Identification And Mutational Analysis Of Rice RMV1 Homologous Genes

Rice is one of the most important food crops in the world.The total rice production of China ranks first in the world,and 60%of the population eats rice.Weeds can seriously affect the yield and quality of rice.Paraquat is a fast-killing herbicide that can be quickly absorbed by plant green tissue and eventually kills plants.The Arabidopsis RMV1 gene encodes an amino acid transporter that helps green plants uptake and transport polyamines in the environment.Paraquat is a polyamine analogue and shares the transport system with polyamine substances,the mutation of RMV1 gene can impair paraquat absorption and its transport in cells,and consequently results in resistance of Arabidopsis to paraquat.Three homologous genes of RMV1were identified in rice and knocked out using CRISPR/Cas9 to create new rice germplasm resistant to paraquat in the present study.The main results are as follows:Firstly,three homologous genes of RMV1 in rice and named as OsPUT1,OsPUT2 and OsPUT3.OsPUT1/2/3 and RMV1 were very similar in gene structure and transcript length,and their encoded proteins have similar number of amino acids and similar number of transmembrane domains at similar locations.Using the multi-target gene editing system p YLCRISPR/Cas9,we successfully obtained triple knockout mutants,with proteins encoded by OsPUT1,OsPUT12 and OsPUT3 were all truncated.Two mutant lines OsPUT1/2/3-KO-1and OsPUT1/2/3-KO-6,were generated.The mutations in these two lines lead to deletion of transmembrane domain in their encoded proteins.Through the investigation into agronomic traits,it was found that the agronomic and yield-related traits of the triple knockout mutant lines OsPUT1/2/3-KO-1 and OsPUT1/2/3-KO-6 were not significantly different from those of the wild-type parents,indicating that the deletion of OsPUT1/2/3 did not affect the normal growth and yield of rice.In addition,the transcription level of OsPUT1/2/3 was determined by RT-PCR under paraquat treatment.It was found that the transcription level of OsPUT1/2/3 was significantly increased by exogenous paraquat.Secondly,the resistance of OsPUT1/2/3 triple knockout mutants to paraquat were verified.Under the treatment of 1.0 mmol·L^-1paraquat foliar spraying,the wild-type rice plants showed a sensitive lethal phenotype,while the mutant rice plants could still maintain normal growth and development.When cultured 1/2MS solid medium supplemented with 0.1μmol·L^-1paraquat,the growth and development of above ground parts of wild-type rice were seriously affected:the elongation of main roots and the growth of adventitious roots were significantly inhibited.OsPUT1/2/3-KO-1 and OsPUT1/2/3-KO-6 mutant lines showed significant resistance to paraquat,and maintained normal growth and development in the concentration range of 0.1-2.0μmol·L^-1.In addition,the transcription level of OsPUT1 in the OsPUT1/2/3 triple gene knockout mutant was no longer induced by paraquat treatment,and remained at a low and stable level.With the increase in paraquat treatment time,the transcription level of OsPUT3 in the triple gene knockout mutant was significantly up-regulated but only about 57%of that in the wild type.Thirdly,the physiological basis of resistance to paraquat in OsPUT1/2/3 triple knockout mutants were studied.By observing and measuring the chloroplast structure and chlorophyll content with OsPUT1/2/3-KO-1 and OsPUT1/2/3-KO-6 mutant lines and wild-type under paraquat treatment,it was found that the chloroplast structure of wild-type rice leaves was significantly damaged and the chlorophyll was largely lost under 1.0μmol·L^-1paraquat treatment.The deletion mutation of OsPUT1/2/3 significantly reduced the toxicity of chloroplasts in mutant rice plants under 0.1–2.0μmol·L^-1paraquat treatment.In addition,the paraquat contents in leaves and roots of OsPUT1/2/3-KO-1 and OsPUT1/2/3-KO-6 mutants and wild-type plants under different concentrations of paraquat were measured,respectively.It was found that no paraquat was detected in rice plants under normal culture conditions.Compared with plant roots,rice mainly transported to leaf tissues after uptake of exogenous paraquat.The paraquat contents in leaves and roots of wild-type rice plants increased significantly from the increase in exogenous paraquat concentration.Compared with wild type,the accumulation of exogenous paraquat in leaves and roots of OsPUT1/2/3-KO-1 and OsPUT1/2/3-KO-6 mutants decreased significantly and remained relatively stable at a low level.Finally,the two mutants and the wild type were investigated for their resistance to other types of herbicide with different targets.It was demonstrated that the mutant plants also had a certain degree of resistance to four types of herbicides,i.e.,enoxadone,quinclorac,imidazole-ethylnicotinic acid and ethoxyfluorfen.Under the treatment of the first three herbicides,compared with the wild type,the mutant plants showed late sensitive phenotypes.Under the condition of acetofluorfen treatment,the wild-type plants were sensitive to death within 4 days,while the mutant plants showed significant resistance and maintained normal growth and development within 7 days.The main conclusions of this study are as follows.（1）There are three genes highly homologous to RMV1 in rice:OsPUT1,OsPUT2 and OsPUT3,and OsPUT1/2/3 have transmembrane domain very similar to the protein encoded by RMV1.（2）Exogenous paraquat could significantly increase the transcription level of OsPUT1/2/3 genes.（3）Knockout of OsPUT1/2/3 can significantly improve rice resistance to paraquat.（4）The knockout of OsPUT1/2/3 could significantly inhibit the absorption of exogenous paraquat and its accumulation in plants,thereby protecting chloroplast structures from damage.（5）The knockout of OsPUT1/2/3 could significantly improve the resistance of rice to herbicides other than paraquat（etenophenone,quinclorac,imidazole-ethylnicotinic acid,and acetofluorfen）.The above research results provide a theoretical basis for further study on the gene functions of OsPUT1/2/3 and the molecular mechanism of paraquat transport from rice in vitro to in vivo.OsPUT1/2/3 triple gene knockout mutant lines can also be used as new rice germplasms with other types of herbicide resistance（etenophenone,quinclorac,imidazole-ethylnicotinic acid,and acetofluorfen）in actual agricultural production.