Plant inducible promoter and application thereof
A promoter and inducible technology, which is applied in the field of plant genetic engineering, can solve the problems such as the lack of systematic mining and utilization of dark inducible promoters, and achieve the effects of enhanced inducibility, broad application prospects and improved luminous intensity.
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Embodiment 1
[0054] Example 1 Identification of dark-inducible promoters
[0055] 1. Through the analysis of rapeseed dark-treated transcriptome data, a dark-inducible promoter (darkinduce element, DIE) was screened out, the nucleotide sequence of which is shown in SEQ ID NO.1.
[0056] Utilize PCR technology to amplify this promoter sequence, used F primer: 5'-TTATTGCCTAAGATTGATG-3', R primer: 5'-TTTTCTTATTTAATGCAAAGT-3', the promoter element that amplifies is connected into promoter analysis vector ( figure 1 ), after the sequencing was verified to be correct, it was transferred into Agrobacterium EHA105, and the EHA105 bacterial solution containing the verified correct vector plasmid was respectively streaked and activated on the LA+Kana+Rif plate, cultured at 28°C for 36h, and picked from the plate Colonies, transferred to LB+Kana+Rif+15μMAs medium, cultured at 28°C, 200rpm to OD 600 =0.8-1.0, 4000rpm, 10min to collect the bacterial cells, suspend the Agrobacterium bacterial cells wit...
Embodiment 2
[0059] Embodiment 2 Transformation of dark inducible promoter
[0060] We performed core sequence analysis on the Dark induce element (DIE) promoter screened in Example 1. According to the existing results, it was shown that the promoter part has a core element Gbox (CACGTG), which may be the core region of dark inducement. The core elements GATACCACGTGTATGAC and ATACAACACGTGGCAACA are designed to be repeated multiple times. The promoter element is named Engineered dark induce element (eDIE), and its nucleotide sequence is shown in SEQ ID NO.2.
[0061] According to the above sequence, entrust the company to synthesize the corresponding promoter fragment, and connect it into the promoter analysis vector ( image 3 ), after the sequencing verification was correct, it was transferred into Agrobacterium, and verified by the transient transformation experiment of tobacco leaves (the operation method is the same as that in Example 1).
[0062] The result is as Figure 4 As shown,...
Embodiment 3
[0063] Example 3 Construction of Dark-Inducible Luminescent Plant Transgenic Vector
[0064] According to the above results, the modified dark-enhanced promoter element eDIE was used to drive the expression of the Luz gene in the plant self-illumination system. In this operation, the TransGeneStacking II system developed by Academician Liu Yaoguang's research group of South China Agricultural University was used for multi-gene assembly, see application number It is a Chinese patent of 2017103841977, and pYL322d1, pYL322d2 and pYLTAC380GW were donated by the laboratory of Professor Liu Yaoguang of South China Agricultural University.
[0065] The NPGA (4'-phosphopantetheinyltransferase) gene in the Aspergillus nidulans genome, the H3H (hispidin-3-hydroxylase) gene in the Neonothopanus nambi genome and the Hisps (Hispidin synthase) gene in the genome of Aspergillus nidulans ), caffeoyl pyruvate hydrolase gene CPH (caffeoyl pyruvate hydrolase), and fungal luciferase (Luz) gene we...
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