Salt Tolerance And Physiological Mechanisms Of Yheml Transgenic Oilseed Rape Plants

Oilseed rape(Brassica napus L.) is a important member of the Brassicaceae family. It is important for the edible oil and protein feed source, also be our country s significant industry raw materials. It played a vital leading position to the development of national economy. However, in recent years, Salinity is one of the major abiotic stress factors which affects the growth and productivity of oilseed rape. It is restricted to enlarge the oilseed rape cultivation area and reduce the yield, so how to improve the salt stress of oilseed rape is very important.5-Aminolevulinic acid (ALA) is the essential precursor of porphyrin compounds in all bodies, and it has been showed to be able to apply in agriculture production, which has been suggested being ability to improve plant the salinity resistant. However, most of the previous works in the field were based on exogenous ALA application, and no transgenic oilseed rape plants that could over-produce ALA under the salt stress had been reported up to now. This thesis takes trans-YHeml gene rape and the wild type rape as the research materials, studying the mechanism of salt tolerance in different aspects. The main results were as follows.1. YHeml is a yeast (Saccharomyces cerevisiae) Heml gene controlled by the light-sensitive promoter sequence of Arabidopsis thaliana HemAl gene. After it was transformed into oilseed plants, the transgenic plants were used to study the effect of5-aminolevulinic acid (ALA) metabolism under different concentrations of NaCl stress. The results demonstrated that ALA synthase (ALAS) activity and the exogenous YHeml gene expression could be detected in the transgenic plants, which was absent in the wild type. The ALA dehydrase (ALAD) activity and the expression of its coding gene in both of the transgenic and the wild type plants reduced as the increase of the NaCl concentrations, however, they kept much higher in the transgenic plant than that of the wild type plants. Therefore, the transgenic plants possessed higher ability of ALA biosynthesis and catabolism under either salt stress or the control condition. The chlorophyll a, chlorophyll b and SPAD in transgenic oilseed rape leaves, compared with the wild type, were obviously increased with higher Ch1b/a ratios. These results suggested that YHeml gene transformation into oilseed could over-produce ALA, which might be involved in regulation of chlorophyll biosynthesis in transgenic oilseed rape plants.2. In this part of work, the diurnal variations of photosynthesis and the diurnal dynamics of fast chlorophyll fluorescence of transgenic YHem1and wild type oilseed rape were compared by a portable Ciras-2photosynthetic system and a Plant Efficiency Analyzer (PEA) under450mmol·L-1NaCl treatment. The results showed that the net photosynthetic rates (Pn) in transgenic plants were still greatly higher than that of wild type either in normal or under salt stress. JIP-test analysis demonstrated that the performance index on an absorption basis (PIABS) and the PSII maximal photochemical efficiency(φPo) in transgenic plants were also higher than that of wild type. However, the amplitude of the K step (Wk), which reflects the inhibition level of oxygen evolving complex activity at the donor side of PSII reaction center, and the approximate initial slope of the fluorescence transient (Mo), which reflects the close level of PSII reaction center, were lower in transgenic than that in wild type, especially after salt stress. Additionally, the expression of the gene coding Rubisco small subunit of transgenic leaves was higher than that of wild type under NaCl stress. Furthermore, the soluble sugar content of the former was also higher than that of the later in different tissues. Therefore, the higher leaf photosynthetic capacity of transgenic was presumably related with greater conversion efficiency of light energy into biochemical energy during photosynthetic light reaction, and as well as the CO2fixation during photosynthetic dark reaction. It suggested that transgenic plants have stronger salt tolerance.3. In this part of work, the activities and the expressions of the gene of leaf anti-oxidative enzymes, including superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX) and catalase (CAT) were compared between YHeml transgenic oilseed rape and wid type at4days after treatments of NaCl solutions with different concentrations. The results indicated that these activities and the expressions at gene transcription level of antioxidant enzymes in both plants increases along with the augment of the concentrations of NaCl and the activities in transgenic plant were higher than that of the wild type all the time. On the other hand, the O2production rate and malondialdehyde (MDA) content were increased after salt treatment, which was lower in transgenic plants than that in the WT. The date suggested that the recombinant YHeml transgenic plants have the stronger salt tolerance, which was related with the increase of antioxdative ability. Additionally, the H2O2content was generally higher in transgenic plants after salt treatment, which might act as a signaling molecule involved in cellular regulation.4. In this part of the thesis, the YHeml transgenic and the WT oilseed rape plants were used to study the changes of the inorganic ions including Na+,K+and Cl-and organic solutes such as proline, protein and amino acids in different tissues under NaCl stress. The results indicated that either the dry weight or fresh weight, the transgenic plants were always much higher than the wild type at the same salinity, suggesting that YHeml transformation tended to improve salt tolerance of oilseed rape. When the plants were divided into different tissues, including leaf blade, leaf vein, stem, taproot and fibrous root, the measurements of Na+and K+content showed no close relationship with salt tolerance, however, the Cl-content increased as NaCl concentrations increased, and that in the transgenic plant were lower than the wild type, which might an index of salt tolerance in oilseed rape. Furthermore, there was no significant proline accumulation under salt stress in the most tissues of oilseed rape plants, which was different from most of other species of plants. As to soluable proteins, they increased firstly, and then dreased, which was higher in the transgenic than that of wild type. Additionaly, the free amino acid content decreased along with the augment of salinity, which was higher in the transgenic than that of the wild type, suggesting that amino acid content could act as an index to reflect the salt stress tolerance of oilseed rape.5. The YHeml transgenic oilseed rape was used to research the different tissues of mineral elements distribution. The results showed that N, P contents were the highest in stems, and the S content was the same in the leaves. Transgenic and wild type N, P and S trended vary in various tissues with the increase of the concentration of NaCl. N, P contents were the first elevated after reducing to wild type and were rising to transgenic in the leaf and leaf vein, excepting in450Mm as the leaves of wild type were withered to the losing of nutrients. The ability of storage and absorbing N, P in transgenic was higher than the wild rape. In leaf, leaf vein, stem:S content was first decreased then increased to the wild type, which was gradually decreasing to transgenic. At the same salt concentration levels the wild type was higher than transgenic in leaf, leaf vein, however both difference was not significant (P>0.05) instem. In different tissues of Fe content of transgenic in different variation tendency was higher than that of wild type. Both of Ca content gradually declined along with the augment of the concentration of NaCl. The difference is significant (P<0.05) in the fibrous roots in which transgenic was higher than that of the wild type; Mg ion distribution rule was not clear; Cu, Zn in different tissues of different genotypes had different change tendency. The salt stress on different genotypes of oilseed rape in different mineral elements in different tissues of the distribution law of the influence was different, where Fe, Ca content in different tissue content may be the salt tolerance index.