Eucommia ulmoides laccase EuLAC1 gene and application thereof

[0031] Example 1 Cloning and analysis of Eucommia laccase EuLAC1 gene

[0032] Use the RNA extraction kit RNA pure Kit of Kangwei Century Company to extract Eucommia RNA according to the instructions, measure the concentration of RNA with a spectrophotometer, select the RNA with a value of about 2.0, and synthesize cDNA according to the instructions of the reverse transcription kit of Takara Company , and then perform PCR amplification of the target fragment. The PCR system is shown in Table 1.

[0033] Table 1 PCR amplification system

[0034]

[0035] Wherein, forward primer and reverse primer are:

[0036] EuLAC1-F: 5'-ATGGGTTCTTGTATTCTTAAGGCAT-3',

[0037] EuLAC1-R: 5'-CTAACCTGGATTGTCTGCCC-3'.

[0038] Obtained PCR products were analyzed by agarose gel electrophoresis, as figure 1 As shown, a specific amplification band can be observed. Purify the target band according to the glue recovery kit of Omega Company, connect it with the pTOPO-Blunt vector according to t...

[0042] Example 2 Construction of expression vector and genetic transformation mediated by Agrobacterium

[0043] The above-mentioned recombinant plasmids were transformed into Agrobacterium LBA4404 competent cells by freeze-thaw method, and the plasmids of positive Agrobacterium colonies were extracted using a plasmid extraction kit and verified by KpnI and XbaI double enzyme digestion. The agarose gel was used for detection, and the electrophoresis result showed that the target band with the expected size of 1617bp was obtained, indicating that the recombinant plasmid had been contained in the Agrobacterium. The EuLAC1 gene overexpression vector pCAMBIA1300-35S-EuLAC1 was designed by the project team and constructed by Transduction Advanced (Wuhan) Biotechnology Co., Ltd. ( figure 2 ).

[0044] Transfer into Samsung tobacco by Agrobacterium-mediated leaf disc method, the steps are as follows: Prepare 100mL of Agrobacterium strains carrying the pCAMBIA1300-35S-EuLAC1 plasmid t...

[0045] Example 3 EuLAC1 Gene Tissue Expression Characteristics

[0046] Primer 5.0 software was selected, the cloned EuLAC1 gene sequence was used as a template, and primers suitable for EuLAC1 were selected according to the design principles of fluorescent quantitative PCR primers, and they were named EuLAC1-qtF and EuLAC1-qtR and sent to Huada Gene Technology Co., Ltd. Company Synthesis. EuLAC1-qtF: 5'-GCCCTATTCAGAAAGACCAGAC-3'; EuLAC1-qtR: 5'-TTGCCCTAAGCCACGATATG-3'. Eucommia Eucommia EuACTIN was selected as an internal reference gene to test the expression of Eucommia laccase gene EuLAC1 in the roots, stems and leaves of Eucommia ulmoides young trees. The expression of laccase gene EuLAC1 in Eucommia ulmoides plants was measured and analyzed by real-time fluorescent quantitative dye technology, using Select Master Mix kit, according to the kit instructions, carry out Real-time PCR step by step, calculate and analyze the expression level of genes according to the ΔΔCT me...