The Primary Study Of An Alkaline/Neutral Invertase NINV1 Gene Function In Cassava (Manihot Esculenta Crantz)

Cassava is rich of starch in roots,which is an important food crop in the tropic countries,also is broadly used as industrial raw materials and new energy sources.It could be used in medicine,textile,food,fermentation industry and so on.However,the carbohydrate storage in the cassava roots is much lesser than the synthetic photosynthetic products in leaves.Therefore,it is very important to increase the conversion rate of photosynthetic products between the source and organ as well as the accumulation of starch in roots of cassava.During the sucrose metabolism,the invertases as the key enzymes play an important role in sucrose metabolism in source and sink organs.Our preliminary study found that the activity of the alkaline/neutral invertase in roots is the highest.q RT-PCR analysis found that the expression of nINV1 was the highest during the stage of starch accumulation among all alkaline/neutral invertase gene family(a total of 11 members)in cassava.,Thus,it is meanful to study the function of alkaline/neutral invertase nINV1 gene.In this study,the yeast functional complementation experiment has been used to study the nINV1 ability to metabolize sucrose and its enzymatic properties.The nINV1 gene promoter has been cloned in the cassava genome;and the effect of the homone cis-acting elements including in the promoter sequence on the regulation of gene expression has been studied.The subcellular localization of nINV1 in tobacco mesophyll cells,the root specific expression of nINV1 gene through transgenic technology have been investaged.Futhermore,the nINV1 gene expression and the activity of the enzyme in transgenic cassava have been preliminarily investeraged.The main results are as follows:1.By using the invertase yeast mutant strain SEY2102,we have identified that the alkaline/neutral invertase nINV1 is able to hydrolyze the sucrose;and the optimum p H value is 6.5,temperature is 45?;its specific substrate is sucrose with the concentration lower than 400 m M.The activity of nINV1 invertase could be inhibited by fructoses;strongly inhibited by Tris;and some metal ions such as Ca2+,Mg2+,Mn2+,Zn2+,Pb2+ and chemicals such as EDTA,DTT,VB6 could also inhibit its activity.2.1291 bp fragment of nINV1 gene promoter sequences has been isolated through cassava genome.Bioinformatic analysis the cis-acting elements in the promoter sequence found that TATA boxes,CAAT box,light response elements and hormone-related response elements are included in the promoter sequence.Futher investergation found that the expression of nINV1 gene could be regulated by hormones of abscisic acid(ABA),gibberellin(GA3),salicylic acid(SA)and ethephon in the cassava tissue culture seedlings..3.Bioinformatic analysis finds that the location of the alkaline/neutral invertase nINV1 is in cytoplasms.A nINV1 fused with GFP was transiently expessed in tobacco leaves,and the result found that nINV1 is located in cytoplasms,which was observed with the laser confocal scanning microscopy.4.The alkaline/neutral invertase nINV1 gene was transformed in cassava roots through root specific promoter;and six transgenic lines have been obtained.The expression of nINV1 gene and the activity of alkaline/neutral invertase have been increased in the transgenic and non-transgenic cassava tissue culture roots,which suggests that nINV1 gene has been successfully expressed in cassava roots.It will be a base for further studying the function of nINV1 gene in regulation of cassava starch accumulation.