Preliminary Functional Analysis Of BHLH Transcription Factor Os11g39000 In Rice

The bHLH transcription factors are a large family of eukaryotic proteins and its members are involved in a variety of important functions in plants and animals. At present, the function of many members of bHLH transcription factor family is still not clear in plants. In present paper, in order to investigate the effect of bHLH transcription factors on the growth and development of rice (Oryza sativa L.), a bHLH transcription factor Os11g39000 was specially selected to study its function via the engineering method of hybrid transcription factors. The main research results are as follows:(1) Bioinformatics analysis revealed that the transcription factor Os11g39000 contains a typical HLH domain but not a basic domain, indicating that Os1139000 belongs to a member of the atypical bHLH transcription factor family. Promoter sequence analysis found that the promoter region of Osllg39000 contains ABA, GA and IAA hormone response elements.(2) Yeast two-hybrid and X-gal chromogenic experiments found that the transcription factor Osllg39000 can form a homologous protein polymer. Random binding site selection demonstrated that Os11g3900O can bind to DNA, and its binding site was preliminarily identified as TT/CG/CACC/GT/C.(3) The wild type plants Kittake were treated by NaCl, PEG6000 and various hormones, respectively, and then the transcriptional levels of Osllg39000 were detected. It was found that the expression amount of Osl lg39000 changed most significantly responding to IAA treatment, indicating that the gene Osllg39000 may be involved in the auxin response pathways. Analysis of the expression pattern at rice seedling and heading stage discovered that the gene Osllg39000 mainly expressed in leaves and roots, which speculated that the gene may play a role in the developmental regulation of leaves and roots.(4) The recombinant plasmids Ubi::EAR-Osllg39000,35S::Os11g39000 and ProOs11g39000::Gus were constructed and transformed into rice via Agrobacterium-mediated transformation method, and finally the corresponding transgenic plants were obtained. After the molecular identification at the level of DNA and RNA, two Os11g39000 transcription-inhibited type positive transgenic lines, Ubi::EAR-Os11g39000-10 and Ubi::EAR-Os11g39000-11, were successfully screened. The hydroponic experiments found that, compared with wild-type plants, the transgenic plants Ubi::EAR-Os11g39000 exhibited much shorter roots and obvious defect in root development, which suggested that Os11g39000 was involved in the developmental regulation of roots.(5) The wild type and transcription-inhibited type transgenic plants Ubi::EAR-Os11g39000 were treated with various concentration of IAA, and the transgenic plants were found not to be sensitive to high concentration of IAA, indicating that inhibition of Os11g39000 transcriptional activity can result in the damage in rice response to auxin, and the transcription factor Os11g39000 have an important role in auxin signal transduction pathway. Q-PCR analysis further revealed that the transcriptional levels of auxin biosynthesis and signal transduction related genes, OsYUCCAl, OsYUCCA4, OsARF23 and OsARF24, increased significantly in transgenic plants, but no obvious change in polar auxin transport related genes OsPIN1b and OsPIN2. The results further demonstrated that the functional defect in Os11g39000 may affect the auxin biosynthesis and signal transduction, and but not for the polar auxin transport.In conclusion, the present results preliminarily suggested that transcription factor Os11g39000 is involved in the regulation of auxin biosynthesis and signal transduction, especially plays an important role in regulation of root development in rice.