Analysis Of OsRSRI Gene Sequence And Its Expression Regulation In Japonica Transgressive Variants

With the improvement of people's quality of life,people's demand for rice quality is increasing.Because rice is consumed mainly in the form of rice,cooking and eating quality is the most important among rice quality contents.The content and proportion of amylose and amylopectin in endosperm and the fine structure of amylopectin played an important role in the formation of cooking and eating quality of rice.The process of starch synthesis and accumulation is complex,involving a series of enzymatic reactions,and OsRSR1(Rice Starch Regulator1)transcription factors regulate the expression of these enzyme genes.The expression level of OsRSR1 is closely related to that of the key enzymes in starch synthesis.On the other hand,the biological genetic information stored in the DNA base sequence is ultimately expressed by the transcription and the translation of proteins,and the changes in one base in the gene coding region can alter the function of corresponding proteins.The crossbreeding of sexual hybrids between crop varieties is a common phenomenon of biological genetic variation and an important source of variation for breeding new crop varieties.Moreover,interspecific hybridization is still the main way to select new varieties.RNA interference technique is a reliable molecular technique for the identification of genes with dual functions.Thus,further study of OsRSR structure,variation pathway and transcriptional and expression characteristics by the techniques such as RNA interference and quantitative expression analysis has important theoretical and practical significance for elucidating the molecular regulation mechanism of starch synthesis and improving starch quality.The transgressive variants Dongnong 1701 and Dongnong 1724 and their parent varieties Xixuan 1 and Tong 769 with significant difference in AC of endosperm,transformation line Dongnong 1724,indica rice variety Bashier with extremely high AC and glutinous rice variety Longdao 8 with extremely low AC were used as test materials.Analysis of starch components content,OsRSR1 expression by pot experiment at different filling stages,test the characteristic values of rice RVA spectra and cloning OsRSRI of six materials.Analysis of the changes of amino acid sequence,domain and function caused by the changes of DNA and mRNA base sequence,the changes of quality traits and expression of gene interference materials and the response to nitrogen nutrition.The findings are as follows :(1)In the process of grain filling,the synthes and accumulation of amylose,amylopectin and total starch were mainly in the prophase and middle stage.The accumulation of amylose between varieties in different filling stage showed a trend of glutinous rice < japonica?indica rice;The higher the amylose content,the smaller the breakdown value,the higher the setback value and the consistence value.(2)The expression of OsRSR1 was large in the initial stage and late stage of grouting,but small in the middle stage,and showed a V-shape change;The expression of OsRSR1 in six test materials in different periods from high to low were Longdao 8,Dongnong 1724,Tong 769,Xixuan1,Dongnong 1701 and Bashier,was opposite to the order of amylose content in grain,and there was a negative relationship between of them.Moreover,the expression of OsRSR in the interspecific hybrid progenies showed super-parent variation.(3)OsRSR1 DNA and mRNA base sequence and amino acid sequence of different amylose content varieties were not identical,and compared with parents,DNA base sequence changed in exon region and intron.Moreover,some base changes occurred in the process of DNA transcription mRNA,which resulted in the formation of mRNA base sequence that did not match the DNA base sequence;OsRSR1 is composed of 8 exons and 7 introns.Functional analysis shows that the main function of OsRSR1 is to regulate the expression of other genes;Amino acid differences did not result in changes in the basic components and functions of the OsRSR1 encoding protein,which is highly conserved.(4)The expression level of the key enzymes in starch synthesis was opposite to that of OsRSR1;OsRSR1 interference significantly increased the expression of SS1,GBSS1,AGPL2,SBEIIb and ISAI during grain filling.The effect on the peak value of expression from high to low was GBSS1,SBEIIb,ISAI,SS1 and AGPL2;The expression of the key enzymes in starch synthes increased,increased the content of amylose in endosperm,increased the setback value and,decreased the breakdown value,and finally decreased the cooking and eating quality of rice;Increasing the nitrogen nutrition at the filling stage decreased the expression of OsRSR1 and downstream structural genes of SS1,GBSS1,AGPL2,SBEIIb and ISAI,especially the expression of OsRSR1 decreased most significantly in the middle of grain filling stage,which affected starch synthesis and accumulation,and finally affected yield and quality.