Codon Optimization Of Cry1Ah1 Gene In Rice

Rice is an important crop,perennial planting and total account for the top of the crop.Rice production occupies a very important role in the national economy.At home and abroad transgenic Bt rice has made great breakthrough in research and development.Our government in 2009 approved the transgenic rice commercial application of biological safety certificate.It indicates that Bt transgenic rice and other crops in agricultural production will play an important role.crylAhl,one of holo-type cry genes,cloned in this laboratory from Bacillus thuringiensis strain has been patented in China,and it encoded a protein with strong insecticidal activity against certain lepidopteran insect pests,such as Chilo suppressalis.It is superior to the current commercialized cry genes of crylAb and crylAc.crylAhl gene is exhibiting good application prospects.Numerous studies showed that wild-type Bt gene transferred directly to the plants,its expression is very low or no expression and do not have the practical effect of the insect.It is important to obtain a foreign gene codon optimization,and such other modification according to the codon preference of the target species.In this study,according to rice amino acid codon usage frequency,crylAhl critical region of the gene insecticidal five different sequences and the necessary modification were optimized.At the molecular level to assess the five programs in the transformed Var nippobare.The first time in the level of transcription and translation compared the difference between the different optimization,while the best optimization.This provides a reference for Bt and other prokaryotic gene optimization and efficient expression,laid a solid foundation for the study of Bt insect-resistant transgenic plants.The main results were as follows:In the case to ensure that the same gene amino acid sequence,we use five different program optimization and transformation of rice codon usage frequency on the critical areas of crylAhl gene insecticidal.We improved the G+C content and removed unstable factors such as shear signal of intron.Meanwhile,? sequence,Kozak sequence,double termination of the recognition sequence and signal for endoplasmic reticulum retention were fixed around cry1Ah1 gene.Final synthesis 2077bp sequence is connected to the pUC57 vector.The sequencing results with the expected sequence.Programs in the synthesis of optimized gene prokaryotic expression vector pEB1Ah(pEB1Ah1,pEB1Ah2,pEB1Ah3,pEB1Ah4 and pEB1Ah5).IPTG induction of the same size with the expected 65-kDa protein expressed in E.coli Rosetta(DE3)and insoluble components.It showed that the optimized gene normally expressed in E.coli.All of these expressed polypeptides showed insecticidal activity against 2nd-instar larvae of Plutella xylostella and neonate of Chilo suppressalis.When the protein concentration of 5 ?g/mL,the mortality rates of Plutella xylostella were more than 33%,the mortality rates of Chilo suppressalis more than 24%.When the protein concentration of 50 ?g/mL,the mortality rate of Plutella xylostella and Chilo suppressalis were 100%.Proteins had good insecticidal activity.The results show that the genetic optimization does not change the amino acid sequence of the optimized gene target pests insecticidal activity did not significantly affect.Programs after the synthetic optimized gene construct plant expression vector pCAMBIA1300UAh(pCAMBIA1300UAh1,pCAMBIA1300UAh2,pCAMBIA1300UAh3,pCAMBIA1300UAh4 and pCAMBIA1300UAh5).After Agrobacterium mediated transformation of Var nippobare,the transgenic rice seedlings were detected by PCR,the positive rate containing target gene was more than 87%.Afterwards,the results of real-time RT-PCR and ELISA assay indicated that the highest expression level of five modified crylAhl genes was that using the highest frequent codons.Average expression amount of CrylAhl polypeptides was 0.104%of total soluble proteins from the positive transgenic rice.