Protein for regulating insect resistance in <i>Arabidopsis thaliana </i>and encoding gene and use thereof

Abstract

The present disclosure relates to use of a lectin receptor-like kinase LecRK-IX.1 as a protein for regulating insect resistance of Arabidopsis thaliana. A. thaliana with high resistance to Bemisia tabaci can be cultivated by reducing the expression of, or knocking out, an encoding gene of the protein. Therefore, Arabidopsis thaliana with the high-level resistance to Bemisia tabaci can be cultivated. The gene and its encoded protein can be applied to plant genetic improvement.

Claims

1. A method of increasing insect resistance of an Arabidopsis thaliana plant, wherein the method comprises introducing into an Arabidopsis thaliana cell a Cas9 endonuclease and a gRNA comprising SEQ ID NO:2 so as to generate a knockout mutation in SEQ ID NO:1; and regenerating a plant from the knocked-out cell.

Description

DETAILED DESCRIPTION

(1) Hereinafter, the present disclosure will be illustrated in detail with reference to the examples. It should be appreciated that the following examples are only intended to further explain the present disclosure, but not to limit the protective scope thereof. Any improvements and adjustments made by those skilled in the art based on the principles of the present disclosure shall fall into the protective scope thereof. The specific process parameters given in the following examples are illustrated examples in an appropriate range. That is, those skilled in the art can make any appropriate choice within the range described in the present disclosure, rather than being limited to the specific data as shown in the followings.

Example 1

(2) With gene editing technology, AT5G10530 gene is knocked out in a gene knockout experiment, to make insect-sensitive A. thaliana have high resistance to B. tabaci.

(3) The sequence of AT5G10530 is as follows:

(4) TABLE-US-00002 (SEQ ID NO: 1) ATGGCCAACTCAATTCTGTTATTTTCCTTTGTTTTGGTTCTCCCTTTTGT CTGTTCAGTTCAATTTAACATATCTCGTTTTGGATCAGATGTTTCTGAAA TAGCATACCAAGGAGATGCAAGAGCAAATGGGGCTGTTGAGCTTACCAAC ATTGACTACACATGCCGTGCCGGTTGGGCTACTTATGGTAAGCAGGTTCC TTTATGGAATCCAGGTACCAGTAAGCCTTCGGATTTTAGTACGCGTTTCT CCTTCAGAATTGATACCCGTAATGTTGGGTATGGTAATTACGGTCATGGG TTTGCTTTCTTTCTAGCTCCAGCGAGAATCCAATTGCCTCCCAACTCAGC TGGTGGTTTCTTGGGTCTATTCAATGGAACCAATAATCAGTCTTCTGCTT TCCCACTTGTTTATGTCGAGTTCGACACATTTACTAATCCAGAATGGGAT CCTCTCGATGTCAAATCCCATGTAGGAATCAACAACAACTCTCTTGTTTC TTCTAACTACACTTCTTGGAATGCAACGTCACACAACCAAGATATAGGCC GTGTCCTGATATTCTATGATTCCGCTAGAAGAAACTTGAGTGTCTCTTGG ACTTACGACTTAACATCTGATCCTCTGGAGAATTCAAGCCTATCTTACAT CATTGATCTCTCAAAGGTACTGCCATCAGAAGTTACAATTGGGTTTTCTG CGACATCTGGAGGGGTCACCGAGGGAAATAGACTTCTGTCATGGGAGTTC AGTTCAAGCCTGGAGCTAATAGATATAAAGAAAAGTCAGAATGACAAGAA GGGGATGATAATTGGTATTTCAGTTTCTGGGTTCGTTTTGCTGACCTTTT TTATTACCTCGCTCATCGTCTTCTTGAAACGGAAGCAGCAGAAGAAGAAA GCAGAGGAGACAGAAAACTTAACATCGATAAATGAAGATCTCGAAAGAGG AGCAGGACCAAGAAAGTTTACTTATAAAGATCTTGCATCAGCTGCAAACA ATTTCGCAGATGATAGGAAGCTAGGGGAAGGAGGGTTTGGAGCGGTTTAT AGAGGGTACTTAAACAGCTTAGATATGATGGTTGCGATAAAGAAGTTTGC GGGTGGGTCTAAGCAGGGAAAAAGAGAGTTCGTAACCGAAGTAAAGATAA TCAGCAGTTTGAGACATCGAAACCTTGTGCAACTCATTGGTTGGTGCCAT GAGAAAGATGAGTTTCTAATGATATACGAGTTCATGCCAAATGGTAGCCT TGACGCCCATCTATTTGGTAAAAAGCCGCATCTCGCTTGGCATGTGAGAT GCAAAATAACTCTCGGTCTCGCCTCTGCACTGCTTTATCTTCACGAGGAG TGGGAGCAGTGTGTTGTACACAGAGACATCAAGGCGAGTAATGTGATGCT CGACTCCAATTTCAATGCCAAGCTTGGTGATTTCGGGTTGGCTAGATTGA TGGACCACGAGCTAGGTCCACAGACTACAGGGTTAGCAGGAACATTTGGT TACATGGCTCCTGAATACATAAGCACCGGAAGGGCGAGCAAAGAATCTGA TGTGTATAGCTTTGGAGTGGTTACATTAGAGATTGTTACAGGAAGAAAAT CTGTGGATCGAAGACAAGGAAGAGTAGAGCCTGTAACAAACCTTGTAGAG AAAATGTGGGACCTTTATGGAAAAGGAGAAGTTATTACAGCTATCGACGA GAAACTCAGGATCGGTGGTTTCGATGAGAAACAAGCAGAATGTCTCATGA TTGTAGGATTATGGTGTGCTCATCCTGATGTAAACACGAGGCCTTCAATA AAACAAGCAATCCAAGTCTTGAATCTTGAAGCACCAGTGCCTCATCTTCC GACCAAAATGCCTGTCGCAACATATCATGTATCCTCTTCGAATACTACAT CGGTAAGCTCTGGTGGAGCTACGGTAACGTTTTCAAGTGCTCAACATGGT CGTTGA.

(5) 1. Construction of a Knock-Out Vector for a Gene of A. thaliana

(6) According to the cDNA sequence of AT5G10530 gene, the 5′-end fragment was selected as a target sequence, and a gRNA (guide RNA) sequence was designed and synthesized. The target and corresponding gRNA sequences are shown below, but not limited thereto. The gRNA sequence fragments are reassembled into a H2S-cas9pl(AT) vector containing a hygromycin (Hyg) resistant tag. One or more nucleotides were mutated in the target sequence, that is not divisible by three, by means of the CRISPR/Cas9 genome-editing vector system to delete or insert nucleotide(s) in the target sequence. As a result, the cDNA sequence of AT5G10530 gene had a frameshift mutation, and produced a different amino acid product from the original one. That is, the AT5G10530 gene was knocked out.

(7) TABLE-US-00003 gRNA sequence 1: (SEQ ID NO: 2) 5′-TCCGAAGGCTTACTGGTACC-3′;; Target sequence 1: (SEQ ID NO: 3) 5′-TCCGAAGGCTTACTGGTACCTGG-3′..

(8) 2. Obtain A. thaliana Knockout Seedlings Through Genetic Transformation

(9) 1) Induce calluses by using mature embryos of insect-sensitive A. thaliana as raw materials: Take cultured EHA105 Agrobacterium solution and place it in a centrifuge tube, centrifugate and pipette supernatant, to make Agrobacterium suspension. Select calluses of a certain size, infect with the Agrobacterium suspension, and place the infected callus on a co-cultivation medium.
2) Screen: Take out the infected calluses, air dry, and then transfer the calluses to a screening medium for a first screening process. Transfer initial calluses with resistant calluses to a new screening medium for a second screening process.
3) Induce the differentiation and rooting of the resistant calluses. Pick up the resistant calluses, and transfer to a petri dish with a differentiation medium, seal with Parafilm, and incubate in a constant temperature incubator to differentiate into seedlings. Move the seedlings of about 1 cm to a rooting medium in order to obtain strong seedlings.
4) PCR detection of the Hyg-resistant gene. Detect the presence of the Hyg-resistant gene in A. thaliana seedlings by a conventional PCR amplification method using specific primers of the Hyg-resistant gene. If the resistant gene is detected, the corresponding A. thaliana seedlings would be positively transformed seedlings.
Specific Primers of the Resistant Genes:

(10) TABLE-US-00004 Hyg-f: (SEQ ID NO: 4) 5′-ACGGTGTCGTCCATCACAGTTTGCC-3′,, Hyg-r: (SEQ ID NO: 5) 5′-TTCCGGAAGTGCTTGACATTGGGA-3′.,
5) Knock-out test for positive seedlings. Design detection primers targeting near the target sequence to perform PCR application reactions, then sequence the PCR products, and determine whether the target gene was knocked out (or whether a homozygous knockout seedling was obtained). Finally, the seedlings, which were homozygous for the knocked out AT5G10530 gene, were successfully obtained from B. tabaci-sensitive A. thaliana.

(11) 3. Identification of Insect Resistance of Knockout Seedlings of A. thaliana

(12) The seedlings, which were homozygous for the knocked out AT5G10530 gene, of insect-sensitive A. thaliana, were tested for their resistance to B. tabaci. It was demonstrated that these homozygous seedlings having knockout of AT5G10530 gene had strong resistance to Bemisia tabaci with Antibiosis Scores between 30 and 45. Antibiosis Score is one of the parameters for judging the antibiosis level of a plant, and a plant is considered to be a high-resistant variety when it has an Antibiosis Score of less than 60. Thus, the result indicated that the insect-sensitive A. thaliana was conferred with high insect resistance by knocking out AT5G10530 gene.