Construction of a yeast cDNA library using Centaurea cyanus petals of different colors
[Objective]The molecular regulation mechanism of both blue petal coloration and petal color variation among cornflower cultivars remains unclear.In order to further screen the key interaction proteins involved in regulating petal coloration by Y1H or Y2H method in the near future,the yeast cDNA library from cornflower petals of six cultivars with different colors was established by Gateway technology in the present study.[Method]The white,pink,red,blue,mauve and black cornflower petals were used to extract total RNA,followed by mRNA isolation and purification.After the synthesis of double-strand cDNA,the BP recombination and LR recombination were performed to obtain the primary and secondary library,respectively.Finally,the plasmids from secondary library were transformed into yeast Y187 competent cells to build the yeast cDNA library from cornflower petals of different colors.[Result]The quality identification of both the primary and the secondary library revealed that the library capacities were 1.3×107 CFU and 1.6×107 CFU,respectively,the recombination rate was both 100%,and the average length of insert fragment was both more than 1 000 bp.After transforming into yeast,the obtained cDNA library titer was 3.5×107 CFU/mL.A total of 24 yeast clones were chosen randomly for PCR detection and showed bright bands,i.e.,the recombinant rate was 100%.Moreover,the length of inserted cDNAs was longer than 1 000 bp.[Conclusion]A high quality of yeast cDNA library using cornflower petals of different colors is established,satisfying the standard of yeast library screen,which will provide material basis for research the molecular mechanism of both the blue petal coloration and the petal color variation among cultivars in the near future.
万方数据
维普
