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Application and Realization of Rice Bleeding Purple Line Gene osmyb76

A gene and rice technology, applied in the field of genetic engineering, can solve problems such as false positives and complicated detection process, and achieve the effects of reducing false positives, accurate detection results, and obvious purple traits

Active Publication Date: 2021-10-22
YUNNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the reporter genes used in transgenics are mainly exogenous GUS genes or some fluorescent protein genes. These genes have the following insurmountable shortcomings when used as reporter genes: the detection process is complicated, and special detection reagents (such as GUS staining solution) need to be prepared or Using special detection equipment (such as fluorescent emitters), prone to false positives, etc.

Method used

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  • Application and Realization of Rice Bleeding Purple Line Gene osmyb76
  • Application and Realization of Rice Bleeding Purple Line Gene osmyb76
  • Application and Realization of Rice Bleeding Purple Line Gene osmyb76

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Obtaining and Morphological Observation of Embodiment 1, OsMYB76 Gene Mutant Plant

[0041] The OsMYB76 gene mutant is derived from the mutation of indica rice R25 (purple lines on the coleoptile, referred to as purple lines on the coleoptile). OsMYB76 mutant crossed with R25, F l Generations are all buds with purple lines, self-crossed F 2 Segregation occurred in the generation, and the ratio of purple line and no purple line was 3:1, which indicated that the phenotype of the mutant without purple line was caused by a nuclear gene mutation. Morphological observation of OsMYB76 mutant plants: the main difference between the mutant and the wild type is that there is an obvious purple line ( figure 1 , A), while the shoots of the OsMYB76 mutant had no purple lines ( figure 1 , B).

Embodiment 2

[0042] Embodiment 2, location and cloning of OsMYB76 gene

[0043] 1. Positioning groups

[0044] The OsMYB76 mutant was crossed with II-32B with purple lines, and at the same time Zhongjiu B without purple lines was crossed with wild-type R25 to obtain F 1 F 2 generation, planting F 2 Get targeting groups.

[0045] 2. Extraction of rice DNA

[0046]Parents were extracted using the improved CTAB method, including the following steps: Take 0.1-0.2 grams (about half a piece) of leaves and put them in a small mortar, add an appropriate amount of liquid nitrogen, grind them to powder immediately, put them into a 2m1 centrifuge tube, and add 700μL Put the 1.5×CTAB solution preheated at 100°C in a centrifuge tube, mix it carefully, put it in a 65°C water bath, take out the centrifuge tube after 20 minutes, add an equal volume of chloroform / isoamyl alcohol, mix vigorously, centrifuge at 13000rpm for 10 minutes, and take Put the supernatant in a new tube, add 900 μL of absolute e...

Embodiment 3

[0053] Example 3, Functional analysis of OsMYB76 gene and its application in the identification of transgenic positive offspring

[0054] In order to further confirm that the OsMYB76 gene is the gene that causes the no purple line phenotype, and to detect whether the gene can be used as a reporter gene for the detection of transgenic positive offspring, the CDS of the OsMYB76 gene and the modified OsMYB76 gene (SEQID NO.1) were respectively The GUS gene in pCAMBIA1301 was replaced by enzyme digestion and homologous recombination to obtain vectors p1 (pOsMYB76) and p2 (pOsMYB76R), and the complementary fragment of the gene OsABCG15 (Wu et al., 2014) related to rice pollen fertility and development was loaded Enter the multiple cloning sites in the p1(pOsMYB76) and p2(pOsMYB76R) vectors to obtain the vectors pX1(pOsABCG15-OsMYB76) and pX2(pOsABCG15-OsMYB76R), and transfer the vectors into related materials to investigate the purple and fertility traits. The specific operation as...

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Abstract

The invention discloses the application and realization method of the rice coleoptile purple line gene OsMYB76. The nucleotide sequence of the gene is shown in SEQ ID NO.2, and the modified sequence is shown in SEQ ID NO.1. The gene's The sequence of the interference fragment is shown in SEQ ID NO.3; the gene, the modified gene or the down-regulated expression of the gene can regulate the synthesis of anthocyanins in rice bud sheaths and other parts, so it can be used as a reporter gene in transgene identification, and The method for identifying transgene-positive offspring is simple and accurate, and plays an important role in simplifying the identification of gametophytic male sterility, identifying the ratio of transgene components transferred with pollen, removing transgene contamination, and improving its accuracy.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to the identification of the rice budding purple line gene OsMYB76, its modified gene OsMYB76R and expression down-regulating elements in transgene-positive strains, the identification of gametophyte male sterility, the identification and removal of transgene pollution, and the implementation method of the application. Background technique [0002] The MYB transcription factor family is one of the most abundant transcription factor families in plants. There are more than 150 MYB transcription factors in rice (Katiyar et al., 2012), among which the OsMYB76 gene (Loc_Os06g10350, C gene or OsC1, hereinafter collectively referred to as OsMYB76) was found to be necessary for the regulation of anthocyanin synthesis in rice coleoptiles, etc. (Saitoh et al., 2004; Zhang Yi 2009; Zhao Shasha 2014). At present, the research on this gene mainly focuses on evolution analysis, gene mapping and ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12N15/29C12N15/82A01H5/00A01H5/02A01H6/46
CPCC07K14/415C12N15/8212C12N15/8218C12N15/825C12N15/8265C12N15/8289C12Q1/6895C12Q2600/13
Inventor 张毅陈云杜双林
Owner YUNNAN UNIV
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