Preliminary Function Identification Of OsRac5 In Rice Growth And Development

The rice ROP gene OsRac5 is a functionally unknown gene isolated from rice panicles during pistil and stamen formation.Previous studies have found that this gene is associated with photoperiod fertility conversion of photoperiod-sensitive genic male sterile rice cultivar 58 S,and is widely expressed in rice growth and development,and has the highest expression in young panicles;In order to identify the function of this gene,RNA interference rice has been constructed in the early stage of the laboratory.In this study,the knockout rice of this gene was further constructed by using CRISPR/Cas9 gene editing technology,and the wild type Nipponbare rice(WT),RNA interference rice(RNAi-OsRac5)and knockout rice(Cas9-OsRac5)of the gene were used as materials to identify the function of this gene in rice growth and development.The PCR and qRT-PCR of RNAi-OsRac5 rice showed that compared with WT rice,the 18 lines tested were all transgenic lines,and the expression level of OsRac5 gene in 15 transgenic lines was significantly down-regulated.The down-regulation range is 0.15~0.78 times of the control group,which can be used as a material for subsequent experiments.The OsRac5 gene was edited by CRISPR/Cas9 technology,and the constructed Cas9-OsRac5 vector was transformed into Nipponbare rice.PCR and sequencing confirmed that 6 hybrid transgenic lines were obtained in T0 generation,and 10 homozygous knockouts lines were obtained in T1 generation,the nature of the mutation is the deletion and/or insertion of the base;Multiple sequence alignments are performed on the amino acid sequences of the OsRac5 gene-encoded protein in different homozygous knockout lines,and the results showed that in 10 homozygous knockout lines,frameshift mutations and nonsense mutations occurred in the OsRac5 sequence,causing amino acid sequence changes,which led to the production of OsRac5 truncated protein.Due to the loss of the conserved GTP/GDP binding site of the truncated protein,the GTPase domain is incomplete,so it is expected that there will be no normal OsRac5 protein production in the 10 homozygous knockout lines.The germination experiments of rice seeds showed that the seed germination completion time of RNAi-OsRac5 and Cas9-OsRac5 rice was 12 h and 24 h earlier than that of WT rice.At 3d,5 d and 7d of rice seed germination,compared with WT rice,the radicle elongation of RNAi-OsRac5 rice increased significantly,which increased by 1.95 times,1.09 times and 1.14 times,respectively,and the germ elongation of RNAi-OsRac5 rice increased significantly,which increased by 0.61 times,0.71 times and 0.49 times respectively;the radicle elongation of Cas9-OsRac5 rice increased significantly,which increased by 2.14 times,1.2 times and 1.22 times,respectively,and the germ elongation of Cas9-OsRac5 increased significantly,which increased by 0.81 times,0.77 times,and 0.59 times,respectively.It indicated that the knockout or down-regulation of OsRac5 gene promoted rice seed germination and seedling growth.The cytological section experiments of 8 typical stages of rice microspore development showed that compared with WT rice,there was no significant difference between the first and sixth stages of RNAi-OsRac5 and Cas9-OsRac5 rice,atrophy and abnormal vacuolization of microspores appeared in the seventh stage(Late microspore stage),more abnormal pollen grains appeared in the eighth stage(Mature pollen stage);The mature pollen grains were stained and the number of statistically active pollen was observed.The results showed that the fermented pollen ratios of RNAi-OsRac5 and Cas9-OsRac5 rice were significantly reduced compared with WT rice,which decreased by about 13.4% and 20.2%,respectively.It indicated that the knockout or down-regulation of OsRac5 gene affected the late development of microspores,which in turn affected the pollen maturation process and led to a decrease in fertility pollen ratio.At the heading stage,the physiological indexes of flag leaves were measured.The results showed that the net photosynthetic rate of RNAi-OsRac5 and Cas9-OsRac5 rice was significantly reduced compared with WT rice,which decreased by about 9.7% and 32.7%,respectively,while there was no significant change in the transpiration rate of RNAi-OsRac5,and the transpiration rate of Cas9-OsRac5 rice was significantly reduced,which decreased by about 27.2%.At the maturity stage,the agronomic traits of rice were counted.The results showed that compared with WT rice,the plant heights of RNAi-OsRac5 and Cas9-OsRac5 rice significantly increased,increasing by 6.6%,6.0%,respectively;The effective tiller number of RNAi-OsRac5 and Cas9-OsRac5 rice significantly reduced,which decreased by about 37.5%,40.5%,respectively;The spikes were significantly reduced,which decreased by about 8.5%,15.1%,respectively;The seed setting rate of RNAi-OsRac5 was significantly reduced,which decreased by about 6.0%;The seed setting rate of Cas9-OsRac5 rice was significantly reduced,which decreased by about 19.7%;The 1000-grain weight of RNAi-OsRac5 rice did not change significantly;The 1000-grain weight of Cas9-OsRac5 rice was significantly reduced,which decreased by about 12.4%;There were no significant changes in the numbers of the first branch,the total grain size,the grain length and the grain width.Based on the preliminary work of the laboratory,this study constructed 10 rice OsRac5 knockout homozygous lines using CRISPR/Cas9 technology.The wild type Nipponbare rice(WT),RNA interference rice(RNAi-OsRac5)and knockout rice(Cas9-OsRac5)of the OsRac5 gene were used as experimental materials to study the function of OsRac5 gene.The function of this gene was found to involve rice seed germination,seedling growth,fertility and important agronomic traits such as plant height,tiller number and seed setting rate,it laid a foundation for further study of the function of this gene,and also provided an important basis for exploring the value of this gene in rice molecular design and breeding.