Identification Of Heat Tolerance And Construction Of Plant Expression Vector Of Small Heat Shock Protein Gene HSP17.4 From Soybean

Soybean is the major grain and oilseed crops in China,the development potential extremely.The transpiration coefficient,water demand is bigger,high temperature resistant,weak ability to resist drought,growth period in high temperature,drought,soybean production is an important factor.Phenomenon gradually enhanced in recent years,the greenhouse effect and global climate warming,high temperature caused by production problems cannot be ignored,for the study of the heat resistance of plants is also more and more.Heat Shock Protein(Heat Shock Protein HSP)is an organism from outside stress stimuli(high temperature,drought,frost,metal ion,etc.)when the synthesis of Protein of biology protection.Small molecules including Heat Shock Protein(Small Heat Shock Protein,sHSP)is one of most species,the most important category.Synthesis of sHSP under stress stimulus can quickly,reduce the stress stimulus to the damage of the organism,enhance the capacity of an organism's tolerance,adversity stress resistance have important significance for organisms.Test the cloned from soybean JN18 HSP17.4 gene constructed by herbicides in selection markers of plant expression vector pCPB-HSP17.4 and RNA interference expression vector pCPB-HSP17.4-RNAi,mediated by agrobacterium tumefaciens and pollen tube channel method soybean import receptors in "qinong" 18.Molecular biology detection in the their offspring plants and physiological and biochemical tests,so as to verify HSP17.4 genes related to soybean high temperature resistant ability and overexpression of HSP17.4 genes can increase the ability of soybean resistance to high temperature.This experiment mainly the results of the study are as follows:1.Successfully constructed with herbicide by selection markers of plant expression vector pCPB-HSP17.4 and RNAi interference carrier pCPB-HSP17.4-RNAi.2.Using the method of agrobacterium mediated putting the plant expression vector into receptor soybean varieties "JN18" by PCR detection T0 obtain the T0 generation plant 13 plants,T1 generation plants 26 strains,T2 generation plants 39 strains,the conversion rate of 2.6%;Using the method of pollen tube channel transformation of T1 generation plants from 28 strains,T2 generation plant 47 plants,conversion rate reached 5.83%.3.The use of Southern blotting identified 7 strains of T1 generation plants,2 strains were obtained by agrobacterium mediated method,5 strains were obtained by pollen tube pathway.And T2 generation plant eight plants were positive,3 strains were obtained by agrobacterium mediated method,5 strains were obtained by pollen tube pathway.All in a single copy form integrated into the soybean genome.4.Fluorescence quantitative PCR results showed that the genes in T1 and T2 generation plant leaves,stems and roots are the expression,the expression of different parts and different transformation plants,quantity are different,the highest expression of leaf.5.T1,T2 generation transformation plant high temperature resistant identification results show that in 42?under high temperature stress,compared with not transformed plants,T1,T2 turned expression vector plant relative gene expression quantity increased significantly;Relative conductivity,mda content decreased obviously,increased significantly peroxidase activity.Leaf blade recovery effect is good,does not appear obviously wilting,curly,and so on and so forth;Turn RNAi expression vector relative expression of plant gene below conversion plant,relative conductivity,mda content increased obviously,peroxidase activity significantly decreased,leaf apparent wilting,curly,and so on and so forth.