| Cotton is important cash crops in China, moderate salt-tolerant species and also a pioneer crop to development and utilization of saline-alkali soil. Many salt tolerant genes have been transformed into cotton and improved salt tolerance of such transgenic cotton in different degree. But most salt tolerance related gene engineering was focused on single-gene transformation, the researches of improved plant tolerance to abiotic stress by multi-genes co-transformation were less. Furthermore, those few researches were pyramiding the same mechanism and metabolic pathway genes. The plant tolerance to abiotic stress was complex genetic traits which was controlled by many genes, and depend on the coordination between multiple genes. So the aggregate number of salt-related genes to the same plant may further enhance the salt stress of transgenic plants.Enhancement of salt tolerance in cotton by transgene pyramiding of betA/TsVPThe betA gene was from E. coli, encoding choline dehydrogenase (CDH) which catalyzes oxidizing reaction from choline to betaine, the transformation of this gene into plant increased internal levels of glycine betaine. The TsVP gene from Thellungiella salsuginea, encoding V-H+-PPase protein, can drive protons (hydrogen ions) to move across a membrane against their concentration gradient, thereby improve the ability of proton transport. Many researches have confirmed that transformation of each gene can effectively improve the tolerance to abiotic stress.In this study, the betA gene and TsVP gene transformed cotton lines were named BL and TL, respectively. The gene pyramiding cotton plants were obtained by sexual crossing of these two single transgenic lines (betA×TsVP, named BT); which possessed two genes that effectively improve salt tolerance via different mechanisms. The PCR and RT-PCR results, determination of betaine content and V-H+-PPase activity indicated that the two genes stably integrated into the cotton genome and expressed functionally in gene pyramiding plants. Then we analyzed the salt tolerance of gene pyramiding lines and single gene transgenic plants. Physiological analysis results indicated that the gene pyramiding plants showed higher salt-tolerant character than single gene transgenic plants in some indexes.Salt stress can cause water loss of cotton leaf, then plant appear wilting symptoms. After salt treatment for 14 days, RWC decreased in all cotton lines. The wild-type (WT) plants wilted seriously while most of the transgenic plants grew well with slight wilting symptoms. RWC in the WT plants was 71.12%, while that of transgenic lines was 76.20～78.25%, with gene pyramiding line BT1 was highest. Salt stress also caused the decrease in solute potential of plant cell. Comparing with the solute potential of WT plants under salt stress, transgenic lines BL and BT1 accumulated significantly more solutes under salt stress. This would help to absorb more water and to maintain cell turgor under osmotic stress.After salt treatment for 14 days, the chlorophyll content in transgenic lines was 1.66～1.75 mg g-1 FW, which was significantly higher than that in WT plants (1.29 mg g-1 FW). Moreover, the content in gene pyramiding lines was significantly higher than that in BL. With the duration of salt stress, the Pn and Gs were both decreased in all plants. After salt treatment for 14 days, the gene pyramiding plants maintained higher photosynthesis rate. The Gs in gene pyramiding plants was also significantly higher than that of single transgenic and WT plants. These results indicated that the photosynthesis was inhibited in all plants under salt stress. Comparing with WT plants, each transgenic lines was less inhibited, with gene pyramiding plants (BT1) maintained significantly better photosynthesis than the single transgenic lines. So pyramiding of these two genes can further protected photosystem and mitigating the photoinhibition induced by salt stress. Fv/Fm, the maximum photochemical potential, can reflect the destroy degree of reaction center in PSII. It was also decreased under salt stress while transgenic lines all maintained significantly higher values, with BT1 was highest.Salt stress can also cause membrane system damage, which exhibited in the form of the increase lelvel of both MDA content and ion leakage. After 7,14 and 21 days of salt treatment, the two indexes in transgenic lines were both lower than the WT plants; this difference was significantly at 14 days, with gene pyramiding lines BT1 maintained better membrane stability. So under same salt stress conditions, BT1 suffered less membrane damage.Plant generates much reactive oxygen and free radical under both normal growth and abiotic stress conditions. Meanwhile plants also evolve a serious of anti-oxidative systems to protect themselves from oxidative stress, including maintained higher antioxidant enzymes activities. SOD activity was first increased in all plants with the duration of salt stress, then decreased after 7 days. During the salt stress, transgenic lines always maintained higher activity than that in WT plants, with that in BT1 (2905.75 unit g-1 FW) was also significantly higher than BL (2610.05 unit g-1 FW) and TL (2675.36 unit g-1 FW) at 7th day. The transgenic lines were also maintained significantly higher SOD activity after 14 days of salt treatment except BL, but there was no significant difference between them. The transgenic lines maintained higher SOD activity, and possessed stronger oxygen radical scavenging capacity, so enhanced the oxidative stress tolerance under salt stress. The activities of POD and CAT also had the same variation tendency. Transgenic lines possessed significantly higher level than WT, with gene pyramiding plants also maintained significantly higher activities than single gene transgenic plants at 7th days of treatment.Salt stress cause damage to plant via osmotic stress and ion toxic action, ultimately inhibited the plant growth. After 21 days of salt stress, the biomass result indicated that the cotton growth were really inhibited by salt stress, with transgenic lines kept higher level and BT1 also had significantly higher dry weight than single transgenic lines.All the above physiological results indicated that gene pyramiding plants maintained lower osmotic potential and better leaf water retaining capacity; it also possessed higher antioxidant enzymes activities to better scavenging free radicals, ultimately protected photosystem and membrane system. So gene pyramiding can further increased plant tolerance to abiotic stress, but this degree was not simple additive effect of the pymading genes. Therefore, it is necessary to construct careful experiment design and further understanding the mechanism of plant salt damage. Then dramatically improve crop tolerance to stress through the nichetargeting plant gene engineering.Production of gene polymerization cotton by co-transformationThe genes in the co-transformation plasmid were transferred into the shoot apical meristem of cotton cultivar LM19 by agrobacterium-mediated transformation. The T-DNA district including the bet A gene (encoding CDH in Escherichia Coli), ZmPIS gene (encoding phosphatidylinositol synthase in maize), and the TsVP gene (encoding V-H+-PPase in Thellungiella salsuginea). The selected marker was EPSP gene, which endow plant the glyphosate resistance. After herbicides screening, the live plants was further for the PCR verification to obtain transgenic positive plants. The results showed that some transgenic plants had strong positive signal; it suggested that foreign genes have been integrated into cotton genome, and expressed correctly.Southern blotting results with positive plants of PCR showed that PIS have been integrated into the cotton genome. Further southern blotting experiments should be carry out to verified transgenic plants. The next work is obtaining good Southern hybridization results and stable transgenic lines, analyzing the abiotic stress tolerance of transgenic lines, and comparing it to WT plants and single transgenic plants. To evaluate whether their resistance to stress conditions is further strengthened. |
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