Identification and application of two novel cis-acting elements induced by Rhizoctonia solani
A technology of cis-acting elements and corn sheath blight bacteria, which is applied in the field of plant genetic engineering, can solve the problems that the properties and functions cannot be revealed, and achieve the effect of broadening applications
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Embodiment 1
[0024]Example 1 Construction of recombinant vectors of different truncated sequences from -1243bp to -1228bp and GFP gene and transient expression on Nicotiana benthamiana
[0025] The truncated promoter involved in this example is derived from the GRMZM2G315413 promoter sequence, which can be obtained according to the technology disclosed in ZL201410104914.2, in which the truncated sequence from -1243 to -1228 has been obtained. In the embodiment, it is the further research and analysis of the region from -1243 to -1228.
[0026] This segment was truncated again in order to identify the response element of sheath blight from -1243 to -1228. Using the 35S basic promoter as a template, use forward primers respectively, the sequences of which are shown in SEQ ID NO.3, 4, 5, and 6
[0027] (5-GTTGAGGCACTTATTTCGCAAGACCCTTCCTCTATAA-3,
[0028] 5-GGCACTTATTTCGCAAGACCCTTCCTCTATAA-3,
[0029] 5-TATTTCGCAAGACCCTTCCTCTATATAA-3,
[0030] 5-CGCAAGACCCTTCCTCTATATAA-3) and reverse prime...
Embodiment 2
[0035] Example 2 Detection of responses to sheath blight after transient expression of different truncated promoters from -1243bp to -1228bp on Nicotiana benthamiana
[0036] Cut off the leaves of Bunsen tobacco 5 days after the injection of Agrobacterium, spread them flat on the inoculation tray, remove the round bacterial blocks of the cultivated corn sheath blight bacteria YWK196 with a puncher, and place them upside down on the tobacco leaves, and keep them moist. After 24 hours of treatment at 25°C, the leaves at the inoculation point were taken for analysis.
[0037] The GFP fluorescence intensity results of the above-mentioned inoculated tobacco leaves are as follows: figure 1 As shown, after inoculation of YWK196 for 24 hours, compared with -1243 to -1228, the fluorescence intensity after deletion of GTTGA was reduced by half, while the fluorescence intensity of deletion of GGTGAGGCACT was comparable to that of deletion of GTTGA, and the remaining sequence TATTT mainta...
Embodiment 3
[0038] Example 3 Construction of recombinant vectors of GTTGA and TATTT tandem repeats and GFP gene and transient expression on Nicotiana benthamiana
[0039] In order to verify whether the individual GTTGA and TATTT can respond to the infection of sheath blight, these two elements were repeated twice in series, using the 35S basic promoter as a template, respectively using forward primers, the sequences of which are shown in SEQ ID NO.12 , 13, as shown in 6
[0040] (5-GTTGAGTTGACGCAAGACCCTTCCTCTATAA-3,
[0041] 5-TATTTTATTTCGCAAGACCCTTCCTCTATAA-3,
[0042] 5-CGCAAGACCCTTCCTCTATATAA-3) and reverse primer, its sequence is shown in SEQ ID NO.7
[0043] (5-GGGACTGACCTACCCGGG-3) amplifies the promoter fragment; at the same time, using the corn B73 genome as a template, respectively use forward primers, the sequence of which is shown in SEQ ID NO.14 (5-TTCTATGGCAAAATCAATGAAGG-3) and reverse primers, which The sequence is shown in SEQ ID NO.15 (5-TGGCGGTGACGATGGTAA-3) to amplify...
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