Establishment method of caffeine synthetase CRISPR/Cas9 genome editing vector

Abstract

The invention relates to an establishment method of a caffeine synthetase CRISPR/Cas9 genome editing vector. The method comprises the following steps: designing two target sequences T1 and T2 on the basis of tea tree caffeine synthetase genes; respectively establishing sgRNA expression cassettes of the target sequences T1 and T2; and carrying out enzyme digestion-coupling reaction on a double-element expression vector pYLCRISPR/Cas9P35S-H, the T1sgRNA expression cassette and the T2sgRNA expression cassette so that the T1sgRNA expression cassette and the T2sgRNA expression cassette are connected and inserted into the double-element expression vector, thereby obtaining the CRISPR/Cas9 genome editing vector. By taking the tea tree caffeine synthetase as the example, conventional PCR (polymerase chain reaction), Overlapping PCR and Golden Gate Cloning techniques are combined to establish the CRISPR/Cas9 gene editing vector comprising the tea tree caffeine synthetase double targets, thereby laying a solid foundation for application of the CRISPR/Cas9 gene editing technique in tea trees, and providing technical supports for the establishment of CRISPR/Cas9 gene editing vectors of other plants.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for constructing a tea tree caffeine synthase CRISPR / Cas9 genome editing vector. Background technique [0002] Tea tree is an important woody economic crop in my country and occupies an important position in China's national economy. my country is not only the country with the richest tea resources in the world, but also the largest producer, consumer and trader of tea resources in the world. However, there are not many excellent tea tree varieties with multi-resistance and high quality in my country. Although my country has always attached great importance to the improvement of tea tree varieties, due to the long growth cycle and self-incompatibility of tea trees, it is difficult to achieve breakthroughs in tea tree breeding using conventional breeding techniques. In recent years, modern molecular biology techniques such as molecular breeding and genetic engineering have ...

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Problems solved by technology

[0007] However, so far, there have been no reports on the application of CRISPR / Cas9 technology in tea trees. One of the important reasons is that the construction technology of CRISPR / Cas9 gene editing vectors for tea trees is not yet perfect.

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