Sugarcane flowering regulatory protein ScFT-3 and encoding gene thereof
A technology of flowering regulation and sugarcane, applied in the fields of genetic engineering, plant genetic improvement, angiosperms/flowering plants, etc.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0086] Example 1 Homologous Cloning of Sugarcane Flowering Regulatory Protein Gene ScFT-3
[0087] 1. Extraction of total RNA
[0088] Using sugarcane variety Yuetang 93-159 as the material, take the first fully developed mature leaf (i.e. +1 leaf, the same below) outside the tip core leaf at the mature stage (October 2015), and cut 0.1-0.2 g Middle leaves, ground to powder with liquid nitrogen, using Trans Zol TM Plant kit (ET121), extract sugarcane total RNA according to the instruction manual.
[0089] 2. Synthesis of the first strand of cDNA
[0090] Using sugarcane total RNA as a template, Trans One-Step gDNA Removal and cDNASynthesis Super MIX Reverse Transcription Kit (AE311) synthesizes the first strand of cDNA. System components: Take 1 μg of sugarcane total RNA, 1 μl of Anchored Oligo (dT) 18 Primer (0.5 μg / μl), 10 μl of 2×ES Reaction Mix, RT / RI Enzyme Mix 1μl, gDNA Remover 1μl, RNase-free Water to make up to 20μl. Incubate reverse transcription at 42°C for ...
Embodiment 2
[0101] The acquisition of embodiment 2 sugarcane flowering regulation protein gene ScFT-3 3 ' and 5 ' UTR
[0102] Design 3'RACE outer gene-specific primers, 3'RACE inner gene-specific primers, 5'RACE outer gene-specific primers and 5'RACE inner gene-specific primers based on the determined target gene fragments, all using The RACE 5' / 3' Kit (Clontech Laboratories, Inc) kit was used to amplify the 3' and 5' ends of the target gene, and the specific operation steps refer to the RACE kit instructions. Methods as below:
[0103] The extraction method of total RNA was as described in Example 1. The synthesis of the first strand of cDNA was performed according to the instructions of the RACE kit, and the specific steps were as follows:
[0104] 3' cDNA first-strand synthesis
[0105] ①Prepare 3’reverse transcription synthesis cDNA reaction solution A (5.5μl): 5×First-Strand Buffer 4.0μl, DTT (100mM) 0.5μl, dNTPs (20mM) 1.0μl.
[0106] ②Prepare reverse transcription cDNA synthe...
Embodiment 3
[0132] Example 3 The full-length cDNA of the sugarcane flowering regulatory protein gene ScFT-3 and the acquisition of genomic DNA
[0133] Design full-length cDNA and genomic DNA primers according to the spliced sequence, and obtain a 858bp cDNA product (SEQ ID No.2) and 2924bp genomic DNA comprising a start codon (ATG) and a stop codon (TGA) by PCR amplification Sequence (SEQ ID No.3 in the sequence table), the PCR amplification product is detected by agarose gel electrophoresis with a mass percent concentration of 1.2%, and the target product is purified and recovered from the gel, and 4 μl of the recovered and purified PCR product is mixed with 1 μl Gently mix, react at room temperature for 5 minutes, connect with a PCR instrument at 25°C for 30 minutes, put the connection product in 50 μl Trans-T1 competent cells (add the connection product when the competent cells are just thawed), flick and mix, and ice-bath for 25 minutes , heat shock at 42°C for 40s, immediately pu...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More