Studies On The Genetic Variability And Regulation Mechanism Of Transcription Factor RSR1 In Rice

Nity percent of composition of the rice in our daily diet is starch.Amylose content was one of the main factors which affects and improves the cooking and eating quality,and study on the amylose biosynthesis and regulation mechanism is an important basis for improving the quality of rice.The grain filling period is the most important period for starch accumulation.Starch synthesis and accumulation are caused by a series of enzymatic reactions,these four enzymes involved in starch synthesis are pyrophosphorylase,soluble starch synthase,starch branching enzyme and starch debranching enzyme.The rice starch synthesis regulator RSR1 belongs to the AP2 / EREBP transcript that can regulate the expression of starch synthase gene.In this paper,we select the hybrid progeny and parents whose amylose content showed transgressive variation and similar genetic background by directional selection,analysises of filling in different periods of endosperm amylose content,ADGP,SSS and SBE starch synthase activity and m RNA expression level of isoform enzyme gene and RSR1,and clones the RSRS1 gene of parents and progeny,compares to the base sequence and structure.Using RNA interference technology,the study obtains Os RSRS1-RNAi transgenic rice material,researches the influence of Os RSR1 on the expression level of rice starch synthesis related enzyme gene,and the influence of grain amylose content,explores the formation characteristics of amylose content difference,and molecular regulatory mechanism of genetic variation,provides supplement on the molecular mechanism of amylose content quantitative and traits producing transgressive variation,and hybrid progeny RSR1 genetic variation and regulation mechanism,provides a molecular basis to rice starch quality formation.The main results are as follows:Following the filling process,grain amylose content increases gradually.20 d after heading,tested varieties have been accumulated to 71.35%~84.63% of mature rice amylose content,and 30 d after heading,they have reached 95.37%~99.05%,and the high amylose content variety accumulation is greater than the low amylose content variety from heading to 20 d after heading straight.The results show that the accumulation of grain amylose is almost end at 30 d after heading,and the accumulation rate is closely related to amylose content.In each filling stage,the amylose content of progeny and parents with high grain amylose content are higher than those with low amylose content.In the parents and the transgressive variation strains,grain ADPG,SSS and SBE enzymes activity changing trend are consistent in the filling process,they all show a single peak curve.Follow the filling process,the activity of starch synthesis related enzyme gradually increases,and gradually decreases after reaching the peak,the peak appears at 20 d after heading.The peak values of the transgressive variation strains and parent grain pyrophosphorylase,the soluble starch synthase and the starch branching enzyme shows a super high or super low transgressive phenomenon.The Os AGPL1,OsAGPL2,Os AGPS2,Os GBSSI,OsSSSI,Os SSSIIV and Os SBE3 genes of the parent and transgressive variation strains with different amylose content have abundance expression.The expression time of four key genes of starch synthesis basically synchronizes with the rice filling time,and shows a low-high-low single-peak trend with the change of filling process,however,there is a difference in the peak time of expression level between the genes,the peak value of Os AGPS2 gene transcription expression level appears at 15 days after heading,and the peak value of other gene expression level appears at 20 days after heading.The m RNA expression levels of ADPG,SSS and SBE isoform enzyme gene are closely related to the grain amylose content,and the transcript expression level could produce transgressive variation.In the parents and the transgressive variation strains,the transcription expression level changes of grain Os RSR1 gene are almost same with upward curve in the filling process that is mRNA expression of grain RSR1 gene increase gradually,Os RSR1 gene expression level significant difference occurs in the different filling period between transgressive variation strains and parents.The sequence length of cloned RSR1 gene are 1539 bp,encoding 512 amino acids.The homology of parents and progeny are 99.94%,99.94%,99.87% and 99.94% respectively compared with the Nipponbare nucleotide sequence in Gen Bank.Rice varieties starch regulatory factors with different amylose content are very conservative,but there are differences in individual bases among varieties;in the process of gene exchange and separation of varieties hybrid progeny,there are changes of base and triplet code,which could result in the changes of the amino acid sequence and the physical and chemical properties and functional domain of the enzyme protein.Sequence comparison shows that Os RSR1 and other AP2/EREBP transcription factors are highly conserved in the DNA binding domain and have the typical characteristics of transcription factors.Adopting Golden Gate cloning technology to construct hp RNA interference vector of RSR1 transcription factor,the Os RSR1-RNAi transgenic rice plants are obtained.The results show that the transcription expression level of grain Os RSRl in transgenic rice is about 71% of that of wild type,gene silencing effect is remarkable.In this study,the transgenic rice T1 separates different strains and shows different separation ratio to Hygresistance.In the investigation,13 strains of the 15 strains are in line with the separation proportion of 3:1,2 strains are 1:1 separation proportion.In starch synthase gene test of transgenic strains and wild-type strains,the expression levels of Os AGPL1,Os AGPL2,Os AGPS2,GBSSI,SBE3,SSSI and SSSIV-2 increased,and the difference reaches significant level among the comprisons,the expression level of Os AGPS2 gene in AGPase is higher than that of Os AGPL1 and Os AGPL2,and the expression level of SSSI gene in SS is higher than that of SSSIV-2,the transcription expression level of GBSSI gene is the highest in all genes.The amylose content of wild type and transgenic rice grain is significantly different,and the amylose content of transgenic rice is significantly higher than that of wild type.Interfering the expression of RSR1 gene could significantly increase the grain weight of rice,and the grain weight per plant increased by 7.5% compared with that of the wild type.At the same time,it can significantly improve the grain amylose content,affecting the formation of starch quality.