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Bacillus thuringiensis is a very important class of insect pathogens, it can produce a specific insecticidal crystal proteins, many pests on agricultural and biomedical cytotoxicity. Bt toxin protein (or toxin Bt-toxin) gene is widely used in the world within the insect-resistant gene, the 2005 global acreage of transgenic Bt crops 263,000,000 hm 2 . Pests poplar the afforestation production on facing a major problem. Although the use of Bt genes into Populus nigra, male Chinese white poplar, Populus deltoides, Populus have been successful, but influenced by many factors, so far no turn the Bt Transgenic Young for afforestation production reports, Bt Transgenic poplar poplar insect resistance breeding hotspots. The Zhongjia Young superior clones of Hubei Province, focusing on the promotion of poplar, but the more serious insect pest. By Agrobacterium tumefaciens-mediated leaf disc method to the study, the artificial modification of Bacillus thuringiensis insecticidal crystal protein gene BtCrylA successfully imported CNS Yang and plant regeneration of transgenic poplar. Detected by PCR, ELISA detection, SDS-PAGE identification and MTT assay confirmed BtCrylA integration and expression of the gene in the CNS Yang. CNS No. 8 Young transgenic Bt gene leaves fed anachoreta larvae, results that Young plants of transgenic insect activity. Opened up a new way to explore the nurturing of new lines of poplar insect-resistant. The main conclusions of the study are as follows: to the budding leaves hydroponics as explants CNS tissue culture techniques in the 8th Yang. The best medium for callus induction: of MS BA1.0 mg / L 2,4-D 0.1 mg / L sucrose 30g / L agar 6.5 g / L. ,2,4-D and BA factors adventitious buds callus by analysis of variance are not significant, but the impact of 2,4-D than BA. Callus best medium for adventitious buds: MS BA 0.5 mg / L 2,4-D 0.1mg / L sucrose 30g / L agar 6.5 g / L, average differentiation rate of 71.1% adventitious buds. The best medium for the blade Directly to Differentiate Buds: (1/2N) MS BA 0.2mg / L NAA0.02mg / L sucrose 25g / L agar 6.5 g / L, the single explants resulted in an average of 3.9 effective bud average differentiation rate of more than 85%. The low concentrations KT poplar plantlets rooting has a certain role in promoting best rooting medium: MS KT0.05mg / L NAA0.02mg / L agar 6 ~~ 7g / L sucrose 30g / L. Rooting plantlets indoor Lianmiao 1 to 2 weeks, dipping 0.1% ABT ABT solution, sterilized garden soil: Mountain sand (4:1) to do matrix rooting plantlets survival rate of transplanted up to 90 % or more. Agrobacterium tumefaciens strain LBA4404 antibacterial test and Zhongjia Populus differentiation of adventitious buds affected by several antibiotics, to establish a more effective CNS direct differentiation of the No. 8 Young leaves bud gene transformation receptor system. The results show that: cefotaxime (CEF) Preferably, the inhibitory effect of the Agrobacterium tumefaciens strain LBA4404 optimum dosage is 150 ~ 200mg / L, Agrobacterium correction inhibition rate of 81.8% to 100%, leaf differentiation more adventitious buds frequencies of up to 73.3%. Kanamycin (Km) dosage of 60mg / L completely inhibited callus and adventitious buds occurred; the Km on shoot elongation growth inhibition, Km = 60mg / L, the survival rate of bud 0 . In the screening of the transformants, Km concentration on Sprouts preferably 30mg / L. Rooting screening Km concentration of 15mg / L more appropriate. Streptomycin (str) the poplar leaves differentiation adventitious buds auxin-like effect. With Str10mg / L instead of the NAA 0.02mg / L and BA with adventitious buds differentiation rate was 71.1%: the Str 20 mg / L, NAA 0.02 mg / L and BA0.2 mg / L with the adventitious bud differentiation rate of up to 100% . In addition, a certain concentration of streptomycin affect parts of the leaves budding on medium containing streptomycin, some leaves, petioles punishment of adventitious buds or the petiole leaf margin while Differentiate Buds, and control of adventitious buds only from the leaf margin occur. Affect Agrobacterium tumefaciens-mediated the BtCry1A genes into the Zhongjia Yang certain factors Zhongjia Yang simple, effective genetic transformation system. Optimal transformation conditions: the preculture 12h, broth (OD 600 = 0.3 ~ 0.4) disseminated explants body 10 to 15min, co-culture 4d, co-culture medium supplemented with acetosyringone (AS ) 150μmol / L and co-cultured delay 10d choose the the Km r bud average induction rate up to 28.9%. All affect the conversion factor discussed in this test, select the time of greatest impact, followed by co-culture time. Transformation strategy, selected after the first regeneration screening transformants false transformants, the late high rate of elimination, homozygous difficult, but can be overcome due to the low concentration of Km No. 8 Young leaves callus induced inhibition of CNS and adventitious bud differentiation led to the transformation of the problem difficult to complete. This study Cotransformation the explant 1936, differentiation of the Km r bud outside explants 171, Km r bud induction rate of 8.8% on average. Via adventitious bud growth and the rooting stage kanamycin continuous screening, 17 the Km r plants, the average conversion rate of 0.88%. Molecular Detection and Identification of insect resistance to CNS Yang to turn BtCry1A transgenic plants in the 8th. PCR positive plants genetically BtCry1A gene from 17 CNS Yang Km r plants were screened by molecular detection 6, 35.3%; enzyme-linked immunosorbent test and SDS-PAGE electrophoresis results show that the BtCry1A gene expression has six lines in the PCR positive plants Bt toxic protein content magnitude to 3.88 ~ 2.36ppb, and average 3.32ppb,. The test results show that the the BtCry1A gene has been integrated into the, Yang genome, CNS and expressed. The MTF assay results show that the Zhongjia No. 8 Young PCR positive plants protein processing Trichoplusia ni cells, a higher mortality rate, pathological changes similar to the Bt toxin protein Trichoplusia ni cells lesions to prove BtCry1A gene expression in transgenic plants, and transgenic plants on insects vitro cell cytotoxicity. Indoor insect resistance test results show that, feeding insects 8 Tian, ??the tested three transfected gene strains Yang Fan Moth larvae growth inhibition rate were 66.13%, 60.94% and 46.65%; insect feeding 10 Tian, ??3 transferred gene strain larvae relative mortality were 75.99%, 36% and 55.99%, respectively, at this time, the control untransfected survival gene leaves fed larvae all pupate, while the survival of the three test lines leaf fed pupation 33.3%, 18.8%, 54.5%, and not the survival of the larvae pupate move slowly, smaller, still unable to 12 days of feeding insects pupate. Outdoor planting PCR positive plants showed the obvious insect resistance. The worm measured results show that the three PCR-positive strains have significantly inhibited the growth and development of larvae anachoreta which strains X1 works best. No. 8 Young to turn BtCry1A gene leaves anachoreta larvae Zhongjia, pathological changes of the intestinal epithelial cells the typical Bt crystal toxin acts on the pathological features of Lepidoptera. Therefore, this study Zhongjia Transgenic Populus on BtCry1A transgenic plants insect-resistant plants, further testing and promotion of value.
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