Cabbage type rape BnKCS1-2 gene, vector constructed therefrom and application thereof
A cabbage rape, gene technology, applied in the application, genetic engineering, plant genetic improvement and other directions
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Embodiment 1
[0038] Embodiment 1: the cloning of rape BnKCS1-2 gene
[0039]1. Extraction of Rapeseed RNA
[0040] Select 100 mg of fresh leaves of Brassica napus Zhongshuang 11 (preserved by the Rapeseed Engineering Technology Center of Southwest University), grind with liquid nitrogen, and extract total RNA using TRIzol reagent method. The operation steps refer to the kit instructions of TransGen Biological Company.
[0041] 2. Synthesis of the first strand of cDNA using PrimeScript TM RT reagent Kit (TaKaRa) was used for synthesis, and the operation was carried out according to the instructions of the kit used.
[0042] 3. PCR amplification
[0043] Using Arabidopsis thaliana KCS1 gene sequence (AT1G02205) as a probe, searched for homologous sequences in the Brassica napus genome database, and obtained 4 related homologous sequences (SEQ ID NO:2, SEQ ID NO:3 , SEQ ID NO:4, SEQ ID NO:5). The following primers were designed according to the sequence of SEQ ID NO:2.
[0044] Forward ...
Embodiment 2
[0053] Example 2: Expression Analysis of Rapeseed BnKCS1-2 Gene
[0054] 1. Expression analysis of BnKCS1-2 gene in various tissues and organs of rapeseed
[0055] Extract the total RNA of each tissue and organ of double 11 in Brassica napus, then reverse transcribe the cDNA (the method is the same as in Example 1), use the first strand of the cDNA synthesized as a template, and express it using a real-time quantitative kit (Bio-Rad) analyze.
[0056] The primers used in fluorescent quantitative PCR are as follows:
[0057] The upstream primer is: 5'-AGTAAACTGCAGCTTATTCAATCCGAC-3',
[0058] The downstream primer is: 5'-TGTTGGCGAGATCGATTGAGATTAGT-3'.
[0059] The reaction system is as follows:
[0060]
[0061] The fluorescent quantitative PCR reaction program was: 95°C for 30s; 95°C for 10s, 54°C for 30s, 45 cycles; 65°C for 5s, 95°C for 30s. Actin7 (AT5G09810) was used as an internal reference gene to detect the expression level. The upstream internal reference primer...
Embodiment 3
[0067] Embodiment 3: Construction of BnKCS1-2 gene plant overexpression vector
[0068] Use BamHI and SacI to excise the above-mentioned BnKCS1-2 sequence verified by sequencing from the pMD-19T vector, and simultaneously use BamHI and SacI to digest the plant expression vector pCambia2301M1DPB (preserved by Rapeseed Engineering Technology Center of Southwest University), and then use T4 ligase to connect. The plant expression vector contains 1 set of plant expression elements of CaMV35S promoter controlling NPTII gene, 1 set of plant expression elements of CaMV35S promoter controlling reporter gene GUS, 1 set of plant expression elements of MAS promoter controlling BAR gene and a set of CaMV35S promoter Plant expression elements controlling target genes, enabling triple marker screening of Kan, GUS and Basta. Inserting exogenous genes into multiple cloning sites can achieve overexpression of exogenous genes. The recombinant plant overexpression vector of the successfully co...
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