FUNCTIONAL ANALYSIS OF HSBP AND TWO OTHER STRESS RELATED GENE FAMILIES IN RICE(Oryza sativa L.)

摘要

Rice (Oryza sativa L) is considered as the most important food crop of the word.It has been the main source of human nutrition for more than 10 thousand years.Several environmental factors may affect rice yield, among which heat stress is the most important.Enormous increase in ambient temperature can cause significant reduction in grain yield in rice.Global warming has been proved to be a serious threat to the crop plants during previous century and an increase in global mean temperatures is expected by the end of current century in various regions of the world.This situation has propelled the attention of crop scientists towards the development of heat-tolerance, especially in case of cereals.In addition to being a major cereal crop, rice is considered as a model monocots plant for molecular studies, as well.Its genome has been fully sequenced, so it is very reliable to study stress responsive genes/proteins for understanding their involvement in stress response mechanism through molecular characterization and transgenic development.The results can be then simulated for other crops with great confidence.We characterized three gene families involved in stress mechanism of rice, viz.heat shock factor binding protein (HSBP), the Bcl-2-associated athanogene (BAG) and autophagy protein 6 (ATG6).A detailed functional analysis of rice HSBP genes was done in transgenic and mutant lines of these genes to elucidate their involvement in stress response mechanism.Furthermore, six BAG genes and three ATG6 genes were identified in rice using different bioinformatic tools and their expression profile was also studied under heat stress.The main results are described as follows:The role of HSBP genes in rice (OsHSBP) is first unveiled in the present study.Two homologs of OsHSBP (described as OsHSBP1 and OsHSBP2 in proceedings) were identified in preliminary study.Both proteins were predicted to contain potential coiledcoils, as well as highly conserved α-helix region.OsHSBP1 and OsHSBP2 proteins were found to be localized in cytoplasm, as well as in nucleus.Yeast two-hybrid assay revealed that both the genes showed self-binding ability, probably making trimer and hexamer.Both the genes exhibited similar functions as both were induced by heat stress.Heat shock (HS) of 42℃ for 1 h slightly upregulated the expression of OsHSBP1 and OsHSBP2.Their expression was significantly increased during the recovery phase and reached its maximum level at 2 h after recovery; however, it returned to the basal level at 4 h after recovery.Both genes were ubiquitously expressed in all the tissues.However, maximum expression of both the genes was observed in panicle.Furthermore, knock-down lines of both the genes showed significant seed abortion, hence can be considered to play an important role during seed development.The survival of over-expressed seedlings of both genes was significantly decreased, whereas those of knocked-down and mutant seedlings significantly increased, as compared to their respective wild-types during thermotolerance assay.Expression analysis of heat specific HSPs showed significant increase in the expression of OsHSP16.9, OsHSP17.5 and OsHSP70 in OsHSBP1 knockdown lines, while OsHSP82 in OsHSBP2 knock-down lines.This result indicated the negative regulation of heat tolerance by OHSBP1 and OsHSBP2.Furthermore, antioxidant activity was determined as an indicator of thermotolerance, which revealed a significant increase in case of knock-down lines.Catalase (CAT) and Peroxidase (POD) activity was significantly increased in knock-down lines of both the genes.Similar results were observed when expression of CA T and POD genes was analyzed.This result further confirms the involvement of OHSBP1 and OsHSBP2 in thermotolerance as negative regulators.Overall, these studies provide an insight into functional basis of HSBP homologs in rice and reveal that both genes are negative regulators of HSR and required for seed development in rice.In the present study, we identified six BAG genes in rice (OsBAG).These genes showed conservation in HSP70 binding domain region.Expression analysis of OsBAG genes through microarray data showed that OsBAG genes are up or down regulated under different biotic and abiotic stresses.OsBAG genes were analyzed for their expression under heat stress, as well as in different tissues in rice through real-time PCR.All OsBAG genes were expressed differentially in different tissues.The expression of OsBAG1, OsBAG4 and OsBAG6 genes was observed at maximum level in culm, while OsBAG2 expressed at very low level with maximum expression in panicle.Expression level of OsBAG3 was observed at maximum level in node and panicle, while OsBAG5 expressed only in root and leaf with highest expression in leaf.Analysis of OsBAG expression under heat stress showed that maximum expression of all the genes was observed at 1 h of heat stress treatment (42℃), after which the expression kept on decreasing, and it returned to the basal level at 24 h treatment (42℃).These results suggest that OsBAG genes might play an important role at the onset of heat stress.Further detailed study will explore the exact function of the members of this gene family and help to make understanding their role in HSR.Accumulation of misfolded proteins in abundance resulting from severe stress triggers autophagosome mediated autophagy, and autophagy related protein 6 (ATG6) plays a key role in autophagosome formation.Our analysis of ATG6/Beclin-1 proteins, revealed significant functional divergence while assessing across the species phylogenetic and evolutionary relationship.Presence of important stress related cis-acting elements in the promoters region of rice ATG6 genes depicted their involvement in abiotic stress responses.Real-time PCR analysis of ATG6 genes showed that OsATG6a showed maximum expression in panicle; OsATG6b expressed equally in all the tissues; while, OsATG6c showed maximum expression in root, intemode and panicle.Furthermore, the expression profiling of rice ATG6 genes based on microarray data, as well as by real-time PCR showed differential expression under different stresses suggesting their involvement in abiotic stresses (heat, cold and drought) responses.Analysis of co-expressed genes showed that most of them annotated to DNA repair pathways and proteolysis, etc.Collectively, these results suggest the involvement of OsATG6 genes in different stresses, and provide basis for further functional studies to investigate the biological mechanism of action of these genes under abiotic stresses in rice.Conclusively, we studied three gene families involved in stress response mechanism.HSBPs modulate the expression of HSPs by binding to HSFs, while BAG proteins modulate HSPs negatively or positively during stresses.If damage caused by stress is beyond the limits of protein repair mechanism, autophagy related proteins (ATG) are activated, ATG6 take part in the formation of autophagosome, where protein degradation takes place.Our studies revealed the involvement of these three gene families in stress response mechanism.OsHSBPs are found as negative regulators of thermotolerance as well as essential for seed development.In silico studies, phylogeny, protein structural modeling and expression analysis of OsBAG and OsATG6 gene families revealed that these families might be functional homologs in rice, and play important role in stress response mechanism.Overall, our results will provide an insight and understanding of the complex stress response mechanism in rice.

目录

声明

Table of Contents

ABSTRACT

List of Abbreviations

ChapterⅠ A cross talk between cell death and life：Protein repair，recycling and degradation

Abstract

1．1．Plant stress reponse

1．2．Heat shock proteins as molecular chaperones

1．3．Heat Shock Factors

1．4．Regulation of heat shock response

1．5．HSBP as HSF Regulator

1．6．BAG as co-chaperones

1．7．Unfolded Protein Response and Autophagy

1．8．ATG6／Beclin1 proteins

1．10．Recent research advances in rice on HSR

1．11．Study objectives

ChapterⅡ Heat Shock Factor Binding Protein 1 and 2 as negative regulators of heat shock response in rice

Abstract

2．1．Introduction

2．2．Materials and methods

2．2．1．Plant materials，growth conditions and treatments

2．2．2．Bioinformatic analysis of OsHSBP1 and OsHSBP2

2．2．3．RNA extraction and first-strand cDNA synthesis

2．2．4．Gene cloning and vector construction

2．2．5．Semi-quantitative RT-PCR

2．2．6．Quantitative real-time RT-PCR

2．2．7．Sub-cellular localization

2．2．8．Trans-activation and self-binding assay through yeast two-hybrid

2．2．9．Transgenic rice development and identification

2．2．10．Determination of antioxidant (CAT，POD and SOD) activity

2．2．11．Identification of OsHSBP1 and OsHSBP2 mutants

2．2．12．Phenotypic data

2．2．13．Primers

2．3．Results

2．3．1．OsHSBP1 and OsHSBP2 are functional homologs among plants and animals

2．3．2．Cloning of target gene

2．3．3．Expression analysis of OsHSBP1 and OsHSBP2 in rice tissues

2．3．4．Expression analysis of OsHSBP1 and OsHSBP2 under heat stress

2．3．5．Subcellular localization of OsHSBP1 and OsHSBP2

2．3．6．Trans-activation and self-binding assay of OsHSBP1 and OsHSBP2

2．3．7．Vector construction for over-expression and knock-down

2．3．8．Confirmation of transgenic and mutants

2．3．9．Thermotolerance assay

2．3．10．Analysis of antioxidant activity

2．3．11．Expression analysis of antoxidatnt (CAT and POD) and heat specific HSP genes in transgenic lines

2．3．12．Premature seed abortion

2．4．Discussion

ChapterⅢ Identification and characterization of The Bcl-2-associated athanogene (BAG) family in rice

Abstract

3．1．Introduction

3．2．Materials and Methods

3．2．1 Plant materials，growth conditions and stress treatments

3．2．2 Identification of BAG family in rice

3．2．3 Multiple sequence alignment and phylogenetic relationship

3．2．4 Protein structure model

3．2．5 “Digital Northern”analysis of BAG gene expression in rice

3．2．6 Expression analysis of OsBAG family in rice based on microarray data

3．2．7 Expression of OsBAG gene family in rice by real-time RT-PCR

3．3．Results

3．3．1．Identification of BAG family in rice

3．3．2．Genomic organization，chromosomal distribution and subcellular localization

3．3．3．Alignment and phylogenetic relationship

3．3．4．Comparison of the rice BAG BDs with template model

3．3．5．Digital northern analysis

3．3．6．Expression analysis of OsBAG family in rice based on microarray data

3．3．7．Expression pattern of six OsBAG genes in rice organs

3．3．8 Expression analysis of six OsBAG genes in rice seedlings under heat stress

3．4．Discussion

ChapterⅣ Identification and characterization of ATG6／Beclin-1 homologs in rice

Abstract

4．1．Introduction

4．2．Materials and Methods

4．2．1．Plant materials，growth conditions and stress treatments

4．2．2．Identification of ATG6 domain in rice

4．2．3．Multiple sequence alignment and phylogenetic relationship

4．2．4．Test for selection and functional divergence analysis

4．2．5．Expression analysis of OsATG6 gene family and co-expressed genes in rice based on microarray data

4．2．6．Quantitative real-time PCR

4．2．7．Analysis of stress-related cis-acting elements

4．3．Results

4．3．1．ATG6 homologs in rice genome

4．3．2．Evolution of ATG6 family

4．3．3．Expression profile of OsATG6 genes

4．3．4．Stress related Cis-acting elements and co-expressed genes

4．4．Discussion

Summary

Main Findings

Appendix

References

List of Publications

ACKNOWLEDGEMENT