Functional Analysis Of OrCrZFl And OrCrGHl In Chaling Common Wild Rice

Chaling common wild rice(Oryza rufipogon Griff.),distributed in Chaling County,Hunan Province,China,is one of the two northernmost populations of wild rice in the world.It has strong cold tolerance and certain freezing tolerance as it can safely overwinter under natural conditions in Changsha City and Chaling County,Hunan Province.In our studies before,two cold-tolerant candidate genes OrCrZFl(Oryza rufipogon cold responsive zinc finger like gene)and OrCrGHl(Oryza rufipogon cold responsive glucose hydrolase like gene)were screened from the genome of Chaling common wild rice by using gene expression microarray analysis and bioinformatics analysis.One of them,OrCrZFl,may encode a zinc finger protein,and overexpression in japonica rice can improve cold tolerance of transgenic rice,the other,OrCrGHl,may encode a glycosyl hydrolase.The aim of this study was to analyze the function of CRISPR/Cas9 knockout mutants of OsCrZFl,most similar gene of OrCrZFl in rice,and to clone and analyze the overexpression function of OrCrGHl in order to find out whether they have cold tolerance.The results are as follows:1.Bioinformatics analysis showed that the homologous gene of OrCrZFl gene in cultivated rice is located on rice chromosome 8.It encodes a putative zinc finger protein transcription factor containing both domains of BBOX and CCT.BBOX domain usually combines with some domains of other proteins,such as cyclic zinc finger domain,NHL domain and RFP domain,to mediate protein-protein interaction;CCT domain is found in many plant proteins,which is rich in basic amino acids and contains nuclear localization signal in the second half.Several putative cis-acting regulatory elements related to stress response are found in its promoter region.2.OsCrZFl-Cas9,the knockout vector of the OsCrZFl,most similar gene of OrCrZFl in rice,was constructed.The transformation of OsCrZFl-Cas9 into rice Zhonghua 11 was carried out by Agrobacterium mediated method,and two pure knockout mutants were obtained.3.After cold stress,the seedling survival rate of OsCrZFl knockout mutants was significantly lower than that of wild-type rice Zhonghua 11,and in vitro leaf water loss rate,relative conductivity,MDA content of OsCrZFl knockout mutants were significantly higher than those of wild-type rice,and photosynthetic pigment content and free proline content were significantly lower than those of wild-type rice,and the active oxygen scavenging enzyme(superoxide dismutase,SOD,peroxidase,POD,catalase,CAT)activity was not significantly than that of wild-type rice.4.The c DNA of OrCrGHl gene was cloned from Chaling wild rice by RT-PCR.5.Bioinformatics analysis showed that the OrCrGHl gene encoded 558 amino acids with a theoretical molecular weight of 59.25 KD and an isoelectric point of 4.94.It is predicted that the gene may encode glucoan endo-1,3-beta-glucosidase GII isoform X2,which belongs to glycosyl hydrolase superfamily.Several putative cis-acting elements related to stress response,especially abscisic acid response element,were found in its promoter region.6.The expression pattern analysis of OrCrGHl gene showed that OrCrGHl gene expresses mainly in the leaves and roots of Chaling wild rice.The expression level of OrCrGHl gene was up-regulated after cold treatment in Chaling wild rice,especially at 3 h after cold,however,the homologous gene in cultivated rice PA64 S was not induced by cold.7.The overexpression binary vector OrCrGHl-PHB was constructed and successfully transformed into rice Zhonghua 11,and several independent transgenic events were obtained.8.After cold stress,the seedling survival rate of transgenic rice with OrCrGHl overexpression was significantly higher than that of wild-type rice Zhonghua 11,and in vitro leaf water loss rate of transgenic rice with OrCrGHl overexpression was slightly lower than that of wild-type rice,and relative conductivity,MDA content were significantly lower than those of wild-type rice,and free proline content was significantly higher than that of wild-type rice,and the activity of SOD and POD were slightly higher than that of wild-type rice.These results indicate that OrCrZFl and OrCrGHl genes can improve the cold tolerance of rice.