Galactosyl transferase and encoding genes and application thereof
A technology of galactosyl and transferase, which is applied in the field of genetic engineering, can solve the problems of no biological activity and insolubility, and achieve the effect of improving resistance
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Embodiment 1
[0034] Example 1 Isolation and Cloning of Dendrobium officinale DoGALT Gene
[0035] (1) Extraction of total RNA from Dendrobium officinale and synthesis of the first strand of cDNA
[0036] Take 0.1 g of just-harvested stems of Dendrobium officinale (Dendrobium officinale, planted in the South China Botanical Garden, Chinese Academy of Sciences, Dendrobium officinale, N23°10′, E113°21′, Guangzhou, China), cut into 1-2mm segments, and place in liquid nitrogen Grind it into powder. The total RNA of Dendrobium officinale was extracted using the column plant RNAout2.0 kit (Beijing Tianenze Gene Technology Co., Ltd., article number: 90404-50). Using NanoDrop TM 2000c ultramicro spectrophotometer (Thermo Scientific, Wisconsin, USA) and 1.0% agarose gel electrophoresis (Biorad, California, USA) were used to determine the content and purity of total RNA. Aspirate 2 μg of purified total RNA, and synthesize the first strand of cDNA according to the instructions of the Reverse Trans...
Embodiment 2
[0046] Embodiment 2 Dendrobium officinale DoGALT gene expression pattern analysis
[0047] Harvest the roots, stems, leaves and flowers of Dendrobium candidum seedlings in the growth period and flowering period, and perform RNA extraction and reverse transcription reaction with reference to the method of Example 1-(1). Fluorescent quantitative PCR reaction designed specific primers RTGALTF and RTGALTR according to the conserved region of the DoGALT gene, using SYBR Premix Ex Taq TMKit was purchased from Takara Company (product number: DRR420A) for PCR amplification. The reaction program was denaturation at 95°C for 2 min, followed by 40 cycles of reaction (95°C for 15 s, 60°C for 1 min). The internal reference gene is the DoActin gene of Dendrobium officinale, and the NCBI accession number is JX294908. The data were obtained after the reaction was run by ABI 7500 real-time fluorescent quantitative PCR (Applied Biosystems, California, U.S.), using 2 -ΔΔCt The method is calcu...
Embodiment 3
[0050] Example 3 The acquisition and identification of transgalactosyltransferase gene DoGALT Arabidopsis
[0051] (1) Construction of Dendrobium officinale galactosyltransferase gene DoGALT overexpression vector
[0052] According to the sequence on the pCAMBIA1302 vector and the full length of the galactosyltransferase gene DoGALT, a pair of primers OEGALTF and OEGALTR were designed, the specific sequences of which are shown in SEQ ID NO: 13-14.
[0053] Using the cDNA of Dendrobium officinale as a template, the full-length galactosyltransferase gene DoGALT was amplified with high fidelity. After the amplified product was purified, the target fragment was recovered, and the expression vector pCAMBIA1302( Figure 7 ). Using Takara's The HD Cloning Kit kit is used to construct plant expression vectors, and the specific operation method is detailed in the operation manual. The pCAMBIA1302 vector contains the CaMV 35S promoter, the galactosyltransferase gene DoGALT (as show...
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