Yeast Two-hybrid Analysis On Cryptochromes Of Soybean

Cryptochromes are photoreceptors which can absorbe near-UV/blue light to regulate the morphology,metabolic and phototropism changes.Cryptochromes widely exist in organisms.The study shown that many of the cryptochromes were proteins which had a molecular weight of 70-80 kD.There were two identifiable regions in cryptochromes:one is the N-terminal which is similar in sequence and structure to photolyases but lack DNA repair activity,the other one is C-terminal which is different from photolyases.The C-terminal is a conservative region for binding proteins so that the blu-light signal is passed downstream.The signal transformation of blue light involves the direct or indirect interactions between cryptochromes and other proteins.At present,the cryptochromes in many plants had been isolated and identifed primarily.Two cryptochrome genes in soybean had also been cloned and identifed primarily by 613 room of crop genmplasm reserch crop breeding in CAAS.Yeast two-hybrid is a new method used to study on the interactions between proteins in vivo.In order to study the functions and downstream signal transduction mechanisms of these two genes,yeast two-hybrid analysis of the two genes was made in this paper.Firstly,six vectors were constructed with the N-terminals,C-terminals and the full-length sequences of the two genes by PCR,enzyme digest and ligation. These vectors were named respectively as pGBKT7-CRY1N,pGBKT7-CRY1C, pGBKT7-CRY1Q,pGBKT7-CRY2N,pGBKT7-CRY2C,pGBKT7-CRY2Q. Matched with the empty AD vector pGADT7,these recombinants were transformed into yeast strain AH109 to assay their self-activation.The results showed that only pGBKT7-CRY2N + pGADT7could activate the expression of the reporter genes, therefore it could not be used for the yeast two-hybrid analysis.Secondly,the yeast two-hybrid cDNA library was screened with the bait of pGBKT7-CRY1C.After transforming sequencedly the bait and library plasmids into the AH 109 yeast cells by LiAc method,the transformed cells were amplified on plates of SD/-Leu-Trp.Then the tansformed cells were analyzed to see if they could activat all the three reporter genes of HIS3,ADE2,lacZ.The results showed that seventeen clones could activat all the three reporter genes.After that,PCR and sequencing were made about the seventeen positive clones.The results showed that five clones of them had right open reading frames and four of them had homologous sequences with the genes in photosysterms.At last,the five putative positive clones were transformed into AH109 yeast cells again to confirm the interactions.The results showed that there was possibility that these five positives could interact with the C-terminal of the cryptochrome one of soybean.However,the results still needed to be confirmed again.