Study On The Salt-tolerant Physiological Characteristics And Proline Metabolism-related Genes Of Kosteletzkya Virginica

Soil salinization has become one of the resource and ecological problems all over the world, and it is one of the important abiotic stress factors that affect crop production. In order to fully develop the saline soil resources and saline soil agriculture, the selection and breeding of salt-tolerant plants with economic values has been attracting more and more attention. Salt-tolerant plants have evolved into complicated salt-tolerant mechanisms which enable them to thrive in high-salt environments and they can be either domesticated into new salt-tolerant crops or used as a potent genetic source for improving the salt tolerance of conventional crops through genetic engineering methodology. So it is very important to study the salt-tolerant physiological mechanisms and determine the key genes of salt tolerance for breeding the new salt-tolerant species by use of genetic engineering, which is of extremely important theoretical and practical significance for the sustainable development of saline soil amelioration and saline agriculture. This work firstly studied the physiological response of Kosteletzkya virginica to salt stress and proline accumulation was found in the leaf of seedlings under salt stress. To explore the cause behind proline accumulation, we first cloned proline metabolism-related genes and investigated their expression and function. The main results are as follows:(1) After treatment by different concentrations of Na Cl for 20 days, the growth parameters of Kosteletzkya virginica seedlings were almostly unaffected under 100 and 200 mmol/L Na Cl stress. Although Na+ content in the leaves and roots was significantly increased, more Na+ was compartmented into roots and the leaves had higher K+/Na+ ratio and higher proline content than roots, indicating that both inorganic ions and organic solutes of seedlings were involved in the osmotic adjustment to counter salt stress. Compared with the control, no significant difference was found in chlorophyll fluorescence parameters, MDA content and cell membrane permeability. Maybe the increased activitiy of antioxidant enzymes protected the seedlings from oxidative damage caused by reactive oxygen species. Under 300 and400 mmol/L Na Cl stress, the growth of seedlings was severely inhibited. The physiological indexes showed that the treated seedlings were subjected to severe osmotic stress and ion toxicity which damaged their photosynthetic apparatus and cell membrane system. In summary, Kosteletzkya virginica seedlings had good salt tolerance due to the combination of osmotic adjustment, ion balance and antioxidant capacities, which enable them to grow well under less than 200 mmol/L Na Cl stress.(2) To explore the causes behind proline accumulation induced by salt stress, we cloned four proline metabolism-related genes including Kv P5 CS, Kv OAT, Kv PDH,and Kv Pro T from Kosteletzkya virginica by means of RT-PCR and RACE methods.Bioinformatics analysis showed that these genes and their encoded amino acid sequences shared high homology with other known homologous sequences from other plants. Their expression profiles under salt stress revealed that proline accumulation in leaves under salt stress was due to the up-regulated expression of Kv P5 CS and Kv OAT which promoted the biosynthesis of proline. In this process, the up-regulated expression of Kv P5 CS played a more important role than Kv OAT. Similar to the Kv P5 CS expression, the Kv Pro T expression was also significantly up-regulated by salt stress. It was speculated that Kv Pro T might be involved in the proline transport from other parts to the leaves, but this needs to be confirmed.(3) The expression vector p BI121-Kv P5 CS was constructed and transformed into tobacco by Agrobacterium-mediated method to obtain transgenic tobacco. The T1 generation transgenic seedlings and wild tobacco were treated by 200 mmol/L Na Cl stress for 14 days. The results showed that transgenic seedlings had better salt tolerance than wild type. The chlorophyll content, proline content and antioxidant enzyme activity of transgenic seedlings were significantly higher than that of wild type, indicating that increased proline content induced by over-expression Kv P5 CS might play an important role in their responses to salt stress.