| The insect-resistant transgenic sugarcane clones keep genetic and insect-resistant stability of exogenous genes in asexual generations,could directly affects the prospects of insect-resistant transgenic sugarcane.This paper,basis with the obtained transgenic Cry1Ac gene in the early research on sugarcane,we intends to analyze the integration mechanism,genetic expression and insect-resistance of Cry1Ac gene in transgenic sugarcane,and analytical the generation,insect-resistant stability of sugarcane asexual with transgenic Cry1Ac gene.In order to provide a theoretical basis and breeding material for selecting effective insect-resistance,good characters sugarcane clones.The main results of this study as follows:1.The Cry1Ac gene prokaryotic expression vector pCznl-Cry1Ac was constructed and the prokaryotic expression found that Cry1Ac recombinant protein was mainly in the form of inclusion bodies in E.coli.and the recombinant protein was purified for antibody preparation in late stage.2.Transgenic plants with Cry1Ac gene were detected by PCR,48 positive plants were obtained and the positive rate was 88.89%.Positive plants were detected by Bar immune/detection test strip,and there were 44 expressed Bar protein.Also the Southern blot was carried out on positive plants,all plants had hybridization signal,and the Cry1Ac gene is mainly in 1-3 low copy inserted into the sugarcane genome,then the Cry1Ac expression in transgenic sugarcane were detected by RT-PCR and Bt immune/detection test strips,a total of 28 plants expression of Cry1Ac protein.3.The insect-resistantce of 28 transgenic sugarcane were identificated in indoor and field,and 6 transgenic plants with good insect-resistantce.4.T1 generation of the insect-resistant transgenic strains were detected by PCR,the positive rate was 1000%and Southern blot analysis revealed that the copy number and integration site of T1 generation were in conformity with the TO generation results.It showed that the Cry1Ac gene not losed in the clonal propagation of sugarcane.5.T1 generation of the insect-resistant transgenic strains are analyzed by RT-qPCR and the results show that the expression of Cry1Ac in different transgenic strains was different.The expression level of Cry1Ac gene was consistent with the plant insect-resistant ability.Among them the relative expression of the 36 strain was the highest,which was 7.83 times of that of the susceptible strain.36 strain were used as research objects,for RT-qPCR analysis of Cry1Ac gene in different tissues of transgenic sugarcane and the results show that Cry1Ac was impressed in immature leaves,mature leaves,immature stem,mature stem and roots.Cry 1 Ac has higer expression in leaves and stems than roots.Leaves and stems of mature parts has higer expression than immature parts.6.T1 generation of the insect-resistant transgenic strains are analyzed by Western blot and the results show that all resistant transgenic strains had hybrid bands,it was proved that the Cry1Ac protein can expressed in all the insect-resistant transgenic strains.The protein expression of the resistant transgenic strains was analyzed by ELISA,the results show that there were significant differences in protein expression between the resistant transgenic strains and the protein expression levels significantly higher than the susceptible strains.Transgenic strains insect-resistant ability was consistent with the expression of Cry1Ac protein.The 36 transgenic strain protein expression is 245.33?pg/L which is 7.46 times than the susceptible 37 transgenic strain.7.The insect-resistantce of T1 generation of the transgenic strains were identificated in indoor and field,the results of indoor and field were consistent.All strains were maintained with the parent of insect-resistantce.8.Through agronomic traits measured and comprehensive index analysis for T1 generation of the insect-resistant transgenic strains,3 transgenic strains with potential for production and application were obtained. |
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