Indica rice target line for gene stacking of specific sites mediated by recombinase
A technology of recombination sites and recombinases, applied in the field of indica rice target lines, can solve problems such as genetic transformation difficulties, achieve the effects of reducing separation sites, reducing breeding workload, and reducing release evaluation costs
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Embodiment 1
[0044] Embodiment 1 is suitable for the carrier of gene stacking
[0045] The structures of vectors pZH109, pZH108 and pZH93 are as follows figure 1 As shown, its construction refers to the standard DNA recombination method. All PCR reactions used Phusion high-fidelity polymerase (NEB Beijing, USA). The sugarcane baculovirus (ScBV) promoter and terminator octopine synthase (ocs) control the initiation and termination of the green fluorescent reporter gene gfp, respectively. The rice actin promoter and terminator cauliflower virus (CaMV35s) control the initiation and termination of the hygromycin gene hpt, respectively. The CaMV 35S promoter and terminator control the initiation and termination of the herbicide resistance gene bar, respectively. The promoter of the cre recombinase gene is the CaMV35S promoter, and the terminator is the nopaline synthase (nos) terminator.
[0046] This application adopts two strategies to obtain the starting target line of indica superposi...
Embodiment 2
[0049] The screening of embodiment 2 initial target lines
[0050] method:
[0051] Agrobacterium-mediated transformation
[0052] About 2000 immature embryos of indica rice Huanghuazhan or 500 indica rice R900 were infected with Agrobacterium. One part was infected with Agrobacterium EHA105 containing the target line vector pZH109, and the other part was co-transformed with Agrobacterium containing pZH108 and Agrobacterium containing pZH93. For specific transformation methods, please refer to Hiei Y. and Komari T.2008. Agrobacterium-mediated transformation of rice using immature embryos or calli induced from mature seed. Nature Protocol (3) 824–834.
[0053] GFP observation
[0054] GFP expression was observed by a Leica inverted fluorescence microscope DMI6000B (Leica, Wetzlar, Germany). The excitation filter wavelength range used is 440-520nm, and the barrier filter wavelength is 510LP.
[0055] PCR analysis
[0056] About 100 mg of fresh or frozen GFP-expressed you...
Embodiment 3
[0091] Example 3 Initial Target Line Genome Location Map
[0092] figure 1 The insertion position shown in C is obtained by comparing the left and right side DNA sequences with the rice genome database (Wang, J., Kong, L., Zhao, S., Zhang, H., Tang, L., Li, Z., Gu , X., Luo, J., and Gao, G.2011. Rice-Map: a new-generation rice genome browser. BMC Genomics.12, 165) comparison and determination. For detailed DNA sequence see Figure 4 and Figure 5 . Important features of each insertion site are listed here (insertion sites are figure 1 A and 1B3 are described from the T-DNA left border to the right border), that is, five genome sites suitable for indica rice gene stacking of the present invention:
[0093] The target site of the target line H11: located between 24,920,581 and 24,921,595 of the long arm of the 12th chromosome, the 21bp genome sequence from 24,921,594 to 24,920,581 was deleted. At the left end of the T-DNA, the first 24 bp of the 26 bp LB was deleted. At t...
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