CN110846322A

CN110846322A - Corn small-grain mutant and application thereof

Info

Publication number: CN110846322A
Application number: CN201810850808.7A
Authority: CN
Inventors: 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
Original assignee: Maize Research Institute of Shandong Academy of Agricultural Sciences
Current assignee: Maize Research Institute of Shandong Academy of Agricultural Sciences
Priority date: 2018-07-29
Filing date: 2018-07-29
Publication date: 2020-02-28
Anticipated expiration: 2038-07-29
Also published as: CN110846322B

Abstract

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.

Description

Corn small-grain mutant and application thereof

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

Example 1 phenotypic analysis of maize granule mutant mn2-m1

The maize small kernel mutant mn2-m1 was discovered by the applicant in a pool of rejected breeding material and was examined to spontaneously express small kernels from early pollination, after maturation, the kernels appeared small and shriveled (as shown in fig. 1), and its embryo development was also affected (as shown in fig. 2), significantly less compared to the wild type.

The experiments as shown in table 1 show that mutant mn2-m1 kernel has reduced length, width, thickness and weight-average of its hundred grains compared to wild type, and its seedling development is also affected (as shown in fig. 3).

The a/A, B/B, c/C, D/D referred to in FIGS. 1-3 and Table 1 are respectively from the F2 population of mn2-m1 in combination with maize inbred lines B73, Chang 7-2, Zheng 58 and Cheng319.

TABLE 1 analysis of grain traits

^**The significance difference level P is less than or equal to 0.01, the length, width and thickness of the grains are respectively measured for 30 grains, and the three steps are repeated. Hundred kernel weights were measured for 100 kernels, and repeated three times.

Example 2 genetic analysis

The small-grain maize mutant mn2-m1 is respectively hybridized with a common maize inbred line B73, Chang 7-2, Zheng 58 and Qiqi 319 to obtain F1S, F1S is inbred to obtain F2S, and genetic analysis is carried out on an F2 segregation population, so that the result shows that the small-grain phenotype of mn2-m1 is controlled by a recessive single gene (shown in the following table 2).

TABLE 2 analysis of the phenotypic segregation of the five parents and their F1, F2 populations

WT, wild type; x2_(0.05,1)＝3.84.

Example 3 allelic variants

Mn2-m2 was obtained in 2016 from obsolete breeding material and had a phenotype similar to mn2-m 1. To verify whether they are derived from the same site mutation, we hybridized mn2-m2 with mn2-m1/B73 and found that their F1 exhibits 1: 1, which indicates that MN2-M1 and MN2-M2 are both from MN2-M mutations (see table 3).

TABLE 3 hybridization F of mn2-m1/B73 with mn2-m2₁Grain phenotype isolation

Hybrid ear	Wild type	Mutant forms	Separation ratio	Chi-square value	P-value
						Ear-1	154	139	1.11	0.77	0.3<P<0.5
Ear-2	121	108	1.12	0.74	0.3<P<0.5
						Ear-3	168	146	1.15	1.54	0.2<P<0.3
Ear-4	157	133	1.18	1.99	0.1<P<0.2
						Ear-5	143	127	1.13	0.95	0.3<P<0.5

Chi-square value_(0.05,1)＝3.84.

Example 4 location of the mn2-m1 site of the mutant Gene

2 polymorphic molecular markers P1 and P2 linked with a target gene are found by using 188 pairs of core SSR primers through a BSA separation population segregation analysis method, are positioned on the short arm of No. 7 chromosome, and the region is not reported related to known functional genes related to corn kernel mutation, so that the gene for controlling the trait is preliminarily judged to be a novel gene with unknown functions (detailed in Table 4 and A in FIG. 4).

The 218 recessive mutant plants in the BC1 population were genotyped with P1 and P2, and 7 and 3 recombinant individuals were identified, respectively. The 10 recombinant individuals were genotyped by using 7 pairs of SSR primers P3-P9 with polymorphism in the region reported in the previous literature, and 7, 7, 2, 2, 3, 3 recombinant individuals were identified, respectively, wherein P3, P4 and P5 are located on the P1 side near the chromosome end arm, and P6, P7, P8 and P9 are located on the P2 side near the centromere end (see Table 4 and A in FIG. 4 for details) (Xuet al, 2013). Thus, the mn2-m1 gene is located between P5 and P6 at a physical distance of about 11.78 Mb.

For fine mapping of mn2-m1 gene, 4347 recessive individuals in BC1 population were genotyped with P5 and P6, while 9 polymorphic SSR markers P10, P11, P12, P13, P14, P15, P16, P17, P18 were newly identified (see table 4 and B in fig. 4 for details). P5 and P6 identified 31 recombinant individuals together, 8 on P5 side and 23 on P6 side. The 9 polymorphic markers P10-P18 were used to identify 35(31+2+2) crossover individuals identified by P5 and P6, and 9,9,9,7,3,0,4 and 5 recombinant individuals were identified, respectively. P10, P11, P12, P13, P14 and P15 are located on the side of P5, and P17 and P18 are located on the side of P6. Thus, the target gene is currently located between two molecular markers P15 and P17 (shown as B in FIG. 4) that are physically separated by about 1.34 Mb. P15 and P17 were used to identify 4611 recessive individuals, 5 and 12 recombinant individuals, respectively, in the BC1 population.

To further refine the localization of mn2-m1, this example was developed with 3 SSR markers P19-P21 (detailed in Table 4 and C in FIG. 4). A total of 24 (17+3+4) recombinant individuals between the P15 and P17 markers were identified using four SSR markers, P16 and P19-P21, each with 2 crossover individuals. The desired gene mn2-m1 was finally located between the markers P19 and P20 at a physical distance of 209.9kb (see FIG. 4 for details, C).

Gene analysis and prediction software (www.softberry.com) is used to carry out gene prediction on the 209.9kb, and only one candidate gene Zm00001d019294 is located in the interval by combining an annotation gene published by a mailedDB website, and the gene is predicted to code nitrotranporter NRT1.5 (see C in figure 4 in detail).

TABLE 4 primers for map-based cloning of mn2-m1 genes

F and R represent the upstream and downstream primers, respectively.^aSSR primers were from the maizeGDB database;^bSSR markers are from the pre-human literature (Xu et al, 2013);^cSSR markers are our own development.)

Example 5 cloning of the mutant Gene mn2-m1

The candidate gene Zm00001d019294 genome DNA and cDNA sequence thereof use the following primers: G1-2F-G1-3R (F: 5 '-CACTACCGCCCTAGCAAA; R: 5' -TGCAAAAGTCAAAAACATACAAC) and GR1-6F-GR1-6R1 (F: 5 '-ATGGCTGAGGGTAGCTG; R: 5' -GCCTTAGAATGGATTGGTAT) were amplified from wild-type B73 and mutant (mn2-m1 and mn2-m2) leaf genomes and seed cDNAs, directly ligated to cloning vectors, sequenced, and then aligned with the sequence analysis software DNAStar.

The PCR amplification program specifically comprises: 5min at 95 ℃, 45s at 65 ℃, 90s at 72 ℃ for 32 cycles, and finally 10min at 72 ℃ and forever at 4 ℃.

And (3) detecting that the corn small grain mutant mn2-m1 comprises an amino acid sequence shown as SEQ ID No. 1.

The maize small grain mutant mn2-m2 comprises an amino acid sequence shown as SEQ ID No. 2.

Sequence analysis revealed that deletion of a G at ORF1395 in the mutant gene mn2-m1(SEQ ID No.3) caused a frameshift mutation in the coding region resulting in premature translation termination (as shown in D in FIG. 4 and FIG. 5). Deletion of the mutant gene mn2-m2(SEQ ID NO.4) at ORF1455-1500 by 46bp caused a frameshift mutation in the coding region resulting in premature translation termination (as shown in FIG. 4D and FIG. 5).

By sequencing candidate genes of key recombinant individuals, only missense mutation caused by ORF1379(A/G) and deletion mutation thereof were found to be located in the candidate region (as shown in FIG. 6). This also indicates that Zm00001d019294 is the gene responsible for the mn2-m1 and mn2-m2 mutant phenotypes.

Example 6 expression analysis

Expression analysis of MN2-M was performed by semi-quantitative RT-PCR, and MN2-M showed the highest expression level in the seeds and trace expression in the leaves (see A in FIG. 7). In addition, gene expression analysis is carried out on seed materials obtained 15 days and 21 days after mn2-m1 mutant plants and wild type pollination respectively, and the result shows that the expression of the gene in the mn2-m1 mutant is obviously reduced (as shown in B in figure 7). In addition, the expression levels of genes Meg-1, BETL-2, BETL-10, TCRR-1 and ESR-6 related to the development of basal transfer layer of corn endosperm and periembryonic region thereof in mutant grains are all remarkably reduced (as shown in B in FIG. 7).

Example 7

The following PCR primers were designed, respectively:

GR-2NINDEL2F-GR-2NINDEL2R：

F-TTCCTCATGCTCGGTAAGTCAA；

R-AAAAAATCTAAAACTGGCAGAGGAG; and the combination of (a) and (b),

46F-46R：

F-CGCTCCGATCAGCAGGTA；

R-TTCTGCATCGTCAGGGTCAT。

the amplification from the wild type and mn2-m1 and mn2-m2 mutant leaf genomes using the primers is shown in FIG. 8.

The PCR amplification system comprises: ddH₂O5.75 ul; 10x Easy Taq Buffer 1 ul; primer F (10uM)0.4ul, primer R (10uM)0.4 ul; dNTP_S(2.5mM)0.25 ul; easy Taq 0.2ul; genomic DNA2ul, reagents used were purchased from whole gold.

The PCR amplification conditions include: 5min at 95 ℃, 30s at 60 ℃ and 30s at 72 ℃ for 30 cycles, and finally 10min at 72 ℃ and forever at 4 ℃.

Therefore, the designed GR-2NINDEL2F-GR-2NINDEL2R and 46F-46R marker primers can distinguish mn2-m1 mutants and mn2-m2 mutants and can be used for molecular marker-assisted selection.

Example 8 application of mutant gene mn2-m1 and mn2-m2 thereof in assisted breeding

The mn2-m1 amylose content was determined to be 34.75%, significantly higher than 24.55% for the conventional corn B73 (27.9%). The prior art shows that the content of the direct-linked starch in the common corn is about 25 percent, and mn2-m1 provides gene resources for cultivating the high amylose corn.

In addition, after the homologous gene in the arabidopsis thaliana is mutated, the stress resistance (salt resistance, drought resistance, heavy metal cadmium poison resistance and the like) is improved. In the future, we can culture new nitrogen-efficient corn varieties through gene transformation or gene editing or molecular marker-assisted breeding.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

Sequence listing

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Claims

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.