阅读说明：本技术 一种玉米小籽粒突变体及其应用 (Corn small-grain mutant and application thereof ) 是由 关海英 汪黎明 刘铁山 董永斌 鲁守平 何春梅 刘春晓 董瑞 刘强 王娟 于 2018-07-29 设计创作，主要内容包括：本发明属于基因工程和分子生物学技术领域,具体涉及一种玉米小籽粒突变体及其应用。本发明提供了一种玉米籽粒突变基因mn2-m1和mn2-m2,并通过图位克隆获得Zm00001d019294为籽粒突变体mn2-m1和mn2-m2的候选基因,明确Zm00001d019294调控玉米籽粒发育,并验证了该基因可以影响并调控玉米籽粒的大小,可以获得比野生型植株产量更高的转基因作物,从而为该基因在培育大籽粒转基因经济作物领域中的应用提供理论依据。本发明所获得突变体可为培育农作物新品种提供理论依据和基因来源,在玉米种质资源的遗传改良育种中作用重大。(The invention belongs to the technical field of genetic engineering and molecular biology, and particularly relates to a corn small grain mutant and application thereof. The invention provides a corn kernel mutant gene mn2-m1 and mn2-m2, obtains Zm00001d019294 as candidate genes of kernel mutants mn2-m1 and mn2-m2 through map location cloning, confirms that Zm00001d019294 regulates and controls corn kernel development, verifies that the gene can influence and regulate the size of corn kernels, can obtain transgenic crops with higher yield than wild plants, and provides a theoretical basis for the application of the gene in the field of cultivating large-kernel transgenic cash crops. The mutant obtained by the invention can provide theoretical basis and gene source for cultivating new crop species, and has great effect in genetic improvement and breeding of corn germplasm resources.)

1. A mutant gene coding nitrate transporter NRT1.5 is characterized by having a nucleotide sequence shown as SEQ ID No.3 or SEQ ID No. 4.

2. Use of the mutant gene of claim 1 encoding nitrate transporter NRT1.5 in the construction of maize mini-kernel mutants.

3. A corn small grain mutant mn2-m1 is characterized by comprising an amino acid sequence shown as SEQ ID No. 1.

4. A mutant gene encoding the maize kernel mutant mn2-m1 of claim 3, which comprises the nucleotide sequence shown in SEQ ID No. 3.

5. A corn small grain mutant mn2-m2 is characterized by comprising an amino acid sequence shown as SEQ ID No. 2.

6. A mutant gene encoding the maize small grain mutant mn2-m2 of claim 5, which comprises the nucleotide sequence shown in SEQ ID No. 4.

7. The use of the corn kernel volume mutant of claim 3 or 5 in the field of breeding large kernel transgenic commercial crops.

8. The use of the maize grain volume mutant gene of claim 4 or 6 in the field of breeding large grain transgenic commercial crops.

9. The use of the corn kernel volume mutant of claim 3 or 5 in the field of commercial crop assisted breeding.

10. The application of the corn kernel volume mutant gene of claim 4 or 6 in the field of auxiliary breeding of commercial crops.

Technical Field

The invention belongs to the technical field of genetic engineering and molecular biology, and particularly relates to a corn small grain mutant and application thereof.

Background

Corn is an important food crop and a typical C4 type model plant in China, and plays an important role in food production and monocotyledon functional genomics research. With the rapid increase of global corn demand, the position of corn in national economy is increasingly prominent. The demand of various countries in the world for corn increases year by year, and the corn consumption structure is changed fundamentally, namely, the corn is gradually developed into diversified patterns of livestock feed, industrial raw materials, dining table subsidiary food and energy crops from main grain crops which solve the problem of satiety. Particularly, in recent years, the renewable energy and fine and further processing fields endow new connotation to the corn, so that the industrial processing proportion of the corn is rapidly increased, and the corn becomes a strategic resource which plays a great role in the 21 st century due to the multiple demands. Therefore, the corn yield directly influences the development of livestock raising, light industry, energy and related industries, is related to the improvement of national food safety and people living standard, and has a particularly important position in economic development.

How to improve the yield of the corn is a major subject which needs to be solved urgently in China at present. Besides optimizing planting environment and conditions, the method analyzes genetic factors influencing yield and action molecular mechanisms thereof starting from the genetic factors of crops, and is also an important basis for carrying out genetic improvement on crop yield. High yield is an eternal topic of corn genetic improvement research, and grain (seed) size is an important trait related to corn yield index. Therefore, the research on the molecular mechanism for regulating and controlling the grain size and the search for the gene for controlling the grain size have remarkable significance for improving the corn yield.

In recent years, with the rapid development of molecular biology and genomics research technologies, some genes for controlling corn kernel development have been identified and cloned by methods such as map-based cloning and transposon tagging. In 1996, Cheng et al reported that miniture 1 encodes an isozyme of a cell wall-converting enzyme with increased activity in endosperm development, and that the grain weight of the mutant was reduced by more than 30% compared to the wild type (Cheng et al, 1996). Another gene rgf1 affecting seed size, whose mutation would affect filling of the seed, partial dysplasia with the basal portion of the seed connected to the mother, reduced gene expression in the cells of the metastatic layer, and ultimately smaller endosperm, yielding small seeds (Maitz et al, 2000). In addition, the PPR2263 gene encodes a DYW domain-containing PPR protein that plays a role in RNA editing after transcription of nad5 and cob, and mutation of the gene also leads to the production of small kernels (Sosso et al, 2012). Li et al also found that the maize Smk1 gene encodes a class E PPR protein localized in the mitochondria, and that mutation of this gene represses the development of the embryo and endosperm, leading to seed miniaturization (Li et al, 2014). In addition, tomato fw 2.2 gene homologous gene family CNRs (cell number regulation) in maize also affects seed development, and this family of genes contains a conserved cysteine-rich motif that affects seed organ size by affecting cell number. Overexpression of the CNR1 gene of this family results in smaller kernels, while suppression or knockout of the gene can result in significantly larger kernels (Guo et al, 2010).

There have also been some studies on genes controlling seed size in model plants, Arabidopsis and rice. The ANT and ARGOS genes of Arabidopsis are two relatively well-understood genes studied and influence the size of the seed organ by altering cell number. Wherein ANT is a transcription factor containing AP2 domain, and AGROS gene affects the size of seed organ by acting on ANT gene located at the upstream. Overexpression of both ANT and ARGOS leads to enlargement of the seed organs (Elliott et al, 1996; Krizek et al, 1999). In addition, unlike ARGOS, the ARGOS-LIKE gene affects the size of seeds mainly by changing the size of cells (Hu et al, 2006). In recent years, genes controlling seed size have also been cloned in rice, for example: GS3(Fan et al, 2006,2009; Li et al, 2004), GW2(Song et al, 2007), qSW5/GW5(Wan et al, 2008; Weng et al, 2008; Shomura et al, 2008), GL3.1(Qiet al, 2012), and the like, which can regulate the size of rice seed organs through different mechanisms, but most of them are negative regulators. The homologous genes ZmGS3 and ZmGW2 of GS3 and GW2 in maize have also been shown to be linked to maize yield determinants (Li et al, 2010 a; Li et al, 2010 b). Recently, the GS5 gene cloned by Zhang Yao Shi laboratory plays an important role in regulating the size and yield of rice seeds, and experiments prove that the factor is a positive regulation factor and encodes a predicted serine carboxypeptidase, and the seeds of over-expressed lines of the gene are obviously increased compared with a control (Liet al, 2011).

Nitrate nitrogen is the main nitrogen source absorbed by plants, and nitrate transporters play an important role in their absorption from the soil to the roots, and their transport from the roots to the stalks or other organs. The arabidopsis thaliana nitre transporter NRT1.5 is mainly expressed at roots, the expression quantity of the roots in the mutant is obviously reduced, and NO transferred from the roots to stalks₃ ^-A significant decrease (linear, 2008). However, there is no report in arabidopsis that NRT1.5 mutation affects grain development. However, NRT1.5 is predominantly expressed in grain in maize, and its mutant grain shows reduced shrinkage, which may be due to differences in NRT1.5 function between monocots and dicots. By researching the function of the mutant gene and analyzing the expression rule of the gene, a new thought can be provided for the genetic improvement of the corn yield, and a gene element can be provided for molecular breeding.

Disclosure of Invention

Therefore, the technical problem to be solved by the invention is to provide a corn small-kernel mutant and further disclose the application of the mutant in the field of commercial crop assisted breeding.

In order to solve the technical problem, the mutant gene for coding nitrate transporter NRT1.5 has a nucleotide sequence shown as SEQ ID No.3 or SEQ ID No. 4.

The invention also discloses application of the mutant gene of the nitrate transport protein NRT1.5 in constructing a corn small grain mutant.

The invention also discloses a corn small grain mutant mn2-m1, which comprises an amino acid sequence shown as SEQ ID No. 1.

The invention also discloses a mutant gene for coding the corn small grain mutant mn2-m1, which comprises a mutant gene of a nucleotide sequence shown as SEQ ID No. 3.

The invention also discloses a corn small grain mutant mn2-m2, which comprises an amino acid sequence shown as SEQ ID No. 2.

The invention also discloses a mutant gene for coding the corn small grain mutant mn2-m2, which comprises a mutant gene of a nucleotide sequence shown as SEQ ID No. 4.

The invention also discloses application of the corn kernel volume mutant in the field of large kernel transgenic cash crop cultivation.

The invention also discloses application of the corn kernel volume mutant gene in the field of cultivation of large-kernel transgenic cash crops.

The invention also discloses application of the corn kernel volume mutant in the field of economic crop auxiliary breeding.

The invention also discloses application of the corn kernel volume mutant gene in the field of commercial crop assisted breeding.

The invention provides a corn kernel mutant gene mn2-m1 and mn2-m2, obtains Zm00001d019294 as candidate genes of kernel mutants mn2-m1 and mn2-m2 through map location cloning, confirms that Zm00001d019294 regulates and controls corn kernel development, verifies that the gene can influence and regulate the size of corn kernels, can obtain transgenic crops with higher yield than wild plants, and provides a theoretical basis for the application of the gene in the field of cultivating large-kernel transgenic cash crops. The mutant obtained by the invention can provide theoretical basis and gene source for cultivating new crop species, and has great effect in genetic improvement and breeding of corn germplasm resources.

Drawings

In order that the present disclosure may be more readily and clearly understood, the following detailed description of the present disclosure is provided in connection with specific embodiments thereof and the accompanying drawings, in which,

FIG. 1 is the grain phenotype of mutants mn2-m1 of example 1;

FIG. 2 is the development of embryos of mutants mn2-m1 after pollination as in example 1;

FIG. 3 is a graph of the mn2-m1 seedling phenotype of the four F2 isolates in example 1;

FIG. 4 is a map-based clone of mn2-m1 gene from example 4;

FIG. 5 is an alignment of the amino acids encoded by Zm00001d 019294;

FIG. 6 shows the sequencing result of the key recombinant individual Zm00001d 019294;

FIG. 7 shows the results of expression analysis of MN2-M

FIG. 8 shows the result of identifying a specific genotype.

Detailed Description