Silver Staining MRNA Differential Display Analysis Under Aluminum Stress Condition In Chinese Fir Different Clones

Aluminium is the most widely distributed metal elements in the earth. Aluminium presents in abundance as variety of insoluble silicate minerals or alumina in crust.Usually,it is not toxic to plants,but under acidic conditions(pH<5),the aluminum of solubility(major is Al³⁺)produces for the most plant metropolises to poison.In recent years,such reasons of air pollution arouse acid subsides,using the chemical fertilizer of physiological acidity in large quantities and absorb overcast cation unbalanced etc have aggravated soil acid melts,make acid soil area and acid degree further raising.There are 3,950,000,000 hm² acid soils according to statistical whole world,in which,may plough soil area for 179,000,000 hm² spreading mainly in tropics, subtropical zone and the region of temperate zone,especially in developing countries. The distribution of Chinese acid soil extends all over 15 provinces districts,gross area takes the 21%of nation-wide land gross area approximately for 20,300,000 hm².Rich of aluminum of particular acid soft in chinese south have created congenital condition for south forest aluminum poisoning in the south of Chinese.Chinese fir is the particular quickly growth and highyield tree of the south of our country,it is important timbering tree,most of the chinese fir forest is artificial pure forest. According to the long period of forestry production,the growth quantity of chinese fir drops generally along with the increase of the planting generation.The aluminum poisons of chinese fir be considered as the one of important reasons of the decline of soil fertility chinese fir even plants.On the condition of different fundamental culture medium and different concentration and ratio of IBA and NAA and adding activated carbon or not,the mixed orthogonal design was applied to analyze the effect of the rate of rooting of Chinese fir tissue-cultured seedling of FS01 and FS05 clones to screen the optimum induction root culture medium,discuss the significant influence on different factors and levels.Using sand to cultivade,each clone removes the outside disturbing factors on the condition of tissue-cultured.According to observe and statistical analysis of shape symptom of each clone,screen enduring aluminum clone and aluminum sensitive clone. This experiment uses optimized and improved mRNA DDRT-PCR systerm and silver-staining techinique,discussing enduring aluminum clone FS10,intermediate clone FS02 and aluminum sensitive clone FS01 of aluminum coerced different expresssion of the gene,and separating,cloning and measureing order of the genes,in order to screen genes related to endure aluminum.In order to establish theoretic foundatiois of expression pattern of enduring aluminum genes,regulation the enduring ability of resistant aluminum of chinese fir by the way of Gene engineering, further to cultivate endure aluminum superior clones of chinese fir.The result shows that:1.The better combination of culture medium is 1/4MS + IBA1.4mg/L+ NAA0.75 mg/L.Variance analysis shows that the rate of rooting was high on 1/4MS fundamental culture medium,IBA significantly affect the rate of rooting of FS01, NAA had remarkable influence of the rate of rooting of FS05.When adding activated carbon into culture medium,the rate,number and length of rooting of Chinese fir tissue-cultured seedling are obviously reduced.2.During the same period observation of the index of each Chinese fir clone on, such as brown and rotten degree of the top bud department,the brown spot number of stem and leaf,and the yellow degree of leaves ect,have gotten enduring aluminum clone FS10,intermediate clone FS02 and aluminum sensitive clone FS01,from the clone of FS01,FS02,FS03,FS04,FS05,FS10,FS16,FS18,FS19andFS23.3.The orthogonal design was applied to optimize the major factors that influences PCR result.Polyacrylamide Gel Electrophoresis showsthe effect of amplifying is better in the PCR system that MgCl₂ is 2.0ul,dNTP is 0.33ul,annealing temperature is 38℃.4.There are 45 primer combinations between three stripe anchor primers of 3'end and 6 stripe random primers of 5'end,which is 2 stripe for a group. Polyacrylamide Gel Electrophoresis shows that there are 8 pairs of effective primers combinations.5.Different recovery methods have been tested,and the electrophoresis pattern showed that the effect of recovery is better by using the combination of water-boiling method and sodium acetate purification.Agarose Gel Electrophoresis shows that recovery bands have not taken off end and is more distinct.6.Using the effective primers combinations for PCR,the production carry out for Polyacrylamide Gel Electrophoresis and silver-staining,getting 25 strip differential bands.According to the distribution of differential bands,36 percent of the gene bands is inhibitted expression,64 percent of the gene bands is induced expression,which is in the majority of the differential expressed gene bands.7.By identifying their homology of the getting differential gene bands,there are 2 differential bands AB1(313bp)and AB2(329bp)have high homology with some known gene sequences.AB1 has 73.0 percent of homology with enduring dry related gene of white poplar,and AB2 has 59.7 percent of homology with Triticum aestivum high-affinity nitrate transporter cDNA.