Silencing Of Starch Branching Enzyme Genes(sbe Ⅰ And Sbe Ⅱb) In Maize By RNA Interference (RNAi)

Amylose is an important industry and functional food material. The cost of amylose from maize is expensive,and high amylose maize lines havenot widely been planted in China, So the study of high amylose maize lines is valuable.In the study RNA interference(RNAi) method was used to silence starch branching enzyme genes in maize, sbeâ… and sbeâ…¡b, in order to reduce synthesis of amylopectin and increase amylose content. Meanwhile, application prospect of RNAi in the crop was also discussed.As the targeted sequences of RNAi, parts of sbeâ… and sbeâ…¡b genes encoding starch branching enzyme in maize was ascertained by searching internet database http://jura.wi.mit.edu/bioc/siRNAext/. Segments of sbeâ… and sbeâ…¡b and their promoters existed in maize inbred line Zong 31 were cloned by PCR.The xylA gene from Escherichia coli encoding xylose isomerase which is used as selective marker was cloned by PCR. Expression vector pBAC418 containing CaMV 35S promotor driving xylA gene and reverse repeat sequence of starch branching enzyme gene (sbeâ… ) drived by its corresponding promotor was successfully constructed. The other vector named pBAC413,including the same selective marker gene as pBAC418 and reverse repeat sequence of starch branching enzyme gene (sbeâ…¡b) drived by its promotor.19 maize inbreds were studied to determine the influence of genotypes and media on embryo-derived callus induction.8 genotypes such as B73,Huang C, Zong 31 and so on ,were identified with higher performance both in induction of embryonic callus and plant regeneration.Otherwise N6 medium without proline and CH (casein hydrolysate) and with 4mg/L 2,4-D was proved better than other media tested for callus quality and reducing frequency of embryo shooting.Expression vectors pBAC413 were used for the bombardment of biolistic.PDS1000/He from BioRad was used to bombard maize callus induced from the elite inbred lines. The selection was on media containing serial concentrations of xylose from 0% to 100%. Calli and regeneration plants were obtained from all selective media.But the results showed media containing 50% and 100% D-xylose were better,and the effect mainly depends on maize genotype.68 transformed plantlets were obtained. Dot blotting,PCR and PCR-Southern hybridization analysis of genomic DNA from transformed plantlets were performed.24 regeneration plants transplanted in Hainan were analysed by Dot blotting,PCR and PCR-Southern hybridization. Successful integration of xylA gene and FAD2 intron in 14 regeneration plants was confirmed, 5 regeneration plants were transformed pBAC418,others were transformed pBAC413.24 regeneration plants were transplanted in Hainan, and 19 plants were fertile to seed. Seeds of Some plants is too little,so it is possible to get contents of all kinds of ingredient. The change of total protein, oil and starch contents of seeds of conformed regeneration plants were measured by Foodï¹ Feed Analyzer. The total protein and starch contents of seeds of conformed regeneration plants were lower, oil contents of them were higer than negative maize. The amylose content of 9 regeneration plants confirmed was detected. The increment of amylose ranged from 3% to 10.4% in transplants transformed pBAC418 and the increment of amylose ranged from 0 to 15.6% in transplants transformed pBAC413.