阅读说明：本技术 Ub2/ub3基因在调控玉米多果穗发育中的应用 (Application of UB2/UB3 gene in regulation and control of multiple-ear development of corn ) 是由 王海洋 孔德鑫 魏洪彬 王宝宝 赵永平 刘宇婷 赵斌斌 李全权 薛伟聪 于 2019-08-28 设计创作，主要内容包括：本发明公开了UB2/UB3基因在调控玉米多果穗发育中的应用,具体为UB2和UB3基因双突变在调控玉米多果穗发育中的应用。本发明利用CRISPR/Cas9转基因技术对玉米中的UB2和UB3这两个靶点进行了基因编辑,使UB2基因的靶位点有碱基的缺失,导致移码突变,而UB3基因则大片段的删除；结果显示,UB2和UB3双突变体植株的雄穗分枝数减少,并首次观察到UB2和UB3双突变体的果穗数增加,主穗的穗柄上长出新的雌穗,表现出多穗性,下部的腋生分生组织也脱抑制发育成雌穗,并且伴有气生根增多的表型；表明敲除UB2和UB3基因后,可使玉米突变体植株表现出多果穗表型,本发明对玉米多穗性的遗传改良提供了理论依据,具有较大的应用前景。(The invention discloses an application of UB2/UB3 genes in regulation and control of multiple-ear development of corn, and particularly relates to an application of UB2 and UB3 gene double mutation in regulation and control of multiple-ear development of corn. The invention utilizes CRISPR/Cas9 transgenic technology to edit two target points of UB2 and UB3 in corn, so that the target point of UB2 gene has base deletion, thereby causing frame shift mutation, and deleting the large fragment of UB3 gene; the results show that the number of tassel branches of UB2 and UB3 double mutant plants is reduced, the number of ears of UB2 and UB3 double mutants is increased for the first time, new ears grow on the ear stems of the main ears, the ears are shown to be multi-tassel, the axillary meristem at the lower part is also removed to inhibit the development of the ears, and the phenotype of increased aerial root is accompanied; the deletion of UB2 and UB3 genes shows that the maize mutant plant can show a multi-spike phenotype, the invention provides theoretical basis for genetic improvement of maize multi-spike property, and has great application prospect.)

The application of UB2/UB3 gene in regulating and controlling the development of multiple ears of corn.

2. the use of claim 1, wherein the nucleotide sequence of UB2 gene is shown in SEQ ID NO. 1, the nucleotide sequence of UB3 gene is shown in SEQ ID NO. 2,

3. The use of claim 1 or 2, wherein the use of double mutations in the UB2 and UB3 genes for modulating multi-ear development in maize.

4. The use of claim 3, wherein the knockout mutation is of the UB2 and UB3 genes for modulating multi-ear development in maize.

5. The use of claim 4, wherein the UB2 gene mutation of SEQ ID NO. 7 and the UB3 gene mutation of SEQ ID NO. 8 are used to modulate maize multi-ear development.

The application of UB2/UB3 gene in multi-ear corn breeding, which is characterized in that UB2 and UB3 genes in corn are knocked out and mutated.

7. The use according to claim 6, characterized in that the UB2 gene of SEQ ID NO. 1 is deleted in maize in 2420-2423 bp and 3197bp and UB3 gene of SEQ ID NO. 2 in 2980-3308 bp.

8. The use of claim 6 or 7, wherein CRISPR/Cas9 knockout vectors of UB2 and UB3 genes are constructed and transformed into maize to obtain UB2/UB3 transgenic maize plants.

9. The use of claim 8, wherein the sgRNA sequence of UB2 gene is shown in SEQ ID NO 3-4, and the sgRNA sequence of UB3 gene is shown in SEQ ID NO 5-6.

Technical Field

The invention relates to the technical field of corn genetic breeding, in particular to application of UB2/UB3 genes in regulation and control of corn multi-ear development.

Background

Corn (Latin's name: Zea mays L.) is an annual herbaceous plant of Zea of Gramineae, corn is an annual cross-pollinated plant with the same plants, namely male and female, is tall and strong in stem, is an important food crop and feed crop, is also a crop with the highest total yield all over the world, and has the planting area and the total yield only second to rice and wheat. Reasonable increase of corn planting density is one of effective ways for improving corn yield per unit, but light quality change (low R/FR ratio) caused by close planting can excite a series of shade-avoiding reactions of plants: such as higher plant height and ear height, poor lodging resistance, reduced leaf area, reduced branching, longer interval between male and female flowers, reduced fruiting rate, etc., which ultimately leads to reduced yield. Therefore, the cultivation of the ideal plant type variety which is resistant to close planting, high in light efficiency and suitable for mechanized operation is the most important technical measure for improving the unit yield of the corn in China.

SPL (SQUAMOSA promoter-binding protein-like) is a transcription factor specific to plants and widely exists in green plants. SPL plays an important regulatory role in a number of physiological and biochemical processes such as plant morphogenesis, developmental stage conversion, sporulation, flower and fruit development, anthocyanin accumulation, adverse stress response, and hormone signal transduction (Gouet al, 2011). For example, arabidopsis thaliana AtSPL3, AtSPL4, AtSPL5, AtSPL9, AtSPL10, and the like, can control juvenile to adult and flowering transitions in plants; the arabidopsis AtSPL8 not only participates in regulation and control of GA signal transduction, but also plays an important role in maintaining plant fertility; arabidopsis AtSPL9 and AtSPL10 can regulate anthocyanin anabolism through MYB-bHLH-WD40 complex and DFR, and over-expression of AtSPL9 and AtSPL10 can inhibit anthocyanin accumulation; the rice OsSPL14 is a key gene for controlling the plant type of rice, can reduce the tillering number of the rice and increase the inflorescence branch, the grain number per spike and the thousand grain weight, thereby improving the rice yield; OsSPL16 can control rice size, shape and color; tomato LeSPL-CNR is a key gene for controlling fruit ripening, and the mutation of the gene can inhibit the normal ripening of fruits.

After simultaneous mutation of two SPL genes UNBRANCHED2(UB2, encoding the ZmSBP8 protein) and UNBRANCHED3(UB3, encoding the ZmSBP30 protein) which are directly homologous to rice IPA1/OsSPL14 in maize, the plants show a phenotype of significantly reduced tassel branching and increased ear row number (Chuck et al, 2014; Liu et al, 2015 a). However, it is not clear that ZmUB2/UB3 regulates ear number and how to respond to close-planting cultivation conditions.

Disclosure of Invention

The invention aims to overcome the defects and shortcomings in the prior art and provides application of a UB2/UB3 gene in regulation and control of multiple-ear development of corn. According to the invention, after UB2 and UB3 genes are knocked out by using a CRISPR/Cas9 transgenic technology, a multi-ear phenotype is observed in a maize mutant plant, and the research result provides a theoretical basis for genetic improvement of maize polycarpity.

The second purpose of the invention is to provide the application of the UB2/UB3 gene in multi-ear corn breeding.

The above object of the present invention is achieved by the following technical solutions:

The invention utilizes CRISPR/Cas9 transgenic technology to carry out gene editing on two target points of UB2 and UB3 genes in corn, so that the target point of the UB2 gene has base deletion, thereby causing frameshift mutation, and the UB3 gene is deleted in large segment, thereby obtaining double mutants of UB2 and UB3 genes with sequences respectively shown as SEQ ID NO 3 and SEQ ID NO 4 after gene editing; the results show that the number of tassel branches of UB2 and UB3 double mutant plants is reduced, the number of ears of UB2 and UB3 double mutants is increased for the first time, new ears grow on the ear stems of the main ears, the ears are shown to be multi-tassel, the axillary meristem at the lower part is also deprived of inhibition to develop into the ears, and the phenotype of increased aerial roots is accompanied, so that the UB2 and UB3 genes are knocked out, the maize mutant plants can be shown to be multi-tassel phenotype, and theoretical basis is provided for genetic improvement of maize multi-tassel.

The invention firstly protects the application of the UB2/UB3 gene in regulating and controlling the development of multiple ears of corn.

Specifically, the nucleotide sequence of the UB2 gene is shown as SEQ ID NO. 1, the nucleotide sequence of the UB3 gene is shown as SEQ ID NO. 2,

In particular to application of double mutation of UB2 and UB3 genes in regulation of maize multi-ear development.

In particular to application of the UB2 and UB3 gene knockout mutation in regulation of maize multi-ear development.

In particular to the application of UB2 gene mutation type described by SEQ ID NO. 3 and UB3 gene mutation described by SEQ ID NO. 4 in regulating and controlling the development of multiple ears of corn.

Meanwhile, the invention also provides application of the UB2/UB3 gene in multi-ear corn breeding, which is to knock out and mutate UB2 and UB3 genes in corn to obtain UB2 and UB3 gene double-mutation corn plants (multi-ear corn plants).

Specifically, the UB2 gene 2420-2423 bp and 3197bp shown in SEQ ID NO. 1 and the UB3 gene 2980-3308 bp shown in SEQ ID NO. 2 in the corn are deleted, so that a UB2 and UB3 gene double mutation corn plant (multi-ear corn plant) shown in SEQ ID NO. 3 and SEQ ID NO. 4 is obtained.

Preferably, the knockout mutation is performed by using a CRISPR/Cas9 gene editing technology, specifically, a CRISPR/Cas9 gene editing vector of UB2 and UB3 genes is used for transforming corn to obtain a UB2/UB3 transgenic corn plant (a multi-ear corn plant).

Preferably, the sgRNA sequence of UB2 gene in the CRISPR/Cas9 gene editing vector is shown as SEQ ID NO 3-4, and the sgRNA sequence of UB3 gene is shown as SEQ ID NO 5-6; specifically, a CRISPR/Cas9 gene editing vector containing sgRNA shown in SEQ ID NO. 3-6 is used for transforming corn through an agrobacterium-mediated method to obtain a UB2/UB3 transgenic corn plant (multi-ear corn plant).

Compared with the prior art, the invention has the following beneficial effects:

The invention provides application of a UB2/UB3 gene in regulation and control of multiple-ear development of corn, and particularly provides application of double mutants of UB2 and UB3 genes in regulation and control of multiple-ear development of corn. The invention utilizes CRISPR/Cas9 transgenic technology to edit two target points of UB2 and UB3 in corn, so that the target point of UB2 gene has base deletion, thereby causing frame shift mutation, and deleting the large fragment of UB3 gene; the results show that the number of tassel branches of UB2 and UB3 double mutant plants is reduced, the number of ears of UB2 and UB3 double mutants is increased for the first time, new ears grow on the ear stems of the main ears, the ears are shown to be multi-tassel, the axillary meristem at the lower part is also removed to inhibit the development of the ears, and the phenotype of increased aerial root is accompanied; the UB2 and UB3 genes are knocked out to enable a corn mutant plant to show a multi-ear phenotype, and the double-mutation UB2 and UB3 genes can be applied to cultivation of multi-ear corn varieties and have a wide application prospect.

Drawings

FIG. 1 shows the genotype identification of UB2/UB3 transgenic plants, and the PCR products were sequenced and aligned with the wild-type sequence.

FIG. 2 shows UB2/UB3 regulating the phenotypes of maize, such as tassel branch number, female ear spiciness, aerial root, etc.

FIG. 3 shows that the ears of maize UB2/UB3 double mutants differed significantly at stage V14.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.