Chimonanthus praecox CpWRI-L4 gene as well as encoded protein and application thereof
A technology of cpwri-l4 and wintersweet, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problems of late discovery and less research
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0036] Example 1 Cloning and Molecular Characterization of Wintersweet CpWRI-L4 Gene
[0037] Sequence analysis was carried out on the CpWRI-L4 gene obtained from the transcriptome to design specific primers, and the first strand of Chimera cDNA was used as a template to amplify the CpWRI-L4 gene. The primer sequences for PCR reactions were as follows:
[0038]
[0039]
[0040] Using CpWRI-L4 specific primers to carry out PCR amplification, the results of PCR amplification of the open reading frame of Wintersweet CpWRI-L4 gene were detected by electrophoresis, and the results showed that the target band was specific (such as figure 1 ). The PCR product was cloned into the pMD19-T vector, transformed into Escherichia coli, detected with CpWRI-L4-F (10 μM) and CpWRI-L4-R (10 μM) specific primers, and sequenced the single clone detected as positive by PCR. The sequence is as follows: Shown in SEQ ID No.1.
[0041] Online analysis with EditSeq and ProtParam showed that th...
Embodiment 2
[0043] Example 2 Analysis of the expression characteristics of Wintersweet CpWRI-L4 gene
[0044] The plant material used in this experiment was 'Suxin' wintersweet (C. praecox 'Concolor'). The stems, leaves, fruits, and various flower organ materials used are all collected from adult Wintersweet plants, and the plant materials treated with abiotic stress use four-leaf stage Wintersweet seedlings with roughly the same growth. The above wintersweet plants were all planted on the campus of Southwest University, and all were managed routinely.
[0045] Total RNA of each sample was extracted according to a commercially available kit, and reverse-transcribed into cDNA.
[0046] The Actin and Tublin genes of Wintersweet were selected as the double internal reference genes for qPCR analysis of Wintersweet CpWRI-L4 gene, and Primer Premier 5.0 software was used to design the internal reference gene and qPCR specific primers for the target gene CpWRI-L4, and then sent to Beijing Liuhe...
Embodiment 3
[0059] Example 3 Genetic Transformation and Functional Analysis of Wintersweet CpWRI-L4 Gene in Arabidopsis
[0060] The most suitable enzymes KpnI and SalI were selected by combining the restriction enzyme cutting sites and distribution characteristics contained in the CpWRI-L4 gene ORF box sequence itself and the characteristics of the multiple cloning sites contained in the plant overexpression vector pCAMBIA1300 used. Restriction sites and corresponding protective bases are added upstream and downstream of the primers, which are used to amplify the CpWRI-L4 gene coding region carrying suitable restriction sites and clone it into the multiple cloning site of the plant expression vector. The primer names and sequences are as follows:
[0061] p-CpWRI-L4-F:
[0062] p-CpWRI-L4-R:
[0063] Using the bacteria liquid sequenced correctly in Example 1 as a template, PCR amplification was carried out with specific primers with enzyme cutting sites (KpnI and SalI respectiv...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More