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Embedded RbcS cTP gene and expression vector and application thereof

A chimeric, genetic technology, applied in the field of genetic engineering, can solve problems such as not very clear

Active Publication Date: 2016-09-21
江苏南京农大科技开发有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the functions of the domains and partial motifs of the cTP of Arabidopsis RbcS are relatively clear, but in the cTP of monocot RbcS, how the domains and different motifs function are not very clear, especially Whether the cTP of monocotyledonous and dicotyledonous plants has species specificity has been rarely reported so far

Method used

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  • Embedded RbcS cTP gene and expression vector and application thereof
  • Embedded RbcS cTP gene and expression vector and application thereof
  • Embedded RbcS cTP gene and expression vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Cloning of RbcScTP gene and vector construction

[0030] Maize is a monocot and its genome has been sequenced. The maize RbcScTP gene was identified by the first applicant (inventor) Zhang Wenli according to mDNase-seq (modified DNase-seq) to identify DHSs (DNase I hypersensitive sites), combined with RNA-seq analysis to obtain candidate promoters / enhancers, and then use As a reporter gene, GFP was screened from these candidate promoters / enhancers by protoplast transformation. Use blast to find the homologous genes of Arabidopsis on NCBI.

[0031] Firstly, primers F1CGATAAGGCGACAAGTGGTG (SEQ ID NO.5) / R1: CTGCATGCACCGGATCCTT (SEQ ID NO.6) and F2: AGTAGTAATGGCTTCCTCTATGC (SEQ ID NO.7) / R2: ACCTTCATGCAGCTAACTCTTC (SEQ ID NO.8) were obtained from maize and A. The RbcScTP gene in the mustard genome was cloned into the pMD19-T vector respectively, and the sequences were compared by NCBI after sequencing. 然后用玉米RbcScTP基因引物F3:GCCCTTGCTCACCATGGATCCATGGCGCCCACCGTGATGAT(...

Embodiment 2

[0032] Example 2 Protoplast Extraction and Transformation

[0033] The extraction and transformation methods of protoplasts mainly refer to published methods (references: Yang Zhang, Jianbin Su, etc., A highly efficient rice green tissue protoplastsystem for transient gene expression and studying light / chloroplast-related processes, Plant Methods, 2011), and Protoplasts from maize, rice, wheat and Arabidopsis were extracted by this method with appropriate modifications.

[0034] step:

[0035] 1) Cultivate the green seedlings of normal growth, choose the seedlings of corn, rice and wheat for about ten days, and the seedlings of about three weeks in the south, and purify and transform two kinds of transient expression plasmids 20ul (1ug / ul) constructed in Example 1 simultaneously. ).

[0036] 2) Prepare 10ml of enzyme solution and heat at 55°C for 10min.

[0037] 3) Cut the leaves (corn, wheat and Arabidopsis) or stems (rice) to about 0.5-1mm with a blade, and immediately im...

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Abstract

The invention discloses an embedded RbcS cTP gene and an expression vector and application thereof. The gene is formed in the mode that the N-end of arabidopsis RbcS cTP is inserted between the N-end of corn RbcS cTP and an intermediate structure domain, and the cDNA sequence is shown as SEQ ID NO.2. The cDNA sequence of a corn RbcS cTP gene is shown as SEQ ID NO.1. The embedded RbcS cTP gene can be applied when exogenous genes are transferred into chloroplast of monocotyledon and / or dicotyledon.

Description

technical field [0001] The invention belongs to the field of genetic engineering and discloses a chimeric RbcS cTP gene and its expression vector and application. Background technique [0002] Chloroplasts are important organelles for green plants to convert light energy into chemical energy. Chloroplasts can only synthesize part of the proteins they need. Many chloroplast proteins are encoded by nuclear genes, which are translated into mature functional proteins or proteins in the cytoplasm Precursors are then imported into chloroplasts to perform their respective functions. Peptides play a very important role in this process. The transport of most chloroplast proteins from the cytoplasm to chloroplasts is mediated by transit peptides (TP). The transit peptide is generally located at the N-terminus of the transported protein. In the primary structure of amino acid sequence, TP can be divided into three structural domains, which are N-terminal domain, intermediate domain ...

Claims

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Application Information

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IPC IPC(8): C12N15/60C12N9/88C12N15/82A01H5/00
CPCC12N9/88C12N15/8214C12Y401/01039
Inventor 张文利汪西蒙方圆王磊潘秀才
Owner 江苏南京农大科技开发有限责任公司
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