Protein for regulating insect resistance in Arabidopsis thaliana and encoding gene and use thereof

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.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is based on and claims the benefit of priority from Chinese Patent Application No. 201911049321.X, filed on Oct. 31, 2019, the entirety of which is incorporated by reference herein.

INCORPORATION BY REFERENCE

This application includes a sequence listing in computer readable form (a “txt” file) that is submitted herewith. This sequence listing is incorporated by reference herein as

TECHNICAL FIELD

The present disclosure relates to the field of molecular biology, particularly to a protein for regulating insect resistance inArabidopsis thaliana, and the encoding gene and uses thereof.

BACKGROUND

Bemisia tabaci(Gennadius) is a worldwide insect pest, and distributes in all continents except Antarctica.B. tabacihas an extremely wide host range and attacks substantially all tropical and temperate crops, resulting in serious harm. It has high population density and food intake. During sucking up host plant juices,B. tabaciproduces a lot of honeydew, which can induce sooty molds and then seriously affect crop photosynthesis. In addition,B. tabaciis a main vector of various plant viruses, which will cause deformity, atrophy, chlorotic leaves, and wilting in host plants. It is one of the most harmful invasive species in the world, and causes destructive damages to important crops in many countries and regions during the invasion.

Though chemical control ofB. tabacihas been effective to some extent (except in severe outbreak years), it has a high economic and ecological cost. Further,B. tabacihas evolved resistance to insecticides resulting from over-reliance and use of the insecticides. For example, in 2010, differentB. tabacipopulations in various regions of China developed moderate to high levels of resistance to neonicotinoids, such as the resistance to imidacloprid increasing by 28-1900 times, and the resistance to thiamethoxam increasing by 29-1200 times. By 2018, the resistance ofB. tabacito thiamethoxam has increased by 192-1040 times.

Genetic engineering of crops for improving insect resistance is the most economical and effective method in integrated control of agricultural pests. However, there are still many unknown fields to be explored in the study ofB. tabaciresistance-related genes. As moving into the post-genomic era, functional genomic research has become the frontier of life science.

Arabidopsis thalianais considered as a model plant, because it has relatively easy transgenic approaches, and has collinearity with the genome of other dicotyledon-type plants. The research onA. thalianahas great reference and practical significance for social-economic development and biological studies. Now, the detailed genetic and physical maps ofA. thalianagenome have been completed, which lay a solid foundation for further studying on functional genes by usingA. thalianaas a model plant. It also has great significance for developing new crop varieties with high resistance to pests, by usingA. thalianaas a model plant to screen and study resistant genetic materials, find new resistance-related genes, and achieve mapping and cloning of such genes

Lectin receptor-like kinases (LecRKs) belong to a class of subfamilies of receptor-like kinases in plants, and play important roles in plant physiological reactions, such as regulating growth and development, and protecting against pathogens. LecRKs can perceive different external stimulus mainly through extracellular domains, and convert extracellular signals to intracellular signals through intracellular kinase properties, so as to regulate cellular physiological and biochemical reactions. By now, 75 members of LecRKs have been found inA. thaliana, and can be divided into three categories: 32 G-type, 1 C-type and 42 L-type LecRKs. Among them, the G-type LecRKs play an important role in self-incompatibility. The C-type LecRKs are Ca2+-dependent proteins, exist extensively in mammals, and involve in pathogen recognition and immune responses. However, the C-type LecRKs are rare in plants, and only one C-type LecRK was found inA. thaliana. The L-type LecRKs were named for the extreme similarity of its extracellular receptor domain with soluble lectin proteins, which are ubiquitous in leguminous seeds. While, very little is known about the L-type LecRKs.

In LecRKs gene family, the promoter region of LecRK-IX.1 (At5g10530) contains cis-regulatory elements, such as light-, ABA-, GA-, drought-, heat shock-, damage-, anaerobic-, activator-, endosperm expression-, lactose expression-, zein-responsive elements. However, there is no defense- and stress responsive element, which indicates that LecRK-IX.1 has no active defense or stress response. The expression of LecRK-IX.1 (At5g10530) has little variations at different growth stages ofA. thaliana, and maintains at a low level. It indicates that this gene may be induced by some factors, especially by pathogens or adverse situations, and thus has a potential to be a disease-resistant gene. It has been reported thatArabidopsisshowsPhytophthoraresistance when LecRK-IX.1 (At5g10530) is highly expressed, which further implies that LecRK-IX.1 (At5g10530) is a disease resistant gene.

SUMMARY

One objective of the present disclosure is to provide a protein for regulating insect resistance ofArabidopsis thaliana, and its encoding gene and uses thereof.

In the first aspect, the present disclosure provides use of a lectin receptor-like kinase LecRK-IX.1 as a protein for regulating insect resistance ofA. thaliana.

Further, the insect resistance refers to resistance toBemisia tabaci.

In the second aspect, the present disclosure provides use of a gene AT5G10530 encoding a lectin receptor-like kinase LecRK-IX.1, as a gene for regulating insect resistance ofA. thaliana.

Further, the insect resistance refers to resistance toB. tabaci.

In the third aspect, the present disclosure provides a method of increasing insect resistance ofA. thaliana, including a step of reducing an expression of a lectin receptor-like kinase LecRK-IX.1 inA. thaliana.

Further, the insect resistance refers to resistance toBemisia tabaci.

Further, the expression of the lectin receptor-like kinase LecRK-IX.1 may be reduced by knocking out, or reducing the expression of, a gene AT5G10530 encoding the lectin receptor-like kinase LecRK-IX.1.

In the fourth aspect, the present disclosure provides a breeding method of insect-resistantA. thaliana, including a step of reducing an expression of a lectin receptor-like kinase LecRK-IX.1 inA. thaliana, so as to increase insect resistance ofA. thaliana.

Further, the insect resistance refers to resistance toB. tabaci.

Further, the expression of the lectin receptor-like kinase LecRK-IX.1 may be reduced by knocking out, or reducing the expression of, a gene encoding the lectin receptor-like kinase LecRK-IX.1.

The present disclosure relates to use of the lectin receptor-like kinase LecRK-IX.1 as a protein for regulating insect resistance ofA. thaliana. It allows insect-sensitiveA. thalianato have high resistance toBemisia tabaciby reducing, or knocking out the expression of, the gene encoding the lectin receptor-like kinase LecRK-IX.1. Therefore,A. thalianawith high resistance toB. tabacican be cultivated, and the above gene and encoded protein thereof can be applied to plant genetic improvement.

DETAILED DESCRIPTION

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.

With gene editing technology, AT5G10530 gene is knocked out in a gene knockout experiment, to make insect-sensitiveA. thalianahave high resistance toB. tabaci.

The sequence of AT5G10530 is as follows:

1. Construction of a Knock-Out Vector for a Gene ofA. thaliana

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.

1) Induce calluses by using mature embryos of insect-sensitiveA. thalianaas raw materials: Take cultured EHA105Agrobacteriumsolution and place it in a centrifuge tube, centrifugate and pipette supernatant, to makeAgrobacteriumsuspension. Select calluses of a certain size, infect with theAgrobacteriumsuspension, 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 inA. thalianaseedlings by a conventional PCR amplification method using specific primers of the Hyg-resistant gene. If the resistant gene is detected, the correspondingA. thalianaseedlings would be positively transformed seedlings.
Specific Primers of the Resistant Genes:

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 fromB. tabaci-sensitiveA. thaliana.

3. Identification of Insect Resistance of Knockout Seedlings ofA. thaliana

The seedlings, which were homozygous for the knocked out AT5G10530 gene, of insect-sensitiveA. thaliana, were tested for their resistance toB. tabaci. It was demonstrated that these homozygous seedlings having knockout of AT5G10530 gene had strong resistance toBemisia tabaciwith 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-sensitiveA. thalianawas conferred with high insect resistance by knocking out AT5G10530 gene.