CN115976051B - Potato StRTP gene and application thereof in disease-resistant breeding - Google Patents
Potato StRTP gene and application thereof in disease-resistant breeding Download PDFInfo
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Abstract
The invention provides a potato StRTP gene and a specific gene fragment contained in the gene, wherein potato StRTP contains two homologous genes in a potato genome, which are respectively named as StRTP a and StRTP b, and the nucleotide sequences are respectively shown in SEQ ID NO:1 and SEQ ID NO: 2. Homologous genes StRTP a and StRTP b have common specific fragments, and the nucleotide sequence is shown in SEQ ID NO: 3. The invention also provides application of the potato StRTP gene and the common specificity fragment in disease-resistant breeding of potatoes. The RNA interference technology is utilized to silence StRTP gene in the atlantic ocean of potato cultivar, which can obviously improve the resistance of the potato cultivar to phytophthora infestans. The StRTP gene can also be used for regulating the height of potato plants. The invention provides a certain theoretical and practical basis for applying the gene to potato disease-resistant breeding.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, relates to a plant immune regulation mechanism, and in particular relates to StRTP gene and application thereof in disease resistance breeding of potatoes.
Background
In the process of affinity interaction of plants with pathogenic bacteria, pathogenic bacteria successfully achieve infestation and colonization by means of a series of nutrient transfer, molecular exchange and hormonal signal transduction processes involving plant regulatory factors. When plants lack the necessary immune negative regulatory factors for infection by these pathogenic bacteria, the plants can be made to exhibit broad-spectrum and durable resistance to pathogenic bacteria. Therefore, the negative immune regulatory factor is knocked out from plants by biotechnology means to improve the durable resistance of the plants to pathogenic bacteria, and the method has become a new strategy for crop breeding.
Phytophthora (Phytophthora) is an oomycete (Oomycetes) which is a eukaryotic microorganism of similar morphology to fungi but which is homologous to algae. Phytophthora is an important plant pathogenic oomycete, can infect various grains and economic crops, often causes destructive crop diseases and seriously threatens grain production. Due to strong genetic variation and toxicity evolution capability of phytophthora, the problem of disease resistance loss of crops is outstanding, so that the control difficulty of the phytophthora of crops is high, and the phytophthora is highly dependent on chemical pesticides. Therefore, the genetic basis of plants on phytophthora susceptibility is known, new disease-resistant strategies are explored, immune regulation genes are identified, molecular mechanisms of the immune regulation genes are analyzed, and the method has important significance for molecular design of new disease-resistant genes and crop disease-resistant breeding and guaranteeing of grain safety.
At present, the immune negative regulatory factor has been explored in the aspect of enhancing potato resistance: resistance of potato to phytophthora is enhanced by silencing orthologous genes of other species' immune negative regulatory factors. Six known orthologous genes of plant immune negative regulatory factors including DMR1 and DMR6 are respectively silenced on potatoes by reverse genetics means in 2016 Sun et al through RNAi technology, the resistance (Sun et al.2016.Silencing of six susceptibility genes results in potato late blight resistance.Transgenic Res,25(5):731-742.). of the potatoes to phytophthora infestans is improved, the homologous gene of arabidopsis DND1 is silenced in the potatoes, the resistance (Sun et al.2016.Down-regulation of Arabidopsis DND1 orthologs in potato and tomato leads to broad-spectrum resistance to late blight and powdery mildew.Transgenic Res,25(2):123-138.). of the potatoes to phytophthora infestans and downy mildew can be improved, and excavation and immune mechanism research of the oomycete immune negative regulatory factors can be carried out more quickly by combining forward or reverse genetics means. Meanwhile, the identification and development of the research of the plant immunity negative regulation factor can not only improve the understanding of human beings on pathogenic bacteria pathogenic mechanism, but also have important application value.
Disclosure of Invention
Because the phytophthora has strong toxicity variation capability and causes the problem of crop variety resistance loss to be prominent, the molecular mechanism of plant-phytophthora interaction is explored, and the development of new plant immunoregulatory genes for phytophthora has important guiding significance for plant disease resistance breeding. The invention aims to improve the disease resistance of potatoes by utilizing molecular biology and genetic engineering means, explores functional genes with the function of regulating and controlling the disease resistance of potatoes, and expands the application of the functional genes in the aspect of improving the disease resistance of potatoes.
In order to achieve the technical purpose of the invention, the invention provides a potato StRTP gene. Silencing the StRTP gene and the common specificity fragment, the potato plant shows stronger resistance to phytophthora infestans. The StRTP gene has two homologous genes StRTP a and StRTP7b, stRTP a in the genome of potato, and the nucleotide sequence of the gene is shown as SEQ ID NO:1, and the nucleotide sequence of StRTP b gene is shown as SEQ ID NO: 2. The homologous genes StRTP a and StRTP b have a common specific fragment, and the nucleotide sequence of the common specific fragment is shown in SEQ ID NO: 3. The invention provides a certain theoretical and practical basis for applying the gene to potato disease-resistant breeding.
Further molecular mechanism studies showed that the StRTP gene was involved in intron splicing of Stnad7, reducing the accumulation of Stnad mature transcripts. Stnad7 has the nucleotide sequence shown in SEQ ID NO: 4.
Based on the description of the invention, the invention provides the application of the StRTP gene and the common specificity fragment of the potato in disease-resistant breeding of the potato for the first time. Specifically, silencing the StRTP7 gene (or reducing the expression of the StRTP gene by other means possible) enhances resistance of potatoes to phytophthora infestans. In another preferred embodiment, silencing the common specificity fragment of StRTP gene (homologous genes StRTP a and StRTP b) which has the nucleotide sequence set forth in SEQ ID NO: 3. Based on the description of the invention, the enhancement of the resistance of potato plants to phytophthora infestans can be realized by reducing the expression abundance (expression quantity) of the StRTP gene and the common specificity fragment.
Furthermore, the invention provides a method for increasing resistance of potatoes to phytophthora infestans. It should be noted that, the present invention is not limited to a specific implementation manner for cultivating the disease-resistant potato plant, and based on StRTP gene and common specificity fragment disclosed in the present invention, a person skilled in the art combines with the prior art to realize the cultivation of the disease-resistant potato plant. The methods of the invention include silencing the potato StRTP gene, or silencing a common specificity fragment of the potato StRTP gene (homologous genes StRTP a and StRTP b). The nucleotide sequence of StRTP a gene is shown as SEQ ID NO:1, and the nucleotide sequence of StRTP b gene is shown as SEQ ID NO: 2. The homologous genes StRTP a and StRTP b have a common specific fragment, and the nucleotide sequence of the common specific fragment is shown in SEQ ID NO: 3.
As described above, the method for improving the disease resistance of the potato and obtaining the potato plant resistant to phytophthora capsici based on StRTP gene and common specificity fragment provides an effective technical path for solving the research problem of disease resistance breeding of the potato, so to speak, the method for controlling potato phytophthora capsici is the most economical, effective, green and durable method so far, and simultaneously effectively overcomes the technical defects of the potato phytophthora capsici control method mainly comprising agricultural control and chemical control.
Furthermore, the invention provides application of the StRTP gene and the common specificity fragment in regulation and control of potato growth (plant height). It was found by experiment that silencing the potato StRTP gene (or reducing the expression level of the StRTP gene by other possible means) can reduce the height of the potato plant. In another preferred embodiment, silencing a common specificity fragment of the potato StRTP gene, having a nucleotide sequence set forth in SEQ ID NO: 3. The present invention surprisingly found that silencing the potato StRTP gene, or silencing a common specific fragment of the potato StRTP gene, has a specific effect on potato plant growth, manifesting as inhibition of potato plant internode elongation.
Compared with the prior art, the potato StRTP gene and the application thereof in disease-resistant breeding have the following beneficial effects or advantages:
1) The invention discloses two homologous genes StRTP a and StRTP b of potato StRTP7 genes in a potato genome for the first time, wherein the nucleotide sequences of the two homologous genes are respectively shown as SEQ ID NO:1 and SEQ ID NO: 2. Homologous genes StRTP a and StRTP b have common specific fragments, and the nucleotide sequence is shown in SEQ ID NO: 3. By regulating and controlling the expression of the gene of the potato StRTP, the potato plant shows stronger resistance to phytophthora infestans. The invention provides an effective technical scheme for solving the research problem of disease-resistant breeding of potatoes, and can provide technical support for creating new disease-resistant germplasm of potatoes.
2) The invention has great significance in improving the disease resistance of potato plants to phytophthora infestans. By means of genetic engineering technology, the resistance of potato plants to phytophthora infestans can be improved through gene silencing of potato StRTP, a new solution idea is provided for preventing and treating potato diseases from the aspect of molecular biology, and a certain theoretical and practical basis is provided for applying the gene to potato disease-resistant breeding.
3) The invention further discloses that the potato StRTP gene and the specific fragment thereof have a certain regulation and control effect on the potato plant height, and are used for inhibiting the internode elongation of potato plants, but not influencing the leaf size of the potato plants.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following description will simply make reference to the accompanying drawings, which are listed as being only a part of the contents of the embodiments of the present invention.
FIG. 1 is a schematic diagram of the structure of potato mitochondria Stnad gene. In FIG. 1, intron2, intron3 represent introns, and Exon1, exon2, exon3, and Exon4 represent exons, respectively.
FIG. 2 is a semi-quantitative PCR assay for the accumulation of Stnad gene maturation transcripts in RiSt lines and Atlantic potato leaves. In FIG. 2, stEF1 alpha was used as an internal reference gene to show consistent loading, atlantic represents an Atlantic potato variety, riSt-1, riSt-2, riSt-3, riSt-4 represent potato StRTP7 gene-silenced lines, respectively.
FIG. 3 shows the RT-qPCR assay StRTP for expression of the gene in RiSt lines and in Atlantic potato leaves. In FIG. 3, atlantic represents an Atlantic potato variety, riSt-1, riSt-2, riSt-3, riSt-4 represent potato StRTP gene silencing strains, respectively. The results in fig. 3 are expressed as mean ± standard error of 3 biological replicates, with P <0.01 and P <0.001 analyzed for statistical significance by t-test.
FIG. 4 is a qPCR assay RiSt strain and splicing efficiencies of Atlantic potato leaves Stnad Intron1 and Intron 3. In FIG. 4, atlantic represents an Atlantic potato variety, riSt-1, riSt-2, riSt-3, riSt-4 represent potato StRTP gene silencing strains, respectively. The results in fig. 4 are expressed as mean ± standard error of 3 biological replicates, with statistically significant analysis by t-test, × P <0.001.
FIG. 5 is a graph of the diameter observation of Atlantic potato leaf spot and trypan blue staining results 3 days after inoculation with Phytophthora infestans. In FIG. 5, atlantic represents an Atlantic potato variety, riSt-1, riSt-2 represent potato StRTP gene-silenced lines, respectively.
FIG. 6 is a statistical result of the diameter of the phytophthora infestans spot of StRTP silent plants. The plaque diameter results of fig. 6 are expressed as mean ± standard error from at least 5 leaf plaques, statistically significant analyzed by t-test, P <0.001.
FIG. 7 is a quantitative PCR assay for potato leaf pathogen colonization. The results in fig. 7 are expressed as mean ± standard error of 3 biological replicates, statistically significant analysis was performed with t-test, <0.05.
FIG. 8 is a3 day leaf count of Phytophthora infestans after inoculation. Fig. 8 sporangia statistics expressed as mean ± standard error of 3 biological replicates, statistically significant analysis was performed with t-test, × P <0.001.
FIG. 9 is a view of trypan blue staining of Phytophthora infestans inoculation leaves of StRTP silent plants. In FIG. 9, atlantic represents a potato Atlantic variety, riSt-1, riSt-2 represent potato StRTP gene-silenced lines, respectively. In FIG. 9A, riSt-1 and RiSt-2 silence plant leaves in which epidermic cells infected with Phytophthora infestans exhibit stronger cell necrosis, black arrows indicate aspirator-like structures, and asterisks indicate necrotic cells. In fig. 9B, phytophthora infestans the aschersonia, and the black arrow indicates the aschersonia.
FIG. 10 is a phenotype plot of RiSt plants grown after 4 weeks of culture in substrate soil. In FIG. 10, atlantic represents an Atlantic potato variety, riSt-1, riSt-2 represent potato StRTP gene-silenced lines, respectively.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described in detail with reference to the following technical route diagram shown in fig. 1 and the embodiment. It should be understood that the detailed description and specific examples are intended for purposes of illustration only and are not intended to limit the scope of the invention.
Example 1
Using the Solanaceae plant genome information website (https:// solgenomics. Net /), genome alignment analysis revealed that the StRTP gene contained two homologous genes with highly similar nucleotide sequences in the potato genome, designated StRTP a (Gene No. PGSC0003DMC 400008531) and StRTP b (Gene No. PGSC0003DMC 400052354), respectively. In view of the nucleotide sequence similarity of up to 94%, the present patent uses both as test genes and considers that both have similar biological functions. Coding (CDS) sequences for homologous genes StRTP a and StRTP b are available from the Solanaceae plant genome information website (https:// solgenomics. Net /).
The potato mitochondrial Stnad gene sequence can be obtained from NCBI query under the gene number GenBank: MF989958.1.Stnad7 has the nucleotide sequence shown in SEQ ID NO: 4.
Example 2
In order to provide a more exact theoretical and practical basis for applying the gene to potato disease-resistant breeding, the embodiment gives the experimental result that the gene StRTP of potato participates in the intron splicing of Stnad.
Since the sequences of StRTP a and StRTP7b are highly similar, there may be functional redundancy, by selecting segments of similar nucleotide sequences as silencing segments, the two genes are co-silenced. A gene fragment (in the present invention, a specific fragment and a common specific fragment have the same meaning as the expression) which is highly conserved by StRTP a and StRTP b and has a length of 470bp and has a sequence similarity of up to 94.89% in the StRTP a and StRTP7b genes, and only a small single nucleotide difference is considered to be present, so that it is considered that co-silencing of StRTP a and StRTP7b genes can be achieved by using the fragment. And the sequence comparison proves that the fragment is unique to StRTP gene, and the nucleotide sequence is shown as SEQ ID NO: 3.
Designing a specific primer, amplifying the StRTP gene specific fragment by taking potato cDNA as a template, and respectively connecting the fragment to two ends of a pKANNIBAL carrier (intermediate carrier) Intron by enzyme digestion by utilizing different enzyme digestion sites to form a hairpin structure. The primer sequences designed in this example were as follows:
RiStRTP7-F1:CCGCTCGAGTTCGACAACAGTGAAGTCCACAA；
RiStRTP7-R1:CGGAATTCTCAAAGACTCAGCTTTAGCAGTG；
RiStRTP7-F2:GCTCTAGATTCGACAACAGTGAAGTCCACAA；
RiStRTP7-R2:CGGGATCCTCAAAGACTCAGCTTTAGCAGTG。
the correct pKANNIBAL vector was sequenced using Not1 restriction enzyme and ligated into the pART27 plant expression vector.
The StRTP gene was silenced in the atlantic potato cultivar using RNA interference (RNAi) technology. Potato transformation was performed using a stable transformation system mediated by Agrobacterium using potato stem sections grown for approximately 4-5 weeks, and specific transformation methods were described in published literature (Kong Xianglan.2021. Construction of vector for genetic transformation of potato and immunorelated gene StCAD gene editing [ Shushi ]. China Yang Ling: university of agriculture and forestry science and technology, north-West). Four transformants (labeled RiSt-1 to 4) were finally obtained by stable transformation of potato.
The expression level of StRTP gene was found to be significantly down-regulated in all four RNAi strains by real-time fluorescent quantitative PCR detection (fig. 1-4), with expression levels of about 20% of the atlantic potato leaf.
The number and position of the intron of Stnad were obtained by sequence analysis of the Stnad gene, see in particular FIG. 1. The levels of intron splicing of Stnad intron1 and intron3 in the RiSt-1 to 4 RNAi strain leaves were examined using RT-qPCR. The experimental results showed (fig. 4) that the intronic splicing efficiency of Stnad intron1 and intron3 was significantly reduced to about 15% of the control group in all four RiSt lines. Meanwhile, the accumulation of Stnad mature transcripts in RiSt strain was detected by semi-quantitative PCR, and the result shows that the accumulation of Stnad mature transcripts in RiSt plant leaves is also obviously reduced (figures 2 and 3). The above results indicate that the StRTP gene is involved in intron splicing of Stnad and results in a decrease in the accumulation of Stnad7 mature transcripts.
Example 3
This example describes the resistance performance of RNAi plants of potato StRTP against Phytophthora infestans. Phytophthora infestans (P.infestans) strain 88069 was used in this example.
Culturing phytophthora infestans: the phytophthora infestans silk blocks with the side length of about 0.5cm are scratched by using a sterile toothpick, transferred onto a new rye culture medium plate and cultured in a dark inversion mode for 10-15 days at the temperature of 16 ℃.
Production of zoospores of Phytophthora infestans: 4mL of sterile water was added to the dish and the surface of the medium was gently rubbed with a blue gun head. After 1 hour of treatment at 4℃for 30 minutes on ice, a large amount of zoospores were observed to be produced.
Inoculation of phytophthora infestans: the fully flat potato leaves are cut, and the petioles are wrapped on cotton strips soaked by sterile water for moisture preservation. A drop of zoospore liquid of Phytophthora infestans (about 800 zoospores) was inoculated at the widest center point of the left and right side leaves of the main vein. Dark culture at 18℃for 2-3 days, and spot diameter was measured and counted.
The leaves of the two StRTP lines RiSt-1 and RiSt-2 were subjected to Phytophthora infestans inoculation test analysis. Experimental results indicate that the plaque diameters of both RiSt-1, riSt-2 RNAi strains are significantly smaller than that of Atlantic potato leaves (FIG. 5). The quantitative PCR assay of hyphal colonization further showed that the phytophthora infestans biomass of RiSt-1, riSt-2 plant leaves was significantly lower than that of the atlantic ocean (FIGS. 6-8). Counting the number of phytophthora infestans sporangia on the leaves of both lines RiSt-1, riSt-2 was found to be significantly lower than in the atlantic ocean (figure 8). Staining and observing the bacterial leaves by trypan blue, and finding that a large number of sporangia are generated on the atlantic leaves; almost no aerial hyphae and sporangia were observed on RiSt-1 leaves; a small amount of aerial hyphae and sporangia were visible in RiSt-2 leaves (FIG. 9). Upon observation of trypan blue stained leaves, riSt-1 and RiSt-2 leaf epidermal cells infected with phytophthora infestans were found to undergo massive necrosis, but little epidermal cell necrosis was observed in the atlantic ocean (fig. 9). The above results demonstrate that StRTP RNAi strain exhibits greater resistance to phytophthora infestans.
Example 4
This example describes the growth of two StRTP7 lines RiSt-1 and RiSt-2.
After 4 weeks of culture in matrix soil, the plant growth phenotypes RiSt-1 and RiSt-2 are shown in FIG. 10. It was further observed that StRTP RNAi plants were shorter than atlantic potato plants, but did not affect leaf size. The results of this example show that StRTP gene can regulate potato growth and inhibit internode elongation of potato plants.
The foregoing is merely illustrative of specific embodiments of the present invention, and the scope of the invention is not limited thereto, but any modifications, equivalents, improvements and alternatives falling within the spirit and principles of the present invention will be apparent to those skilled in the art within the scope of the present invention.
Claims (2)
1. Use of a homologous gene of potato gene StRTP in potato genome in breeding of potato against phytophthora infestans (Phytophthora infestans), characterized in that the homologous genes are StRTP a and StRTP b, and the nucleotide sequence of StRTP a is as shown in SEQ ID NO:1, wherein the nucleotide sequence of StRTP b is shown as SEQ ID NO:2 is shown in the figure;
silencing the StRTP a and StRTP b common specificity fragment to enhance resistance of potato to phytophthora infestans, the nucleotide sequence of the specificity fragment being as set forth in SEQ ID NO: 3.
2. Use of a homologous gene of potato gene StRTP in potato genome for reducing height of potato plants, wherein said homologous genes are StRTP a and StRTP b, and the nucleotide sequence of StRTP a is as set forth in SEQ ID NO:1, wherein the nucleotide sequence of StRTP b is shown as SEQ ID NO:2 is shown in the figure;
the StRTP a and the StRTP b have a common specific fragment, and the nucleotide sequence of the common specific fragment is shown in SEQ ID NO:3, silencing the common specificity fragment to reduce the height of potato plants; inhibiting the internode elongation of potato plants reduces the plant height.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104583409A (en) * | 2012-08-08 | 2015-04-29 | Kws种子股份有限公司 | Transgenic plant of the species solanum tuberosum with resistance to phytophthora |
| CN110938637A (en) * | 2019-11-26 | 2020-03-31 | 西北农林科技大学 | Homologous gene of phytophthora resistance negative regulatory factor StMKK1 and application thereof |
| CN116515861A (en) * | 2023-06-27 | 2023-08-01 | 西北农林科技大学深圳研究院 | Gene StLTPa and application of encoding protein thereof in disease resistance of potatoes |
| CN116987730A (en) * | 2023-09-22 | 2023-11-03 | 西北农林科技大学深圳研究院 | Application of thioredoxin StCDSP32 in plant disease resistance |
| CN117660478A (en) * | 2023-11-17 | 2024-03-08 | 重庆市农业科学院 | Gene for improving resistance of potatoes to late blight and application thereof |
-
2022
- 2022-11-16 CN CN202211459529.0A patent/CN115976051B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104583409A (en) * | 2012-08-08 | 2015-04-29 | Kws种子股份有限公司 | Transgenic plant of the species solanum tuberosum with resistance to phytophthora |
| CN110938637A (en) * | 2019-11-26 | 2020-03-31 | 西北农林科技大学 | Homologous gene of phytophthora resistance negative regulatory factor StMKK1 and application thereof |
| CN116515861A (en) * | 2023-06-27 | 2023-08-01 | 西北农林科技大学深圳研究院 | Gene StLTPa and application of encoding protein thereof in disease resistance of potatoes |
| CN116987730A (en) * | 2023-09-22 | 2023-11-03 | 西北农林科技大学深圳研究院 | Application of thioredoxin StCDSP32 in plant disease resistance |
| CN117660478A (en) * | 2023-11-17 | 2024-03-08 | 重庆市农业科学院 | Gene for improving resistance of potatoes to late blight and application thereof |
Non-Patent Citations (7)
| Title |
|---|
| A mitochondrial RNA processing protein mediates plant immunity to a broad spectrum of pathogens by modulating the mitochondrial oxidative burst;Yang Yang等;Plant Cell;20220309;第34卷;第2344页，第2346页右栏最后一段，第2356页左栏第4-5段、右栏第1段，第2358页右栏第1-2段，第2359页左栏第5段，图S24 * |
| AtRTP5 negatively regulates plant resistance to Phytophthora pathogens by modulating the biosynthesis of endogenous jasmonic acid and salicylic acid;WEIWEI LI等;Molecular Plant Pathology;20200131;第21卷;第95–108页 * |
| PREDICTED: Solanum tuberosum pentatricopeptide repeat-containing protein At4g02820, mitochondrial-like (LOC102586806), mRNA.GenBank.2016,Accession No. XM_006366065.2. * |
| PREDICTED: Solanum tuberosum pentatricopeptide repeat-containing protein At4g02820,mitochondrial-like (LOC102591148), mRNA.GenBank.2016,Accession No. XM_015312378.1. * |
| 一个马铃薯种质资源圃致病疫霉群体的分析;马云芳等;菌物学报;20130915(第05期);第802-811页 * |
| 马铃薯StBI-1基因鉴定及其抗疫霉菌的功能探索;高贤贤等;西北农业学报;20221019;第31卷;第1329-1336页 * |
| 马铃薯StCYP83 基因家族鉴定及其抗晚疫病的功能分析;孔乐辉等;中国农业科学;20230816;第56卷;第3124-3139页 * |
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