| Nelumbo nucifera is a large emergent aquatic plant belonging to Nelumbo of the Nympheaceae. And it is one of the top ten traditional Chinese flowers having highly ornamental and economic value, rich flower type and color and strong ability of anti-pollution. In our study, we cloned two valuable genes about anti-heavy metal and flower development, analyzed their temporal and spatial expression and further studied their function by transforming them into Arabidopsis thaliana. We can not only investigate genetic variation mechanism about anti-heavy metal and flower development but also provide a theoretical basis and technical support to improve amental characters and anti-pollution ability by using plant genetic engineering in Nelumbo nucifera.1. NnPCSl (GenBank accession AB360381) was cloned from Nelumbo nucifera leaf by RACE-PCR. We found that the length of NnPCSl was 2208bp and the putative protein contained 505 amino acids and two typical subfamily domains which had four adjacent Cys-Cys element in the site of 331-332,352-353,370-371,417-418 and four PCS protein feature sites in Cys-56, Cys-90/91, Cys-109, Cys-113. The most closely related amino acids sequence (70.41%identity) was Lactuca sativa PCS.2. NnAP2 (GenBank accession AB622392) was cloned from Nelumbo nucifera petal finding that the length of NnAP2 was 2048bp and the putative protein contained 511 amino acids. Addition to a completely conserved nuclear localization signal (KKSR) sequences, NnAP2 had the typical AP2 domain belonging to AP2 subfamily which was the character of AP2 family, the identity of it compared to Arabidopsis thaliana was 91.03%and 85.29% respectively. The phylogeny of plant AP2 polypeptides suggested that NnAP2 had the the closest orthologs relationship with VvAP2.3. The NnPCS1 expression was analyzed by Real-Time PCR in different organs and under different heavy metal stress in Nelumbo nucifera. NnPCS1 was constitutively expressing and the transcription level of NnPCS1 was induced by the presence of either CdCl2 or CUSO4·5H2O in the leaf. Zn can effectively relieve Cd toxicity to a certain extent, but different concentrations of Zn were different on the alleviation of Cd toxicity.From our results, NnPCS1 expression was not diurnally regulated in root and leaf within 24 h, but the expression in leaf was higher than in root. The leaf expression level increased 1.7 fold after exposure to 400μmol/L CdCl2 for 1h, and by 2.7 fold after 3h; and by 2.4 fold after being exposed to 500μmol/L CUSO4·5H2O for 1h. These enhanced levels of transcription fell subsequently, although transcript abundance increased again between 6h and 12h of the CdCl2 treatment and between 3h and 12h of the CUSO4·5H2O treatment. In the root, NnPCS1 expression initially rose slightly after exposure to CdCl2, but fell back after 1h and was no longer detectable by 12h. When exposed to CUSO4·5H2O, its expression was stable over the first 3h, thereafter decreasing to below the level of detection by 6h. These showed that Cd2+ had the higher regulation capacity in regulating NnPCS1 transcription than Cu2+. The NnPCS1 expression was inhibited that can not be detected in the leaf by exposure to 400 μmol/L CdCl2 after 48h, but the NnPCS1 expressed after exposure to 400 μmol/L CdCl2 plus 200 μmol/L ZnSO4 with the control of no expression under 400μmol/L CdCl2 and 200μmol/L ZnSO4 exposure.4. The NnAP2 expression was analyzed in different organizations, flower organs and growth stages. From simple petal flower cultivar to double-petalled flower cultivar and then to thousand petal flower cultivar, the NnAP2 expression was always the highest in petal, however, the expression in non-simple petal flower cultivar was higher than in simple petal flower cultivar.NnAP2 expression was detected in root, stem, leaf and flower, and the expression was the highest in flower followed by leaf. In bud, early blossoming and full bloom stages. NnAP2 was expressed in all floral organ:receptacle, sepal, petal, stamen and gynoecium. During the three phases of flowering, the expression level of NnAP2 was highest in petal, followed by stamen and lowest in sepal. Between five representative species in the early blossoming stage, the expression level of NnAP2 was also the highest in petal followed by stamen in’Zhongguogudailian’,’Yaoniangyujiao’and’Hongtailian’. In’Yiliangqianban’, the expression in petal was the highest followed by sepal and receptacle. We can concluded that regardless of flower type, the NnAP2 expression was always the highest in petal. In early blossoming stage, the stamen and gynoecium are degenerated to petals. In other four flower type cultivars, the NnAP2 expression in stamen was only lower than in petal. In the longitudinal comparison of five representative cultivars, order of the expression in petal was that double-petalled flower ’Yaoniangyujiao’> proliferation flower ’Hongtailian’> thousand petal flower’Yiliangqianban’> multiplicate flower’Jinxia’> simple petal flower ’Zhongguogudailian’.5. We constructed the NnPCSl heterologous expression vector and transformed it to Arabidopsis thaliana. The transgenic plants had differently tolerant to Cd toxicity. Under excessive Cd stress, they can mitigate the heavy metal ions toxicity.The NnPCSl was transformed to Arabidopsis thaliana by the floral dip method. Eight plants were selected as being carriers of the transgene on the basis of the Tl hygromycm selection, GUS assay and PCR and these were designated pcsl through pcs8. The NnPCS1 expression in pcs2, pcs5, pcs6 was higher than in other five lines and there is no expression in wild-type by Real-time PCR. At 100μmol/L CdCl2, there was no significant difference with respect to the content of GSH between the transgenic and the wild type plants, but the level of NPT was 1.3 to 1.5 fold higher than than of wild type and the level of PC was 1.6 to 1.9 fold higher. The size of the two indicator values was pcs2> pcs5> pcs6>WT (control), however, the difference between the transgenic plants was not significant. After one week exposure of 50μmol/L CdCl2 and 100μmol/L CdCl2, the Cd content of three transgenic lines in shoot was 2 to 2.5 fold higher than that in wild type. The pcs2 Cd content was slightly higher than in pcs5 and pcs6 under two CdCl2 concentration. Increased NPT contents promoted the biosynthesis of PC and increased Cd tolerance.6. NnAP2 gene was transformed into Arabidopsis thaliana. The transgenic plants had higher stem height than wild type and were early flowering. However, we can not see any change in the petal number, stamen and pistil of transgenic plants.We constructed the heterologous expression vector and Arabidopsis thaliana Col-0 was transformed by the floral dip method. Eleven plants were selected and these were designated AP2-1 through AP2-11. The NnPCSl expression in AP2-2, AP2-6 was higher than in other nine lines and there was no expression in wild-type by Real-time PCR. The stem height of transgenic plants AP2-2 and AP2-6 was higher than that in the wild type after growth for seven weeks. Moreover we analyzed eleven GA related genes finding that the expression of GA2ox3 in transgenic plants was 2-3 fold greater than that of wild type, while that in the expression of GA2ox7, was 4-5 fold higher. The GA2ox3 and GA2ox7 had significant differences compared to wild type, while the other nine genes had no significant difference. These showed that the decrease of GA2ox3 and GA2ox7 promoted the biosynthesis of endogenous GA when NnAP2 was transformed to Arabidopsis thaliana and that may be why the transgenic plants had higher stem height. In addition, two transgenic lines were early flowering compared to wild-type. We analyzed the expression of four flowering relative genes (LFY, FT, CO, FLC) after sowing for 12 and 18 days. The transcription level of LFY and FT in AP2-2 and AP2-6 plants was higher than that in wild type after sowing for 12 days, however, there was no significant difference in CO and FLC expression. After sowing for 18 days, the transcription level of LFY and FT in AP2-2 and AP2-6 plants was still higher than that in wild type. But the expression of LFY increased rapidly, there was no significant difference in CO gene expression between transgenic lines and wild type, however, the expression of FLC in wild type was higher that in two transgenic lines. Results showed that the expression of flowering genes were regulated when endogenous GA when NnAP2 was transformed to A. thaliana. The decrease of FLC and the increase of FT and LFY promoted the flowering, however, in this process CO had no significant effect. |
