| Preharvest sprouting (PHS) is the precocious germination of grains breaking dormancyin the spike before harvesting in damp weather. PHS is one of the major disasters that greatlyinfluence grain quality and yield. There are many factors leading to PHS, which mainlyinclude seed dormancy, α-amylase activity, spike character, grain color, germination inhibitorsin glume and testa and so on. PHS has always been considered to be associated with red coatcolor which is controlled by R-1gene. Besides, red-grained wheat varieties are usually moretolerant to PHS than white-grained wheat varieties. The pigment of red wheat grain isidentified to be anthocyanin, proanthocyanidin and catechin which are all synthesized throughflavonoid biosynthetic pathway. Therefore, research on related genes in flavonoidbiosynthetic pathway as well as R-1gene will help figure out the relationship between graincolor and preharvest sprouting, and lay a basis for cultivating PHS-resisitant wheat varieties.Particle bombardment and Agrobacterium-mediated transformations are the two mostwidely employed methods in plant genetic transformation. Vector system plays an importantrole in development of an efficient transgenic technology. At present, RNAi andoverexpression vectors are widely used for gene function analysis. Gateway is a efficient andhigh throughput cloning technology, which is based on the site-specific recombinationcharacteristics of lambda phage. Using Gateway technology, large numbers of genes can becloned into the desired vectors quickly and conveniently.Detailed research contents are as follows:1. To amplify genomic DNA of120wheat varieties using genome specific primers ofDFR-B, and two kinds of PCR products were obtained with the difference of whethercontained8bp insertion sequence. The insertion sequence and the upstream adjacent sequencejust formed HinfⅠrestriction enzyme sites. Therefore, the genotypes of DFR-B gene in120wheat varieties were determined thereby digesting amplified promoter region of DFR-B genewith HinfⅠ. Correlation analysis between PHS and DFR-B genotypes indicated that wheatvarieties with8bp insertion (the average spike germination rate is23.6%) were more tolerant to PHS than those without the insertion (the average spike germination rate is69.5%) and thedifference was extremely significant (P<0.01). In addition, transient assay of DFR-B geneusing Chinese Spring coleoptiles revealed that solely increasing DFR gene expression cannotinduce the synthesis of anthocyanins.2. The genotypes of R-1gene in119wheat varieties were determined using PCR-basedmarkers of Myb10gene. Correlation analysis between PHS and R-1genotypes demonstratedthat the differences between genotypes baa and aab,baa and bbb were extremely significant(P<0.01), and the differences between genotypes baa and bba,baa and abb,aba and bbbwere significant (P<0.05). It can be concluded that there is an association between R-1genotypes and PHS and R-D1gene might have a great influence on PHS resistance, followedby R-B1gene and R-A1gene.3. In this study, the overexpression vectors pAHC-PSK-OE and pClean-G185-OE, andthe RNAi vectors pAHC-PSK-RNAi and pClean-G185-RNAi for biolistic or Agrobacterium-mediated transformations were constructed using traditional enzyme digestion and ligationmethods and gateway technology. These vectors are likely useful for high throughput genefunction studies in monocotyledonous plants in the future. |
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