Lecular Colning And Expression Analysis Of B-class MADS-box Genes From Ribes (Ribes Mandshuricum Maxim.)

Ribes mandshuricum, pertains to Dicotyledon Rosales Ribesiaceae, which are widely distributed in heilongjiang, jilin. Gansu province.The fruit has the value of high nutritional, rich in vitamin C, vitamin B2 and other vitamins and mineral trace elements zinc, iron, tin, potassium, etc., which are good for our immunity and anti-cancer. To explain the molecμLar mechanism of floral organ development, Three full length cDNA sequences of Ribes mandshuricum MADS-box genes from Ribes diacanthum as RdDAP3-1,RdDAP3-2, and RdDPI was isolated and characterized by using RACE. In addition, investigation was also made to analyze sequence characteristics and phylogenetic relationships based on bioinformatics methods. We analyzed the expression pattern of these genes in different organs of Ribes mandshuricum according to the semi-quantitative RT-PCR and real-time fluorescence quantitative PCR. The results of this study laid a solid foundation for further interpreting the action mechanism of Ribesiaceae MADS-box genes and molecular mechanism of floral development.(1)In this study we used Ribes mandshuricum flowers as test materials, obtained four fμLl length cDNA sequences contained open reading frames (ORF) by the method of RACE and named RdDAP3-1,RdDAP3-2,and RdDPI.(2)In order to interpretate the homology of the three genes to established MADS-box genes, We analyzed sequence characteristics and phylogenetic relationships of Ribes mandshuricum acid sequences corresponding to the genes using softwares of bioinformatics. The result of sequence characteristics analysis showed that RdDAP3-1,RdDAP3-2 and RdDPI encoded deduced proteins with230,236 and 216 amino acids residues respectively, all of the amino acid sequences of B class genes have a typical MIKC-type structural domain, Phylogenetic analysis indicated that MDAP3-1 and RdDAP3-2 belongs to the euuap3/DEF-lineage of B-function gene family. RbDPI belongs to the, PI/GIO-lineage of B-function gene family.(1)The results of Ribes mandshuricum qRT-pPCR show that RbDAP1 expression showed a trend of decline, RbDPI and RbDAG gene expression quantity increase gradually from the early stages of development until the end stage. RbDSVP gene low expressed during all stages of flower development. RbDAP3, RbDPI and RbDSVP gene were detected in the leaves and petioles.(2) The results of Ribes diacanthum Pall. qRT-pPCR show that RdFAP1 and RdMAP1 showed a trend of decreasing amount in five development period; RdFAG and RdMAG,RdFPI and RdMPI gene expression quantity increased, PI gene in male flowers expressed higher than female flowers, AG gene in female flowers expressed higher than male flowers. RdFAP3 gene expression rised from flowering early stage to inflorescence development early stage then reduced, gene expressed highest during the early stage of inflorescence development; RdMAP3 gene expression rised from flowering early stage to inflorescence development early stage then reduced until all the flowers completed development expression rised. PI gene was expressed different in two kinds of Ribes, in the Ribes mandshuricum (Maxim.) expression decreased in all the stage but rised in the Ribes diacanthum Pall.