Studies On A Blue-light Oversensitive Mutant Har1 In Sorghum And The Involvement Of Cryptchrome 2 In The Blue-light Response

Using wild type (R111) and a sorghum mutant har1, which was obtained through space flight, we investigated photomorphogenesis of the sorghum seedlings under blue light. Cryptochrome 2 (CRY2) gene was cloned, identified and analyzed from both Rllland harl. The 3-end of sorghum CRY2 cDNA was inserted to expression vector pET32a and the fused protein was expressed in E.coli. Polyclonal antibody against sorghum CRY2 protein was created. The mechanism of R111 and harl seedlings responding to blue light was investigated by expression analysis using antibody against CRY2. The results are as follows:1. Photomorphogenesis of R111 and harlThe seeds of R111 and harl were germinated in darkness and 3-day-old etiolated seedlings of R111 and harl were transferred into blue light (20molm-2s-1) under 12L/12D cycles and grown under this condition for a period of time before harvesting.1). The elongation of mesocotyl and coleoptile of har1 were inhibited under blue light and the length of mesocotyl and coleoptile in har1 were decreased 31.2% and 22.5% respectively. The leaves of harl were emerged earlier than those of R111 and the length, width and surface area of the 1st leaves in harl were decreased 29.3%, 18% and 34.9%, respectively after 7 d irradiation under blue light. The 2nd leaves of harl showed a less inhibition in leaf growth comparing to the 1st leaves and the leaf area had no significant inhibition after 7 d irradiation.2). The anthocyanin accumulation of harl mesocotyl and coleoptile was higher than that of R under blue or white light. But red light had no effect. R had no significant changes in anthocyanin content after blue light irradiation but anthocyanin content in harl mesocotyl reached the peak at 48 h after blue light irradiation and was 6.57 fold as those in R.3). The above differences of harl seedlings from R could be minimized when seedlings were treated with GA3.4) Chlorophyll content was increased in harlunder blue light and the development of chloroplast was faster in har1 than that in R during de-etiolation.All the phenotypes observed above provided evidence that harl was oversensitive to blue light and the blue light responses might be mediated by cryptochrome.2. Cloning and sequence analysis of CRY2 from sorghum.1). The sorghum CRY2 cDNA was 2460 bp in length and it contained an open reading frame that encoded a deduced 690-amino acid protein with a calculated mass of 75.9 kD (GenBank accession No. AF545572).2). The CRY2 genomic DNA from sorghum was 3490 bp. Compared the genomic sequence with the cDNA sequence, the sorghum CRY2 gene contained three introns and four exons at the following nucleotide positions of the gene: 34-382, 954-1172, 1490-2196, 2299-3096. Intron splice donor/acceptor sequences were found as expected.3). Sorghum CRY2 showed 87% similarity with rice CRY2, 57% with tomato CRY2 and 45.5% with Arabidopsis CRY2. The similarity between sorghum CRY2 and rice CRY2 was much higher than that with tomato and Arabidopsis CRY2.4). No mutation was found in CRY2 gene of harl when compared the harl genomic and the cDNA sequence of CRY2 gene with that of R.3. Expression of CRY2 in E. coli and preparation of anti-CRY2 antibodyPartial CRY2 cDNA fragment corresponding to amino acids 472 to 690 was inserted into the vector pET32a. The resulting HIS-tagged protein was expressed efficiently in E. coli. strain DE3. The fused protein was purified from bacteria by SDS-PAGE, and used to prepare anti-CRY2 antibody in rabbits. The indirect ELISA result showed that the anti-CRY2 antibody was highly efficient.4. CRY2 was expressed in different organs in sorghum1) RT-PCR and Western blotting results showed that CRY2 gene was transcripted and CRY2 protein was expressed in mesocotyl, leaf and root in the dark.2) Western blotting result showed that CRY2 protein was degraded under blue light. After 7 hours in blue light, the level of CRY2 protein in the seedlings was very low.3) We detected CRY2 protein level in a light-dark-light cycle. Sorghum seedlings were grown for 7d under 12hlV12hD blue light (20mol m-2s-1). S...