Correlation Of Male Sterility Induced By Chemical Hybridizing Agent With AHAS Activity In Brassica Napus L. And Functional Analysis Of BnERF114.A1 In Plant Architechture Control

Rapeseed is one of the most important oil crops in China.The pollination control system in utilization of rapeseed heterosis and ideal plant architechture suitable for mechanized harvesting are both important goals in rapeseed breeding.Chemical hybridizing agent（CHA）-induced male sterility is an efficient pollination control system in the utilization of rapeseed heterosis.In hybrid seed production by using CHA,the procedure can be simplified by using CHA-resistant lines as male parents,which doesn’t need shelters to prevent male parent from injuring of CHA.Moreover,the herbicides on the basis of which CHA were developed can be used for both weeds and hybrid purity control in rapeseed field.Therefore,establishment and characterization of herbicide-resistant rapeseed accessions is of fundmental importance.Plant arhitechture consists of plant height,branching habit,and branching angle etc.Ideal plant arhitechture is the major goal of rapeseed breeding for mechnized harvesting.It has been well documented that plant hormonesis have great effects on plant architecture.Arabidopsis AtERF114,belonging to ethylene response factor,can regulate cell proliferation,facilitate the emergence of axillary buds and promote branch growth.BnERF114.A1 is a homologous gene of AtERF114 in Brassica napus L.So far,BnERF114.A1 was not characterized.A novel tribenuron-methyl（TBM）-resistant mutant（K5）from rapeseed（B.napus）cultivar “Zhongshuang No.9”（ZS9）were previously obtained in our research group through ethyl methanesulfonate（EMS）mutagenesis and screened out by foliar-sprayed TBM.In this investigation,the K5 and wild type ZS9 were used to carry out the following researches: 1)to determin the relationship of male sterility induced by chemical hybridizing agent with acetohydroxyacid synthase（AHAS）activity in B.napus;2)to reveal the mechanism of TBMresistance underlying the mutant K5;3)to uncover the target of TBM acting as CHA.Mover over,BnERF114.A1 was chosen as a candidate target gene for investigating ideal plant architechture.Identification and functional characterization of BnERF114.A1 were explored,in order to provide useful imformation for rapseed architecture breeding.Main results obtained were as follows:1.The mutant K5 showed comparable seed yield per plant,but flowered eight days later and its growth period was extended for five days,compared with ZS9.At bolting stage,ZS9 showed 96.7 % of male sterile plants and no significant change in main agronomic traits after 0.10 mg/L TBM treated.While fertile pollens were observed in K5 plants treated with the same concentration of TBM,suggesting K5 could serve as male parent without shelters in TBM-induced hybrid seed production.K5 plants treated with 20 mg/L TBM and above performed male sterility thoroughly,indicating that the resistance to CHA-TBM of K5 was approximately 200-fold compared to ZS9.2.TBM-induced male sterility（Y）associated with the relative AHAS activity of inflorescences（X）in the both ZS9 and the mutant K5,and their relationship could be described as a modified logistic function,Y=100-A/(1+Be^-KX),although the obtained constants A,B,and K in logistic function were different between ZS9 and K5.A C-to-T transition at 544 position from the translation start site was identified in BnAHAS1S in K5,which resulted in a substitution of proline to serine at 182 amino acid（reference to Pro197 in Arabidopsis thaliana AHAS）.Ectopic expression of BnAHAS1S^544Tsuggested that BnAHAS1S^544T should be responsible for the resistance to TBM of mutant K5.3.BnERF114.A1,a homologous gene of AtERF114 in B.napus was cloned,which encoded a putative protein of 252 aa,consisting of an AP2/ERF domain and a CMX-1 motif.Yeast self-activation experiment proved that BnERF114.A1 has transcriptional activation activity and its functional region located in 142 aa～252 aa of its C-terminus.Subcellular localization experiment proved that BnERF114.A1 was located in nucleus.Further,the GUS staining analysis of transgenic Arabidopsis thaliana（homozygous transgenic plants obtained from A.thaliana using the BnERF114.A1 promoter to convert BnERF114.A1-eGFP-GUS fusion gene）revealed that it highly expressed in leaf primordia,shoot apical meristem,leaf marginal meristem,and reproductive organs,including pistils and anthers,suggesting that BnERF114.A1 may be involed in cell proliferation in these organs.4.The ectopic expression of BnERF114.A1 in A.thaliana showed that over expression of BnERF114.A1 inhibited elongation of main inflorescence,promoted branching of lateral branches and rosette,resulted in loss of apical dominance.Determination of the expression level of 16 polar auxin transporters in main inflorescence of transgenic Arabidopsis plants showed the expression of eight PINs family genes（PIN1-PIN8）,four AUX/LAXs family genes（AUX1,LAX1-LAX3）,as well as two intercellular transport carriers（PGP2 and PGP19）were significantly down-regulated,while the expression of PGP4 was significantly up-regulated,and the expression of PGP1 did not change signicantly.These results indicated that ectopic expression of BnERF114.A1 lead to decrease in the expression level of most auxin transporters in transgenic A.thaliana.Determination of the content of endogenous auxin（IAA）in the main inflorescence of wild type Arabidopsis and transgenic lines showed that,compared with wild type A.thaliana,the endogenous IAA content in the main inflorescence of transgenic lines increased significantly.It is speculated that the ectopic expression of BnERF114.A1 inhibited the elongation of the main inflorescence of the transgenic A.thaliana,and promoted the branching of the lateral branches and rosette.The main reason for the loss of the apical dominance of the transgenic A.thaliana may be the influence of the expression level of the auxin transport protein genes,thus affecting the transport and distribution of endogenous growth factors,resulting in accumulation of IAA in the main inflorescence.Taken together,the present study provided a novel valuable TBM-resistant rapeseed mutant line K5(with BnAHAS1S^544T allele)for rapeseed breeding.The TBM-resistance of male reproductive organs in K5 attributed to the mutation of BnAHAS1S at Pro-182-Ser,and it was 200-fold of wild type rapeseed ZS9 aproximately.TBM-induced male sterility associated with the relative AHAS activity of inflorescences in both rapeseed lines.BnAHAS1S^544T transgenic A.thaliana plants also showed a higher TBM-resistance of male reproductive organs.Our results supported that AHAS should be the target of the AHAS-inhibiting herbicide TBM when used as CHA in rapeseed.BnERF114.A1,a homologous gene of AtERF114 in B.napus,was cloned.It encoded a putative protein,which had a conserved AP2/ERF domain and a conserved CMX-1 motif,and belonged to the Group Xa of ethylene response factor family.Its transcriptional activation region is located at its C-terminus.This gene was expressed in leaf primordia,shoot apical meristem,leaf marginal meristem and reproductive organs.It may cause plant dwarfing,branch increasing and apical dominance loss through affecting the distribution of auxin in plants.Changes of plant phenotype in transgenic BnERF114.A1 Arabidopsis were likely associated with the changed distribution of auxin in plant.These results suggested BnERF114.A1 was probably a valuable gene for rapeseed breeding.