TS2 And Its Homologues In Maize Complemented Aba2 Mutant In Arabidposis

[Objective] In recent years,the cloning and functional analysis of the key jasmonic acid(JA)biosynthesis genes TS1 and OPR7/8 in maize indicate that JA is a necessary signal for maize sex determination.TS2,working downstream of TS1,was cloned in 1993.It encodes a short-chain alcohol dehydrogenase(SDR),biochemical analysis of TS2 recombinant protein showed that the substrate of TS2 may be 3β-/17β-hydroxysteroid,carbonyl compounds or quinones.Acosta(2009)infered that TS2 was an enzyme in JA biosynthesis pathway due to the tasselseed phenotype of ts2 mutant could be recovered by exogenous applicaton of JA to the mutant,but the substrate of TS2 is still unknown.In this study,the substrates of TS2 were identified by transgenic compensation method,and TS2 was proved to be involved in the synthesis of which hormones.This study is of great significance for elucidating the molecular mechanism of sex determination of tassel in maize.[Methods] The cDNA sequences of four TS2 family genes were cloned.Here using the vector p ROK2,we constructed four plasmids by homologous recombination method.We transformed ZmTS2,ZmTS2 H,ZmTS2L1,ZmTS2L2 into aba2-1 mutant of Arabidopsis by Agrobacterium-mediated transformation,floral dipping.The phenotypic traits,adversity tolerance,and gene expression level relevant to ABA signal were assayed by specific methods.[Results] Using the amino acid sequence of the ZmTS2 as the search query to blast maize genome B73＿AGP＿v3,four homologous genes,GRMZM2G455809,GRMZM2G308351,GRMZM2G332976,GRMZM2G156739 were obtained,named TS2,TS2 H,TS2L1,TS2L2,collectively called TS2 Gene family.In Arabidopsis genome,11 homologous genes were obtained,called ABA2 gene family.The homologous phylogenetic tree of TS2 gene family and ABA2 gene family was constructed by Clustal.And the tree showed that the 15maize/Arabidopsis homologous genes are divided into 4 clades.Among them,ZmTS2 and At ABA2 are located in the clade 2 and 3,respectively,suggesting that they may have similar molecular function as ABA2 does.ABA2 protein catalyzes the conversion of xanthoxin to ABA-aldehyde in the ABA synthesis pathway.The main results of transformation experiments are listed below:(1)aba2-1 mutant showed dark green leaves which have much higher chlorophyll content(1.5mg/g)compared with WT(1.1mg/g).Transgenic plants with TS2,TS2 H,TS2L1,TS2L2 in aba2-1 background have 1.20mg/g,1.28mg/g,1.16mg/g and 1.32mg/g chlorophyll,respectively,of which TS2 and TS2L1 have significant differences with aba2-1;(2)The measured values of leaf and floral traits of ZmTS2s-transgenic plants were significantly higher than aba2-1;(3)DAB and NBT staining are used to detect the content of reactive oxygen species in the leaves.WT,aba2-1and transgenic lines have the same degree of staining under normal conditions.However,under Mannitol and Na Cl,the staining of WT and transgenic lines was much lighter than aba2-1.The staining blackness of the leaves of transgenic lines was ZmTS2L2 > ZmTS2 H >ZmTS2 > ZmTS2L1.The lighter the color,the less the accumulation of reactive oxygen species in the leaves;(4)The root length of WT and aba2-1 are significantly different under different hyper-osmotic stresses(Na Cl,Mannitol,and Sucrose).Transgenic plants with ZmTS2 or ZmTS2L1 were recovered to WT length,while the plants with ZmTS2 H was recovered a little bit;the plant with ZmTS2L2 is basically the same as aba2-1;(5)The results of q PCR detection of the expression of ABA-responsive genes showed that ABI3,ABI5 and RD29 A of ABA signaling pathway were significantly up-regulated in ZmTS2,ZmTS2 H and ZmTS2L1 transgenic plant,compared to aba2-1,and Sn RK2.6 was down-regulated in transgenic plants;the gene expression level of ABA pathway in ZmTS2L2 transgenic plants was not changed significantly.[Conclusion] Compared with WT,aba2-1 shows slower plant growth.aba2-1 mutant has deep leaf color and sensitive to abiotic stress;its seed germination rate was higher under hyper-osmotic stresses.Transformed ZmTS2L1 can restore the phenotype of aba2-1 very well,and ZmTS2 can also restore the phenotypic traits of aba2-1 as well;ZmTS2 H can partially restore the phenotypic traits of aba2-1;The recovery degree of ZmTS2L2 is relatively weak,and phenotypic traits are almost same between ZmTS2L2 transgenic plants and aba2-1 mutant.The experimental results indicate that four members of the ZmTS2 family can fully or partially restore the phenotypic traits of aba2-1 mutant,indicating that TS2 family genes have similar function with At ABA2 gene of Arabidopsis,suggesting that TS2 family genes involve in ABA biosynthesis in maize.Taking together,this study concluded that ABA plays an important role in the sex determination of maize.