Research On The Salt Stress And Regulation Effect About Onion Based On Patch Clamp Technique

Salinization of soil is one of the primary agricultural crises in the world.Na⁺,as the main caution in the saline soil,can almost disserve all the life processes of plants which greatly limits the growth and output of the plants.So how to effectively study the salt stress regulation effect and develop agricultural machinery becomes an urgent problem for modern agriculture development.The cusp of a microelectrode with the diameter of about one micron is combined with the cell membrane where a giga-ohm resistance is formed by using the patch clamp technique according to different Na⁺ concentration.And the influence that Ca²⁺outside the cell imposes to the Na⁺ current is studied and then a mathematics model of sodiumcalcium concentration is set using the inhibition as a bridge which gives us a reference for the precise application of calcium fertilizer for the macro sense.A parallel equipment for fertilization is designed by using this model and wenqiuli principle that provides a hardware for the proper concentration of calcium including its spray.The main contents are as follows:(1)Cell wall of onion epidermal is a major obstacle to achieve the giga- resistance seal between the tip of microelectrode and membrane.This paper used "one step enzymatic hydrolysis method",and by improving the existing combination of centrifugal speed and time to purify the enzyme mixture,we get the access to a greater yield,better protoplast activity,which makes the giga- resistance seal easier to achieve.(2)A standpoint that K⁺ and Ca²⁺selective channels in the Arabidopsis and rye take very few contributions for large numbers of Na⁺ fluxing into the cytoplasm.In this paper,onion skin cell protoplast is choosed as material to research the change of Na⁺ current by using the TEA⁺(K⁺ channel inhibitor)and verapamil(Ca²⁺channel inhibitor).A conclusion is reached that K⁺ and Ca²⁺selective channels are not main ways to make lots of Na⁺ flux in.That's to say,their contribution is very few.(3)An anti-sodium process about single cell is studied by using the patch clamp technique.Giga-resistance seal is formed between the microelectrode tip and the cell membrane,and then some Na⁺ current curves are record and measured by using the multiclamp software when there are different Na⁺ concentrations.The results show that the Na⁺ current formed by its fluxing into the cell is depend on the concentration of outside Na⁺:When the concentration of extracellular Na⁺ increases from 25 mmol/L to 50 mmol/L,the ion current also grows with little growth rate(14.1 percent).When the concentration of increases from 50 mmol/L to 75,the ion current is much greater and grows very fast(36.1 percent).And the ion current with the Na⁺ concentration 100mmol/L increases 43.2 percent comparing the 75 mmol/L situation.So,with the growth of Na⁺ concentration,the current increases and the growth in high Na⁺ environment is faster than that in the low Na⁺ environment.(4)Ion currents are record and measured with all kinds of Na⁺ and Ca²⁺ concentration combination at certain voltage.Also the inhibition rate that Ca²⁺impacts the Na⁺ currents is calculated,and as a bridge(inhibition rate is above 60 percent), Na-Ca Concentration model is established:a good method that guides us to find the proper Ca²⁺concentration which can effectively relieve the ion stress is given,including a curve relation between the Na⁺ concentration and relative[Ca²⁺]_max,[Ca²⁺]_minand [Ca²⁺]_optimal,also with their curves fitting equations.The establishment of Na-Ca concentration model strongly provides us many theoretical basis and data reference for the fact that imposing certain concentration calcium improver can effectively relieve the salt stress,alsot it is of great theoretical value for study the anti-stress mechanism of crops at the level of ion channels.(5)In this paper,by using the mathematics model of sodium- calcium concentration,Ca²⁺concentration is firstly calculated,and then a parallel equipment for fertilization is designed referring to other equipment.Two obliquities of wenqiuli tubes are achieved,when the concentration of fertilizer is 6.72percent with the volume of 50L,it can be steady absorbed by regulating the pressure of wenqiuli tube.The proper sodium concentration is between 52.5 and 88.2 mmol/L.When the concentration of Na⁺ is 25～52.5 mmol/L or 88.2～100 mmol/L proper spray can be accomplished by fixing the liquid fertilizer concentration to 4.50 percent and 8.50 percent.The calcium ameliorant and the water are together sprayed onto the leaf surface of the crops which directly enter the crops and inhibit the entrance of Na⁺.It provides a hardware security for the salt relief by spraying the fertilizer onto the surface of the leaf.