Functional Analysis Of Rice Sucrose Phosphate Synthase

Non-structural carbohydrate(NSC)in rice stem is very important for increasing rice grain sink strength and stability of yield under adverse conditions.The transformation of stem NSC into grain involves degradation of starch and resynthesis of sucrose during grain-filling stage.Sucrose synthesis in rice is mainly catalyzed by sucrose phosphate syn-thase(SPS,EC 2.4.1.14).Therefore,it is of great significance to study the function of SPS in rice for yield increase and stress resistance.The knockout mutants of rice SPS2,SPS6,SPS8 and SPS11 were obtained by CRSPR/Cas9 technology.And the characteristics of the mutants were studied.The results are as followings:(1)Subcellular localization studies using green fluorescent protein(GFP)fusion con-structs indicate that SPS1,SPS2,SPS6,SPS8,and SPS11 are all localized in the cytosol.(2)Real-time q-PCR shown that SPS1 and SPS11 genes were mainly expressed in the leaves,and SPS1 was also highly expressed in anthers.The expression of SPS2 and SPS6did not have obvious tissue specificity.Both real-time q-PCR and GUS staining showed that SPS8 gene was highly expressed in the stems of rice.(3)The knockout mutants of rice SPS2,SPS6,SPS8 and SPS11 were obtained by using CRSPR/Cas9.Real-time q-PCR showed that the expression level of theses genes in the mu-tant line decreased significantly.Western blot assay showed that there were no protein sig-nals of SPS8 could not be detected in two sps8 lines,while protein signals could be detected in complementary lines.(4)Through two years of field observation,sps2,sps6 and sps11 mutants displayed WT vegetative and reproductive morphology.it was found that the superior grains filling rate of sps8 lines was significantly lower than that of WT during rapid grain filling period.The grain filling rate of the inferior grains was lower than that of WT in the whole grain filling stage.(5)The statistics of yield showed that the yield composition of mutant lines sps2,sps6 and sps11 had not significantly difference with WT.The percentage of ripened grain of-SPS8-1 decreased significantly compared with WT,and the yield decreased by 15-17%.Shading treatment can increase the yield difference between WT and sps8.(6)By measuring SPS enzyme activity of different mutants in different tissues,it was found that there was no significant difference in V_max and V_limit among sps2,sps6 and sps11.In the stem of sps8 mutant,V_max and V_limit were lower than that of WT.(7)By measuring the sugar content in different tissues,we found that the sucrose,starch,glucose and fructose content in sps2,sps6 and sps11 were not significantly different from that of WT.However,sucrose content in stem of sps8 decreased,starch accumulation in-creased,and glucose and fructose content also increased.(8)The SPS enzyme activity in leaf,stem and endosperm of sps8 lines and WT during the grain filling stage was determined.It was found that the level of V_max and V_limit in stem were lower than that of WT during the whole grain filling stage.we determined the sugar content in leaves,stem and endosperm of sps8 and WT during the grain filling.We found that the content of starch in sps8 shown great accumulation.?NSC of sps8 was significant-ly lower than that of WT.The ¹³C labeling experiment showed that the labeled carbohydrate before heading could not be effectively converted into grains.(9)Among the enzymes involved in sucrose metabolism,the expression of Susy1 and UGP2 gene was up-regulated and Susy activity increased in the stems of sps8.Among the enzymes involved in starch degradation,the expression of Amy3D and BAM were up-regulated,and the activity of?-amylase and?-amylase was increased.The expression of SUT4 was up-regulated.(10)The interaction between Os SPS8 protein and 14-3-3 protein was confirmed by im-munoprecipitation experiment and yeast double-hybrid experiment.