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Application of micro RNA529a in adjusting and controlling rice plant types

A rice plant type and gene technology, applied in the field of plant genetic engineering, can solve problems such as unknown biological functions

Inactive Publication Date: 2017-05-24
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Many miRNAs are encoded in rice, but the biological functions of most of them are still unknown, and more work is urgently needed to elucidate the role of these small RNAs and their utilization value in rice genetic improvement

Method used

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  • Application of micro RNA529a in adjusting and controlling rice plant types
  • Application of micro RNA529a in adjusting and controlling rice plant types
  • Application of micro RNA529a in adjusting and controlling rice plant types

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Isolation of DNA fragments comprising the precursor gene pri-miR529a of miR529a

[0042] Using Rice Genome Annotation Project (http: / / rice.plantbiology.msu.edu / ) and miRBase (http: / / www.mirbase.org / ) two websites to search, get miR529a (miRBase database accession number: MIMAT0002885) and The nucleotide sequence of its precursor gene pri-miR529a (miRBase database accession number: MI0003202). The miR529a mature body sequence is shown in SEQ ID NO:1, and the sequence of its precursor gene pri-miR529a is shown in SEQ ID NO:2; the formed secondary structure is shown in figure 1 shown.

[0043]According to the analysis results of the database sequence, in order to amplify the gene fragment of pri-miR529a, the following primers were designed:

[0044] MiR529aOEF (forward primer): 5'- GGTACC TGAAAGAGATGAGTGTGCT-3' (the underlined sequence is the KpnI recognition site);

[0045] MiR529aOER (reverse primer): 5'- GGATCC TGGGGTAACTAGCGTTTTGCA-3' (the underlined ...

Embodiment 2

[0047] Embodiment 2: Construction of the overexpression vector of miR529a

[0048] KpnI and BamHI were used to double-digest plasmid TA-miR529a (the KpnI and BamHI were purchased from Bao Bioengineering Dalian Co., Ltd., and the specific usage and dosage refer to the instructions of the corresponding products of the company. The plasmid TA-miR529a is from Example 1, such as figure 2 (shown in Figure A), recover the 371bp DNA fragment containing pri-miR529a, and then combine it with the vector plasmid pCAMBIA1301S [the vector was publicly reported: Zhou et al., Over-expression of aspartate aminotransferase after KpnI and BamHI double digestion genes in riceresulted in altered nitrogen metabolism and increased amino acid content in seeds. Theor Appl Genet, 2009, 118:1381-1390; its basic skeleton originated from CAMBIA laboratory in Australia (http: / / www.cambia.org / daisy / cambia / materials / overview.html) of pCAMBIA1301, through the addition of 35S promoter to realize the expressi...

Embodiment 3

[0049] Embodiment 3: the acquisition of transgenic rice

[0050] The expression vector 35S:miR529aOE (from Example 2) was introduced into the callus of the rice variety Zhonghua 11 through the genetic transformation method mediated by Agrobacterium EHA105, and after pre-cultivation, infection, co-cultivation, and screening with hygromycin (using A kind of antibiotic used in the screened positive transgenic callus was purchased from Roche Pharmaceuticals Co., Ltd. of Denmark), and the resistant callus was differentiated, rooted and hardened and transplanted in the field to obtain transgenic plants. The method of genetic transformation of Agrobacterium and the reagents and formulas used were optimized according to the report of Hiei et al. (Hiei et al., Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA . Plant J, 1994, 6: 271-282; Lin and Zhang, Optimizing the tissue culture conditions for high effic...

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Abstract

The invention relates to the technical field of plant genetic engineering, in particular to application of micro RNA529a in adjusting and controlling rice plant types, namely non-coding RNA miR 529a for regulating and controlling rice plant heights, tiller numbers and spike types and isolation clone, functional verification and application of a precursor gene pri-micro RNA529a (pri-miR529a) of the non-coding RNA miR529a. A nucleotide sequence of the miR529a is shown as SEQ ID NO. 1, and a nucleotide sequence of the prim-miR529a is shown as SEQ ID NO. 2. As the pri-miR529a is expressed through transgenosis excessively, the rice plant height is shortened, tiller is increased, the spike length is shortened, and spike branches and spike kernels are reduced. The invention not only provides an active fragment of the miR529a and a genetic transformation carrier thereof , but also provides a novel rice plant type improving method.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering. It specifically relates to the application of a microRNA529a (abbreviated as miR529a hereinafter) and its precursor gene pri-miR529a in regulating rice plant type. Changing the expression abundance of miR529a and its precursor gene pri-miR529a through genetic engineering technology can change the agronomic traits of plants such as plant height, number of tillers, number of spikes and branches, and number of grains per spike. Background technique [0002] Rice is one of the most important food crops in the world, and more than half of the population depends on it as a staple food. Therefore, the yield of rice has an important impact on social development. With the continuous growth of the world population, the continuous reduction of arable land and the increasing environmental pollution, the cultivation of new rice varieties with higher yields and environmental friendliness has...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/84A01H5/00
Inventor 张启发王磊
Owner HUAZHONG AGRI UNIV