Cloning And Functional Identification Of Four Stress-Induced Genes In Rice

Rice is one of the most important cereal crops in China. Environmental stresses, such as cold, drought and salinity, adversely affect the yield and quality of rice. Improvement of rice stress tolerance through genetic engineering has been proved to be an effective apparoach. Consequently, in this paper, four stress-related genes, such as CRK10、 OSK24、LRK1 and GT74E2, were screened from a cold stress expression gene-chip and RT-PCR under several abiotic stresses were also undertaken in Nipponbare variety. After that, their overexpression transgenic T1 plants and CPK10 T-DNA insertion mutant have been obtained through Agrobacterium-mediated method, followed by the abiotic stress tolerance identification under the cold, drought, and salinity stresses. The research results are as follow:1. Protein sequence homology analysis exhibited the four candidate genes induced by stresses, CRK10 encoded a Cysteine-rich receptor-like kinase, OSK24 encoded a SnRK1b subclass protein kinase, LRK1 encoded a Leucine-rich receptor-like kinase, GT74E2 encoded a auxin glycosylation transferase.2. Gene expression pattern analysis of rice seedling under stress treatments by RT-PCR indicated that, CRK10, GT74E2 were significantly induced by 4℃, NaCl and H2O2 stress while OSK24 showed a consistent expression pattern under NaCl, PEG and H2O2 stresses. LRK1 is induced only by NaCl stress.3. Over-expression transgenic plants through Agrobacterium-mediated method were developed, the T1 transgenic plants were confimed by using both RT-PCR and hygromycin-resistance tests. Both overexpression transgenic plants were as normal as wild type during tillering stage and heading stages in greenhouse culture conditions.4. Sensitivity assays of CRK10 T-DNA mutant responses to H2O2 and ABA stresses suggested that, the T-DNA mutant lines showed the more sensitive growth of roots to H2O2 stress while the over-expression lines are more tolerant than did the wild type. Meanwhile, ABA treatment shows no obvious impact on the germination of the T-DNA mutants and wild type, suggesting its involvement in the H2O2 signaling transduction process.5. Abiotic stress tolerance identification of the over-expression transgenic and T-DNA mutant seedlings revealed that, over-expression of CRK10 and OSK24 can enhance the tolerance of rice seedling to salinity and drought stresses. On the contrary, over-expression of GT74E2 decreases the tolerance of rice seedlings to cold, salinity and drought stresses. As a result, CRK10 and OSK24 positively regulate salinity and drought stresses whereas the GT74E2 gene had a negative effect.6. Inductions of GST-fused CRK10, OSK24 or LRK1 proteins demonstrated that, they all can be strongly induced. However, they formed the inclusion bodies by SDS-PAGE gel electrophoresis. Therefore, the induction condition required modification in the future.