DNA (deoxyribonucleic acid) insertion fragment for down-regulating tomato YFT1 allele expression and application of DNA insertion fragment in tomato quality improvement
A technology for inserting fragments and tomatoes, applied in the field of molecular biology and genetics, to achieve the effect of improving tomato quality
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Embodiment 1
[0047] Example 1: Analysis of YFT1 / YFT1 allele promoter activity
[0048] To dissect 2 events of genetic damage leading to downregulated expression of the YFT1 allele gene in yft1 - IF 573 (Sequence shown in SEQ ID NO: 2) The biological role played by insertion and Del 13 deletion, the promoter pYFT1 of YFT1 and the promoter pyft1 of YFT1 allele of yft1 were respectively cloned from cv.M82 / yft1 by PCR technology, A series of promoter mutation vectors were constructed in order to analyze and clarify the impact of two mutation events on the down-regulated expression of YFT1 allele. The specific steps are as follows:
[0049] 1. Construction of detection promoter activity vector
[0050] Based on the upstream sequence of the YFT1 gene, primers [pYFT1-F: 5'-gatgttatcggggaggaagaaac-3' (SEQ ID NO: 13), pYFT1-R: 5'-tatttgatttaaatggttagcaagc-3' (SEQ ID NO: 14)] were designed and synthesized to amplify The upstream promoter sequence of YFT1 gene in cv.M82 and mutant yft1 was added,...
Embodiment 2
[0056] Example 2: Analysis of YFT1 allele genetic damage in mutant yft1
[0057] The significant down-regulation of the expression level of the yft1 tomato mutant YFT1 allele is due to the genetic damage of the gene, which occurred at -318bp upstream of the start codon (ATG): 1) a 573bp DNA fragment (sequence such as Shown in SEQ ID NO: 2) insertion and 2) a 13bp DNA sequence deletion (Del 13) caused by two mutation events, and in Gao L, Zhao W, Qu H, Wang Q and Zhao L.The yellow-fruited tomato 1 (yft1)mutanthas altered fruit carotenoid accumulation and reduced ethylene production asa result of a genetic lesion in ETHYLENE INSENSITIVE2. Theoretical and AppliedGenetics, 2016, 129:717-728 elaborated, while YFT1 allele genetic lesion in IF 573 and Del 13's role is not revealed; meanwhile, the IF 573 The source is also lack of experimental evidence, whether the yellow fruit phenotype of yft1 mutant tomato is caused by insertion in the regulatory region of YFT1 allele gene, or d...
Embodiment 3
[0071] Example 3: Functional complementation experiment of YFT1 gene
[0072] 1. Vector construction
[0073] 1) Construction of the overexpression vector YFT1-CDS: PCR technology was used to clone the CDS sequence of YFT1 [YFT1-CDS-F: 5′-atggagtctgaaactctgactagag-3′ (SEQ ID NO: 15); YFT1-CDS-R: 5′- caagacgaaagggggtgat-3' (SEQ ID NO: 16)]
[0074] The sequences of the amplification primers used are shown in and), which were constructed on the pHB plasmid (gifted by the laboratory of Professor Hongquan Yang of Shanghai Jiaotong University, Mao J, Zhang YC, Sang Y, Li QH, Yang HQ.2005.A role for Arabidopsiscryptochromes and COP1 in the regulation of stomatal opening. Proceedings of the National Academy of Sciences, USA 102, 12270-12275.) BamH I and Xba I sites, and named YFT1-CDS;
[0075] 2) Construction of knockdown vector YFT1-RNAi: design primers (RNAi-F: 5′-cacctaagccatccagatttatcaac-3′( SEQ ID NO: 18); RNAi-R: 5'-catgtcccccttcaattctgag-3' (SEQ ID NO: 19)), amplified w...
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