Cabbage rape BnBDC1 protein coding sequence
A technology of Brassica napus and its coding sequence, which is applied in the field of protein coding sequence and Brassica napus BnBDC1 protein coding sequence, and can solve the problems of unclear gene salt tolerance, drought resistance and insect resistance, and ineffectiveness.
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Embodiment 1
[0068] Cloning of BnBDC1 Gene in Brassica napus
[0069] 1. Organization separation (isolation)
[0070] Brassica napus (variety "Huyou 15") was purchased from the market. The Brassica napus was germinated at 28°C for 24 hours, and then sown in the greenhouse. When the Brassica napus leaves were 3-5 slices, prepare to extract DNA or RNA.
[0071] 2. RNA isolation (RNA isolation)
[0072] Take part of the tissue, grind it with a mortar, add a 1.5mL EP tube containing the lysis solution, shake it enough, and then move it into a glass homogenizer. After homogenization, transfer to a 1.5mL EP tube to extract total RNA (TRIzol Reagents, GIBCO BRL, USA). Use formaldehyde denaturing gel electrophoresis to identify the total RNA quality, and then measure the RNA content on a spectrophotometer.
[0073] 3. Cloning of Full-length cDNA
[0074] According to the amino acid conservative sequence of Arabidopsis AtRD22 gene, using the principle of homologous gene cloning, the RACE method (GibcoB...
Embodiment 2
[0086] Sequence information and homology analysis of BnBDC1 gene in Brassica napus
[0087] The length of the new Brassica napus BnBDC1 full-length cDNA is 1326 bp, the detailed sequence is shown in SEQ ID NO. 3, and the open reading frame is located at nucleotides 42-1205 (1164 nucleotides). According to the full-length cDNA, the amino acid sequence of Brassica napus BnBDC1 was deduced, with a total of 387 amino acid residues, a molecular weight of 41411.51 Daltons, and an isoelectric point (pI) of 9.30. The detailed sequence is shown in SEQ ID NO.3.
[0088] The full-length cDNA sequence of Brassica napus BnBDC1 and its encoded protein were used BLAST program to carry out nucleotide and protein in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDStranslations+PDB+SwissProt+Superdate+PIR database A homology search revealed that it has 84% identity with the Arabidopsis gene AtRD22 at the nucleotide level (Table 2); at the amino acid level, it has 85 percent identi...
Embodiment 3
[0090] Eukaryotic expression of Brassica napus gene BnBDC1 protein or polypeptide in Arabidopsis thaliana and identification of drought and salt tolerance of transgenic plants
[0091] Construction of expression vector containing target gene (Brassica napus gene BnBDC1 gene)
[0092] According to the full-length sequence of Brassica napus gene BnBDC1 (SEQ ID NO.3), the primers that amplify the complete coding reading frame were designed, and restriction endonuclease sites were introduced on the upstream and downstream primers respectively (this can be optional Depending on the vector) in order to construct an expression vector. Using the amplified product obtained in Example 1 as a template, after PCR amplification, the Brassica napus gene BnBDC1 gene cDNA was cloned into an intermediate vector (such as pBluescript), and further cloned into a binary expression vector (such as pBI121 and improved pCAMBIA2300). ), under the premise of ensuring the correct reading frame, the identifi...
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