasts of cassava,it can be seen that the fluorescence of GFP overlaps with the autofluorescence of chloroplasts,which indicates that MeFtsZ2 protein is localized on chloroplasts.The Me ERF2 gene was obtained by screening yeast library.By verificating the results showed that Me ERF2 can bind to the MeFtsZ2 promoter region.It was further verified that Me ERF2 can inhibit the transcription of MeFtsZ2 gene,Me ERF2 is a negative regulatory transcription factor upstream of MeFtsZ2.Because Me ERF2 is an ethylene responsive transcription factor,in order to explore whether MeFtsZ2 is involved in ethylene signal response,we performed quantitative expression analysis on the transcription level of cassava MeFtsZ2.After treatment,the expression of MeFtsZ2 gene showed a downward trend over time.In leaves,the expression began to decline at 3h,reached the lowest point at 12 h,and then gradually increased;while in roots,it showed a very significant downward trend,reaching the lowest point at 0.5h,and then in a downward trend,gradually starting at 12 h elevated.It can be inferred that the response of MeFtsZ2 gene to ethylene.In conclusion,this study proves that the plastid division protein MeFtsZ2 plays a key role in the process of chloroplast and amyloplast division,and also lays a theoretical foundation on the regulatory pathway for research in the future.