The Cloning, Analysis Of The Full-length CDNA Of Plant Defensins From "Shuza No,9" Of Brassica Napus And Its Expression In Escherichia Coil

Plant defensins, a kind of low MW cationic peptides widely distributed in many plant seeds, have strong ability to inhibit pathogen. As multifunctional effetor molecules of non-specificity innate immunity, defensins are the key components in plant immunity system. Plant defensins are thought to interact with specific receptor harboring in the cell membrane and inhibit the growth of fungi, especially filamentous fungi. Plant defensins have conserva(?)e eight Cys residues and form four intramolecular cysteine disulfide bonds.According to the known sequences of Cruciferae plant defensin genes from Gen-Bank, two primers, 5' -GCGGAATTCAT GGCTA AGTT TGCT T CC- '3 and 5'-CGC TCTAGATTA ACATGG GAA A TAACAGATAC- '3, were designed. The total RNA was extracted from Brassica Napus(Shuza No, 9) seeds and reverted into cDNA, then amplifyied plant defensin gene by using PCR. The plant defensin cDNA sequence, with the length of 243bp coding 80 amino acids, was successfully cloned from Brassica Napus.The target DNA was inserted into the expression vector, GTK, which is derived from the pGEX—2T, then transformed to E. coli BL21. Having been cultivated in 2 X YTA medium and induced by 0. lmM IPTG at 18 °C for 2 hours, the E. coli BL21 effectively expressed the prospective 34kD fussion protein which could be detected by SDS-PAGE. Moreover, the result of Western-blot proved this conclusion.In the course of the fussion protein purification , the fussion protein was expressed as the form of inclusion bodies in E. coli either varying concentration of IPTG or adjusting the cultivated temperature. While IPTG is toxicity to cells and expression system has relative higher expression efficiency induced at 18°C, we selected 0.1 mM IPTG and induction at 18°C as the condition of the fussion protein purification. Then a series of purification steps were done, including supersonic breaking cell, inclusion bodies washing, inclusion bodies soluting in a buffer containing 8M urea, 50mM Tris- HCl(PH8.0) and 0.4% ME. Generally, this work gave the prerequisite conditions to further purification of Brassica napus defensins and took the first step of more advanced research aimed at regulators of plant defensin genes.