Functional Identification Of A Vacuolar Invertase Gene MeVINV1 From Cassava (Manihot Esculenta Crantz) In Response To Stresses

Plant vacuolar invertase is one of the key enzymes in sucrose catabolism,which can decompose sucrose into glucose and fructose,and plays an important role in regulating plant organ development and stress resistance.There are three members of cassava vacuolar invertase gene family: MeVINV1,MeVINV2 and MeVINV3.The biological functions of different members of vacuolar invertase gene in cassava need to be verified.In this thesis,MeVINV1 was used as the research object to identify the function of sucrose catabolism regulated by MeVINV1 in response to low temperature,drought and salt stress by means of fluorescence quantitative PCR,promoter activity study,enzyme activity determination,sugar content analysis,transgenic Arabidopsis thaliana,gene overexpression in cassava,CRISPR/Cas9 system gene editing and so on.The main results are as follows:1.Cassava tissue culture seedlings were treated with 4℃ low temperature,20%PEG-6000 solution drought and 300 m MNa Cl solution stress,respectively.Fluorescence quantitative PCR technique was employed to analyze the expression of MeVINV1.The results showed that the expression of this gene was activated by low temperature,drought and salt stress.2.The promoter of MeVINV1 was cloned,and it was found that there were no cis-acting elements related to low temperature,drought and salt stress in the promoter of the gene.The plant expression vector pCAMBIA1304-p MeVINV1-GFP:GUS with GFP:GUS expression driven by MeVINV1 promoter was constructed and transformed into cassava variety SC8.The transgenic calli were obtained and further studied.It was proved that low temperature,drought and salt stress could activate MeVINV1 promoter activity.3.The overexpression vector pCAMBIA1300-35S-MeVINV1:GFP was constructed and transformed in Arabidopsis thaliana.The activity of vacuolar invertase in transgenic A.thaliana was enhanced,which proved that the cassava MeVINV1 protein could catalyze the decomposition of sucrose.Stress resistance analysis revealed that MeVINV1 gene promoted the accumulation of glucose and fructose in A.thaliana,and enhanced the ability of cold tolerance,drought tolerance and salt tolerance of A.thaliana.At the same time,it was found that the overexpression of MeVINV1 gene promoted the flowering of A.thaliana.4.Five transgenic lines were obtained by genetic transformation of pCAMBIA1300-35S-MeVINV1:GFP into SC8 cassava.The activity of vacuolar invertase in transgenic cassava with overexpression of MeVINV1 gene was increased;the CRISPR/Cas9 gene editing vector of MeVINV1 gene was constructed,and the effectiveness and specificity of the target were verified;five homozygous mutants of MeVINV1 gene were obtained.The activity of vacuolar invertase decreased in the mutant of MeVINV1 gene.The results of this study laid a foundation for further identification of the function of MeVINV1 gene.The above results indicate that cassava vacuole converting enzyme MeVINV1 actually participates the response of cassava to low temperature,drought and salt stress,according to its enzyme activity variation resulting in the regulating of the contents of glucose and fructose.