Mulberry micromolecule heat shock protein gene Mn16.8-CI promoter and recombinant expression vector and application thereof
A technology of mn16.8-ci, heat shock protein, applied in the field of molecular biology, can solve the problem of less research on the mechanism of mulberry stress resistance
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Embodiment 1
[0019] Example 1, Acquisition of Mn16.8-C Ⅰ promoter of Morus alba small molecule heat shock protein gene
[0020] 29 sHSPs were identified from the mulberry genome database. After analyzing the gene structure, transcriptional regulatory elements and tissue expression profiles of the 29 sHSPs, it was found that the promoter of the Mn16.8-C Ⅰ gene contained a complete heat shock element (HSE). Gene expression was upregulated under high temperature, low temperature, drought and high salt stress. In order to study the activity of the Mn16.8-C Ⅰ promoter, primers were designed from the upstream 1500bp promoter sequence of the Mn16.8-C Ⅰ gene, cloned and verified by sequencing. Using plant transgenic technology, insert it into the plant expression vector pBI121, the specific method is as follows:
[0021] According to the genome sequence of Chuanmulberry, the upstream 1500bp sequence of the Mn16.8-C Ⅰ gene was intercepted to design primers as primers for cloning the Mn16.8-C Ⅰ pro...
Embodiment 2
[0026] Example 2. Obtaining and Identification of Mn16.8-C Ⅰ Promoter Transgenic Tobacco
[0027] The constructed pBI121-Mn16.8-C Ⅰ expression vector was transformed into tobacco by leaf disk transformation method, and 5 transgenic tobacco plants were obtained after kan resistance screening, named T1-5 respectively. Genomic DNA was extracted from the leaves of 5 transgenic tobacco plants as a template, and the wild-type tobacco genome was used as a control, and PCR amplification was performed with the upstream primers of the Mn16.8-C Ⅰ promoter and the specific primers of the GUS gene. The sequences of the primers are as follows:
[0028] 5'-cgaggtacggtagaggttgg-3' (SEQ ID NO. 4);
[0029] PCR amplification conditions were pre-denaturation at 94°C for 4 minutes; denaturation at 94°C for 40 seconds, annealing at 60°C for 40 seconds, extension at 72°C for 2 minutes, and 30 cycles; final extension at 72°C for 10 minutes, and storage at 4°C; Agarose gel electrophoresis, the resu...
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