Effects Of StPP2C And StABF Genes On Potato Tuberization

Potato tubers, reproductive organs, are also important productive organs. Tuber formation is influenced by many factors, such as photoperiod, hormones, temperature and sucrose, implying many genes may be involved. In our lab, screening differentially expressed genes with light and sucrose treatment by RNA sequencing (RNA-seq) technology. However, factors that on tuber formation are still not very clear. In this study, four StPP2C responded to photoperid or sucrose and one StABF were cloned. And we investigated the relationship among five genes and microtuber formation. Then, it was performed to verify the function of StPP2C and StABF on potato microtuber formation. The main results are as follows:1. We screened four StPP2C from eight differential expression genes and one StABF by RT-qPCR. StPP2C1, StPP2C4are responded to sucrose, StPP2C6, StPP2C7, StABF are responded to photoperid. Then the genes were cloned. ORF was about1200bp. Cluster analysis showed the relationship of two sucrose-induced StPP2C genes was closed, the other two photoperid-induced StPP2C genes had close relationship.2. To analysis the responses of StPP2Cl, StPP2C4and StABF to sucrose, the E26microplant was cultivated in MS medium with2%sucrose in SD (8h/d) in vitro for3weeks and then added2%sucrose and10%sucrose liquid MS medium. The first day, we collected the materials every four hours, and then we collected them every one day to analysis the expression levels of the three genes. In our results, StPP2C1and StPP2C4presented an up-regulated expression in8%sucrose in the first day. It emerged difference at4hours under sucrose treatment between8%sucrose and2%sucrose. StPP2Cl’s peak difference appeared at one day under sucrose treatment, while StPP2C4’s difference reached the peak at20hours under sucrose treatment. After one day, difference was also very clear, but both of the genes expressions were decreased. So, it was proved that StPP2C1and StPP2C4were responded to sucrose very early. StABF displayed difference between2%sucrose and8%sucrose in the first day, but not in the second day. Therefore, we thought that it was responded to sucrose early.3. To analysis the responses of StPP2C6, StPP2C7and StABF to photoperiod, the E26microplant was cultivated in LD (16h/d) in vitro for3weeks and then transferred to LD and SD respectively. In two days, we collected the materials every4hours to analysis the expression levels of the three genes. In our results, StPP2C6and StPP2C7displayed circadian rhythms,8hours under SD treatment was the moment that entering into the dark, the expression of StPP2C6became increasing and to the peak at16hours then decreased. StPP2C7showed an increase in first8hours and a decrease in the second8hours in dark. While, StABF showed clear circadian rhythms. In LD, the expression of StABF increased in the light, while decreased when entering in the dark. In SD, the expression of StABF increased in the light, and then the level of expression began to decrease after12hours in the dark. What’s more, the trend of the expression in two days was the same.4. In order to investigate whether StPP2C had activities in vitro, we constructed expression vectors PP2C-GEX which could express StPP2C protein in vitro. StPP2C protein was expressed and purified. Protein phosphatases activities were measured. Analysis demonstrated that StPP2C1, StPP2C4, StPP2C6, StPP2C7could let substrate generate free phosphate about2.21nmol,1.05nmol,1.22nmol,2.62nmol. So we concluded that StPP2C had protein phosphatases activity.5. To understand the subcelleular localization of StPP2C, we constructed GFP-fusion vectors PP2C-RI. The results revealed that StPP2C1was maily localized in the Membrane, StPP2C4was mainly in the Nucleus, StPP2C6and StPP2C7have not been completed.6. We constructed expression vectors PP2C-GBK and ABF-GBK to analysis the interaction of StPP2C, StABF and SnRK1. Using Y2H proved that StPP2C6and StABF were interacted with SnRK1.7. To understand the effect of five genes on the potato microtuber formation, we constructed expression vectors PP2Ci and ABFi by RNAi and conserved sequence of genes with the constitutive promoter CaMV35S, then transformed them into potato E3. Finally, we have obtained positive transgenic lines. Every transgenic lines presented different down-regulated expression, the repression rate was up to99%. Treating the transgenic line and E3with sucrose and photoperid, and then six positive transgenic lines of each gene were selected to detect the No. of microtuber per plant and mean weight per plant. Compared with E3, StPP2C1and StPP2C4transgenic lines showed that the No. of microtuber per plant and mean weight per plant were decreased. And they were also increasd with the increasing of sucrose concentration. It was suggested that the two genes were induced by sucrose, and they might participate in the tuber formation. In StPP2C6, StPP2C7, StABF transgenic lines, the No. of microtuber per plant were lower than E3in SD while not in LD, thus, it indicated they might just play important roles in potato formation in SD.