The Study Of Salt Tolerance In Cotton By Transgene Of ZmPLC1and Transgene Pyramiding Of ZmPLC1/betA

Salt stress is one of important abiotic stresses that seriously affects the yield and quality of crops in china and wordwide. Cotton is a kind of important economic crops, which occupies an important position in the national economy. Although cotton is the moderate salt-tolerant species, its sensitivity to salt stress varies greatly among cultivars, even if more resistant cultivars planted in moderately saline-alkali soil, its production will be greatly affected. Therefore, utilizing genetic engineering to improve cotton stress resistance and yield and broaden the planting area are of great importance.This work is about the preliminary study on salt tolerance of cotton by transfering ZmPLC1gene and transgene pyramiding of ZmPLC1/betA.Transgenic ZmPLC1cotton lines and wild type line (Lumianyan19) were grown under water culture and sand culture to investgate their physiological reponses and the expression of key enzyme genes in proline metabolism to salinity. The effect of tolerance to salinity in the transgenic cotton lines was also studied.After250mM NaCl treatment, transgenic ZmPLC1cotton lines compared with wild type line, which were cultured in Hoagland solution, exhibited higher green of leaf color, lower wilting and demage of photosystem II, higher net photosynthetic rate and relative water content, to maintain a lower osmotic potential.Cottons in flowerpots with sand were treated with250mM NaCl, after21days of salt stress, the RWC, net photosynthetic rate (Pn), stomatal conductance(Gs), intercellular CO2concentration, Fv/Fm, activity of antioxidant enzymes, solute sugars content, proline content and ion content were significantly higher in transgenic ZmPLC1cotton than that of wild type line, the cell membrane ion leakage and MDA level in transgenic lines were significantly lower than that of wild type line. The RT-PCR and Real-TimePCR results indicated that the expression of ZmPLCl gene in transgenic ZmPLC1cotton was significantly increased, the expression of P5cs gene encoded the key enzymes of proline synthesis was higher than wild type line, the expression of ProDH gene encoded the key enzymes of proline degradation was lower than wild type line. The yield of unginned cotton in transgenic ZmPLCl cotton in saline-alkali soil was much higher than wild type line. These results suggested that the salt tolerance of transgenic ZmPLCl cotton was much higher than wild type line. Therefore, we predicted that heterologous expression of ZmPLCl gene not only enhanced the expression of P5cs gene, decreased the expression of ProDH gene, but also participated in the antioxidant defense and ionic regulation, protected photosynthetic system and improved the photosynthesis and the salt tolorance of transgenic cotton.The transgenic ZmPLC1cotton lines (named C), transgenic betA cotton lines(named B) and the gene pyramiding cotton lines (ZmPLC1/betA, named BC) were grown under water culture and sand culture to investgate their physiological reponses, betaine content and the expression of key enzyme gene in proline metabolism after salt stress.The study will help us to understand the mechanism of stress resistance of ZmPLCl and betA genes.The PCR and RT-PCR results indicated that the ZmPLCl and betA genes were stably integrated into the cotton genome, even transcripted and expressed actively in gene pyramiding plants.After7days of250mM NaCl treatment, the yellowing degree of cotyledon, the wilting degree of euphylla and the rolling degree of first leaf in gene pyramiding cotton lines cultured in Hoagland solution were lesser than single gene transgenic lines and wild type line. The RWC, net photosynthetic rate, stomatal conductance, intercellular CO2concentration and Fv/Fm in gene pyramiding cotton lines were significantly higher than single gene transgenic lines and wild type line, osmotic potential were significantly lower than single gene transgenic lines and wild type line.Cottons in flowerpots with sand were treatment with250mM NaCl, after21days of salt stress, the plant height of gene pyramiding cotton lines compared with the single gene transgenic lines and wild type line was higher, the leaf size lagger, extent fo damage lower.The RWC, chlorophyll content, antioxidant enzymes activity, solute sugars content, proline content, betaine content, ion content (K+、Na+、Ca2+、Cl-), K+/Na+rate, net photosynthetic rate, stomatal conductance, intercellular CO2concentration, Fv/Fm in gene pyramiding cotton lines were significantly higher than single gene transgenic lines and wild type line, the cell membrane ion leakage, MDA level and osmotic potential in gene pyramiding lines were significantly lower than single gene transgenic lines and wild type line. The RT-PCR and Real-TimePCR results indicated that expression of ZmPLCl gene and betA gene increased and significantly higher than single gene transgenic lines and wild type line. The RT-PCR results indicated the expression of P5cs gene encoded the key enzymes of proline synthesis was higher than single gene transgenic lines an wild type line, the expression of ProDH gene encoded the key enzymes of proline degradation was lower than single gene transgenic lines an wild type line.The yield of unginned cotton in gene pyramiding cotton lines was significantly higher than the transgenic ZmPLC1cotton line, transgenic betA cotton line and wild type line.These results suggested that the salt tolerance of gene pyramiding cotton lines and single gene transgenic lines were greater than wild type line, specilally, the salt tolerance of gene pyramiding cotton lines were significantly high than single gene transgenic lines. Therefore, we predicted that pyramiding of ZmPLC1and betA gene could greatly increase the ion content to maitain the ionic balance; greatly improve the antioxidant enzymes activity, strenghting the eliminating of active oxygen and free radicals, slowing down the damage to cell membrane, then reduce the ion leakage and malondialdehyde content; gene pyramiding also could slow down the damage of PSII, enhancing the photosynthetic capacity; induce downstream gene expression, promoting the accumulation of proline,which was osmotic adjustment and protective substance, then enhance the salt tolerance of cotton.The work suggests that transfering ZmPLC1gene could improve the salt resistance of cotton to a certain extent, and the salt tolerance of gene pyramiding cotton is higher than single gene transgenic cotton, therefore, gene pyramiding means is feasible, which will provide excellent gernplasm resources for the cultivation fo new cotton carueties and new experimental materials for resesrch the mechanisms of multigenes interaction and regulatory.