Physiological Responses Of Leymus Secalinus Seedling Under Saline-alkali Stress And The Clone And Function Analysis Of LsCDPK Gene

Saline-alkali stresses is the main environmental problem throughout the world. With development of industry, growth of population and decreasing of arable land, selecting and breading plant material for salt resistance and rationally using saline-alkali land are very significant. Leymus secalinus is more tolerant to drought, leanness as well as saline-alkali, so it is first choice to improve saline-alkali land. The paper studied on protective enzyme activity, photosynthetic characteristics, ions content and ultrastructure of mesophyll cells of Leymus secalinus seedling by conducted under different concentration of NaCl(0,100,200,300,400,500,600,700), Na2CO3 and NaHCO3 (0,50,75,100,150,200,300,400)stress in the greenhouse experiment. Primarily damage and salt tolerance mechanisms of Leymus secalinus seedling was discussed and analyzed under the saline-alkali stress. And Leymus secalinus LsCDPK (calcium-dependent protein kinase) was cloned,and further analyzed bioinformatics. The research laid a foundation to further study the LsCDPK function and the signal transduction pathway of saline-alkal resistance mechanism in Leymus secalinus. The main study results were as follows:1 Influences of salt-alkali stress on physiological and biochemical substances of Leymus secalinus seedlings(1) There was a significant negative correlation between the concentration of salt-alkali and water content of leafs, growth rate after stress.The growth rate after stress obvious decrease, descending speed of Na2CO3 stress(0.59 cm/d) was much faster than that NaHCO3 and NaCl stress. R/T was positive correlated with concentration of salt-alkali. R/T increasing speed of Na2CO3 stress was the fastest than NaHCO3 and NaCl stress, there was a significant difference(P<0.05).(2) With increase of salt-alkali concentration, the activity of SOD and POD first increase and then decrease. The activity of SOD and POD were the maximum at 400 mM,700 mM NaCl; 150 mM NaHCO3; 100 mM and150 mM Na2CO3. But the content of MDA in leaf and root was not decreased on the contrary it was inereased. The results indicate that Leymus secalinus had stronger ability of toleranting NaCl than stress.(3) The results show that proline content, betain content, soluble sugar decreased slightly after a increase and tends to the peak. The proline content was the highest at 600 mM NaCl,300 mM NaHCO3 and 200 mM Na2CO3. But betain content and soluble sugar were the maximum at 500 mM NaCl,200 mM NaHCO3 and Na2CO3. Three osmotic matters reaches the maximum under different concentration, which is possible a reasone for Leymus secalinus to tolerant salt-alkali.(4) Photosynthetic capacity has the relationship with the chlorophyll content. The chlorophyll a, chlorophyll b and stomatal conductance of Leymus secalinus seedling decreased significant under high concentration.Water use efficiency of leaf first increased and then decreased with increase of salt-alkali concentration. The net photosynthetic rate and water use efficiency of leaf were drcreased with time of stress.but transpiration rate and stomatal conductance first increased and then decreased. It was suggested that non stomatal factors were the major causes of the decline in photosynthesis.With the increase of the concentration of NaCl, NaHCO3 and Na2CO3, Fo increased first and decreased afterwards and Fv/Fm, Fv/Fo decreased. Fv/Fm value decreased obviously, falled 38%,19% and 38% respectively due to fluorescence quenching. NPQ is one of the important indicators of photoprotective. NPQ increased with the increase of the PAR, NPQ have not reached a stable state within the scope of PAR. Under low concentration the seedling was not hurt due to the heat dissipation, with the increase of the concentration, the damage degree increased.(5) Na+ of acrial part was higher than roots but the changes of Cl- was opposite to Na+ under NaCl,Na2CO3 and NaHCO3 stress. Na+ restriction capability of roots increased with increasing of NaCl, Na2CO3 and NaHCO3 stress concentration. Seedlings of Leymus secalinus could normal growth under 300 mM of NaCl concentration, but it was inhibited at NaHCO3 concentration more than 200mM. Under 150 mM of Na2CO3 concentration, Seedling of Leymus secalinus could accumulated K+、Ca2+, in order to inhibit increased of Na+ and Cl-. Na/K ratio increased with increasing of NaCl, Na2CO3, and NaHCO3 stress concentration, but increasing amount of roots were higher than shoots. The results also reflected that roots inhibit Na+ transporting to acrial part was salt-tolerant mechanism of Leymus secalinus.Leymus secalinus has different ability to tolerant NaCl,NaHCO3 and Na2CO3 stress. The analysis from protective enzyme activity, photosynthetic characteristics, ions content results showed that Leymus secalinus was seriously affected by Na2CO3 than NaHCO3 and NaCl.2 Effects of salt-alkali stress on the ultrastructure of mesophyll cells in Leymus secalinusNo alteration of ultrastructure was found by mild salt-alkali stress (NaCl 100 mM,NaHCO3 50mM and Na2CO3 50mM). While under moderate salt-alkali stress (NaCl 400mM,NaHCO3 150mM and Na2CO3 150mM), the most conspicuous change was the enlargement of the thylakoid membranes gap, disorganization of grana lamella, a marked increase in the number of plastoglobulus, and a few starch grains in chloroplasts. The break dowm of cristae was observed in some mitochondria. Under severe salt-alkali stress (NaCl 700mM,400mM NaHCO3 and Na2CO3), the chloroplast membrane and the lamellar structure were almost disorganized with a mass of plastoglobulus, few starch grains in chloroplasts,mitochondria became disintegrated. But mitochondria did not disintegrated and had cavity under 400mM NaHCO3.The whole mesophyll cells was damaged under 400 mM NaCl and 150 mM NaHCO3,Na2CO3. But Leymus secalinus was seriously affected by Na2CO3 than NaHCO3 under the same concentration.3 Gene cloning and expression of LsCDPKLsCDPK was isolater by RT-PCR and RACE methods from Leymus secalinus.The gene has 2480bp in length which encode 520 amino acids, named LsCDPK. The amino acids sequences of LsCDPK showed significant sequence similarity with Brachypodium distachyon, LsCDPK protein was located in the cell nucleus.Analyze specificity expression of LsCDPK gene in different organization and various abiotic stresses by Real-time PCR. Result show that LsCDPK gene was detected in all Leymus secalinus organs tested, but their expression patterns of them-were significantly different. It was expressed strongly in roots. Expression of LsCDPK gene not only be induced by NaCl, Na2CO3, but also induced by PEG and low temperature. But the strong relative expression were expressed under different treatment time. So primary inference that LsCDPK gene may be involved in the defense responses against saline-alkali,low temperature and drought stresses.