Study On The Expression Of Cry2 Genes From Bacillus Thuringiensis

Bacillus thuringiensis Cry2 proteins have insecticidal activity against a variety of lepidopteran pests,and have no cross-resistance with Cry1 proteins widely used in transgenic crops,and they are important candidate proteins for lepidopteran pests resistance management of transgenic crops.At present,there are 94 cry2 genes published,which are classified into 13 subgroups(cry2Aa-cry2Al,cry2Ba).Cry2 proteins have high sequence similarity but with different characteristics of insecticidal activity,spectrum and expression.This project focused on the expression characteristics of cry2 genes and analysis of the effects that factors pose on Cry2 proteins.Specifically,seven cloned cry2 genes(cry2Aa9,cry2Aa17,cry2Ab4,cry2Ab29,cry2Ad2,cry2Ba2,cry2Ah1)from five subgroups are expressed in E.coli and B.thuringiensis.The main results were below:1.Analysis of the expression patterns of different cry2 genes in E.coli under the same induction conditions.The expression of Cry2 protein was induced at 20°C and 30°C respectively,and the expression amount of Cry2Ah1 protein was the highest.By comparing the difference of amino acid sequence and three-dimensional structure among Cry2Ah1 protein and other proteins,it was found that Cry2Ah1 protein was deleted an amino acid at the position 355 on the loop between DomainIIβ4 andβ5.The increased amino acid mutants Cry2Ahs(354v-sp)and Cry2Ahv(354v-vp)of Cry2Ah1 protein were induced under the same conditions,and the expression amount of the mutants was significantly lower than Cry2Ah1 protein,further confirming the expression amount of Cry2 proteins were determinded by the number of amino acids on the loop between DomainⅡβ4 andβ5.2.Comparison of insecticidal activities against Spodoptera exigua among seven Cry2 proteins expressed in E.coli induced at different temperatures.Four of them(Cry2Aa17,Cry2Ab4,Cry2Ab29,Cry2Ah1)were active against Spodoptera exigua.Under the induced condition of 20°C,when the protein concentration was 100μg/mL,the weight-inhibitory activities of Cry2Aa17,Cry2Ab29,Cry2Ab4 and Cry2Ah1 against Spodoptera exigua were 42.74%,89.18%,87.02%,85.28%,respectively;under the induced condition of 30°C,when the protein concentration was 100μg/mL,the body weight inhibitory activities of Cry2Aa17,Cry2Ab29,Cry2Ab4 and Cry2Ah1 on Spodoptera exigua were 0,43.72%,47.48%and 70.54%,respectively.The results indicated that the insecticidal activity of Cry2 proteins were affected by temperature,and the activity of Cry2Ah1 protein was less affected by temperature.3.The expression vector pHT315-cry2AΔo1 previously constructed of Cry2Aa protein expressed in Bt had been modified.The ClaI and NcoI restriction enzyme sites were inserted into the two ends of the cry2Aa gene by site-directed mutagenesis,and the Bt recombinant expression vectors of nine cry2genes was constructed by seamless cloning.After transformed into Bt HD73~-acrystalliferous mutants,9 proteins were expressed.Each recombinant strain was bioassayed against Helicoverpa armigera,Plutella xylostella,Mythimna separata,and Ostrinia furnacalis.Except for the insecticidal activity of Cry2Aa9 recombinant strain,other recombinant strains had no significant insecticidal activities.4.Comparison of the effects of helper proteins ORF1 and ORF2 on the expression amount of Cry2 proteins and protein solubility in Bt expression vector.In the presence of the helper proteins ORF1 and ORF2,the expression quantity of Cry2 proteins was significantly higher than that of ORF2alone,but the simultaneous presence of ORF1 and ORF2 had no effect on the solubility of Cry2protein in alkaline solution.These advances lay the foundation for further transformation of Cry2 protein sequences,protein expression and analysis of insecticidal activity.