Isolation And Functional Identification Of The Glutenin Gene Regulatory Region From Triticum Aestivum L.

Glutenins are heterogeneous polypeptides that provide the unique viscoelastic properties in dough from bread wheat(Triticum aestivum L.). Much of glutenin research has focused on characterizing protein subunits, defining chromosomal location, and describing properties in bread making. However, little is known regarding the full-length promoter sequence of glutenin genes. In this research, we isolated the full-length glutenin gene promoter sequences from model wheat(T. aestivum L. cv. Chinese Spring) using a genomic PCR strategy with allele-specific primers. We found ten distinct amplicons, six of which belonged to high-molecular-weight glutenin subunit (HMW-GS) gene promoter, while four to low-molecular-weight glutenin subunit (LMW-GS). These sequence lengths varied from1361to2554bp. Major sequence differences between HMW and LMW were identified as indels and base substitutions. We show that the glutenin gene promoter motifs are conserved in diverse sequences, with HMW-GS and LMW-GS gene promoters characterized by distinct conserved motif combinations. Our findings show HMW-GS promoters contain more functional motifs in the distal region of the glutenin gene promoter (>-700bp) compared with LMW-GS. In addition, we found conservation between the x-and y-type HMW glutenin gene promoters. We also identified several important motifs associated with seed storage protein genes also found in other species. These motifs were found in the distal region of the wheat promoter and included a5’-UTR Py-rich stretch-like motif and an as-2box-like motif. Alignment analysis using nulli-tetrasomic lines showed promoter locations were all in accordance with expected results, except the Glu-B3-1promoter. Taken together, these results offer insight into the regulatory mechanisms of glutenin and support efforts to design molecular breeding strategies aiming to improve wheat quality.Two sequences of3’untranslated region from high molecular weight glutenin subunits were also isolated. In order to investigate the difference in efficiency of full length and partial glutenin gene promoter and the difference in efficiency of glutenin gene terminator and nos terminator, we constructed five kinds of expression vectors, using different combinations of promoters (full length/partial glutenin promoter or ubiquitin promoter) and terminators (glutenin terminator or nos terminator) to drive the expression of β-glucuronidase (GUS) gene. The five expression vectors were used to perform transient expression test with gene gun bombardment. The result of transient expression experiment showed that the full length glutenin gene promoter used in analysis was more efficient than partial one. No notable differences of GUS expression were detected between vecotrs using glutenin terminator and nos terminator. We also transform those5vectors into wheat using shoot tip transformation technique and got positive strains of all the5vectors. This laid the foundation of the systemic analysis of the5expression vectors.