Editing Of Waxy Gene In Rice To Regulate Rice Quality

Rice（Oryza sativa L.）is an important food crop in my country.With the rapid economic development and people’s continuous pursuit of high-quality life,the taste quality of rice has received great attention in breeding projects.Amylose content（AC）is the main indicator of rice eating quality and cooking quality,and is controlled by the Wx gene in rice,and its encoding protein is GBSSⅠ.So far,studies have found that the N-terminal domain of Wx can greatly affect the AC of rice grains,but the number of natural mutations in this region is limited,and whether the structure of other regions of Wx affects AC needs further research to prove.In this study,the 7th and 10th exon regions of Wx were edited by the adenine/cytosine base editor.According to the nucleotide substitution changes of 15 amino acids in Wx,a series of new Wx alleles with AC 0.3-29.43%（control AC 19.87%）were produced.Among them,the rice grains of the waxy^abe2editing line（amino acid change type is T237A）showed AC similar to"soft rice".After research,the mutant strain has improved eating and cooking quality（ECQ）and grain appearance,but there is no significant difference in other agronomic traits from the control group.In addition,we successfully removed foreign genes from waxy^abe2offspring.The discovery of waxy^abe2alleles provides a new allele material for rice breeding that can improve the quality of rice,and meets the diversified needs of crop breeding.The specific results are as follows:1.Ten vectors of Waxy-ABE1,Waxy-ABE2,Waxy-ABE3,Waxy-CBE4,Waxy-ABE5,Waxy-CBE6,Waxy-CBE7,Waxy-CBE8,Waxy-ABE9 and Waxy-ABE10were successfully constructed,and the target fragments were transformed into the recipient callus.The target gene-edited transgenic plants were obtained through testing,which preliminarily proved the feasibility of base editing technology in crop breeding.2.Extract genomic DNA from T₀generation transgenic plants,and use Sanger sequencing method to observe and count the editing type of each plant.A total of 448transgenic plants were obtained,of which 151 rice plants were edited,with a total editing efficiency of 34.00%.The base editing efficiency of the vectors Waxy-ABE10and Waxy-CBE7 is very high,68.63%and 66.67%,respectively.ABE and CBE produced 34.29%and 40.00%homozygous mutants at the ABE10 and CBE4 sites,respectively.According to statistics,a total of 18 Wx gene nucleotide substitution sites were obtained in this experiment,resulting in 15 amino acid substitutions in the GBSSⅠprotein.A total of 18 novel Wx alleles were created in this study.3.The AC measurement of homozygous T₂generation showed that the change of amino acid affected the amylose content of rice endosperm.The starch AC at amino acid mutation sites of Q427R and T237A were significantly increased and moderately decreased,and some sites（H236R,E410K/Q and E411K）decreased to the level of glutinous rice.And using Sanger sequencing to analyze E411K,T237A mutant plants,no gene off-target phenomenon was found.These results indicate that the amino acid changes of the GBSSⅠprotein in the grain fine-tune AC.4.The endosperm iodine staining of the mature rice waxy^cbe9.1and waxy^abe2edited at E411K and T237A sites found that,consistent with the data of AC,the ratio of base-edited kernel endosperm amylose to amylopectin was lower than that of the wild-type parent.AC value is negatively correlated with transparency.The transparency of rice changed by H236R,E410K/Q and E411k amino acids is 5,which is white and completely opaque.5.Through the quality analysis of mature rice at waxy^cbe9.1and waxy^abe2sites,it is found that the amylose content of transgenic rice is closely related to the gel consistency.The gel consistency is significantly higher than that of the wild-type parent,indicating that the rice quality of the transgenic rice has become softer.In the starch RVA profile,the gelatinization temperature of waxy^cbe9.1and waxy^abe2locus-edited rice was lower than that of the wild type.The waxy^abe2transgenic starch showed a higher disintegration value（2155.00 c P）and a lower reduction value（-1177.00 c P）,indicating that its ECQ is better.6.Observing the morphology of starch granules under a scanning electron microscope,it can be found that the waxy^abe2editing line and the wild-type endosperm native starch granules are basically the same in morphology.They are all polygons with sharp edges and corners,and the surface is smooth,flat or slightly recessed,without structural debris.However,the waxy^cbe9.1editing site particles are relatively irregular,and the starch granule core has many small pores.It is speculated that the transparency value of the endosperm of waxy^abe2and waxy^cbe9.1increased,which may be due to the small space in the center of the starch granules.7.Analysis of the agronomic traits of homozygous edited rice showed that there was no significant difference between the transgenic rice and the parent in plant height,plant shape,grain width,grain length,tiller number,and seed setting rate.These results are necessary prerequisites for achieving crop improvement goals without affecting the agronomic characteristics of the superior variety（XS134）.8.Use specific primers to perform primer analysis on Tad A box,hygromycin box and UBI box,and obtain 5 foreign DNA-free expression lines in waxy^abe2alleles.Therefore,we speculate that waxy^abe2may constitute a new type of"soft rice"with good breeding potential.