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Protein GSW8 for regulating and controlling grain shape and thousand grain weight of rice, and coding gene and application of protein GSW8

A thousand-grain weight and protein technology, applied in the field of genetic engineering, can solve problems such as unclear molecular regulatory network, unused genes in rice breeding, complicated rice grain shape and thousand-grain weight

Active Publication Date: 2021-09-14
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] So far, rice grain shape and 1,000-grain weight related genes have been cloned, but rice grain shape and 1,000-grain weight are complex quantitative traits, and their molecular regulatory network is still unclear, and many genes have not been used in rice breeding

Method used

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  • Protein GSW8 for regulating and controlling grain shape and thousand grain weight of rice, and coding gene and application of protein GSW8
  • Protein GSW8 for regulating and controlling grain shape and thousand grain weight of rice, and coding gene and application of protein GSW8
  • Protein GSW8 for regulating and controlling grain shape and thousand grain weight of rice, and coding gene and application of protein GSW8

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1: Analysis of the expression pattern of the GSW8 gene in rice

[0020] 1. Quantitative PCR primer design

[0021] The present invention uses the quantitative PCR primer online design website (https: / / quantprime.mpimp-golm.mpg.de / ) to design a pair of quantitative PCR primers spanning the 1st and 2nd exon in the coding region of GSW8, the specific sequence as follows:

[0022] Y1879-F: 5'-GGTGGTTTCATTCTTGGAG-3' (SEQ ID NO.3);

[0023] Y1880-R: 5'-CACATCTTGGGACCCTTTG-3' (SEQ ID NO. 4).

[0024] 2. RNA extraction and reverse transcription

[0025] In order to analyze the expression pattern of GSW8, materials from different developmental stages and different tissues of Shuhui 498 (including roots and leaves at the seedling stage, stems and flag leaves at the booting stage, young panicles with different lengths, glumes, and caryopsis at different days after fertilization were collected) ) were stored in a -80°C refrigerator for later use. Using OMEGA’s Plant RN...

Embodiment 2

[0035] Example 2: CRISPR / Cas9 knockout vector construction and genetic transformation of GSW8 gene

[0036] 1. CRISPR / Cas9 Knockout Target Site Selection

[0037] In order to obtain a mutant with complete loss of function, we designed a knockout target site at the position of the first exon of GSW8 near ATG, the target site sequence is: 5'-CCAACTAAGCAATGGCCTTT-3' (SEQ ID NO.7 )( figure 2 ).

[0038] 2. CRISPR / Cas9 knockout vector construction

[0039] The present invention utilizes the CRISPR / Cas carrier construction kit of Baige Biotechnology Co., Ltd., selects the CRISPR / Cas carrier BGK03 suitable for rice, and constructs the knockout carrier GSW8-BGK03 of GSW8. The specific process is as follows:

[0040] (1) Synthesize Oligo sequences and prepare Oligo dimers

[0041] According to the above target sequence, the Oligo-F and Oligo-R sequences corresponding to the kit were designed. The sequences are as follows: Oligo-F: 5'-TGTGTGCCAACTAAGCAATGGCCTTT-3' (SEQ ID NO.8);

...

Embodiment 3

[0057] Example 3: Identification and Phenotypic Analysis of Knockout Plants of GSW8 Gene

[0058] 1. Identification of knockout plants

[0059] After obtaining the positive transgenic plants in Example 2, extract DNA from the leaves of individual plants, and design amplification and sequencing primers Y2118-F (5'-TTCGTAGTGCGATTGTTTC-3', SEQ ID NO.11) and Y2119 across the knockout target site -R (5'-ACCGACCAAGAGCATTAGA-3', SEQ ID NO.12), the mutation was confirmed by sequencing after amplification. Such as figure 2 As shown, a total of three independent knockout lines with different mutation methods were obtained, named KO1-KO3. Among them, KO1 and KO2 were deleted and inserted 1 base A respectively, and KO3 was deleted by 4 bp (CTAA), both of which resulted in frameshift mutations and premature termination of protein translation ( Figure 4 ), therefore, the obtained GSW8 knockout mutant is a complete loss-of-function mutant.

[0060] 2. Phenotype analysis of knockout pla...

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Abstract

The invention discloses a protein GSW8 for regulating and controlling the grain shape and the thousand grain weight of rice, and a coding gene and application of the protein GSW8. The amino acid sequence of the protein GSW8 is shown as SEQ ID NO.2; and the nucleotide sequence of the coding gene thereof is as shown in SEQ ID NO. 1. The gene has a biological function in the aspect of regulating and controlling the grain shape and the thousand grain weight of the rice, can be applied to improvement of the grain shape and the thousand grain weight of the rice and increase of the yield of the rice, has an important breeding utilization value, and provides a new thought for researching a regulation and control mechanism of the grain shape and the thousand grain weight of the rice.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a protein GSW8 for regulating rice grain shape and thousand-grain weight, its coding gene and application. Background technique [0002] Rice is one of the most important food crops in the world, providing food and nutrition sources for more than half of the population, and it is also the most important food crop in my country. According to statistics, my country's population will reach 1.6 billion by 2030, and the total grain demand will increase from 580 million tons in 2010 to 740 million tons. Under the influence of population growth, decreasing arable land area, and environmental degradation, food production is still facing enormous pressure. Therefore, food security issues have attracted more and more attention. Coupled with the occurrence of the global new crown epidemic in the past two years, it is of great strategic significance to further increase rice prod...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/10A01H6/46
CPCC07K14/415C12N15/8261Y02A40/146C12N15/8242
Inventor 袁华许正艳高鹏袁敏陈薇兰李仕贵涂斌马炳田王玉平钦鹏
Owner SICHUAN AGRI UNIV
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