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CN-121294445-B - Soybean bidirectional promoter, recombinant vector, construction method and application thereof

CN121294445BCN 121294445 BCN121294445 BCN 121294445BCN-121294445-B

Abstract

The invention discloses a soybean bidirectional promoter, a recombinant vector and a construction method and application thereof, belonging to the technical field of plant genetic engineering. The soybean bidirectional promoter can drive gene expression in two directions simultaneously, and three genes are respectively connected in series at the front end and the rear end of the bidirectional promoter, so that six genes can be driven to express simultaneously by one promoter. And a method for obviously improving the expression level of more than six genes simultaneously is developed by combining the bidirectional promoter with a ZQ6 enhancement sequence and a 35S: mdMYB10 expression cassette, and a method for making plants emit obvious self-luminescence under dark conditions is developed based on the method. The soybean bidirectional promoter-mediated multi-gene efficient synergistic transformation method provided by the invention can be applied to the construction of a soybean multi-gene synergistic transformation system, and realizes the aspects of rapid polymerization of excellent characters, cultivation of new soybean germplasm with excellent characters and the like.

Inventors

  • CAI YUPENG
  • HOU WENSHENG
  • CHEN LI

Assignees

  • 中国农业科学院作物科学研究所

Dates

Publication Date
20260505
Application Date
20251202

Claims (7)

  1. 1. The soybean bi-directional promoter SXPRO is characterized in that the nucleotide sequence of the soybean bi-directional promoter SXPRO10 is shown in SEQ ID NO. 1.
  2. 2.A recombinant vector containing the soybean bi-directional promoter SXPRO as claimed in claim 1, which is characterized by further comprising a ZQ6 enhancement sequence and a 35S MdMYB10 expression cassette, wherein the nucleotide sequence of the ZQ6 enhancement sequence is shown as SEQ ID NO.4, and the nucleotide sequence of the 35S MdMYB10 expression cassette is shown as SEQ ID NO. 5.
  3. 3. The method for constructing a recombinant vector according to claim 2, comprising the steps of: (1) Double enzyme digestion is carried out on the vector PTF101-GFP-RUBY and the vector Blunt-Zero-SXPRO by using restriction enzymes SpeI and NcoI respectively, and the target fragment is recovered by gel electrophoresis; (2) The target fragment obtained in the step (1) is connected by using T4 DNA ligase and then is transformed into escherichia coli to prepare a recombinant expression vector PTF 101-GFP-SXPRO-RUBY; (3) Integrating ZQ6 enhancement sequences at two ends of SXPRO promoter of PTF101-GFP-SXPRO10-RUBY vector to obtain intermediate vector SXPRO-ZQ 6-FAGUANG; (4) Respectively introducing AscI and PmeI enzyme cutting sites at two ends of a 35S: mdMYB10 expression cassette sequence, then carrying out double enzyme cutting on the two ends by using restriction enzymes AscI and PmeI, and inserting the enzyme-cut 35S: mdMYB10 expression cassette sequence into a SXPRO-ZQ 6-FAGUANG vector by using T4 DNA ligase to prepare the vector; the preparation method of the carrier Blunt-Zero-SXPRO (Blunt-Zero-SXPRO) 10 comprises the following steps: ① Amplifying the soybean bidirectional promoter SXPRO by using DNA of soybean yellow 13 variety as a template and using primers SXPRO-F and SXPRO-R, and introducing enzyme cutting sites NcoI and SpeI at two ends of SXPRO sequence respectively; ② Separating the product obtained in the step ① by adopting 1% agarose gel electrophoresis, recovering a target band by using the gel, connecting the target band to a Cloning vector of TA/Blunt-Zero Cloning Kit, converting escherichia coli Fast-T1, and determining that the target sequence on the connection is correct after sequencing to obtain a Blunt-Zero-SXPRO vector; the nucleotide sequence of the soybean bi-directional promoter SXPRO is shown as SEQ ID NO. 1; the nucleotide sequences of the primer SXPRO-F and the primer SXPRO-R are respectively shown as SEQ ID NO. 2-3.
  4. 4. Use of the recombinant vector of claim 2 for driving expression of a gene of interest in plants.
  5. 5. A method for driving expression of a gene of interest in a plant, comprising the steps of: (1) Constructing a recombinant vector by the method of claim 3, and then performing double digestion; (2) Connecting target genes in series through P2A connecting peptide, respectively introducing enzyme cutting sites at two ends of a target gene sequence, and then carrying out enzyme cutting; (3) Respectively connecting the enzyme digestion products of the step (2) to the front end and the rear end of the SXPRO promoter of the recombinant vector of the step (1), wherein the nucleotide sequence of the SXPRO promoter is shown as SEQ ID NO. 1; (4) And (3) transferring the product obtained in the step (3) into agrobacterium tumefaciens by a freeze thawing method, and then carrying out agrobacterium injection to infect plants.
  6. 6. Use of the recombinant vector of claim 2 for improving plant traits, growing transgenic plants or growing new varieties of plants.
  7. 7. A method for preparing luminous tobacco, which is characterized by comprising the following steps: (1) The BnC' H1, anNPGA and NnH H-v2 genes are connected in series through P2A connecting peptide, then NcoI and StuI enzyme cutting sites are respectively introduced at two ends of the series sequence, after the PTF 101-GFP-SXPRO-RUBY vector of claim 3 is subjected to double enzyme cutting by adopting NcoI and StuI, the series genes are connected to the front end of SXPRO promoter of the PTF 101-GFP-SXPRO-RUBY vector; (2) Connecting NnLuz-v4, nnCPH and MCITHISPS genes in series through P2A connecting peptide, and then respectively introducing XbaI and HpaI enzyme cutting sites at two ends of the series sequence, wherein after the PTF 101-GFP-SXPRO-RUBY vector obtained in the step (1) is subjected to double enzyme cutting by using XbaI and HpaI, the series genes are connected to the rear end of a SXPRO promoter of the PTF 101-GFP-SXPRO-RUBY vector; (3) Transferring the recombinant vector obtained in the step (2) into agrobacterium tumefaciens by a freeze thawing method, and then dip-dying tobacco to obtain the recombinant vector; Wherein the nucleotide sequence of the SXPRO promoter is shown as SEQ ID NO. 1.

Description

Soybean bidirectional promoter, recombinant vector, construction method and application thereof Technical Field The invention relates to the technical field of plant genetic engineering, in particular to a soybean bidirectional promoter, a recombinant vector, a construction method and application thereof. Background In crop molecular breeding and functional genome research, the development of a polygenic cooperative transformation and character rapid polymerization technology is a core direction for breaking through the bottleneck of the traditional genetic improvement technology. The traditional single-gene transformation system is difficult to adapt to the multi-gene regulation network characteristics of complex agronomic characters such as crop yield, stress resistance, quality and the like, and synchronous introduction and collaborative expression of a plurality of functional genes cannot be realized. At present, the cauliflower mosaic virus CaMV 35S promoter and the corn UBI promoter are used as stable and efficient exogenous gene expression regulatory elements, and are still common tools in the field of plant genetic engineering. However, in transgenic operation, repeated DNA sequences are prone to cause gene silencing, and the phenomenon is remarkably aggravated in polygenic transformation, so that the acquisition of transgenic plants and the genetic stability of offspring are seriously affected. When two or more exogenous genes are introduced, the genes are ensured to be consistent in expression level and space-time expression mode, so that the normal biological functions of the genes can be ensured. The prior researches prove that the CaMV 35S promoter and the corn UBI promoter are difficult to meet the regulation requirement, and the related technical difficulty becomes a key bottleneck which needs to be broken through in the plant genetic engineering field. The development of genomics has driven the completion of a variety of biological genomic sequencing, and bioinformatics analysis has confirmed the presence of bi-directional promoters. This provides the possibility to exploit plant endogenous bi-directional promoters to develop transgenic operations and to circumvent gene silencing by multiple gene insertion. The bidirectional promoter can drive two genes to reversely transcribe only by 1 element, thus greatly reducing the repeated sequence in the vector and reducing the gene silencing risk caused by the repeated sequence. The characteristics of the cis-regulatory elements are shared, so that the high synchronization of the double genes on the expression level and the time-space mode can be ensured, and the requirement of the coordinated expression of multiple genes in metabolic pathways or signal pathways can be met. In the construction of the vector, the promoter does not need to construct an expression cassette for a plurality of genes independently, can synchronously drive the expression of the plurality of genes, obviously shortens the length of the vector, and improves the stability of the vector and the transformation efficiency of agrobacterium. Part of bidirectional promoters also have tissue-specific or induced response characteristics, and can realize the synergistic expression of double genes under specific conditions. The soybean is used as an important grain and oil crop, the endogenous bidirectional promoter of the soybean is mined and applied to the efficient synergistic expression of multiple genes, and the soybean has important significance for promoting the biological breeding process. Disclosure of Invention In order to solve the defects in the prior art, the invention aims to provide a soybean bidirectional promoter, a recombinant vector, a construction method and application thereof, so as to provide a soybean endogenous bidirectional promoter which is applied to efficient collaborative expression of multiple genes and improve the stability of the vector and the transformation efficiency of agrobacterium. The technical scheme for solving the technical problems is as follows, and the soybean bi-directional promoter SXPRO is provided, and the nucleotide sequence of the soybean bi-directional promoter is shown as SEQ ID NO. 1. The invention provides a recombinant vector containing the soybean bi-directional promoter SXPRO, which also comprises a ZQ6 enhancement sequence and a 35S: mdMYB10 expression cassette. Further, the nucleotide sequence of the ZQ6 enhancement sequence is shown as SEQ ID NO.4, and the nucleotide sequence of the 35S: mdMYB10 expression cassette is shown as SEQ ID NO. 5. The invention provides a construction method of the recombinant vector, which comprises the following steps: (1) Double enzyme digestion is carried out on the vector PTF101-GFP-RUBY and the vector Blunt-Zero-SXPRO by using restriction enzymes SpeI and NcoI respectively, and the target fragment is recovered by gel electrophoresis; (2) The target fragment obtained in the step (1) is connecte