Construction And Identification Of Amylopectin Synthesis Pathway Genes In Rice

Rice?Oryza sativa?is an important food crop in the world.With the impro vement of people's living standards,there is a higher demand for the rice quality.Starch is the main component of rice endosperm and one of the main factors a-ffecting rice quality.The synthesis of starch is a network regulation process inv olving a variety of enzymes.The study of enzymes in its biosynthetic pathway i s an important strategy to optimize rice quality.The ratio of amylopectin to amy lose in rice endosperm is about 3:1,and the ratio of the two is an important fa ctor in the quality of rice.In this study,a highly efficient CRISPR-Cas9 system was used to construct a targeted vector for the key genes editing of the amylope ctin synthesis pathway.Through agrobacterium-mediated transformation,the target site mutated T₀ plants were obtained.Furthermore,T₁ generation of the diallele with the target site mutated material were obtained,which no exogenous fragme nt insertion was found.And with our method the mutant library of the amylopec tin synthesis pathway-related gene was quickly obtained.Thereby further clarifyin g the function of each gene and its effect on amylopectin synthesis in the amylo pectin synthesis pathway contribute to improve the starch content and structure o f endosperm,providing a theoretical basis for quality rice breeding.The main fin dings are as follows:1.A total of 13 directed editing vectors pQL05-pQL11,pQL13-pQL14 and pQL16-pQL19 were constructed for the SS family,SBE family and DBE family of rice amylopectin synthesis pathway genes.Two adjacent sgRNA1 and sgRNA2 sites were designed in the coding region of each gene to increase the probability of gene mutation and ensure the successful creation of mutant materials.2.Through Agrobacterium mediated genetic transformation,22 strains,21 strains,23 strains and 26 transgenic mutant plants were obtained for the SS gene family,include OsSSI gene,OsSSIIa gene,OsSSIIIa gene and OsSSIVa gene,respectively.The detection and identification of the target sites showed that 21 strains,15 strains,19strains and 18 mutant materials were obtained,and the mutation rate was71.43%-95.48%.The mutation efficiency of a single sgRNA was 28.57%-90.90%.3.The regenerated plants obtained from the stable transformation of the SBEs gene family were identified.The transgenic positive rate of OsSBEI,OsSBEIIa and OsSBEIIb was 100%,and 16 strains,16 strains and 15 transgenic positive plants were obtained,respectively.The results showed that the mutation rate was 56.25%-93.75%,and the mutation efficiency of single sgRNA was 43.75%-93.75%.9 strains,15 strains and 14mutant materials were obtained,among which the OsSBEI gene was regenerated.4.After stable transformation of rice,15 individuals were detected in the OsISA1 in the DBE enzyme gene family,and 14 transgenic positive individuals were obtained.The positive rate of transgene was 100%.The target sites were identified,and the total mutation efficiency was 100%,therefore a total of 15 mutant materials were obtained.A total of 23 individuals were detected by OsPUL,and the positive rate of transgene was100%.Two mutant sites were identified and analyzed,and 21 mutant materials were obtained with a mutation efficiency of 95.45%.5.By observing the appearance traits of the T₁ seeds of the genes OsSSI,OsSBEI,OsSBEIIa and OsPUL,it was found that the brown seed size of the gene OsSBEIIa T₀mutant pQL08-16-1 was smaller and the transparency was significantly reduced;OsSSI,OsPUL,OsSBEI,the brown seed length of them T₀ mutant strain was significantly larger than that of WT,and the grain width became smaller.Further analysis and identification of T₂ mutations will be carried out in the future.6.The T₁ plants of the mutant materials of the OsSSI,OsSBEI,OsSBEIIa and OsPUL were identified and screened,we finally obtained diallele mutations at the target site,which did not contain the exogenous fragment,and the mutant library of SS,SBE,and DBE gene family members of amylopectin was successfully created.The synthetic pathway gene mutant library provides a basis for further study of the function of starch synthesis genes.