Establishment Of Agrobacterium-mediated Transformation System Of Limonium Sinense And The Functional Research Of NKCC Gene Silence

Plants are inevitable to suffer from environmental stresses during their whole lives. Stresses might impede the normal growth and development of plants and lead to the decrease of products' quantity, quality and economic values. Researches about the mechanism plants respond to stresses are of great importance for people to avoid the adverse effect of stresses on plants and improve the agricultural production. High salinity in soil is a common stress factor and can hinder plant's growth and development seriously, which eventually results in the decrease of product's quality and quantity. Salt stress is closely correlated to the ion transport in plants. Therefore, it is quite meaningful to study the ion transport in order to make clear the mechanisms of salt tolerance and corresponding regulation. Researches in animals reveal that NKCC is a key kind of ion transportor that is tightly related to the salt secretion function of salt glands, companied with Cl~---Channel and other members in the same gene family such as KCC, NCC, NKCC plays a significant role in salt secreting of animals salt glands. In plants, salt secretion is a vital strategy of halophytes to resist salt stress, but studies concerning the salt secretion of plant on the molecular level are far from enough now. As a consequence, in order to elucidate the mechanisms of salt secretion in plants and demonstrate the function of genes that is similar to animals, it is practically worthwhile to analyze the function of NKCC in plants, especially in halophytes.The Limonium sinense Kuntze is a perennial plants secreting salt of family Plumbaginaceae and genus Limonium. It has the peculiar secreting salt structure- salt gland , may will absorb salt ions from in vitro to in vivo, avoiding salinity hurt thereby. The Limonium sinense Kuntze can use solonchak desalination , improve soil structure, so the "pioneer plant for transformation of saline and alkaline land"is praised. The Limonium sinense Kuntze is Fresh cut flowers cultivated species ,and also is the ideal material to doing dry flower. It is popular named "stars flower ", and is commercially cultivatde in a lot of area of world. The Limonium sinense Kuntze has large amount of sterile branch , at the same time, it has the features of the same type of hybrid infertility, and of Less strong seed, therefore, professional production is limited. We can consider using the transgenic technology to improve the variety of Limonium sinense Kuntze. Study by the fact that the process and mechanism of the gland secreting salt , what form of salt gland structure and their key factor secreting salt process and rules already have the first step knows, but the reason why Limonium sinense Kuntze can adapt to high-salt environment is unknown. To solve the problem, the gene knocksout or the RNAi technology is a forceful implement undoubtedly. At the first we must require building the high-effect inheritance system of the Limonium sinense Kuntze.This study is on the basis of regenerating system of Limonium sinense Kuntze already built. In this experimental study, we study the relevant factors of agrobacteriutum tumefaciens-mediated genetic transformation. We find that:1) Limonium sinense Kuntze leaf is sensitive to kanamycin. 50 mg/L kanamycin is the most proper concentration to the Limonium sinense Kuntze leaf.2) Bacteriostat effect is different between Ceftriaxone Sodium (Rocephin) and Timentin. 400 mg/L spit of Timentin have the bacteriostat effect, and the effect can last three or four weeks. That not only can decrease by amount of work, but also improve conversion rate.3) When inoculated with EHA105 bacterial strain, the Limonium sinense Kuntze conversion rate is overtoped to inoculating with LBA4404 and GV3101 bacterial strain, it can amounts to 15%.4) The OD600 of bacterium liquid being 0.7-1.0 is suitable, conversion rate may amount to 15%.5)Three or four days of co-cultivation have no obvious effect, but four days is better, conversion rate may amount to 15%.In this study, silent carrier of the NKCC gene have been successfully built, the thransgenic plants have been obtained, and have done the molecule identifies . The molecular identification of these thransgenic plants are as below:1)PCR result showed that all kanamycin -resistant plants had strong positive signals, and no signal was shown in wild type plants.2) The Southern hybrid result further confirmed that exogenous gene have already conformity to thegenome of the Limonium sinense Kuntze.3) Real time PCR results showed that the NKCC RNA decreased in different transformed plants to different degree.