The Function Identification Of The ABRE Element In The Promoter Of Arabidopsis At3g21380 Gene

Seed-specific promoters can drive large amounts of foreign genes to be expressed only in plant seeds.The level of tissue-specific transcriptional activity of foreign genes is usually regulated by some cis-acting elements present on the promoters.Studies have shown that the ABRE is an element related to seed-specific expression.So a preliminary analysis of the function of the ABRE can lay the foundation for a more in-depth study of the regulation mechanism of seed-specific promoters.The At3g21380 gene is a type of mannose-binding protein coding gene.Previous analysis results have shown that At3g21380 promoter is a seed-specific promoter.The analysis of the cis-acting elements on the promoter showed that,there were ABRE elements as well as RY elements,skn-1 elements,GCN4 elements and so on.These cis-acting elements are related to seed-specific expression in some plant promoters.In this study,in order to clarify the effect of the ABRE elements on the seed-specific transcription activity of the At3g21380 promoter,the mutation sits of the ABRE elements and specific PCR amplification primers were designed,and by PCR amplification,the mutant promoter fragments were obtained.The target fragments were ligated to the plant gene expression vectors respectively.The verified expression vectors were transformed into wild-type Arabidopsis thaliana respectively by Agrobacterium-mediated flower dipping method.The infected Arabidopsis seeds were harvested and tested for hygromycin resistance.The transgenic pure lines of Arabidopsis plants obtained above was subjected to PCR identification.And the transgenic pure lines Arabidopsis containing the correct mutant promoter fragments were obtained.Finally,GUS staining,GUS enzyme activity and semi-quantitative RT-PCR analysis experiments were performed on the transgenic pure line seeds containing wild-type and mutant promoter fragments respectively.GUS staining analysis was also performed on other tissues including seedlings,flowers and seedlings.The main results of this study are as follows:(1)Successful cloning of At3g21380 mutant promoter fragments.At3g21380 promoter sequences analysis results have showed that there are two ABRE elements in the promoter,named them ABRE1 and ABRE2 respectively.Based on this,we designed corresponding mutation primers for PCR amplification.After the amplified products were detected correctly by electrophoresis,the target fragments were purified,and the two correct mutant promoter fragments containing the ABRE element mutations were obtained respectively.In this study,they were designated as MUTABRE1 and MUTABRE2 respectively.(2)The expression vectors of mutant At3g21380 promoter fragments were constructed,and the homozygous transgenic lines were obtained successfully.The MUTABRE1 and MUTABRE2 promoter fragments obtained above were respectively connected to the corresponding expression vectors.Screened by antibiotics and double enzyme digestion,positive clones were obtained.The sequencing results showed that the sequences of the amplified MUTABRE1 and MUTABRE2 promoter fragments were consistent with the target sequences,so the two recombinant plant expression vectors with mutant ABRE elements were successfully constructed.Then,they were transformed into wild-type Arabidopsis thaliana by the flower dipping method mediated by Agrobacterium.Through hygromycin resistance screening and PCR identification experiments,the correct mutant homozygous lines were finally obtained.(3)The function of the ABRE elements of At3g21380 promoter have been clarified preliminarily.GUS histochemical staining analysis was performed on the seeds of transgenic homozygous lines containing ABRE element mutant promoter and wild-type promoter respectively.As a result,no visible staining was found in the seeds of the transgenic lines containing the mutant promoter fragments,compared with the seeds of the transgenic lines containing wild-type promoter fragments which have showed obvious staining.Therefore,the staining results showed that the ABRE elements can promote the seed-specific expression activity of the At3g21380 promoter.Further quantitative determination of GUS protein expression activity was performed in the seeds of the transgenic lines respectively containing the mutant promoters and the wild-type promoters in the seeds.It was found that GUS protein expression activity in the transgenic seeds containing the mutant promoters were significantly lower than that of the wild-type promoter,and the GUS protein expression activity in the seeds of the ABRE1 element mutant promoter is lower than that of the ABRE2 element mutant promoter.These results not only showed that the ABRE elements can play positive roles in regulating the seed-specific transcription activity of the At3g21380 promoter,but also indicated that the ABRE1 element has a stronger promotion effect than the ABRE2 element in terms of regulating the seed-specific transcription activity of the At3g21380 promoter.Semi-quantitative RT-PCR experiment was used to detect the expression level of GUS gene.It was found that the expression levels of GUS gene in seeds activated by two mutant promoters were significantly lower than that of wild-type promoter,and the expression level of GUS gene in seeds activated by mutant promoter of ABRE1 element was lower than that in mutant promoter of ABRE2 element.These results further showed the correctness of the above experimental conclusions.