Mapping-based Cloning And Functional Study Of Boron-deficiency Mutant Gene In Rice

Boron is an essential trace element for higher plant,directly involve in the synthesis of cell wall,and regulate a variety of physiological processes in plant cells.Boron is so important for plant,but the mechanism of boron absorption and transport is not clear.Rice is a main grain crops in the world,and the research for the mechanism of boron absorption and transshipment in rice,is useful to improve the tolerance of rice in different boron environment by molecular biology techniques.In our study,we identified a dwarf and tiller-enhancing mutant,dtel,in rice.The target gene was localized by map-based cloning.Further experiments proved that it is a boron deficiency mutant,and the biological function had analyzed by molecular biology experiments.The research results are as follows:1.Phenotype characteristic,map-based cloning and complementation analysis of dtelWe isolated a rice dwarf and tiller-enhancing mutant,was the No.19 family line of the chromosome segment substitution lines(CSSL)with the genetic background of japonica variety Asominori as the recurrent and indica variety IR24 as the donor,named dtel.Compared to the wild-type plants,the dte1 mutant exhibited shorter plant height,increasing tillers and the dead of leave tip at the tillering stage;showed smaller spike,seriously low pollen fertility and sustained tillering at heading stage;showed seriously low spikelet fertility and a large number of high-node tillers at maturation stage.The dwarf phenotype of dtel was due to the shortening intemodes.Further scanning electron microscopy(SEM)analysis revealed that,compared with the wild type,cell sizes of intemodes were significantly reduced and the cells become flaccid in the dtel mutant,indicating that it is cell size resulting in the dwarf phenotypes of dtel plants.The genetic analysis indicated that the dtel mutant phenotypes were inherited as a single nuclear recessive mutation.Replacement fragment screening in CSSL 19 and linkage analysis showed that the mutant phenotype was not caused by the fragment displacement.A map-based cloning approach was used to isolate the target gene.And the target gene was mapped to a 14.4-kb interval between insertion/deletion markers L13 and L14 on the long arm of rice chromosome 10.There is only one predicted open reading frames in this region,and genomic sequencing revealed that a 445-bp retrotransposon was inserted into the 2nd exon of LOC_Os10g36924.This insertion resulted in a frame-shift mutation and premature translation.Further expression analysis showed that the expression of LOC_OslOg36924 was sharply down-regulated in dtel mutant.Genome complementary confirmed that LOC_Os10g36924 is the DTE1 gene that can regulate dwarf and tiller-enhancing in rice.Predict found that DTE1 encoding a nodulin 26-like protein.Homologous analysis showed that DTE1 protein belongs to NIPs protein family,is homologous with AtNIP5;1 in Arabidopsis thaliana and the same protein with OsNIP3;1.2.dtel is a boron deficiency mutantIt is interesting to note that severe growth defects were easily observed in dtel mutant but not in wild-type plants when they were grown in a filed(here labeled as field abnormal,FA).However,in another filed(here labeled as field normal,FN),the growth of rice plants were comparable in the dtel mutant and the wild type,indicating that the dtel mutant may be a nutrition-deficient mutant.Because AtNIP5;1 as a key boric acid channel proteins involved in B uptake,of which mutant exhibited growth retardation in low B conditions.We cultivated wild type and mutant with no boron nutrient solution,found that the mutant can't normal growth,but wild type normal,suggested that dtel is a boron deficiency mutant.Furthermore,we measured the B contents of the FA and FN,shown that the B content of the FA was significantly lower than FN,indicated that dtel is indeed a rice low B-sensitive mutant.RNAi transgenic plants growth in low B conditions exhibited the growth retardation phenotypes,compared with the wild type.Wild type and mutant were cultivated with a series of boron concentrations,showed that the mutant was sensitive to the low boron condition,and the critical concentration was between 0.1-1 ?M.The growth of dtel mutant was restrained under low boron(0,0.01,0.1 ?M)condition,and was normal under high boron(1,10,100 ?M)conditions.Wild type was normal growth for 30 days without boron provided,indicated that there has a boron transfer mechanism to transport boron from old tissues to new organs in the wild type;and the growth of mutant is restrained after be transferred to low or no boron condition,indicated that the mechanism of boron transport is damaged in mutant.The dry matter accumulation were determinated and showed that all of the amounts of dry matter in dte1 mutant were significantly lower than the wild type under different boron concentrations.In addition,the dtel mutant displayed severe growth retardation at the first week indicated the ability of mutant to adapt to the changes of environmental was destroyed.Determined the content of boron in wild type and mutant showed that the total boron content in mutant were significant lower than wild type under low boron condition,indicated that the capacity of boron absorption under low boron condition was decreased significantly in mutant;and the boron contents of unit dry weight in mutant were no less or even higher than wild type,suggested that the capacity of boron distribution is defective in mutant.3.DTE1 protein is located on plasma membraneDTE1-GFP binary vector,which is started by a strong CaMV35S promoter,was built and transformed into rice.The DTE1-GFP fusion protein was located on plasma membrane in the root tip of GFP transgenic rice.The plasma membrane was markered by membrane dye FM4-64,and the fluorescence signal of DTE1-GFP fusion protein was according with the fluorescent signal of FM4-64.OsBOR1 is a marker gene of plasma membrane,and we built BOR1-mCherry carrier,transformed into DTE1-GFP transgenic rice.Laser confocal microscope showed the fluorescence signal of DTE 1-GFP fusion protein was according with the fluorescent signal of BOR1-mCherry fusion protein.These results indicated that DTE1 is a boron channel protein on plasma membrance.4.The tissue specific expression of DTE1GUS staining analysis showed that DTE1 had high expression in root and leaf collar,and this result was verified through the analysis of quantitative RT-PCR in various organizations at different periods.The semithin section of GUS stained shown that the expression of DTE1 was found in root hair,epidermis,exodermis and column in the root of seven-days seedling;and was specific found in exodermis in the root of thirty-days seedlings.These results suggested that DTE1 protein is involved in boron absortion in the rice root,and may be associated with the morphogenesis of rice at seedling period.The analysis of leaf collar shown that the activity of DTE1 promoter was lower in young leaves,and higher in the mature leaves,suggested that DTE1 protein may pay a role to transfer boron out from leaves.5.Promoter activity of DTE1 was induced enhanced by low boronGUS transgenic seedlings were induced by low boron condition for 6 h,12 h,24 h,48 h and 72 h,and dyeing experiment showed that the promoter activity of DTE1 was induced enhancement under low boron condition.LUC gene carrier,which is starting by DTE1 promoter,was built and transformed into rice protoplast.Then dealed with W5 culture solution,which contained 20 or 100 pM boron acid,for 16h,and detected the instantaneous expression level of LUC protein.The results suggested that the promoter activity of DTE1 was 1.7 times under low boron(20 pM)than that of high boron(100 p,M).These results indicated that the promoter activity of DTE1 is regulated by boron concerntration,and is upregulation by low boron induced.6.mRNA level of DTE1 was induced up by low boron,and was induced sharply downby high boronQuantitative RT-PCR analysis in wild type showed that the mRNA levels of DTE1 were high in low boron(0,0.01,0.01 ?M)conditions,and were low in high boron(1,10,100 ?M)conditions.Low boron-induced experiments showed that the mRNA levels of DTE1 were induced increased in roots under low boron condition,and to 5 times than comparison after 12 h;in shoots,the mRNA levels were down-regulation first and then up-regulation,reached 5 times than comparison after 6 h.Suggested that the mRNA level of DTE1 was induced up-regulation by low boron.High boron-induced experiments showed that the mRNA level of DTE1 were sharp down-regulation both in roots and shoots in 1 h,illustrated the mRNA of DTE1 was sharp degradation by the high boron induced.7.DTE1 protein was regulated by boron concentrationDTE1-LUC carrier was started by strong promoter 35S,transformed into rice protoplast,dealed with W5 culture solution which contained 20 or 100 p,M boron acid for 16 h,and then detected the instantaneous expression level of DTE1-LUC fusion protein.The results showed that the protein level of DTE 1-LUC was 4.9 times under low boron than that of high boron,suggested DTE1 protein is regulated by boron concentration and with high protein level under low boron condition.