马铃薯脂氧合酶POTLX-1基因RNA干扰载体的构建及遗传转化
发布时间：2021-08-19 07:38
植物脂氧合酶基因是一个多基因家族,它参与植物体内茉莉酸等生长调节剂的生物合成。植物对逆境胁迫的响应及其组织器官的膨大均可诱导多种脂氧合酶基因的表达。前人研究表明,马铃薯脂氧合酶基因POTLX-1参与对马铃薯块茎生长和发育的调节,但该基因是否参与马铃薯块茎发芽和块茎休眠过程仍不清楚。因此,研究POTLX-1基因在马铃薯生长发育阶段尤其是块茎发芽过程中的功能对阐明POTLX-1基因功能的多样性和复杂性具有重要的意义。为了研究脂氧合酶在马铃薯发芽过程中的功能,本研究构建了POTLX-1基因RNA干扰载体,并进行了农杆菌介导的马铃薯茎段遗传转化实验,同时,使用萘普生（脂氧合酶的抑制剂）和茉莉酸（脂氧合酶途径的中间产物）,预测脂氧合酶在马铃薯块茎发芽和薯块形成中的功能。获得的主要实验结果如下:1.通过TOPO克隆,将POTLX-1基因干扰片段连入pENTR,成功构建入门载体pENTRPOTLX-1。2.根据GATEWAY CLONING TECHNOLOGY进行LR重组,将入门载体pENTR-POTLX-1连入pHELLSGATE12,成功构建干扰表达载体pPOTLX-1-RNAi。3.采用冷融...
【文章来源】：兰州理工大学甘肃省
【文章页数】：80 页
【学位级别】：硕士
【文章目录】：
ABSTRACT
摘要
ABBREVIATIONS
CHAPTER1.INTRODUCTION
1.The function of lipoxygenase gene family in solanaceae plants development
1.1.Studies on lipoxygenase gene function in solanaceae plants
1.2.Solanaceae lipoxygenase gene identity and the analysis of motifs organization of lipoxygenase gene system in solanaceae
1.3.Lipoxygenase pathway and products
1.4.Lipoxygenase gene family is involved in maturation and development of solanaceae plants
1.5.Lipoxygenase function in defense and environmental stress of solanaceae plants
1.6.Previous studies on lipoxygenase(POTLX-1 Gene)in potato sprouting
2.RNAi technology
2.1.RNAi mechanism
2.2.Methods for constructing RNAi vectors
2.2.1.Constructions of RNAi vector by traditional restriction enzyme ligation
2.2.2.Gateway cloning methods to construct RNAi vectors
3.Plant genetic transformation technology
3.1.Binary agrobacterium tumefaciens vectors
3.2.Hormone regulates tissue regeneration during potato genetic transformation
3.3.Antibiotic concentration affects transgenic plants regeneration and agrobacterium elimination.
4.Naproxen(lipoxygenase inhibitor),JA and methyl jasmonate inhibits sprouting of potato tuber disc and affect tuberization
5.The objective and significance of this study
6.Research experimental procedure
CHAPTER2.MATERIALS AND METHODS
2.1.Experimental methods
2.1.1 Potato tissue culture
2.1.2 Extraction of total RNA from the potato tissue
2.1.3 cDNA was obtained by reverse transcript kit
2.1.4 Amplification of the interference cloning fragment and RNA template
2.2.Construction of RNA interference expression vectors for POTLX-1 gene
2.2.1.Downregulation of POTLX-1 with RNAi
2.2.2.LR Cloning or LR Reaction(Entry clone(pENTR-POTLX-1) +Destination Vector(pHELLSGATE12) –Expression clone(pPOTLX-1-RNAi)
2.2.3.E.coli transformation
2.3.Potato genetic transformation
2.3.1.Transformation of potato tuber stem with antibacterial genes containing our silenced gene of interest
2.3.2.Preparation of freeze/thaw-competent agrobacterium cells
2.3.3.Transformation of agrobacterium through freeze/thaw method
2.3.4.Freeze-thaw method:p POTLX-1-RNAi vector was transferred to agrobacterium tumefaciens GV
2.3.5.Studying the transgenic plants
2.4.Prediction of the transgenic plant’s behavior by naproxen(LOX activity inhibitor)
2.4.1.Stock solutions preparation
2.4.2.The tuber disc eyes treatment with naproxen(LOX inhibitor),methyl jasmonate,gibberellic acid
2.4.3.Atlantic potato stems cultured in MS medium supplemented with jasmonic acid,methyl Jasmonates and naproxen
CHAPTER3.RESULTS AND ANALYSIS
3.1.Construction of pPOTLX-1-RNAi vector
3.1.1.Extraction of total RNA from plants
3.1.2.RNAi interference fragment amplification
3.1.3.pENTR-POTLX-1 entry Clone vector construction
3.1.4.Ligation of digested pENTR-POTLX-1 and pHELLSGATE12 carrier vector
3.1.5.PCR detection of recombinants
3.1.6.Restriction enzyme analysis of the expression vector
3.2.Potato transformation
3.2.1.Detection of pPOTLX-1-RNAi in agrobacterium tumefaciens
3.2.2 Callus regeneration from the potato stems
3.3.Prediction of the transgenic plant’s behavior by naproxen(LOX activity inhibitor)
3.4.Jasmonic acid,methyl jasmonate and naproxen affect tuberization of favorita potato stolons cultured in vitro
CHAPTER4.DISCUSSION AND CONCLUSION
4.1.DISCUSSION
4.1.1.Construction of pPOTLX-1-RNAi vector
4.1.2.Potato transformation
4.1.3.Preliminary studies about POTLX-1 and what they indicated
4.1.4.Naproxen(Lipoxygenase Inhibitor)and methyl jasmonate inhibits sprouting of potato tuber disc
4.1.5.LOX activity as well as JA expression affect tuber initiation in vitro and the tuber skin color
4.2.CONCLUSION
CHAPTER5.SUMMARY AND FUTURE DIRECTIONS
5.1.Summary of the results
5.2.Future directions
REFERENCES
DEDICATION
ACKNOWLEDGEMENTS
本文编号：3351003
【文章来源】：兰州理工大学甘肃省
【文章页数】：80 页
【学位级别】：硕士
【文章目录】：
ABSTRACT
摘要
ABBREVIATIONS
CHAPTER1.INTRODUCTION
1.The function of lipoxygenase gene family in solanaceae plants development
1.1.Studies on lipoxygenase gene function in solanaceae plants
1.2.Solanaceae lipoxygenase gene identity and the analysis of motifs organization of lipoxygenase gene system in solanaceae
1.3.Lipoxygenase pathway and products
1.4.Lipoxygenase gene family is involved in maturation and development of solanaceae plants
1.5.Lipoxygenase function in defense and environmental stress of solanaceae plants
1.6.Previous studies on lipoxygenase(POTLX-1 Gene)in potato sprouting
2.RNAi technology
2.1.RNAi mechanism
2.2.Methods for constructing RNAi vectors
2.2.1.Constructions of RNAi vector by traditional restriction enzyme ligation
2.2.2.Gateway cloning methods to construct RNAi vectors
3.Plant genetic transformation technology
3.1.Binary agrobacterium tumefaciens vectors
3.2.Hormone regulates tissue regeneration during potato genetic transformation
3.3.Antibiotic concentration affects transgenic plants regeneration and agrobacterium elimination.
4.Naproxen(lipoxygenase inhibitor),JA and methyl jasmonate inhibits sprouting of potato tuber disc and affect tuberization
5.The objective and significance of this study
6.Research experimental procedure
CHAPTER2.MATERIALS AND METHODS
2.1.Experimental methods
2.1.1 Potato tissue culture
2.1.2 Extraction of total RNA from the potato tissue
2.1.3 cDNA was obtained by reverse transcript kit
2.1.4 Amplification of the interference cloning fragment and RNA template
2.2.Construction of RNA interference expression vectors for POTLX-1 gene
2.2.1.Downregulation of POTLX-1 with RNAi
2.2.2.LR Cloning or LR Reaction(Entry clone(pENTR-POTLX-1) +Destination Vector(pHELLSGATE12) –Expression clone(pPOTLX-1-RNAi)
2.2.3.E.coli transformation
2.3.Potato genetic transformation
2.3.1.Transformation of potato tuber stem with antibacterial genes containing our silenced gene of interest
2.3.2.Preparation of freeze/thaw-competent agrobacterium cells
2.3.3.Transformation of agrobacterium through freeze/thaw method
2.3.4.Freeze-thaw method:p POTLX-1-RNAi vector was transferred to agrobacterium tumefaciens GV
2.3.5.Studying the transgenic plants
2.4.Prediction of the transgenic plant’s behavior by naproxen(LOX activity inhibitor)
2.4.1.Stock solutions preparation
2.4.2.The tuber disc eyes treatment with naproxen(LOX inhibitor),methyl jasmonate,gibberellic acid
2.4.3.Atlantic potato stems cultured in MS medium supplemented with jasmonic acid,methyl Jasmonates and naproxen
CHAPTER3.RESULTS AND ANALYSIS
3.1.Construction of pPOTLX-1-RNAi vector
3.1.1.Extraction of total RNA from plants
3.1.2.RNAi interference fragment amplification
3.1.3.pENTR-POTLX-1 entry Clone vector construction
3.1.4.Ligation of digested pENTR-POTLX-1 and pHELLSGATE12 carrier vector
3.1.5.PCR detection of recombinants
3.1.6.Restriction enzyme analysis of the expression vector
3.2.Potato transformation
3.2.1.Detection of pPOTLX-1-RNAi in agrobacterium tumefaciens
3.2.2 Callus regeneration from the potato stems
3.3.Prediction of the transgenic plant’s behavior by naproxen(LOX activity inhibitor)
3.4.Jasmonic acid,methyl jasmonate and naproxen affect tuberization of favorita potato stolons cultured in vitro
CHAPTER4.DISCUSSION AND CONCLUSION
4.1.DISCUSSION
4.1.1.Construction of pPOTLX-1-RNAi vector
4.1.2.Potato transformation
4.1.3.Preliminary studies about POTLX-1 and what they indicated
4.1.4.Naproxen(Lipoxygenase Inhibitor)and methyl jasmonate inhibits sprouting of potato tuber disc
4.1.5.LOX activity as well as JA expression affect tuber initiation in vitro and the tuber skin color
4.2.CONCLUSION
CHAPTER5.SUMMARY AND FUTURE DIRECTIONS
5.1.Summary of the results
5.2.Future directions
REFERENCES
DEDICATION
ACKNOWLEDGEMENTS
本文编号：3351003
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