Cloning And Preliminary Functional Verification Of Cassava MeAnn2 Gen

Cassava（Manihot esculenta Crantz）is an important starch,cash and energy crop grown in tropical and subtropical regions,and it plays a vital role in the national economy of all countries.At present,the increasing market demand for cassava is far higher than the supply of cassava raw materials.The domestic cassava output is in short supply,and the cassava industry is in urgent need of expansion.Cassava itself has the advantages of drought and barren tolerance,but also has the disadvantages of being sensitive to low temperature and salt,which limits its development to a certain extent.Ca²⁺signals are related to many physiological functions of plants.Annexin is one of the Ca²⁺signal receptor proteins and the first stop in the signal transduction pathway.It plays an important role in plant biological development and response to adversity stress.Arabidopsis At Ann1 has been confirmed to play an important role in plant resistance to abiotic stress.It has not been systematically studied whether the homologous gene MeAnn2 in cassava has similar functions.Therefore,this study intends to study the function of At Ann1 orthologous gene MeAnn2 in cassava.Use bioinformatics to analyze the relevant information of the gene,and study the subcellular localization of the gene through the transient expression of tobacco leaves;heterologous expression of MeAnn2 in Arabidopsis and overexpression of MeAnn2 in cassava,observation of the phenotype and resistance of the two transgenic materials Inverse capacity changes.The main findings are as follows:1.The cassava MeAnn2 gene was cloned from SC8 cassava.The CDS region of the gene is 951 bp and encodes 316 amino acids.Phylogenetic analysis shows that the cassava The genetic relationship of MeAnn2 gene is closest to the Ricinus communis L.Rc Ann2,Populus trichocarpa Ptr Ann1,Salix sinopurpurea Spu Ann Gb3,and Theobroma cacao Tc Ann1 genes,and the homology to At Ann1 of the Arabidopsis annexin family is the highest.2.Multiple sequence alignment and protein structure analysis show that cassava MeAnn2 and its homologous proteins have 4 typical repeat domains（Domain I-Domain IV）,and Domain I and Domain III contain typical type II Ca²⁺ion binding sites,belonging to Typical plant annexin.3.Subcellular localization analysis showed that MeAnn2 gene is mainly located on the cell membrane and Cytoplasmic.4.Analysis of specific expression levels in cassava tissues showed that MeAnn2 gene was expressed in all test organs/tissues of cassava,among which the expression level in tubers was highest and the expression level in leaves was lowest.5.Phenotype analysis of transgenic Arabidopsis showed that overexpression of MeAnn2 promoted the growth of Arabidopsis leaves and roots.The stress resistance analysis of transgenic Arabidopsis showed that the root system of transgenic Arabidopsis was more developed than the wild type under the treatments of 0 and 10 mmol/L low Ca²⁺concentration and 40 and 50 mmol/L high Ca²⁺concentration;at 150 mmol/L Na Cl and300 mmol/L mannitol environment,the transgenic lines are more developed than the wild type;MeAnn2 gene responds to the induction of plant hormones IAA,GA3,ABA,Me JA;transgenic Arabidopsis roots under the concentration of 50μmol/L IAA and 100μmol/L GA3 Developed,wild-type root growth is inhibited;transgenic Arabidopsis root hairs are developed well at each concentration of ABA treatment;transgenic Arabidopsis root hairs are developed well than wild-type at concentrations of 100 and 150μmol/L Me JA.There is no difference in phenotype between the transgene treated with 4℃low temperature and SA and wild-type Arabidopsis.6.Overexpression of MeAnn2 makes cassava leaves slender,the distance between stem nodes is shorter,the root phenotype presents emission,the root length becomes shorter,and the growth is slow;the cells of leaves and root tissues become shorter,the average cell area becomes smaller,and the stem tissues The length and average area of parenchyma cells are similar to those of SC8,but the cell direction is changed;the submicroscopic structure of plants such as chloroplasts and mitochondria has changed;MeAnn2 gene is expressed in large quantities on the cell membranes of cassava roots and leaves.7.The expression analysis results of MeAnn2 and the interaction protein found that overexpression of the MeAnn2 gene also activated the high expression of calpain,and decreased the expression of glutathione hydrolase（GGH）.It is speculated that these two interaction proteins are related to the phenotype and function of transgenic cassava have played an important role.