Cloning And Genetic Transformation Of FcSOC1 Gene Homologous Gene From Fortunella Crassifolia Swingle

MADS-box transcription factor plays a crucial role in plant development especially controllng ulthe formation and development of floral organs. SOC1, encoding a MADS box transcription factor, integrates mulultiple flowering signals derived from photoperiod,temperature, hormone, and age-related signals, is an important flowering related genes.In recent years, for the study of SOC1 in Pisum sativum L.、Betula platyphylla、Zea mays、Oryza sativa、Vitis vinifera、Fragaria x ananassa Duch、apple、Paeonia delavayi、 Arabidopsis thaliana and other plant gradually founded and cloned successfully, to verify its function. But for aspects of citrus plant, at present the research were only on Mangifera indica L, study on SOC1 gene of Fortunella crassifolia Swingle has not been reported yet. In order to discuss the function of SOC1gene of Fortunella crassifolia Swingle,we cloned a pair of homologous genes from inflorecence of Rong’an kumquatwith homology-based cloning and analyzed their biological informations.(1) Biological information analysis showed that the cDNA of this pair of homologous genes were 660bp and 636bp, encode 220 and 212 amino acids, respectively, named FcSOC1a and FcSOC1b. The physical and chemical properties of this pair of homologous gene were analyzed and the results are as follows.The molecular weight of FcSOC1a is 25204.7 Da,theotical pI is 9.36,the total number of atoms are 3561,formula is C1085H1796N332O339S9, instability index is 53.23, aliphatic index is 76.73, grand average of hydropathicity (GRAVY) is-0.831. The molecular weight of FcSOC1b is 24829.24Da, theotical pI is 8.9, the total number of atoms are 3488, formula is C1067H1755N319O333S14, instability index is 53.73,aliphatic index is 77.31, grand average of hydropathicity (GRAVY) is-0.823.The N-terminal of the sequences of FcSOC1a and FcSOC1b considered is M(Met).Both FcSOC1a and FcSOC1bs’estimated half-life are 30 hours (mammalian reticulocytes, in vitro).The similarity of the amino acid sequences between this pair of homologous gene of Rong’an kumquatand the SOC1 gene of Citrus clementina is the highest and reached 98%. The amino acid similarity with Citrus sinensis can reach more than 90%.The similarity of FcSOC1a and FcSOC1b between Vitis vinifera can reach repectively 75% and 65%.The amino acid similarity of SOC1 gene between F. crassifolia.With Jatropha curcas, Tarenaya hassleriana, Arabidopsis thaliana are all above 50%.Phylogenetic tree analysis revealed that FcSOC1a clustered with Citrus Clementina, FcSOC1b clustered with Vitis vinifera. But FcSOC1a and FcSOC1b do not cluster together.Sequence alignment and phylogenetic tree anaysis indicated that both FcSOC1a and FcSOC1b deduced protein contained a conservative MADS-box and semi-conservative K domain.(2) Real-time fluorescent quantitative PCR indicated that FcSOC1a and FcSOC1b could been expressed in both vegetative and reproductive organs. The expression levels of FcSOC1a and FcSOC1b in vegetative organs is different, they both have a high level expression in flowers. In the vegetative,this pair of homologous gene are high expression in tender leaveas,tender stems,old leaves and old stems.However,there is almost no expression in stems and leaves of young trees. In the reproduction organs,the expression of FcSOC1a is significantly higher than that of FcSOC1b, but they both highly express in flower 1(1.0mm), flower 2(2.0-3.0), flower 6 (7.0mm), flower 7(8.0-9.0mm),flower bud,petal of flower 6 (petal 7),overy of the initial open flower(overy 8),torus of flower 7(torus 7). FcSOCla is expressed apparently in both overy of the initial open flower(overy 8) and torus of flower 7(torus 7), but FcSOC1b is only exprssed apparently in overy of flower 7(overy 6).The expression of this pair of homologous gene is not obvious in fruit. In 24 hours, FcSOC1 was expresssed in various of Rong’an kumquat. highe expression in flower than stems and leaveas, higher expression in the day than the night. It also showed that the expression in the flower has lower stability than that in the stems and leaves. Thieir expression characteristics indicated that they may play an important role in dominating the growth of flower, and this pair of homologous genes may have gene differentiation because they have different function in the growth of vegetative organs and different bioinformatics features.This pair of homologous gene have almost the same bioinformatics features, but there are also some difference. Fluorescence quantitative analysis showed that FcSOC1a and FcSOC1b were expressed in both vegetative organs and reproductive organs, and high expression in the stems,leaves,flower bud,inforescences,torus and ovary of flowers.Both FcSOC1a and FcSOC1b were expressed significantly in flowers, and the expresion rised sharply at the early stage (flower 2) and later stage (flower 6) of the development of flower. Thieir expression characteristics indicated that they may play an important role in dominating the growth of flower, and this pair of homologous genes may have gene differentiation because they have different function in the growth of vegetative organs and different bioinformatics features.(3) The eukaryotic expression vector Cam35S-gfp was successfully constructed.At present, the homologous genes have been transforming into Arabidopsis thalianaby Agrobacteriumtumefaciens-mediated method to screen its transgenic plants with resistance and conduct PCR detection and GUS staining.