The Functional Analysis Of Leaf Trichome Gene OsGL6 And Related Gene In Rice

Trichome,an outgrowth from a plant epidermal cell and may take a wide variety if forms,has a variety of important physiological functions in plants.It can interact with environments and play an important role in enhancing the light refraction,adjusting the leaf heat balance,radiation interception and resisting the invasion of pathogens.So far,the developmental models of trichome in dicots have been studied and great progresses have been made,meanwhile,the pattern of trichome development with the similar molecular mechanism for most dicots has been found.A recently published study,however,indicated that the molecular mechanism in dicots is not suitable for monocotyledonous plants.Therefore,studies on the trichome development and regulation in rice will be help to furtherly analyze the initiating of trichome and the development as well as in monocotyledonous has of great significance.Based on the gene Os GL6 which previously mapped in chromosome 6 of rice and functional analysis,we furtherly identify and elucidate the regulatory mechanism of the formation of trichome on the gene Os GL6 for rice.Additionally,functional analysis of another candidate gene Os DUF6 related to drought tolerance was also performed on the regulation mechanism related to drought tolerance.The main findings are follows:1.The F2 population was contructed with 75-1-127 and 93-11 to verify the prior results which fine mapping crossing 75-1-127 and MH63.The 7 predicted genes in fine mapping regions were found by bioinformatics methods.Based on the gene annotation,phylogenetic analysis,quantitative real-time PCR expression analysis,DNA sequence alignment and comprehensive bioinformatics analysis,the Loc＿Os06g44750 and Loc＿Os06g44810 as possible candidate gene for Os GL6 was identified.2.For furtherly identified the target gene of trichome formation,the corresponding vectors including over-expression vector,CRISPR/Cas9 system,tissue specific expression vector and subcellular localization vectors of two candidate gene were constructed respectively.The results indicated that Loc＿Os06g44750 could be involved in regulating the initiation and development of trichome.The subcellular localization assay showed that Os GL6 was only expressed in the cell nucleus and expressed in whole plant.3.Trichome formation was always regulated by multiple genes under the genetic complex regulatory networks in plants.To explore the gene Os GL6 on how to regulate the trichome formation in rice,the yeast two-hybrid system was used to screening the proteins interacted with Os GL6 and co-immunoprecipotation(Co-IP)was applied to confirm the interaction candidate proteins with Os GL6.The results indicated that 6possible interacting proteins have been achieved by preliminary selection.Additionally,Os GL6 was confirmed to be able to interact with Os WOX3 B by Co-IP.4.To further explore the possible function of the function unknown gene Loc＿Os06g44810(Os DUF6)in mapping interval regions,the expression pattern of the gene was also analyzed.Screening interaction protein of Os DUF6 by yeast-two-hybrid system was also undertaken.The findings suggested that Os DUF6 is a membrane protein which located on the cell membrane and expressed in whole plant.Four candidate genes were selected from yeast two-hybrid and demonstrated that the Os DUF6 might play a role in the regulation of abiotic stresses.The Os DUF6 was confirmed to be able to interact with Os NAC78 by Co-IP technique.5.In order to further validate that the OsDUF6 can enhance the drought resistance under drought stress.The effect of drought on the Os DUF6 over-expression transgenic lines was studied under the drought stress simulated with polyethylene glycol(PEG-6000)treatment.To observe the change of morphology and differentially expressed gene in different time,we analyzed the phenotypic changes,gene expression and determination the antioxidant activities of antioxidants.The results showed that the genes expression in Os DUF6 was greatly enhanced,the stomata aperture reduced significantly,the ROS elimination ability were also enhanced.It also has a significant drought tolerance phenotype,indicated that Os DUF6 it can enhance the drought resistance of plants under drought stress.The results demonstrated the Loc＿Os06g44750 was a new gene regulated the trichome formation for rice.Meanwhile,six Os GL6-interacting proteins were identified by yeast-two-hybrid system and Co-immunopreciptation method.These studies also find a gene Os DUF6 might be a stress related gene and its drought tolerance were verified.Taking together,the above results are beneficial to reveal the regulatory mechanism of trichome formation in monocotyledon plants and responses to abiotic stresses.This provides an important theoretical foundation for subsequent research.