Gene for regulating number of nodules of nodule plants and application of gene to efficient nitrogen fixation aspect
A nodule, plant technology, applied in the fields of biotechnology and botany, can solve problems such as acidification, destruction of ecological balance, and polluted soil
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[0063] As one embodiment of the present invention, an interfering molecule that specifically interferes with the transcription of the Rac1 gene is used to down-regulate the expression of the gene. Methods of small molecule interference include but are not limited to: gene silencing regulated by miRNA, cosuppression caused by positive sense RNA (Cosuppression), antisense RNA suppression, virus-mediated gene silencing (Virus Induced Gene Silencing, VIGS), shRNA, dsRNA, Small interfering RNA, gene silencing mediated by hairpin RNA (hairpinRNA, hpRNA), etc., can also be applied in the present invention. In a preferred example, the interference molecule is targeted at positions 135-472 of the gene encoding the polypeptide according to claim 1 .
[0064] As an embodiment of the present invention, the gene encoding Rac1 is knocked out by knocking out the Rac1 gene. For example, the CRISPR / Cas9 system is used for gene editing to knock out the Rac1 gene. As another embodiment of the ...
Embodiment 1
[0117] Cloning of embodiment 1, Rac1 gene
[0118] In order to obtain genes that regulate the root nodules of legumes, the inventors screened a large number of genes, and after repeated research and testing, found that the Rac1 gene has a regulatory effect on controlling the number of soybean root nodules.
[0119] The nucleotide sequence of the Rac1a gene is shown in SEQ ID NO:1, and the encoded amino acid is shown in SEQ ID NO:3. The nucleotide sequence of the Raclb gene is shown in SEQ ID NO:2, and the encoded amino acid is shown in SEQ ID NO:4. Both have a high degree of sequence identity.
[0120] SEQ ID NO:1
[0121]ATGATGAATGCTTCAAAGTTCATTAAATGTGTTACTGTTGGAGATGGAGCTGTTGGGAAAACCTGCATGCTCATTTGCTACACCAGCAACAAGTTCCCCACTGATTACATACCAACAGTATTTGATAATTTTAGTGCCAATGTTGCTGTGGATGGAAGCATTGTCAATTTGGGGCTATGGGACACAGCAGGCCAGGAAGACTACAGCAGGTTGAGGCCATTGAGTTATAGAGGAGCAGACATTTTTGTCTTAGCATTCTCACTGATTAGCAGAGCTAGCTATGAAAATGTTCTCAAGAAGTGGATGCCGGAATTGCGTAGATTTGCACCTAATGTTCCAATTGTTCTTGTTGGTACAAA...
Embodiment 2
[0128] Example 2, Nodule phenotype analysis after silencing the Rac1 gene
[0129] Since the nucleotide sequences of Rac1a and Rac1b have a high degree of homology, when designing RNAi primers, the inventors obtained a target position for simultaneously silencing both Rac1a and Rac1b.
[0130] The constructed recombinant plasmid vector was transferred into Agrobacterium rhizogenes A.rhizogenes K599 to transform soybean hairy roots, and the co-cultivated soybean plants were transplanted into sterilized vermiculite for rooting and growth for 7 days, and the rhizobia B. japonicumUSDA110 Soybean plants were infected, and the number of nodules on soybean hairy roots 21 days after inoculation was counted, and the difference in the number of nodules was tested using the Student T-test method using Microsoft Excel 2010 software.
[0131] After infection with rhizobia B. japonicum USDA110, the phenotypes of Rac1-RNAi and empty vector plants were as follows: figure 1 As shown, A is the...
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