Construction method and application of rolling-circle replication recombinant vector for heterologous protein expression

Abstract

The invention belongs to the field of biotechnology and botany, and particularly provides a construction method of a rolling-circle replication recombinant vector for heterologous protein expression,the recombinant vector and an application of the recombinant vector. According to the construction method provided by the invention, the high-efficiency rolling-circle replication transient expressionvector mainly based on the sequence of a sweet potato leaf curl virus (SPLCV) is constructed, so that duration time and expression abundance of heterologous nucleic acids and proteins on plants are remarkably improved, and the results of the vector have high repeatability and reliability. The recombinant vector provided by the invention has a molecular tool vector which has an extremely long expression time to heterologous proteins (longer than 20 day compared with the expression time of a common transient expression T vector) and has an extremely high expression abundance (larger than 30-fold expression compared with a common transient expression T vector), and the recombinant vector endows different plants (tobaccos, sweet potatoes and morning glory) with heterologous protein expressioncapability, and makes up the blank of related plants for utilizing transient expression to study gene functions.

Description

technical field [0001] The invention belongs to the fields of biotechnology and botany, and more specifically relates to the development of a construction method, recombinant vector and application of a rolling circle replication recombinant vector for efficient and transient expression of heterologous proteins in different plants. Background technique [0002] Using plants to express heterologous proteins is a general research technique for studying plants. For example, in the study of plant functional genes, model plants (Arabidopsis, Nicotiana benthamiana) are used to express unknown genes of non-model plants to verify their gene functions; Plant reactors (spinach, tobacco) express certain medicinal proteins and separate and purify the target proteins. The best way to express heterologous proteins is to obtain transgenic plants capable of sustained and stable expression of exogenous genes through molecular cloning; however, due to factors such as complex vector constructi...

AI Technical Summary

Problems solved by technology

The best way to express heterologous proteins is to obtain transgenic plants capable of sustained and stable expression of exogenous genes through molecular cloning; however, due to factors such as complex vector construction and long transformation procedures (often 6-10 months) impact, which directly limits the use of the method

[0003] The construction of a transient expression vector with a T-DNA insertion region is an alternative for the study of plant heterologous protein expression. By transforming the constructed vector into Agrobacterium and directly infecting the plant with a syringe, the injection area can be kept within a period of time. Stable and continuous expression of the target protein, this method is simple to operate and highly time-sensitive, and the entire process may only take about 10 days to complete; however, this method also has some inherent defects, which directly limit the use of this method: (1) continuous The time is short, and the longest expression time of the protein can only be maintained for about 5-7 days; (2). The ability to express proteins with larger molecular weights is poor; (3) Excessive injection of bacteria can easily lead to hypersensitivity reactions and cause bruises in the injection area. Withered necrosis

Images