Gene for regulating and controlling sugar content of tomato fruits and application thereof
A technology for sugar content, tomato, applied in the fields of biotechnology and genetic breeding
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Embodiment 1
[0028] Construction of RNAi vector and recombinant Agrobacterium
[0029] The SlCIN2-RNAi vector was constructed using gateway technology.
[0030] 1. Extract tomato RNA, and reverse transcribe it into cDNA, use cDNA as template to amplify the target fragment SlCIN2 gene, the target fragment size is 170bp ( figure 1 A), its nucleotide sequence is shown in SEQ ID NO.1;
[0031] The primer sequences are as follows:
[0032] SlCIN2-F: 5'-CGCACCTGGTCCATTGTGTCGTCTT-3', shown in SEQ ID NO.2.
[0033] SlCIN2-R: 5'-GGTGTGTTCTTAGGGTCGCT-3', shown in SEQ ID NO.3.
[0034] The amplification reaction system is as follows:
[0035] High-fidelity DNA polymerase 10 μL, SlCIN2-F 1 μL, SlCIN2-R 1 μL, cDNA 2 μL, RNase-FreeddH2O 6 μL.
[0036] The amplification reaction procedure is as follows:
[0037] 98°C for 30s, 1 cycle; 98°C for 10s, 55°C for 20s, 72°C for 10s, 35 cycles; 72°C for 7min.
[0038] 2. Through TOPO reaction, take 4 μL of the amplification product of the target fragment,...
Embodiment 2
[0040] Transformation of tomato with recombinant Agrobacterium
[0041] Using Micro-Tom tomato as a test material, the recombinant vector plasmid was transformed into tomato by the leaf disc method to obtain stable genetic transformation materials.
[0042] When the tomato seedlings in the medium grow until the cotyledons are fully stretched ( figure 2 A), cut the 0.5cm long cotyledons and place them in the pre-medium for dark culture for 2 days, use OD 600 Agrobacteria reaching 0.6-0.8 infect the tomato cotyledons, and after infection, the back of the leaf is placed in the co-culture medium ( figure 2 B), after dark culture for 2 days, the explants were transferred to antibacterial medium for cultivation. When the explants were grown therein for two weeks, they were transferred to the germination medium and cultivated ( figure 2 C), after which the culture medium was changed every two weeks. When the adventitious buds grow to 2-3cm, cut the adventitious buds from the b...
Embodiment 3
[0044] Identification of Transgenic Tomato Plants Silencing SlCIN2
[0045] The DNA of the transgenic tomato leaves was extracted, and the screened silent SlCIN2 transgenic plants were identified by PCR. SlCIN2 silenced plants were detected using the Bar gene site on the pB7GWIWG2 vector plasmid to design specific primers.
[0046] The specific primer sequences are as follows:
[0047] Bar-F: 5'-GAAGTCCAGCTGCCAGAAA-3', as shown in SEQ ID NO.4.
[0048] Bar-R: 5'-CACCATCGTCAACCACTACA-3'T, as shown in SEQ ID NO.5.
[0049] Using DNA as a template for PCR amplification and electrophoresis detection, the detected specific fragment length is 439bp. As a result, it was found that a specific fragment of 439bp was detected in the 10 transgenic lines ( image 3 ), while negative controls (ddH2O and untransformed wild-type Micro-Tom) did not amplify any bands. These 10 lines are all transgenic positive plants and can be used for subsequent test analysis.
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