Construction, Expression Of The Fusion Gene Cry1Ac-cmg1 In Escherichia Coli

Bacillus thuringierrsis(Bt) is one of the most widely exploited insecticidal microbes. Bt genes coding insecticidal proteins have been transferred into a variety of important grain and cotton crops.Bt crops are significantly resistant to major insect pests,but they are still susceptible to pathogenic diseases.β-1,3-glucanase is an important resistant compound in plants.It can inhibit the growth and reproduction of plant fungal pathogens,making it an effective agent to control fungal pathogens in developing transgenic disease resistant plant.Therefore,it would be interesting to create transgenic crop strains that harbor both insect resistant gene and disease resistant gene.In order to construct the recombinant strain with toxicity and resistance to both insect pests and fungal pathogens,we construct a fusion gene of Bacillus thuringierrsis(crylAc) andβ-1,3-glucanase(cmgI).We intend to develop the trangenic rice with this fusion gene.The main results as follows:1.Specific primers with the enzymes restriction sites were designed to the cryIAc gene sequence.Using this pair of primers,the cry1Ac gene was amplified from the plasmid pCR2.1-cry1Ac.The PCR fragment of cryIAc and the plasmid pET-22b(+) were both digested by Ndeâ… /Hindâ…¢.Then they were linked by T4 DNA ligase and transformed into E.coli to construct the recombinant plasmid pET-22b-Ac.2.Specific primers with the enzymes restriction sites were designed to the cmg1 gene sequence.Using this pair of primers,the cmg1 gene was amplified from the plasmid pGEM-T-CMG.The PCR fragment of cmg1 and the plasmid pET-22b(+) were both digested by Hindâ…¢/Xhoâ… .Then they were linked by T4 DNA ligase and transformed into E.coli to construct the recombinant plasmid pET-22b-cmg1.3.The fragment of cryIAc digested by Ndeâ… /Hindâ…¢was linked to the plasmid of pET-22b-cmg1 which was also digested by Ndeâ… /Hindâ…¢with cmg1 gene.They were transformed into E.coli to construct the recombinant plasmid pET-22b-Ac-cmg1.4.We separately transformed the plasmids of pET-22b-Ac,pET-22b-cmg1 and pET-22b-Ac-cmg1 into Rosetta(DE3).The results of SDS-PAGE confirmed that these three genes could be expressed.5.We purified the proteins of cry1Ac-6His and cmg1-6His at denatured state with Ni²⁺ affinity chromatography.Then we immunizatied the male New Zealand rabbit with the purified proteins as the antigen to make polyclonal specific antiserums.6.The expressing product of fusion Ac-cmg1 was analysis by Western blot with the polyclonal specific antiserums.The results showed that:pET-22b-Ac-cmg1 expressed the cry1Ac protein of 69.9 kDa,but also expressed the glucanase protein of 99.0 kDa.It confirmed that the proteins of cry1Ac and glucanase were both expressed in the fusion gene Ac-cmg17.We made a preliminary study of the renaturation of inclusion body protein Ac-cmg1.We purified the Ac-cmg1 protein with the Ni²⁺ affinity chromatography to obtain a large number of purification protein in order to lay the foundation for following study.