Artificially Synthesizing And Transformation Researching Into Rice Of CryIBa3

Bacillus thuringiensis (Bt) was a spore forming Gram-positive bacteria, and its parasporal crystal protein is toxic to a variety of insect pests, every parasporal crystal protein insecticidal spectrum is specificity. CryIBa2 gene consists of 1228 amino acid residues, and its core region is located from 22 to655AA.Based on the original sequence of CryIBa3 gene, CryIBa3 gene was reprogrammed and synthetized using maize codon preferences in this study. The full length fragment and the core region of CryIBa3 gene were transformed into rice callus by Agrobacterium-mediated method. To transformed seeds were obtained by tissue culture, and then we gained some results about T1 plants through genetic transformation tests. The main results are as follows:1)Codon modification was carried out according to maize codon preference, making GC content to 56.58%. The protein sequences of synthetized gene were same as the original gene, and the difference of the DNA sequences was up to 19%.2) Two plant expression vectors, full length fragment of L CryIBa3 and the core region of SCryIBa3, were constructed.13 positive plants were obtained through genetic transformation in rice by Agrobacterium-mediated method. Eight lines of Ti transformed plants were obtained through screening tests.3) Bt toxic protein contents in leaves at tillering stage and heading stage were measured. It was found that Bt toxic protein content was 630 ng/g at tillering stage, while 710 ng/g at heading stage was apparently higher than tillering stage. It stated Bt toxic protein content showed spatiotemporal dynamic change rule along with different growth stage.4) After determination of Bt toxic protein content in leaves and heads at heading stage, we have found that Bt content in leaves was 710 ng/g, which was higher than 650 ng/g in heads. It showed that the expression quantity of Bt were different in different organs.5) By comparing Bt content in transgenic plants containing full-length with the core region of CryIBa3 gene, we have found that expression quantity of Bt in transgenic plants SCryIBa3 was higher than that of LCryIBa3 both in leaves and heads. It showed that the core region of CryIBa3 was much easier to express in rice.