| ABSTRACT:Maize (Zea mays L.) is domesticated from Mexico of south American, a plant requiring short day photoperiods to flower. Although the majority of maize varieties are generally characterized as day-neutral plants through centuries of domestication and artificial selection, numerous tropical landraces of maize remain sensitive to photoperiod. Photoperiod sensitivity was the major obstacle to using tropical for breeding programs of temperate maize. Therefore, it is vital for maize genetics and breeding to understand the genetic basis of photoperiod sensitivity (PS).Dark period is the main facter in flowering induce of short-day plants (SDPs). A short exposure to light in the middle of the night causes inhibition of flowering in SDPs, this phenomenon is called night break (NB) and has been used as a tool to study the phothperiodic control of flowering. So, NB is also an important method to study maize PS.In this study, the genetic basis of maize photoperiod sensitivity were carried out from the aspects of phenotype and photoperiod signaling passway by using NB. Firstly, tropical and temperate maize inbred lines were conducted by NB to analyse characters of development traits. Secondly, phyB2as a main photoreceptor of maize were cloned, and their expression patterns were analysed before and after NB within48h by real-time RT-PCR. Finally, expression of circadian clock genes Hd6, ZmCCA1and signaling output pathway genes Gigantea (GI), Constans(CO), and Flowering Locus T (FT) were addressed to investigate the relationship between their expression patterns and maize flowering. The main results obtained-in this research were as following:1. A tropical maize inbred line CML288and two temperate inbred line B73, Huangzaosi were grown under inductive short-day (SD)9h light/15h dark cycles, continuous night break (NB) under SD condition were conducted in the most photoperiod sensitive developmental stage. Changes in day to tassel (DTT), day to pollen (DTP), day to silking (DTS) and plant height were measured after NB. The results indicated that DTT, DTP, DTS were delayed, and the plant height was decreased in CML288after NB, so is in B73with lower degree, while DTT, DTP, DTS were advanced, and the plant height was increased in Huangzaisi. All these demonstrated that different lines produce different NB responses and tropical photoperiod sensitive inbred line is the most sensitive to NB. NB effects were compared from4-leaf period to8-leaf period, the results showed that traits in flowering time and plant height of tropical photoperiod sensitive inbred line CML288were similar to that of SD condition when NB conducted at4-leaf period. The largest effects of NB on these traits occurred when NB were conducted at5th "leaf period, demonstrating that the5th-leaf stage is the most sensitive to NB. The dalay of DTT, DTP, DTS and inhibition in plant height were positively correlated to the days of NB treatment when continuous NB were conducted at the photoperiod sensitive developmental5-leaf to8-leaf period, indicated that NB treatment has additive effects.Red light(R) and Far Red light(FR) irradiation in the middle of night period also delayed flowering time of CML288,effect of R on inhibition of flowering was larger than that of FR, and FR could partly reverse these effect.These results implied that phytochrome was the photoreceptor in the NB effects on growth and development of maize.2.CDS of phyB2was cloned by homologous amplification in maize inbred lines CML288and Huangzaisi, a3501bp open reading encoded1166amino acids which has six conserved domains.There are seventeen single DNA letter differences in phyB2CDS of CML288and Huangzaisi. Multiple alignments of the predicted complete amino acid sequences of these two lines indicated that there are seven amino acid diversity, three of seven were located in conserved domains of PHYB2. Expression pattern of phyB2in48h under SD and NB conditions were conducted using real-time fluorescence quantitative PCR in the stem apexes and leaves of CML288and Huangzaisi. Under SD conditions, phyB2exprssion patterns in leaves and SAs of CML288peaked twice, the first peak occurred at dawn,the second peak occurred at6h after dusk. NB treatment brought forward the peak of phyB2in leaves of CML288. phyB2exprssion patterns in leaves and SAs of Huangzaisi.also peaked twice under SD conditions, the peaks occurred at3h before dawn and dusk, expression patterns hardly changed after NB.3.Expression pattern of circadian clock genes Hd6、ZmCCAl in24h under SD and NB conditions were conducted in the stem apexes and leaves of CML288and-Huangzaisi. Under SD conditions, Hd6exprssion levels was lower in leaves and SAs during light period, then increased in leaves after entering dark period, while peaked at4h before dawn. Hd6exprssion levels decreased after NB treatment. Under SD conditions, ZmCCAl exprssion levels peaked at at4h after dawn. NB treatment did not change this gene exprssion pattern but enhanced its level. Under SD conditions, Hd6exprssion levels in leaves and SAs of Huangzaisi increased at4h after dawn, then peaked at3h after dusk in leaves, while peaked at4h before dawn in SAs. NB treatment decreased its exprssion levels in leaves and SAs, then recovered to normal SD expression pattern after dawn. ZmCCAI exprssion patterns in leaves and SAs of Huangzaisi was similar to CML288, NB treatment increased its exprssion levels during dark period, but recovered to normal level after dawn.4.Under SD conditions, ZmGI exprssion peaked at3h before dawn in leaves and SAs of CML288. NB treatment did not change its exprssion pattern, but enhanced its exprssion levels in leaves, while brought forward its peak in SAs. conzl exprssion levels in leaves and SAs peaked at6h after dusk under SD conditions, conzl expression in leaves exhibited a diurnal expression pattern.with two peaks per day while its levels in SAs were lower all the time by NB treatment. ZCN8exprssion levels in leaves and SAs peaked at dawn under SD conditions, NB treatment dramatically inhibited its levels,but recovered to normal at the second cycle.Under SD conditions, ZmGI exprssion levels in leaves and SAs of Huangzaisi peaked at3h before dusk. NB treatment delayed its peak and enhanced its amplitude in leaves,while decreased its amplitude in SAs with normal expression pattern, conzl exprssion patterns in leaves and SAs was similar to that under SD conditions and NB treatment. ZCN8exprssion levels peaked at dawn under SD conditions. NB treatment decreased its amplitude,but did not changed its normal expression pattern.5.After NB treatment, phyB2peaked earlier in tropical photoperiod sensitive inbred line CML288,this result decreased circadian clock genes Hd6level, and increased ZmCCA1level,giving rise to longer circadian clock period. Longer circadian clock period induced higher ZmGI exprssion levels to produce two peaks expression pattern of conzl gene, inhibit ZCN8exprssion, resulting in flowering delay in the end. |
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