Application of miR172d and its target gene SNB1 in regulating plant height and panicle neck length in rice

Abstract

The application relates to the technical field of agricultural bioengineering, and particularly provides application of miR172d and a target negative regulation gene SNB1 in regulation of rice plant height and panicle neck length. Knocking out of miR172d can reduce the rice plant height and the panicle neck length, and the stem node number is significantly reduced; overexpression of miR172d can increase the plant height and the panicle neck length. The SNB1 gene is a target of miR172d, and miR172d reduces the expression of the target coding gene SNB1 at a transcriptional level. Through a genetic transformation experiment, it is proved that overexpression of SNB1 can reduce the plant height, and a snb1 mutant has an increased plant height. The miR172d and the target gene SNB1 provided by the application can be applied to rice breeding work, and have a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of agricultural bioengineering technology, and relates to the application of miR172d and its target negative regulatory gene SNB1 in regulating rice plant height and panicle length. Background Technology

[0002] Rice is one of the world's most important food crops, playing a vital role in national food security. Plant height is one of the most important agronomic traits, closely related to rice yield. Excessive plant height can lead to lodging, while reducing plant height can sometimes result in reduced biomass and yield. Currently, the commonly used sd1 gene relies on high water and high nitrogen fertilizer to ensure stable yield; however, high nitrogen fertilizer negatively impacts the environment and the cost of rice cultivation. Therefore, screening for more genes that regulate plant height as a background for trait improvement is essential.

[0003] MicroRNAs are a class of non-coding RNAs approximately 19-22 nucleotides in length, and are key regulators in plant growth and development. Studies have shown that microRNAs participate in regulating various aspects of plant growth and development, as well as biological processes such as responses to abiotic stress. Changes in the expression of microRNAs and their target genes in plants can affect a variety of genetic traits. Therefore, functional research on rice microRNAs can provide new theoretical basis and research methods for breeding and improving rice plant height. Summary of the Invention

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

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] The first aspect of the present invention provides a miR172d that targets and negatively regulates the SNB1 gene, wherein the primary sequence of the miR172d precursor is shown in SEQ ID No. 1 and the SNB1 gene sequence is shown in SEQ ID No. 4.

[0007] Furthermore, the precursor of miR172d is processed by the DCL complex to form a functional mature sequence in vivo as shown in SEQ ID No. 2.

[0008] Furthermore, the amino acid sequence of the protein encoded by the SNB1 gene is shown in SEQ ID No. 5.

[0009] The second aspect of the present invention provides the application of miR172d in regulating rice plant height and panicle length, wherein the application is carried out by knocking out or silencing miR172d to reduce rice plant height and panicle length; and by overexpressing miR172d to slightly increase plant height and panicle length.

[0010] Furthermore, the promoter of miR172d has the characteristic of internode-specific expression, and the promoter sequence is shown in SEQ ID No. 3.

[0011] Furthermore, the operation method of the application is as follows: 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 changes in panicle neck length.

[0012] Furthermore, the rice miR172d overexpression vector, knockout vector, and RNAi vector were transformed into Agrobacterium, and transgenic rice plants were obtained by screening using Agrobacterium-mediated genetic transformation.

[0013] Furthermore, the primers for PCR amplification of miR172d overexpression are shown in SEQ ID No. 6 and 7, and the primers for PCR amplification of RNAi miR172d are shown in SEQ ID No. 8 and 9.

[0014] After miR172d is processed into a mature sequence in the plant, it moves to its downstream target gene SNB1 (LOC_Os07g13170) mRNA, forming a complementary pair, resulting in SNB1 cleavage and a decrease in SNB1 abundance. In a specific embodiment, it was found that... snb1 The mutants did indeed show increased plant height, while SNB1 overexpression resulted in decreased plant height.

[0015] A third aspect of the present invention provides a method for reducing rice plant height by overexpressing the rice SNB1 gene, the sequence of which is shown in SEQ ID No. 4.

[0016] The fourth aspect of the present invention provides a method for increasing rice plant height by knocking out or silencing the rice SNB1 gene, the sequence of which is shown in SEQ ID No. 4.

[0017] The beneficial effects of this invention are:

[0018] The rice plant height and panicle 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 were obtained to reduce or increase rice plant height while simultaneously regulating panicle length. The miR172d RNAi and knockout vectors described in this invention, after transformation into rice, significantly reduced miR172d expression, resulting in a significant decrease in plant height and panicle length in the transformed plants. Overexpression of miR172d significantly increased panicle length. Overexpression of the downstream targeted negative regulatory gene SNB1 described in this invention reduced plant height, while mutants showed increased plant height. Therefore, the complete set of genes and gene modification vectors provided by this invention can be used for the improvement of new varieties, allowing for relatively free regulation of rice plant height, providing new gene resources and methods for modern agricultural molecular breeding. Attached Figure Description

[0019] Figure 1 Phenotypic characteristics of panicle length and plant height in miR172d overexpression, knockout, and STTM interference materials. (A) From left to right: panicle length phenotype of Nipponbare NIP, the main Northeast cultivar Longjing 46 / LG46, and the short-growth material Kitaake as background for 172dKO knockout, STTM interference, and OE overexpression materials. (B) Plant height phenotype of Nipponbare NIP 172dKO knockout and STTM interference materials. (D) DICM cell size detection of the lower internode of wild-type Nipponbare NIP and 172dKO knockout materials.

[0020] Figure 2 Detection of SNB1 protein accumulation levels suppressed by miR172. (A) Changes in SNB1 protein accumulation levels were detected by co-expressing miR172 and 1305-SNB1-GFP with pCambia background in tobacco leaves compared to the empty vector. (B) Example regions of complementary and interacting miR172d mature sequence and SNB1 CDS.

[0021] Figure 3 For rice snb1 Plant height and panicle length phenotypes of mutants. (A) Plant height and panicle length phenotypes of rice snb1 mutant. (B) DICM cell size detection of the lower internode of wild-type Tc65 and snb1 mutant.

[0022] Figure 4 To detect the transcriptional activation of Eui by SNB1 in rice. (A) The methylation level of histone H3K27me3 on the promoter of Eui was significantly increased in rice SNB1 OE overexpression material. (B) SNB binds to a downstream region of the Eui genome 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 Implementation

[0023] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0024] Example 1: Creation of miR172d knockout, STTM RNAi silencing, and miR172 overexpression materials

[0025] Rice leaves were rapidly frozen in liquid nitrogen, shaken and broken, and then extracted with 600 μl of CTAB extract at 65°C for 1 hour. The supernatant was collected by centrifugation, and 200 μl of chloroform was added to extract the protein. The supernatant was collected by centrifugation, and 1 volume of isopropanol was added. The mixture was incubated at -80°C for 1 hour, and the supernatant was removed by centrifugation. The mixture was rinsed twice with 70% alcohol, dried, and dissolved in sterile ultrapure water.

[0026] Using SEQ ID Nos. 6, 7 and SEQ ID Nos. 8, 9 as primers, an STTM silencing fragment based on the miR172d precursor and mature sequences was amplified from genomic DNA. This fragment was then constructed into a vector with a pCambia1300 backbone, yielding miR172d silencing and overexpression vectors. The knockout vector was constructed using the Cas9 / CRISPR system developed by Liu Yaoguang.

[0027] After transforming the vector plasmid into Agrobacterium, the Agrobacterium was used to infect rice callus tissue, and positive plants were obtained after testing.

[0028] Example 2: Detection of the biochemical regulatory relationship between miR172d and SNB1

[0029] The SNB1 CDS sequence was constructed into a vector with the pCambia1305 backbone to obtain an expression vector with the fluorescent tag GFP.

[0030] After transforming the vector plasmid into Agrobacterium, tobacco seedlings were infected. The following combinations from Example 1 were used to infect tobacco: ① 1300 and 1305-SNB1-GFP, and ② 1300-miR172d and 1305-SNB1-GFP. Confocal microscopy was used to observe the fluorescence of the GFP tag. The miR172d combination showed significantly weaker fluorescence than the 1300 empty vector control, indicating that miR172d inhibits SNB1-GFP expression. Western blotting results also confirmed this. Figure 2 A).

[0031] The mature sequence of miR172d is inversely complementary to a segment at the end of SNB1 mRNA, causing SNB1 mRNA cleavage and degradation, thus inhibiting SNB1 expression. Figure 2 B).

[0032] Example 3: Wild-type and miR172d KO knockout materials, STTM RNAi silencing, miR172 overexpression, snb1 Comparison of spikelet length and plant height in mutant materials

[0033] Using the genetic materials created in Example 1, wild-type Nipponbare NIP, miR172d KO knockout material, and STTMRNAi silencing material were used; miR172 overexpression material and STTMRNAi silencing material from the main cultivated variety Longjing 46 in Northeast China and Longjing 46 background; early-maturing variety Kitaake and Kitaake background miR172 overexpression material; and Tc65 and Tc65 background materials were used. snb1 The mutant was planted in a field in Shunyi District, Beijing. After grain filling, plant height was photographed and panicle neck length was measured. The results showed that compared with the background, the panicle neck length of miR172d KO knockout material and STTM RNAi silencing material was significantly higher. Figure 1 A) and plant height ( Figure 1 B) Both were significantly reduced, miR172 OE and snb1 The mutant showed a significant increase in both ear neck length and plant height. Figure 1 A, B, C and Figure 3 A). By observing the number of stem segments and the length between stem segments ( Figure 1 C and Figure 3 A) It was found that the number of stem nodes was significantly reduced in miR172d KO knockout material. snb1 The mutant has an increased number of stem nodes; the internode length of the miR172d KO knockout material is significantly shorter. snb1 The internode length in the lower part of the stem was shortened in the mutant. Further observation of the cell length in the lower nodes of the spike revealed that the cell length of the miR172d KO knockout material was significantly shorter than that of the control. snb1 The length of the lower segment cells in the mutant ear is slightly smaller than that of the wild-type Tc65, but not significantly. Figure 1 (D) This indicates that the miR172d KO knockout material reduces spikelet length and plant height by decreasing the number of internodes and the internode distance. snb1 The mutants mainly increase the length of the panicle neck and the plant height by increasing the number of internodes and the internode spacing at the lower part.

[0034] Example 4: Analysis of the regulatory relationship between SNB1 and the spike neck gene EUI1

[0035] Through ChIP qPCR experiments, the present inventors found that the H3K27me3 methylation level at multiple locations on the EUI1 genome was significantly increased in SNB1 overexpression materials, indicating that SNB1 may bind to the EUI1 gene sequence and lead to a decrease in its transcription level. Figure 4 A). Further expression of three fragment-driven LUC reporter genes from the 1300-SNB1 and EUI1 genomes in tobacco revealed that SNB1 could only bind to and repress a 3940-4383 bp genomic sequence downstream of the EUI1 genome. Figure 4 B). Therefore, this sequence is considered to be the main sequence that binds to SNB1 and causes methylation and transcriptional repression of EUI1 genomic DNA. Using this sequence, it may also be possible to create rice materials with different plant heights and panicle neck lengths.

Claims

1. An application of miR172d in regulating rice panicle neck length, characterized in that, The mature sequence of miR172d is shown in SEQ ID No. 2, and the precursor sequence is shown in SEQ ID No.

1. The application methods are: knocking out or silencing miR172d to reduce the length of the rice panicle neck; and overexpressing miR172d to increase the length of the panicle neck.

2. The application according to claim 1, characterized in that, The promoter of miR172d is characterized by internode-specific expression, and the promoter sequence is shown in SEQ ID No.

3.

3. The application according to claim 1, characterized in that, The operation method of the application is as follows: constructing rice miR172d overexpression vector, knockout vector, and 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 changes in panicle neck length.

4. The application according to claim 3, characterized in that, The rice miR172d overexpression vector, knockout vector, and RNAi vector were transformed into Agrobacterium, and transgenic rice plants were obtained by screening using Agrobacterium-mediated genetic transformation.

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