| Phytochromes are the most important photoreceptors that plants perceive light signaling. As a one of the important phytochrome family members, Phytochrome B(PHYB) plays an important role in many physiological processes in plants. In this study, cloning and expression analysis of PhyB in Triticum aestivum and Zea mays were studied. The main results were described as following:1. Cloning and expression analysis of TaPhyB in Triticum aestivum1.1 A BAC library of the long arm of 4'D'group chromosomes in hexaploid Chinese spring (CS) was screened with probe of Maize ZmPhyBl fragment. We obtained three positive BAC clones, which named BACA17-2, BE02, F02, respectively. According to EST sequence of TaPhyB3, we designed specific primers to amplification and obtained a DNA fragment of 3.5kb. Sequencing result showed that the cDNA sequence and in GenBank logged in a phytochrome gene from wheat (registry number AY888046) at the nucleotide level of 99%consistency, which Indicated that the cDNA cloning and AY888046 were the wheat gene phytochrome B in the corresponding copy of the long arm of 4D. The cDNA sequence named TaPhyB3. According to the principle of the GT-AG were intron boundaries, genomic sequence of TaPhyB3 was compared with the cDNA and the redundant sequences were deducted. Genome sequences in the introns, exons and untranslated regions of TaPhyB3 were obtained, which contained four extrons and three introns. The four extrons E-1 to E-4 were located in 1-2174,2500-3308,4313-4605 and 7246-7472bp and the three introns 1-1 to 1-3 were located in 2174-2500,3308-4313 and 4605-7246bp, respectively.1.2 TaPhyB3 gene coded region of full-length cDNA sequence of 3501bp and predicted that the gene encodes 1165 amino acids. By homology comparison revealed that the protein contains conservative domains that existed in a number of other common species. The similarity among the protein with rice, corn and Arabidopsis were 93%,90%and 73%, respectively.1.3 The seedlings growed seven days under dark, far-red light, red light, blue light, respectively. The fastest growth of coleoptile was in the dark, which was a length of 10 mm or so. While under the far-red light, red, blue and white, its length decreased along with decreasing light intensity. After treatment wheat seedlings under different light intensities seven days, TaPhyB3 expression analysis results showed that the lowest expression level was in the dark and the highest levels was under the white light. The expression levels of TaPhyB3 under far-red light,red light,blue light and white light were 2.2,7.7,7.4 and 37.3 times than that in the dark, respectively. TaPhyB3 gene tissue-specific expression results showed that expression level of TaPhyB3 gene in the ground was significantly higher than that in the root, in which the expression level of the leaves was 11.4 times than that in the root.2. Cloning and preliminary function analysis of ZmPhyB in Zea mays2.1 Extracting RNA in maize inbred line B73 seedlings and RNA was reverse transcription cDNA. According to EST sequences of ZmPhyBl and ZmPhyB2, we designed specific primers to amplification the two parts of ZmPhyBl and ZmPhyB2, respectively. We obtained four DNA fragments of 1.7kb. After these sequences matched, DNA fragments of ZmPhyBl and ZmPhyB2 were sequenced. Sequencing result showed that the cDNA sequence and in GenBank logged in phytochrome genes from maize (registry number AY234827 and AY234828) at the nucleotide level above 99%consistency, respectively, which indicated that the cDNA clonings were the maize gene phytochrome B in the corresponding copy of ZmPhyBl and ZmPhyB2.2.2 Apply to analysis function of the conserved domain in NCBI database, the function of conserved domains in ZmPHYBl and ZmPHYB2 were analyzed. The result showed that ZmPHYB1 and ZmPHYB2 possessed six conserved domains that widespread in other species. These domains named DAF DOMAIN PHYTOCHROME REGION,PAS-A DOMAIN PAS-B DOMAIN,HISTIDINE KINASE RELATED DOMAIN 1 and HISTIDINE KINASE RELATED DOMAIN 2. We also found that ZmPHYB1 protein contains a few motifs that did not existed in ZmPHYB2 protein, which named two putative active sites and two heme-binding sites.2.3 Different Light-induced ZmPhyBl and ZmPhyB2 expression results showed that in the inbred line B73, the highest expression level of ZmPhyBl was under far-red light and the expression level under red light was the lowest, which half of the amount of that under white light. The lowest expression level of ZmPhyB2 was under red light and the highest expression level was under far-red light, which was 1.7 times to that under red light. In the inbred line Mo 17, the highest expression level of ZmPhyBl was in the dark and the lowest expression level was under far-red light, which about half of the amount of that in the dark. Under red light and white light, the expression levels of ZmPhyB2 were higher than other conditions. The lowest expression level was under far-red light, which thirty percents than that in the dark.2.4 Tissue-specific expression of ZmPhyBl and ZmPhyB2 results showed that the highest expression level of leaf in ZmPhyBl was under white light, followed by that in the pedicle. The lowest expression level was in the root. The highest expression level of ZmPhyB2 was also in the leaf, followed by that in the sheath and that in the pedicle. In the stem, bract leaf and root, both of the expression level of ZmPhyB2 were low. In the dark, the highest expression level of ZmPhyBl was in the spike, followed by that in the leaf. The lowest expression level of ZmPhyBl was in the root. As to ZmPhyB2, the highest expression level was in the sheath, followed by that in the stem, leaf, bract leaf and flower. The lowest expression level of ZmPhyB2 was in the root.2.5 The intensely interaction signaling between the C terminal of ZmPHYA1, the C terminal of ZmPHYA2, the full-length of ZmPHYA2 and the full-length of ZmPHYB1 have been detected, respectively, and the relatively weak interaction signaling have also been detected between the C terminal of ZmPHYA1 and the N terminal of ZmPHYB1. But the interaction signaling have not been detected between other protein domains. The relatively intense interaction signaling between the N terminal of AtCOPl and the full-length of ZmPHYB1 have been detected and that has also been detected between the C terminal of ZmPHYB2 and the full-length of AtSPA1.2.6 Transgenic plants TO generation of pJIM19-Hyg-HA-ZmPhyB1 showed petiole and root elongation, and transgenic plants TO generation of pJIM19-Kana-myc-ZmPhyS2 showed normal growth in resistance plate and seedlings of Arabdopsis turned dark green. PCR detection transgenic plants result showed that ZmPhyBl and ZmPhyB2 have been transformed into wide type Col-0 and mutant PhyB-9, respectively. Resistance screening Tl generation transgenic plants, separation ratio of resistance plants to nonresistance plants was 3:1. The hypocotyl of T1 generation transgenic plants that pJIM19-Hyg-HA-ZmPhyB7 transformed mutant PhyB-9 turned short and cotyledon opened up, which showed that ZmPhyBl has been complemented the phenotype of mutant PhyB-9.The T1 generation transgenic plants of pJIM19-Kana-myc-ZmPhyB2 transformation mutant PhyB-9 showed not only for the shorter hypocotyl, cotyledon opening, but petiole elongation, which indicated that ZmPhyB2 has also been complemented the phenotype of mutant PhyB-9. The T1 generation transgenic plants of pJIM19-Hyg-HA-ZmPhyBl transformation wild type Col-0 showed increasing cotyledon area and the angle between cotyledons. The Phenomenon indicated that transgenic over-expression plants showed more evident photomorphogenesis features comparing with wild type Col-0. The transgenic over-expression plants of pJIM19-Kana-myc-ZmPhyB2 transformation wild type Col-0 also showed cotyledon opening, increasing the angle between cotyledon and hypocotyl intensely shorter, which indicated that transgenic over-expression plants also showed more evident photomorphogenesis features comparing with wild type Col-0.
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