Preliminary Study On OsBBX Genes Under Heat Stress In Rice

High temperature is an important factor restricting the rice growth and yield. It is important means to discover regulation of rice thermotolerance element and foster thermotolerance rice varieties for guarantee the stable yield of rice. B-box zinc finger protein (BBX) is a kind of important transcription factor, containing a B-box domain in amino-terminus and also feature a CCT (CONTANS, CO-like, and TOC1) domain in carboxyl-terminus, and play an important role in regulation of plant resistance. This study aimed at BBX zinc finger protein gene in rice (OsBBX) type ? subfamily, which only contains two B-box motif in amino-terminus, and carried out bioinformatics analysis and the preliminary study on the heat related functions, obtained the following results:(1) At first, through bioinformatics analysis we gained 10 OsBBX homologous genes, and found that several genes nucleic acid and protein sequences there is obvious difference in indica and japonica. By the cis-element analysis in promoter region of those this subfamily gene, we found that there are 5 members (OsBBX 1 OsBBX 13, OsBBX 16, OsBBX22, OsBBX30) contains a heat stress related components HSE, and out of 10 members containing ABRE, TCA and TGA hormones such as related elements and circadian rhythms related elements.(2) Secondly, we also analyzed the expression pattern of OsBBX family gene under the treatment of abiotic stress and light through chip data:when treated with the different photoperiod and temperature, the expression level of OsBBX22, OsBBX16, OsBBX30, OsBBX6, OsBBX13have no significant change. But interestingly, OsBBX4, OsBBX11, OsBBX14 have obviously circadian rhythmicity in that treatment, which suggested that these genes maybe critical for circadian rhythm and light response signalling in plant.By analyzed the chip results of heat shock, we found that the expression levels of OsBBX 14, OsBBX29, OsBBX22 were up-regulated, but OsBBX1 and OsBBX16 were down-regulated, revealed that these genes have high sensitivity in heat stock. Moreover, in the chip of drought stress, we found that the expression levels of OsBBX1 and OsBBX13 have obvious difference between drought-resistent and contral rice lines. Furthermore, there are no significant changes in expression quantity in this family under salt stress, means that this gene subfamily has low sensitivity responsed to salt stress.(3) Thirdly, by real-time quantitative PCR analysis of three members (OsBBX1, OsBBX13 and OsBBX30)of OsBBX, contain HSE element, under different heat treatment time in Nipponbare,9311 and N22 rice lines.The expression of OsBBX13 and OsBBX30 increased significantlyin N22 under heat stress, but decreased in Nipponbare and 9311. However, the expression of OsBBX1 increased under 12 h heat treatment in Nipponbare, decreased in 9311 decreases with heat treatment, but no significantly change in N22.(4)Moreover, by construdtion of theprokaryotic expression vectorpET-a-30 contained OsBBX30-CDS, we found that the survival rate of E.coli contain pET-a-30-OsBBX30 is higher than pET-a-30 under 50"C heat treatment. This result showed that OsBBX30 was associated with heat stress, and increased thermaltolerance.(5) Lastly, by constructed the expression OsBBX1-PUC1305 OsBBX genes associated with heat stress and OsBBX13-PUC1305 excessive interference and RNAi expression vector, and agrobacterium mediated transformation, we have obtained the T1 generation transgenic rice.