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Mutant gene identification, variation and molecular markers of maize brown midrib5 (bm5) mutant

A gene and corn technology, applied in the application field of BM5 gene of Poaceae Zea genus, can solve the problems of reducing lignin content, unclear mutation sites and mutation mechanisms, etc.

Active Publication Date: 2022-06-21
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Early identification of corn bm5 Mutations of mutants significantly reduced lignin content[T Mechin V, Laluc A, Legee F, CezardL, Denoue D, Barriere Y, Lapierre C. Impact of the brown-midribbm5 mutation on maize liggins. J Agr Food Chem. 2014; 62: 5102-5107.], but its mutation site and mutation mechanism are not clear

Method used

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  • Mutant gene identification, variation and molecular markers of maize brown midrib5 (bm5) mutant
  • Mutant gene identification, variation and molecular markers of maize brown midrib5 (bm5) mutant
  • Mutant gene identification, variation and molecular markers of maize brown midrib5 (bm5) mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Corn bm5 Mutant phenotyping

[0027] corn bm5 The mutant seeds were derived from the mutant library in maizegdb (www.maizegdb.org), and their StockIDs were 504I ( bm5-PI251930 ), 504J ( bm5-PI262480 ) and 505J ( bm5-PI251893 ). Use ordinary nutrient soil, seed and cultivate, and continue to observe the phenotype. When the seedlings grow to the six-leaf stage, the veins and stems appear reddish-brown phenotype ( figure 1 B, D and F).

Embodiment 2

[0028] Example 2: bm5 Mutant gene cloning and molecular identification

[0029] Based on the results of the exon capture analysis, we narrowed down the mutated region to the region of chromosome 5 80.8-120.7 Mb ( figure 2 A). This region contains only two genes for lignin synthesis: cinnamyl alcohol dehydrogenase 2 ( CAD2 , GRMZM5G844562 ) and 4-coumaric acid: coenzyme A ligase 1 ( Zm4CL1 , GRMZM2G075333 ). in view of bm1 and bm5 is independent bm mutants, so presumably Zm4CL1 Yes BM5 Gene.

[0030] Subsequently, we extracted control plants (B73) and mutant plants ( bm5 ) of genomic DNA (CTAB method), using primers Zm4CL1-S1 and Zm4CL1-S2, for conventional PCR amplification Zm4CL1 Partial fragment of gene, PCR reaction system: 2 μL DNA, 5 μL 10×Buffer, 4 μL dNTP (2.5 mM), 1 μL forward / reverse primer (10 μM) each, 0.5 μL Taq enzyme (5 U / μL) and 36.5 μL ddH 2 O. Mix well after adding samples on ice. PCR reaction conditions are: 94 o C 5 min; 94 o C 3...

Embodiment 3

[0046] Example 3: Effect of Ac transposon on 4CL enzyme activity

[0047] Based on the example shown in Example 1 bm5 -504J mutant Zm4CL1 There was no significant change in the expression level and that shown in Example 2 Zm4CL1 Ac transposon inserted in the exon region of the gene, we analyzed the insertion pair of Ac transposon Zm4CL1 Effects of post-transcriptional translation on protein activity. The method used is to express the mutated protein in vitro, and the vector used is pET32a. First, we put the B73 obtained in Example 2 into Zm4CL1 The mutated transcripts were recovered using a gel recovery kit (Promega), and then ligated into the pET32a vector using In-fusion enzyme (abm) to form Zm4CL1-pET32a, Zm4CL1-L-pET32a, Zm4CL1-S- pET32a recombinant plasmid, transformed into E. coli Escherichia coli (BL21). The protein expression was induced by IPTG (0.2 mM) in vitro, and the enzymatic activity of the expressed protein was detected. The results showed that t...

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Abstract

This application involves bm5 Identification of mutant genes associated with mutant phenotypes, variation and their molecular markers. It is specifically divided into the following four aspects: the content of the present invention focuses on specific naturally occurring mutants of maize bm5 , cloned its mutant gene BM5 , the gene causes the phenotype of lignin composition change in a specific maize line due to the insertion of transposons (Mu elements and Ac elements); the present invention provides a rapid detection method for the identification of the transposons; 4-coumaric acid : The enzyme activity level of coenzyme A ligase (Zm4CL1) directly affects the lignin composition and saccharification efficiency of maize; Zm4CL1 It can be used as one of the target genes for directional molecular genetic breeding of lignin, providing new targets for molecular breeding in the future, and providing practical guidance for directional molecular genetic breeding.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, in particular to a maize reddish brown leaf vein mutant bm5 related BM5 gene mutation site Zm4CL1 and molecular markers thereof, the altered genes contribute to changes in maize lignin components, thereby increasing maize cell wall digestibility, involving the genus Zea BM5 applications of genes. Background technique [0002] corn( Zea mays L.) is an annual C4 herbaceous plant belonging to the genus Maize, which is an important source of food, animal feed, and industrial and agricultural biomass energy raw materials. Plant cell walls are mainly composed of lignin, cellulose and hemicellulose, which are important factors in determining biomass energy conversion efficiency and forage quality. Analyzing the regulatory mechanism related to the lignin metabolic pathway of maize, changing the lignin composition and increasing the digestibility of the cell wall has important ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/52C12N9/00C12Q1/6895C12N15/11C12N15/82A01H5/00A01H6/46
Inventor 付春祥吴振映熊王丹刘雨辰李玉苏昆龙姜珊珊
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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