Ovule-specific Down-regulation Of GhCKX Gene Promoted Seed Development Of Cotton (Gossypium Hirsutum L.)

Cotton（Gossypium hirsutum L.）,is the most important fiber crop in the world,as well as an important source of plant proteins and oil.Although great progress has been made in the improvement of fiber qualities and yield by traditional breeding methods,unfortunately,cotton seeds get smaller and smaller with the increasing fiber yield,presubly because the increased lint percentage is sacrificed of seed size.It is big challenge for cotton breeders to simutanously improve the cotton seeds and fiber productivity.Cytokinins（Cytokinin,CTK）,chemically N⁶-substituted purine derivatives,are a kind of important phytohormone.Active CTKs are involved in numerous important biological processes throughout the whole life cycle.For example,CTK can facilitate fruit setting,promote assimilate accumulation during the development of fruit and seed, and enhance the growth of endosperm.Cytokinin oxidase that irreveribly degrades cytokinin by cleaving the N⁶-side chain from the adenine/adenosine moity has been thought to play a key role in regulating endogenous cytokinin levels in plants.To investigate the possibility of improvement of seed yield by regulation GhCKX in cotton seed development,two ovule-specific promoter（D113 and Pv） was fused to the sense and antisense GhCKX gene respectively, and introduced into cotton via Agrobacterium- mediated transformation method.The main results are as followings:1.Characterization of Pv and D113 as ovule-specific promoters in cotton.To tissue-specificlly control the target gene expression in cotton ovule,the patterns of tow promoters,Pv and D113 were investigated by real time PCR.It was shown that the two promoters could specifically drive foreign genes expression in ovule,especially, in 30 DPA ovule. 2.Obtaining transgenic plants down- or up-regulating GhCKX34 transgenic lines were identified by GUS screening.8 transgenic lines were planted to generate T₁ population.All transgenic Cotton plants showed normal growth. Peal-time PCR analysis showed that the expression of GhCKX in Pv:antiGhCKX and D113:antiGhCKX transgenic ovule（30 DPA） were decreased 3-5 fold;whole that in overexpression GhCKX transgenic plants were increased 2-4 fold over the wild-type.3.Weight per 100 seed and germination rate increased in transgenic cottons down-regulating Gh CKXIt was found that weight per 100 fuzz-free seeds of D113:antiGhCKX（lines 41^#,42^#,43^#） and Pv:antiGhCKX（line 13^# ） transgenic cotton were 10 to 20%higher than wild-type,suggesting that down-regulation of GhCKX in ovules promoted seed development.When measured at 48 h after imbibition,the seed germination rates in all D113:antiGhCKX（lines 41^#,42^# and 43^#） and Pv:antiGhCKX（line 13^#） transgenic plants were increased to 2.4-4.6-fold of wild-type plants,indicating that downregulation of GhCKX in ovules promoted seed gemination.4.The content of Trans-Zeatin Riboside and carbohydrate increased in 30-DPA ovules of down-regulating Gh CKX cottonTrans-Zeatin Riboside is a kind of cytokinin.It’s levels in T₂ ovules of D113:antiGhCKX（lines 42^# and 43^#） and Pv:antiGhCKX（line 13^#） were assayed at 30 DPA,and were found to be 1.3-1.5 times as wild-type.We also analysed the contents of carbohydrate in mature transgenic viable seeds by gas chromatography-mass spectrometry.The carbohydrate contents of D 113:antiGhCKX （lines 43^# and 42^#） mature viable seeds were significantly increased.5.The influence of seed development of overexpression GhCKX transgenic plantsAithough the GhCKX expression level in Pv:GhCKX and D113:GhCKX ovules were increased obviously and the Trans-Zeatin Riboside contents decreased,the carbohydrate contents in mature viable seeds showed no detectable changes compared with wild-type. The present data collectively demonstrated that GhCKX functioned as a negative regulator of CTK level and seed development.Down-regulation of GhCKX increased the seed size,and promoted carbohydrate accumulation,which suggested a novel method to improve the cotton seeds without sacrificed of fiber productivity through genetic manipulation.