CN115948458A - Method for regulating and controlling corn tassel branch number - Google Patents

Method for regulating and controlling corn tassel branch number Download PDF

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CN115948458A
CN115948458A CN202211146740.7A CN202211146740A CN115948458A CN 115948458 A CN115948458 A CN 115948458A CN 202211146740 A CN202211146740 A CN 202211146740A CN 115948458 A CN115948458 A CN 115948458A
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mir529
vector
tassel
overexpression
corn
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刘琳
张睿
宋巧巧
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Shenzhen University
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    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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Abstract

The embodiment of the invention discloses a method for regulating and controlling the number of tassels of corn, which comprises the following steps: constructing miR529 over-expression vectors of two tandem repetitive hairpin structures by utilizing a T-DNA insertion transgenic technology; transferring the miR529 overexpression vector into immature embryos of a maize inbred line C01 through an agrobacterium EHA105 strain to obtain a plurality of transgenic events; and verifying a plurality of transgenic events by PCR, and selecting independent events with the expression quantity of miR529 in the tassel increased by a Northern blotting method. According to the invention, the expression quantity of miR529 is increased by using an overexpression transgenic technology, the tassel branch number is reduced in a close planting population of corn, and the yield per unit area is finally increased.

Description

Method for regulating and controlling maize tassel branch number
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for regulating and controlling the number of tassels of corn.
Background
Corn is one of three major food crops in the world and is also an important raw material in the industries of food, chemical industry, fuel, medicine and the like. At present, corn becomes the first crop with the widest planting area and the highest yield per unit in China. In order to promote national food safety, guarantee the quality of life of people and deal with the pressure brought by global population growth and global environment and climate change, the improvement of the corn yield is always the central importance of national development, and the improvement of the corn yield per unit area is one of the preferable ways.
A large number of researches show that the improvement of the field planting density is the key for improving the yield per unit of corn. For example, the corn planting density of the united states, a country of corn planting and production, has doubled from the middle of the last century, reaching the current 62000-104000 plants/hectare; meanwhile, the yield per unit is also increased from 1287 kg/hectare to 9595 kg/hectare.
In order to improve the close planting resistance of corn varieties, the selection of proper tassel branch number and size, ear position of the female ear, plant height, optimal growth period, leaf angle and the like is a key index for obtaining ideal plant types, yield and biomass, and is also a basis for cultivating varieties suitable for mechanized operations. The tassel size mainly comprises tassel branch number, spikelet number, branch included angle and length, and is an important target character for corn domestication and genetic improvement. The overlarge tassel affects the canopy structure of the plant type, reduces the whole light energy utilization rate of the plant, and shields the illumination which can reach the female tassel originally. And the development of the maize inflorescence is approximately in the same period, and the tassel robs the female ear for photosynthetic products and other nutrients required for development. Under close-planting conditions, smaller tassels or smaller amounts of tassels (detasseling techniques) can produce sufficient pollen to pollinate the female ear without affecting seed set, and therefore, moderately reduced tassels are an important maize breeding goal.
The number of tassel branches of maize is a typical quantitative trait, and the differentiation and development process is synergistically regulated by hormone, genetic and environmental conditions. A plurality of pathways have been identified to participate in regulating maize tassel development, including the CLV/WUS pathway, the auxin pathway, the RAMOSA pathway, the SPL pathway, and the like. Since tassel branch is a quantitative trait-regulated phenotype, more regulatory factors need to be deeply excavated.
Disclosure of Invention
The embodiment of the invention provides a method for regulating and controlling the number of tassels of corn.
A method of modulating maize tassel branch number comprising:
constructing miR529 over-expression vectors of two tandem repetitive hairpin structures by utilizing a T-DNA insertion transgenic technology;
transferring the miR529 overexpression vector into immature embryos of a maize inbred line C01 through an agrobacterium EHA105 strain to obtain a plurality of transgenic events;
and (3) verifying the multiple transgenic events through PCR molecules respectively, and selecting independent events with the increased expression level of miR529 in the tassel by a Northern blotting method.
Specifically, the miR529 overexpression vector of the hairpin structure comprises: artificial miRNA constructs driven using the maize ubiquitin gene UBI and BAR gene constructs driven using the enhanced CaMV 35S promoter.
Specifically, the miR529 overexpression vector with two tandem repeat hairpin structures is constructed by using a T-DNA insertion transgenic technology, and comprises the following steps:
in a pOT2-Polycis-UN vector, two tandem repeats of miR529 with a hairpin structure are cloned into a main stem at the downstream of a UBI promoter of a maize ubiquitin gene.
Specifically, the method further comprises the following steps:
sequencing and verifying an miR529 over-expression vector of a hairpin structure, and carrying out double digestion on the obtained pOT2-Polycis-UN vector by PacI and MluI restriction enzyme;
cloning the UBI-miR529-NOS fragment recovered after digestion into a binary vector of pZZ 00026-PM;
the sanger sequencing verified binary vector was introduced into agrobacterium strain EHA105.
Specifically, primers adopted in constructing the miR529 overexpression vector are as follows: OE529-f and OE529-r.
Specifically, the structure of OE529-f is: GTCGACTCTAGAGGATCCAAGCT.
Specifically, the structure of OE529-r is: AGAGAGCGTACAGCCTCCAATCC.
The embodiment of the invention has the beneficial effects that: according to the invention, the expression quantity of miR529 is increased by using an overexpression transgenic technology, the tassel branch number is reduced in a corn close planting population, and the unit area yield is finally increased. The research result provides a theoretical basis for genetic improvement of the tassel form constitution under the condition of the maize density-resistant planting.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is a diagram of vector construction and tassel phenotype identification for miR529 overexpression provided by an embodiment of the invention.
FIG. 2 is a statistical chart of ear phenotype of miR529 overexpression provided by the embodiment of the invention.
FIG. 3 is a plot of a field density planting experiment of miR529 over-expressing strains provided by an embodiment of the invention.
Detailed Description
In order to make those skilled in the art better understand the technical solution of the present invention, the technical solution in the embodiment of the present invention will be clearly and completely described below with reference to the drawings in the embodiment of the present invention.
The embodiment of the invention provides a method for regulating and controlling the number of tassels of corn, which comprises the following steps:
step one, constructing miR529 overexpression vectors of two tandem repetitive hairpin structures by utilizing a T-DNA insertion transgenic technology;
step two, transferring the miR529 overexpression vector into immature embryos of a maize inbred line C01 through an agrobacterium EHA105 strain to obtain a plurality of transgenic events;
and thirdly, verifying the multiple transgenic events through PCR molecular identification respectively, and selecting the independent event with the increased expression level of miR529 in the tassel by a Northern blotting method.
Specifically, the miR529 overexpression vector of the hairpin structure comprises: artificial miRNA constructs driven using the maize ubiquitin gene UBI and BAR gene constructs driven using the enhanced CaMV 35S promoter. Wherein, a miR529 overexpression vector of two tandem repeat hairpin structures is constructed by utilizing a T-DNA insertion transgenic technology, and comprises the following steps: in a pOT2-Polycis-UN vector, two tandem repeats of miR529 with hairpin structure are cloned into the stem downstream of the UBI promoter of the maize ubiquitin gene.
The embodiment of the invention also comprises the following steps: sequencing and verifying an miR529 over-expression vector of a hairpin structure, and carrying out double digestion on the obtained pOT2-Polycis-UN vector by PacI and MluI restriction enzyme; cloning the UBI-miR529-NOS fragment recovered after digestion into a binary vector of pZZ 00026-PM; the sanger sequencing verified binary vector was introduced into agrobacterium strain EHA105.
The embodiment of the invention provides a construction method of a corn overexpression miR529 vector, which comprises the following steps: the over-expression miR529 is realized by integrating T-DNA insertion, an artificial miRNA structure driven by a maize Ubiquitin gene Ubiquitin (UBI) and a BAR (Streptomyces hygroscopicus phosphorylase acetyltransferase) gene driven by a CaMV 35S promoter are used, and a structure of two series-repetitive hairpin structure miR529 over-expression vectors is constructed by utilizing a T-DNA insertion transgenic technology as shown in A in figure 1. In the pOT2-Polycis-UN vector, two tandem repeats of miR529 hairpin structure were cloned into the UBI promoter downstream stem. After sequencing verification, the resulting pOT2-Polycis-UN vector was double digested with PacI and MluI restriction enzymes. And then cloning the recovered UBI-miR529-NOS fragment into a binary vector of pZZ 00026-PM. The binary vector verified by sanger sequencing was introduced into agrobacterium strain EHA105 and transformed into the immature embryo genome of maize inbred line C01. Successful transgenic events were identified by PCR and miR529 expression levels were further quantified by Northern blotting. Wherein, the primers for vector construction (miR 529-OE-p 26) and genotyping (OE 529-f/r) are as follows:
>OE529-f
GTCGACTCTAGAGGATCCAAGCT
>OE529-r
AGAGAGCGTACAGCCTCCAATCC
the size of the product obtained by the embodiment of the invention is 488bp.
In the embodiment of the invention, the over-expression phenotype of the corn miRNA529 is very prominent, and the influence on the corn morphogenesis is concentrated on the tassel without influencing other agronomic traits such as the female ear or tillering. Compared with the genes which are found to control the number of tassel branches in the past, such as unbrnched 2 (ub 2) and unbrnched 3 (ub 3), the single gene has no obvious control on the number of tassel branches, and a double-mutant homozygote needs to be obtained by genetic improvement or a triple-mutant homozygote needs to be created by combining with the gene Tassel shear 4 (tsh 4). Moreover, the mutants of the three genes have more tillers, the whole plant type is not suitable for agricultural production, the female ears are relatively small, and the improvement of the final yield is not facilitated. Therefore, a single main control genetic factor of important traits, such as miR529, regulates tassel morphogenesis, and has very important genetic breeding and molecular improvement values.
In order to discover genetic factors for regulating the corn tassel morphology, previous research mainly focuses on exploring unknown genes, and small-molecule RNAs such as microRNAs are less emphasized. The innovation of the invention is that a micro RNA (micro RNA 529) is definitely disclosed to play a decisive role in the size of the corn tassel, and further field experiments prove that the micro RNA529 can effectively improve the yield of the corn in unit area under the condition of close planting.
As shown in figure 1, the vector construction and tassel phenotype identification results of miR529 overexpression are shown. Wherein A is a vector for over-expressing miR 529; b, identifying two effective events for over-expressing miR529 for Northern blotting; c is a tassel phenotype of overexpression miR 529; d is the tassel phenotype statistics of the over-expression miR529, including the number of primary branches, the number of secondary branches and the number of spikelet pairs. OE529: over-expressing miR529 plants; wt: wild type plants. As shown in figure 1B, more than 20 transgenic events are successfully obtained by transferring an agrobacterium EHA105 strain into an immature embryo of a maize inbred line C01, and two independent events with the expression level of miR529 in tassels being increased are selected from the events by a Northern blotting method through PCR molecular verification. The results are shown in C and D, the first-stage branch number, the second-stage branch number and the spikelet pair number of the tassel of the maize plant over-expressing miR529 are reduced, and the dry tassel weight of the tassel is reduced. As shown in FIG. 2, the table shows the statistics of the ear phenotype of miR529 overexpression, including ear row number, seed number and ear weight. OE529: over-expressing miR529 plants; wt: wild type plant. The plant has no obvious phenotype difference in the ears, including ear row number, seed number and ear weight. As shown in fig. 3, statistics of field density planting results for miR 529-overexpressing lines. And (4) performing phenotype statistics on the female ears in the density planting experiment, and obtaining the yield per unit area under the conditions of different densities. OE529: over-expressing miR529 plants; wt: wild type plants. Through close planting experiments in fields of Torpedo and Hainan san, it is shown that when the planting density is more than 112500 plants per hectare, the yield per unit area of miR529 over-expressed is far higher than that of a wild type, and the high yield is not caused by the characteristics of the female ear including ear row number, row grain number, ear grain weight and hundred grain weight.
In the embodiment of the invention, the miR529 overexpression vector is miR529-OE-p26, and the full-length sequence thereof is as follows:
pZZ00026-UN-dModuZmamiR529 (miR 529-OE-p26 full-length sequence)
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.
The foregoing is only a partial embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (7)

1. A method for regulating and controlling the tassel branch number of corn is characterized by comprising the following steps:
constructing miR529 over-expression vectors of two tandem repetitive hairpin structures by utilizing a T-DNA insertion transgenic technology;
transferring the miR529 overexpression vector into immature embryos of a maize inbred line C01 through an agrobacterium EHA105 strain to obtain a plurality of transgenic events;
and respectively verifying the multiple transgenic events by PCR, and selecting independent events with the expression quantity of miR529 in the tassel by a Northern blotting method.
2. The method of claim 1, wherein the miR529 overexpression vector of the hairpin structure comprises: artificial miRNA structures driven using the maize ubiquitin gene UBI and BAR gene structures driven using the enhanced CaMV 35S promoter.
3. The method according to claim 2, wherein the miR529 overexpression vector of two tandem repeat hairpin structures is constructed by using a T-DNA insertion transgenic technology, and comprises the following steps:
in a pOT2-Polycis-UN vector, two tandem repeats of miR529 with a hairpin structure are cloned into a main stem at the downstream of a UBI promoter of a maize ubiquitin gene.
4. The method of claim 1, further comprising:
sequencing and verifying the miR529 overexpression vector of the hairpin structure, and carrying out double digestion on the obtained pOT2-Polycis-UN vector by PacI and MluI restriction endonucleases;
cloning the UBI-miR529-NOS fragment recovered after digestion into a binary vector of pZZ 00026-PM;
the binary vector verified by Sanger sequencing was introduced into agrobacterium strain EHA105.
5. The method according to claim 1, wherein primers used in constructing the miR529 overexpression vector are: OE529-f and OE529-r.
6. The method of claim 5, wherein the sequence of OE529-f is: GTCGACTCTAGAGGATCCAAGCT.
7. The method of claim 5, wherein the sequence of OE529-r is: AGAGAGCGTACAGCCTCCAATCC.
CN202211146740.7A 2022-09-21 2022-09-21 Method for regulating and controlling corn tassel branch number Pending CN115948458A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116548300A (en) * 2023-06-26 2023-08-08 中国农业科学院生物技术研究所 Method for improving density-resistant yield of corn and application thereof

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US10683513B2 (en) * 2013-12-31 2020-06-16 Dow Agrosciences Llc Tissue-specific expression and hybrid plant production
CN109161558B (en) * 2018-09-19 2022-05-06 深圳大学 Construction method of monocotyledon miRNA high-efficiency overexpression vector
WO2021150469A1 (en) * 2020-01-21 2021-07-29 Pioneer Hi-Bred International, Inc. Targeting microrna to regulate native gene function by genome editing

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116548300A (en) * 2023-06-26 2023-08-08 中国农业科学院生物技术研究所 Method for improving density-resistant yield of corn and application thereof
CN116548300B (en) * 2023-06-26 2023-12-19 中国农业科学院生物技术研究所 Method for improving density-resistant yield of corn and application thereof

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