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Controlling sites of miR528 and applications thereof

A technology of os03g03724, rice, applied in the direction of application, DNA/RNA fragments, use of vectors to introduce foreign genetic material, etc.

Inactive Publication Date: 2016-03-30
INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the process of pest control, modern agriculture is facing a huge contradiction: on the one hand, the use of pesticides plays an important role in ensuring the stable production of crops; on the other hand, excessive use of pesticides also brings great risks to the environment and food safety

Method used

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  • Controlling sites of miR528 and applications thereof
  • Controlling sites of miR528 and applications thereof
  • Controlling sites of miR528 and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1. Acquisition of miR528 recognition site

[0028] Using the principle of sequence complementarity between plant miRNA and target mRNA, we obtained the recognition site of miR528 in rice through genome-wide scanning of rice. There are five target mRNAs of miR528 in rice, which are transcribed from Os06g06050, Os06g37150, Os08g04310, Os08g46240, and Os07g38290 genes, respectively. The recognition site of miR528 is a nucleotide sequence with a length of about 21 nt, and there is a good sequence match between miR528 and the target mRNA at the corresponding recognition site, see the attached figure 1 .

Embodiment 2

[0029] Embodiment 2, MIR528 Construction of gene overexpression vector

[0030] This example is about pCAMBIA2300- pACTIN1-MIR528 Vector builds a generic description.

[0031] First, using Nipponbare rice DNA as a template, design primers to match it, and add PstI restriction site sequences to the 5' ends of the forward and reverse primers respectively, and use high-fidelity DNA polymerase PCR amplification MIR528 gene sequence. The PCR product was recovered by electrophoresis, connected to the cloning vector P-EASYblunt, transformed into Escherichia coli DH5α, and plated to obtain a single clone. Select clones with correct sequencing to extract plasmids, digest with PstI, and recover MIR528 fragment; at the same time, the vector pCAMBIA2300 was digested overnight with PstI, and the end was dephosphorylated and recovered. will be recycled MIR528 Fragments and carrier fragments are connected at a molar ratio of about 3:1 (T 4 Ligase (NEB?)) overnight. The next da...

Embodiment 3

[0032] Example 3, Identification of miR528 Knockout and Overexpression Rice Lines

[0033] By searching the rice database RiceGE (http: / / signal.salk.edu / cgi-bin / RiceGE), we found a rice line whose T-DNA insertion site was located in the miR528 precursor (pre-miR528), we will its named mir528 . Small RNA Northern hybridization results showed that, mir528 miR528 was completely knocked out in the strain, see appendix image 3 . In addition, we will contain a rice endogenous strong promoter ACTIN1 drive MIR528 sequential pCAMBIA2300 carrier ( pCAMBIA2300-pACTIN1-MIR528 ) was transformed into rice, and multiple independent transgenic lines were identified and obtained. The mature results showed that the expression level of miR528 in the transgenic lines was up-regulated, see attached image 3 .

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Abstract

The invention relates to applications of a coding gene MIR528 of rice OsmiR528 and OsmiR528 controlling sites (Os06g06050, Os06g37150, Os08g04310, Os08g42640 and Os07g38290) in aspects of controlling rice growth stages and virus resistance. By researching OsmiR528 overexpression and rice strain knockout, the inventor found that the OsmiR528 influences rice heading stages and has a capability of resisting a rice stripe virus (RSV), namely overexpression of the OsmiR528 can result in an earlier heading stage and sensitivity to the RSV, and knockout of the OsmiR528 can result in a delayed heading stage and an anti-RSV capability. The MIR528 exists in different rice varieties and directly influences regulatory region and sequence polymorphism of an expression level of the OsmiR528. The above-mentioned results can be applied for rice crossbreeding and marker-assisted selection.

Description

Technical field: [0001] The present invention relates to plant breeding, in particular to rice miR528 coding gene MIR528 And the application of miR528 regulatory sites (Os06g06050, Os06g37150, Os08g04310, Os08g42640 and Os07g38290) in cultivating crop varieties with suitable growth period and high virus resistance. technical background: [0002] Flowering time (or heading in cereal crops) is the transition of a plant from vegetative to reproductive growth, and in order to reproduce successfully, a plant needs to flower and complete its life cycle before the arrival of extreme environments (such as heat, freezing, etc.). Therefore, flowering time directly determines whether a plant can complete its life cycle in its growing area, and is also one of the important agronomic traits that determine crop yield. Cultivating crop varieties with suitable growth periods is also an important factor to be considered in the process of crop breeding. Take rice, a food crop, as an exam...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/82A01H5/00
CPCC12N15/8241C12N15/113C12N15/82
Inventor 曹晓风杨荣新吴建国李毅
Owner INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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