Drought-resistance related key enzyme derived from Leymus chinensis (Trin.) Tzvel. as well as coding gene and application thereof
A technology of encoding genes and genes, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problems of difficult plant drought resistance and complicated plant drought resistance mechanism, and achieve the effects of improving drought resistance, broad application prospects, and high application value
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Embodiment 1
[0045] Example 1. Obtaining the full-length cDNA sequence of LcSAMDC1 gene of Leymus chinensis thioadenosylmethionine decarboxylase
[0046] 1. Cloning of the 3' end sequence of LcSAMDC1 from Leymus chinensis thioadenosylmethionine decarboxylase gene
[0047] 1. Plant material processing and total RNA extraction
[0048] Using Leymus chinensis (Jisheng No. 1, Jilin Province Jisheng Leymus chinensis Fine Variety Station, registration form for disclosure of genetic resources sources is shown in Table 2) seedlings as materials, total RNA was extracted after 6 hours of PEG treatment, and detected by 1% agarose gel electrophoresis. Such as figure 1 shown. The extracted RNA has two obvious electrophoresis bands, which are 28S RNA and 18S RNA from top to bottom, indicating that the total RNA with higher purity and integrity has been obtained.
[0049] 2. Cloning of the 3'-end sequence of LcSAMDC1 from Leymus chinensis thioadenosylmethionine decarboxylase gene
[0050] Find the co...
Embodiment 2
[0065] Example 2, Bioinformatics analysis of LcSAMDC1 and its encoded protein
[0066] 1. Sequence analysis of LcSAMDC1 gene and prediction of structure and function of its encoded protein
[0067] Utilize DNAMAN and OMIGA software to carry out bioinformatics analysis to the full-length cDNA sequence of LcSAMDC1 obtained in Example 1, this sequence full-length 1905bp, from the 5' end 555th-1733rd is ORF, coding consists of 392 amino acid residues The composition of the protein LcSAMDC1, the schematic diagram of the structure of LcSAMDC1 is shown in Figure 5 shown. It is estimated that its molecular weight is 42.881kDa, and its isoelectric point pI value is 4.617. Using the online Blast tool (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi) to analyze the domain of LcSAMDC1, the results show that the protein contains a typical SAM_decarbox domain, that is, sequence 1 in the sequence table from the amino group The 5th to the 339th amino acid residue of the terminal, such as Figure...
Embodiment 3
[0070] Example 3, Analysis of the expression pattern of LcSAMDC1 under drought conditions
[0071] Leymus chinensis seedlings grown normally for 8 weeks were treated with drought (20% polyethylene glycol 6000) for different time (0, 1, 3, 6, 12, 24 hours). The total RNAs of Leymus chinensis seedlings treated above were extracted, respectively, by RT-PCR (primers: LcSAMDC1:1:5'-AGGCATACGACTGCAACAACG-3'; 2:5'-ACGC-AGCAAACCACCTAGAGCT-3'; internal control Actin: 1:5 ′-TGGACTCTG-GTGATGGTGTGAG-3′; 2: 5′-GTGCTAAGGGAGGCAAGGATG-3′, the reaction conditions are: 94°C 4min; 94°C 30s, 60°C 30s, 72°C 30s, 25 cycles) and real-time quantitative PCR (primers are : LcSAMDC1: 1: 5′-CAACATTGTGGAGCAGGAGC-3′; 2: 5′-CAGAAAATCATCGCATCACT-CG-3′ internal reference Actin: 1: 5′-CCCATGCTATC-CTTCGTCTCGACCT-3′; 2: 5′-TCGTAGCTCTTTCTCACGGAGGAGC-3′, reaction The conditions are: 95°C for 10s; 95°C for 5s, 60°C for 20s, 72°C for 30s, 40 cycles) method to analyze the expression pattern of LcSAMDC1 gene under dr...
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