Cloning And Study On Rice OsMY1 Gene

The OsMY1(GenBank DQ641916)gene was previously unknown in this laboratory by yeast two-hybrid screening and was isolated from the cDNA library of young panicles during the formation of rice and rice.Sequence analysis showed that the 5' end of the cDNA coding region of the gene was incomplete.In this study,the full-length cDNA sequence of the gene was cloned,and the CRISPR/Cas9 genome editing technology was used to construct the OsMY1 knockout rice.At the same time,the OsMY1 RNAi rice T1 generation constructed in the laboratory was used as a material to conduct research,aiming to identify the gene in the The function of rice growth and development.To obtain the 5?-end sequence of the OsMY1 gene,the full-length cDNA sequence of the gene was cloned from the panicle tissue of rice using 5?-RACE binding RT-PCR technique.Firstly,the 5?-end sequence was obtained by 5?-RACE.After splicing with the original 3?-end sequence,the full-length 1953 bp OsMY1 cDNA sequence was obtained.Primers were designed and the c DNA sequence was amplified by RT-PCR.The sequencing results showed that the OsMY1 gene cDNA sequence contained a 1140 bp ORF encoding 379 amino acids.Bioinformatics analysis showed that OsMY1 protein contains only two conserved CC domains and belongs to the rice ICR protein.OsMY1 is 90.7% homologous to the ICR sequence of O officinalis.In order to analyze the expression pattern of OsMY1 gene in rice,qRT-PCR technique was used to detect OsMY1 gene at different stages of development: seedling stage(root,leaf),tillering stage(root,leaf),and spikelet differentiation stage.The expression level.The expression of OsMY1 in roots was higher at the seedling stage than in the roots,and was higher in the roots of the tillering stage than in the leaves.The expression level of OsMY1 in the panicles at the heading stage was significantly higher than that of the other two developmental stages.The expression level was high,among which the highest expression level was in spikelets with 3-5mm spikelet length.To identify the function of the rice OsMY1 gene,a CRISPR/Cas9 genome editing technique was used to construct knockout rice.Through sequence comparison,two editing targets were screened and a CRISPR/Cas9 plant expression vector was constructed.Using Nipponbare rice as a material,Agrobacterium-mediated method was used to genetically transform rice callus and obtain regenerated plants.Screening for hygromycin resistance genes and sequencing of PCR target sites confirmed that two homozygous knockout rice lines(Cas9-OsMY1)were obtained.The agronomic traits of the Cas9-OsMY1 rice T1 generation were statistically analyzed.The results showed that compared with the control,the plant height was significantly reduced,with an average decrease of 5.3%,and the spike length was significantly increased with an average increase of 18.6%,but effective.There were no significant differences in agronomic traits such as tiller number,panicle number,seed setting rate,and 1000-grain weight.Continuous screening and identification of the T2 and T3 generations of the OsMY1 gene RNAi rice(RNAi-OsMY1)constructed earlier in the laboratory yielded six OsMY1 gene expression down-regulated lines.The RNAi-OsMY1 rice phenotype observation revealed that the height of RNAi rice plants was significantly reduced at the seedling stage and the reproductive growth stage compared with the control.Statistical analysis of agronomic traits showed that plant height,panicle length,seed setting rate,and 1000-grain weight all decreased significantly.Among them,the average plant height decreased by 21.25%,the panicle length decreased by an average of 17.1%,the seed setting rate decreased by an average of 10.5%,and the grain weight decreased by an average of 13.24%.In order to explore the reasons for the reduced height of the OsMY1 RNAi rice and knockout rice plants,qRT-PCR was used to detect the expression of the gibberellin(GA)synthesis pathway and signal transduction pathway related genes in the leaves.In rice or knockout rice,gibberellin synthesis genes(OsCPS,OsKO2,OsKAO)and signal forward-regulated gene(OsGID1,OsGID2)were down-regulated compared to the control,while the negative control signaling gene OsSLR1 was compared with the control.Increase in the number of expressions.Based on the previous work of the laboratory,this study cloned the full-length cDNA sequence of OsMY1 gene by 5?-RACE combined with RT-PCR technology.Through sequence alignment analysis,it was confirmed that this gene belongs to rice ICR protein.RNAi and CRISPR/Cas9 genome editing techniques were used to perform functional identification of this gene,respectively,and it was confirmed that the gene was knocked out or the expression was inhibited.In order to understand the molecular mechanism of plant height reduction in transgenic rice,the GA signal pathway was used as the starting point to prove that the reason for the decrease of plant height was the down-regulation of the key gene expression pathways of GA synthesis pathway and signal transduction pathway.This will further study the OsMY1 gene in rice plant height.The functions in trait control laid the foundation,and also provided new potential candidate genes for rice dwarf molecular breeding.