Cloning And Expressing Of Cyclin-dependent Kinase Subunits In Malusⅹdomestica Borkh

The size and weight of fruit is two important economic characters,these two phenotype is by environmental and genetic coaction. The fruit size of apple is mainly effected by genotypes, besides, the size has a relationship with variety characteristics, fruit setting, water and fertizer level, and the potential of the fruit tree. Studying the development rules of fruit size and the genes related to fruit size has improtant theoretical and practical value to reveal the fruit size development rule, affecting factors of fruit size, and regulation measurements.In this survey, two varieties"Fuji"and"Ralls"as test materials. The fruits from different stages after the flower were collected, and the lengthways and transverse diameter and single fruit weight were measured. The results were as follows: (1) The growth curves of lengthways and transverse diameter in the two varieties were the same which looked like the'S'shape. The longitudinal elongation showed priority to in the early stage of fruit development, and the transverse diameter grew rapidly in the later stage of fruit development which resulted in the index of fruit shape was less than 1.0 from 56d after flower in the two varietyies. In the two varieties, the rapid increasement rates of lengthways diameter occurred in the 3-5 weeks after flower, the rapid increasement rate of transverse diameter occurred after the 8 weeks after flower; the index of fruit shape of Fuji was higher than of Ralls. (2) At the same stage, the fruit weight of Fuji was larger than Ralls. When the fruits were mature, the average fruit weight of Fuji was 250.38g, and that of Ralls was 150.91g. The rapidest increasement rates of fruit weight in Fuji and Ralls occurred in 1-3 week after flower, hereafter, Ralls had a rapider increasement rate of fruit weight in 5-8 week after flower, and Fuji had a rapider increasement rates of fruit weight in 11-14 weeks and three weeks before ripeness.Based on the CKS sequences in GenBank, the primers were designed for PCR amplication in this study. As the result, two 421bp amplication sequences were cloned from the carpels after the aflower in Fuji and Ralls, by blast in GenBank, their maximum identidy genes were CKS genes of plant. The two genes had high sequence identity with CO868566(CKS1;1) which came from apple variety Goldrush leaf, and CV466287(CKS1;2) from Goldrush fruit. So, the two sequences were named CKS1, the ORFs had 267 bp and coded 88 amino acids, the accession number for Ralls CKS1 was HQ 68473, and for Fuji was HQ 684734.The biologic informatics forecast that the two genes belong to CKS super family, their anterior amino acids from 5'end were highly conserved and the functional regions for CKS. The putative protein sequences had no transmembrane helices or signal peptide, so they were non-transmembrane proteins and non-secretory proteins.Quantitative real-time PCR showed that: The highest express quantity of MdCKS in Fuji and Ralls occurred in 21 days after flower, and higher in 21d-35d afer flower, then descended gradually, but in Fuji the express quantity at 161d after flower enhanced to the amount as the same as that of 35d afer flower, in Ralls at 98d and 161d after flower the express quantity enhanced to the amount as the same as that of 35d afer flower. In addition, in the same stage of fruit development, the express amount of Fuji was higher than of Ralls, and was 1.3-2.0 times of Ralls.The MdCKS expression vector was constructed, and would be used to test the function of MdCKS in fruit.