Construction Of A Salt Gland Specific CDNA Library From Limonium

Excessive soluble salt in soil has different bad effects on plants. About twenty percent of the soil is saline soil all over the world, and nearly a half of the irrigated land suffers secondary salinization. The damage of salinization to arable land impacts and constrains social economic development. Therefore it is particularly important to study the mechanisms of salt tolerance. According to the different physiological mechanisms, morphological structures and ecological characteristics, Breckle ( 1990 ) divided the halophytes into three types: euhalophyte, pseudohalophyte and recretohalophyte.There are many kinds of recretohalophyte, and they also have a wide range of distribution in our country. Seashore, salt marsh, around the salt lake, desert, meadow and bosk etc are their places of residence. Salt secretion of the plant is an active physiological process, and recretohalophytes can excrete the excessive salt outside by salt gland or salt bladder. Limonium sinense Kuntze, a kind of perennial plants secreting salt, belongs to family Plumbaginaceae and genus Limonium. It has the peculiar secreting salt structure-salt gland , which can secrete salt ions to avoid injury thereby.Based on Laser Microdissection(LMD), we got cells of salt gland from the lower epidermis of Limonium sinense Kuntze,and then isolated the total RNA from salt gland cells. At last, we gained amplified aRNA by reverse transcription in vitro and will use it to construct a salt gland specific cDNA library from Limonium. In the experiment, we knew that:(1)The material fixed by Carnoy's fluid can be decolorized well, and RNA can be stored perfectly. Even you can get high-quantity RNA from the material fixed for three months(.2)The leaves of Limonium sinense Kuntze became fleshy and brittle, so it was nearly impossible to get the lower epidermis. So you can only get the material at special time(.3)The lysis buffer containing guanidine isothiocyanate was used to treat the cells of salt gland, but we can only get little RNA and even can not find RNA bands by electrophoresis. After this, we used improved CTAB to get high-quality RNA. (4)During the experiment, we found that the amount of RNA isolated from the material treated with 300mM NaCl is more than that from normal material. Although we could not make sure if salt treatment is the real reason which resulted in this phenomenon, but it seems to be better to treat Limonium with 300mM NaCl. Summarily, in this experiment, only primary research was made on construction of a salt gland specific cDNA library from Limonium because of the limited time and material. Secondly, we studied the development of the salt gland in Limonium sinense Kuntze by Scanning Electron Microscope. We found that:(1)Between the salt treated and non-salt treated Limonium sinense Kuntze, the number of salt gland is different while the development time is nearly the same.(2)On the same young leaf, there are developed and developing salt glands. It means that salt glands which belong to different developmental stages can be on the same leaf.(3)The development time of the salt gland in Limonium sinense Kuntze is much earlier than that of stoma.In addition, we also studied the influence of ouabain combining the new fluorescent Na~+ indicator CoroNa Green. Ouabain is an inhibitor of Na~+/K~+-ATPase in animals. We found that Limonium sinense Kuntze planted in 200mM NaCl and 100μM ouabain showed drastically higher sodium-specific fluorescence in the root tip, which told us that the content of Na~+ significantly increased. So we concluded that Na~+ accumulated in vivo when the plant was treated by ouabain and presumed that there was some Na~+ transporter which was sensitive to Na~+ and it played an important role in the normal growth of Limonium sinense Kuntze under salt stress conditions.