The Function Of The Single Nucleotide Polymorphism SNP₁₀^C/T On Exon 10 Of Wx Gene In Rice

The amylose content(AC)in rice endosperm is considered as the most important determinant influencing rice cooking and eating quality.The Waxy(Wx)gene,controlling the synthesis of amylose in endosperm,is the major locus affecting rice grain quality.Multiple alleles of Wx gene have been identified,which might be the main reason for the diversity of rice grain quality.It is very important to clone new genes or alleles controlling starch biosynthesis and regulation mechanism of the rice grain quality formation.In previous study,we have identified a new Wx allele,Wxlsv,which is responsible for a high AC and low viscosity profiles.Based on the sequence alignment,just a SNP C-T in the exon 10(SNP10C/T)was found between Wxlsv and Wxa.With the exception of Wxa with T nucleotide,we found that all the other Wx alleles had C atspecific site.In order to verify the direct effects of the SNP10C/T on rice quality,we have constructed a serious plasmid vectors that carried the SNP10C/T in several Wx alleles and the effect of the SNP10C/T on rice grain quality were analyzed.The main results are as following:1.The full-length genomic DNA of Wxlsv and Wxa from Q11 and GC2,respectively,was introduced into a near-isogenic line(NIL)under the Nipponbare(Nip)background carrying the null wx allele,Nip(wx).The data showed that both the Wxlsv and Wxa allele contributed to a high AC level.However,Wxlsv transgenic rice(Nip(wx)-Wxlsv)showed softer and more viscous gel and significantly lower starch viscosity compared to Wxa transformants(Nip(wx)-Wxa).The Wxlsv gene was further transformed into the NIL line Nip(Wxa);the mature grains of the transgenic line Nip(Wxa)-Wxlsv showed higher AC,softer GC,and lower RVA profile compared to the wild-type Nip(Wxa).These results demonstrate that the SNP10C/T is truly responsible for the high AC and low starch viscosity.2.When developing the seeds of NILs with Wxlsv and Wxa alleles,both the Wxlsv and Wxa alleles produced a similar level of mature Wx mRNAs.In addition,the levels of both total and starch granule-bound GBSSI proteins in the mature seeds were similar between the NILs with Wxlsv and Wxa alleles.However,the activity of Wxlsv-encoded GBSSI in the developing seeds was obviously higher than that of Wxa-encoded GBSSI during the entire grain-filling stage.By using homology modeling,we found that the the 415th proline-to-serine substitution by the C-T substitution at the SNP10C/T site of Wx gene did not change the alpha-helix structure of Wxa-encoded GBSSI compared to that of Wxlsv-encoded GBSSI.Then we identified the phosphorylation sites of GBSSI and found that at least nine residues,including the 415th serine residue,were phosphorylated in Wxa encoded GBSSI.In contrast,the 415th proline residue in Wxlsv-encoded GBSSI was not phosphorylated.This implies that the serine-to-proline substitution at the 415th residue might change the phosphorylation level of GBSSI,thereby modulating its enzyme activity.3.With the purpose of knowing the effects of the SNP10C/T under the background of other Wx allels,we further generated several transgenic constructs,carrying the full Wxmp,Wxb,Wxin alleles and their point-mutant Wmmp-10T,Wxb-10T and Wxin-10T with a C-T substitution.All transgenic constructs were also introduced into the waxy NIL line Nip(wx).The results showed that this SNP caused a lower AC level and thus lower grain transparency,while the effect on RVA profiles differed between different alleles.