Banana NBS-LRR type wilt-resistant gene and application thereof

Abstract

The invention discloses banana NBS-LRR wilt-resistant genes and application thereof, and belongs to the field of plant genetic engineering. The invention firstly provides a MaRRS1 gene obtained by cloning from a banana corm, and the nucleotide sequence of the MaRRS1 gene is shown as SEQ ID No.1. According to the invention, the MaRRS1 gene is further transferred into banana seedlings for functional verification. Results show that the over-expression of the MaRRS1 gene can significantly enhance the resistance of susceptible varieties to fusarium oxysporum. The separated MaRRS1 gene is an important gene for regulating and controlling the fusarium wilt resistance of the bananas, can improve the fusarium wilt resistance of the bananas, and has an important application prospect in the aspects of banana fusarium wilt resistance molecular breeding and the like. The banana NBS-LRR disease-resistant gene cloned by the invention can improve the resistance of bananas to fusarium wilt, and has important application value in genetic improvement of banana fusarium wilt resistance and cultivation of new fusarium wilt-resistant banana varieties (lines).

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to banana NBS-LRR wilt-resistance genes and applications thereof. Background Art

[0002] Bananas are a common, convenient, and high-calorie fruit grown in tropical and subtropical countries. They are a staple starch and a source of food for millions of people worldwide. They are the world's second-largest fruit crop and the most traded fruit. The United Nations Food and Agriculture Organization considers bananas the fourth-largest food crop after rice, wheat, and corn.

[0003] However, bananas face the challenge of Fusarium oxysporum f. sp. cubense (Foc), a soil-borne disease caused by the fungus Fusarium oxysporum. Fusarium oxysporum destroys vascular tissue, preventing the plant from properly absorbing water and nutrients, leading to plant wilt and death. It is currently one of the most devastating diseases of the crop. The pathogen, tropical race 4 (Foc TR4), can cause disease in the Cavendish banana, a major internationally traded variety, under any environmental conditions. Once infected, no physical or chemical control measures, including biological control, chemical control, and the use of fungicides, can effectively control F. oxysporum. Due to the complex genetic structure of the banana genome, long breeding cycles, and high space requirements, breeding new varieties through mutation breeding and mutant screening is inherently risky and requires a long development cycle. Effective, long-term management of banana Fusarium wilt remains a challenge, and genetic modification may be the only way to improve banana varieties. Therefore, breeding new disease-resistant banana varieties is currently the most effective approach to controlling banana Fusarium wilt. One effective approach to combating banana Fusarium wilt is to identify disease-resistance genes in bananas. This discovery provides valuable genetic resources for transgenic banana breeding. However, the biological functions of banana genes are largely unknown, requiring extensive transcriptome and proteome data to identify genes associated with Fusarium wilt resistance. Summary of the Invention

[0004] The present invention aims to address the technical deficiencies of the prior art and provide banana NBS-LRR wilt-resistant genes and applications thereof.

[0005] To achieve the above object, the present invention adopts the following technical solutions: The banana NBS-LRR type wilt disease resistance gene is characterized in that the amino acid sequence of the protein encoded by the banana NBS-LRR type gene is shown in SEQ ID NO: 1.

[0006] The application of banana NBS-LRR wilt-resistant genes is characterized by: application in cultivating banana wilt-resistant plant germplasm.

[0007] The banana NBS-LRR gene recognizes the invasion of Fusarium oxysporum and initiates downstream programmed cell death, thereby achieving a disease resistance effect.

[0008] The banana NBS-LRR gene is cloned from banana corm.

[0009] The invention relates to an application of a recombinant vector or recombinant bacteria containing banana NBS-LRR genes in banana resistance to Fusarium wilt disease. The amino acid sequence of the protein encoded by the banana NBS-LRR genes is shown in SEQ ID NO: 1. Specifically, the invention relates to an application of a recombinant vector or recombinant bacteria containing banana NBS-LRR genes in banana resistance to Fusarium wilt disease.

[0010] Compared with the prior art, the present invention has the following characteristics and beneficial effects: The banana NBS-LRR disease-resistant gene cloned in the present invention can improve the resistance of banana to Fusarium wilt, and has important application value for genetic improvement of banana resistance to Fusarium wilt and breeding new wilt-resistant banana varieties (lines). BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 RACE obtained the 3' unknown sequence and 5' unknown sequence of the MaRRS1 gene as well as the ORF frame electrophoresis pattern; Figure 2 Identification of the T0 generation of MaRRS1-overexpressing transgenic tobacco. DETAILED DESCRIPTION

[0012] In order to make the technical means, innovative features, objectives and effects achieved by the present invention easier to understand, the present invention is further described below.

[0013] The embodiments described herein are specific embodiments of the present invention and are used to illustrate the concept of the present invention. They are illustrative and exemplary and should not be construed as limiting the embodiments and scope of the present invention. In addition to the embodiments described herein, those skilled in the art can also adopt other obvious technical solutions based on the claims and the disclosure of the specification, including technical solutions that adopt any obvious substitutions and modifications to the embodiments described herein.

[0014] Nucleotide-binding site-leucine-rich repeat (NBS-LRR) proteins, the largest family of plant resistance (R) proteins, play a crucial role in pathogen defense. The completion and refinement of the banana reference sequence has enabled a systematic understanding of the banana NBS-LRR protein family. In this study, a total of 98 NBS-LRR proteins were identified from the banana genome and grouped into eight classes in a phylogenetic tree. NBS-LRR genes are unevenly distributed across all 11 banana chromosomes. Typical NB-ARC (nucleotide-binding APAF-1, disease resistance protein, CED-4) and LRR motifs were found in all NBS-LRR proteins, with a small number of introns present in most NBS-LRR genes. The transcriptional profiles of NBS-LRR proteins were investigated in Fusarium oxysporum f. fusarium wilt (FOC)-susceptible and -resistant banana cultivars BX and HDJ were challenged with FOC race 1 (FOC 1) and tropical race 4 (FOC TR4). Of the 31 representative NBS-LRRs detected in this study, 12 members showed stronger transcriptional stimulation in HDJ than in BX, while conversely, 8 members showed higher expression in BX than in HDJ. This study improves our understanding of the role of banana NBS-LRRs in defense against Fusarium wilt and will aid in the genetic improvement of banana resistance. Sequence SEQ ID NO:1 was selected for cloning and further investigation of banana disease resistance. The specific steps were as follows: PCR amplification products of the 3' and 5' unknown sequences of the NBS-LRR-type Fusarium wilt resistance gene (hereinafter referred to as Ma-RRS1) were separated by electrophoresis on a 2% agarose gel. The products were selectively recovered based on the predicted target band size using bioinformatics and ligated into the pMD18-T vector. The products were then transformed into competent Escherichia coli DH5ɑ. Single colonies were isolated and tested by PCR. Positive clones were then sent to BGI for sequencing. The obtained 5' and 3' unknown sequences were spliced ​​to obtain the full-length cDNA sequence, namely the sequence shown in SEQ ID NO: 1. The electrophoresis diagram of the 3' unknown sequence, 5' unknown sequence and ORF frame sequence of the MaRRS1 gene is shown in FIG. Figure 1PCR amplification products were separated by electrophoresis on a 1.5% agarose gel. Products were recovered based on the bioinformatically predicted target band size and ligated into a vector for transformation into competent Escherichia coli. Using enzyme digestion and ligation, forward and reverse inserts of the Ma-RRS1 gene were amplified from a banana corm cDNA template using a forward insertion primer pair and a reverse insertion primer pair, respectively. These inserts were then inserted into a digested RNAi expression vector to construct the expression vector for genetic transformation of banana corms. The constructed vectors were then transformed into Agrobacterium using the liquid nitrogen freeze-thaw method for transgenic banana production. For genetic transformation of banana corms, PCR identification of transgenic tobacco bananas, and PCR detection of overexpressed transgenic bananas, a pair of primers (primers 35S-F) were designed based on the 35S promoter and MaRRS1 gene sequences. DNA from transgenic and non-transgenic bananas was extracted using the CTAB method. PCR amplification was performed using DNA from transgenic plants as templates and DNA from untransformed bananas as controls using primers 35S-F and MaRRS1-R. The PCR reaction system consisted of 2.0 μl of 10× buffer, 1.5 μl of dNTPs, 0.5 μl of 35S-F, 0.5 μl of MaRRS1-R, 0.1 μl of Taq enzyme, 14.4 μl of ddH2O, and 1.0 μl of template DNA, for a total volume of 20 μl. The reaction procedure was: 94°C for 5 min → (94°C for 30 s → 58°C for 30 s → 72°C for 60 s) for 40 cycles → 72°C for 10 min → hold at 4°C. The results showed that a 1650-bp band was amplified in positive plants, and 73 positive plants were identified from 84 transgenic plants. To understand whether the transgene was expressed in the positive plants, total RNA was extracted from the positive plants using the CTAB method and reverse transcribed into single-stranded cDNA using PrimeScript reverse transcriptase. Primers Ma RRS 1 -F and Ma RRS 1 -R were designed based on the MaRRS1 gene sequence for RT-PCR analysis. All 73 positive plants analyzed showed expression bands, with a positive rate of 87% ( Figure 2 ).

[0015] To identify transgenic plants resistant to Fusarium wilt, functional leaves of wild-type banana plants, transgenic banana plants overexpressing the gene, and RNAi-transgenic banana plants were inoculated with Fusarium oxysporum along the leaf veins using a disposable syringe for resistance testing. Three biological replicates were used for each treatment combination. The disease index was used to evaluate the overall disease resistance of the inoculated plants. Twenty days after inoculation, the transgenic plants maintained normal growth, with obvious necrotic spots appearing at the inoculated sites on the leaves. However, the stems showed no infection or only mild infection in some cases, whereas the control group showed signs of death.

[0016] 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, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A banana NBS-LRR wilt resistance gene, characterized by: The amino acid sequence of the protein encoded by the banana NBS-LRR gene is shown in SEQ ID NO:

1.

2. Application of banana NBS-LRR wilt resistance genes, characterized by: Application in cultivating banana wilt-resistant plant germplasm.

3. The use according to claim 1, wherein: The banana NBS-LRR gene recognizes the invasion of Fusarium oxysporum and initiates downstream programmed cell death, thereby achieving a disease resistance effect.

4. The use according to claim 1, wherein: The banana NBS-LRR gene is cloned from banana corm.

5. Use of a recombinant vector or recombinant bacteria containing banana NBS-LRR genes in resisting banana wilt disease, wherein the amino acid sequence of the protein encoded by the banana NBS-LRR genes is shown in SEQ ID NO:

1.

6. The use according to claim 5, characterized in that It is used in the cultivation of banana wilt-resistant plant germplasm.

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