Preliminary Functional Identification Of An Alkaline/Neutral Invertase MeNINV1 From Cassava (Manihot Esculenta Crantz)

Cassava roots are rich in starch, as the main food, to feed 500 million of the population in tropical. Cassava has high photosynthetic efficiency and strong base, but the accumulation of carbohydrate content in cassava is far less than its theoretical yield. The study on the transport efficiency of cassava starch in the source and sink has great significance for the cultivation of cassava varieties with high yield and high starch content.Invertase is a key enzyme for sucrose metabolism; the enzymes regulating photosynthate from phloem transport to the roots, in the transformation rate of source sink effect of starch.The high expression of alkaline/neutral invertase gene MeNINV1 accumulation in cassava starch of each period, so we speculate that it is the key gene to push the cassava sucrose metabolism. The alkaline/neutral invertase MeNINV1 function for an in-depth study will help us to analyze alkaline/neutral invertase regulation of cassava sucrose metabolism mechanism, and provide a theoretical basis for the cultivation of high yield and high starch content of cassava varieties, but also for screening high starch yield of transgenic cassava.This study uses the PCR technology, the cloned MeNINV1 promoter, bio-informatic analysis of promoter, and study the regulation of the expression of different hormones on MeNINV1, further the invertase activity was verified in the yeast invertase mutant and analyzed the characteristics of MeNINV1. Finally over-expression of MeNINVl gene in cassava and its subcellular analysis the position and effect of alkaline/neutral invertase activity have been investergated. The main results are performed as follows:1, The MeNINV1 gene promoter 1714 bp sequence was amplified by PCR using cassava genomic DNA as template and specific primers designed according to the CDS sequence of cassava alkaline/neutral invertase gene MeNINV1 and the predicted gene sequence in cassava genome database. PLACE software analysis indicated that the promoter sequence contains both typical TATA box and CAAT box and other cis-acting elements that response to light and hormones, such as P-box (gibberellin responsive),ABRE (abscisic acid responsive), ERE (ethylene responsive) and TAC-element (salicylic acid responsive).2, Online bio-informatic analyses of the promoter sequence showed some cis-acting elements that relate to stress responses in the promoter of MeNINV1. The expression mode to different stresses of MeNINV1 including four hormone treatments: 30 ?M GA3, 30 ?M ABA, 100 ?M SA and 1% ethephon were studied in tissue culture seedling of cassava plants by Real-time PCR. The results showed that MeNINV1 gene expressions were regulated by hormones, which indicate that the alkaline/neutral invertase promoter of MeNINV1 might have cis-elements that ralate to adversity response.3, The yeast invertase mutant SEY2102 was used to verify the invertase function of MeNINV1. The expression of MeNINV1 in yeast was studied, and the optimum pH of the enzyme was 6.5, the optimum reaction temperature was 40?,and the enzyme activity was not significantly inhibited in the concentration of sucrose in 200mM. The specificity of MeNINV1 was confirmed by sucrose, maltose, lactose and trehalose. MeNINV1 enzyme activity was inhibited by fructose. Organic compounds Tris, calcium, magnesium,manganese, zinc and lead metal ions and some chemical reagents have obvious inhibition on the enzyme activity.4, Prediction of the subcellular localization of MeNINV1 in mitochondria or plastids,the plant expression vector 1300-MeNINV1 was transformed to tobacco leaves and cassava. The results that showed that MeNINV1 localized in chloroplast of the transgenic tobacco leaf and transgenic cassava leaf cells. Over-expression of MeNINVl gene in cassava,qRT-PCR analysis showed that the expression of MeNINV1 in transgenic cassava leaves and roots was increased,the simultaneous determination of transgenic cassava leaves and roots in alkaline/neutral invertase activity, reducing sugar and glucose content,the results show that the alkaline/neutral invertase activity, reducing sugar and glucose content of leaves and roots were increased.