阅读说明：本技术 小麦TaARF12基因及其应用 (Wheat TaARF12 gene and application thereof ) 是由 李爱丽 王芳 耿帅锋 毛龙 于 2019-11-07 设计创作，主要内容包括：本发明提供了小麦TaARF12基因及其应用,属于作物分子生物学领域。本发明设计引物从中国春幼穗的cDNA中克隆TaARF12的编码区序列,TaARF12基因编码区序列在A、B、D同源染色体中的序列分别如SEQ ID NO.1-3所示。本发明选择TaARF12特异性的RNAi片段,构建RNAi载体并转化Fielder。同时,使用CRISPR/Cas9载体构建包含两个靶位点的基因编辑载体并转化野生型Fielder。获得的RNAi转基因株系和基因编辑株系具有类似的表型,表现为株高变矮,穗子变长。说明小麦TaARF12基因能够调控植株高度和穗子长度,该基因被干扰表达后植株表现为植株变矮,穗子增长。(The invention provides a wheat TaARF12 gene and application thereof, belonging to the field of crop molecular biology. The invention designs a primer to clone a coding region sequence of TaARF12 from cDNA of young ears in spring of China, and the sequences of the coding region sequence of the TaARF12 gene in A, B, D homologous chromosomes are respectively shown in SEQ ID NO. 1-3. The invention selects a specific RNAi fragment of TaARF12, constructs an RNAi vector and converts the RNAi vector into Fielder. Meanwhile, a gene editing vector containing two target sites is constructed by using the CRISPR/Cas9 vector and the wild-type Fielder is transformed. The obtained RNAi transgenic line and the gene editing line have similar phenotypes and show that the plant height is shortened and the spike is lengthened. The wheat TaARF12 gene can regulate and control the height and the ear length of the plant, and the plant is shown to be dwarfed and the ear is increased after the gene is interfered to express.)

1. Any one of the following applications of wheat TaARF12 gene,

(1) the application in wheat breeding;

(2) the application in regulating and controlling plant height;

(3) the application in regulating the wheat head length;

(4) the application in preparing transgenic plants;

(5) application in plant germplasm resource improvement.

2. The use according to claim 1, wherein the wheat TaARF12 gene is located on a second wheat homologue, which has the sequence on the wheat 2A homologue as shown in SEQ ID No.1, on the wheat 2B homologue as shown in SEQ ID No.2, and on the wheat 2D homologue as shown in SEQ ID No. 3.

3. The primer group for cloning the wheat TaARF12 gene is characterized in that the nucleotide sequence is shown as SEQ ID NO. 4-5.

4. The cloning method of the wheat TaARF12 gene is characterized in that a specific primer group is utilized, wheat young ear cDNA is taken as a template, the PCR amplification is carried out to obtain the wheat TaARF12 gene, and the nucleotide sequence of the specific primer group is shown as SEQ ID NO. 4-5.

5. An RNAi fragment of wheat TaARF12 gene, characterized in that the nucleotide sequence is shown in SEQ ID NO. 12.

6. An RNAi vector of wheat TaARF12 gene, comprising the RNAi fragment of claim 5, wherein the RNAi vector is prepared by a method comprising the steps of:

(1) amplifying an RNAi fragment of a TaARF12 gene by using a pair of primers with BamH I and Kpn I enzyme cutting sites respectively, and connecting the amplified RNAi fragment to a linearized pWMB006 vector after enzyme cutting to obtain a new linearized vector; the nucleotide sequence of the primer is shown as SEQ ID NO. 6-7;

(2) utilizing a pair of primers with Sac I and Spe I enzyme cutting sites respectively to amplify a reverse complementary fragment of the TaARF12 RNAi fragment, connecting the amplified reverse complementary fragment to the new linearized vector after enzyme cutting, and constructing an intermediate vector pWMB006-TaARF12, wherein the nucleotide sequence of the primers is shown as SEQ ID NO. 9-10;

(3) HindIII is used for digesting pWMB006-TaARF12 and pWMB111, a band with a TaARF12 interference fragment is recovered by glue and is connected to a linearized pWMB111 vector to obtain a pWMB111-TaARF12 RNAi vector.

7. The identification method of wheat TaARF12 RNAi transgenic positive plants is characterized in that PCR amplification is carried out by using primers for detecting Bar genes, and the nucleotide sequences of the primers are shown as SEQ ID NO. 13-14.

8. The sgRNA of the specific targeting wheat TaARF12 gene is characterized in that the DNA sequence of the sgRNA is shown in SEQ ID NO.17 or SEQ ID NO. 18.

9. A CRISPR/Cas9 vector containing the DNA sequence of the sgRNA of claim 8.

10. Use of an expression inhibitor of wheat TaARF12 gene of claim 1, an RNAi fragment of claim 5, an RNAi vector of claim 6, or a CRISPR/Cas9 vector of claim 9 for reducing plant height or promoting plant ear lengthening.

Technical Field

The invention relates to the field of crop molecular biology, in particular to cloning of a wheat TaARF12 gene, a construction method of a TaARF12 RNAi (RNA interference) vector, application of a CRISPR/Cas9-TaARF12 vector and application of a TaARF12 gene in regulation of plant height and spike length.

Background

Common wheat is an important food crop, and wheat is taken as a staple food in people with more than 1/3 all over the world. The wheat plant height and the wheat ear length have important influence on the yield formation, and the research on the development genetic rule of the wheat plant height and the wheat ear length is helpful for understanding the yield formation mechanism. In the 70 s of the 20 th century, the green revolution dominated by the utilization of the semi-dwarf gene of wheat brought great increase to the global wheat yield. The lodging resistance of wheat can be improved by reducing the plant height, and lodging-resistant, big ear and high yield varieties can be bred by fully utilizing the short stalk genes in breeding. The ear length is an important agronomic character of wheat, the ear length character of the wheat is closely related to three yield forming factors of the wheat, and longer ears provide possibility for increasing the number of small ears, the number of grains per ear and the thousand grain weight, thereby being beneficial to improving the yield of the wheat.

Auxin is a ubiquitous plant hormone in plants and plays an important role in the growth and development stages of plants such as apical dominance and root and stem morphogenesis. Auxin Response Factors (ARF) are a family of transcription factors that bind cis-response elements and mediate auxin signaling responses. The ARF protein comprises 3 conserved structural domains, an N-terminal B3 DNA binding structural domain and has the function of binding downstream genes; an intermediate Activation Domain (AD) and an inhibition domain (RD) that determine whether ARF exerts an activating or inhibitory effect; the CTD domain at the C-terminus determines the interaction of ARF with other proteins. At present, the research on ARF genes generally focuses on root systems, such as OsARF12, transcription activator of auxin response genes, and regulation of root elongation in rice. However, research on ARF genes in wheat has been rare so far, especially for functional research and production application of TaARF12 in wheat. Therefore, RNAi and knockout of TaARF12 in wheat are necessary, and the role of TaARF12 in wheat is deeply researched.

Disclosure of Invention

The invention aims to provide a wheat TaARF12 gene and application thereof.

The TaARF12 gene is amplified in the young ear of hexaploid wheat in Chinese spring to obtain a gene sequence on A, B, D homologous chromosomes, which are respectively shown as SEQ ID NO.1-3, and the gene is named as TaARF12 and is positioned on a second chromosome homologous group.

The invention provides application of a wheat TaARF12 gene in wheat breeding.

The invention provides application of a wheat TaARF12 gene in regulating and controlling plant height.

The invention provides application of a wheat TaARF12 gene in preparation of transgenic plants.

The invention provides application of a wheat TaARF12 gene in regulation of ear elongation.

The invention provides application of a wheat TaARF12 gene in improvement of plant germplasm resources.

Such plants include, but are not limited to, wheat.

Specifically, the wheat TaARF12 gene is located on a second wheat homologous group, the sequence of the wheat TaARF12 gene on a wheat 2A homologous chromosome is shown as SEQ ID NO.1, the sequence of the wheat 2B homologous chromosome is shown as SEQ ID NO.2, and the sequence of the wheat TaARF12 gene on a wheat 2D homologous chromosome is shown as SEQ ID NO. 3.

The invention provides a primer combination for cloning a wheat TaARF12 gene, and the nucleotide sequence of the primer combination is shown as SEQ ID NO. 4-5.

Correspondingly, the invention provides a cloning method of a wheat TaARF12 gene, which utilizes a specific primer combination shown in SEQ ID NO.4-5 and takes wheat spike cDNA as a template to obtain the wheat TaARF12 gene by PCR amplification.

The invention provides an RNAi fragment of a wheat TaARF12 gene, and the nucleotide sequence of the RNAi fragment is shown in SEQ ID NO. 12.

The invention provides an RNAi vector containing the RNAi fragment of the wheat TaARF12 gene.

The invention also provides a construction method of the wheat TaARF12 RNAi vector, which is constructed by the following method and comprises the following steps:

(1) carrying out PCR amplification by using SEQ ID NO.4-5 as a primer and Chinese spring young ear cDNA as a template, and recovering a PCR product for sequencing;

(2) using SEQ ID NO.6-7 as a primer and the correct sequencing plasmid of (1) as a template to perform PCR amplification, recovering a PCR product, and using SEQ ID NO.8 to perform sequencing;

(3) performing double enzyme digestion on the recovered product in the step (2) and the pWMB006 vector by using BamH I and Kpn I, recovering, and performing connecting sequencing;

(4) using SEQ ID NO.9-10 as a primer and the correct sequencing plasmid of (1) as a template to perform PCR amplification, recovering a PCR product, and using SEQ ID NO.11 to perform sequencing;

(5) carrying out double enzyme digestion on the recovered product in the step (4) and the vector successfully subjected to the ligation sequencing in the step (3) by using Sac I and Spe I, recovering, and then performing ligation sequencing to construct an intermediate vector pWMB006-TaARF 12;

(6) hind III is used for digesting pWMB006-TaARF12 and pWMB111 vectors, a fragment with a TaARF12 gene sequence and pWMB111 are recovered, and the pWMB111-TaARF12 RNAi vector is constructed through ligation detection.

The invention provides a method for identifying wheat RNAi transgenic plants, which uses primers SEQ ID NO.13-14, takes DNA of the transgenic plants as a template, and obtains positive plants with 430bp bands through PCR amplification.

The invention provides a method for identifying the expression quantity of a TaARF12 gene in an RNAi plant, wherein the used fluorescent quantitative primer is SEQ ID NO. 15-16.

Compared with the wild type, the plant height of the TaARF12 RNAi transgenic plant provided by the invention is obviously reduced.

Compared with the wild type, the spike phenotype of the TaARF12 RNAi transgenic plant provided by the invention is obviously longer.

The invention provides a target site for constructing a TaARF12 CRISPR/Cas9 vector and application thereof.

The invention provides a target site sequence of a wheat TaARF12 CRISPR/Cas9 vector, and the nucleotide sequence of the target site is SEQ ID NO.17 and SEQ ID NO. 18.

The CRISPR/Cas9 vector containing the target site sequence belongs to the protection scope of the invention.

The invention provides a method for identifying a wheat TaARF12 CRISPR/Cas9 transgenic plant, which uses a primer SEQID NO.13-14, takes DNA of the transgenic plant as a template, and obtains a positive plant with a 430bp strip by PCR amplification.

The invention provides a TaARF12 gene CRISPR/Cas9 transgenic plant which has a phenotype that the plant height is obviously reduced.

The invention provides a TaARF12 gene CRISPR/Cas9 transgenic plant which has a phenotype that the ear is obviously lengthened.

The invention provides a cloning method of a wheat TaARF12 gene and a biological function of the wheat TaARF12 gene in regulating and controlling plant height and spike length of a plant, and experiments show that after the wheat TaARF12 gene is silenced or is subjected to interference expression, the plant shows that the plant height is reduced and the spike length is lengthened. The wheat TaARF12 gene can be widely applied to the fields of wheat genetic breeding, germplasm resource improvement and cultivation of TaARF12 gene-transformed plants, and has an important effect on improving and improving the germplasm resources of crops such as wheat.

Drawings

FIG. 1 is a plasmid map of pWMB006 vector.

FIG. 2 is a plasmid map of the pWMB111 vector.

FIG. 3 shows the identification of positive plants of Fielder T3 transformed with TaARF12 RNAi. M is Marker, WT is wild-type Fielder as negative control, RNAi-L2, RNAi-L6, RNAi-L3 represent TaARF12-ARNAi wheat T3 generation plant (the same below), and the used template is DNA of three-leaf stage seedling. The 430bp band in the figure indicates that the transgenic plant is a positive plant.

FIG. 4 shows the expression level of TaARF12 in ears of wild-type Fielder and RNAi plants.

FIG. 5 is a plant height phenotype diagram of TaARF12 RNAi transgenic plant.

Fig. 6 is a statistical plot of plant heights of TaARF12 RNAi transgenic plants, which represent very significant differences.

FIG. 7 is a graph of spike length phenotype of TaARF12 RNAi transgenic plants.

Fig. 8 is a statistical plot of spike length of TaARF12 RNAi transgenic plants, representing very significant differences.

FIG. 9 shows the identification of positive plants of Fielder T1 generation transformed by TaARF12 CRISPR/Cas 9. M is Marker, WT is wild-type Fielder as negative control arf12-2, arf12-5, arf12-11 and arf12-9 represent TaARF12 CRISPR/Cas9 wheat T1 generation plant (the same below), and the used template is DNA of trefoil seedling. The 430bp band in the figure indicates that the transgenic plant is a positive plant.

FIG. 10 is gene editing site information of TaARF12 CRISPR/Cas9 transgenic plant.

FIG. 11 is a plant height phenotype diagram of TaARF12 CRISPR/Cas9 transgenic plant, Bar is 10 cm.

Fig. 12 is a statistical graph of plant heights of TaARF12 CRISPR/Cas9 transgenic plants, which represents the differences are very significant.

FIG. 13 is a spike length phenotype diagram of TaARF12 CRISPR/Cas9 transgenic plant, Bar 1 cm.

Fig. 14 is a statistical plot of tasrf 12 CRISPR/Cas9 transgenic plant spike length, which represents very significant differences.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. It is within the scope of the present invention to modify or replace methods, steps or conditions of the present invention without departing from the spirit and substance of the present invention.

Unless otherwise specified, the chemical reagents used in the examples are all conventional commercially available reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.