Effects Of Down-regulating Of Starch Synthase?-1 And Pullulanase On The Starch Synthase Gene Expression And The Content Of Starch In Rice

Rice is the one of major crops.The requirement of the quality become much higher in rice when the improvement of people's life.Starch is the main storage material of rice,the content of amylose and amylopectin will directly result in the quality of rice cooking and eating quality,which also decided the economic value.Therefore,improving rice quality is very important in breeding.In the starch synthesis pathway,different genes respectively control the synthesis of amylose and amylopectin.SSIII-1 and PUL are two key genes in amylopectin synthesis pathway.SSIII-1 is one of the soluble starch synthases,which is mainly involved in extending the amylopectin in starch synthesis;PUL is one of the branching enzymes which is responsible for cutting the branches to the appropriate length.SSIII-1 and PUL genes have different effects on the eating and cooking quality of rice and the process of amylopectin synthesis.Through the expression of SSIII-1 and PUL genes in RNA interference plants,understand the change of other related genes in biosynthesis process of starch and the ECQ in rice,when down-regulated of SSIII-1or PUL.In this study,expression vector of SSIII-1 and PUL gene target sequences were established by using RNAi.Agrobacterium-mediated transformation was used to transfect low amylose material indica rice R372 callus,down-regulated SSIII-1 and PUL with the purpose to reduce the synthesis of amylopectin that increase the content of amylose.It studied the regulation of post-transcriptional level of starch synthase during amylopectin synthesis in down-regulated plants with RNAi.In addition,the starch content of the seeds between transgenitic plants and contrast plants were measured,and we deeply understand the relation between the genes in the starch synthesis process.The specific results are as follows:1.According to the SSIII1 and PUL gene sequences in rice were reviewed on the NCBI database,the RNAi target sequence of starch synthaseIII-1(SSIII-1)and Pullulanase(PUL)was selected and designed PCR primers.The positive and reverse RNAi fragments of SSIII-1 and PUL genes were respectively cloned from the cDNA of indica rice R372,and respectively constructed the interference vectors of SSIII-1 and PUL genes,which called as PTCK303-SSIII-1 and PTCK303-PUL.2.The Agrobacterium with PTCK303-SSIII-1 or PTCK303-PUL vector was respectively transfected into embryogenic callus of indica R372,after the selection of hygromycin resistance,regeneration,we gained transfected plants.3.The transgenic plants were identified by Hygromycin Resistance Genes PCR and GUS tissue stain,the RNAi interference fragment of SSIII-1 and PUL genes has been integrated into receptor material in the chromosome of indica rice R372,identification of positive plants.2 strains of SSIII-1-RNAi and 49 strains of PUL-RNAi plants were obtained.4.Cultivating transformation materials in Hainan field,Real-time PCR was used to analyze the positive plants endosperm of T0 generation,we found that the expression of SSIII-1 gene in SSIII-1-RNAi-positive plants was decreased 50% than those control material,and the expression of GBSSI,GBSSII,SSII-1,sbe4 were up-regulated,and the expression of GBSSI was increased 7 times.AGPlar,AGPiso,AGPsma,SSI,SSII-2,SSII-3,SSIII-2,SSIV-1,sbe1,Sbe3,ISA,PUL expression were down-regulated.The expression of PUL gene in PUL-RNAi-positive plants was down regulated 80%,the expression of AGPiso,GBSSI,SSSIII-1,SSIII-2,sbe1,sbe3,sbe4 were up-regulated,the expression of GBSSI increased 2 times,and the expression of starch branching enzyme gene(sbe1,sbe3,sbe4)also obviously increased.The expression of AGPlar?AGPsma?GBSSII?SSI?SSII-1?SSII-2?SSII-3?SSIV-2?ISA?PUL were down regulated.Those results indicate that down-regulation of SSIII-1 and PUL genes can improve the expression of amylose synthesis gene GBSSI.5.We respectively measured the content of starch and amylose in the plant of SSIII-1-RNAi,PUL-RNAi and the control.We found that the content of starch in SSIII-1-RNAi and PUL-RNAi plants increased obviously,the amount of starch respectively increased 330-400 mg/g and 70-170 mg/g in PUL-RNAi plant and SSIII-1-RNAi plant.But the content of amylose in transgenic plants changed not obviously,the content of amylose respectively increased 43-61.5 mg/g and 55-56 mg/g in PUL-RNAi plant and SSIII-1-RNAi plant.