| Resistant starch is a kind of beneficial carbohydrates for human health. The study about resistantstarch mainly focused on the establishment of preparation and determination methods andphysiological effect, etc. But there were few reports on the research of its formation mechanisms andenzymological mechanisms. Barley stripe mosaic virus-based virus-induced gene silencing (BSMV-VIGS),as an efficient and rapid reverse-genetics tool has been developed and applied on studying gene function inwheat.In this study, BSMV-VIGS technique was applied to research the relationship between wheat starchkey regulatory enzyme genes SBEIIa and SSIIa with resistant starch, expecting to explain itsformation mechanisms in the molecular level. The main results were summarized as follows:1, The construction of VIGS recombinant vectors.The VIGS recombinant vectors were built usingmethods of homology-based cloning. The results of enzyme digesting showed that γ-PDS, γ-SSⅡa, γ-SBEⅡa, γ-SBEⅡa-SSⅡa recombinant vectors were successfully constructed.2, The establishment of BSMV-VIGS system in wheat spikes and grain. Wheat spikes were inoculatedwith BSMV:PDS using the spike-rub method at heading stage and photo-bleaching appeared5-6dpi,became distinct15-16dpi, and reached its highest intensity25-27dpi. Photo-bleaching was also observedon grain25dpi with BSMV:PDS at heading stage. No photo-bleaching was observed on spikes or grainwhen spikes were inoculated with BSMV:00at heading stage. The qRT-PCR analysis showed thatcompared with BMSV:00, the relative expression of PDS gene was obviously down-regulated inphoto-bleaching spikes and grains that infected with BMSV:PDS.3, Separate-silencing of SBEⅡa and SSⅡa genes in wheat grain and its influence on the biosynthesisof amylose and resistant starch. The results of qRT-PCR indicated that the relative expression of SBEⅡagene showed significant reduction in the grains of BSMV:SBE Ⅱa infected spikes compared withBSMV:00. Similarly, the relative expression of SSⅡa was also obvious decline in the grains ofBSMV:SSⅡa infected spikes compared with BSMV:00. To evaluate the influence of separate-silencing ofSBEⅡa and SSⅡa genes on the biosynthesis of amylose and resistant starch, spikes were inoculated withBSMV:SBEⅡa and BSMV:SSⅡa respectively at heading stage. The amylose contents were18.62%higherand resistant starch contents were11.61%higher in grain from SBEⅡa-silenced spikes than from thecontrol (BSMV:00).Similarly, SSⅡa-silenced spikes showed the amylose contents were24.48%higher andresistant starch contents were16.67%higher than from the control (BSMV:00).4, Co-silencing of SBEⅡa and SSⅡa genes in wheat grain and its influence on the biosynthesis ofamylose and resistant starch. The qRT-PCR results showed that the relative expression of SBEⅡa and SSⅡa were both significantly down-regulated in the grains of BSMV:SBEⅡa-SSⅡa inoculated spikescompared with BSMV:00. To evaluate the influence of co-silencing of SBEⅡa and SSⅡa genes on thebiosynthesis of amylose and resistant starch, spikes were inoculated with BSMV:SBEⅡa-SSⅡarespectively at heading stage. The amylose contents were32.02%higher and resistant starch contents were22.33%higher in grain from co-silencing of SBEⅡa and SSⅡa genes spikes than from the control(BSMV:00)..In this study, BSMV-VIGS technique was applied to silence SBEⅡa and SSⅡa genes, separately andsimultaneously, in wheat grain. Amylose and resistant starch content were both increased whether silencingtwo genes sparately or simultaneously. SSIIa showed greater impact than SBEⅡa and two genes havepositive synergistic effect on the biosynthesis of amylose and resistant starch.These results indicate thatSBEⅡa and SSⅡa genes were closely related to the biosynthesis of amylose and resistant starch. |
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