Cloning And Functional Analyses Of LEA Gene Promoters Drought-Inducible In Barley And Rice

Drought and water deficit are a globle problem for agricultural production. To breed and utilize crop varieties resistant or tolerant to drought is the most economical approach for water-saving farming and buffering drought disaster. It is very important and necessary to investigate the gene promoters drought-inducible for enhancing efficiency of molecular breeding through transgenic technology, especially in evaluation, screening, modification and application of promoter. In this thesis, the isolation, cloning and functional analyses of promoters belonging to LEA (late embryogensis abundant protein) gene have been carried out from barley (Horcleum vulgare L.) and rice (Orizy sativa L.) by using molecular cloning and transient expression technique. The results are as following:1. Five LEA gene promoters have been cloned from barley variety Sahara and rice variety Jarrah using PCR techonolgy, including HVAls、Dhn4s、Dhn8s、rabl6Bj、wsil8j. Sequencing analysis showed that there are95.3%～99.8%consensus identities between these five and those of HVA1、Dhn4and Dhn8gene from barley varieties "Himalaya" and "Dicktoo" as well as from rice varieties "Toride"and "Josaeng Tongil" reported previously. Sequence differences were found between those promoters from different varieties. These five promoters are various in their number and type of cis-acting elements stress-responsive.2. In order to determine the expressional levels of the cloned promoters and their mutants,15expression vector constructs have been constructed,5for GFP reporter,5for GUS reporter;3for ABRE sequence base-substitution mutation and reporter as well as1for DRE/CRT sequence base-substitution mutation and reporter of HVA1promoter;1for ABRE sequence base-substitution mutation and reporter of wsi18js promoter.3. Barley seedlings, cultured barley calli and some organs were used for gene-gun transformation receptor so as to select the best candidate material for transient experiment. Studies on DNA concentration of the promoter expression vector for transient experiment, factors and treatments for inducing expression have also been conducted. A methodology has been established for rapid test and determination of expression level of the promoters stress-inducible, based on qualitative and quantitative analyses.4. Dry, ABA, NaCl, cold as treatments for inducing the different promoter’s transient expression and their effects have been investigated, the results indicated:1) Dhn8s showed non-specific and highest expression under dry, ABA and control treatments, which means it is a constitution promoter without specific requirement. Dhn4s、HVAls、rab16Bj and wsi18j promoters were induce-dependent and differed in their expression levels. Under ABA induction, their expression levels were in such an order: Dhn4s> HVAls> wsi18j> rabl6Bj. Response to dry treatment, they were in Dhn4s> HVAls> wsi18j> rab16Bj.2) HVA1s、Dhn4s、rab16Bj and wsi18j promoter being weak in response to cold and NaCl, they were different from each other. Comparatively, HVAls was strongest, wsi18j and Dhn4s weaker, and rabl6Bj without response to cold..5. DRE/CRT sequence base-substitution mutation of HVAls promoter showed that it was reduced in expression level (7.7%和10.9%down) under ABA and dry treatments. While under cold treatment, the mutant promoter expression level dropped to37.9%in comparison with the wild one, indicating the responsibility of DRE/CRT mainly for cold stress response. But DRE/CRT was still related to ABA and dry response.6. Base-substitution mutations on the responsive elements ABRE1、ABRE2和ABRE3sequences containing ACGT core motif in HVA1promoter showed that:the expression level of ABRE3mutant dropped by40%～50%under ABA and dry treatments; ABRE2+ABRE3mutant decreased by89%～92%; ABRE1+ABRE2+ABRE3mutant was the same as ABRE2+ABRE3mutant. These results proved that just ABRE2and/or ABRE3were high-efficient elements among the above three, ABRE1was not a real ABRE cis-acting element.7. Base-substitution mutation on the responsive element in wsi18j promoter showed that the wild and the mutant did not expressed under control treatment, while after ABA induction both of them expressed in way that the mutant was only1/4of the wild in expression level. This result proved that CE3-ABRE complex in Wsi18j played a major role in responsive expression. Analysis on responsive elements in wsi18j and wsi18promoters revealed that both have the same number and type of elements related to dry, cold and ABA response. Base deletion and distance change between CE3与ABRE elements in wsi18promoter may be the reason for why they are completely different in response to ABA. There is respectively one C base deletion flanking the ABRE sequence of CE3-ABRE complex.8. The innovation and usefulness on this research:1) The room of the methodology has been broadened for rapid test and determination of expression level of the promoters stress-inducible, which could be used for the efficient selection of promoters for transgenic molecular breeding. The method can be applied to study the relation and crosstalk between pathways in the transcription factor regulatory networks. It can also be utilized for investigate the function of and the interaction between sic-acting elements. Finally, it would be useful for confirmation of bioinformatic prediction on a promoter’s function.2) The characteristics and differences of LEA gene promoters have objectively been compared under the same backgroud.3) It was found that there be promoter like Dhn8s not inducible by stress but constitutively expressional in LEA gene family.