Functional identification and analysis of SoSnRK2.1 and SoACLa-lgenes from sugarcane

摘要

干旱是影响甘蔗生长、发育、产量和质量的一种重要因素。ATP-柠檬酸裂解酶(ACL)和非酵解型蛋白激酶(SnRK2)是植物正常生长和发育关键调控酶。ACL催化作用柠檬酸在细胞质中转变成乙酰辅酶A，SnRK2在响应多种非生物胁迫方面发挥着重要作用。在ABA信号转导途径中，这两个基因参与植物非生物胁迫下的发育。我们研究了SoACLA-1与SoSnRK2.1在原核生物和真核生物中的调控机制。主要研究结果如下:　　1.根据SoSnRK2.1和SoACLA-1基因序列，我们克隆了这两个基因。使用特异性的引物扩增SoSnRK2.1和SoACLA-1基因的全长并且根据原核表达载体pET30a(+)和植物表达载体pRI101-ON，pRI101-AN和pUBTC设计限制性酶切位点的。基于序列分析表明SoACLA-1和SoSnRK2.1基因的完整开放阅读框长度分别为1272 bp和1002 bp。氨基酸序列分析表明，SoACLA-1和SoSnRK2.1与玉米和栽培稻中的同源基因有很高的同源性。　　2.SoSnRK2.1和SoACLA-1均在原核生物获得表达。将没有内含子的SoSnRK2.1和SoACLA-1基因插入到pET30a(+)载体，其中该载体包含T7启动子进行原核表达。原核表达结果显示重组的pET-SoACLA-1和SoSnRK2.1分别为46 kDa和38 kDa。此外pET-SoACLA-1和SoSnRK2.1重组蛋白以包涵体形式存在。SoACLA-1和SoSnRK2.1重组蛋白通过Ni2+ NTA柱亲和层析纯化和透析浓缩。此外，SoACLA-1和SoSnRK2.1进行响应干旱(PEG)处理，鉴定它们的干旱耐受性。　　3.利用模式植物烟草鉴定SoSnRK2.1的功能。将受CaMV35S启动子控制的SoSnRK2.1-GFP-pBI121转入烟草中。植株转化率达到75％。结果表明，转基因烟草植株比非转基因烟草有较低水平的离子泄漏(IL)，低浓度丙二醛(MDA)和H2O2含量。而且，转基因烟草植株体内超氧化物歧化酶(SOD)，过氧化物酶(POD)和过氧化氢酶(CAT)的三种抗氧化酶活性较高，叶绿素含量和叶片相对含水量（RWC）也比非转基因烟草高。SoSnRK2.1能够通过有性繁殖稳定传播到下一代。研究结果表明，转SoSnRK2.1基因的烟草的生长和形态学显示该基因的过量表达提高了烟草的耐旱性，说明SoSnRK2.1是甘蔗响应非生物胁迫并且是调控生长和发育的关键基因。　　4.通过转基因烟草鉴定甘蔗SoACLA-1基因的耐旱功能。将受CaMV35S启动子控制的SoACLA-1-pRI101-AN表达载体转入农杆菌，再转入野生型烟草植株。采用PCR方法检测转基因烟草的转化率达到56％。观察转基因烟草的形态和多种生理特性和生化指标，结果与转SoSnRK2.1基因的相似，过量表达甘蔗SoACLA-1基因可以增强烟草植株的耐旱性。结果显示，SoACLA-1基因与植物的耐旱性相关。　　5.成功获得转SoSnRK2.1基因甘蔗。将SoSnRK2.1连接到pRI101-ON载体，利用农杆菌介导转入ROC22和YL6胚性愈伤组织。通过PCR检测目标片段和NPTⅡ标记基因，ROC22和YL6转基因植株率分别为26.5％和16.7％。初步证明，转基因甘蔗植株对干旱胁迫有可能增强。　　6.成功获得转SoACLA-1基因甘蔗。甘蔗植株表达载体pUBTC与SoACLA-1构建成功并且使用用Ubi启动子控制，ROC22愈伤组织作为受体，使用农杆菌介导的遗传转化体系，转化率达到16％。转基因植株与对照植株进行表型的观察和对比。结果表明在干旱胁迫下，转基因植株的耐旱性明显比对照植株提高。

目录

声明

摘要

ABSTRACT

Abbreviations

Table of Contents

1．Introduction

1．1 Effecting of drought on plants

1．2 Genetic modification improved drought tolerance in sugarcane

1．2．1 Sugarcane conventional breeding situation

1．2．2 Developments and application of transgenic technology on sugarcane

1．3 Overview of SnRKs gene in plants

1．3．1 Structural Analysis of SnRKs family

1．3．2 Regulation and activity of SnRK2 gene in plants

1．3．3 Functional of SnRK2 in plant

1．4 Overview of ACLs gene in plants

1．4．1 Mechanism of ACLs

1．4．2 Functional of ACLs in plant

1．5 Objective

2．Cloning，prokaryotic expression，purification and function identify of SoACLA-1 and SoSnRK2．1 from sugarcane and preparation of antiserum

2．1 Materials and methods

2．1．1 Bacterial strain，media，and vector

2．1．2 Main reagent and equipment

2．1．3 Cloning the full-length SoACLA-1 and SoSnRK2．1 gene

2．1．4 Construction of expression vectors for SoACLA-1 and SoSnRK2．1 genes

2．1．5 Expression of the recombinant protein from E．coli

2．1．6 Purification of the recombinant protein from E．coli

2．1．7 Protein Concentration and monoclonai antibody preparation

2．1．8 Recombinant under PEG treatment

2．3 Results

2．3．1 Isolation and identification of SoACLA-J and SoSnRK2．1 genes

2．3．2 Molecular characterization of SoACLA-1 and SoSnRK2．1

2．3．3 Construction of the pET-SoACLA-1 and pET-SoSnRK2．1 expression Vectors

2．3．4 Expression and purification of pET-SoACLA-1 and pET-SoSnRK2．1

2．3．5 Concentration protein and preparation for monoclonal antibodies

2．3．6 Epression of SoACLAI and SoSnRK2．1 genes enhances PEG stress

2．3 Discussion

3．Overexpression of SoSnRK2．1 improved drought tolerance in tobacco

3．1 Materials and method

3．1．1 Plant material and bacteria stain

3．1．2 Experimental equipments and reagents

3．1．3 Transformation tissue cultures

3．1．4 Transgenic vector construction

3．1．5 Plant transformation and transgenic tobacco generation

3．1．6 PCR analysis

3．1．7 RT-PCR analysis expression of SoSnRK2．1 in transgenic tobacco

3．1．8 Southern blot analysis

3．1．9 Drought tolerance experiment of WT and transgenic tobacco plants

3．1．10 Measurement and analysis of RWC，IL，MDA accumulation in WT and transgenic plants

3．1．11 Measurement of SOD，POD and CAT activities，H2O2 and Chlorophyll content

3．1．12 Sub-cellular Localization of SoSnRK2．1

3．1．13 Transgene inheritance experiment

3．1．14 Statistical analysis

3．2 Results

3．2．1 Transgene construction

3．2．2 Transgenic tobacco plants generation

3．2．3 Detection of transgenic tobacco plants

3．2．4 OVer-expression of SoSnRK2．1 improves drought toleranee in transgenic plants

3．2．5 Transgenic plants via over-expression of SoSnRK2．1 increased RWC and declined MDA and IL content under drought stress condition

3．2．6 Over-expression of SoSnRK2．1 enhances chlorophyll content and reduces H2O2 concentration under drought stress

3．2．7 Over-expression of SoSnRK2．1 in transgenic tobacco plants increases activities of three antioxidants enzymes under drought stress

3．2．8 Transgenic plants inheritance experiment

3．2．9 Over-expression of SoSnRK2．1 in T2 transgenic plants improves drought tolerance

3．3 Discussion

4．SoSnRK2．1 gene transferred in Sugarcane improving drought tolerance

4．1 Materials and methods

4．1．1 Plant material and bacteria stain

4．1．2 Plasmid

4．1．3 Experimental equipments and reagents

4．1．4 Transformation tissue cultures conditions

4．1．5 Expressed vector construction

4．1．6 Tissue culture of sugarcane

4．1．7 Kill curve experiment

4．1．8 Agro bacterial infection，Co-cultivation and Sugarcane transformation

4．1．9 Drought tolerance of the WT and sugarcane transgenic plants

4．1．10 Statistical analysis

4．2 Results

4．2．1 Transgene construction of pRI-SoSnRK2．1 and its introduction into A．tumefaciens

4．2．2 The kill curve of G418 of callus

4．2．3 The kill curve of G418 of shoots

4．2．4 The kill curve of G418 of roots

4．2．5 Injection and CO-culture and generation of sugarcane transformation by Agrobacterium

4．2．6 Selection of putative transgenic sugarcane

4．2．7 Molecular analysis of putative sugarcane transgenic

4．2．8 Drought assay

4．3 Discussion

5．Overexpression ofACLA-1 gene from sugarcane and its enhanced drought tolerance in tobacco transgenic tobacco

5．1 Materials and methods

5．1．1 Plant material and bacteria strain

5．1．2 Experimental equipments and reagents

5．1．3 Transformation tissue cultures

5．1．4 Transgenic vector construction

5．1．5 Regeneration of transgenic tobacco

5．1．6 PCR analysis

5．1．7 RT-PCR analysis expression of SoACLA-1 in transgenic tobacco

5．1．8 Drought tolerance experiment of WT and transgenic tobacco plants

5．1．9 Statistical Analysis

5．2 Results

5．2．1 Transgenic vector construction and tobacco transformation

5．2．2 Transgene inheritance assay of SoACLA-1 transgenic tobacco

5．2．3 Drought stress assay for overexpresion of SoACLA-j gene improved drought tolerance

5．2．4 Overexpresion of SoACLA-1 gene enhances the RWC and decreases MDA and IL content under drought stress

5．2．5 OVerexpression of SoACLA-1 improved the activities of SOD，POD and CAT under drought stress

5．2．6 Overexpression of SoACLA-1 affect the contents of chlorophyll and H2O2 in sense transgenic plants under drought stress

5．3 Discussion

6．ACLA gene transfer mediated by Agrobaeterium tumefaci ensenhances drought tolerance in sugarcane

6．1 Materials and methods

6．1．1 Plasmid and bacteria strain

6．1．2 Plants material

6．1．3 Experimental equipments and reagents

6．1．4 Culture condition

6．1．5 Kill curve experiment

6．1．6 Transgenic vector construction

6．1．7 Preparation for A．tumefaciens suspension

6．1．8 Sugarcane transformation and selection assay

6．1．9 Analysis ofexpression of SoACLA-1 in transgenic sugarcane by PCR and RT-PCR

6．1．10 Drought tolerance analysis of WT and sugarcane transgenic plants

6．1．11 Statistical analysis

6．2 Results

6．2．1 The construction of pUBTC-SoACLA-1-GFP and its introduction into A．tumefaciens strain

6．2．2 The kill curve of PPT for shoots and roots

6．2．3 Sugarcane transformation system mediated by A．tumefaciens strain

6．2．4 Sugarcane transformation PCR analysis

6．2．5 SoACLA-1 transgenic plants improve tolerance drought under drought stress condition

6．3 Discussion

7．Conclusions and prospects

7．1 Conclusion

7．2 Innovation points

7．3 Suggestions and future prospects

References

Acknowledgements

Publication

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