Mechanism Of Cold Sweetening Of Stored Potato Tubers And Cloning And Functional Identification Of Invertase Inhibitor Gene

The potato (Solarium tuberosum L.) is the fourth food crop with multiple uses in the world, of which potato chips and fries are fashionable. The harvested tubers of potato are stored at low temperature to prevent losses caused by spoilage spreading, sprouting and shrinkage, and to extend the processing period of industries. Cold storage, however, induces the accumulation of reducing sugar (RS) due to the conversion of starch to reducing sugar that can react with the free amino acid of tubers at high temperature frying. This results in a dark-coloured, bitter-tasting and undesirable product unfiting for human consumption. The statch-sugar conversion involves multiple metabolic pathway with multi-regulaing and -feedback regulating factors. Therefore, mechanism of "low temperature sweetening (LTS) " is a basic research for improving potato post-harvest quality. The aim of the present research reported is exploit the dynamic relationship between the content of RS and activities of the main enzymes involved in the starch-sugar conversion of the tubers stored at different temperatures and clarify the key factors affecting the accumulation of RS. Moreover under the basis of the present analysis and previous research, to clone the genes regulating of key factors and to elucidate its function by transformation in order to obtain further understanding of the starch-sugar conversion mechanism with purpose to provide academic evidences and technological supports for improving the processing quality of potato tubers. The main results obtained are as following:1. The experiment was designed, via storing potato tubers of cvs. E-Potato 1 and E-Potato 3 in different temperature, to explore the variation patterns of reducing sugar and total sugar (TS) contents and enzyme activities that involved in the pathway of starch-sugar metabolism aiming at to identify the main factors that influence the chip color. The results showed that low temperature in storage was a main factor that accelerated the accumulation of RS of the stored tubers and there was a very significant linear relationship existed between RS content and chip color index of the tubers. Further analysis elucidated that when tubers stored at 4℃, the activities of ADP glucose pyrophosphorylase (AGPase), UDP glucose pyrophosphorylase (UGPase) and sucrose synthase (SuSy) were negatively exponential to the RS content significantly while that of acid invertase and alkaline invertase were significantly linear to RS content. It suggested that these enzymes could play main roles in the cold sweetening of potato tubers through regulating starch-sugar metabolism.2. The invertase inhibitor is a protein family with low homology. Activity regulation of the invertase inhibitor to invertase may bu efficient to reduce theaccumulation of the reducing sugars through its effect on the activities of invertases. A full cDNA sequence of vacuolar invertase inhibitor gene was amplified from leaves of tobacco Tl strain by RT-PCR and using the primers designed on the base of published sequence of NCBI. Nt-inhh (vacuolar invertase inhibitor), assession number in GenBank AY594179. Analysis of Blast showed that the cloned cDNA shares 100% homology with Nt-inhh of tobacco. The sense-orientation vectors containing Nt-inhh cDNA regulated by 35S or patatin promoter were constructed and introduced successfully into potato E3 by the PCR detection and northern blot analysis. The transgenic tubers were harvested and stored at 4°C or 20 "C for 1 month in order to analyse the variation in reducing sugar content and vaculoar invertase (VI) activity. The results showed that VI activity are inhibited by range from 22.7% (strain A-3) to 78.7% (strain B-13) and RS content decrease by range from 20% (strain A-17) to 80% (strain A-30). This experiment further identified that the activity of invertase was significantly linear to RS content.3. Invertase inhibitor is a protein family found in higher plant speices. A full length cDNA encoding invertase inhibitor was isolated by RT-PCR combined with 5' RACE from potato tubers of cv. JH and designated as St-inh. The encoding region of St-inh is of 663bp encoding a protein of 221 amino acids. The DNA fragment including St-inh cDNA was cloned into the vector pET28a (+) and expressed successfully in E. coli. Co-incubation of the proteins produced by St-inh in E. coli and the invertase extracts from potato tubers of cv. El and JH and tomato fruits showed that the invertase activities of potato tubers and tomato fruits decreased by 34.3%, 21% and 33.8% respectively. Moreover, the reaction between expression proteins and CWI and VI extracts from cv. E3 leaves resulted in the decrease of CWI and VI activities. These results indicated that products of St-inh protein had a function of invertase inhibitors. The analysises of the nucleotide and amino acid sequences using T-COFFEE demonstrated that St-inh cDNA was of over 95% homologous to Kunitz-type C and there was a typical domain of Kunitz-type protein [L, I, V, M]-X-D-X-[E, D, N, T, Y]-[D, G]-[R, K, H, D, E, N, Q]-X-[L, I, V, M]-X (5) -Y-X-[L, I, V, M]. Therefore, it was conjectured that St-inh could be a member of Kunitz- type gene family. Clustalw analysis for amino acid sequences showed that invertase inhibitor is a protein family with very low homology. St-inh has 14.2% to 21.0% homologous to Ara-inh^ Nt-inhh^ Nt-inh^ To-inh^ Pa-inh^ Ip-inh^ Ci-inh and Zm-inh, indicating that St-inh is a new invertase inhibitor gene from potato.4. Low temperature promoter (LTP) was isolated from E3 genomic DNA by PCR. Two Agrobacterium transformation vectors were constructed, which contained a sense St-inh gene under the regulation of 35S and LTP and introduced successfully into potatoE3 by the PCR detection and northern blot analysis. The analysis of RS content and invertase activities from the tubers stored at 4°C and 20 °C for 1 month indicated that the maximal extent of RS decreased by 80.3% (Strain E-l) and VI activity decreased by 54.2% (strain D-7) with average reduction in RS content by 21.0% and VI activity by 19.0%. This experiments further identified that the activity of VI was significantly linear to RS content. The results reconfirmed that invertase is a main factor influencing low temperature sweetening which can be altered by regulating the expression of the invertase inhibitor, hence reducing the content of RS. Northern blot results revealed that the expression of St-inh increase especially in the the control of LTP but which did not lead to the difference of RS inhibition compared with 35S, suggesting that either constitutive or induced expression of St-inh had similar function in regulating RS accumulation of stored potato tubers.