Functional Analysis Of Glutenin Gene And Methionine Sulfoxide Reductase Gene Promoters

The technology of producing important foreign protein using plant bioreactor has now attracted increasing attention in the field of Life science, now one topic of the research that is crucial and needs to be solved urgently is how to drive foreign genes to express efficiently and specifically in certain plant tissues or organs such as endosperm, so the work of searching for effective promoters become a key factor. Glutelin is the main storage protein of cereals crops and express richly and specifically in endosperm. So the glutelin gene promoter is a promising candidate one that can be applied in plant bioreactors. But wheat does not get a lot of research progress in this field because of the limitation of its poor transformation efficiency.The glutenin gene promoter consists of core promoter and regular elements. Only when they are combining together can the gene being driven to express efficiently and specifically. Our work firstly explored the GUS transient expression under the control of glutenin promoter using gene gun in immature wheat seeds, GUS staining indicated that the pericarp, testa and endosperm periphery of wheat grain can be stained. Then we constructed GUS expression vectors of 9 full-length Chinese Spring glutenin gene promoters (including 5 high molecular weight glutenin subunit(HMW-GS) genes and 4 low molecular weight glutenin subunit (LMW-GS) ones), using gene gun transient transformation to transform immature wheat seeds, GUS staining revealed that these promoters all can drive GUS expression in exterior cells of seeds. Among HMW-GS genes, the function of Bx7 promoter is stronger than that of other HMW-GS promoters, but Ay promoter is the weakest one in all these promoters. Among LMW-GS promoters, Glu-A3-1 and-B3-1 are the stronger promoters yet-D3-2 is the weakest one. It is necessary to point out that Ay subunit is silent in common wheat, but its promoter can drive GUS to express weakly. To study the function of the conserved element RY-element in the By8 subunit promoter, we introduced mutation into the conserved motif by the technology of overlapping primers and constructed the expression vector consists of mutational version of By8 promoter and GUS. The gene gun transient transformation showed that the mutation of this element can result in the declined function of this promoter to drive GUS expression. The bioinformatics analysis of the 4 LMW-GS promoter found no cis-acting element in the distal 1 kb sequence to the 5’-end. So we also constructed the GUS expression vector of the full-length sequence and 972 bp sequence of the Glu-D3-1 promoter respectively to ensure whether there is any sequence that can affect expression exist in die distal end of the promoter. Transient transformation indicated that 972 bp sequence can also initiate efficient expression of GUS gene, the change of strength is not notable compared with the full-length promoter.Methionine sulfoxide reductases (MSRs) can specifically reduce the oxidation of methionine(Met), thus it can help us to understand the significance of MSR genes in the organism defense mechanism to study the function of their promoters. Our research group had cloned several MSR genes from wheat, existing phenotype experiments showed that Arabidopsis thaliana lines overexpressing TaMSR A4.1 and-B3.1 displayed notable anti-stress phenotype. To deeply comprehend their function, we obtained electronically assembled sequences of these two genes by virtue of the bioinformatics method and cloned them from the genome of the wheat cultivar SR3 with gradient PCR strategy by designing promoter-specific primers. They were 1898 and 1787 bp respectively. We analysed the conserved cis-acting elements of the two promoters with the help of the bioinformatics method and we found that these elements included ABA-responsive elements ABRE, GA-responsive elements GARE-box and P-box, auxin-responsive element TGA, the binding site of MYB transcription factors MBS and some other elements being responsible for tissue-specific expression such as Skn-1 motif, GCN4 and as-2 box, etc. At the same time, we constructed the two genes’GUS expression vectors and transformed the model plant species Arabidopsis thaliana. The selection of positive To lines is now in progress. We planned to study the tissue-specific expression pattern using X-gluc staining.