Changes Of Physiological Characteristics Of The Obtained Trangenic YHem1 Plants With Ability To Over-Produce 5-Aminolevulinic Acid

5-Aminolevulinic acid (ALA) is the essential precursor of porphyrin compounds in all living bodies, and it has been suggested to be able to apply in agriculture production. However, most of the previous works in the field were based on exogenous ALA application, and no transgenic plants that could over-produce ALA under natural light condition had been reported up to now. In this work, the promoter sequence of Arabidopsis thaliana HemAl gene and yeast(Saccharomyces cerevisiae) Hem1 gene were cloned to recombine a new light-responsive gene YHem1, which was then transformed into tobacco, tomato, oilrape and Arabidopsis thaliana mediated by Agrobacterium tumefaciens. The transgenic plants could grow under natural light condition and synthesize more endogenous ALA than the wild type. After several generation selections, the homogenous lines were used to study the effects of YHeml transformation on leaf photosynthetic characteristics, antioxidate enzymes, and stress tolerance, biological and economic mass. Agilent Arabidopsis Oligo Microarray (V4) (4*44K) was also used to compare the transcription profile between transgenic YHeml Arabidopsis and wild type. The achievement was considered significant for determination ALA’s physiological function and mechanism, and exogenous ALA application in agriculture. The main results were as follows.1. The HemAl promoter in Arabidopsis thaliana is a light responsive promoter that regulates the diurnal rhythmic biosynthesis of 5-aminolevulinic acid (ALA) in plants. A binary recombinant gene comprising A.thaliana HemAl promoter and yeast Heml, encoding ALA synthase in the microbe, was transformed into tobacco explants. The To generation seeds of transgenic tobacco were distinguished by series concentrations of kanamycin (Km) solutions, and the Km-resistant plants from 1000 mg·L-1 Km solution were transplanted in pots and inspected by GUS. The results showed that 92% of the Km-resistant tobacco plants were GUS-positive. Further inspection by PCR with specific primers showed that 92.5% of GUS-positive plants contained,4. thaliana hemAl promoter and 88%contained yeast Heml, and therefore,84.2% contained the complete binary recombinant gene. The expression of pYHeml-EGFP showed that the ALA synthase protein was located in mitochondrion of transgenic plants. RT-PCR detection demonstrated that the expression of yeast Hem1 gene was conditioned because the relative abundance of Heml mRNA transcript was higher under light than that at dark, which was corresponded with the light-responsive property of the HemAl promoter. Furthermore, the endogenous ALA biosynthesis and the chlorophyll content in transgenic tobacco leaves, compared with the wild type, were obviously increased with higher ratios of Chl b/a. The leaf SPAD values in the base leaves of the wild type plants dropped distinctly whereas those in transgenic ones were kept in much higher levels. These results suggested that both exogenous genes could be inherited in transgenic tobacco, and the translated ALA synthase protein was distributed in the mitochondrion of plants, which grew normally under natural condition and over-expressed YHeml genes to overproduce endogenous ALA when exposed to light.2. Transgenic YHeml tobacco plants had significantly rise of the net photosynthetic rate (Pn), stomatal conductance (Gs) and transpiration rate (Tr) of the leaves. Measurement of chlorophyll fluorescence of the dark-adapted leaves revealed that the minimal fluorescence (Fo) was not obviously different among leaf positions in the transgenic plants, but that of the base leaves raised distinctly in the wild type ones. Meanwhile, the maximum fluorescence (Fm), the variable fluorescence (Fv) and the PS II maximal photochemical efficiency (Fv/Fm) of the base leaves in transgenic tobacco were all significantly higher than that of the wild type. When seedlings were exposed to light, it was then observed that, the chlorophyll fluorescence parameters including the photochemical efficiency of PS Ⅱ (Fv’/Fm’), PS Ⅱ actual photochemical efficiency (ΦPSⅡ), photochemical quench (qP), electronic transfer rate (ETR), photochemistry (Pc), and energy entering PS II center (Pc+Ex) were generally higher than that of the wild type, whereas the antenna heat dissipation (Hd), non-photochemical quench (NPQ) were much lower than that of the wild type. These differences were most significant in the base leaves, suggesting that the over-production of ALA could prolong leaf photosynthetic life, enhance photochemical conversion efficiency, increase photosynthate accumulation and promote plant growth.3. Comparison of the chlorophyll biosynthesis showed that the porphobilinogen (PBG) content was rather stable between the wild type and the transgenic plants whether under light condition or dark, while the content of uroporphyrinogen Ⅲ, coproporphyrinogen Ⅲ, proto IX, Mg-proto IX and Pchlide was decreased when the plants were switched to light condition. The content of chlorophyll a and chlorophyll b was generally higher when plants were grown under light than at dark, suggesting that light promoted metabolism of porphyrin intermediate products and chlorophyll biosynthesis. The transformation of YHeml gene into tobacco did not affect the content of porphyrin intermediate products, however, the expression of ALA dehydratase gene Hem B, coprophyrinogene oxidative decarboxylase gene Hem F, Mg-protoporphyrinogen IX methyltransferase gene Chl M and chlorophyll synthase gene Chl G was significantly up-regulated under light condition, suggesting that ALA over-production was involved in regulation of chlorophyll biosynthesis at gene expression level.4. The activities of leaf anti-oxidative enzymes, including superoxide dismutase (SOD), peroxidase (POD) and ascorbate peroxidase (APX) were compared between YHeml transgenic tobacco and wild type during their development from May to Augest. It was found that the increase of POD activity was most closely correlated with transgene among the three enzymes. Several new POD isoenzyme bands were observed in the polyacrylamide gel electrophoresis in transgenic plants, but absent in the wild type. Observation with electron microscope showed that more POD staining in the chloroplast in the transgenic tobacco than the wild type. RT-PCR detection demonstrated that many antioxidative enzyme genes were up-regulatedly expressed at different levles. Therefore, it could be concluded that YHem1 gene transformation might promote antioxidative enzyme activity in tobacco plants, where the most important was POD. It was related with superoxidate anion production rate, as well as leaf senescence.5. The binary recombinant gene YHeml comprising Arabidopsis thalinanian HemAl promoter and yeast Hem1 was transformed into explant of tomato mediated by Agrobacterium tumefaciens LBA4404 line. The resistant regenerated plants were obtained after Km selection and GUS dyeing. The detection of PCR and RT-PCR showed that YHeml gene had been transformed into tomato and expressed in the transgenic plants. ALA was over-biosynthesized in the transgenic tomato under light condition, which led to higher levels of chlorophyll in leaves.6. The gas exchange characteristics of the transgenic tomato with ability to over-produce ALA were analyzed with a Li-6400 photo synthetic system. The results showed that the net photo synthetic rate (Pn) in the transgenic plants was 55% higher than that of the wild type, and the stomatol conductance (Gs) and transpiration rate (Tr) were also increased significantly. The apparent quantum yield (AQY) in the transgenic tomato was 0.0363, while AQY was 0.0313 in the wild type. Therefore, the former was significantly higher than the latter, which suggests the ALA over-production could improve light energy utility and photosynthate accumulation.7. The fast chlorophyll induction fluorescence characteristics in YHeml transgenic tomato plants were compared with a Plant Efficiency Analyzer (PEA) at a series of saturated pulse light intensity from 90 to 2400 μmol·m-2·s-1. The results showed the fluorescence curves increased as the light intensity. The maximum photochemical efficiency (φpo) was a rather stable parameter, which was significantly higher in transgenic tomato than that in the wild type. The possibility of a trapped exciton moves an electron into the electron transport chain beyond QA- ((?)o) and quantum yield of electron transport of PSII reaction center (φEo) were declined as the pulse light increased, which were also higher in the transgenic plants than that of wild type. The photosynthetic performance index whether based on absorption (PIABs) or cross section (PIcs) were always higher in the transgenic plants than that of wild type, suggesting that over-production of ALA in YHeml transgenic tomato might improve PSII reaction center activity and electron transfer ability, especially at the side of electron reception.8. The diurnal dynamics of fast chlorophyll fluorescence in transgenic YHeml tomato with ability to over-produce 5-aminolevulinic acid were compared between two environmental light intensities. It was found that the OJIP curves were various among the measurement times when the plants were grown under low light intensity (the maxmium light intensity was about 80 μmol·m-2·s-1, while the OJIP curves distorted greatly at noon time when the plants were under high light environment (the maxmium light intensity was 1700 μmol·m-2·s-1. The maxmium fluorescence at noon decreased dramatically, and therefore the photosynthetic performance index (PIABs) dropped greatly. Since the temperature in two environementa was consistant, the midday depression of photosynthesis in tomato was due to the high light stress. The maxmium fluorescence and photosynthetic performance index in transgenic tomato were significantly higher than that of wild type, suggesting ALA over-production was benificial for alleviation of midday depression in tomato plants.9. A binary recombinant gene comprising A. thaliana hemAl promoter and yeast Heml, encoding ALA synthase in yeast, was transformed into oilrape explants mediated by Agrobacterium tumefaciens LBA4404. The detection by PCR and RT-PCR demonstrated that the exogenous YHeml had been transformed into oilrape genome and could be regulatedly expressed by light exposure. Analysis with plants at anthesis or fruiting period showed that both the endogenous ALA turnover rate and chlorophyll content in the transgenic plants were higher than the wild type. Measurement with a PEA appeared that the chlorophyll fast fluorescence induction in the transgenic plants was obviuos higher that of the wild type, where the parameters including leaf photosynthetic performance index (PIABs), light trapping capacity (PTR), electron transfer capacity (PET), PSII maxmium photochemical effeciency (φpo), quantum yield for electron transport (φEo), and possibility of a trapped exciton moves an electron into the electron transport chain beyond QA-(()?o) were significantly higher, while the approximated initial slope of the fluorescence transient (Mo) and relative variable fluorescence intensity at the J-step (Vj) were significanlty lower. Additionally, ALA over-production induced higher of the density of active reaction center (RC/CS) in transgenic oilrape. The yield of potted oilrape in the transgenic plants was about 80% higher than the wild type without any bad effect on the rape seed quality. The results above suggested that the combinant yeast Heml gene had been successfully integrated into oilrape genome, which could be expressed under light condition and induce over ALA production and chlorophyll accumulation. The electron acceptance capacity in the accepting side of PSII reaction center was improved, even in the older leaves. Therefore, the photisynthetic accumulation capacity and yield were both increased because of YHeml gene transformation.10. The recombinant gene YHeml encoding aminolevulinate synthase (ALAS) in yeast was introduced into the genome of Arabidopsis controlled by the Arabidopsis thaliana HemAl gene promoter by the floral dip method. All transgenic lines were able to transcribe the YHeml gene, especially under light condition. ALAS activity could be detected in the transgenic plants, which was absent in the wild type. Measurements with a PEA showed that the fluorescence parameters positively related with photosynthetic energy conversion were higher in the transgenic plants while a lower level of those with non-photochemical efficiency was found. Furthermore, the biomass in transgenic plants was much higher than the wild type. This suggested that ALA over-production was beneficial to photosynthetic accumulation. The transgenic Arabidopsis thaliana can be used to elucidate the the regulatory mechanism of ALA on plant growth and development.11. Salt tolerance of the YHeml transgenic Arabidopsis thaliana was studied during seed germination and seedling growth. The results showed that transformation of Yheml did not affect the endogenous ALA content; however, it increased the ability of ALA biosynthesis and catabolism. Under salt stress, seed germination and seedling growth were seriously inhibited in the wild type, while the transgenic plants maintained higher vitality. Both YHeml gene transformation and salt stress stimulated antioxidative enzyme activity, including SOD, POD, CAT and APX at least at gene transcription level, which was beneficial to decrease active oxygen production and malonaldehyde (MDA) accumulation. These data suggested that the recombinant YHeml transformation could improve plant salt tolerance, which was related with the increase of antioxdative ability in transgenic plants.12. Agilent Arabidopsis Oligo Microarray (V4) (4*44K) was used to study gene expression profile difference between transgenic YHeml plant and wild type, and 43604 gene expressions prolife was obtained. After analysis by Agilent GeneSpring GX software in SBC bioinformatic online ebioservice, NCBI, Refseq, TIAR, TIGR and Unigene online database, the differential expression genes were selected and annotated. Then Gene Ontology and Pathway of the genes were analyzed, and 16321 genes were observed in differential expression, where 9048 genes were up-regulated and 7273 were down. The foldchanges of 3651 genes were significant. These genes covered the signal transduction, metabolisms, transcriptional factors, functional proteins, and others. The suggestion that ALA was related with promotion of plant photosynthesis, respiration, stress tolerance and delaying senescence could be found the molecular traits in the genechips.