Interactions Among Starch Systhesis Related Genes And Enzymes And Their Relations To The Formation Of The Eating And Cooking Qualities Of Rice

Rice(Oryza sativa L.)is one of the most important crops,rice varieties with high yield,good eating and nutritional quality is closely related to the improvement of people's living standard.Starch is the predominant rice seed reserves.Starch biosysthesis and physiochemical properties influence rice yield and cooking and eating quality.Currently,there are more research concerning genetic basic of the formation of rice quality.But few reports concerning genes mutual effect or protein-protein interaction that control rice quality has been published.In this study,the genetic network that control rice quality was studied with Wx(G/A)and SSIIa(GC/TT)allelic gene as covariate.The polymorphic forms of SSI and PUL were discovered for the first time by zymographic analysis,and the polymorphic forms of SSI may be caused by SNP(A/G).The expression levels of SSRGs and the activity of starch systhesis related enzymes(SSREs)were altered in endosperm mutant,and the interaction of SSREs was different between wild and mutant.The results will enhance our understanding of starch biosynthesis process and provide helpful information to breed high yield and good quality rice.The research results are as follows:(1)Starch physiochemical properties were mainly controlled by Wx and SS?a,and were influenced by other SSRGs with Wx(G/A)and SS?a(GC/TT)allelic gene as covariate by association mapping using 416 rice seeds.143 markers were used for association mapping for 20 starch physicochemical property parameters.A total of 64 main-effect loci were detected.Wx locus was a major main-effect locus for AAC,pasting viscosity,gel texture,and retrogradation property(P<0.0001).SS?a locus was a major main-effect locus for pasting temperature,thermal and retrogradation properties(P<0.0001),but it was a minor main-effect locus for some pasting viscosity parameters,such as breakdown,consistency,stability and setback ratio.In addition,56 and 62 loci were identified with Wx(G/A)and SS?a(GC/TT)as covariate,respectively.Four other SSRGs,SS?a,BE1,SS?c and GBSS? were detected for AAC with Wx(G/A)as covariate.Wx was detected for Tc and ?Hg with SS?a(GC/TT)as covariate.PUL was detected for hardness as main-effect locus and under SS?a(GC/TT)as covariate.AGPL2 and ISA1 were detected respectively for enthalpy and retrogradation as main-effect loci as well as with Wx(G/A)and SS?a(GC/TT)as covariate.This study suggested that retrogradation properties were mainly controlled by Wx,SS?a and ISA1 with the relative effects in the order of SS?a>Wx>ISA1.(2)The polymorphic forms of SSI and PUL were discovered for the first time by zymographic analysis,and the polymorphic forms of SSI may be caused by SNP(A/G).Here,two forms of starch synthase I(SSI)and pullulanase(PUL)in 390 developing rice seeds,defined SS?-1/SS?-2 and PUL-1/PUL-2,were discovered for the first time by zymographic analysis.Association mapping revealed that SS? and PUL polymorphisms were strongly associated with the SS? microsatellite marker(P= 3.6 × 10-37)and PUL-InDel markers(P<3.6 × 10-51).Western blotting and mass spectrometric analysis confirmed that the polymorphic bands were truly the SSI and PUL enzyme.To elucidate the molecular mechanisms underlying the zymographic diversity,further analyses of DNA and amino acid sequences were conducted.Only one non-synonymous variation in DNA sequence(SNP,A/G)causing change of the amino acid K 438(Nipponbare)to E438(93-11)in SSI was observed,which coincided well with the polymorphic forms of SSI.It suggested that the polymorphic forms of SSI in zymogram resulted from the non-synonymous variation(K438 to E438).Nine non-synonymous variations were found between PUL-1(Nipponbare)and PUL-2(93-11).Two non-synonymous variations of PUL(F316 to L316,D770 to E770)were identified by mass spectrometric analysis,but all the variations didn't change the structure of PUL.The co-immunoprecipitation results revealed the differences in protein-protein interaction patterns,i.e.strong or weaker signals of SS?-BE? and SS?-BE?b between the two forms of SS?.The results will enhance our understanding of SS? and PUL properties and provide helpful information to understand their functions in starch biosynthesis in rice endosperm.(3)The expression levels of SSRGs and the activity of starch systhesis related enzymes(SSREs)were altered in endosperm mutant,and the interaction of SSREs was different between wild and mutant.The expression levels of SSRGs(SSREs),enzyme activity and protein-protein interaction in the endosperm development of GLA4 and two endosperm mutants(GM077 and GM645)were studied.The results showed that GM077 was waxy mutant with highly expression levels of 11 SSRGs during endosperm development,and the activity of SS? and SS?a was higher than that of GLA4.The expression levels of SSRGs and SSREs were decreased during endosperm development of GM645,but the changes of SS? and SS?a activities were not obvious.The co-immunoprecipitation results found that the interaction of SSs-BEs,SSs-PUL and BEs-PUL in 3 developing rice seeds were detected,but the weak signal of SS?-SS?a and no signal of SS?-SS?a in GM077 endosperm were detected.The weak signals of SS?a-SS?,SS?a-BE?,SS?a-BE?b,BE?-SS?,BE?-SS?a in GM645 endosperm were also detected.The results can provide molecular theoretical basis for the breeding and application of rice mutant materials.