CN114480341A

CN114480341A - Application of poncirus trifoliata protein kinase PtrSnRK2.4 in drought-resistant genetic improvement of plants

Info

Publication number: CN114480341A
Application number: CN202210162283.4A
Authority: CN
Inventors: 刘继红; 宋杰; 李春龙
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2022-02-22
Filing date: 2022-02-22
Publication date: 2022-05-13

Abstract

The invention belongs to the field of plant genetic engineering, and discloses application of a trifoliate orange protein kinase PtrSnRK2.4 in drought-resistant genetic improvement of plants.PtrSnRK2.4The gene is from Zhizhi (Poncirus trifoliata) The protein kinase separated and cloned from Zhizhi (A) Hovenia dulcis.), (poncirustrifoliata) Separated and cloned, and the sequence of the gene is shown as SEQ ID NO. 1. After the gene is used for constructing an over-expression vector, the gene is transferred into the lemon through agrobacterium-mediated genetic transformation, and simultaneously, an interference vector is constructed and then transferred into the trifoliate orange. After obtaining transgenic plants respectively, carrying out biological function verificationShowing that cloned in the present inventionPtrSnRK2.4The gene has the function of controlling the drought resistance of plants. The development and utilization of the genetic resources are beneficial to reducing the agricultural production cost and realizing the green and environment-friendly agricultural target.

Description

Application of poncirus trifoliata protein kinase PtrSnRK2.4 in drought-resistant genetic improvement of plants

Technical Field

The invention belongs to the field of plant genetic engineering, and particularly relates to application of a Poncirus trifoliata protein kinase PtrSnRK2.4 in drought-resistant genetic improvement of plants, wherein an applicant obtains a protein kinase PtrSnRK2.4 by separating and cloning from Poncirus trifoliata (Poncirus trifoliata), and the drought resistance of a transgenic plant is obviously increased after the gene is over-expressed in lemon; after the gene is interfered in the trifoliate orange, the drought resistance of a transgenic plant is obviously reduced.

Background

The plants can be continuously subjected to environmental changes in the growing processExtreme survival conditions and various stress stimuli are brought, wherein drought stress causes different degrees of harm to plant growth and development, geographical distribution, yield and the like (maetal, 2017). Plants have developed various mechanisms for adapting to drought stress under long-term evolution, such as plant external morphological changes, various physiological and biochemical reactions, enrichment of stress proteins and triggering of various signal transduction mechanisms, which ultimately help plants to obtain better drought tolerance (Tan et al, 2018). Research shows that during the process of responding to stress, plants are involved with a plurality of signal molecules such as MA K kinase, Ca²⁺bZIP transcription factors, MYB/MYC transcription factors, etc., encompassing the MAK kinase cascade pathway, the AB A signaling pathway and Ca²⁺Abiotic stress signal transduction pathways (zhuetal, 2016), such as the second messenger pathway, of which ABA signal pathways play an important role in plant response to biotic and abiotic stresses, especially drought stress; on the other hand, plants can regulate the metabolic processes in vivo, accumulate a series of metabolites which can be used as osmoprotectants or oxide scavengers, thereby protecting cells from damage caused by stress and finally improving the drought tolerance of the plants (Vigeoles et al, 2008).

The SnRK2 protein kinase is a core element and an important component of an ABA signal path, and the SnRK2-AREB/ABF regulation path plays an important role in plant response to osmotic stress. The SnRK2 protein kinase can be induced and expressed by ABA and drought/osmotic stress, and the three have a relationship: the ABA signaling pathway enables signaling through SnRK2 protein kinase to respond to osmotic stress, while SnRK2 protein kinase in turn enables plant functions to resist stress, especially drought stress, by being expressed induced by ABA and osmotic stress (Fujii & Zhu 2012). Currently, SnRK2 protein kinases have been identified separately in various plants and have been shown to be involved in the ABA signaling pathway. The SnRK2 transgenic plants of different species all show improved growth vigor and resistance function. The model plant arabidopsis thaliana (arabidopsis thaliana) SnRK2 family has 10 members in total, all members except snrk2.9 are induced by hyperosmotic stress, and snrk2.6 is induced most strongly (Boudsocq et al, 2004); also found in rice (Oryzasativa) are 10 SnRK2 family members, SAPK1-SAPK10, all of which are responsive to hyperosmotic stress, where SAPK8, SAPK9 and SAPK10 are inducible by AB a (kobayashiet, 2004); further research shows that the drought resistance of Sn RK2 transgenic plants in Arabidopsis, wheat, poplar, rice and other plants is obviously enhanced (Zhang et al, 2010; Du et al, 2013; Song et al, 2016; Tan et al, 2018).

In addition, polyamines, one of the important metabolites in plants, play an important role in abiotic stress response. Polyamines are a class of physiologically active low molecular weight aliphatic nitrogenous bases, mainly including putrescine, spermine, spermidine, which are part of a complex signaling network for plant resistance (Berberich et al, 2015; Liu et al, 2015). Research shows that when plants are stressed by environment, polyamines in the plants can be rapidly accumulated, so that key enzyme genes for synthesizing or decomposing polyamines are subjected to different induced expressions under different stress conditions of drought, low temperature, high salt, ABA treatment and the like, and the endogenous polyamine content of the plants is changed, and the resistance is changed (Alcazar et al, 2010; Capell et al, 2004). The researches show that the ABA signal, the polyamine synthesis and the plant drought resistance function are related, however, the aspects of regulating the polyamine synthesis and further regulating the drought resistance by SnRK2 protein kinase and the like are rarely reported at present. Therefore, the research on the resistance action mechanism has important significance on the genetic improvement of the crops.

Trifoliate orange (Poncirus trifoliataal. Raf) is a plant of the genus Poncirus of the family Rutaceae, has good resistance and high affinity, is a stock widely applied to citrus planting, and is also an important germplasm resource for researching plant resistance breeding and genetic improvement. Therefore, cloning the drought-resistant related gene of trifoliate orange is the key and the basis of the drought-resistant gene engineering.

Disclosure of Invention

The invention aims to provide application of poncirus trifoliata protein kinase PtrSnRK2.4 in drought-resistant genetic improvement of plants, and the drought resistance of obtained transgenic plants is obviously increased after the gene is over-expressed in lemon; after the gene is subjected to interference in a Hovenia dulcis, the drought resistance of a transgenic plant is obviously reduced, the corresponding nucleotide sequence of the gene is shown as SEQ ID NO.1, and the coded protein sequence is shown as SEQ ID NO. 2.

In order to achieve the above object, the present invention adopts the following technical measures

The applicant identifies and clones a new gene PtrSnRK2.4 from Hovenia dulcis based on a plant gene cloning technology, the sequence of the new gene is shown as SEQ ID NO.1, and the encoded protein is shown as SEQ ID NO. 2; the gene contains an Open Reading Frame (ORF) of 1344bp in total, codes 448 amino acids with a sequence shown as SEQ ID NO.2, and codes the protein shown as SEQ ID NO. 1. The molecular weight prediction shows that the protein has the molecular weight of 48.3kDa and the isoelectric point of 9.65.

The applicant analyzes the relative expression quantity of the PtrSnRK2.4 gene under different stress conditions through qRT-PCR technology, and the result shows that the relative expression quantity of the gene is induced most obviously under dehydration treatment. In addition, the transgenic plants of PtrSnRK2.4 before and after drought treatment and related physiological data are analyzed, and the results show that: compared with the wild type, the drought resistance of the PtrSnRK2.4 transgenic lemon plant is higher, and simultaneously, the activity of Fv/Fm and ADC enzyme and the content of putrescine are obviously increased, the conductivity, the MDA content and the H content are obviously increased₂O₂Content and O₂ ^·-The content is relatively low. However, PtrSnRK2.4 silencing is the opposite of Hovenia aurantiaca phenotype and physiological indexes. The results show that PtrSnRK2.4 is a potential breeding gene for positively regulating drought resistance of trifoliate orange.

The application of the drought-resistant gene PtrSnRK2.4 of the trifoliate orange in plant drought resistance is characterized in that an overexpression and interference vector is constructed through the gene provided by the invention, the gene is respectively introduced into the lemon or the trifoliate orange through agrobacterium-mediated genetic transformation, and the obtained transgenic plant is verified through biological functions.

Compared with the prior art, the invention has the following advantages:

the discovery and successful cloning of the gene provide new gene resources for plant stress-resistant molecule design breeding, provide new genetic resources for implementing green agriculture and water-saving agriculture, and the development and utilization of the genetic resources are beneficial to reducing the agricultural production cost and realizing environmental friendliness.

Drawings

FIG. 1 is a technical flow diagram of the present invention.

FIG. 2 is a schematic representation of the expression pattern of PtrSnRK2.4 of the invention in response to different treatments;

wherein: in FIG. 2A is ABA treatment; in FIG. 2B is a dehydration treatment; in FIG. 2, C is the dehydration + Flu treatment;

FIG. 3 is a schematic diagram of the subcellular localization of the PtrSnRK2.4 gene of the invention.

FIG. 4 is a schematic diagram of PtrSnRK2.4 gene transgenic lemon identification and relative expression analysis;

wherein: FIG. 4A is a diagram of the identification of the PtrSnRK2.4 gene of the invention as lemon; in FIG. 4, B is the relative expression level of PtrSnRK2.4.

FIG. 5 is a schematic diagram of the determination of the phenotype and physiological index of the drought treatment of PtrSnRK2.4 transgenic lemon of the invention;

wherein: in FIG. 5, A is the phenotype of transgenic lemons (#2, #6) and wild-type lemons before and after drought treatment; FIG. 5B is a chart of chlorophyll fluorescence phenotype before and after drought treatment of lemon; in fig. 5C is the relative conductivity before and after the lemon treatment; d in FIG. 5 is Fv/Fm value before and after lemon treatment, and E in FIG. 5 is MDA content before and after lemon drought treatment; FIG. 5F is DAB and NBT staining pattern after lemon treatment; in FIG. 5G is the arginine decarboxylase ADC enzyme activity before and after the treatment of lemon; in FIG. 5H is the polyamine content before and after the lemon treatment.

FIG. 6 is a schematic representation of the identification and relative expression quantification of VIGS silencing material according to the invention;

wherein: in FIG. 6A is the identification of PtrSnRK2.4 interference material (PtrSnRK2.4-TRV2), "M" represents marker, "P" represents positive plasmid, and "TRV" represents TRV empty particle; in FIG. 6, B is the expression level of PtrSnR K2.4 determined by randomly selecting 6 positive materials.

FIG. 7 is a schematic diagram of drought resistance analysis of Hovenia dulcis-silenced PtrSnRK2.4 gene plants (TRV-PtrSnRK2.4 for short);

wherein: FIG. 7A is the phenotype of empty TRV and the interference plant TRV-PtrSnRK2.4 before and after drought treatment; in FIG. 7B is a chlorophyll fluorescence phenotype image before and after drought treatment of the intervened raisin tree; c in FIG. 7 is the Fv/Fm value before and after drought treatment of the intervention Hovenia dulcis; in FIG. 7D is the relative conductivity of the intervened citrus before and after drought treatment; in FIG. 7E is the MDA content before and after drought treatment of the intervened citrus; in FIG. 7, F is the staining pattern of DAB and NBT after the treatment of Zhi; in FIG. 7, G is the activity of arginine decarboxylase ADC enzyme activity before and after intervention of Citrus aurantium; in FIG. 7H is the polyamine content before and after the intervention treatment.

Detailed Description

The present invention will be described in detail with reference to specific examples. From the following description and examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. In the embodiment of the invention, the PtrSnRK2.4 gene is over-expressed in lemon, which shows that the PtrSnRK2.4 gene can improve the cold resistance of plants. For example, the ptrsnrk2.4 gene is overexpressed in trifoliate orange to obtain transgenic plant trifoliate orange with enhanced cold resistance, and thus, the expression is not repeated in the examples.

Example 1: cloning of full-length cDNA of Poncirus trifoliata PtrSnRK2.4 gene

The method comprises the following steps of (1) performing amplification by using a high-fidelity enzyme and taking trifoliate orange cDNA as a template, wherein the amplification primer sequence is as follows: a forward primer: 5'-TGG ATTGAGATGGAAGAGAGA-3', reverse primer: 5'-CGGATCATGGCTCATATAATC-3' are provided.

The amplified product was purified and recovered by AxyPrep-96 DNA gel recovery kit (Axygene, USA), and then the purified product was ligated to pEASY-Blunt vector (all-type gold, China) in the ligation system, and E.coli competent DH 5. alpha. was transformed after incubation for 5min at room temperature. Coating a plate, carrying out inverted culture at 37 ℃, selecting plaque and shaking bacteria, carrying out positive detection, sending the obtained positive clone to Wuhan engine department biology company for sequencing, and obtaining the full-length gene sequence of PtrSnRK2.4 according to the sequencing result.

The sequencing result shows that the gene contains an Open Reading Frame (ORF) and codes 448 amino acids with 1344 bp. The molecular weight prediction shows that the protein has the molecular weight of 48.3kDa and the isoelectric point of 9.65. The gene is named as PtrSnRK2.4, the nucleotide sequence is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2.

Example 2: expression analysis of PtrSnRK2.4 gene under different conditions

Immature bitter orange seedlings which are 2 months old are planted in a constant-temperature sunlight incubator. Picking up the seedlings with consistent growth vigor, pulling up the seedlings with roots, cleaning the seedlings with the roots by using clear water, inserting the seedlings into a triangular flask filled with deionized water, and putting the triangular flask into an incubator with the temperature of 25 ℃ and the photoperiod of 16h/8h alternately for water culture for three days. For dehydration treatment, the seedlings after water culture were wiped clean of residual water droplets with filter paper, and dehydrated on filter paper of 90X 90mm for 0h, 3h, 6h, 9h, 12h, 24h, and 36 h. In ABA treatment, water-cultured seedlings are placed in 100 mu M ABA solution, and the treatment sampling time is 0h, 3h, 6h, 9h, 12h, 24h and 36h respectively. Dehydration treatment + FLU (ABA inhibitor) treatment hydroponic seedlings were pre-cultured in 100. mu.M FLU solution for three days, followed by dehydration treatment. At least 10 seedlings were randomly sampled and mixed for each treatment, and the leaves were quickly frozen in liquid nitrogen and stored in an ultra-low temperature freezer at-80 ℃ for subsequent gene expression pattern analysis.

The expression pattern of the PtrSnRK2.4 gene was analyzed by real-time fluorescent quantitative PCR (qRT-PCR) using AceQ qPCR SYBR Green Master Mix (Novozam, Germany) according to the instructions. The prepared reaction system adopts QuantStudio^TMThe 7Flex Real-Time system (Applied biosystems, USA) system. Calculation of relative expression level of Gene 2^-ΔΔCTThe method comprises the steps of taking citrus Actin as an internal reference gene (a forward primer: 5'-CCGACCGTATGAGCAAGGAAA-3'; a reverse primer: 5'-TTCCTGTGGA CAATGGATGGA-3') and taking a PtrSnRK2.4 real-time quantitative primer (a forward primer: 5'-AAGCAGGACGTGACCAA CCAA-3'; a reverse primer: 5'-TCATATAATCACCGCTGGCAT-3').

The result of the experiment shows that PtrSnRK2.4 is induced and expressed by ABA, and the highest induction multiple is 14 times (A in figure 2). Meanwhile, the expression level of the PtrSnRK2.4 gene is also induced by dehydration stress, and the expression level is highest at 9h and 12h and is increased by about 5 times compared with that before treatment, and then is slowly reduced (B in figure 2). When dehydration and a concurrent treatment with fluazinone (an ABA synthesis inhibitor), the gene-induced expression was inhibited (C in fig. 2). In conclusion, PtrSnRK2.4 is induced by dehydration stress and ABA at the same time, and may play an important role in drought stress resistance of plants.

Example 3: subcellular localization of PtrSnRK2.4 Gene

The ORF region of PtrSnRK2.4 (without stop codon) was amplified, amplification primers (F: 5'-GGACTAGT TGG ATT GAG ATGGAAGAGAGAT-3' and R: 5'-ACGCGTCGAC CGGATCATGGCTCATA TA-3') were designed and fused to the vector p101LYFP, the YFP protein was located at the C-terminus of the gene, and expression was driven by the CaMV35S promoter. Then, controls 35S: YFP +35S: OFP-HDEL and 35S: PtrSnRK2.4-YFP +35S: OFP-HDEL are respectively and instantaneously transformed into leaf epidermal cells of Nicotiana benthamiana, and fluorescence observed by a laser confocal microscope shows that the fluorescence of the controls fills the whole epidermal cells including cell membranes and cell nuclei, compared with that, yellow fluorescence can also be observed in the cell membranes and the cell nuclei after the fusion expression vector 35S: PtrSnRK2.4-YFP is transformed into Nicotiana benthamiana, and the fluorescence on the cell membranes is coincided with the red fluorescence of the membrane positioning marker 35S: OFP-HDEL, which indicates that PtrRKSn2.4 is positioned in the cell membranes and the cell membranes (see figure 3).

Example 4: construction of plant transformation vector, genetic transformation of lemon and identification of positive seedling

1. Construction of plant transformation vectors

Amplifying the full-length sequence of the gene of PtrSnRK2.4, designing amplification primers (F: 5'-GCTCTAGAATTGAG ATGGAAG AGAGATATG-3' and R: 5'-TCCCCCGGGTCACGGATCATGGCTCAT-3') and fusing the amplification primers to the vector pBI121, extracting plasmids after obtaining positive clone and transferring the plasmids into agrobacterium-infected GV3101 for later use.

2. Genetic transformation of lemon

1) Plant material preparation

Cutting lemon fruit stored at 4 deg.C, taking out seed, and washing with distilled water. Preparing 1mol/L NaOH solution to soak the seeds for 15-20min to remove pectin on the surfaces of the seeds, and washing the seeds clean with distilled water. Soaking and sterilizing in 2% sodium hypochlorite on a superclean bench for 15-20min, and washing with sterile water for 3-5 times. Peeling seed coat under aseptic condition, sowing on MT solid culture medium, dark culturing for one month, and light culturing for 5-7 days for transformation.

2) Preparation of Agrobacterium infection solution

Taking target gene vector agrobacterium liquid stored in an ultra-low temperature refrigerator, streaking on an LB (lysogeny broth) plate added with corresponding antibiotics (the pBI121 vector uses kanamycin) for 2-3d at 30 ℃, then selecting a monoclonal, streaking again, carrying out dark culture at 30 ℃ for 2-3d for conversion, scraping plaque streaked for the second time, placing the scraped plaque in a proper amount of liquid suspension MT +20mg/L Acetosyringone (AS) culture medium, and carrying out shaking culture at 28 ℃ for 180r/min for about 20min so AS to suspend and uniformly distribute the bacterial blocks. Taking a plurality of bacteria liquid spectrophotometer to measure OD₆₀₀Value, and adjusting OD using MT +20mg/L AS Medium in liquid suspension₆₀₀Values between 0.6 and 0.8 are used as infestation.

3) Explant preparation

Taking out the lemon seedlings, placing the lemon seedlings in a sterilized large culture dish paved with filter paper for sectioning, cutting the lemon seedlings into 1-1.5cm stem sections at an angle of about 45 degrees by using a scalpel, and adding a small amount of MT liquid culture medium into a sterilized triangular flask to immerse the cut stem sections for keeping moisture for later use.

4) Infection and co-culture: and (3) placing the stem segments in the prepared agrobacterium tumefaciens bacterial liquid, and oscillating for 20 min. Taking the infected strain section, sucking dry surface bacteria liquid on sterile filter paper, then flatly placing the strain section on a co-culture medium paved with a layer of filter paper in advance, and carrying out dark culture at 25 ℃ for 3 d.

5) Screening and culturing: co-culturing the stem segments for 3d, taking out, washing with sterile water for 3-5 times, transferring into a screening culture medium added with 400mg/L of cefuroxime for dark culture for one month, transferring into a screening culture medium added with 400mg/L of cefuroxime and Kan 50mg/L for culture after callus or regeneration buds are induced. Cutting the regenerated bud when the regenerated bud grows to about 1cm, putting the regenerated bud into an elongation proliferation culture medium, and transferring the regenerated bud into a rooting culture medium after rooting.

The media formulations used are shown in table 1 below. The prepared culture medium is autoclaved at 121 ℃ for 15min, the antibiotic subjected to filtration sterilization is added into a super clean bench, and then the culture medium is subpackaged into triangular bottles or can bottles which are autoclaved at 121 ℃ for 15min and sealed for later use.

TABLE 1 culture Medium formulation

3. Positive seedling identification

The relative expression quantity of the PtrSnRK2.4 gene in the transgenic lemon is quantitatively analyzed through real-time fluorescence, the result shows that the relative expression quantity of the PtrSnRK2.4 gene in the PtrSnRK2.4 transgenic lemon (#2, #6) is obviously increased compared with the wild type WT, and the obtained positive transgenic lemon is used for subsequent drought resistance analysis.

Example 5: transgenic lemon drought resistance analysis

Before the drought treatment, the growth state of wild lemons was not significantly different from that of the over-expressed lemons (#2, #5), but after the drought treatment, the wild lemons were severely damaged, leaves were significantly withered, and the transgenic lemons were less damaged (a in fig. 5). Chlorophyll fluorescence imaging results and Fv/Fm of over-expressed lemons were significantly better than wild-type (B in fig. 5 and D in fig. 5). Furthermore, the relative conductivity of the transgenic plants was also significantly lower than that of the wild type (C in fig. 5), while the transgenic plants accumulated less MDA content (E in fig. 5). Meanwhile, the ADC enzyme activity and the putrescine content of the transgenic lemons and the wild lemons are increased to different degrees after drought treatment, but the increase range of the enzyme activity and the putrescine content of the transgenic lemons is larger (G in a figure 5 and H in a figure 5). The increase of the putrescine content in the plants is beneficial to promoting the elimination of active oxygen, so that less H is accumulated in the transgenic lemons after treatment compared with wild lemons₂O₂And O₂ ^·-(F in FIG. 5).

In conclusion, transgenic lemons exhibit higher putrescine content and stronger active oxygen scavenging capacity under drought treatment, which may be a significant cause of their enhanced drought resistance. By using the method, the gene is overexpressed in the plant, and the drought resistance of the plant can be obviously enhanced.

Example 6: identification of VIGS interfering Hovenia dulcis and positive seedlings

1. Vector construction

Takes trifoliate orange cDNA as a template, designs a specific primer to amplify a segment with about 474bp of a non-conservative region in the CDS of the PtrSnRK2.4 gene, and adoptsTrelief^TMThe SoSoSoo Cloning Kit (Ongji, China) is inserted and connected between two enzyme cutting sites BamH I and SmaI on the pTRV2 vector by one-step method, and the constructed vector is transferred into GV3101 competence after being sequenced correctly. The primers for constructing the vector were as follows:

pTRV2-PtrSnRK2.4-F(BamH I):

5’-AGAAGGCCTCCATGGGGATCCGTTTGGTCCTGTGGAGTGA-3’；

pTRV2-PtrSnRK2.4-R(Sma I):

5’-TGTCTTCGGGACATGCCCGGGCGGATCATGGCTCAT-3’。

infection with VIGS

Separating seeds from the fruits of trifoliate orange, soaking for 15min by using 1mol/L NaOH solution to remove pectin, washing for 2 times by using sterile water, paving on a wet clean gauze, putting in an incubator at 28 ℃ under the dark condition for accelerating germination, and using for infection of VIGS when the sprouts of the seeds germinate to 1-2cm long. The operation is as follows:

1) marking Agrobacterium such as pTRV1, pTRV2, target gene recombinant vector and the like on LB (containing 50mg/L Rif and 50mg/L Kan) solid culture medium respectively, and obtaining single clone after inverted culture for 2-3 days at 28 ℃;

2) respectively selecting one monoclonal antibody in 5mL LB liquid culture medium containing the same antibiotic, shaking for 24-48h at 28 deg.C and 220r/min, and fully activating thallus;

3) inoculating the activated Agrobacterium liquid into LB liquid culture medium at a ratio of 1:100, culturing at 28 deg.C for 10-12 hr, centrifuging at 4000r/min, collecting thallus, adding MES buffer (10mm ol/L MES,10mmol/L MgCl)₂150. mu. mol/L AS, pH 5.6-5.7), OD₆₀₀Adjusting to 1.0;

4) mixing heavy suspensions of pTRV1 and pTRV2 and pTRV1 and target recombinant bacteria respectively according to the ratio of 1:1, mixing uniformly, and performing dark culture in a 28 ℃ incubator for 2-3h to prepare an infection solution;

5) slightly pricking some small holes on the germinated buds with a syringe needle, completely soaking in the prepared agrobacteria infection solution, vacuum pumping for 10min, rapidly deflating to immerse the agrobacteria in the germinated seeds, and repeating for 3 times. Standing for 15min, taking out the infected seeds, airing on dry filter paper, standing for 2-3min, spreading in a large dish with sterile water for wetting the filter paper, and standing in a room-temperature culture chamber in a dark place for 2-3d in a dark place;

6) the seeds subjected to dark culture were washed with clear water, the residual bacterial solution was removed, and the seeds were sown in a medium (soil: vermiculite 3:1), and positive identification is carried out after the growth of about one month in a room-temperature illumination incubator.

3. Positive material identification

Identifying positive plants of the trifoliate orange with VIGS silencing, taking the extracted DNA of the trifoliate orange as a template, adopting two pairs of primers to carry out PCR identification on the positive plants, wherein the sequences of the primers are respectively as follows (A in a picture 6 and B in a picture 6) and the reverse primer constructed by the pTRV2 forward primer and a target gene recombination vector, and simultaneously detecting the expression quantity of the PtrSnRK2.4 gene in the positive plants through real-time fluorescence quantification (C in the picture 6).

TRV1-F：5’-ATTGAGGCGAAGTACGATGG-3’

TRV1-R：5’-CCATCCACAATTATTTTCCGC-3’

TRV2-F：5’-ATTCACTGGGAGATGATACGCT-3’。

Example 7: interference drought resistance identification of PtrSnRK2.4 trifoliate orange

Drought resistance analysis is carried out on the identified TRV-PtrSnRK2.4 positive interference plants, under the normal growth condition, the interference plants and the no-load control plants have no obvious difference on phenotype, but after drought treatment (25 ℃,20d), the leaf wilting and curling degree of the interference plants is found to be far higher than that of a control group, and the TRV-PtrSnRK2.4 interference plants externally supplemented and sprayed with Put (10mM) hardly have any change (A in figure 7), which indicates that the interference plants are higher in degree of being damaged by drought stress. Also, chlorophyll fluorescence imaging showed that the leaves of the interfering plants almost all turned green after treatment, while the control and Put-on-spray interfering groups still appeared blue (B in fig. 7), and the maximum photosynthetic rate Fv/Fm values also agreed with the imaging results (C in fig. 7). Compared with the control, the ptrsnrk2.4 interference plants have higher relative conductivity and MDA content after drought treatment, while the interference group supplemented with Put has no significant difference from the control (D in fig. 7, E in fig. 7). In addition, after drought treatment, the ADC enzyme activity and endogenous putrescine (Put) content of the interference plants are obviously lower than those of the interference plants sprayed with Put in a control group (G in figure 7 and H in figure 7), and DAB and NBT dyeing of leaves of the interference plants is also obviously deeper than those of the interference plants sprayed with Put in a TRV control group (F in figure 7). In conclusion, the interference of the PtrSnRK2.4 gene enhances the sensitivity of the plant to drought stress, and the exogenous putrescine supplementation can relieve and restore the drought stress resistance of the plant, thereby further proving the important regulation and control function of the PtrSnRK2.4 in improving the drought resistance of the plant.

Sequence listing

<110> university of agriculture in Huazhong

Application of <120> Hovenia dulcis protein kinase PtrSnRK2.4 in drought-resistant genetic improvement of plants

<160> 17

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1026

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atggaagaga gatatgagcc agtgaaggag cttgggtctg gaaattttgg ggtggcaaga 60

ttggtcagag ataagaggac taaggaactt gttgctgtca aatacataga aagaggcaag 120

aagattgatg agaatgtcca gagggaaatc atcaaccaca gatctctgag gcatccaaac 180

attatcaggt tcaaagaggt gttgttgact ccaactcact tagctattgt catggaatat 240

gctgctggtg gtgaactctt tgcaagaata tgcagtgctg gtcgatttag cgaagatgag 300

gctagatttt tcttccagca gctaatatct ggcgtcagct actgtcattc tatggaaatt 360

tgtcacaggg atctgaagtt ggaaaacact ctattggatg gaagtccaca accacggctg 420

aagatatgcg actttggtta ctcaaagtca ggattgttgc actcgcaacc aaaatcaaca 480

gttggtactc cagcatacat tgcccctgag gtcctagcaa gaaaggaata tgatggcaag 540

agttcagatg tttggtcctg tggagtgaca ctgtatgtga tgttggtggg tggatatcca 600

tttgaggatc ctgaagatcc aagaaacttc cgcaagacaa ttgatagaat aaggaatgtt 660

cagtacttca tgcccgacta tgtacgtgta tctgcggatt gcaggcatct gctttctcag 720

atttttgttg ctgatccctc aaagaggatc cccattccag agatcaaaag gcatccttgg 780

ttcctgaaga atttgccgaa agagataatt gaaattgaga aaacaaatta caaggaagca 840

ggacgtgacc aaccaactca gagtgttgaa gaaataatgc gtatcataca agaggcaaag 900

atgccgggtg aagcaacgaa agttgccggg caaagtgctg ctggggcatc ggaccctgat 960

gacatggagg atgacataga atctgagatt gatgccagcg gtgattatat gagccatgat 1020

ccgtga 1026

<210> 2

<211> 341

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Glu Glu Arg Tyr Glu Pro Val Lys Glu Leu Gly Ser Gly Asn Phe

1 5 10 15

Gly Val Ala Arg Leu Val Arg Asp Lys Arg Thr Lys Glu Leu Val Ala

20 25 30

Val Lys Tyr Ile Glu Arg Gly Lys Lys Ile Asp Glu Asn Val Gln Arg

35 40 45

Glu Ile Ile Asn His Arg Ser Leu Arg His Pro Asn Ile Ile Arg Phe

50 55 60

Lys Glu Val Leu Leu Thr Pro Thr His Leu Ala Ile Val Met Glu Tyr

65 70 75 80

Ala Ala Gly Gly Glu Leu Phe Ala Arg Ile Cys Ser Ala Gly Arg Phe

85 90 95

Ser Glu Asp Glu Ala Arg Phe Phe Phe Gln Gln Leu Ile Ser Gly Val

100 105 110

Ser Tyr Cys His Ser Met Glu Ile Cys His Arg Asp Leu Lys Leu Glu

115 120 125

Asn Thr Leu Leu Asp Gly Ser Pro Gln Pro Arg Leu Lys Ile Cys Asp

130 135 140

Phe Gly Tyr Ser Lys Ser Gly Leu Leu His Ser Gln Pro Lys Ser Thr

145 150 155 160

Val Gly Thr Pro Ala Tyr Ile Ala Pro Glu Val Leu Ala Arg Lys Glu

165 170 175

Tyr Asp Gly Lys Ser Ser Asp Val Trp Ser Cys Gly Val Thr Leu Tyr

180 185 190

Val Met Leu Val Gly Gly Tyr Pro Phe Glu Asp Pro Glu Asp Pro Arg

195 200 205

Asn Phe Arg Lys Thr Ile Asp Arg Ile Arg Asn Val Gln Tyr Phe Met

210 215 220

Pro Asp Tyr Val Arg Val Ser Ala Asp Cys Arg His Leu Leu Ser Gln

225 230 235 240

Ile Phe Val Ala Asp Pro Ser Lys Arg Ile Pro Ile Pro Glu Ile Lys

245 250 255

Arg His Pro Trp Phe Leu Lys Asn Leu Pro Lys Glu Ile Ile Glu Ile

260 265 270

Glu Lys Thr Asn Tyr Lys Glu Ala Gly Arg Asp Gln Pro Thr Gln Ser

275 280 285

Val Glu Glu Ile Met Arg Ile Ile Gln Glu Ala Lys Met Pro Gly Glu

290 295 300

Ala Thr Lys Val Ala Gly Gln Ser Ala Ala Gly Ala Ser Asp Pro Asp

305 310 315 320

Asp Met Glu Asp Asp Ile Glu Ser Glu Ile Asp Ala Ser Gly Asp Tyr

325 330 335

Met Ser His Asp Pro

340

<210> 3

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

tggattgaga tggaagagag a 21

<210> 4

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

cggatcatgg ctcatataat c 21

<210> 5

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ccgaccgtat gagcaaggaa a 21

<210> 6

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ttcctgtgga caatggatgg a 21

<210> 7

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

aagcaggacg tgaccaacca a 21

<210> 8

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

tcatataatc accgctggca t 21

<210> 9

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ggactagttg gattgagatg gaagagagat 30

<210> 10

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

acgcgtcgac cggatcatgg ctcatata 28

<210> 11

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

gctctagaat tgagatggaa gagagatatg 30

<210> 12

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

tcccccgggt cacggatcat ggctcat 27

<210> 13

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

agaaggcctc catggggatc cgtttggtcc tgtggagtga 40

<210> 14

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

tgtcttcggg acatgcccgg gcggatcatg gctcat 36

<210> 15

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

attgaggcga agtacgatgg 20

<210> 16

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

ccatccacaa ttattttccg c 21

<210> 17

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

attcactggg agatgatacg ct 22

Claims

The application of the protein shown in SEQ ID NO.2 or the gene for coding the protein shown in SEQ ID NO.2 in controlling the drought resistance of plants, wherein the plants are lemon or trifoliate orange.
2. The application of claim 1, wherein the application process comprises: constructing a plant overexpression or interference vector containing a gene for coding the protein shown in SEQ ID NO.2, and introducing the plant overexpression or interference vector into lemon or trifoliate orange.