The Study Of Enhancement Of Drought Tolerance In Cotton By Transgene Of ZmPIS And Transgene Pyramiding Of BetA/TsVP

In the face of a global scarcity of water resources, drought has already become a primary factor in limiting crop production worldwide. With the increase of world population and the demand for biomass resources, increasing food and cash crops production has become a top priority and it is of important strategic significance to breed drought tolerant crop varieties by biotechnology. Cotton, classified as a drought tolerant crop, is suitable for moderate drought conditions. However, its sensitivity to drought varies greatly among genotypes, and the cotton yield can't be satisfying under severe drought conditions. Therefore, it is of great importance for enhancing cotton yield and utilizing the arid and semi-arid soils to further improve the drought tolerance of cotton.Studies on drought tolerance of transgenic ZmPIS genePlants have evolved many complicated mechanisms in response to drought through a variety of signaling pathways. Previous studies have indicated that phosphoinositide-signaling pathway plays key roles in plant growth, development and response to environmental stresses. Phosphatidylinositol (PtdIns) is synthesized from CDP-DG and myo-inositol, catalyzed by phosphatidylinositol synthase (PIS), and is one of the most important phospholipids and a major precursor of molecules involved in phosphoinositide-signaling pathways.In this study, tolerance to drought stress of T3 generations of transgenic ZmPIS lines from Lumianyanl9 was investigated at three developmental stages, including seedlings, squaring stage and anthesis stage. Cotton seedlings at five-leaf stage were subject to osmotic stress in Hoagland solution supplemented with 12%(w/v) PEG-6000 for 24 h. After treatment, wild-type (WT) plants wilted seriously because of large lose of water, while most of the transgenic plants grew well with comparatively slight symptoms of water stress. After osmotic stress, net photosynthesis and stomatal conductance of the four transgenic lines were significantly higher than that of WT. The solute potential of cotton seedlings decreased greatly after osmotic stress, with transgenic lines decreasing more than that of WT. The lower solute potential, which implied that more solutes were accumulated in transgenic plants than in WT plants, could help plant cells absorb more water and maintain cell turgor under osmotic stress.Cotton plants cultured in flowerpots were exposed to drought stress by withholding water at the squaring stage for 8 days. At the fourth day of drought stress, the plants of WT started to wilt and the leaf RWC decreased dramatically. After 8 days of drought stress, the cell membrane ion leakage and MDA level in transgenic lines were significantly lower than that of WT, which suggested less membrane damage and lipid peroxidation occurred in these transgenic lines. In addition, the chlorophyll content and photosynthesis were significantly higher in transgenic cotton plants than in wild type. Results of measurement of solute sugars, proline content and solute potential indicated that, solute sugars and proline content of all cotton lines had no significant difference; transgenic plants exhibited a larger decrease inΨs, and therefore had stronger osmotic adjustment ability.Experiment results from cotton plants subject to a long-term water stress at anthesis for 4 weeks indicated that the number of lateral branches, flowers and bolls per plant were affected by water stress to different extents. No significant difference was observed in number of lateral branches, flowers and bolls per plant after drought stress between wide type and transgenic plants. But, water stress at anthesis led to a 16.51 and 23.53% reduction in seedcotton yield and lint yield per plant respectively in WT. However, this reduction ranged 10.33～11.59% and 15.89%～19.69% in the three transgenic lines. It is important to note that the seedcotton yield and lint yield in transgenic line 3 was significantly higher than that in WT after 4 weeks drought stress at anthesis (34.07% and 30.77%, respectively). In transgenic lines S1 and S2, the seedcotton yield and lint yield were, respectively,4.95～10.44% and 14.10%～15.38% higher than that in WT.Results of drought stress experiments above indicated that over-expression of ZmPIS in cotton significantly improved the drought tolerance of transgenic plants at three important developmental stages. What is important is that, in our study, the seedcotton yield of transgenic line 3 was significantly higher than that of WT after a long-term drought stress at anthesis, which was of great value in cotton breeding and production. In addition, this work provides valuable reference materials for a better understanding of the relationship between plant adaptation to drought stress and phosphatidylinositol signal pathway.Studies on drought tolerance of betA×TsVP (BT) pyramiding cottonGenetic engineering has become one of the most effective strategies to improving the drought tolerance of crops. Until now, enhancement of stress tolerance of plants has been achieved by genetic transformation of stress tolerance-related genes. In view that the tolerance to stress of plants is a complex trait and is modulated by multiple genes, requires the coordination of many genes, the transformation of single gene into a plant can only improve stress tolerance partially. Therefore, introducing more than one stress tolerance-related genes into a single plant by gene pyramiding is considered as an efficient approach for engineering. The betA gene from Escherichia coli, which encodes chroline dehydrogenase (CDH), could increase the betaine content in transgenic plants; the TsVP gene from salt cress (Thellungiella halophila), which encodes vacuolar H+-translocating inorganic pyrophosphatase, would enhance the ion transport across the vacuolar membrane. It has been reported that overexpressing each gene alone can improve the drought tolerance of plants.In this study, the betA gene and TsVP gene transformed cottons were named BL and TL, respectively. The transgene pyramiding cotton plants, obtained by sexual crossing, bearing two genes that undergo different mechanisms of drought tolerance (betA×TsVP, named BT). Results from PCR, the determination of betaine content and V-H+-PPase hydrolytic activity indicated that the two genes were integrated into the genome and expressed functionally in transgene pyramiding cotton plants. The analysis of drought tolerance at the physiological level and biochemical level were performed. The transgene pyramiding plants showed higher drought tolerance than single gene transgenic plants. Cotton seedlings at five-leaf stage were subject to osmotic stress in Hoagland solution supplemented with 12%(w/v) PEG-6000 for 24 h. After treatment, wild-type (WT) plants wilted seriously because of sever lose of water, while most of the transgenic plants grew well with comparatively slight symptoms of water stress. After osmotic stress,RWC decreased in all cotton lines. Transgenic lines lose less water comparing WT, with transgene pyramiding line BT1(73.00%) significantly higher than that of single gene transgenic lines (68.61～69.31%). The decrease in solute potential of plant cell would help absorb more water and to maintain cell turgor under osmotic stress. Comparing the values in solute potential of transgene pyramiding and single gene transgenic lines under osmotic stress, transgenic pyramiding lines BT1 accumulated more solutes under osmotis stress.Cotton plants cultured in flowerpots were exposed to drought stress by withholding water at the squaring stage for 8 days. During the drought treatment, RWC of all lines decreased gradually, with transgenic lines decreased less than WT. On the 8th day of drought stress, RWC of transgene pyramiding line BT1 was higher than single gene transgenic lines without significant difference. Photosynthetic parameters of all lines decreased after drought stress treatment, with net photosynthesis rate and Fv/Fm higher in transgenic lines than in WT. Drought stress resulted in membrane injury and caused MDA content and electrolyte leakage rising in both transgenic and WT plants. The wild-type cotton lines exhibited higher MDA content and ion leakage than the transgenic lines under drought conditions, suggesting the higher drought tolerance in transgenic cotton lines. The transgene pyramiding plants showed the highest drought tolerance with the lowest MDA content and ion leakage. In addition, transgene pyramiding cotton lines maintained higher SOD activity, which would help clean of radical oxygen and protect cells from drought stress.Experiment results from cotton plants subject to a long-term water stress at anthesis for 4 weeks indicated that the plant height, number of lateral branches and drought weight per plant were higher in transgenic lines than in wide type before or after drought stress. After drought stress, transgene pyramiding plants exhibited higher values in plant height, number of lateral branches and drought weight, but no significant difference was observed.In transgene pyramiding plants, although the two exogenous genes contributed to improved drought tolerance of cotton through different tolerance mechanisms, the improved drought tolerance of transgene pyramiding plants was much less than the additive effects of the two genes alone. It is presumably because plant drought tolerance is a polygenic trait which requires the cooperation of many genes, and some key elements or polygenic interactions restricted the sensitivity of plants to drought stress. Therefore, it is necessary to deeply understand the stress-related polygenic interactions and its regulatory mechanisms, which will help in making strategies for stress-tolerance genetic engineering in plants.