Cloning And Functional Identification Of ADP-glucose Pyrophosphorylase Small Subunit Gene Of Potato

The potato {Solarium tuberosum L.) is the fourth food crop with multiple uses in the world. Among the precessing potato products, chips and fries are very fashionable. The harvested tubers of potato are normally 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) that can react with the free amino acids of the tubers when frying at high temperature. This resulted in a dark-colored, bitter-tasting and undesirable product unfitting for human consumption. Therefore, mechanism of "low temperature sweetening" (LTS) has become one of the basic topics for improving potato post-harvest quality. AGPase, which consists of two "regulatory" subunits and two slightly smaller "catalytic" subunits, is considered to be a key rate-limiting enzyme of suger-starch metabolism. The results of former research showed that the curve of starch synthesis was consistent with that of AGPase accumulating. If AGPase activity was inhibited, starch synthesis would consequently decrease, even cutout. However the relationship between RS and AGPase activity is still remaind unknow, particallarly, little information is available about the effects of AGPase activity on on the LTS. Therefore it has important theoretic and practical significance in regulating metabolism of starch and sugar to further elucidate the relationship between AGPase activity and starch and RS contents in potato tuber and the relationship between AGPase activity and LTS.The main purpose of the present research is to look into this aspect through cloning a subunit gene of AGPase (sAGP) and regulating AGPase activity via over-expression or inhibition of sAGP in potatoes by using sense or antisense/RNAi strategies governed by either constitutive CaMV 35S or tuber-speific CIPP promoter. Te main results obtained are as following:1. The complete sAGP cDNA, a specific band with length of about 1.9 kb, was amplified from the low temperature tubers of potato clone JH using RT-PCR combined with rapid amplification of cDNA end (RACE) strategy according to the fragment of sAGP conservative site. The sequence analysis showed that the cloned cDNA had a length of 1 854 bp, encoding a protein of 521 amino acids and had a 65 bp 5' untranslated sequence and a 219 bp 3' non-coding region including tow putative polyadenylation signals AATAA at 12bp and 19bp upstream of the poly(A) (Genbank accession no. AY 186620). The clone was subcloned into the expression vector pMETa-B after PCR mutation. The recombinant plasmid was transformed into the Pichia methabolica pMAD16. The results of SDS-PAGE analysis indicated that sAGP expressed in the receptor and the AGPase activities of the induced recombinant yeast were increased significantly. All of these proved that the sAGP clone was a gene with full function, and its expression product had AGPase activity.2. Vectors of sense and antisense orientated sAGP gene controlled by either CaMV 35S or CIPP promoter, as well as the RNAi vector of sAGP were introduced into potato cultivars "E3-Pototo" and "Nanzhong552" to investigate the function of sAGP on regulating the starch-sugar metabolism in potato tubers. The analysis of 93 lines, which were selected from more than 140 transgenic lines, indicated that AGPase activity and starch content increased while RS content decreased in the transformed lines with over-expression of sAGP. In 19 out of 23 transgenic lines with sense sAGP under the CaMV 35S promoter, the average increase in AGPase activity and starch content wasabout 25% and 5-6%, respectively, but the RS content decreased by 20%. Similarly, AGPase activity and starch content of the 20 transgenic lines with sense sAGP under the control the of CIPP promoter were increased by 10% and 13%, respectively, while the RS content reduced by more than 35%. On the contrary, repression of sAGP resulted in decrease of AGPase activiy and starch content and increase in RS content. Antisense transformation of sAGP controlled by CaMV 35S led to average decline of both AGPaseactivity and starch content by 22% and increase of RS by about 36% in all of the 15 lines. When the antisense orientation was governed by CIPP promoter, the corresponding data were 20% decrease for both AGPase activity and starch content and even more than 65% increase for the RS content. The significance of sAGP in regulating AGPase activity was further confirmed by inhibition of sAGP through RNAi which reduced AGPase activity by up to 46% in all of the 27 transgenic lines. The present results prove that the AGPase activity is largely controlled by sAGP gene and modulation of sAGP expression can directly influence strach-suger conversion in potato tubers.3. In order to investigate the function of sAGP in controlling LTS, potato tubers, produced by the sense and antisense sAGP transformed potato lines with CIPP promoter, were analyzed after stored 60 d at 4°C and 20°C in 2003 and 2004. Under 4°C condition, the average increase in AGPase activity of the sense lines was 9.3% and decrease in RS content was 11.4%. At the same time, the average of activity decreased 8%, and RS content raised up by 60% in the antisense lines. These patterns were also true when the tubers stored at 20°C. The above findings indicate that sAGP had function under low temperature condition in improving that of LTS by regulating AGPase activity.4. According to the above results and the integrated characteristics of the plants, the transgenic lines of CA-11, CA-12 and CA-21 were selected for further test of processing quality, the common features shared by these lines were increase in AGPase activity and slowly accumulation of RS that allowed a direct frying of the stored tubers under 4°C for 60d (the up-limit of RS content for frying is < 0.4 g/100 g FW), and the plants with almost the same morphology identical to the receiptant variety. The selected lines have been put into the trials of the national transgenic plants safety assessment.5. Compared with the tuber-specific promoter CIPP lines, CaMV 35S promoter was superior in enhancing AGPase activity, and the occurrence frequency of lines with good trait was more than 3 times of the latter, and the former impacted on the RS directly. But CIPP seemed more efficient in regulating the RS content than CaMV 35S and about 3 times more desired plants were obtained from the CIPP driver transformation in relation to CaMV 35S. It is suggested that the tuber-specific expression of sAGP regulated the metabolism of tuber starch-sugar specifically, without changing the synthesis and transportation of photo- asiminates produce above ground.6. The analysis of Northern blot showed that the gene expression level of sense transgenic lines was increased differently and had no significant relativity with the AGPase activity. It conferred that both transcriptional and post-transcriptional modulation systems may involved in regulation of AGPase activity. The analysis of Southern blot showed that the anticipative results usually represented by single copy lines, and multiple-copy lines normally performed reversely. It was presumed that multiple-copy could affect on the post transcriptional regulation, and the single copy lines should be selected preferentially in early stage.