Patatin SK24-1Gene Effects On Tuber Development

Patatin is a major storage protein of potato tubers. Its lipid acyl hydrolase (LAH) activity and oxidation resistance activity unlike the other tuber proteins. Previous studies showed that the LAH activity of Patatin could non-specificly catalyze kinds of acyl lipid hydrolysis reactions. When plants are infected by diseases or attacked by insects, patatin will be over-expressed to activate the defense system. Patatin has activities of anti-oxidation and anti-free radical, so it can also scavenge free radical and inhibit DNA damage mediated by hydroxyl free radical. In addition, expression of some members of the patatin gene family, it can elevate the tolerance of plant to abiotic stesses.In this study, we cloned a patatin gene, SK24-1. The results of genetic transformation demonstrated that SK24-1gene could affect potato tuber formation. The further studies by ELISA revealed that this phenomenon may have close relationship with the balance of jasmonic acid (JA) and GA3. Since the substrate, alpha linolenic acid, for jasmonic acid biosynthesis is released from plastid and protoplast membrane due to the existence of lipoxygenase. Many patatin proteins have lipid acyl hydrolase activity. Reasonably, a question raised is whether patatin is involved in the synthesis of JA by which to influence potato tuber formation?Based on the previous studies, present research is intended to clarify if SK24-1gene is involved in the process of tuber formation and to elucidate its potential regulatory mechanism. Firstly, we successfully isolated SK24-1protein in vitro and confirmed its acyl hydrolase activity. Through over-expression, and RNAi silencing of SK24-1gene or the gene family, in potato cv. E-potato3(E3), the variations in contents of MeJA and GA3and LAH activity were investigated to establish a relationship between SK24-1expression and tuberization.1. Constructed pGEX-SK24-1vector which could express SK24-1protein in vitro. SK24-1protein was expressed and purified. LAH activity was measured three times. Analysis demonstrated that SK24-1has LAH activity. The result of specific activity is73.228. 2. Constructed expression vectors pHellsgate-SAT24-1and pHellsgate-patatin by RNAi SK24-1gene and conserved sequence of patatin family with the constitutive promoter CaMV35S. Next, transformed potato cv. E-potato3. Finally,22transgenic lines by overexpression SK24-1gene,13transgenic lines by RNAi SK24-1gene and15transgenic lines by RNAi conserved sequence of patatin family were obtained.3. Use RT-qPCR technology analysis showed that the expression of SK24-1gene in overexpression transgenic lines more than E3potato. Transgenic lines by RNAi SK24-1gene had a slight decrease for SK24-1-Ri-E314line.There are significantly upregulated in seven transgenic lines.The transgenic lines by RNAi conserved sequence of patatin family showed that significantly lowered expression. The growth of some transgenic lines were effected, such as growth retardation, weak for pants.4. Determination of endogenous Hormones include MeJA and GA3showed that level of MeJA synthesis can be improved in tuber formation by SK24-1gene overexpressed. Compared with E3, the ratio of MeJA/GA3of2weeks,3weeks, in overexpression transgenic lines had significantly improved. Transgenic lines by RNAi SK24-1gene had no significant effect for MeJA synthesis. Jasmonic acid synthesis levels in transgenic lines by RNAi conserved sequence of patatin family had comparatively large change, but it had no significant change law.5. LAH activity of the transgenic lines were determined by extraction plants crude protein. Compared with E3, LAH activity increased in overexpression lines. LAH activity had no significant effect in all RNAi lines.6. Tuberization were observed and recored. Compared with E3, number of tubers, mean tuber weight, number of tubers per plantlet and percent of plantlets formed tubers had significant increased in overexpression transgenic lines. It had no significant effect in RNAi SK24-1gene lines, while, lines of RNAi patatin familiy showed that they had significant decrease. All transgenic lines tuberization were early than E3.7. By import or knockout SK24-1gene, regression analysis showed that it can affect the balance of MeJA and GA3to affect tuberization. It caused the content of endogenous MeJA change, but little effect on tuber formation. LAH activity were changed in transgenic lines, and were highly related to tuberization. The changing of LAH activity influence on the synthesis of MeJA, but but between them has low correlation. SK24-1gene can effect the synthesis of MeJA to a part extent.In this study, the results confirmed that SK24-1gene is involved in the process of tuber formation and elucidated its potential regulatory mechanism. SK24-1gene has LAH activity to mediate the biosynthesis of MeJA, and it involve in regulating tuber formation.