Studies On Physiological And Biochemical Responses Of Halocnermum Strobilaceum To Salt Stress And Thecloning Of Its Betaine Aldehyde Dehydrogenase Gene

Soil salinity is the one of the main limitation factors affecting plants growth in the word, and it becomes more and more serious as pollution mounts up, which effect the growth and development of plants. Study on mechanism of salinity tolerance in plants is very important. There are different types of ions in soil. It is different for the effects of ions in soil on seed germination and seedling growth of plants. Most studies concerning effects of salts on seed germination and seedling growth were carried out by using NaCl, which may limit the accuracy of the conclusions, especially under natural conditions. In this work, we have studied the effect of NaCl or mixed salt from the soil on Halocnermum strobilaceum. Firstly, we analyzed the seed germination and early growth of H. strobilaceum, and measured seed accumulated germination rate, seedling length and seedling fresh weight under different temperature and concentrations of salts. Three night-day temperature regimes5～15℃,15～25℃25～35℃, and NaCl and mixed salt solutions at nine concentrations were applied and distilled water was used as the controled solution. Secondly, the effect of salt stress on physiological characteristics of H. strobilaceum were studied using four different salt concentrations,0%,0.9%,2.7%,5.4%of NaCl and mixed salt. The contents of Na+, K+, Glycine Betaine (GB), Proline (Pro), Malonaldehyde (MDA) and activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) were measured. Thirdly, we designed primers according to BADH completed reading frame of Suaeda liaotungensis (AF359282), Spinacia oleracea (AY156694), Atriplex centralasiatic (AY093682), Beta vulgaris(X58463). In order to clone HsBADH, we make objective cDNA to pMD18-T and got the BADH sequence.We blast analyzed HsBADH of cDNA using online database (http://www.ncbi.Nlm.Nih.Gov/) and analyzed the protein isoelectric point and the molecular weight with online tools SOPMA. HsBADH gene sequence of protein cluster was analyzed using DNAMAN.The results showed that the night-day temperature range of15～25℃was optimum for seed germination, seedling length and seedling fresh weight. In the three night-day temperatures conditions, seed germination, seedling length and seedling fresh weight were increased firstly and then decreased as the concentration of NaCl and mixed salt increased. Low-concentration of NaCl or mixed salt could promote germination of seeds and growth of seedings. In5～15℃, seed germination, seedling length and seedling fresh weight were much higher than those of control when concentration NaCl or mixed salt was below0.9%or1.5%(P<0.05). Under15～25℃, seed germination, seedling length and seedling fresh weight were much higher than those of control when concentration of NaCl or mixed salt was below0.6%or1.2%(P<0.05). At25～35℃, seed germination, seedling length and seedling fresh weight were much higher than those of control when concentration NaCl or mixed salt was below0.6%or0.9%(P<0.05). Seed germination rate, seedling length and seedling fresh weight were higher in mixed salt than in NaCl (P<0.05). At15～25℃, seed germination, seedling length and seedling fresh weight in mixed salt were23%,27%and28%higher than those in NaCl. This experiment showed that NaCl was more harmful than mixed salt solution to accumulated germination, seedling length and seedling fresh weight of H.Strobilaceum. The accumulated seed germination rates, height and fresh weight of seedlings under NaCl treatment were higher than under mixed salt treatment. As salt concentration increased, seed germination was delayed under5～15℃,15～25℃,25～35℃conditions. So concentration of NaCl and mixed salt was one of key factors to seed germination. All that may lead to different results of salt-tolerant behaviors of plants between natural environments and laboratories. Potassium ion, calcium ion and silicon ion in mixed salt solutions may improve seed germination and promoted early growth of H. strobilaceum seedlings.Under NaCl and mixed salt stress, after treatment of20d and60d, K+/Na+and the content of K+, MDA, Pro and antioxidant enzyme activities (SOD, POD, CAT) of H. Strobilaceum first rose and then declined with increased concentration, which were highest when salt concentration was2.7%and then decreased. The content of Na+, K+MDA, Pro all gradually increased, but contents of MDA and antioxidant enzyme activities gradually decreased as prolonged growth time. At the concentration of NaCl and mixed salt, the content of K+, Pro and antioxidant enzyme activities were higher in mixed salt than in NaCl, however, content of MDA was lower in mixed salt than in NaCl. Potassium ion, calcium ion and silicon ion (mixed-irons) from mixed salt may decrease the content of MDA, and enhanced K+, K+/Na+, Pro, GB and antioxidant enzyme activities. The effect potassium ion, calcium ion and silicon ionalleviates salt deficit of H. strobilaceum by preventing the oxidative membrance damge and may be associated with plant osmotic adjustment.In this work we isolated cDNAs of betaine aldehyde dehydrogenase from H. Strobilaceum. We named the betaine aldehyde dehydrogenase of H. strobilaceum was HsBADH. The HsBADH DNA contained a completed open reading frame of1848bp, coding for a polypepitde of549amino acids. GenBank landing number is EF4382. we would forecast that HsBADH molecular weight of protein was6.46KD, isoelectric point of9.26. It included Alanine3.75%, Glutamic3.03%, Glycine5.89%, Leucine7.14%and Valine4.82%. HsBADH protein peptide contains26.12%of the alpha helix structure,27.01%of the extension,36.14%of no rules of chain curly. The homology of H. Strobilaceum are90%of Halostachys Caspica, and53%of Rice Latin. So the equivalent of plant evolution degree is similar in the plants evolving process.The study could provide primary reference for mixed salt tolerance of H. strobilaceum. We are looking forword to getting new way and method to salt resistance of plant. The HsBADH gene would enrich the resources of salt resistance of plant. It is very important to improve salt tolerance in plants by making good use of gene resoureces of the species.