Cloning And Characterization Of The Promoters Of Key Starch Synthesis Enzyme Genes In Wheat (Triticum Aestivum L.)

Starch is the major component of wheat grain. It is possible andeffective to improve the quality of wheat starch using transgenictechniques. Development of suitable endosperm-specific promoters isimportant for regulation of specific gene expression and furtherdevelopment of new crop cultivars. Promoters which control expressionspecifically in wheat endosperm tissue, and whose expression pattern(both time of expression and intensity) is synchronous with starchsynthesis are being cloned and analyzed. There is an urgent need do findor construct endosperm-specific promoters with high expression activity. Starch synthesis is a complex biochemical pathway. There are four keyenzymes in the starch synthesis process: ADP-glucose pyrophosphorylase(AGP), starch synthase (SS), starch branching enzyme (SBE) anddebranching enzyme (DBE). During the maturing of wheat grain, these fourkey enzymes have a major influence on starch biosynthesis, including thecontent and ratio of amylose and amylopectin, and affect the quality andusage of starch. Building on results from previous research on wheat starchbio-synthesis, this thesis describes the following research conducted to A河南农业大学博士学位论文gain a deeper understanding of the regulation of gene expression andthe endosperm-specificity of the key enzymes AGP, GBSS and SBE.1. Cloning of the gbss, sbeila and agp-s promoters In this thesis, wheat (Yu 18 cultivar) gbss, sbella and agp-spromoters have been cloned using an APCR strategy, and the basesequences of the gbss promoter (4.1Kb, GenBank AY278458) and the sbellapromoter (3.09Kb, GenBank AY357072) have been sequenced andanalyzed. Partial cloning of an agp promoter sequence has also beenconducted. During the cloning of these unknown fragments, APCR and IPCRmethods were used. Nested PCR was used to optimize the PCR reactionconditions. APCR was found to be a more effective method and was thusused to clone the wheat gbss and sbella promoter sequences.2. Setup of a transient assay system in wheat endosperm A transient expression system in wheat endosperm was investigatedusing a CaMV 35S-gus vector. GUS activity was influenced by manyfactors in wheat endosperm, including endosperm age, pre-culturing theendosperm and bombardment pressure, etc. Though endosperm from 6-16DPA(day post anthesis) grain could all be used for the assay of specificgene expression, 8-10 DPA endosperm were found to be most suitable.Cell activity of younger and older endosperm was evidently low andaffected the GUS expression assay. Pre-culture of endosperms afterbeing extruded only decreased the cell activity of the triploid B河南农业大学博士学位论文endosperm tissue and thus did not favor GUS expression or its assay.Therefore, it is better to conduct bombardment on freshly extrudedendosperm tissue. Bombardment pressures ranging from 650-1100psiwere found to be effective for gene transformation. When bombardmentpressure was decreased to 900psi, the number of blue foci on someendosperms increased, suggesting that lower bombardment pressuresfavor expression and assay of GUS activity. GUS activity was evaluatedin different wheat cultivars, but no significant relationship wasfound between cultivar and GUS activity.3. Loss of function analysis using sbella and pbf promoter deletions To evaluate the promoting functions and activity of the clonedpromoters of sbella and the regulator pbf relative to the above threekey enzyme genes, a transient assay was used to assess sbe.a-g andpbf.a-d deletion constructs. Results showed that the 3094bp sequenceof sbella promoter (in sbe.g fusion) has stable promoting activity.Partial 5' deletions (sbe.b) had low activity, however, the activityof sbe.a (deletion of -1210- -lbp) was high; Under the deletion of-1641bp- -lbp, 3' deletion (sbe.c) there were few blue GUS spots withlow GUS activity. Internal deletions in...