Application of miR172d and targeted negative regulation gene SNB1 of miR172d in regulation of plant height and neck length of spike of rice

Abstract

The invention relates to the technical field of agricultural bioengineering, and particularly provides miR172d and application of a targeted negative regulation gene SNB1 of the miR172d in regulation of the plant height and the neck length of a rice spike. The miR172d is knocked out, so that the plant height and the neck length of a rice spike can be reduced, and the number of stem nodes is remarkably reduced; the overexpression of the miR172d can increase the plant height and the neck length of a spike. The SNB1 gene is a target of the miR172d, and the expression of the target coding gene SNB1 is reduced at the transcriptional level by the miR172d. Genetic transformation experiments prove that the plant height can be reduced by over-expression of SNB1, and the plant height of the snb1 mutant is increased. The miR172d and the target gene SNB1 thereof provided by the invention can be applied to rice breeding work and have a good application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural bioengineering, and relates to the application of miR172d and its targeted negative regulatory gene SNB1 in regulating rice plant height and panicle neck length. Background Art

[0002] Rice is one of the most important food crops in the world and is of great significance to national food security. Plant height is one of the most important agronomic traits and is closely related to rice yield. Too high plant height can lead to lodging, and reducing plant height can sometimes lead to reduced biomass and yield. The commonly used sd1 relies on high water and high nitrogen fertilizers to ensure the stability of yield, but high nitrogen fertilizers have a negative impact on the environment and the cost of rice cultivation. Therefore, it is necessary to screen more genes that regulate plant height traits as a background for improving traits.

[0003] MicroRNA is a type of non-coding RNA with a length of about 19-22 nucleotides, and is a key regulatory factor in the growth and development of plants. Studies have shown that microRNA is involved in regulating various aspects of plant growth and development, as well as biological processes such as stress response. Changes in the expression of microRNA and its target genes in plants can affect a variety of genetic traits. Therefore, functional research on rice microRNA can provide a new theoretical basis and research method for rice plant height breeding and improvement. Summary of the invention

[0004] The purpose of the present invention is to overcome the above-mentioned deficiencies in the prior art and provide an application of miR172d and its target gene SNB1 in regulating rice plant height and panicle neck length.

[0005] In order to achieve the above object, the present invention provides the following technical solutions: In a first aspect, the present invention provides a miR172d that targets and negatively regulates the SNB1 gene. The miR172d precursor primary sequence is shown as SEQ ID No.1, and the SNB1 gene sequence is shown as SEQ ID No.4.

[0006] Furthermore, after the precursor of miR172d is processed by the DCL complex, a functional mature sequence is formed in vivo as shown in SEQ ID No.2.

[0007] Furthermore, the amino acid sequence of the protein encoded by the SNB1 gene is shown in SEQ ID No.5.

[0008] The second aspect of the present invention provides the application of miR172d in regulating rice plant height and panicle neck length, wherein the application is as follows: knocking out or silencing miR172d reduces rice plant height and panicle neck length; overexpressing miR172d slightly increases plant height and increases panicle neck length.

[0009] Furthermore, the promoter of miR172d has the characteristic of internode-specific expression, and the promoter sequence is shown in SEQ ID No.3.

[0010] Furthermore, the operating method of the application is: constructing a rice miR172d overexpression vector, a knockout vector, and an RNAi vector; transforming the constructed recombinant plant expression vector into plant tissues or plant cells; and cultivating and screening transgenic plants with increased or decreased plant height and changed panicle neck length.

[0011] Furthermore, the rice miR172d overexpression vector, knockout vector and RNAi vector are transferred into Agrobacterium, and the transgenic rice plants are screened and obtained by using the Agrobacterium-mediated genetic transformation method.

[0012] Furthermore, the PCR amplification primers for miR172d overexpression are shown in SEQ ID Nos. 6 and 7, and the PCR amplification primers for RNAi miR172d are shown in SEQ ID Nos. 8 and 9.

[0013] After being processed into mature sequences in plants, miR172d will move and target its downstream target gene SNB1 (LOC_Os07g13170) mRNA, forming a complementary pair, resulting in the cleavage and decrease of SNB1 abundance. In a specific embodiment, it is found that snb1 The mutant did have increased plant height, while SNB1 overexpression reduced plant height.

[0014] The third aspect of the present invention provides a method for reducing rice plant height, which comprises overexpressing the rice SNB1 gene, wherein the SNB1 gene sequence is shown in SEQ ID No.4.

[0015] A fourth aspect of the present invention provides a method for increasing rice plant height, which comprises knocking out or silencing the rice SNB1 gene, wherein the SNB1 gene sequence is shown in SEQ ID No.4.

[0016] Beneficial effects of the present invention: The rice plant height and panicle neck length regulating gene miR172d was successfully cloned from the rice variety Nipponbare NIP. Using the miR172d gene and its downstream targeted negative regulatory gene SNB1, methods for reducing or increasing rice plant height and regulating rice panicle neck length were obtained respectively. After transforming rice, the miR172d RNAi and knockout vector described in the present invention can significantly reduce the expression level of miR172d, and the plant height and panicle neck length of the transformed plants are significantly reduced. Overexpression of miR172d can significantly increase the panicle neck length. Overexpression of the miR172d downstream targeted negative regulatory gene SNB1 described in the present invention can reduce plant height, and increase mutant plant height. Therefore, the complete set of genes and gene transformation vectors provided by the present invention can be used for the improvement of new varieties, relatively freely regulating rice plant height, and providing new gene resources and methods for modern agricultural molecular breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The panicle length and plant height phenotypes of miR172d overexpression, knockout and STTM interference materials. (A) From left to right, the panicle length phenotypes of Nipponbare NIP, the main variety in Northeast China Longjing 46 / LG46 and the short-growing material Kitaake are the 172dKO knockout materials, STTM interference materials and OE overexpression materials. (B) The plant height phenotypes of the japonica rice variety Nipponbare NIP 172dKO knockout materials and STTM interference materials. (D) The DICM cell size detection of the lower panicle node of the wild-type Nipponbare NIP and 172dKO knockout materials.

[0018] Figure 2 Detection of miR172 inhibiting SNB1 protein accumulation. (A) By co-expressing miR172 and 1305-SNB1-GFP in the pCambia background in tobacco leaves, the changes in SNB1 protein accumulation levels were detected compared with the empty vector. (B) Examples of the complementary and interactive regions between the mature sequence of miR172d and the SNB1 CDS.

[0019] Figure 3 For rice snb1 Plant height and panicle neck length phenotypes of mutants. (A) Plant height and panicle neck length phenotypes of rice snb1 mutant. (B) DICM cell size detection of the lower panicle node of wild-type Tc65 and snb1 mutant.

[0020] Figure 4 Figure 2 shows the detection of transcriptional activation of rice Eui by SNB1. (A) The histone H3K27me3 methylation level on the promoter of Eui was significantly increased in rice SNB1 OE overexpression materials. (B) SNB binds to the downstream region of the Eui genomic sequence and activates its transcription, resulting in a significant increase in the transcription level of the reporter gene LUC, rather than binding to its promoter. DETAILED DESCRIPTION

[0021] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0022] Example 1 Creation of miR172d knockout, STTM RNAi silencing, and miR172 overexpression materials The rice leaves were quickly frozen in liquid nitrogen, shaken and broken, and then extracted with 600 μl CTAB solution in a 65-degree water bath for 1 hour. The supernatant was centrifuged and 200 μl chloroform was added to extract the protein. The supernatant was centrifuged and 1 volume of isopropanol was added. The mixture was mixed and incubated at -80 degrees for 1 hour. The supernatant was removed by centrifugation. The leaves were rinsed twice with 70% alcohol, dried and sterile ultrapure water was added to dissolve the genomic DNA.

[0023] SEQ ID No6, 7 and SEQ ID No8, 9 were used as primers to amplify the STTM silencing fragment with the miR172d precursor sequence and mature sequence as the background from genomic DNA, and constructed into the pCambia1300 backbone vector to obtain the miR172d silencing vector and overexpression vector. Its knockout vector was constructed using the Cas9 / CRISPR system of Liu Yaoguang's experiment.

[0024] After the vector plasmid was transformed into Agrobacterium, the rice callus was infected with Agrobacterium and positive plants were obtained after testing.

[0025] Example 2 Detection of the biochemical regulatory relationship between miR172d and SNB1 The SNB1 CDS sequence was constructed into the pCambia1305 backbone vector to obtain an expression vector with a fluorescent tag GFP.

[0026] After the vector plasmid was transformed into Agrobacterium, tobacco seedlings were infected. ①1300 and 1305-SNB1-GFP, ②1300-miR172d and 1305-SNB1-GFP in Example 1 were infected with tobacco respectively. The fluorescence of GFP tag was observed by confocal confocal microscopy. The fluorescence of the combination of miR172d was significantly weaker than that of the 1300 empty control, indicating that miR172d would inhibit the expression of SNB1-GFP. The results of western detection also proved this point ( Figure 2 A).

[0027] The mature sequence of miR172d is reverse complementary to a segment at the end of SNB1 mRNA, causing the cleavage and degradation of SNB1 mRNA and inhibiting the expression of SNB1 ( Figure 2 B).

[0028] Example 3 Wild-type and miR172d KO knockout materials, STTM RNAi silencing, miR172 overexpression, snb1 Comparison of spike length and plant height among mutant materials Through the genetic materials created in Example 1, the wild-type Nipponbare NIP, miR172d KO knockout materials, STTM RNAi silencing materials; the main varieties of Northeast China Longjing 46 and Longjing 46 background miR172 overexpression materials, STTM RNAi silencing materials; the early-maturing varieties Kitaake and Kitaake background miR172 overexpression materials; Tc65 and Tc65 background snb1 The mutants were planted in the field in Shunyi, Beijing. After the filling was completed, photos were taken to record the plant height and the panicle neck length was calculated. The results showed that compared with the background, the panicle neck length of the miR172d KO knockout material and the STTM RNAi silenced material ( Figure 1 A) and plant height ( Figure 1 B) Both miR172 OE and snb1 The mutant's panicle length and plant height were significantly increased ( Figure 1 A, B, C and Figure 3 A). By observing the number of stem nodes and the length of the internodes ( Figure 1 C and Figure 3 A), we found that the number of stem nodes of miR172d KO knockout materials was significantly reduced. snb1 The number of stem nodes of the mutant increased; the internode length of the miR172d KO knockout material was significantly shortened. snb1 The length of the lower stem internode of the mutant was shortened. Further observation of the length of the cells under the panicle showed that the length of the cells in the miR172d KO knockout material was significantly shorter than that in the control. snb1 The length of the sub-ear node cells of the mutant was slightly reduced compared with the wild-type Tc65, but not significantly ( Figure 1 D) indicates that miR172d KO knockout materials reduce the panicle length and plant height by reducing the number of internodes and internode distance. snb1 The mutant increased the panicle length and plant height mainly by increasing the number of internodes and the lower internode distance.

[0029] Example 4 Analysis of the regulatory relationship between SNB1 and the long spike neck gene EUI1 Through ChIP qPCR experiments, the present invention technicians found that in the SNB1 overexpression material, the H3K27me3 methylation levels at multiple locations on the EUI1 genome were significantly increased, indicating that SNB1 may bind to the gene sequence of EUI1 and cause its transcription level to decrease ( Figure 4 A). We further expressed the LUC reporter gene driven by 1300-SNB1 and three fragments of the EUI1 genome in tobacco and found that SNB1 could only bind to and inhibit the 3940-4383bp genomic sequence downstream of the EUI1 genomic gene ( Figure 4 B). Therefore, this sequence is considered to be the main sequence that SNB1 binds to and causes EUI1 genomic DNA methylation and transcription inhibition. Using this sequence, it is also possible to create rice materials with different plant heights and panicle neck lengths.

Claims

1. A miR172d targeting negative regulation of the SNB1 gene, characterized in that: The miR172d precursor primary sequence is shown in SEQ ID No.1, and the SNB1 gene sequence is shown in SEQ ID No.

4.

2. The miR172d targeting and negatively regulating SNB1 gene according to claim 1, characterized in that: After the precursor of miR172d is processed by the DCL complex, a functional mature sequence is formed in vivo as shown in SEQ ID No.

2.

3. The miR172d targeting and negatively regulating SNB1 gene according to claim 1, characterized in that: The amino acid sequence of the protein encoded by the SNB1 gene is shown in SEQ ID No.

5.

4. The use of miR172d according to claim 1 in regulating rice plant height and panicle neck length, characterized in that: The application method is: knocking out or silencing miR172d reduces rice plant height and panicle neck length; overexpressing miR172d slightly increases plant height and increases panicle neck length.

5. The use according to claim 4, characterized in that: The promoter of miR172d has the characteristic of internode-specific expression, and the promoter sequence is shown in SEQ ID No.

3.

6. The use according to claim 4, characterized in that: The application operation method is: constructing rice miR172d overexpression vector, knockout vector, RNAi vector; transforming the constructed recombinant plant expression vector into plant tissue or plant cells; cultivating and screening to obtain transgenic plants with increased or decreased plant height and changed panicle neck length.

7. The use according to claim 6, characterized in that: The rice miR172d overexpression vector, knockout vector and RNAi vector are transferred into Agrobacterium, and the transgenic rice plants are screened and obtained by using the Agrobacterium-mediated genetic transformation method.

8. A method for reducing rice plant height, characterized in that: The rice SNB1 gene is overexpressed, and the SNB1 gene sequence is shown as SEQ ID No.

4.

9. A method for increasing rice plant height, characterized in that: Knock out or silence the rice SNB1 gene, the SNB1 gene sequence is shown as SEQ ID No.4.

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