Cloning And Characterization Of Photoperiod Sensitive Gene Hd6-like In Maize

Tropical and subtropical germplasms contain abundent hereditary variation to broaden Chinese maize germplasm and increase the breeding level.However,photoperiod sensitivity is the main restriction of its use in temperate zone.Clone and analyse the fuction of photoperiod-sensitive genes is important to the research on the mechanism of Maize photoperiod-sensitive and flowering.Hd6-like gene was cloned by RT-PCR and rapid amplification of cDNA ends(RACE) using a photosensitive tropical maize(Zea mays L.) inbred line CML288.We studied the expression of Hd6-like in shoot apical meristem(SAM) and leaf under long day and short day conditions between 5-leaf and 9-leaf stages,then give the night break to CML288 under SD condition,detect the change of expression of Hd6-like in SAM and leaf.We constructed the overexperssion veter PBI-HD6 then transformed it into Arabidopsis with Floral Dip method,and got the transformant plant hd6-ox.The expression of photoperiod-relatived genes had analysed in hd6-ox and wt,and we conclude the mechanism of Hd6-like delayed flowering based on the results.The main results were as follows.1.Hd6-like gene was cloned,a predicated 999-bp open reading frame was detected in the cloned sequence,which encoded 332 amino acids.By BLAST analysis,we found the gene sequence shared a high similarity with a gene encoding protein kinase CK2 in maize. The amino acid sequence contained two CK2 activity domains,one ATP binding site,one basic stretch(NLS),one N terminal domain,a putative serine/thereonine protein kinase active site,and showed a high homologization with that of protein kinase CK2αin maize, wheat,rice and Arabidopsis.Hd6-like encode the alpha subunit of protein kinase CK2 that closely relates to photoperiod-sensitive.2.We established fluorescence quantitative RT-PCR system with CML288,and found that Hd6-like expressed both in SAM and leaf of CML288.Under the short day treatment,it expressed with the highest level at 7-leaf stage in SAM.,and with the peak at 9-leaf stage in leaf under the long day treatment,the high-level stage meet the photoperiod-sensitive time of CML288.After the night break under SD,the expression of Hd6-like fall down both in SAM and leaf and the peak disappeared.Give the CML288 night break of different days,we fould that lower expression showed while more days of night break treated,we propose that Hd6-like joined the regulation of oscillator by Phytochrome with an unknown mechanism,and keep the sensitivity to dark to promote flowering in maize. 3.We got the overexpression Arabidopsis plant of Hd6-like.It found that hd6-ox plants flowered later than wt about 22 d under LD condition.We examine the effects of Hd6-like overexpression(hd6-ox) on some genes of oscillator and clock output that are modulated by photoperiod.It showed that,Oscillations in expression of Hd6-like were robust in hd6-ox plants under LD condition,reaching maximum levels after lighting 3h.,the expression of TOC1,GI,CO and FT all changed.Our studies suggest a function for Hd6-like very close to the oscillator,as expression of the core component TOC 1 altered in hd6-ox plants.The transfer of Hd6-like lead the oscillator dysfunction and shaked the expression of CO and FT in dark,then late the flowering of Arabidopsis with an unknown way.