CN114525303A - Application of CaM2 gene as regulatory factor in improving insect pest stress resistance of plants - Google Patents

Abstract

The invention discloses application of a CaM2 gene as a regulatory factor in improving insect pest stress resistance of plants, and belongs to the technical field of biology. The nucleotide sequence of the protein coding region of the CaM2 gene is shown as SEQ ID NO. 1. The invention discloses the regulation and control function of the CaM2 gene in improving the resistance of plants to pests, particularly root-knot nematodes for the first time, and the CaM2 gene improves the content of jasmonic acid by inducing the expression of related genes synthesized by jasmonic acid, thereby improving the resistance of plants to the root-knot nematodes. The tomato CaM2 gene up-regulated expression can obviously improve the resistance of the tomato CaM2 gene to root-knot nematodes. The invention provides gene resources for cultivating new species of the nematode-resistant tomato, has certain potential application value, provides more complete theoretical basis for analyzing response mechanism of plant defense against root-knot nematodes, and has important practical value.

Description

Application of CaM2 gene as regulatory factor in improving insect pest stress resistance of plants

Technical Field

The invention relates to the technical field of biology, in particular to application of a CaM2 gene as a regulatory factor in improving insect pest stress resistance of plants.

Background

Specialized, large-scale and intensive production is the development direction of vegetable industry, however, with the increasing of multiple cropping indexes, the continuous cropping obstacle of soil is more serious, soil-borne diseases and insect pests are main factors causing the continuous cropping obstacle, especially root-knot nematode harm, and huge economic loss is caused to the vegetable industry of facilities.

Root knot nematode disease is a common Plant parasitic nematode disease known as "Mol Plant Pathol, 2013, 14(9), 946-61" and results in the formation of root knots in Plant roots (Jones et al, "Top 10 Plant-parasitic nematodes in molecular Plant Pathology). The root-knot nematode has wide host range, can harm more than 3000 plants such as vegetables and grain crops and seriously harm agricultural production. According to statistics, more than 90 kinds of root-knot nematodes are reported worldwide, and the four main types of root-knot nematodes harm crop production in China are as follows: meloidogyne incognita (Meloidogyne incognita), Meloidogyne javanica (Meloidogyne japonica), Meloidogyne arachidis (Meloidogyne arenaria), and Meloidogyne hapla (Meloidogyne hapla), which are types of nematodes that are primarily harmful to greenhouse vegetables.

The tomato (Solanum lycopersicum L) is the vegetable crop with the largest consumption in the world, and is also the vegetable crop with the largest facility cultivation area and the best economic effect in China. Tomatoes are highly sensitive to root-knot nematodes, and various fumigants and nematicides are used as common defense measures in current production, but the problems of low efficiency, environmental pollution, influence on quality of agricultural products and the like exist. Therefore, the method has the advantages of defining the signal transduction path of the plant to the root-knot nematode, analyzing the defense mechanism of the plant root-knot nematode and exploring the resistance gene of the plant root-knot nematode, and has very important scientific and practical significance for developing new economic, effective and environment-friendly measures for efficiently preventing and controlling the nematode.

As sessile organisms, plants are exposed to various environmental changes and even stresses throughout their life, and when local tissues of the plants are stressed, Ca is rapidly induced²⁺And second messengers such as reactive oxygen species, which convey information about environmental changes to distant unstressed tissues and organs (Kollist et al, "Rapid Responses to Abiotic Stress: printing the landmark for the Signal Transmission network," Trends in Plant Science, 2018). The signal molecules do not improve the stress resistance of plants, but can be coupled with hormone signals to trigger stress response genes and activate stress response. It is known that plants are mainly immune-enhanced by Jasmonic Acid (JA), a hormone derived from lipid compounds, whose synthesis requires multiple enzymatic reactions (Wasternack and Song, "Jasmones: biosynthes, metablism, and signalling by proteins activating and reproducing," Journal of Experimental botanic, 2016, 1303) in response to pest infestation including nematodes, but the molecular mechanism for inducing and regulating JA synthesis is not clear.

Calmodulin (CaM) is the major Ca cell²⁺Binding proteins, are present in all eukaryotes and are highly conserved (Yang and Poovatah, "Calcium/calmodulin-mediated signal network in plants," Trends in Plant Science, 2003, 8(10), 505-. CaM itself is biologically inactive, with Ca²⁺After combination, the space structure is changed, the hydrophobic surface is exposed, and the downstream stress-resistant gene is activated by combining with the target protein. Research shows that CaM plays an important role in enhancing plant stress resistance: soybean CaM4 improves salt tolerance of plants by activating R2R3 type MYB2 transcription factor (Yoo et al, "Direct interaction of a Direct CaM isoform and the transformation factor, MYB2, enhancement salt tolerancein arabidopsis, "J Biol Chem, 2005, 280(5), 3697-; arabidopsis CaM3 regulates Plant heat resistance by modulating the transcriptional activity of the heat shock transcription factor HSF1 (Zhang et al, "Molecular and genetic evidence for the key role of AtCaM3 in heat-shock signal transduction in Arabidopsis," Plant Physiol, 2009, 149(4), 1773-84); tomato CaM6 negatively regulates the cold resistance of tomatoes through interaction with COR-like 47 protein (Li Shu Li, the mechanism research of SlGLRs and SlCaMs in regulating the low-temperature resistance of tomatoes, Zhejiang university, 2019). However, the function of the CaM gene in the field of insect pests has not been studied and reported. Therefore, the research on the effect of the CaM2 gene in resisting plant root-knot nematodes by using related mutant materials and combining with a molecular genetic technology has important theoretical and production values for the research on a green efficient prevention and control technology of the vegetable root-knot nematodes.

Disclosure of Invention

The invention aims to discover resistance genes participating in insect pest stress resistance, particularly root-knot nematodes in plants, and provides a theoretical basis for researching and developing a green efficient prevention and control technology of the vegetable root-knot nematodes.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides application of a CaM2 gene as a regulatory factor in improving insect pest stress resistance of plants, wherein a nucleotide sequence of a protein coding region of the CaM2 gene is shown as SEQ ID No.1 or has at least 70% homology with a sequence shown as SEQ ID No.1, and the coded proteins are functionally equivalent.

The CDS region of the CaM2 gene is 450bp in length, the coded protein consists of 149 amino acid residues, and the amino acid sequence of the coded protein is shown as SEQ ID NO. 2.

Further, the application includes: by using a biotechnology means, the CaM2 gene in the plant body is up-regulated and expressed, and the resistance of the plant body to insect pest stress is improved.

Further, the plant is tomato.

Further, the insect pest is root-knot nematode.

According to the invention, tomato CaM2 gene overexpression and mutant transgenic plants are constructed, and through root-knot nematode inoculation experiments, the CaM2 gene is found to play a positive regulation role in tomato nematode stress resistance.

Furthermore, the CaM2 gene is used as a regulatory factor to improve the resistance of plants to insect pest stress by improving the content of plant resistance hormone.

The research of the invention shows that the lower the expression level of the CaM2 gene in the plant, the more the root knot number of the plant is after the inoculation of the root knot nematode, the lower the content of the resistant hormone is, and the more sensitive the root knot nematode is; the higher the expression level of the CaM2 gene in the plant, the less the root knot number of the plant after the root knot nematode is inoculated, the higher the content of the resistant hormone, and the stronger the resistance of the plant to the root knot nematode.

Further, the resistant hormone is Jasmonic Acid (JA).

Furthermore, the CaM2 gene improves the jasmonic acid content by promoting the expression of jasmonic acid synthesis related genes, thereby improving the resistance of the plant to the root-knot nematode.

Specifically, the jasmonic acid synthesis related genes comprise an AOC gene (Allene oxide cycle, Solyc02g085730), an AOS2 gene (Allene oxide synthase 2, Solyc11g069800) and a LoxF gene (lipoxygene F, Solyc01g006560), and the nucleotide sequences of protein coding regions are respectively shown as SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5; allene oxide cyclase, allene oxide synthase and lipoxygenase which are respectively encoded by the three genes are key enzymes for JA synthesis.

The invention also provides a method for improving the resistance of plant root-knot nematodes, which comprises the following steps: inserting a CaM2 gene segment with a nucleotide sequence shown as SEQ ID NO.1 into an overexpression vector to construct a recombinant plasmid, introducing a target gene segment into a receptor through an agrobacterium-mediated technology, and culturing and screening to obtain a functionally-obtained transgenic plant.

Preferably, the overexpression vector is pFGC 1008-HA.

Preferably, the receptor is a tomato cotyledon.

The invention has the following beneficial effects:

the invention discloses the regulation and control function of the CaM2 gene in improving the resistance of plants to pests, particularly root-knot nematodes for the first time, and the CaM2 gene improves the content of jasmonic acid by inducing the expression of related genes synthesized by jasmonic acid, thereby improving the resistance of plants to the root-knot nematodes. The tomato CaM2 gene up-regulated expression can obviously improve the resistance of the tomato CaM2 gene to root-knot nematodes. The invention provides gene resources for cultivating new species of the nematode-resistant tomato, has certain potential application value, provides more complete theoretical basis for analyzing response mechanism of plant defense against root-knot nematodes, and has important practical value.

Drawings

FIG. 1 shows Western Blot detection results of plant proteins of tomato plants over-expressed by CaM2 gene.

FIG. 2 shows CaM2 gene CRISPR/Cas9 knockout tomato plant T₂Sequencing results for generations (homozygous, without exogenous Cas9 fragment). Compared with wild tomatoes, the tomato cam2 mutant is known to have deletion mutation of 4 bases at the position of sgRNA, so that protein translation is terminated early; WT represents wild type tomato, cam2 represents mutant plant, and "- - - - - - -" represents deletion of 4bp sequence.

FIG. 3 is an observation of the root knot phenotype of cam2 mutant and overexpressing plants 4 weeks after inoculation with root knot nematodes. Wherein A is the dyeing result of acid fuchsin at the root of a plant; b is the statistical result of the number of root knots; WT represents wild type tomato, CaM2 represents mutant plant, OE-CaM2 represents over-expressed plant; statistical analysis of the data was performed by analysis of variance (ANOVA), and the significance of the data differences was analyzed using Tukey test, in which the same letter indicates that the mean value has no significant difference at P < 0.05.

FIG. 4 shows the content changes of JA and isoleucine jasmonate (JA-Ile) 24h after inoculating cam2 mutant and overexpression plants to root-knot nematode. Wherein A is JA content, and B is JA-Ile content; WT represents wild type tomato, CaM2 represents mutant plant, OE-CaM2 represents over-expressed plant; statistical analysis of the data was performed by analysis of variance (ANOVA), and the significance of the data differences was analyzed using Tukey test, in which the same letter indicates that the mean value has no significant difference at P < 0.05.

FIG. 5 shows the expression level analysis of the JA synthesis related gene of cam2 mutant and over-expressed plant inoculated with root-knot nematode for 24 h. Wherein the JA synthesis related genes are AOC, AOS2 and LoxF; a is AOC gene expression quantity, B is AOS2 gene expression quantity, and C is LoxF gene expression quantity; WT represents wild type tomato, CaM2 represents mutant plant, OE-CaM2 represents over-expressed plant; statistical analysis of the data was performed by analysis of variance (ANOVA), and the significance of the data differences was analyzed using Tukey test, in which the same letter indicates that the mean value has no significant difference at P < 0.05.

Detailed Description

The invention is further illustrated with reference to the following figures and specific examples. It is to be understood that the following are merely exemplary embodiments of the present invention, and the scope of the present invention is not limited thereto.

Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the experimental materials, reagents and the like used therein are commercially available.

The tomato variety used in the examples below is the tomato conventional variety Ailsa Craig.

Example 1: construction of CaM2 Gene overexpression vector

According to CDS sequence analysis of CaM2 gene, specific primers CaM2-F and CaM2-R are designed, and restriction enzyme sites AscI and KpnI are respectively added to the 5' ends of the primers, and the sequences are as follows: CaM 2-F: 5'-ttacaattaccatggggcgcgccATGGCGGATCAGCTGACGG-3' (SEQ ID NO. 6); CaM 2-R: 5'-aacatcgtatgggtaggtaccTTACTTGGCCATC ATGACCTTAAC-3' (SEQ ID NO. 7);

total RNA of tomato was extracted using a plant Total RNA extraction Kit (RNA simple Total RNA Kit, TIANGEN, China), with reference to Kit instructions. The total RNA of the sample was reverse transcribed using a reverse transcription kit (HiScriptII Q RT Supermix for qPCR, + gDNA wiper, Vazyme, China) according to the kit instructions.

And (3) specifically amplifying the CaM2 gene by PCR by using the obtained cDNA as a template and CaM2-F and CaM2-R as primers to obtain the CDS total length 450bp of the tomato CaM2 gene.

The 35S promoter-driven plant expression vector pFGC1008-HA is digested by restriction enzymes AscI and KpnI to be linearized, and then homologous recombination of a PCR fragment and the vector is carried out to obtain an overexpression vector pFGC1008-CaM 2-HA.

Sequencing is carried out by the recombinant plasmid of Zhejiang Shanghai Biotechnology Limited company, and the sequencing result is shown as SEQ ID NO.1, and the result shows that the cloned sequence is consistent with the sequence (Solyc10g081170) published in Solgenomics. The amino acid sequence of the protein coded by the gene is shown in SEQ ID NO. 2.

Example 2: construction of CaM2 Gene editing vector

The full-length DNA sequence of CaM2 found on SGN website (http:// solgenomics. net /) is shown in SEQ ID NO. 9. The sequence of sgRNA of CaM2 gene is designed by CRISPR-P website (http:// cbi. hzau. edu. cn/cgi-bin/CRISPR) and is shown in SEQ ID NO. 8. The synthetic sgRNA sequence was annealed and ligated with AtU6-sgRNA-AtUBQ-Cas9 intermediate vector that had been single digested with BbsI overnight at 16 ℃ with T4 ligase. The newly obtained AtU6-sgRNA (CaM2) -AtUBQ-Cas9 recombinant plasmid and the vector pCAMBIA1301 are subjected to double enzyme digestion by Hind III and EcoRI respectively, and the two are connected by T4 ligase at 16 ℃ overnight to obtain a gene editing vector pCAMBIA1301-AtU6-sgRNA (CaM2) -AtUBQ-Cas 9. Sequencing and confirmation are carried out by the recombinant plasmid of Zhejiang Shanghai Biotechnology Co.

Example 3: preparation and identification of tomato CaM2 transgenic material

Respectively transforming the overexpression vector pFGC1008-CaM2-HA obtained in the example 1 and the gene editing vector pCAMBIA1301-AtU6-sgRNA (CaM2) -AtUBQ-Cas9 obtained in the example 2 into agrobacterium GV3101, transforming the agrobacterium into the cotyledons of wild tomato Ailsa Craig by using an agrobacterium infection method, and preliminarily screening candidate T by using hygromycin₀Transgenic plants are generated.

1. A positive CaM2 overexpression plant is verified by Western-Blot, and the specific method is as follows:

0.3g of tomato leaf was ground with liquid nitrogen, 600. mu.L of an extract (50mM Tris-HCl, pH8.0, 150mM NaCl, 1mM EDTA, 1% Triton X-100, 1mM phenyl methyl vinyl fluoride (PMSF) and 0.2% beta-mercaptethanol) was added and ground to a slurry, centrifuged at 12000rcf for 20min, and the supernatant was taken. Protein content was determined by Coomassie Brilliant blue. Proteins were denatured and separated on a 12% SDS-PAGE gel. SDS-PAGE gels were separated and transferred to nitrocellulose membranes, blocked with 5% skimmed milk powder in TBST buffer (20mM Tris, pH 7.5,150mM NaCl, 0.1% Tween20) for 1h at room temperature, incubated with murine HA monoclonal antibody (Pierce, 26183) for 1h at room temperature, and then with goat anti-mouse (Millipore, AP124P) HRP secondary antibody for 1h at room temperature, and antigen-antibody complexes were detected using luminol chemiluminescence detection kit (Thermo Fisher Scientific, 34080).

The Western-Blot results are shown in FIG. 1. WT had no protein band, while the over-expressed strain detected a CaM2-HA protein band. WT is wild type tomato Ailsa Craig; OE-CaM2 is CaM2 overexpression plant.

2. PCR detection of CaM2CRISPR/Cas9 mutant plant

Mixing the above T₀Planting the positive plant of CaM2 mutant in growing room, selfing to obtain T₁Seeds, and utilizes PCR and sequencing technology to verify the gene editing condition, thereby obtaining homozygous mutant plants which lack 4 basic groups, and the sequence comparison is shown in figure 2. Will T₁Selfing the plant to obtain stably inherited T₂And (4) generation.

Example 4: effect of tomato CaM2 Gene on nematode resistance

The tomato CaM2 gene overexpression and mutant plants are inoculated with root-knot nematodes, and the method comprises the following steps:

wild type tomato (WT), mutant plants (CaM2) and over-expressed plants (OE-CaM2) were divided into two groups, one control group and one experimental group.

When the tomatoes grow to four leaves and one heart, the experimental group is inoculated with the root-knot nematode, and the inoculated root-knot nematode is the meloidogyne incognita in the J2 stage.

The method comprises the following steps of:

(1) pulping diseased roots infected with root-knot nematodes, fully and uniformly mixing with sandy soil, placing in a water culture box with the thickness of 340mm multiplied by 270mm multiplied by 130mm, planting 6 common cultivated tomatoes in each pot, breeding the nematodes in a greenhouse of an agricultural test station of Zhejiang university, and maintaining the room temperature at 22-26 ℃.

(2) Digging the root system with serious disease, slowly washing the sandy soil of the root with clear water, soaking in 0.5% sodium hypochlorite aqueous solution, stirring, sterilizing for 3min, and washing with clear water for several times.

(3) Cutting root into 1cm root segments, and crushing in a mincing machine. The homogenate was passed through 80 mesh, 200 mesh, 325 mesh and 500 mesh sieves in this order, and finally the residue on the 500 mesh sieve was washed in a beaker to obtain an egg suspension.

(4) The collected egg suspension is poured into a square culture dish of 13cm multiplied by 13cm which is paved with 8 layers of absorbent paper evenly, and the culture dish is placed in a constant temperature incubator at 28 ℃ for incubation for 2 to 3 days.

(6) The culture dish was gently rinsed with a wash bottle to rinse the incubated root knot nematodes into the beaker, and the number of nematodes at stage J2 was recorded by observation under a 50-fold optical microscope (BX 61; Olympus Co., Tokyo, Japan).

(7) Inoculating the nematode suspension to the root soil of the plant to be treated, inoculating about 1000 nematodes of J2 stage to each plant, culturing in a phytotron, and watering normally.

Plants were cultivated in plastic cups containing high temperature sterilized river sand and watered with macroelement water soluble fertilizer (Hangzhou Kangcheng agriculture science and technology Co., Ltd.). Growth conditions are as follows: temperature 25 deg.C/20 deg.C (day/night), photoperiod 12h/12h (day/night), luminous flux density 400 μmol^-2s^-1And the humidity is about 75%.

After 4 weeks of nematode treatment, root knot phenotype observations and root knot number statistics were performed.

The root knot phenotype observation adopts an acid fuchsin dyeing method, and the specific method comprises the following steps:

(1) cleaning root system, and bleaching the root system with 1.5-5% sodium hypochlorite solution for 5-10min to remove impurities in the root system which affect dyeing.

(2) Washing the root system with clear water and soaking for 30 min. Pouring off the soaking solution, sucking off water of root system, completely immersing root system with 3.5% acid fuchsin solution, heating to boil and maintaining for 3-5min, standing at room temperature, and cooling.

(3) After cooling, the root system is washed by clear water to remove the redundant pinkish red liquid on the surface.

(4) Adding acid glycerol, heating to boil, and transferring the root system to normal temperature acid glycerol.

(5) After 24h of storage in acidic glycerol, root knot phenotype photographing and root knot number counting are carried out.

The specific method for counting the root knots is as follows:

cleaning the tomato root system to be counted, sucking surface water, weighing, and carefully recording the root knot number of each root. The nematode sensitivity of the plants was evaluated by counting the number of root knots per gram of fresh and heavy root.

The root knot phenotype observations and the statistics of root knot number are shown in FIG. 3. Compared with WT, the root knot number of the cam2 mutant plant is increased remarkably, which indicates that the resistance of the plant root knot nematode is weakened; the number of root knots of OE-CaM2 plants was significantly reduced, indicating that the plants had increased resistance to root-knot nematodes (FIG. 3B), and a difference in root-knot phenotype was evident from the results of acid fuchsin staining of the root system (FIG. 3A). The CaM2 gene can improve the resistance of tomato to Meloidogyne incognita.

Example 5: effect of tomato CaM2 Gene on resistance hormones JA and JA-Ile

As JA plays an important role in plant response to biotic stress, after Wild Tomato (WT), an over-expression plant (OE-CaM2) and a mutant plant (CaM2) are inoculated with root knot nematode, the content of Jasmonic Acid (JA) and jasmonic acid isoleucine conjugate (JA-Ile) and the expression amount of synthesis-related genes are determined.

1. Inoculating root knot nematode for 24h, taking tomato leaf samples, extracting and determining the content of JA and JA-Ile, wherein the specific method comprises the following steps:

(1) grinding 100mg of liquid nitrogen frozen tomato leaf or root system samples into powder in a sample grinding instrument;

(2) 1mL of chromatographic grade ethyl acetate was added, to which D6-JA (OlChemIm Ltd., Czechloroslavika) and D6-JA-Ile (Quality Control Chemicals INC., USA) had been added as internal standards at final concentrations of 10ng mL each^-1And 40ng mL^-1The standard solution of (4). Placing the sample in a dark place, and extracting at 4 ℃ and 180rpm for 12 hours;

(3) centrifuging at 12000rcf at 4 deg.C for 10min, collecting supernatant, and extracting precipitate with 1mL chromatographic grade ethyl acetate;

(4) combine the two supernatants in a 5mL centrifuge tube, N₂Drying;

(5) 0.5mL of 70% chromatographic grade methanol (v/v) was added, vortexed thoroughly, and the residue in the centrifuge tube was resuspended, centrifuged at 12000rcf at 4 ℃ for 10 min;

(6) the supernatant was transferred to a brown glass bottle and assayed by HPLC-MS/MS (Agilent Technologies, California, USA). The measurement results of JA and JA-Ile contents are shown in FIG. 4.

After the root knot nematode is inoculated for 24 hours, the content of JA (figure 4A) and JA-Ile (figure 4B) induced by the root knot nematode in the cam2 mutant is obviously lower than that of a wild tomato; the content of JA (figure 4A) and JA-Ile (figure 4B) in OE-CaM2 plants is obviously higher than that of wild tomatoes. The CaM2 gene was shown to promote JA synthesis.

2. Inoculating root-knot nematodes for 24h, taking tomato leaf samples to extract RNA, and performing real-time fluorescent quantitative PCR (qRT-PCR) according to primer sequences, wherein the specific method comprises the following steps:

total RNA was extracted using a plant Total RNA extraction Kit (RNA simple Total RNA Kit, TIANGEN, China), with reference to Kit instructions. Total RNA concentration and mass of RNA were determined using a Nano Drop (Thermo Fisher Scientific, USA) and adjusted to approximately the same concentration. The total RNA of the sample was reverse transcribed using a reverse transcription kit (HiScriptII Q RT Supermix for qPCR, + gDNA wiper, Vazyme, China) according to the kit instructions.

Gene expression analysis was performed using SYBR Green RT-PCR Kit fluorescent dye Kit (Vazyme, China) and LightCycler 480 real-time fluorescent detection System (Roche, Switzerland). Tomato ACTIN2 gene was selected as the reference gene and the amplification primer sequences are shown in Table 1.

TABLE 1 real-time fluorescent quantitative PCR primer sequences

Each reaction system was 20 μ L: 10 μ L of dye, 8.2 μ LddH₂O, 1. mu.L of reverse transcription product, 0.4. mu.L of each of forward and reverse primers.

The PCR reaction conditions are as follows: 3min at 95 ℃; at 95 ℃ for 30s, 58 ℃ for 15s and 72 ℃ for 1min, 45 cycles in total.

The calculation of the relative transcription levels is referred to Livak and Schmittgen ("Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCTmethod "Methods, 2001, 25, 402-.

After the root-knot nematode is inoculated for 24 hours, compared with wild tomato, the expression levels of JA synthesis related genes AOC (figure 5A), AOS2 (figure 5B) and LoxF (figure 5C) induced by the root-knot nematode in the cam2 mutant are obviously reduced; the opposite was true for OE-CaM2 plants. The CaM2 gene is shown to be capable of promoting JA synthesis related gene expression.

Sequence listing

<110> Anqing city Ming triangle future industry research institute

ZHEJIANG University

Application of <120> CaM2 gene as regulatory factor in improving insect pest stress resistance of plants

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aaagatgaag cttgtcataa tcttctcttc gctacttgct tcaattcctt cggtgggatg 960

aagattttct tcccgaatat gctgaaatcg atagcgaaag caggggtgga gatccatacc 1020

cgtttagcaa acgagatccg atcggaagta aaatccgccg gcgggaagat cacgatgtcg 1080

gcgatggaga aaatgccgtt gatgaaatca gtagtatatg aagctttacg agttgatcct 1140

ccggtagctt cacaatacgg aagagccaaa caggacctta agatcgaatc acacgacgcc 1200

gttttcgagg tgaaaaaggg tgaaatccta ttcgggtacc aaccatttgc aacgaaggat 1260

ccgaaaattt ttgaccggcc gggagagttc gtcgccgatc ggttcgtcgg agaagaagga 1320

gaaaaattat tgaaacatgt attatggtct aacggaccgg aaacggaaag tcctacagtg 1380

gggaataaac agtgcgccgg aaaagatttc gttgtgatgg tttcgagatt attcgtaacg 1440

gagttttttc tccgttacgg tacactcaac gtcgacgttg gcacgtcggc gttagggtct 1500

tcaattacta ttacttcttt gaaaaaagct taa 1533

<210> 5

<211> 1344

<212> DNA

<213> tomato (Solanum lycopersicum L)

<400> 5

atgactgttg aagaggcggt tcaacaaaag aagttgttca tcttagatta tcatgatttg 60

ctattgccat ttgtgaacaa agtgaatgaa ctcaaaggga cagtacttta tggatcaaga 120

actttgttct atttgacacc taatggcaca ttgagacctt tggcgattga gctaactagg 180

ccaccagtag atgataagcc tcaatggaag caagtttatt gcccaacttg gtatgccact 240

ggtgcttggc tatggaggat tgctaaagct catgttcttg ctcatgactc tggctatcac 300

caactagtta gtcattggct aagaactcat tgttgtacag agccatacat tatagcatct 360

aataggcaac taagtgcaat gcatccaata tatagattat tatttcctca ttttagatat 420

acaatggaga taaatggtac agctagagaa gcacttatta atgcaaatgg tgttattgag 480

agttcatttt cccctggcaa gtattcaatg gagttgagtt ctgttgccta tgatcttgag 540

tggaggtttg atagagaagc actccctgag gaccttatta gtaggggatt ggcagaggaa 600

gatccaaatg caccatatgg attgagacta acaatagaag attacccttt tgctagtgat 660

ggtttagtac tttgggacat acttaaacaa tgggtaacaa attatgtcaa ccattattat 720

ccacaagcaa atctcattga atgtgatgaa gaactccaag cttggtggtt agagattaaa 780

aatgttggac atggtgacaa gaaagatgag ccatggtggc cagagttaaa taccccaaat 840

gacttaattg gtattgtcac aacaataatt tgggtaactt ctggccatca tgcagctgta 900

aactttggcc aatacagcta tgcaggctat tttccaaata ggccaacaat tgctaggtca 960

aaaatgccaa ctgaagatcc aacagatgaa gaatgggagg attttttgaa caaacctgag 1020

gaggcattat taaaatgctt cccttcacaa cttcaagcaa caaaagtaat agcagttttg 1080

gatgttttat caaaccattc tccagatgaa gagtatattg gcacaaatat tgaacctttt 1140

tggaaagatg aacccgtaat taacgcggcg ttcgaggtat tttcggggaa attgaaggag 1200

cttgaaggga taattgatgc cagaaatgct gattgtaatt tgaagaatag aaatggagct 1260

ggagttatgc cttatgaatt attgaaacca ttttctgaac ctggaattac tggaaaaggt 1320

gtaccttata gcatttccat ttga 1344

<210> 6

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ttacaattac catggggcgc gccatggcgg atcagctgac gg 42

<210> 7

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

aacatcgtat gggtaggtac cttacttggc catcatgacc ttaac 45

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ccagatctct gagttcaaag 20

<210> 9

<211> 6191

<212> DNA

<213> tomato (Solanum lycopersicum L)

<400> 9

cgattcatct aatgataata ttcttttatg aagagcagct caaatcaatt tgtaagtacg 60

aacttaactg acttttacat cttaaatatt tatatattta ttttagaaaa atatgatgga 120

ttattttatt tagaaaatcg gaagtcacat ttgatatgat aacttgataa gcacaaaaga 180

gtagagaata ttggaactaa tgaaaaaata tcaagtttgt taaagagata cttatataaa 240

tataatatag aattttataa taaatgtata agatcaatat agattataat gaaactgtct 300

caacgatatt tctttatcta tcttcaaatg ggataacaaa tgagatcatt tttatctaat 360

ctcatatata tatatatata ttataaagta tttatcgatt caacatttat tttcgtatga 420

tattatttaa tctctattga ggggtaaaga gggaaaaacg gacacatcga aataaataaa 480

tcaatcacta tccgtattat aatgtaagga gagagaaaac ttttttctat ttttattatt 540

tattcttctt ttttttaatt caaattcatt tatttttttg aaaattatac tttaaattat 600

atttacaatg aaatgaaatc tgtagttttt ggtataaaag cagcaatctt ctcataattc 660

caaaatccaa acagagctga agagattttc aaaaaaaact aaattgtgtg aaattctaga 720

atcatcttct ctgtgattag tagaagaaga aaaatggcgg atcagctgac ggatgaccag 780

atctctgagt tcaaagaggc tttcagtttg ttcgacaagg acggagatgg tttggcgcct 840

ttttttttct ctttaaatgt gtatattttt ttgtgttttt gatgcttata ggtgtatgta 900

gaatctgttt gttgtagatc tgatgtttga tttgaaattt tgaatgaact ggttgatttt 960

gttttggtga caggttgata tttgagaata ttcagttagt aaatagagga aaattgattt 1020

tgtagcttgt tgaaacgtta aatatgaatt agaaaatatg gaagctatgt agtttgatgt 1080

tacgttggtg aattactgtt ttttgcttat tatttaagat tttttcctgt atcctgtttt 1140

ttttttcttc ctttttggtg aatagagtta tttgatattc gtatccgaag gtttatccgt 1200

ttagttaggt tgagaaccac aaagtatcaa ggtgcttggt tgatagactt acctaaagtg 1260

gttgagagaa tcgaggttca aattccaagt gatttccttc catagagtta cttgatactt 1320

gttgctggta gttgtgggag ttgagaggta ttttatggaa tacgtccaga tgtgtgtagg 1380

ttggttggga tatcacggtt atagagttac cccgtacttg tgttgatggg aggtagtgga 1440

atcagtgaac ttgtaaattg gattatacat ggattgagaa gtgataagtt aatttgatga 1500

aaaatttggc ctgtataact cttaactggc ttcgatttgt gttaaatgct aacaaaatcc 1560

tccttttttt gtaaatctgt agtaacttta tctgaactca ttgattggga cgaacaatta 1620

gatgaatgat tgttgtcatt agttattagt gaaatgcatt ttctgttttc ttctgtggat 1680

aggagcttta ctgatgttag ggtcacaacg tgtagtttgt ttagttttcg ttcagtaagc 1740

taatcgtgaa gctttgtata taggttgcat cacgactaag gagcttggga ctgtgatgag 1800

gtcgttggga cagaacccta ctgaagctga gctccaggac atgataaacg aggtggatgc 1860

agatggtaat ggaaccatcg acttcccaga gtttctaaac cttatggcca ggaagatgaa 1920

ggatactgac tctgaggagg agttgaaaga ggcattcaga gttttcgaca aggatcaaaa 1980

tggcttcatc tccgctgctg agcttcgtca tgtgatgact aaccttggag agaagcttac 2040

tgatgaagaa gtcgatgaga tgattaggga agctgatgtt gatggtgatg gacaaatcaa 2100

ctatgaggag tttgttaagg tcatgatggc caagtaattc cattatcctc ttgttacatt 2160

tgaagtttag acttgtcaaa aatgtgaaaa aatccaaaaa taattcattg ggtaggctct 2220

tgtctagtat aatgttagtc ttaccatctc gatgtattga accttcattg aatgtaatgt 2280

tttattgtat gagtggtgtg atctctactt attttcttta agtttgtgta ctttctttga 2340

tcctcttaga gcctgtttgg ctcagcttaa aaactggtca aactgactta aaaagctggt 2400

ttttgactta tttagctgtt tgacaatact caaaataact tattttaagt taaaaaaaac 2460

ttattttaaa ccaaaagtta aaagctgggg tagggatgtt ttttttttta gcttataagc 2520

tgtttttaat tgaccacatt tttatctttt tgctcttaat atttttatac aatctccaaa 2580

ttacccacat ataaccctaa catttctttc ttccattttt tccttttcac gtttgacata 2640

acaacttcag cacttttatc caaacgcata actgctttca aaagtacttt tttaaagctg 2700

tttttattaa gcctatccaa acgggccctt agtcaatgaa tgcaattcgt catcgtgtat 2760

tgactagctt agtacacaaa gtagtaaaat gcagtttaac tttactgttg ttttaaggtt 2820

ctgttggtat caaccgttac aacgtttaat aacatatcca tttatacaaa ggactatgta 2880

tattgcacat tagagactga tgattgatca tataaaaaaa caagaaagaa aacaactgaa 2940

attatcaatt tcatttatca gggcacaata caaatttaat tctgcaggat tgattctccc 3000

atctatcaaa caaaagcaat aaagaacaaa ggattttagt catattatcc attcaaaaac 3060

aagtttataa tctcttcaag cttttctttc tcgcgtcgat cttctttttg tctgcaggga 3120

gtttcgccaa tccagtttcc ttcttcatag tcagtgatgg tggtgctgct gacccactaa 3180

ccgctgtatc cgcagttagt ggctggtcaa gattgtttcc tccttttaca atgttagctg 3240

taggggacaa cggagaaggc tgttggagag gtgcgactcc agataccaca gtcttctgat 3300

caacgttaca cagtgaagta tgtgtctggc caacatttag ggctgtcgtt gattcggaaa 3360

tagctcctgt ttttaactta gtctttggtt tagcatgaat actggtataa agcctgcatg 3420

tttaaacacc gagggagcat tagacggaca ggtcaggaag tgcattcaaa gtgctaaaca 3480

atacttgaaa ctacaaaggt agcctatttt ttcatctatt aagagtggat gacgcaggca 3540

taagttgaac cttcgaccac ataaactatg ctagtcagcc agagtggctt aagtctgtat 3600

taaaacgtag tcattggatt tacgagcagg aattccaggt ttttacatct acaaagagga 3660

aatgagcatg agtcggttca cttggatctt ctcgttttgg ttaatactag atagatgtat 3720

gatcgtctac caatttgatt tctattgcaa cccttctaaa atgtactgta gcatttacac 3780

ttaatgcaca aaaacatgtt ttcaccataa atataaagac agttatatgt aggataatca 3840

agcaaaacag taccaaacaa atgcaacaaa actcaggcat ggtgatgcca aataattatc 3900

gaggtggcat aaagtgaagc tagtcgggct aactcacctt gcatgtctag catactcatc 3960

ataattatca agaagcatct tgccagcttg ctcatttagc gcagattctg gaaatggctc 4020

gattagtaaa catcttacca cctgttacaa aaccatcaag gaaaggataa tgtcaattca 4080

aacttgtatc tccgtatctc cagaggtgac tcaattttta cttgaaagaa aacagaaatt 4140

taccatgaga acatgtcgta ggcccaaact aggactccaa tcttttttca aagcatttac 4200

acaaatctcg ccattggaag caatgttggg atgaaaaatc ttggtcagaa aataacctag 4260

aaaaaaaaca aaaatgtaaa ttatgcactc agccgtaatt gcttagcaat tggcattgag 4320

agatctccca gagatgccga aagttgcaac gcttcaagtg gtaactgctc ttaaactttt 4380

ccatgagaat tgactaagga attgcagctt atcccagaga gcaatatgct cacaaagata 4440

atgtcaagaa atttatagaa tttaatcaac agctggagag ttcatgtaga accaaggtat 4500

cccagaaagt aaagttactt gacctatttt aatgagtaga aagtgaaaaa aagttcctac 4560

aacgaaataa acggggaaaa ttcaacttag gctcttgaat cgtgatcaat acagaaccaa 4620

ctgattgaaa cataaaaaaa aacccgatac aaggactaca aaacacgtta cactggtaaa 4680

acttgaaaac attgcaatgt tgagacaaac ctttgggtgg ggaatgaggg aaatcatgtg 4740

tcaaaatcaa cttcatgcgg aataccccat tctcataagg agtcccagct gcaaggtaga 4800

tgaggagaaa ttagaacaag tgtcatgtct caagaactga aatatataca acaaacacaa 4860

catacctgga ccctcaatat cagcaaatat ggttgaaaaa tcatcatcat ttacacctac 4920

tttgatgccc tcaggagggg aatcatcaag atttttcaat tccttcgcca attgttttat 4980

cacgtttggt ggcagatttt cattagttgc ctatgccaaa aaaaaacaac caaattttgg 5040

acaaaatcaa ttgacaaatg acaaataagc ataccaaatt acttatcaca agataaggaa 5100

acagaaaaaa agaagtaatt catttggatc caggaagaaa atgacataga gaaacctagt 5160

aatagcaaca ctttgaattc aatgaaatac catagccaaa tataagttga tgtcctagga 5220

gcagttggca actttgctga tgagttatca gtgaagcacg tacttttagc tggtagttat 5280

atgttgataa aatcaagctt ctgaaagaat cctgctaaag ggccagaaaa agatcatcag 5340

aatacaatcc ttcaattgtc caatcaatca caagaaacag gcaagtaaag aaggccatct 5400

tcaagaaaat tgtgtcaatc caccagatgt ggataatatg tcagagaatc acaaaattgt 5460

caatacaagt acaagtccat agactcctct tctccatatt agtccataca ataagccaaa 5520

tgtctaagca agtctctgat actcacatac agctctactt ttctgaactt acactaacta 5580

cttggtgcat atttaccctc acagggtagg ggtaaggctg cgtacacacc accctccctc 5640

gttcccagtt gtgtcaacct aattggtagc actagggcat ggttttttgg ttaacaagct 5700

ccaaagaatg caactcttga tttttaatac tgttccacta cataaataaa attcccttca 5760

taatcggaca tcaaaatcat gaaatatggt agcttgacta gtttcagtgt taagtcaaac 5820

gtaaagacat cacgcagtga tacttataag agaaatgtta gatgccaaag tatctacatc 5880

ctcacttcgt aaaagagtgg tctaaaacta cttgatctca aacatcaaca gaaacaatgg 5940

tagttctgaa gctaacagtg ctacaaagaa tgattagtcg aagaatatga gcgcccagac 6000

aaaagattag tcaagtggac cattgatatc tagaatggaa aaaatcatag tcgatcacat 6060

attccccaat ctggtaaatt ccaaagtgga tattgttaaa cactaaagct cactttggta 6120

cttcaactta gtacacaaga atcaccagta tggtaatcag agactgacag aactaacaaa 6180

attattagga t 6191

Claims (10)

1. The application of the CaM2 gene as a regulatory factor in improving the insect pest stress resistance of plants is characterized in that the nucleotide sequence of the protein coding region of the CaM2 gene is shown as SEQ ID NO.1 or has at least 70% homology with the sequence shown as SEQ ID NO.1, and the coded proteins are functionally equivalent.
2. 2. The use of claim 1, wherein the amino acid sequence of the protein encoded by CaM2 gene is shown in SEQ ID No. 2.
3. 3. The application of claim 1, wherein the application comprises: by using a biotechnology means, the CaM2 gene in the plant body is up-regulated and expressed, and the resistance of the plant body to insect pest stress is improved.
4. 4. Use according to claim 3, wherein the plant is tomato.
5. 5. Use according to claim 3, wherein the insect pest is root-knot nematode.
6. 6. The use of claim 1, wherein the CaM2 gene is used as a regulatory factor to increase the resistance of a plant to insect pest stress by increasing the content of plant resistance hormone.
7. 7. The use of claim 6, wherein the resistance hormone is jasmonic acid.
8. 8. The use of claim 7, wherein the CaM2 gene increases jasmonic acid content by promoting expression of a gene involved in jasmonic acid synthesis; the jasmonic acid synthesis related genes comprise an AOC gene, an AOS2 gene and a LoxF gene, and the nucleotide sequences of protein coding regions of the jasmonic acid synthesis related genes are respectively shown as SEQ ID No.3, SEQ ID No.4 and SEQ ID No. 5.
9. 9. A method of increasing root knot nematode resistance in a plant comprising: inserting a CaM2 gene segment with a nucleotide sequence shown as SEQ ID NO.1 into an overexpression vector to construct a recombinant plasmid, introducing a target gene segment into a receptor through an agrobacterium-mediated technology, and culturing and screening to obtain a functionally-obtained transgenic plant.
10. 10. The method of claim 9, wherein the recipient is a tomato cotyledon.

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