A gene that can increase the number of tillers of rice and its application

Abstract

The application discloses a gene capable of increasing the tiller number of rice and application of the gene.The nucleotide sequence of the gene capable of increasing the tiller number of rice is shown in a sequence from a 134th base to a 738th base in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2.The application first determines that knocking out the LOC_Os01g68500 gene can increase the tiller number of rice, and therefore, the application provides technical support for improving the rice germplasm resource / genetic breeding of the rice for increasing the tiller number and the yield of the rice.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to a gene that can increase the number of tillers in rice and its application. Background Technology

[0002] Rice (Oryza sativa L.) is one of the most important food crops, with over 60% of my country's population relying on it as their staple food. Given my country's large population and limited arable land area, which is showing a declining trend year by year, ensuring total rice production is a crucial issue for guaranteeing food security. Rice yield is closely related to the number of tillers; therefore, identifying genes that regulate rice tillering is beneficial for high-yield molecular breeding. Summary of the Invention

[0003] The first objective of this invention is to provide a functional protein that regulates the number of tillers in rice.

[0004] The functional protein of the present invention that regulates the number of rice tillers has the amino acid sequence shown in SEQ ID NO.2, specifically: MASSSSWWVVMLLMVVAAAGWGGVAAATAAEAAHEVLRAHGLPRGLLPAGIADFRHD EGSGRFEAALGESCTAQFEVGLRYNATVAGVISYGRIASLSGVSAQDLFLWFPVRGIRVDV PSSGVIYFDVGVVFKHFPLAVFEAPPPCTPDPLLLLTQVCEDGSVAGGGAASQ*.

[0005] A second objective of this invention is to provide a gene encoding the aforementioned functional protein that regulates the number of rice tillers.

[0006] Preferably, the nucleotide sequence of the gene regulating the number of rice tillers is as shown in SEQ ID NO. 1, bases 134-738 (containing an 89 bp intron), specifically:

[0007] ATTCCATTTCTTTTCTTTTCTTTCTCCCCCTCAATTATTGCCGCTTCTGTCCATCCCCTCC

[0008] CCCTCCAATTCCATTCCATCTTCATCTCCATATCTTCTCCTCCTCGCGTCGGCAGTTATCT

[0009] CCTCGTTGATAATGGCGTCGTCGTCGTCGTGGTGGGTGGTGATGTTGTTGATGGTGGTG

[0010] GCGGCGGCGGGGTGGGGCGGGGTGGCGGCGGCGACGGCGGCGGAGGCGGCGCACG

[0011] AGGTGCTGCGGGCGCACGGGCTGCCGCGGGGGCTCCTGCCGGCGGGGATCGCGGATT

[0012] TCAGGCACGACGAAGGGAGCGGGAGGTTCGAGGCGGCGCTCGGGGAGTCGTGCACG

[0013] GCGCAGTTCGAGGTCGGGCTGCGGTACAACGCGACGGTGGCCGGGGTCATCAGCTAC

[0014] GGCCGGATCGCGTCGCTGTCGGGCGTCTCCGCGCAGGACCTCTTCCTCTGGTTCCCCG

[0015] TCCGCGGCATCCGCGTCGACGTCCCTTCCTCCGGCGTCATCTACTTCGACGTCGGCGTC

[0016] GTGTTCAAGCACTTCCCCCTCGCCGTCTTCGAGGCCCCGCCGCCCTGCACCCCCGACC

[0017] CCCTCCTCCTCCTCACGCAGGT AATCTAATCCTTCTTCCTCGAACCAATCACGAAGTGA AATCTCCACCCACCTAGTTCATCTCTGAATTGGGCGCTCACGCGGGGCAGGT(This 89bp is an intron)

[0018] GTGTGAGGACGGATCGGTCGCCGGCGGCGGCGCGGCGTCGCAATGAAGGGGACGGA

[0019] CTCGATGGTTGAAATCGGCGAGGCGGAAACCAAAAGAATGCGCAGTTTTGAGGCATG

[0020] GGAGTGGTCGGCCGGTCCGTCGCCGGCGGGCCGGCATCTGTGCAGATAGATAGAATG

[0021] AACATTCCTTTCGTTTTCTACTACTAGTTAGTAACCTTTCATTCCTCATCACTCTGTTCTT

[0022] CTTGCCGTTCTTGTTGTCCAGTGCGATCAAAGCCGGCCGACGGTGGCCGGTAGAGGA

[0023] GGCTGCCGGCCTCCGGTCGCCGCCACGGCGATAATTTTTCGTGGTTATGATGGTTCGGGGATTACAACGTGTTGAA.

[0024] It should be understood that, considering the degeneracy of codons, modifications to the nucleotide sequence of the above-mentioned coding gene without changing the amino acid sequence also fall within the scope of protection of this invention.

[0025] A third objective of this invention is to provide the application of the aforementioned gene regulating rice tiller number in increasing rice tiller number. Preferably, the rice tiller number is increased by interfering with, silencing, or knocking out the gene regulating rice tiller number using transgenic technology.

[0026] A fourth objective of this invention is to provide the application of the aforementioned gene regulating the number of rice tillers in increasing rice yield. Preferably, the number of rice tillers is increased by interfering with, silencing, or knocking out the gene regulating the number of rice tillers using transgenic technology, thereby increasing rice yield.

[0027] The fifth objective of this invention is to provide the application of the above-mentioned gene regulating the number of rice tillers in rice germplasm resource improvement / genetic breeding to increase the number of rice tillers and rice yield.

[0028] The sixth objective of this invention is to provide the application of substances for inhibiting the expression of the genes that regulate the number of rice tillers as described above in rice germplasm resource improvement / genetic breeding to increase the number of rice tillers and rice yield.

[0029] A seventh objective of this invention is to provide a method for increasing the number of rice tillers and yield, comprising the step of rendering the protein regulating the number of rice tillers functional. Specifically, this is achieved by interfering with, silencing, or knocking out the gene regulating the number of rice tillers using transgenic technology, thereby rendering the protein functional.

[0030] This invention is the first to confirm that knocking out the LOC_Os01g68500 gene can increase the number of tillers in rice. This invention utilizes a CRISPR / Cas9 vector to construct a knockout transformation vector for the LOC_Os01g68500 gene. After transformation into rice with this vector, the LOC_Os01g68500 gene is knocked out, and the number of tillers in the transformed plants is significantly higher than that in the wild-type control plants. Therefore, the function of the LOC_Os01g68500 gene in this invention can be applied to molecular breeding for rice yield, increasing rice yield and ensuring food security. Attached Figure Description

[0031] Figure 1 The spectrum of the CRISPR / Cas9 vector.

[0032] Figure 2 The sequencing identification results are for mutant strains cas1 and cas2.

[0033] Figure 3 The results of multiple comparisons of tiller numbers in three groups of materials are shown. Group 1 is wild-type Nipponbare, group 2 is the LOC_Os01g68500 gene knockout line cas1, and group 3 is the LOC_Os01g68500 gene knockout line cas2.

[0034] Figure 4 Duncan's test was used to measure the number of tillers in three groups of materials: 1 was wild-type Nipponbare, 2 was the LOC_Os01g68500 gene knockout line cas1, and 3 was the LOC_Os01g68500 gene knockout line cas2.

[0035] Figure 5 Box plot comparison of tillering number for the three groups of materials, ** indicates p < 0.01. Detailed Implementation

[0036] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0037] Example 1

[0038] 1. Construction of the LOC_Os01g68500 gene knockout vector

[0039] Using the CRISPR-GE software developed by Academician Liu Yaoguang's team, the specific target site 5'-CGGATTTCAGGCACGACGAA-3' (the 3' PAM sequence is GGG) of LOC_Os01g68500 was selected, and knockout primers OsU6aT1F: gccgCGGATTTCAGGCACGACGAA; OsU6aT1R: aaacTTCGTCGTGCCTGAAATCCG were designed. The single-lead RNA (sgRNA) sequence of LOC_Os01g68500 was cloned using the pYLgRNA-OsU3 vector provided by Academician Liu Yaoguang's team (Ma et al., 2015, Molecular Plant, 8:1274-1284). The target site sequence of LOC_Os01g68500 is 5'-CGGATTTCAGGCACGACGAA-3', and its 3' PAM is GGG. Using the CRISPR / Cas9 provided by Academician Liu Yaoguang's team Figure 1 The CRISPR / Cas9 knockout vector LOC_Os01g68500 was constructed.

[0040] 2. Genetic transformation of the LOC_Os01g68500 gene knockout vector

[0041] The LOC_Os01g68500 gene knockout vector was transformed into the normal japonica rice variety Nipponbare using Agrobacterium EHA105-mediated genetic transformation. DNA was extracted from the leaves of T0 and T1 generation transgenic plants. Primers (F: 5'-CCCCCTCAATTATTGCCGCT-3'; R: 5'-AGTAGAGACTTAACCCGCGA-3') were designed for PCR amplification of the LOC_Os01g68500 gene sequence. Homozygous mutants cas1 and cas2, with the LOC_Os01g68500 gene knocked out, were screened using third-generation sequencing. The sequencing identification results of mutants cas1 and cas2 are shown below. Figure 2 As shown.

[0042] 3. Statistics on the number of tillers in LOC_Os01g68500 gene knockout transgenic plants

[0043] Seeds from the two homozygous knockout transgenic T2 generation lines (cas1 and cas2) and wild-type Nipponbare plants were dried at 49℃ for 72 hours, soaked in water at 30℃ for 24 hours, and germinated at 30℃ for 36 hours before being sown in the transgenic field for seedling cultivation. Seedlings with uniform growth were selected and planted in the transgenic field. Normal cultivation and management were carried out continuously.

[0044] At 45 days of plant growth, the number of tillers in the three LOC_Os01g68500 gene knockout lines cas1 and cas2, and the wild-type Nipponbare was measured and counted. The results are shown in the table below. Figure 3-5 .Depend on Figure 3-5 It can be seen that the number of tillers in the transformed plants cas1 and cas2 with the LOC_Os01g68500 gene knocked out was significantly higher than that in the wild-type Nipponbare control plants.

[0045] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Knockout of the regulator of rice tiller number LOC_Os01g68500 The application of genes in increasing the number of rice tillers is characterized by, The regulation of rice tiller number LOC_Os01g68500 The nucleotide sequence of the gene is shown as the sequence of bases 134-738 of SEQ ID NO.

1.

2. A method for increasing the number of tillers in rice, characterized in that, By knocking out the gene that regulates the number of rice tillers, the protein loses its function; the nucleotide sequence of the gene that regulates the number of rice tillers is shown as the sequence of bases 134-738 of SEQ ID NO.1.

Images