Application of smsip1 protein and its related biomaterials in promoting the degradation of squalene synthase
A biomaterial, alkene synthase technology, used in biochemical equipment and methods, cells modified by introducing foreign genetic material, enzymes, etc.
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Embodiment 1
[0080] Example 1. Obtaining and verification of SmSIP1 protein interacting with squalene synthase from Salvia miltiorrhiza
[0081] 1. Construction and evaluation of Salvia miltiorrhiza two-hybrid cDNA library
[0082] 1. Selection of experimental materials
[0083] The present invention selects 2-year-old Salvia miltiorrhiza plants as research materials, collects samples from April 2008, and regularly collects them on the 10th, 20th, and 30th of each month until the end of July, and stores them at -80°C for later use. It was identified as Salvia Miltrorrhiza of Labiatae by researcher Huang Luqi from China Academy of Chinese Medical Sciences.
[0084] 2. Construction and evaluation of cDNA library
[0085] Use the Trizol kit to extract the RNA of the salvia miltiorrhiza collected in each month, mix equal amounts and separate the mRNA, then use the salvia miltiorrhiza total RNA as a template and oligo(dT) as a primer to reverse transcribe and synthesize single-stranded cDNA, ...
Embodiment 2
[0168] Example 2, Prokaryotic expression of SmSIP1 protein
[0169] 1. Design of primers
[0170] The corresponding primers were designed according to the restriction site information of pMAL-c2 vector and the sequence of SmSIP1. The primer sequences are as follows:
[0171] pMAL-c2-SmSIP1-BamHI-F: 5'-TTTTTTGGATCCATGGGAAACACTCTGCAAAAGCT-3', containing BamHI restriction site;
[0172] pMAL-c2-SmSIP1-PstI-R: 5'-TTTTTTCTGCAGTCATCTGAAGCACCATTCGGAAC-3', containing PstI restriction site.
[0173] 2. Construction of expression vector
[0174] Insert the SmSIP1 sequence shown in the 1-729 position of sequence 1 with the transmembrane region removed between the BamHI and PstI restriction sites of the pMAL-c2 vector, and keep other sequences of the pMAL-c2 vector unchanged to obtain the recombinant vector pMAL- c2-SmSIP1DT.
[0175] 3. Expression of mbp-SmSIP1DT fusion protein
[0176] Transfer the recombinant vector pMAL-c2-SmSIP1DT into Escherichia coli E.coli BL21 by the heat s...
Embodiment 3
[0183] Example 3, Analysis of ubiquitination activity of SmSIP1 protein
[0184] In vitro ubiquitination reaction in the presence of ATP energy, wheat E1 (with E1 ubiquitin activating enzyme activity), Arabidopsis E2 (with E2 ubiquitin conjugating enzyme activity) and ubiquitin protein (His-Ub purified protein) , to analyze the ubiquitination activity of SmSIP1 protein. Specific steps are as follows:
[0185] 1. Preparation of ubiquitin system
[0186] The ubiquitin system was prepared and divided into control group, -E1 group, -E2 group, -ub group, -E3 group and experimental group according to the different components in the ubiquitin system. The components of each group are as follows (Table 2):
[0187] (1) Control group: 1uL MBP protein, 1.5uL 20×reaction buffer (1M Tris pH 7.5, 40mMATP (sigma), 100mM MgCl 2 , 40mM DTT), 3uL wheat El (wheat El protein expression crude extract, about 50ng), 3uL Arabidopsis E2 (Arabidopsis E2UBC32 protein expression crude extract, about ...
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