Preliminary Identification Of Gene OsSAM And Related Functions Of Rice Sheath Blight Resistance

Rice sheath blight is a rice disease caused by Rhizoctonia solani infection on plants.Rice resistance to sheath blight is a typical quantitative trait.At present,there is no major gene with resistance or resistance to rice germplasm.Our research group used gene chip technology to analyze the gene expression profile of the susceptible rice variety TP309 12 hours after infection with Rhizoctonia solani,and selected a resistance candidate gene OsSAM,and through the CRISPR/Cas9 knockout and gene overexpression technology constructed two mutant rice,named CrSAM,OeSAM.This study included three consecutive generations of positive screening of the obtained mutant material and related resistance research on sheath blight,the main findings are as follows:(1)Positive screening of T1,T2,T3 generation rice materials.According to the plasmid sequence of the overexpressing mutant OeSAM,specific primers were designed for PCR amplification and identification;based on the CRISPR target of the knockout mutant CrSAM,specific primers were designed for PCR amplification and sequencing,and the detection of the base change of the target fragment was identified as positive.In the T3 generation,7 OeSAM positive lines and 1 CrSAM positive homozygous line were obtained.(2)Toothpick inoculation of Rhizoctonia solani at the end of tillering stage of T3 generation rice.The area of lesions was investigated 7 days after inoculation,the results showed that the area of wild-type lesions was 0.74 cm~2;the area of CrSAM lesions was 4.01 cm~2;the area of OeSAM lesions was 0.52 cm~2,the results are extremely significant(P<0.01).The disease grade of sheath blight was investigated 30 days after rice heading.The results showed that the wild-type disease grade was 6.75;CrSAM disease grade was 8.23;OeSAM disease grade was 4.95,the disease grade results were extremely significant.The above indicates that CrSAM is more susceptible to disease and OeSAM is more resistant to disease,and it is initially proved that the gene OsSAM is positively correlated with rice sheath blight resistance.(3)The basic agronomic traits were investigated at the adult stage of T3 generation rice,and the yield was measured at the mature stage.The results showed that the plant height,chlorophyll content,flag leaf height,and flag leaf length of CrSAM were significantly lower than those of wild type,and the number of tillers and tiller angle were significantly higher than those of wild type;the plant height and number of tillers of OeSAM were significantly higher than that of wild type The height of flag leaves was significantly higher than that of wild type(P<0.05).Correlation analysis of rice agronomic traits and corresponding sheath blight disease grades revealed that the correlation between CrSAM disease grade and plant height was relatively high,and the correlation coefficient r was-0.442,The results are significantly different,indicating that plant height was negatively correlated with sheath blight disease grade in CrSAM relationship.The results of the production test showed that the wild-type yield was the highest,with a yield of 523.23 kg per mu,followed by OeSAM with a yield of 498.65 kg,and CrSAM with a yield of 264.78 kg.The yield of CrSAM per mu is only 0.51 times that of the wild type,indicating that CrSAM disease is serious and leads to a reduction in production.(4)RT-PCR and qPCR were used to detect the expression level of OsSAM gene in wild-type,CrSAM and OeSAM in the four-leaf sheath,three-leaf sheath,three-leaf and two-leaf rice tissues.The results showed that the gene expression was not detected in CrSAM rice tissues;the expression level of the gene in four parts of OeSAM was significantly higher than that of wild type;the expression level in rice leaves was significantly higher than that of leaf sheath.In addition,at 0 h,1 h,24 h,and 72 h after inoculation of CrSAM,OeSAM,and wild-type Rhizoctonia solani,samples were taken and inoculated with tiller-inverted trifoliate sheath,inverted trifoliate,inverted bifoliate sheath,and inverted bifoliate for qPCR The expression of OsPR1b,PDR and E2F genes related to the disease course of rice sheath blight was detected.The results showed that among the three-leaf leaf sheaths and two-leaf leaf sheaths,CrSAM had the highest expression of disease-path genes,followed by wild-type and OeSAM.The expression of disease-path genes had a positive correlation with the degree of rice sheath blight.