Construction Of RNA Interference Vector And Genetic Transformation Of Potato Lipoxygenase POTLX-1 Gene

Lipoxygenase gene in plant is a multigene family,and it is involved in several biosynthesis of growth regulators such as jasmonic acid and traumatin.The enlargement of different plant sections and stress effect on the plant can initiate various types of lipoxygenase gene expression,in this study,we aimed at downregulating POTLX-1 lipoxygenase gene,in order to examine its function during potato tuber seeds sprouting and dormancy mainly because lipoxygenase gene POTLX-1 gene is previously known to be involved in the regulation of potato tuber growth and development.But there is a scarce knowledge that it might be involved in sprouting process or dormancy of the potato tuber.The main objective of this study is to clarify and understand the complexity and functional capacity of lipoxygenase gene in solanaceous plants during potato plant development stages especially throughout sprouting process and tuber dormancy.In this research,the potato POTLX-1 gene RNA interference vector has been constructed and genetically transformed into potato stem,in previous research POTLX-1 was identified as one of lipoxygenase gene responsible of potato tuber growth in size,the current results lead to the understanding of the function analysis of Lipoxygenase enzymes during potato sprouting and tuber dormancy process.The experimental analysis of this research was divided into three main parts: First,the construction of the RNA interference vector using Gateway cloning technology was performed by connecting the entry vector and the destination vector in order to obtain the expression vector.The successful construction of the interference vector was confirmed by sequencing the resulted plasmid.Second,the resulted plasmid was transformed into agrobacterium tumefaciens.Thirdly,using the lipoxygenase inhibitor known as naproxen(best inhibitor of lipoxygenase)and one of the products of lipoxygenase pathway: jasmonic acid,we have been able to predict the transgenic plants behavior during potato tuber seed sprouting,dormancy and tuberization.Overall the main outcomes of this study are as follow:1.Through TOPO Cloning,the interference fragment was transferred into pENTR,and the entry vector pENTR-POTLX-1 was successfully constructed.2.LR recombination was performed according to GATEWAY CLONING TECHNOLOGY.Thus,the interference expression vector pPOTLX-1-RNAi was constructed and inserted to the attL and attR loci of the entry vector and the destination vector pHELLSGATE12.The resulted recombinant plasmid fragment pPOTLX-1-RNAi was identified by restriction enzyme digestion.3.The pPOTLX-1-RNAi vector was transformed into agrobacterium tumefaciens GV3101 by freeze-thawing method.4.The lipoxygenase inhibitor naproxen was found to reduce the sprouting rate of the potato tuber disc of favorita potato species(Naproxen effectively inhibits potato tuber sprouts).5.Naproxen did not fully inhibit potato tuberization instead it stimulated roots development while Jasmonic acid actively stimulated tuber growth and the skin tuber color from JA treatments showed light yellow tuber skin while the tubers from other treatments were of white tuber skin.With the data presented in this study we can conclude that POTLX-1 gene is among the genes that regulate potato tuber dormancy and sprouting process as well as tuberization.