利用转基因番茄验证柚转录因子CgDREB对果实成熟性状的影响

摘要

柑橘是多年生木本作物，在全世界内都具有广泛的经济价值。然而目前关于柑橘果实成熟的研究还较少，其调控机制也了解不多。为了研究转录因子对果实成熟的调节作用，在HB柚中克隆CgDREB基因并且将该基因在番茄(Lycopersicon esculentum)中超量表达进行功能验证。亚细胞定位表明CgDREB蛋白定位在细胞核内。通过实时荧光PCR检测23个转基因系和野生型植株的表达模式，发现其中35S-CgDREBline5，35S-CgDREBline15和35S-CgDREBline19这三个株系该基因表达量较高。因此，本研究以35S-CgDREBline5和35S-CgDREBline19转基因株系为材料，研究番茄果实成熟过程中果肉和果皮中代谢物质的变化。利用GC-MS气质联用仪对转基因株系和野生型番茄的果肉和果皮中的初生代谢物进行了分析，结果表明两个转基因株系果肉和果皮的有机酸、糖类和脂肪酸含量相比野生型番茄显著升高。这些代谢物的显著变化表明CgDREB基因在果实成熟过程中起到了重要作用。相对野生型果实，在两个转CgDREB基因株系果实乙烯释放量也显著增加。本研究发现果实成熟与代谢物具有密切的关系，这些代谢物包括有机酸、糖类、脂肪酸以及乙烯释放，这一发现强调了果实成熟过程中这些代谢物的变化以及乙烯的释放的重要作用。以上结果进一步揭示了CgDREB基因与果实成熟过程的关系。

目录

声明

Contents

Abstract

摘要

Abbreviations

1．Introduction

1．1．Citrus overview

1．2．Citrus and tomato anatomical structure

1．3．Tomato as a model system to examine the fruit ripening processes

1．4．AP2／ERF transcription factors

1．4．1．The role of super family AP2／ERF in fruit ripening

1．4．2．DREB (dehydration responsive element binding) role in fruit ripening

1．5．The behavior of fruit ripening process in climacteric and non climacteric fruits

1．6．Primary metabolism and fruit ripening

1．7．The relation between ethylene production and metabolism process during fruit ripening in both climacteric and non-climacteric fruits

2．Material and methods

2．1．Materials

2．2．Molecular cloning of CgDREB gene

2．3．Plant transformation and regeneration

2．4．Molecular analysis of transgenic plants

2．4．1．DNA Extraction

2．4．2．PCR test

2．4．3．RNA isolation and cDNA synthesis

2．4．4．Semi-quantitative real-time RT-PCR

2．4．5．Southern blot analysis

2．5．Bioinformatics Analysis

2．6．Subcellular localization assay of CgDREB

2．6．1．GFP vector construct

2．7．Experimental design

2．8．Tomato fruit flesh and peel metabolites extracting for GC-MS

2．9．Ethylene measurement

2．10．Buffer and solution components

2．10．1．Components of the plasmid extraction solutions

2．10．2．Components of Agarose and SDS-PAGE Gel

2．10．3 Southern blot solutions

2．10．4．CTAB buffer

3．RESULTS

3．1．Isolation and characterization of CgDREB from Citrus grandis．Hirado Buntan Pummelo (HBP) fruit

3．2．Over-express of CgDREB in tomato plant

3．3．Subcellular localization and expression pattern

3．4．Transgenic plants 35S-CgDREB has a light fruit weight

3．5．Changes in organic acids accumulation in CgDREB overexpressed tomato fruits in flesh and peel

3．6．Changes in sugars，sugar alcohol and sugar derivatatives and other components accumulation in CgDREB overexpressed tomato fruits in flesh and peel

3．7．Changes in fatty acid accumulation in CgDREB tomato fruit in flesh and peel

3．8．Fruit ethylene production rate in transgenic lines and wild type

4．Discussion

4．1．CgDREB characterization，overexpression and phylogenetic analysis

4．2．The changes in organic acid in fruit flesh and peel during tomato fruit ripening

4．3．The pattern changes in sugars，sugar alcohol and sugar derivatatives accumulation in CgDREB overexpressed tomato fruits in flesh and peel

4．4．The Pattern changes in lipids accumulation in CgDREB overexpressed tomato fruits in flesh and peel

4．5．The Pattern changes in ethylene production between CgDREB transgenic tomato and wild type

Conclusion and Future Perspective

References

Annexes

Acknowledgements