Two New Genes OsHSP81 And OsCaMBP From Rice With An Alternative Splicing Mechanism Under Abiotic Stress

Some abiotic stress such as drought, cold and salination can influence the crop's normal growth and development, and reduce its yield. So improve crop's abiotic stress tolerance can play a major role in the increase of yield to ensure food security. With the development of plant molecular biology, finding the new gene associate with abiotic stress has opened up a new era for crop breeding.As rice is considered one of the major crops, the development of new cultivars with enhanced abiotic stress-tolerance will undoubtedly have an important effect on global food production. The transgenic approach offers an attractive alternative to conventional techniques for the genetic improvement of rice cultivars. In recent years, an array of stress-related genes has already been transferred to rice to improve its resistance against abiotic stresses. Many transgenic rice plants with enhanced abiotic stress-tolerance have been obtained. But there still have many genes we do not find and isolate which can improve the rice's tolerance yet. Therefore, we focused on isolating some co-regulation factor of rice which can improve rice stress tolerance by molecular biology technology. The main results are provided as follows:1. Isolate the rice genes which induced by abiotic stress with fluorescent differential display.In order to discover a gene associated with abiotic stress, we utilize the fluorescent differential display (FDD) to screen the differential expressed gene between cold and drought stress in rice.We had obtained 954 differential expression gene fragment which induced by abiotic stress by subcloning and sequencing. About 200 gene fragment were induced by cold specific,～300 gene fragment over-expressed and～400 under-expressed in cold and drought stress. We choose two interesting gene fragment of them for further analysis because of the common character: alternative splicing. 2. Function analysis of alternative splicing gene 0sHSP81(1) A rice HSP90 family gene, OsHSP81, was cloning from rice treated with cold-stress by the fluorescence differential display (FDD) screening method. Its cDNA sequence (3395bp) contains an ORF encoding a 699 amino acids protein (80.2 kD). Meanwhile, we found another transcript OsHSP81-S was produced by alternative splicing. OsHSP81-S and OsHSP81 were located at the same gene locus. Without the ATPase domain, the OsHSP81-S and OsHSP81 encode the same protein sequence.(2) Analysis the OsHSP81 and OsHSP81-S's expression under different stress (low temperature, heat shock, drought, NaCl, GA₃, IAA and ABA) by semi-quantitative RT-PCR. The OsHSP81 and OsHSP81-S mRNA was already detected in control treatment, and the decrease of OsHSP81 and OsHSP81-S mRNA were apparent when rice after being exposed to low temperature, drought, GA3 and ABA, but not sooner than 8 hour. In heat shock, NaCl and IAA treatment, the expression of OsHSP81 increase very quickly, in contrast with OsHSP81-S expression smoothly. Furthermore, the result indicated that the two genes existed different expression level at the same treatment time.(3) By GST fusion protein system, we induced and purified the OsHSP81 and OsHSP81-S fusion proteins. ATPaes assay showed that OsHSP81 possessed ATPase, but OsHSP81-S not to have because of the ATPase domain deletion. Meanwhile, Geldanamycin can prevent OsHSP81 ATPase activity.(4) Screening proteins to interact with OsHSP81 and OsHSP81-S by yeast two-hybrid system. We got three proteins which names were OsPRP1, RBBI3-3, P450, and some unknown protein in rice. The interaction between the OsHSP81, OsHSP81-S and OsPRP1 were confirmed by transfer the insert library from the AD to the DNA-BD vector and vice versa, and then repeat the two-hybrid assay. The result support that the PRR of OsPRP1 interact with OsHSP81 and OsHSP81-S C-terminal.3. Cloning and Tissue-specific expression of the OsCaMBP gene in response to various treatments(1) We isolated OsCaMBP, a rice CaM-binding protein that contains an IQ domain at the N-tenninus. Its cDNA sequence (2094 bp) contains an ORF encoding a 569 amino acids protein, the predicted molecular weight was 63.2 kD. An IQ motif was found at the position of 140-150 amino acid, and IQ calmodulin-binding motif at the position of 133-162 amino acids. These motifs were the most typical for the structures belonging to calcium-free CaM binding proteins family. In addition, we found the alternative splicing of OsCaMBP under heat shock and low temperature treatment.(2) Semi-quantitative RT-PCR analysis showed that the expression of OsCaMBP was remarkably inducible not only with cold treatment, but also with heat-shock treatment. OsCaMBP was undetectable under normal condition, and induced not only with 1h cold treatment (8℃, 1h), but also with heat-shock treatment (42℃, 15 min), suggesting that this gene plays important roles in the signaling pathway in rice under both cold and heat-shock stresses.Using fluorescent differential display, we isolated many rice gene fragments which associated with abiotic stress. In this article, their expression pattern of two new genes OsHSP81 and OsCaMBP from rice with an alternative splicing mechanism under abiotic stress had verified. Because of these two genes' important character, the thorough studies of their stress-resistant and the two genes interacting mechanism will carry on.