Through The Cold Resistance Cotton(Gossypium Hirsutum L. ) Callus Preliminary Screening Cold Resistance Genes From Saussurea Involucrata Kar.et Kirs

Temperature is the main constraints to determine the distribution of plant and crop yield.It is natural disaster that a worldwide prevalence of the problem that the substantial reduction of food crops and cash crops production caused by low temperature stress. Therefore, the study of plant stress tolerance of low-temperature, and improve the plant capacity of low temperature stress tolerance, is extremely important practical significance. With the rapid development of transgenic technology, molecular breeding has become a mean to obtain the plants and crops with high-quality, high-yield, high resistance. Although the molecular breeding of new opened up a new rapid method to cultivate the excellent new varieties of crops, but a large number of studies have shown that transgene silencing has become obstacles to the rapid breeding method. Saussurea involucrata Kar.et Kirs is the unique plant resources of Xinjiang, it has a set of the extreme survival mechanisms in order to adapt to the extremely harsh environment in Tianshan where is the Saussurea involucrata Kar.et Kirs growth. Cloned the genes which can increase plant resistance about the temperature from the Saussurea involucrata Kar.et Kirs, it will significant to improving the productivity of crops.previously, Pro. Zhu Jianbo and his lab members were successfully used the gateway technology constructed the Saussurea involucrata Kar.et Kirs c DNA expression library,and successfully cloned the genes which can increase stress resistance from this c DNA expression library such as:Sik SACPD, Sik COR, Sik DHN, Sik Trxh, Sik LEA and others. In this study,at first we will go to constructed the Saussurea involucrata Kar.et Kirs c DNA library by combining the SMART technology and DSN technology.And than we will go to transformed the genes that were in the c DNA library into the cotton genome by the Agrobacterium-mediated transformation;thirdly,screening the cold resistance cotton callus;finally we will get the Saussurea involucrata Kar.et Kirs cold resistance gene which can normally expressed in transgenic cotton plants.We successfully cloned a cold resistance gene from the Saussurea involucrata Kar.et Kirs. The results showed this gene is a plant metallothionein-like gene,named Sik MT2. Bioinformatics analysis revealed that this gene contains a complete open reading frame(228bp), encodes 75 amino acids. Sik MT2’s molecular weight is 7.47 k Da, the theoretical isoelectric point is 4.44, and is a hydrophilic proteins without signal peptide.Amino acid sequence alignment results showed that Sik MT2 sequence has higher conserved in the N-terminal and C-terminal, this is the typical characteristics of the MT2. Phylogenetic tree analysis between 21 species,the result showed that the Saussurea involucrata Kar.et Kirs and Taraxacum officinale evolutionary relationship is most recently among the 21 species.Methods: Extracted the RNA from the Saussurea involucrata Kar.et Kirs tissue cultureMethods: Extracted the RNA from the Saussurea involucrata Kar.et Kirs tissue culture seedlings that treated by low temperature.Combining the SMART technology and DSN technology constructed the Saussurea involucrata Kar.et Kirs c DNA library.Transformed the cotton hypocotyl by Agrobacterium-mediated transformation,get transgenic cotton callus.Treatment the transgenic cotton callus in the semilethal temperature of the cotton callus.Screening the cold resistance cotton callus and cloned the Saussurea involucrata Kar.et Kirs cold resistance gene from thecold resistance cotton callus.Used the bioinformatics and real-time fluorescence quantitative PCR analysis and forecasting the function of Sik MT2 gene.Results:(1)We successfully constructed the Saussurea involucrata Kar.et Kirs c DNA library by combining the SMART techniques and DSN techniques, test results showed that this c DNA library storage capacity reached 4.26×105cfu, insert fragments mainly concentrated between500 bp to 2000 bp.(2)We got the semilethal temperature(LT50) of the cotton callus by combining the conductance method and Logistc equation measured.(3)We through the Agrobacterium-mediated transformationin troduced the genes that belong to the c DNA library into the cotton genome,and got the transgenic cotton callus.(4)We screening the cold resistance cotton callus by semilethal temperature(LT50) treatment.(5)We successfully cloned Sik MT2 gene from the cold resistance cotton callus.Through analysis, we forecasted the gene encodes a protein involved in the plant antioxidant effects.(6)We through the real-time fluorescence quantitative PCR(q PCR) to detect the expression of Sik MT2,and discovery that Sik MT2 is induced by abiotic stress.Conclusion: Treatment the transgenic cotton callus by semilethal temperature(LT50) of the cotton callus can screen the cold resistance transgenic cotton callus,and than we can clone the genes which can involved in raising cotton cold resistance from the cold resistance transgenic cotton callus.