Molecular Identification Of Cry1Ac Transgenic Sugarcane And Intermediate Test Evaluation

Sugarcane is the most important sugar crop throughout the world. Sugarcane borer is the most serious and universal pests during sugarcane production in China. The sugarcane borer can be harmful to sugarcane planting in the full growing season, at the damage rate up to 85%,. While sugarcane itself lacks resources of resistant to borers, cry gene can effectively improve the insect resistance of sugarcane. Therefore cultivating sugarcane variety of good insect-resistant by genetic engineering, is one of the core technology urgently for China’s current sugarcane industry. In the present study, with six positive crylAc transgenic sugarcane clones(a-1、a-2、a-3、a-4、a-5、a-6), and receptor varieties FN95-1702 plus its tissue culture clone (TC FN95-1702) as experimental material, loop-mediated isothermal amplification (LAMP) was applied to detecct the crylAc regeneration resistant clones. Molecular detection technology for exogenous gene was then used to identify the authenticity and genetic stability of these positive transgenic lines. Photosynthetic characteristics, major agronomic traits and exogenous gene drift to non-target organisms were also studied in positive crylAc transgenic lines and non-transgenic control. Besides, rhizosphere soil microbial community structure and enzymatic characteristics were compared in this study. The primary results are as follows.As a case study in crylAc gene transgenic sugarcane, aim at the technical demand for breeding of transgenic sugarcane, gene composition inspection and supervision, an efficient method based on the principle of LAMP is developed and the results can be judged directly by visual according to the color in the reaction products. In this method, four pairs of specific primers were designed according to the sequence information of six specific regions of crylAc gene and then the compositions of Mg2+ concentration, primers ratio of inside to outside and Bst DNA polymerase in LAMP system were optimized. Besides, several detection methods including precipitation, Calcein and Mn2+(Mn2+ 0.6 mmol/L,0.05 mmol/L) complexation and SYBR Green I methods were evaluated, among which the visual method with SYBR Green I directly added into the reaction tube cap before incubation was proved to be the best. The results can be observed after 1 h of incubation at 65℃. In the sensitivity tests, the optimized LAMP system could detect 43.1 copies of the recombinant plasmid with crylAc gene, which is about tenfold higher than that of conventional PCR. The results showed that the established LAMP technology, which could detect the crylAc gene in transgenic sugarcane with the advantages of fastness, sensitivity, simplicity and efficiency. Besides, the system established requires no special requirements for the detection site, and both the equipment needed and the results judged was simple. This method not only provides the technological support for crylAc transgenic sugarcane breeding and supervision, but also provides a good reference to develop the detection technique for gene composition in other types of transgenic sugarcane.The different tissues from the crylAc transgenic sugarcane during each growth period were detected by LAMP and conventional PCR. The results revealed by LAMP and conventional PCR detection showed that there did not exsit any loss of the exogenous crylAc in all 91 testing crylAc transgenic sugarcane samples. It approved that the genetic stability of cry1Ac performs well in cry1Ac transgenic sugarcane lines.The investigation of insect resistance and yield traits of the cry1Ac transgenic sugarcane lines and control lines indicated that the insect resistance of six testing transgenic lines is apparently much superior to that of non-transgenic lines, while some transgenic lines perform better in yield trait, such as plant height (transgenic lines a-5 and a-1) and the millable stalks per hectare (transgenic lines a-2 and a-1). These results suggests that, cry1Ac transgenic sugarcane, with the improvement of insect resistance, have equal or even better yield and quality characters.In order to detect whether those genes, cry1Ac, bar, and nptⅡ, drift or not,8 typical grass weeds and 5 other species weeds,7 major non-target insects and rhizosphere soil microbial in cry1Ac transgenic sugarcane clones were used as test materials and PCR with specific primers was applied to detect cry1Ac, bar, and nptⅡ gene. The results show that there is no exogenous gene drift to the typical weeds, non-target insects or soil microbial.The rhizosphere soil micro-security and soil enzyme activity of cry1Ac transgenic sugarcane were preliminary studied by plate culture, BIOLOG microbial identification along with soil enzyme activity determination. The result suggests that the exogenous gene crylAc has slight but not significant impact on the culturable soil microbial community and enzyme activity. For soil microbial diversity, the soil microbial diversity indexes were affected by rare cry1Ac sugarcane clones, but on the whole, the impact of cry1Ac transgenic sugarcane on richness and uniformity of soil microbial community species, the microbial species dominance, function diversity index of microbes, and the abundance of diversity in species multidimensional space, is not significant. Changes of the urease, protease, invertase, phosphatase activities in rhizosphere soil were complicated, with no consistent regularity, discontinuity and intermittence, while effect may disappear after a period of time.