Cloning And Characterization Of ZmCOL Gene Related To Photoperiod Sensitivity In Maize

Tropical and subtropical maize germplasm contain abundant hereditary variation, which can broaden the genetic base of Chinese maize germplasm to enhance the breeding level by improving and using of these germplasm. However, photoperiod sensitivity severely limits the use of tropical and subtropical germplasm in temperate zone. In the result, isolating, cloning and analyzing the function of photoperiod-sensitive genes is an important way to research the molecular mechanism of maize's photoperiod-sensitivity.We cloned the ZmCOL gene by the method of orthologus gene cloning in a photoperiod-sensitive tropical maize (Zea mays L.) inbred line CML288 and a photoperiod-insensitive temperate inbred line HuangZao4, which was the cognate of CO gene(Arabidopsis) and OsHd1 gene(rice). Then we studied the circadian rhythm expression pattern of ZmCOL, analyzed the expression pattern of ZmCOL in shoot apical meristems (SAM) and leaves, and researched the relationship among ZmCOL, ZmGI, and ZmFT, in order to investigate the function and mechanism of ZmCOL during the photoperiod induction. We constructed several 35S fusions between GUS and ZmCOL or conserved domain of ZmCOL to test whether ZmCOL L protein is localized to the nucleus, and which domain of ZmCOL is sufficient to localize GUS to the nucleus. Then we constructed the over expression vectors of ZmCOL, transformed it into the Arabidopsis with Floral Dip method, and obtained several transformants. We identified the photoperiod sensitivity of 21 maize inbred lines by photoperiod-sensitive related traits of maize, and investigated the relationship between sequent variations of ZmCOLs and photoperiod-sensitive related traits. The main results are as follows:1. We had cloned the maize ZmCOL gene through orthologus gene cloning. ZmCOL gene sequence has two exons and one introns, and contained a predicated 1197/1188bp open reading frame in the CDS sequence, which could encode a peptide with 398/395 amino acids. By phylogenetic analysis, we found that the ZmCOL and the rice OsHd1 are in the same evolutionary branch with the protein sequence sharing 87.10% homology to rice OsHd1. The predicated protein that especially containing CO/Hd1 gene family is composed of a zinc finger B-box and a CCT domain. That is to say, ZmCOL gene belonged to the member of CO/Hd1 gene family. There is a continuous nine base insertion / deletion mutation variation in the ORF of ZmCOL between photoperiod-sensitive tropical maize inbred line CML288 and photoperiod-insensitive temperate inbred line Haungzao4, and it caused three amino acids differences of ZmCOL protein sequence, suggesting that maybe the variation is closely related to photoperiod sensitivity.2. We used fluorescence quantitative RT-PCR to research the circadian rhythm expression pattern of ZmCOL under long day and short day conditions in CML288 and HuangZao4, analysesed the expression pattern of ZmCOL in shoot apical meristems (SAM) and leaves, and researched the relationship among ZmCOL, ZmGI, and ZmFT. The results showed that similar to it's Arabidopsis and rice homologs(CO and OsHd1), ZmCOL gene exhibited diurnal expression patterns while ZmCOL gene expressed both in SAM and leaf under long day and short day conditions in CML288 and HuangZao4; in different development stages of maize, however, the expression of ZmCOL genes showed different levels or changes. And the peak period of ZmCOL gene expression in SAM and leaves were notably similar to the phase of induction photoperiod-sensitivity, indicating that ZmCOL gene may be joined the regulation of photoperiod, and functional importance in the phase of induction photoperiod-sensitive. And in this period, the main expression parts of ZmCOL is different in different inbred lines: in the photoperiod-insensitive material ZmCOL gene expression higher in leaves, in the photoperiod-sensitive material ZmCOL expression higher in SAM. But the expression level of ZmCOL gene in leaves finally decide whether the maize inbred lines can complete the photoperiod-induction in the phase of induction photoperiod-sensitivity. Analysis by the expression of ZmGI, ZmCOL and ZmFT showed that, ZmGI gene located the upstream of ZmCOL gene, regulating the circadian rhythms expression of ZmCOL gene; ZmCOL gene located the upstream of ZmFT gene, and ZmCOL gene played an important role in the phase of induction photoperiod-sensitivity, while the downstream gene ZmFT functional were of importance in the later phase of corn growth, important to the floral development.3. We had constructed several 35S fusions between GUS and ZmCOL or conserved domain of ZmCOL, a transient expression assay in onion bulb epidermal cells by gene gun bombardment, the result showed that: ZmCOL can localize GUS to the nucleus of the cells, suggesting that CO is a nuclear protein, and the CCT domain is an NLS of ZmCOL, it is sufficient to target GUS to the nucleus. And we constructed the over expression vectors of ZmCOL, transforming it into the Arabidopsis with Floral Dip method. The result of PCR showed that we have got several transformant plants.4. We analyzed the photoperiod-sensitivity related phenotypic traits of 21 maize inbred lines and studied the relationship between sequent variations of ZmCOLs and photoperiod-sensitivity related traits, the results showed that: different maize germplasm groups have different photoperiod sensitivity, specifically, inbred lines of tropical germplasm have the strongest photoperiod sensitivity. Dynamic cluster of phenotypic characteristics divided different maize inbred lines into different sensitive grades:â… ,â…¡,â…¢andâ…£. For maize inbred lines, the higher of the level, the stronger of the photoperiod-sensitivity. The analysis of sequence indicated that only the sequence variation of 2+(523-531)was significantly associated with the photoperiod-sensitivity related traits, however, the relationship between variation among the other six sequence variations and photoperiod-sensitive related traits were not significant.