Cloning And Functional Element Analysis Of Promoter Region Of GPAT Gene In Lilium Regale Wilson

In today's society,the impact of environmental stress on plant growth and development has become a hot topic in biology.Using genetic technology and other means to improve plant resistance has become one of the methods.Promoters are the key to regulating gene expression.Therefore,cloning and construction of environmental inducible promoters are the fundamental ways to cultivate plant resistant varieties.Based on the results of recent years,promoters such as CaMV35S and maize Ubiquitin promoter have been widely used.However,the characteristics of these promoters determine their high level of expression throughout the growth and development of the plant.Not only will cause waste of resources,serious will make the plant dead,can not achieve efficient and adjusted(regular,fixed,quantitative)expression.Inducible promoters can induce plants to express at specific times and conditions,to avoid unnecessary waste of resources,and to reduce the deleterious effects on plants.Therefore,the induction and application of inducible promoters have become a new trend for researchers to chase.Based on the results of previous and research groups,The study selected Lilium regale Wilson as the experimental material,and cloned the promoter of GPAT gene of L.regale Wilson by using effective gene cloning technique.The main results were as follows:1.Based on the study of the GPAT gene sequence of Lilium regale Wilson,the gene specific primers were designed in the coding region of the genus,and the genomic DNA of L.regale Wilson was used as template to clone the GPAT gene promoter,A total of 1007 bp promoter sequence,named GPATp.2.The sequence analysis of the 1007 bp promoter sequence obtained by the step-by-step analysis was carried out by using NewPLACE and PlantCARE.The results showed that the sequence contained many TATA-boxes and CAAT-boxes,Many of the cis-acting elements andtissue-specific elements associated with environmental stress.3.The expression of GPATpl(954 bp),GPATp2(644 bp),GPATp3(386 bp),GPATp4(231 bp),and GPATp5(169 bp)were detected by GUS as the reporter gene.And were used to replace the 35S promoter in the plant expression vector pCAMBIA 3301,and five plant recombinant expression vectors were named GPATp1?5 respectively.4.Tobacco transformation:Agrobacterium tumefaciens-mediated transformation of tobacco leaf discs,the GUS gene as a reporter gene,the results showed:the group of GPATp5(169 bp)of leaf discs were not dyed blue,The expression of GPATp4(231 bp)was the weakest among the other four groups,suggesting that the fragment was the smallest in the other four groups,suggesting that the promoter fragment could not regulate the expression of GUS gene and the promoter was not active.The group of GPATp4(231 bp)was weaker than GPATp3(386 bp),but was weaker than the group of GPATp2(644 bp)and GPATpl(954 bp).The group of GPATp2(644 bp)was deeper than GPATp3(386 bp),GPATpl(954 bp)was the most common in these groups,indicating that the fragment had the strongest activity.By the tobacco transformation of lack-recombinant vector,and at the same time,each group were carried out at room temperature(25 ?)and low temperature treatment(4 ?),The results showed that the GPATpl(954 bp)and GPATp2(644 bp)groups were treated at 4 ?for a period of time,and the color of the leaves was slightly deeper than the same group at the room temperature,but not obviously.The promoter predictive analysis showed that the fragment contains a cold-related cis-element,indicating that the promoter is cold-induced to some extent,but the specific induction needs further study.