Functional Analysis Of OrCrRlkl In Chaling Common Wild Rice

Chaling wild rice is one of the two wild rice polulations which exist northernmost in the world with strong cold tolerance.Recently,A global genome expression profiling of this wild rice has been conducted under cold stress using the GeneChip Rice Genome Array.A gene,designated OrCrRlkl?Oryza rufipogon cold responsive receptor-like protein kinase like gene?,was identified to be highly induced by cold.The study was performed to characterize the gene on the aspects of its expression characteristics,its overexpression function analysis,and its functional analysis of CRISPR/Cas9 knockout mutant.The aim of this study was to make clear whether the gene has cold tolerance or other effects.The following results were obtained:1.The phylogenetic analysis showed that the homologous gene of OrCrRlkl in rice was located on chromosome 1 and was predicted to be a gene encoding a leucine-rich receptor-like protein kinase.2.The full length cDNA fragment of OrCrRlkl gene was obtained by RT-PCR,and the gene was also found in other wild rice accessions and cultivated rice varieties.3.OrCrRlkl is expressed in wild rice and cultivated rice,and it is a stress responsive gene,however,its transcript in Chaling wild rice is higher than in other test cultivars and wild rice accessions,and is faster under stresses than others.4.The cDNA of OrCrRlkl gene was inserted into binary vector pHB and Cas9 and delivered successfully into rice Nipponbare by Agrobacterium-mediated method.Twenty one independent transgenic plants overexpressing this gene and three independent knockout mutant plants of this gene were obtained.5.The survival rate of the seedlings of transgenic Nipponbare rice plants overexpressing OrCrRlkl is 72.6%which was higher than that of its wild type Nipponbare?24.73%?after cold at 6?for 7 days.And the free proline content in transgenic Nipponbare rice plants was significantly higher than that in Nipponbare,on the other hand,the contents of H₂O₂and MDA in transgenic Nipponbare rice plants were significantly lower than those in Nipponbare.The relative conductivity of transgenic Nipponbare rice plants was significantly lower than that of Nipponbare,and the free proline content in transgenic Nipponbare rice plants was significantly higher than that in Nipponbare after cold stress at7??minimum temperature 0??for 7 days at booting stage.6.The survival rate of T₁ seedlings of transgenic Nipponbare rice plants overexpressing OrCrRlkl is 97.22%,not significantly different from that of Nipponbare,after simulated drought treatment with 20%PEG6000 for 7 days.The contents of free proline,chlorophyll and carotenoids in transgenic Nipponbare were 185.01?g/g,2.44 mg/g and0.34 mg/g,respectively,which were significantly higher than those in Nipponbare?the three indicators followed by 120.31?g/g,1.76 mg/g,0.26 mg/g?.The water lose in vitro leaf and the contents of H₂O₂ and MDA in transgenic Nipponbare were significantly lower than those in Nipponbare.7.The Germination velocity of transgenic Nipponbare overexpressing OrCrRlkl were significantly higher than that of Nipponbare.8.The growth rate,plant height and tillering ability of OrCrRlkl knockout mutants were significantly lower or weaker than those of wild type Nipponbare.The results indicated that the OrCrRlkl gene had the effects of enhancing the cold tolerance and drought tolerance of rice,and had important regulation effect on rice growth and development.