Cloning And Function Analysis Of Clock Genes GmLCL1/2 And GmTOC1 In Soybean

The circadian clock genes ,which generate circadian rhythms and maintain its the running, exist in organisms ranging from prokaryotes to mammals. LHY,CCA1 and TOC1 are all key clock genes in Arabidopsis. The oscillator genes compose the feedback loops of circadian system.Three genes that GmLCL1,GmLCL2 and GmTOC1 were cloned from soybean KN18. The cDNA of GmLCL1,GmLCL2 and GmTOC1 are 2250bp,2247 bp and 1677 bp in length which encodes 750,749 and 559 amino acids.GmLCL1,GmLCL2 and GmTOC1 proteins are homologous to LHY,CCA1 and TOC1 in Castanea sativa and Phaseolus vulgaris. Based on the sequence analysis, the phylogenetic tree of GmLCL1,GmLCL2 and GmTOC1 were constructed with the MEGA3 software. The structure of GmLCL1,GmLCL2 and GmTOC1 proteins was also predicted both by internet servers and Spdbv softwares. The results showed that there are abundantα-helix and coils in GmLCL1,GmLCL2 and GmTOC1.Based on having cloned cDNA full length of GmLCL1,GmLCL2 and GmTOC1, several plant expression vectors including 35S::GmLCL1,35S::GmLCL2,35S::GFP:GmLCL1,were constructed and transformed to Arabidopsis. We have identified some positive Arabidopsis transformants by PCR.RNAi fragments of GmLCL1,GmLCL2 and GmTOC1 were cloned to make genes silent, and constructed plant expression vectors including RNAi-GmLCL1,RNAi-GmLCL2 and RNAi-GmTOC1,and then transformed them to Soybean.Moreover, the circadian expression patterns of GmLCL1, GmLCL2 and GmTOC1 in unifoliolates were coincided with that in whole plants. The morning genes of GmLCL1 and GmLCL2 had their peaks in the morning while evening gene GmTOC1 had its peak in evening. This kind of expression patterns of GmLCL1, GmLCL2, and GmTOC1 were kept even though plants were transferred to continuous light. That is reminiscences of the behavior of clock genes in other plants including Arabidopsis and suggested that clock genes may functionally conserved between various plants. Our results suggested that the genes cloned are the candidates of clock genes in soybean. The functions were waited to be uncovered.Futher,specific antibodies were developed for GmLCL2 and GmTOC1,The specific fragments of GmLCL2 and GmTOC1 gene were cloned into plasmid pDEST-17 by LR reaction. pDEST-GmLCL2 and pDEST-GmTOC1 were transformed into E.coliBL21Star. His-GmLCL2 and His-GmTOC1 fusion proteins were expressed under IPTG induction and further purified. Then the purified His-GmLCL2 and His-GmTOC1 fusion proteins were used to immunize rabbits. GmLCL2 specific and GmTOC1 specific rabbit antiserum were identified by Western blot, and results showed the antibodies have high specificity and affinity.All of this helped to study function of GmLCL1/2 and GmTOC1 and widen our views about the mechanisms of photoperiod and circadian clock.