Application of OsmiR156f gene in rice effective tillering increasing

A technology of transgenic rice and genes, applied in the field of agricultural biology, can solve the problems of high proportion and large number of ineffective tillers, and achieve the effect of increasing the number of effective tillers and reducing the proportion of ineffective tillers.

Active Publication Date: 2015-03-25
HUNAN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a kind of function of OsmiR156f gene in increasing the effective tillering of rice in order to solve the problem that the overexpression of miR156 family members in rice can significantly increase the tillering ability of rice but the number of ineffective tillers is too high and the ratio is too high in the prior art. application

Method used

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  • Application of OsmiR156f gene in rice effective tillering increasing
  • Application of OsmiR156f gene in rice effective tillering increasing
  • Application of OsmiR156f gene in rice effective tillering increasing

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Experimental program
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Effect test

Embodiment 1

[0016] Embodiment 1 Amplification of OsMIR156f gene fragment

[0017] According to the Nipponbare genome sequence published on the Internet, Primer 3.0 software was used to design primers (synthesized by a biological company). The primer sequences were: forward primer 5'-CGCCCACCTTTCTTCTCCCA-3' (see SEQ ID No.2), reverse primer 5' - AAGGAGCAGTTAGATAATGGAG-3' (see SEQ ID No. 3). Prepare the synthetic primers with double distilled water to a concentration of 10 μmol L -1 The solution. Genomic DNA was extracted from rice Nipponbare leaves by CTAB method. Using the extracted DNA as a template, high-fidelity pfu Taq enzyme was used for PCR amplification to obtain the pre-precursor pri-miR156f sequence corresponding to OsMIR156f in the OsmiR156 family. The reaction system for PCR amplification was 50 μL: DNA template 1.0 μL, Forward primer 0.5 μL, reverse primer 0.5 μL, 10 μmol L -1 dNTP 0.5 μL, 10X buffer 5.0 μL, pfu Taq enzyme 0.3 μL, double distilled water 42.2 μL. The ampli...

Embodiment 2

[0018] Example 2 Construction of constitutive overexpression vector pWM-UBQpro-OsMIR156f (UbiPro-OsMIR156f)

[0019] The constitutive promoter is preferably Ubiquitin1 promoter. The vector pWM101 was digested with HindⅢ / sal I, the intermediate vector pBS-pUbq was digested with HindⅢ / BamH I, and the intermediate vector pBS-OsMIR156f was digested with BamH I / sal I respectively, and the required fragments were recovered and processed with T4ligase. The three fragments were ligated to obtain the vector. For information about the intermediate vector pBS-pUbq, refer to the literature report (Wei et al. PLoSONE.2013, 8: e59720). The constructed UbiPro-OsMIR156f vector was introduced into Agrobacterium EHA105 strain by freeze-thaw method. The bacterial resistance of this vector is kanamycin and the plant resistance is hygromycin.

Embodiment 3

[0020] Example 3 Construction of stem-specific expression vector pWM-D18p-OsMIR156f (D18Pro-OsMIR156f)

[0021] The stem-specific promoter is preferably the promoter of OsGA3ox2 gene. The Ubiquitin1 promoter in the UbiPro-OsMIR156f vector was replaced by the about 2.1Kb promoter (D18 promoter) of the OsGA3ox2 gene by restriction enzyme ligation, and the related information of the D18 promoter sequence was reported in the literature (Sakamoto et al. Nature Biotechnology. 2003, 21:909-913). The constructed D18Pro-OsMIR156f vector was introduced into Agrobacterium EHA105 strain by freeze-thaw method. The bacterial resistance of this vector is kanamycin and the plant resistance is hygromycin.

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Abstract

Provided is an application of an OsmiR156f gene in rice effective tillering increasing. The OsmiR156f gene which has important regulating effect on rice tillering development is selected from a miR156 family, a precursor pri-miR156f sequence is cloned, through a stem specific promoter vector construction method, the OsmiR156f gene which can increase tillering is guided into rice, specific expression in the stem of rice is carried out, and rice tillering development is regulated. The early tillering capacity of transgenic rice can be improved properly, tillering sprouts at a high section and later-period tillering are still restrained, accordingly, the effective tillering quantity of a single rice plant can be increased, ineffective tillering quantity is not increased, a transgenic line rice ear number is increased properly, influence on yield correlated characters such as rice ear length and thousand kernel weight is small, and finally the purpose of single-plant production increasing is achieved.

Description

technical field [0001] The invention relates to the field of agricultural biotechnology, in particular to the application of an OsmiR156f gene in increasing the effective tillering of rice, in particular to a vector construction method for specifically expressing the tiller-related OsmiR156f gene in stems by using a specific promoter. Background technique [0002] Tillering is one of the important indicators in rice variety breeding. Tillering ability directly affects the number of rice panicles and then affects rice yield. Only the main stem and the primary and secondary tillers that occurred in the early stage of rice have the ability to form panicles, which is an effective tiller; while the secondary tillers in the later period cannot form panicles due to factors such as short growth period, which is an ineffective tiller. In production, only effective tillers contribute to rice yield, while ineffective tillers not only fail to form rice spikes, but also affect the length...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/84A01H5/00
Inventor 肖浪涛刘清王若仲彭克勤李合松童建华苏益
Owner HUNAN AGRICULTURAL UNIV
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