Artificial Design And Functional Analysis Of The Promoters Induced Phytophthora Sojae In Soybean

Soybean is one of the most important oil crops in the world,it plays an important role in agricultural production and people's life.Soybean root rot causes by Phytophthora sojae is a destructive disease of soybean production worldwide.It caused a huge economic losse each year.There are no effective means to control the disease.In recent years,more and more researchers focus on gene engineering.They attempt to promote soybean resistance to downy mildew through cloning resistant related genes.Promoter is a DNA sequence that binds RNA polymerase and regulate the transcription of genes.Inducible promoter is one kinds of promoters that widely exists in plant genes.When stimulated by the specific pathogens,inducible promoter becomes active and the expression level of the gene increases.Inducible promoter can be used in genetic engineering for plant resistance to stress and disease.Therefore,the study of inducible promoter has received more and more attention.In this paper,we got 25 Phytophthora inducible cis elements.We studied the expression of the inducible promoter when it stimulated by the Phytophthora.At the same time,we found that there is a lack of suitable reference gene in soybean.Identification of reference genes is explored.There are many cis elements in the promoter.The combination of different elements and their position influence the properties of promoters.The cis elements are very important to promoters.And they may play an important role in plant response to environmental stress and pathogen infection.At the same time,in gene engineering,cis elements have been widely used in synthetic promoters.Previous work has analyzed a large number of gene chips and got the expression spectrums of all genes in difference kinds of soybeans.And also obtained 180 Phytophthora inducible genes.In my study,we analyzed the promoter of these genes and found a lot of Phytophthora induced structural elements.We also got several synthetic promoter by grouping some of them.I analyzed the expression level of the synthetic promoter by tobacco transient expression system and we also tested the function of the promoter.With this experiment,I got 8 synthetic promoters that are specifically induced by Phytophthora and provided valuable promoters for the genetic engineering of soybean.The conventional PCR detections and analyses the amplified products through the end-point method.That is the detections needs agarose gel electrophoresis.The quantitative real-time PCR can make a real time analysis of the products during the amplification reaction.This significantly improves the efficiency of the experiment.Application of suitable reference genes to normalize qRT-PCR data is critical in analyzing PCR results.According to the reports of soybean reference genes study,I analyzed the expression patterns of 10 housekeeping genes,including Cons4,ACT11,TUA soy,Consl5,ACT20,Cons7,CYP2,Cons6,Tubulin and ELF1B in various tissues of soybean seedings and in soybean roots treated by Phytophthora sojae.The stability of housekeeping genes expression was analyzed by using of 3 software packages,including GeNorm,NormFinder,and BestKeeper.The gene Cons4 was a suitable reference gene for efficient normalization of qRT-PCR data,and ACT11,TUA soy were suitable reference genes for P.sojae induced gene expression research.Tubulin and ELF1B were not suitable for analysis of various tissues of soybean seedings and in soybean roots treated by P.sojae.These results were confirmed by two P.sojae induced genes profiling GmaPPO12 and GmaPR1a expression.This study provides a useful information for reference gene selection in qRT-PCR analysis of gene expression in soybean of pathogen induced gene expression.