A Coral pds gene vigs silencing system and its construction method and application
A construction method and technology of coral vegetable, applied in application, genetic engineering, plant genetic improvement and other directions, can solve the problems of verification, short period, difficult callus induction and tissue culture, etc., and achieve the effect of low cost and short experimental period.
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Embodiment 1
[0029] Example 1: Construction of recombinant viral vector pTRV II-PDS
[0030] (1) Cloning of PDS interference fragment in Coralica
[0031] First, the phytoene dehydrogenase (phytoene desaturase, PDS) mRNA sequence information was obtained according to the gene annotation information of the coral dish transcriptome database, and a specific nucleotide fragment for silencing the PDS gene was designed, the sequence is as shown in SEQ ID NO. 1, and then design the primers used to clone the fragment:
[0032] PDS-F: 5'-GTCGCTAAACTTTATCAATC-3' (SEQ ID NO. 2);
[0033] PDS-R: 5'-CTTCAGTTTCCTGTCGAACC-3' (SEQ ID NO. 3).
[0034] RNA extraction kit (Novizan FastPure Platnt Total RNA Isolation Kit (Polysaccharides&Polyphenolics-rich)) was used to extract the RNA of Coralica leaf (coralica leaf was collected from the germplasm resources of the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province in May 2020). Garden), reversed to cDNA, using Coral cDNA as template, usin...
Embodiment 2
[0037] Example 2: VIGS infection of Coral leaf
[0038] The pTRV II-PDS and pTRV I plasmids were respectively transferred into GV3101 competent cells by electric shock method, and the transformed positive Agrobacterium colonies were picked, and then inserted into 5mL YEP resistant medium (containing 100mg / L rifampicin and 50mg / L) respectively. L kana antibiotics) at 28°C, 200 rpm for overnight cultivation, the bacterial solution was added to 50 mL of liquid resistance medium containing 200 μM acetosyringone (AS) at 1:25, cultured to OD 0.6, the bacterial solution was collected, and the bacterial solution was treated with 10 mM MgCl2 , 10mM MES and 400μMAS resuspension in equal proportions, mix the resuspended pTRV II-PDS and pTRV I bacterial solution 1:1, and let stand in the dark for 2-3 hours to infect the leaves of Coralica seedlings ;
[0039] Liquid resistance medium formula: 9.76g MES, 0.24g NaH2PO4, 5g glucose, 50mL 20×AB salt solution, deionized water to 1L, autoclave...
Embodiment 3
[0041]Example 3: Identification of the PDS expression level of Corsica chinensis after VIGS silencing
[0042] When the leaves of the plants to be infected have the phenotype of albino or white spot ( image 3 ), take fresh leaves, extract total RNA from leaves with RNA kit, use EXP1 as the internal reference gene, and detect the expression level of the target gene after being silenced by fluorescence quantitative PCR.
[0043] The primers used were:
[0044] PDS-qRT-F: 5'-CTTTTCCACTGCTTCCCGTC-3' (SEQ ID NO. 4);
[0045] PDS-qRT-R: 5'-CAGCCACCTTTCCACCTAGA-3' (SEQ ID NO. 5);
[0046] EXP1-RT-F: 5'-AGCCCTCCTGCATGTTTAGT-3' (SEQ ID NO. 6);
[0047] EXP1-RT-R: 5'-ACGAAGCTGGTCACTGTCAG-3' (SEQ ID NO. 7).
[0048] The results of PDS gene expression assay can be found in Figure 4 , compared with the control PDS-CK, the expression of PDS genes in the leaves (PDS-VIGS-1 and PDS-VIGS-2) after VIGS silencing was significantly reduced.
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