Bt Gene Stacking In Rice With Linked Effect Genes And2A Polycistronic Transgene

Bacillus thuringiensis (Bt) have shown great importance in transgenic crops for pest control. However, wide-spread utilization of Bt crops has brought about insect resistance to Bt toxins in field. Bt gene stacking strategy introduces more than one Bt genes into plant, which was proved to be an effective and applicable way to delay the development of insect resistance to Bt toxins.Several methods have been adopted to introduce multiple genes into plant, including hybrid of single gene introduced plants, re-transformation, co-transformation, linked effect genes and polycistronic transgene. First,"linked effect genes" was adopted in this study. Expression cassettes of Bt Cry1Ac gene, Bt Cry1Ig gene (223) and glyphosate-tolerant5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene (1174) were tandemly linked and inserted into a binary T-DNA vector. Then the T-DNA was introduced into rice by Agrobacterium-mediated transformation. From all the100transformants obtained, the single copy inserted transformant with both Ciy1Ac and223genes highly expressed was selected. Later the homozygous transgenic line was selected from the T1generation. The homozygous line showed high expression level of Ciy1Ac,223and1174, which confer high insecticidal activity against cotton bollworm (Helicoverpa armigera), striped stem borer (Chilo suppressalis), cotton leafworm(Spodoptera litura) and rice leaf roller (Cnaphalocrocis medinalis) and high glyphosate-tolerant activity.But Within the experiment of linked effect genes, unbalanced expression of different linked genes were observed and no more genes could be accomodated due to the limit of T-DNA size. To overcome these two limitations, co-expression of different Bt gene was achieved by2A strategy, which is a very important method of polycistronic transgene.2A is an oligopeptide sequence mediating a ribosome "skipping" effect, producing an apparent "cleavage" of polyproteins. When linked by2A peptide coding sequence, different genes could be expressed from one single open reading frame. In this study,2A peptides from Foot and Mouth Disease Virus (F2A) and Porcine teschovirus-1(P2A) were used for co-expression of Cry1Ab and Cry2Ab in rice. Two T-DNA vectors,1300-Ubi-Cry1AbF2ACry2Ab-35Sints-1174(1F2A) and1300-Ubi-Cry1AbP2ACry2Ab-35Sints-1174(1P2A) were constructed. Because C-terminal structure of protein lied upstream of2A peptide may influence the2A activity, position of CrylAb and Cry2Ab were changed. Two other T-DNA vectors,1300-Ubi-Cry2AbF2ACry1Ab-35Sints-1174(2F2A) and1300-Ubi-Cry2AbP2ACry1Ab-35Sints-1174(2P2A) were constructed. The four constructs were separately introduced into rice. Study of transformants obtained from different2A constructs showed that discrete Cry1Ab and Cry2Ab proteins were highly and coordinately expressed while the "Cry-2A-Cry" fusion protein was hardly detectable. High cleavage efficacy of2A peptide was also detected in stem and root part of rice, which is of great importance because some major pest in rice feed mainly on non-green part of rice. Insect bioassay showed that transformants generated from each of2A constructs exhibited high insect-resistance activity. To further examine the2A cleavage efficiency and compare the impact of different upstream proteins on2A efficiency, quantities of CrylAb and Cry2Ab in transformants from different constructs were determined by ELISA and the ratio of upstream protein to downstream protein was analyzed. The result showed F2A was more efficient than P2A and2A peptides were more efficient when CrylAb, instead of Cry2Ab, was placed upstream. What’s more, quantification of CrylAb and Cry2Ab showed that gene expression levels with2A polycistronic transgene were comparable with monocistronic transgene commonly used.On one side,"linked effect genes" was adopted to introduce Cry1Ac,223and1174gene into rice.The homozygous, single copy inserted line was obtained with all the three genes highly expressed. That line exhibited great insect-resistance activity and high glyphosate-tolerant activity, which showed potential for commercial application. On the other side, to overcome the unbalanced expression levels of different genes and limitation of T-DNA size observed in linked effect genes strategy, 2A strategy was used for co-expression of CrylAb and Cry2Ab in rice. The result showed that CrylAb and Cry2Ab could be highly and coordinately expressed by2A strategy in addition to reduce the overall T-DNA size.