Preliminary Study On Role Of Rice SOMNUS Homologous Gene In Photochrome-mediated Photomorphogenesis

In this study,gene profiles were compared between wild-type(WT)and phytochrome B mutant rice(phyB)using Gene Chip.One candidate gene encoding CCCH-type zinc finger protein containing plant-unique TZF(tandem zinc finger)motifs was identified to be regulated by phytochrome B(Phytochrome,phyB)and the phytochrome interaction factor OsPIL15 gene,whose expression level in phyB was significantly higher than than in WT.Bioinformatics analyses revealed that this gene in Oryza Sativa was homologous to the zinc finger protein gene SOMNUS in Arabidopsis Thaliana by analysis of phylogenetic tree and prediction of the promoter region.Thus this gene was named as OsZFL1.In order to study OsZFL1 gene in phytochrome-mediated photomorphogenesis,we investigated the roles of this gene in rice growth and development.In this study,we first analyzed the bioinformatics characteristics of this gene,followed by analysis of the expression patterns of the gene,and then constructed an overexpression vector and CRISPR/Cas9 vector of the gene,and obtained overexpression and CRISPR/Cas9 transgenic rice lines.We initially analyzed the growth and development of transgenic OsZFL1 lines.The results we obtained were as followed:(1)Analysis of OsZFL1 gene sequence,evolutionary tree,and promoter region prediction showed that OsZFL1 was CCCH-type zinc finger protein containing TZF motifs;this gene and reported Arabidopsis thaliana SOMNUS gene were homologous genes;The promoter region of the gene contained the light-regulating element Sp1,elements ABRE,motif IIb related to the hormonal abscisic acid(ABA)response,and the unknown element GC-motif.Therefore,it was speculated that OsZFL1 might be regulated by light and plant hormone ABA.(2)Expression patterns of OsZFL1 showed that its expression was highest in dry seeds compared to seedling roots and leaves.After water absorption of seeds,the expression level of OsZFL1 rapidly decreased,which indicated OsZFL1 might be related to seed germination and development;Photoperiodic assays showed that the expression of OsZFL1 decreased at 14:00 in light period,but there was no significant difference in dark period,indicating that the expression of OsZFL1 gene was not affected by photoperiod.(3)We constructed OsZFL1-overexpression(OsZFL1-OX)vector,successfully obtained OsZFL1-OX transgenic lines,and identified different insertion sites of OsZFL1-OX lines.(4)The expression level of key genes in the ABA metabolic pathway in OsZFL1-OX lines was studied.Overexpression of OsZFL1 could down-regulate the expression of ABA anabolic genes OsZEP,OsNCED1 and OsNCED6,and upregulate the expression of ABA degradation metabolic genes OsABA8ox1 and OsABA8ox2 by RT-qPCR analysis.These results suggested that OsZFL1 might affect ABA metabolic pathway.(5)The expression of key genes in the plant hormone gibberellin(GA)metabolic pathway in OsZFL1-OX lines was studied.Overexpression of OsZFL1 could up-regulate the expression of GA3ox1,GA2ox3,and GA2ox5 genes by RTqPCR analysis.These results suggested that OsZFL1 gene probably played important roles in GA signal pathway in rice.(6)By analyzing the effect of rice phytochrome interaction factors(PILFs)on OsZFL1,we found that the expression of OsZFL1 was up-regulated in all OsPILOX transgenic lines under dark growth conditions.Under light conditions,the expression level of OsZFL1 gene was higher in OsPIL15-OX lines,and the expression level of OsZFL1 in other rice PIFs overexpressed lines was not significantly different from than in WT.The results showed that PIFs positively regulated the expression of OsZFL1,and the light signal eliminated this positive regulation,indicating that OsPIL15 might regulate the expression of OsZFL1.(7)Seed germination of OsPIL15 and OsZFL1 overexpressed lines was studied after 3 days under light and dark conditions respectively,the seed germination of OsPIL15-OX was not good as that of WT in the dark,but there was no difference between OsZFL1-OX and WT.Thus,the bad germination rate of OsPIL15 in the dark was not due to OsZFL1 gene.(8)We constructed OsZFL1-CRISPR/Cas9 vector and successfully obtained three OsZFL1-CRISPR/Cas9 transgenic lines.(9)To analyze light sensitivity of OsZFL1 gene,we detected inhibition of the coleoptile elongation and the first incomplete leaf of WT and OsZFL1 lines under continuous red light and far red light.The coleoptile and the first incomplete leaf of OsZFL1-OX lines were not significantly different from those of WT after red light and far-red light,but the relative length of OsZFL1-CRISPR/Cas9 coleoptile and first incomplete leaves was longer than that in WT.OsZFL1-CRISPR/Cas9 line was less sensitive to red light and far-red light.(10)The resistance of OsZFL1-OX lines to salt and drought stress was analyzed.The results showed that OsZFL1 could transiently increase salt tolerance.(11)By observing the plant height phenotypes of seedling stage in WT and OsZFL1-OX lines,we found that the plant heights of OsZFL1-OX lines were consistently lower than that in WT at the seedling stage.(12)We investigated the important agronomic traits of OsZFL1-CRISPR/Cas9 lines at the mature stage including plant height,grain number,floret number,panicle length,number of primary branches,number of effective tillers,and fruit setting rate.Compared with the WT plants,OsZFL1-CRISPR/Cas9 lines resulted in a reduction in the number of grains per plant,but the number of tillers increased,and the seed setting rate had no difference from the WT.In conclusion,our findings demonstated the important roles of OsZFL1 gene in rice growth and development.At the same time,combined with the reported function of Arabidopsis SOMNUS gene,we preliminary explore the role of the rice SOMNUS homolog gene OsZFL1 gene in the phytochrome-mediated photomorphogenesis.This study lays a foundation for further analysis of the role of OsZFL1 gene in rice growth and development,and provides new information for the further analysis of phytochrome-mediated optical signaling networks.