Cloning And Expressing Analysis Of Cyclin-dependent Kinase B1in Malus X Domestica Borkh.

The fruit size and weight are the important characters in agriculture, and the twophenotypes are depended on the environment and genotype. The apple fruit size is correlativewith the cultivar’s genotype which is determined by the cell number and size. The fruit cellnumber is controlled by cell division in the early development of fruit, in which the cell cyclegenes control the cell number and then contribute to the fruit size. Studying the cell cyclegene expression during the fruit development can contribute to determining the importantgenes and their influencing factors which can regulate the fruit size, and to explaining theregulation rules and influencing factors for fruit size development. In this study, the applecultivars ‘Fuji’ and ‘Ralls’ were used as the materials for cloning the CDKB gene from thecarpel, and the expression for CDKB gene were identified during the fruit development and inthe callus. The main results are as follows:Based on the sequences of plant cell cycle dependent kinases in GenBank, the primerswere designed for amplifying the aimed genes, and the921bp segment was amplified andsequenced. The segment was divided into two kinds of sequence, their open reading framesare915bp coding304amino acids. By Blasting the nucleotide and protein sequences inGenBank, the maximum identity genes are all plant CDKB genes, and the maximum ESTsequences are two ESTs of apple cultivar Gala: CV085424（CDKB1;1）and EB138473（CDKB1;2）; moreover, bioinformatics shows that the two sequences belong to the plantCDKB subfamily, and have the specific motif PPTALRE for CDKB1. Therefore, the twosequences are named MdCDKB1;1and MdCDKB1;2, respectively, and the accessionnumbers in GenBank are JX041699in Fuji and JX041701in Ralls for MdCDKB1;1, andJX041700in Fuji and JX041702in Ralls for MdCDKB1;2.During the different development stages of fruit, Fuji and Ralls have similar expressiontraits for MdCDKB1;1. MdCDKB1;1expression maximum presents at21days after fullbloom (DAFB), up to28DAFB the expression quantity presents higher. After then, theexpression quantities reduce, but during7DAFB-35DAFB, the expression quantity of Rallsis0.33-1times more than that of Fuji which presents distinct difference; meanwhile during56DAFB-77DAFB, the expression quantity of Fuji is0.6-2times more than that of Ralls. Inaddition, Fuji and Ralls have similar expression characteristics for MdCDKB1;2during fruitdevelopment: all have expression maximum at7DAFB, and from7DAFB to35DAFB the expression quantities present higher in which the expression quantity of Ralls is0.16-1timesmore than that of Fuji, after then the expression quantities reduce. At119DAFB theexpression quantity of Fuji rose slightly, and is6times more than that of Ralls which showssignificant difference.The good quality callus was cultured using young leaf of Fuji and Ralls, and used tostudy the expression of MdCDKB1;1, MdCDKB1;2, and MdCKS1under different culturecondition, and the results showed as follows:(1) With NAA,6-BA, and NAA+6-BA on MSmedium, in contrast to basic MS (without exogenous hormones), after the callus was culturedfor one week, the expression of MdCDKB1;1, MdCDKB1;2, and MdCKS1was alteredremarkably. The expression levels of MdCDKB1;1, MdCDKB1;2, and MdCKS1were thelowest on the basic MS medium, and increased on MS medium added with exogenoushormones by0.8-2.5times,1-4times, and0.8-1times, respectively. On the three kinds ofhormone medium, the expression level for the three genes increased most, meanwhilecompared with MS+6-BA medium, on MS+NAA medium the expression levels forMdCDKB1;1and MdCDKB1;2were higher, but the expression level for MdCKS1showed nodifference. The results showed that auxin or cytokinin could increase the expression levels ofthe three genes, and with the coexistence of auxin and cytokinin, the expression levels of thethree genes were higher than that of one of them.(2) On the same medium, the MdCDKB1;1expression had no difference between Fuji and Ralls callus, but the MdCDKB1;2expressionof Ralls callus was higher than that of Fuji.The expression of MdCDKB1;1was restrained when the callus was cultured under lowertemperature (10℃) for28d, but the expression of MdCDKB1;1could come back undernormal temperature (25℃) after lower temperature for7d-14d. The expression ofMdCDKB1;2was not restrained when the callus was cultured under10℃for14d, but couldbe induced higher expression under normal temperature after cultured under10℃for7d-14d。The expression of MdCKS1reduced when the callus was cultured under10℃for7d, andcultured for14d under10℃, the expression of MdCKS1rose to the level of in normaltemperature; in addition, after cultured for7d-14d under10℃and then25℃, the expressionof MdCKS1rose.