Reduction Of Rice Amylose Content By Editing The 5’UTR Of The Wx Gene Via Crispr/Cas9 Technology

Along with the improvement of our national living standard,consumers have higher requirements for the cooking and eating quality of rice,and soft and sticky rice is more favored by consumers.The eating quality of rice is a complex quantitative character,which is closely related to amylose content,GEL consistency,viscosity,cooking quality and appearance quality.The Waxy gene is involved in the biosynthesis of amylose in endosperm of rice,and regulates the amylose content of rice,thus affecting the degree of softness of rice.Therefore,in order to create a batch of soft rice breeding materials,the CRISPR/Cas9 gene editing technology was used in this study to edit the Wx gene of three excellent indica hard rice materials:“Mei Meng B（MMB）”,“Tian Kang B（TKB）”and“DR462”.The main research results are as follows:1.Genetic transformation of rice.According to the sequence of the parent Wx gene（LOC＿Os06g04200）,the CRISPR/Cas9 vector was constructed by targeting the intron splicing site（5’UISS）in the 5’UTR region of Wx.The vector was successfully introduced into MMB,TKB and DR462 rice test materials by agrobacterium-mediated transformation.2.Mutation analysis of rice transformants.24 positive transformed plants were obtained from line MMB with 80.0%of the transformation rate,among which 8 were homozygous ones with 33.3%of the homozygous mutant rate;27positive transformed plants were obtained from line TKB with 81.8%of the transformation rate,among which 9 were homozygous ones with 33.3%of the homozygous mutant rate;and 26 positive transformed plants were obtained from line DR462 with 74.3%of the transformation rate,among which 9 were homozygous mutants with 34.6%of the homozygous mutation rate.The homozygous mutant lines（MMB-10,TKB-21,DR462-9）were selected from T₀generation to receive seeds for T₁generation planting,and then 20representative lines were selected from T₁generation for T-DNA-free detection.The results showed that 7,5 and 4 T-DNA-Free plants were isolated from MMB-10 T₁line（M-10-1～M-10-20）,TKB-21 T₁line（T-21-1～T-21-20）and DR462-9 T₁line（D-9-1～D-9-20）,respectively.3.Quantitative analysis of the expression of Wx gene in edited lines.The results showed that compared with parents MMB,TKB and DR462,the expression of Wx gene in T₂generation was significantly decreased（P<0.01）.4.Analysis of rice eating quality index of the edited lines:（1）Amylose content（AC）.The results showed that the AC values of the endosperm were 27.8%,26.0%and 25.4%in parents MMB,TKB and DR462,respectively.Compared with the AC of parents,AC in the endosperm of the T₂edited lines were significantly decreased（P<0.05）.Among them,the AC of m-10-2,T-21-12 and D-9-9 was 17.2%,16.8%and 17.8%,all reaching the AC standard of first-grade rice（13%～18%）,and the rice quality was significantly softer,indicating that gene editing could effectively reduce the AC content and obtain the line with ideal AC content.（2）Gel consistency（GC）.The results of GC analysis of T₂edited lines M-10-2,T-21-12 and D-9-9 with no exogenous DNA and low AC showed GC were significantly increased up to 78.6 mm,77.4 mm and 79.6 mm from 60.2mm,61.2 mm and 61.4 mm of respective parents,which indicated that the GC of the edited lines was improved significantly.（3）Rapid Viscosity Analysis spectrum（RVA spectrum）.The rapid RVA analysis results of the edited lines showed that the disintegration values（BDV）of the edited lines M-10-2,T-21-12 and D-9-9 increased up to 994.0 cp,1533.6cp and 1293.3 cp from 355.0 cp,641.3 cp and 508.7 cp of the respective parents;The subtractive value（SBV）of the edited line decreased down to 1366.3 cp,729.6 cp and 1444.6 cp from 2353.6 cp,1978.0 cp and 1457.6 cp.The reply values（CSV）of the edited lines M-10-2 and T-21-12 decreased to 2360.3 cp and2263.3 cp from 2708.6 cp and 2619.3 cp of respective parents,while that of the edited line D-9-9 increased to 2738.0 cp from 1966.3 cp of its parent.Analyzed results indicated that the rice quality of edited lines became soft and elastic,and the eating quality of rice was obvious improved.5.Analysis of main agronomic characters of edited lines.The results showed that the plant height,1000-grain weight,panicle length,grains per panicle,seed setting rate and effective panicle number of the T₂edited lines M-10-2,T-21-12 and D-9-9 had no significant changes compared with their parents.The rice chalkiness of T₂edited lines M-10-2 and T-21-12 had no significant difference compared with their parents MMB and TKB,while the chalkiness of D-9-9 was decreased and its mled rice appearance was improved compared with parent DR462.These results suggest that the character changes produced by homozygous editing lines using CRISRP/Cas9 gene editing techniques can be stably passed on to self-crossing progeny.