Wheat secretion carrier related membrane protein TaSCAMP6 and application thereof

Abstract

The invention belongs to the technical field of bioengineering, and relates to a wheat secretion carrier related membrane protein TaSCAMP6 and application thereof. The amino acid sequence of the wheat secretion vector related membrane protein TaSCAMP6 provided by the invention is as shown in SEQ ID NO: 2; the nucleotide sequence for coding the wheat secretion vector related membrane protein TaSCAMP6 gene is as shown in SEQ ID NO: 1; according to the invention, the function of the TaSCAMP6 gene is researched through a genetic means, transgenosis is carried out in a Fielder wild type material, a TaSCAMP6 overexpression plant is obtained, and verification shows that the resistance of TaSCAMP6 overexpression to stripe rust is enhanced, the gene is determined to play a positive regulation role in stripe rust resistance of wheat, and a new gene resource is provided for stripe rust resistance breeding of wheat.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering and relates to a wheat secretion carrier-related membrane protein TaSCAMP6 and an application thereof. Background Art

[0002] Wheat stripe rust, caused by Puccinia striiformis f.sp.tritici, has the characteristics of strong epidemicity, wide range of damage and great destructiveness, and has become one of the most important diseases threatening wheat yield. Puccinia striiformis mainly harms wheat by repeated infection of summer spores. In the early stage of the disease, chlorotic spots appear on the wheat leaves, and then bright yellow dotted powder blisters (summer spores) appear at the diseased parts, hence the name stripe rust. In the later stage, black blisters (winter spores) appear. Frequent outbreaks of stripe rust are caused by the gradual "loss" of wheat varieties' resistance to stripe rust and the continuous production of new pathogenic species by stripe rust. Therefore, it is of great significance to tap into wheat disease-resistant gene resources and accelerate the creation and cultivation of new varieties resistant to stripe rust.

[0003] Secretory carrier membrane proteins (SCAMPs) are a complete family of membrane proteins that can bind to specific receptors on the cell membrane and transport a variety of substances. Structurally, all SCAMPs contain a highly conserved four-transmembrane transmembrane core, called the SCAMP domain, which is located in the center and consists of four hydrophobic transmembrane domains (TMDs) and three transmembrane domain flanking sequences. SCAMPs have multiple functions on the cell membrane. The receptor binding sites on the cell membrane can bind to a variety of ligands including hormones, neurotransmitters, growth factors, etc., triggering signal transduction pathways, thereby affecting a variety of physiological processes in the cell. It has a significant role in regulating stress under adverse conditions and plays an important role in plant immunity.

[0004] At present, the prevention and control methods of wheat stripe rust are mainly chemical prevention and control. The harm caused by the use of chemical pesticides to the environment and food safety has attracted widespread attention. Disease-resistant breeding is one of the most economical and effective measures to prevent and control wheat stripe rust. However, the discovery cycle of disease-resistant genes is long, the breeding of disease-resistant varieties is difficult, and the toxicity of stripe rust fungi mutates rapidly, making it difficult to achieve long-term control of the disease in prevention and control. Therefore, creating broad-spectrum and long-lasting disease-resistant materials is the fundamental way and key technology to prevent and control wheat stripe rust. Summary of the invention

[0005] The purpose of the present invention is to solve the problems of reproductive isolation and distant hybridization incompatibility in traditional disease-resistant breeding, which makes it difficult to achieve targeted improvement of target traits within a short breeding cycle; at the same time, the toxicity of stripe rust fungi mutates rapidly, resulting in many difficulties in the long-term prevention and control of wheat stripe rust under current technical conditions.

[0006] Based on the above purpose, the present invention provides a wheat secretion vector-related membrane protein TaSCAMP6 and its application to solve this need in the field. The present invention aims to further explore the immune defense mechanism of wheat, explore the disease resistance genes that play a positive regulatory role in the defense response of wheat invaded by stripe rust, and provide a new path for using the disease resistance gene to create wheat disease-resistant materials to prevent and control wheat stripe rust by conducting gene function research.

[0007] In order to fully and unambiguously understand the technical solution of the present invention, it is necessary to supplement that the wheat secretion vector-related membrane protein TaSCAMP6 encoding gene of the present invention is represented by the italic font "TaSCAMP6", and the wheat secretion vector-related membrane protein TaSCAMP6 is represented by the non-italic font "TaSCAMP6". Of course, those skilled in the art can clearly and completely understand the meaning and expression of the relevant genes and their encoded proteins based on the description of the present invention.

[0008] In one aspect, the present invention relates to a wheat secretion carrier-associated membrane protein TaSCAMP6, the amino acid sequence of which is shown in SEQ ID NO: 2. The specific sequence of SEQ ID NO: 2 is as follows:

[0009] MHHDPNPFDEGTAGDENPFSNGGGRGGKQQHGFRPTEPVGFGGGGSRGDATVDVPLGNMGDSNGKARELSSWESDLRRREADIKRREESLKNAGVPMEEKNWPPFFPIIHHDIANEIPANVQKLQYLAFASWLGIV LCLSWNFIAVIVCWIKEGDSKLFFLATIYALLGIPLSYLMWYRPLYRAMRTNSAFSFGWFFLCYLIHIGFCIIAAIAPPIVFQGKSLTGILAAIDTFSEHLIIGIFYFVGFALFCLETLLSIGVLQKVYMYFRGHK.

[0010] On the other hand, the present invention relates to a gene encoding the wheat secretory carrier-associated membrane protein TaSCAMP6. The nucleotide sequence of TaSCAMP6 is shown in SEQ ID NO: 1. The sequence SEQ ID NO: 1 is as follows:

[0011] ATGCATCACGACCCCAACCCCTTCGACGAGGGCACCGCCGGCGACGAAAACCCCTTCTCCAATGGAGGAGGCCGCGGCGGGAAGCAGCAGCACGGCTTCCGGCCCACCGAGCCCGTCGGCTTCGGCGGCGGCGGCAGCAGGGGCGACGCCACCGTCGACGTGCCCCTCGGCAACATGGGCGACTCGAATGGCAAGGCGAGGGAGCTCTCGTCGTGGGAATCAGATCTGAGGCGTCGTGAGGCGGATATCAAAAGGAGGGAGGAATCGCTGAAGAATGCTGGAGTGCCCATGGAGGAGAAGAATTGGCCGCCTTTTTTCCCGATTATCCACCATGACATCGCCAATGAGATACCTGCAAACGTGCAGAAGTTACAGTATCTGGCGTTTGCAAGCTGGCTTGGAATTGTGCTCTGCCTCTCCTGGAACTTTATTGCTGTCATAGTCTGCTGGATCAAGGAGGGAGATTCAAAGCTGTTCTTCCTTGCTACAATCTATGCTTTGCTTGGAATTCCCCTTTCTTACTTGATGTGGTATCGACCCCTCTATCGTGCAATGAGGACTAACAGTGCATTCAGTTTTGGATGGTTTTTCCTGTGTTACCTGATCCACATTGGTTTTTGCATAATTGCTGCCATTGCTCCACCAATTGTATTCCAAGGGAAATCATTAACGGGCATATTGGCCGCGATCGACACTTTCTCTGAGCATTTGATAATTGGGATCTTTTACTTCGTGGGGTTTGCACTATTTTGCTTGGAGACACTGCTGAGCATCGGGGTTCTTCAGAAAGTATACATGTACTTCCGAGGGCATAAGTGA。

[0012] On the other hand, the present invention relates to the application of a wheat secretory carrier-associated membrane protein TaSCAMP6 in the cultivation of wheat varieties resistant to stripe rust.

[0013] Furthermore, in the application provided by the present invention, the nucleotide sequence of the wheat secretion vector-associated membrane protein gene TaSCAMP6 is expressed by Agrobacterium-mediated genetic transformation, and has a positive regulatory effect in the interaction between wheat and stripe rust. Overexpression of the nucleotide sequence of the wheat secretion vector-associated membrane protein gene TaSCAMP6 can enhance the resistance of wheat to stripe rust pathogens.

[0014] In addition, the present invention relates to a method for breeding wheat stripe rust resistant varieties, which comprises expressing the wheat secretion vector-associated membrane protein gene TaSCAMP6 in plants or transferring the nucleotide sequence of the wheat secretion vector-associated membrane protein gene TaSCAMP6 into the plants.

[0015] Furthermore, the method for breeding wheat stripe rust-resistant varieties provided by the present invention comprises: transforming the nucleotide sequence of the wheat secretion vector-associated membrane protein gene TaSCAMP6 into the cells of the plant to obtain a plant variety transgenic for the wheat secretion vector-associated membrane protein TaSCAMP6.

[0016] Furthermore, the method for breeding wheat stripe rust-resistant varieties provided by the present invention comprises: constructing an expression vector comprising the nucleotide sequence of the wheat secretion vector-associated membrane protein gene TaSCAMP6; transforming the immature embryos of the plant using an Agrobacterium-mediated genetic transformation method to obtain a plant variety that overexpresses the wheat secretion vector-associated membrane protein TaSCAMP6.

[0017] Furthermore, in the method for breeding wheat stripe rust-resistant varieties provided by the present invention, the plant is a monocotyledonous plant, the monocotyledonous plant is a cereal crop, and the cereal crop is wheat.

[0018] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0019] (1) Compared with traditional disease-resistant breeding technology, plant disease-resistant genetic engineering technology can break through the reproductive isolation and incompatibility of distant hybridization between species, achieve targeted improvement of target traits in a shorter period of time, and provide more comprehensive, continuous and broad-spectrum protection for crops. Through gene function research, the present invention found that wheat secretion vector-related membrane protein TaSCAMP6 plays a positive regulatory role in the defense response of wheat invaded by stripe rust, that is, overexpression of the nucleotide sequence of wheat secretion vector-related membrane protein gene TaSCAMP6 can improve the ability of wheat to resist stripe rust. When the nucleotide sequence of wheat secretion vector-related membrane protein gene TaSCAMP6 is overexpressed in plants, it can give plants certain disease resistance.

[0020] (2) The present invention provides a method for breeding wheat stripe rust-resistant varieties. The method uses genetic engineering technology to overexpress the wheat secretion vector-related membrane protein TaSCAMP6 in wheat plants to enhance the resistance of wheat to stripe rust pathogens. It has been verified that the wheat overexpressing TaSCAMP6 obtained by the method of the present invention shows resistance to the main epidemic subspecies of stripe rust. The present invention provides a new technical idea for breeding wheat stripe rust-resistant varieties from the perspective of molecular biology, and effectively solves the technical problems of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of expression spectrum analysis of TaSCAMP6 gene provided in the embodiment of the present invention. Stripe rust CYR31 (affinity) and CYR23 (non-affinity) races were inoculated. It was found that the expression level of TaSCAMP6 gene reached the highest level in the non-affinity system (inoculation of CYR23) after infection for 24 hours, indicating that TaSCAMP6 gene was induced to express by wheat stripe rust, and TaSCAMP6 gene may be involved in wheat resistance to stripe rust.

[0022] Figure 2 This is the PCR test result of the TaSCAMP6 gene overexpression plant provided in the embodiment of the present invention. L74 and L92 are the strains Line74 and Line92 of the gene overexpression plant; M is DNA Marker; WT is the wild type; H2O is the clear water control group.

[0023] Figure 3 : is a TaSCAMP6 gene expression vector diagram provided in an embodiment of the present invention, wherein LB and RB are homology arms; ZmUbi is a promoter; TaSCAMP6 is a TaSCAMP6 gene; T-NOS is a termination element; and Bar is a herbicide selection marker gene.

[0024] Figure 4 This is a phenotypic result diagram of TaSCAMP6 gene overexpression plants inoculated with stripe rust CYR32 provided in an embodiment of the present invention. TaSCAMP-6-OE#L74 and TaSCAMP-6-OE#-L92 are TaSCAMP6 gene overexpression plants, CYR32 indicates inoculation with stripe rust virulent race CYR32, and Fielder is a wild-type wheat variety. DETAILED DESCRIPTION

[0025] The technical solution of the present invention is described below in conjunction with embodiments; however, the present invention is not limited to the following embodiments.

[0026] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings, but the embodiments are not intended to limit the present invention.

[0027] The experimental methods and detection methods described in the following embodiments are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

[0028] Example 1

[0029] This example provides the application of wheat secretion vector-associated membrane protein gene TaSCAMP6 in improving wheat rust-resistant varieties.

[0030] First, qRT-PCR was used to determine the function of TaSCAMP6 in the interaction between wheat and stripe rust.

[0031] Based on qRT-PCR, the elongation factor gene TaEF1-α was used as an internal reference, and the specific primers of the wheat secretion carrier-related membrane protein TaSCAMP6 gene were used for real-time quantitative PCR to determine the expression level of the TaSCAMP6 gene at different time points when wheat was infected with stripe rust. qRT-PCR was used to detect the induced expression of wild-type wheat Fielder inoculated with stripe rust CYR23 (non-compatible) and stripe rust CYR31 (compatible) at different time points (0h, 24h, 48h, 72h, 96h, 120h, 168h, and 216h of stripe rust infection).

[0032] TaSCAMP6 quantitative primers:

[0033] Forward primer: TaSCAMP6-qRT-F: GCATCACGACCCCAACC;

[0034] Reverse primer: TaSCAMP6-qRT-R: GCCTCACGACGCCTCAG.

[0035] Internal reference TaEF1-α primer:

[0036] Forward primer: TaEF1α-F: TGGTGTCATCAAGCCTGGTATGGT;

[0037] Reverse primer: TaEF1α-R: ACTCATGGTGCATCTCAACGGACT.

[0038] The test results are as follows Figure 1 As shown, the results showed that the expression level of TaSCAMP6 gene reached the highest level 24 hours after infection in the non-compatibility system (inoculated with CYR23), indicating that TaSCAMP6 gene was induced to express by wheat stripe rust, and TaSCAMP6 gene may be involved in wheat resistance to stripe rust.

[0039] The application of the wheat secretion vector-associated membrane protein TaSCAMP6 provided in the embodiment of the present invention in the cultivation and improvement of wheat rust-resistant varieties also includes: constructing an expression vector containing the wheat secretion vector-associated membrane protein gene TaSCAMP6; using the Agrobacterium-mediated genetic transformation method to transform wheat immature embryos to obtain TaSCAMP6 overexpressing wheat varieties.

[0040] The verification method of the application of wheat secretion vector-related membrane protein TaSCAMP6 in cultivating and improving wheat rust-resistant varieties provided by the embodiment of the present invention includes:

[0041] S101, obtaining TaSCAMP6 overexpressing wheat, and performing molecular detection on the obtained TaSCAMP6 overexpressing wheat;

[0042] S102, the T1 generation overexpressing plants were inoculated with the stripe rust epidemic race CYR32, and the resistance of the transgenic plants to the stripe rust epidemic race was identified.

[0043] The TaSCAMP6 gene was overexpressed in the lines Line74 and Line92 using transgenic technology to obtain TaSCAMP6 transgenic Line74 plants and TaSCAMP6 transgenic Line92 plants, respectively. The TaSCAMP6 transgenic positive plants were detected using PCR technology, and the PCR products were detected by 1% agarose gel electrophoresis. The results are as follows Figure 2 As shown, Figure 2 L74 and L92 are TaSCAMP6 transgenic lines Line74 and Line92; M is a DNA marker; WT is the wild type; H2O is the water control group. The results showed that both TaSCAMP6 overexpressing Line74 and TaSCAMP6 overexpressing Line92 plants expressed TaSCAMP6, while TaSCAMP6 was not detected in wild type plants or water.

[0044] The nucleotide sequence of the wheat secretion carrier-associated membrane protein gene TaSCAMP6 of the present invention is shown as SEQ ID NO: 1.

[0045] The amino acid sequence encoded by the wheat secretion carrier-associated membrane protein gene TaSCAMP6 of the present invention is shown in SEQ ID NO:2.

[0046] The plants of the present invention are preferably monocotyledonous plants and cereal crops that can be successfully infected and colonized by wheat stripe rust fungi, and wheat is particularly preferred.

[0047] The application of the wheat secretion vector-associated membrane protein gene TaSCAMP6 provided in the embodiment of the present invention in improving wheat rust-resistant varieties comprises the following steps:

[0048] The TaSCAMP6-overexpressing plants obtained were inoculated with the compatible strain CYR32, and the disease resistance of wheat was enhanced, indicating that TaSCAMP6 plays a positive regulatory role in the interaction between wheat and stripe rust.

[0049] The overexpression plants were inoculated with CYR32 and phenotypic identification showed that TaSCAMP6 overexpression plants showed enhanced resistance to stripe rust CYR32. The expression vector map is shown in Figure 3 shown.

[0050] Forward primer: TaSCAMP6-nucleotide-F: ATGCATCACGACCCCAA;

[0051] Reverse primer: TaSCAMP6-nucleotide-R: CTTATGCCCTCGGAAGTAC.

[0052] The application of the wheat secretion vector-related membrane protein gene TaSCAMP6 in the improvement of wheat rust resistance provided by the embodiment of the present invention is to perform PCR detection on the overexpression plants obtained by Agrobacterium-mediated genetic transformation, and inoculate the main epidemic race CYR32 of stripe rust fungus into the positive strains of wild-type "Fielder" and TaSCAMP6 overexpression plants. After 14 days of inoculation, Figure 4 As shown, wild-type leaves produce spores. The results showed that TaSCAMP6 overexpressing plants had enhanced resistance to stripe rust.

[0053] In summary, the application of wheat secretion vector-associated membrane protein TaSCAMP6 in the improvement of wheat rust-resistant varieties provided by the embodiments of the present invention is to construct a TaSCAMP6 expression vector by using gene recombination technology, and the expression vector is delivered to the recipient wheat Fielder by Agrobacterium-mediated wheat genetic transformation technology. The obtained overexpression plants are confirmed as positive transgenic plants by PCR detection and identification. Two strains of L74 and L92 of the T1 generation of positive transgenic plants are selected and inoculated with the popular race CYR32. After 14 days of observation, it is found that the wheat secretion vector-associated membrane protein TaSCAMP6 overexpression plants have reduced spore production compared with Fielder, showing enhanced resistance.

[0054] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A wheat secretion carrier-associated membrane protein TaSCAMP6, characterized in that: The amino acid sequence of the wheat secretion carrier-associated membrane protein TaSCAMP6 is shown in SEQ ID NO:

2.

2. A wheat secretory carrier-associated membrane protein gene TaSCAMP6, characterized in that: The TaSCAMP6 encodes the wheat secretion carrier-associated membrane protein TaSCAMP6 described in claim 1.

3. The wheat secretory carrier-associated membrane protein gene TaSCAMP6 according to claim 2, characterized in that: The nucleotide sequence of TaSCAMP6 is shown in SEQ ID NO:

1.

4. An expression vector, characterized in that: The expression vector is inserted with the nucleotide sequence described in claim 3, and after transfecting the host cell, the expression vector can make the transfected host cell express the wheat secretion carrier-associated membrane protein TaSCAMP6 described in claim 1.

5. Use of the wheat secretion vector-associated membrane protein TaSCAMP6 according to claim 1 or the wheat secretion vector-associated membrane protein gene TaSCAMP6 according to claim 3 in breeding wheat stripe rust resistant varieties.

6. The use according to claim 5, characterized in that: The nucleotide sequence of the wheat secretion vector-associated membrane protein gene TaSCAMP6 is expressed by Agrobacterium-mediated genetic transformation, and has a positive regulatory effect in the interaction between wheat and stripe rust. Overexpression of the nucleotide sequence of the wheat secretion vector-associated membrane protein gene TaSCAMP6 can enhance wheat resistance to stripe rust pathogens.

7. A method for breeding wheat varieties resistant to stripe rust, characterized in that: The wheat secretion carrier-associated membrane protein TaSCAMP6 described in claim 1 is overexpressed in the plant or the nucleotide sequence of the wheat secretion carrier-associated membrane protein gene TaSCAMP6 described in claim 3 is transferred into the plant.

8. The method for breeding wheat stripe rust resistant varieties according to claim 7, characterized in that: include: The nucleotide sequence of the wheat secretion vector-associated membrane protein gene TaSCAMP6 is transformed into the cells of the plant to obtain a plant variety that overexpresses the wheat secretion vector-associated membrane protein TaSCAMP6.

9. The method for breeding wheat stripe rust resistant varieties according to claim 7, characterized in that: include: An expression vector containing the nucleotide sequence of the wheat secretion vector-associated membrane protein gene TaSCAMP6 is constructed; the immature embryos of the plant are transformed using an Agrobacterium-mediated genetic transformation method to obtain a plant variety that overexpresses the wheat secretion vector-associated membrane protein TaSCAMP6.

10. The method for breeding wheat stripe rust resistant varieties according to claim 7, characterized in that: The plant is a monocotyledonous plant, the monocotyledonous plant is a cereal crop, and the cereal crop is wheat.

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