Screening,Cloning And Preliminary Functional Identification Of Bidirectional Promoter In Maize

The production of recombinant protein with maize as bioreactor has many advantages.Maize is the main food crop in China.A lot of foreign proteins in maize need to express the whole plant,so most of the promoters are constitutive promoter.From the current study,the researchers found that there are a large number of bidirectional gene pairs in the genome of human,microorganism,Arabidopsis,rice and poplar.The promoter has its unique advantages in the application of multi gene transformation,because a promoter of the traditional promoter can start only one gene,and the bidirectional promoter can start two genes at the same time.When we want to express multiple genes in the plant expression vector,we can select the bidirectional promoter so as to avoid the gene silencing caused by repeated use of the promoter.Therefore,we can select the constitutive expression of the bidirectional promoter for the study.Researchers from the Chinese Academy of agricultural sciences have found that a total of 1696 bidirectional transcripts have been found in the whole genome of Maize.This experiment on the basis of manual online screening and 600 of them on the bidirectional transcription,expression of all transcripts of the double contrast spectrum,constitutive expression of the bidirectional transcript pair.A total of 9 pairs of constitutive expressions were screened.Searching the genes ID of bidirectional transcription,the bidirectional promoters for the expression of these two genes was cloned.The GUS and GFP reporter genes were fused at both ends of the 9 bidirectional promoters,to constructed the plant expression vector.8 plant expression vectors were constructed successfully.Using tobacco transient expression system to identify whether the 8 bidirectional promoter has the function of bidirectional initiation.The main results are as follows:We used the conventional PCR method to clone the 8 Maize constitutive expression bidirectional promoter sequences.Sequence analysis was performed by PlantCare and PLACE online software.It was found that each promoter contains the basic cis acting element of the typical promoter,and also contains a drought inducible binding site,a stress inducing cis acting element such as a light induced related element.The plant expression vector was constructed,and the 8 bidirectional promoters were cloned,and named pBD16,pBD23,pBD24,pBD31,pBD34,pBD38,pBD78,pBD80.Then,8 plant expression vectors were constructed by using GUS and GFP gene as the reporter gene,and then transferred into Agrobacterium tumefaciens EHA105 by the three parental hybrid method.The genetic transformation of tobacco was carried out by Agrobacterium mediated injection,and 9 plant expression vectors were transferred into wild type tobacco.The transformation of tobacco by GUS staining and GFP fluorescence observation,we found that can not drive the GUS and GFP gene expression,that constitutively expression of bidirectional promoter has no activity or low activity,can not through the method of transient transgenic tobacco to prove whether the reporter gene expression.Based on the above experimental results,the follow-up study of bidirectional promoter was carried on.