Efficient assembling method of transcription activator-like effectors (TALE) repeating region for editing silkworm genome and framework carrier thereof

Abstract

The invention discloses an efficient assembling method of a transcription activator-like effectors (TALE) repeating region for editing a silkworm genome. The efficient assembling method specifically comprises the steps of firstly numbering random sequences containing X basic groups sequentially and dividing the random sequences into Y groups according to numbers, wherein each group contains 1-4 basic groups; designing a primer pair for amplifying TALE repeating units, and then utilizing the designed primer pair to perform polymerase chain reaction (PCR) amplification with four types of regulatory volume decrease (RVD) repeating units as templates so as to obtain a PCR product library, wherein the primer pair contains II type restriction enzyme recognition sites and specific joints connected with adjacent TALE repeating units in a seamless mode; numbering and grouping target sequences with X basic groups by means of the same method, taking out PCR products with the same numbers as the target sequences and identifying corresponding basic groups from the PCR product library, mixing the PCR products in the same groups, performing GoldenGate connection, and then performing the GoldenGate connection of connection products again to obtain the TALE repeating region containing X repeating units. By utilizing the method, a large amount of deoxyribonucleic acid (DNA) is not required to be synthesized, and the cost is low. The efficient assembling method can be used for preparing the TALE repeating region for editing the silkworm genome.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a high-efficiency assembly method for editing the TALE repeat region of the silkworm genome, and also relates to a skeleton carrier used in the high-efficiency assembly method. Background technique [0002] After more than 5,000 years of artificial breeding and domestication, the silkworm has not only become an important and distinctive economic insect, but also has become an internationally recognized model insect of Lepidoptera with the development of modern molecular biology. In my country, the silk industry developed from the ancient mulberry planting and sericulture has a glorious history, and it has made continuous and great contributions to the economic development and cultural promotion of the Chinese nation. Silk is widely used in emerging biological industries such as textiles, clothing, accessories and even cosmetics due to its unique and excellent qualities in terms o...

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Problems solved by technology

In addition, the silk gland of the silkworm is the biological organ with the strongest protein synthesis ability among known insects. One silkworm can produce as much as 0.5g of silk protein, and its production cost is less than 1 dime

To establish a genome editing technology platform for artificial TALE nucleases and apply it to the large-scale editing and transformation of functional genes in silkworms, it is first necessary to design and synthesize corresponding artificial TALEN nucleic acid tool enzymes according to the sequence and number of target DNA bases, because these tool enzymes contain A large number of repetitive regions are difficult to synthesize through DNA synthesis technology, and it will also increase economic costs, which is not conducive to large-scale gene editing

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