Identification Of Key SNP Controlling Amylose Synthesis By Wx/GBSS I In Rice And Creation Of New Alleles

Rice grain quality,especially appearance quality and eating quality,is an important indicator that determines the value of rice commodities,and it is also a major factor affecting consumer choice.Starch is the most important storage substance in rice,which is divided into amylose and amylopectin.Among them,amylose content(AC)is the most important factor that determines the taste and processing quality of rice.Waxy gene(Waxy,Wx)encode the granuale bound starch synthase I,(GBSSI),which is responsible for the synthesis of amylose in rice endosperm.The natural allelic variation of Wx gene is an important reason for the wide variation of amylose content in cultivated rice,and it is also a major gene for differences in rice grain quality.In previous studies,we have identified a novel Wx allele Wxlv which responsible for high AC as well as low starch viscosity in rice.Compared with Wxa which is also responsible for high AC.We found that only one SNP;T-C in exon 10,which leads to a serine-to-proline substitution(P415S),was found in the Wxlv allele compared with the Wxa allele.In addition,based on the phosphorylation analysis of GBSSI protein,seven possible phosphorylation sites were identified.In order to further clarify the possible role of P415S mutation and related protein phosphorylation sites,we carried out the following study:1.The effect of P415S mutation on rice grain quality.The effect of exon 10 C-T mutation on rice quality in other Wx alleles was investigated.Three complementary expression vector.carrying Wxmw,Wxb and Wxin alleles and three mutant complementary expression vectors carrying the corresponding mutant alleles with site-directed mutation in the 115th base of each Exon 10(Wxin-10T.Wxb-10T and Wxmw-10T)were transformed to glutinous rice line Nip(wx).Through hygromycin resistance screening,the homozygous lines were obtained and then used for grain quality analysis.We found that the mutants carrying alleles with C to T mutation lead to significant decreased AC while the degree of the variation differed among different rice lines.Starch fine structure analysis showed that the modification of the Wx gene would also cause significant changes in amylopectin fine structure.In addition,we found that the C/T SNP would also cause differences in starch pasting profiles.Besides,rice grain from the mutant Wxb and Wxin showed more obvious changes in rice eating quality profiles than that from Wxmw.2.The effect of GBSSI phosphorylation sites editing on rice grain quality.The potential GBSSI phosphorylation sites 57T,168P,283S,298T,349T,415S and 569S were selected as the target sites to do the site-directed gene editing by using nCas-PBE system in the background of japonica rice Nipponbare.A total of 183 independent transgenic lines were obtained from the 7-base editing construction by using agrobacterium-mediated genetic transformation.Gene specific primers were designed based on the target sequence and used for PCR amplification of the the target segments.The decoding results show that all constructions except for 57T and 10CT and 168P sites contain mutation.Among the 183 independent transgenic lines,70 plants with mutant sites were identified by target gene sequence sequencing.And the results showed that the base editing efficiency of different constructions is very different.The editing efficiency of the 168 construct reached to 92%,followed by the 283 construct with 81%.The editing efficiency of the 298 construct is is also very high(57%),while the 349 and 569 constructs show a relative editing efficiency of about 33%.These results indicate that the target sequences used for base editing system has a big influence on the editing efficiency.We then screened the homozygous mutant plants with different mutant sites,and analyzed the gene and protein expression profiles and rice grain from the above plants were prepared and used for grain quality analysis.The results showed that changes in amino acid residues at phosphorylation sites would affect the expression level of Wx as well as the GBSSI enzyme activity,thereby reducing AC and GT.Comparing the effects of the 4 target sites,we found that;in the background of Wxb,rice plants with mutations at positions 283,298,and 349 would fine-tune rice grain AC,and those with mutations at positions 283 and 298 show big influence on AC with changes to about 3%.Besides,the mutants with deletion of individual bases in the Wx coding sequence region lead to glutinous rice grain.To sum up,the above rice lines with site-directed mutation and base editing mutation proved more genetic resources for rice grain quality improvement.