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| Functional Identification of SAHH Gene from Kalanchoe daigremontiana |
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Abstract S-adenosyl-L-homocysteine hydrolase (SAHH) is the only known enzyme to catalyze the breakdown of S-adenosylhomocysteine (SAH) into homocysteine and adenosine. SAH is the inhibitor of most transmethylation reactions which is often compared with S-adenosyl-L-methionine (SAM, one of methyl donor) to evaluate the overall methylation level in plant. In order to identify the function of SAHH in Kalanchoe daigremontiana (KdSAHH), primers were designed according to the full length of KdSAHH cDNA in GenBank (Accession No. KF953475). The KdSAHH gene expression pattern under drought stress (light drought stress(20 d); middle drought stress (35 d); high drought stress (50 d)), subcellular location in cell and ectopic transformation in tobacco (Nicotiana tobacum) were done to unveil the basic biological function. Drought stress was simulated with 20% PEG6000 in the seedling stage between transgenic and wild type tobacco and drought-related physiological indexes like leaf relative water content (RWC), chlorophyll (Chl) content, malondialdehyde (MDA) content, peroxidase (POD) content, superoxide dismutase (SOD) content were measured at 3 different time points (0, 12 and 24 h). The semiquantitative RT-PCR analysis showed that the highest expression level of KdSAHH gene was meatured in high drought stress (50 d), followed by middle drought stress (35 d) and light drought stress (20 d). The result of subcellular location indicated that KdSAHH gene was located both in nucleus and cytoplasm. Nine positive tobacco transgenic line were obtained by PCR. Physiological indicators of transgenic and wild type tobacco under 3 time periods (0, 12 and 24 h) of 20% PEG6000 treatment showed that both of them showed obvious water loss symptoms at 24 h, but the transgenic tobacco of KdSAHH gene had lower wilting degree compared with wild type tobaccos, and some leaves at the upper end of the transgenic plant were still standing, while all leaves of wild type tobaccos showed obvious water loss symptoms. With the increasing of drought stress, both leaf RWC and Chl content of transgenic and wild type tobaccos tended to decreased but the decline extent of transgenic tobacco was relatively slight. Leaf RWC was significantly different between transgenic and wild type tobaccos at 24 h (P＜0.05) and Chl content were significantly different between the 2 types at 12 and 24 h (P＜0.05), which showed that under drought stress, the transgenic tobacco was still able to maintain a stable metabolism. MDA content was increased in transgenic and wild type tobaccos but more MDA content was in the wild type tobacco than that in the transgenic tobacco, and it was significantly different between the 2 types at 24 h (P＜0.05), which showed that transgenic tobacco had a lower degree of membrane lipid peroxidation and suffered lighter injury. The changing trend of SOD and POD content in transgenic and wild type tobaccos were almost the same, however, the wild type tobacco produced more SOD and POD content than the transgenic tobacco with the increasing drought stress. POD content was significantly different between the 2 types at 12 and 24 h (P＜0.05), while there was no significant difference of SOD content. The reason might be analyzed as follows transgenic tobacco had a stronger resistance to drought stress and could not generate too much reactive oxygen. By comparing different physiological indexes between the transgenic and wild type tobaccos under drought stress, we could conclude that transgenic tobacco had a higher resistance than that of wild type tobacco. The result of this study has a great significance that it identified KdSAHH gene was tightly involved in Kalanchoe daigremontiana drought resistance. SAHH widely spreads in various organisms, which is a key enzyme of methyl cycle in organism. The study enriches the function of KdSAHH gene and provides a reference for follow-up study aiming at the function of this gene.
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Received: 24 July 2015
Published: 24 February 2016
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