Abstract
Stacked traits have become an important trend in the current development of genomically modified crops. The bidirectional promoter can not only prevent the co-suppression of multigene expression, but also increase the efficiency of the cultivation of transgenic plants with multigenes. In Gossypium hirsutum, Ghrack1 and Ghuhrf1 are head-to-head gene pairs located on chromosome D09. We cloned the 1429-bp intergenic region between the Ghrack1 and Ghuhrf1 genes from Gossypium hirsutum. The cloned DNA fragment GhZU had the characteristics of a bidirectional promoter, with 38.7% G+C content, three CpG islands and no TATA-box. Using gfp and gus as reporter genes, a series of expression vectors were constructed into young leaves of tobacco. The histochemical GUS (Beta-glucuronidase) assay and GFP (green fluorescence protein) detection results indicated that GhZU could drive the expression of the reporter genes gus and gfp simultaneously in both orientations. Furthermore, we transformed the expression vectors into Arabidopsis and found that GUS was concentrated at vigorous growth sites, such as the leaf tip, the base of the leaves and pod, and the stigma. GFP was also mainly expressed in the epidermis of young leaves. In summary, we determined that the intergenic region GhZU was an orientation-dependent bidirectional promoter, and this is the first report on the bidirectional promoter from Gossypium hirsutum. Our findings in this study are likely to enhance understanding on the regulatory mechanisms of plant bidirectional promoters.
Highlights
Transgenic plants with stacked traits harboring two or more foreign genes can meet the diverse needs of growers and provide multiple benefits; stacked traits have become an important trend in the current development of genomically modified crops
An intergenic region (1258 bp) shared by At4g35985 and At4g35987 in Arabidopsis thaliana is a tissue-specific and stress-inducible bidirectional promoter analyzed in transgenic Arabidopsis and tobacco plants [11]
Yang et al found that 70.8% of human bidirectional promoters have a G+C content exceeding 60%, and that CpG-islands are present in 90% of bidirectional promoters [5,30]
Summary
Transgenic plants with stacked traits harboring two or more foreign genes can meet the diverse needs of growers and provide multiple benefits; stacked traits have become an important trend in the current development of genomically modified crops. In 2015, Liu et al used the promoter::GUS transgenic approach and revealed that the intergenic region of the Arabidopsis thaliana divergent genes At1g71850 and At1g71860 was an asymmetric bidirectional promoter, exhibiting an orientation-dependent expression profile. Based on the expression level and correlation of each adjacent and oppositely transcribed gene pair, they selected four candidate gene pairs, and subsequently found that GUS and GFP assays of the transgenic plants indicated that all the intergenic regions showed bidirectional expression activity in various tissues of rice [12]. The transient expression analysis showed that the intergenic sequence could simultaneously drive the expression of the reporter genes gus and gfp from the forward and reverse orientations. The gfp transcript abundance were assayed by quantitative real-time PCR (qRT-PCR), using gene-specific primers to evaluate the tissue-specific expression of the bidirectional promoter in both orientations. The relative protein abundance of these two reporter genes in various tissues (root, young leaf,Tohldeerrellaetaifv,eflporwoeteri,ndaebvuelnodpainngcesoilfiqthuees, eantwdomraetpuorretesrilgiqeunee)swinevrearaisosuasyteidssbuyesw(reosotetr, nyobulontgulseianfg, oplrdoetreilne-asfp, efcloifiwcearn, tdibeovdeileosptiongevsailliuqautee,thaendtismsuaet-usrpeecsiifilicqeuxep) rwesesrieonaossfatyheedbibdyirewcteisotnearnl pbrloomt outseirnign pbroottheionr-isepnetcaitfiiocnasn. tibodies to evaluate the tissue-specific expression of the bidirectional promoter in both oTrhieenwtaetisotenrsn. blot analysis using an anti-GUS antibody revealed strong GUS expression in the younTghelewaveesste, ronldbelrotleaanvaelsy,sdiseuvesilnogpianng asniltiqi-uGeU, Sanadntfliboordalytirsesvueeasl,edansdtromnogdGerUatSe eexxpprreessssiioonniinntthhee ymouatnugrelesailviqesu,eo. lHdoerwleevaevre,sn,oddeveteelcotpaibnlge GsiUliqSuper,oatneidn fwloarsavl itsiussauliezse,danindthmeordoeorta(tFeigeuxprere7scs)i.oUnsiinngthaen manattiu-GreFsPiliaqnutieb.oHdoywreevveear,lendotdheatteGctFaPbleexGpUreSsspiorontweinaswhaigshveissut ianliztheedyinouthnegrloeaovt e(Fs,igaunrdeh7acd). mUsoidnegraante aenxtpi-rGesFsPioanntinibtohdeyorledvleeaalvedest.hWathGeFrePaesxtphreelsoswioenswt eaxsphriegshsieosnt iwn athsedyeoteucntegdleinavtehse, adnedvehloadpimngodsielirqautee eaxnpdreflsosiroanl tiinsstuhees,oaldndleGavFePs.pWrohteeirneaesxpthreeslsoiownewstaesxnporetsdseiotenctwedasindethteectreodotin(Ftihgeurdee7vde)lo. pTihnegwsielsiqteuren abnldotfrloersaullttsisdsuifefes,readndfrGomFPthperorteesiunletsxporbetsasiinoendwinasthneotfldueotreecstceednicne tqhuearnotoitta(tFivigeuarnea7ldy)s.isT,hpeowteensttiearlnly bbleoctaruesseultthsedtiofftearlepdroftreoimn ftrhoemrethsuelptslaonbttsadineegdraidnetdheduflruinorgeescxetrnaccetiqouna, notrittahteivperoatneainlyesixsp, rpeostseionntiawllays bteocoawuseeakthaentdottahlepsreontesiitnivfirtoymwtahsetopolalnotws ,dmegarkaidngeditdimurpinogsseibxlteratcotidoent,eoctr. the protein expression was too weak and the sensitivity was too low, making it impossible to detect
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
