Application of MaBEL1 in improving cold resistance of fruits

Abstract

The application relates to the technical field of plant stress resistance, and particularly relates to application of a powdery banana BEL1-LIKE HOMEODOMAIN transcription factor MaBEL1 in improving fruit cold resistance, which creates conditions and practical theoretical basis for cultivation of cold-resistant plant varieties and is suitable for large-scale production and application.

Description

Technical Field

[0001] This invention relates to the field of plant stress resistance technology, specifically to the application of the banana BEL1-LIKE HOMEODOMAIN transcription factor MaBEL1 in improving the cold resistance of fruit. Background Technology

[0002] Pink banana ( Musa ABB Pisang Awak, belonging to the Musaceae family, is a newly emerging specialty banana variety. It has a thin peel, thick flesh, a sweet taste, is rich in nutrients, and exhibits excellent disease resistance, making it popular with consumers. It is widely cultivated in southern my country, accounting for approximately 20% of total banana production, and the area under protected cultivation is increasing year by year, making it one of my country's most important fruit crops. ABB Pisang Awak is a tropical and subtropical fruit, and is sensitive to low temperatures. If the growing temperature or storage temperature is below 11℃, the fruit is prone to chilling injury, leading to a decrease in quality and commercial value.

[0003] With the increasing frequency of extreme weather events globally, crop resilience is receiving growing attention. Winters in southern my country frequently see temperatures below 0°C, particularly in northern Guangdong, making bananas more susceptible to chilling injury, severely impacting their quality and commercial value. Therefore, cultivating chilling-resistant banana varieties is crucial to addressing the effects of low-temperature stress on banana fruit.

[0004] Currently, research on the stress resistance mechanisms of crops against low temperatures is relatively lagging, especially in the study of cold resistance in bananas. Not only have significant breakthroughs been made in the measures and methods to solve the cold resistance problem of bananas, but no banana varieties that can withstand low temperatures have also been discovered, which has brought great trouble to the production of bananas. Therefore, it is particularly important to explore the key cold resistance genes of crops such as bananas, analyze the mechanism of fruit resistance to low temperatures, and cultivate low-temperature resistant crops. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide the application of the banana BEL1-LIKEHOMEODOMAIN transcription factor MaBEL1 in improving the cold resistance of fruit, which can significantly improve the cold resistance of transgenic fruit.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] The application of the BEL1-LIKE HOMEODOMAIN transcription factor MaBEL1 in improving the cold resistance of banana fruit is provided, and the sequence of MaBEL1 is shown in SEQ ID NO. 1.

[0008] In some embodiments, the fruit is a banana.

[0009] The application of the protein sequence of banana BEL1-LIKE HOMEODOMAIN transcription factor MaBEL1 in improving plant cold resistance is also provided, and the protein sequence is shown in SEQ ID NO. 2.

[0010] In some embodiments, the fruit is a banana.

[0011] In the above technical solution, based on the genome sequence of the banana plant, specific primers were designed and the BEL1-LIKE HOMEODOMAIN transcription factor MaBEL1 was cloned from the banana plant and transiently overexpressed in the banana plant. The cold resistance of the transgenic fruit was significantly improved. The research results provide a useful method and idea for creating cold-resistant banana plant materials.

[0012] The BEL1-LIKE HOMEODOMAIN transcription factor MaBEL1 (hereinafter referred to as MaBEL1) is 2004 bp in length, encodes 667 amino acids, and has the highest homology with MaBEL1-like4 in bananas and SlBEL1-like4 in tomatoes.

[0013] The method for obtaining the MaBEL1 gene sequence is as follows: using the cDNA of 'Guangfen No. 1' as a template, primers MaBEL1-F and MaBEL1-R are used, and the sequence is obtained after cloning and sequencing.

[0014] The BEL1-LIKE HOMEODOMAIN protein sequence encoded by the MaBEL1 gene has a relative molecular weight of 57.65 kDa, an isoelectric point pI of 7.13, and is slightly alkaline.

[0015] Overexpression of MaBEL1 in banana fruit can induce the ratio of unsaturated fatty acids and the content of flavonoids in banana fruit.

[0016] The aforementioned application of the banana BEL1-LIKE HOMEODOMAIN transcription factor MaBEL1 in improving the cold resistance of bananas involved cloning the 2004 bp, 667 amino acids, from the banana genome using specific primers. Bioinformatics analysis showed the highest homology with MaBEL1-like4 from bananas and SlBEL1-like4 from tomatoes; its location in the cell nucleus and low-temperature stress significantly inhibited its expression level; transient overexpression in banana fruits significantly induced the expression of fatty acid desaturation and flavonoid synthesis genes, thereby increasing the fatty acid unsaturation rate and flavonoid content of banana fruits and improving their cold resistance. This invention provides innovative gene resources for creating cold-resistant banana varieties, creating conditions and a practical theoretical foundation for breeding cold-resistant banana varieties, and also provides new methods and ideas for applied research on the cold resistance of other crop varieties.

[0017] The beneficial effects of the application of the BEL1-LIKE HOMEODOMAIN transcription factor MaBEL1 in bananas in improving fruit cold resistance:

[0018] This invention proposes the application of the BEL1-LIKE HOMEODOMAIN transcription factor MaBEL1 in improving the cold resistance of banana fruits. It innovates the genetic resources for creating cold-resistant plant varieties and creates conditions and a practical theoretical basis for the breeding of cold-resistant plant varieties. In turn, it provides new methods and ideas for the application research of cold resistance of crop varieties. Attached Figure Description

[0019] Figure 1 Homology comparison of the amino acid sequences encoding MaBEL1;

[0020] Figure 2 Phylogenetic analysis of the amino acid sequence of MaBEL1;

[0021] Figure 3 Predicting the protein structure of MaBEL1 amino acids;

[0022] Figure 4 Subcellular localization of MaBEL1;

[0023] Figure 5 For MaBEL1 gene expression;

[0024] Figure 6 Identification of banana fruits with transient MaBEL1 overexpression;

[0025] Figure 7 Phenotypic, physiological and biochemical analysis of banana fruits and unloaded fruits with transient MaBEL1 overexpression under low temperature stress;

[0026] Figure 8 To investigate the expression of fatty acid-related genes and flavonoid genes in banana fruit by transient overexpression of MaBEL1. Detailed Implementation

[0027] Preferred embodiments of the invention will now be described in more detail with reference to the accompanying drawings. While preferred embodiments of the invention are shown in the drawings, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that the invention will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0028] The terminology used in this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The singular forms “a” and “the” as used in this invention and the appended claims are intended to include the plural forms unless the context clearly indicates otherwise. It should also be understood that the term “and / or” as used herein refers to and includes any or all possible combinations of one or more of the associated listed items.

[0029] It should be understood that although the terms "first," "second," "third," etc., may be used in this invention to describe various information, this information should not be limited to these terms. These terms are only used to distinguish information of the same type from one another. For example, without departing from the scope of this invention, first information may also be referred to as second information, and similarly, second information may also be referred to as first information. Thus, features defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this invention, "a plurality of" means two or more, unless otherwise explicitly specified.

[0030] Example 1

[0031] This embodiment discloses the acquisition and analysis of the gene sequence of the BEL1-LIKE HOMEODOMAIN transcription factor MaBEL1:

[0032] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0033] Experimental materials: 'Guangfen No. 1' banana ( MusaABB Pisang Awak (Guangfen No. 1), Escherichia coli strain DH5α, Agrobacterium tumefaciens GV3101, preserved in our laboratory (College of Horticulture, South China Agricultural University); Polysaccharide-Polyphenol / Complex Plant RNA Rapid Extraction Kit (RN53-EASYspin Plus, Beijing Adley Biotechnology Co., Ltd., Beijing, China); Reverse Oxidation Kit (Takara), Gel Extraction Kit (Takara), Primers synthesized by Shanghai Sangon Biotech Co., Ltd., Dual-Luciferase Assay Kit (Promega, Madison, WI, USA).

[0034] Obtaining the gene sequence of the BEL1-LIKE HOMEODOMAIN transcription factor MaBEL1 involves the following steps:

[0035] (1) Template preparation: Extract RNA from banana fruit using a polysaccharide polyphenol / complex plant RNA rapid extraction kit (refer to the kit instructions for specific methods), and then synthesize cDNA using a reverse osmosis kit (Takara) (refer to the kit instructions for specific operation methods).

[0036] (2) Using cDNA from banana pulp as a template, PCR amplification was performed using MaBEL1-F and MaBEL1-R primers. The primer sequences are shown in Appendix Table 1. PCR amplification system: Mix PCR enzyme (25 μl), Primer F (2 µL), Primer R (2 µL), cDNA template 2 µL, and ddH2O added to a final volume of 50 µL. Reaction program: 98 ℃ for 5 min; 98 ℃ for 30 s, 56 ℃ for 30 s, 72 ℃ for 2 min (34 cycles); 72 ℃ for 5 min, and the product was stored at 4 ℃.

[0037] (3) The specific gene fragment obtained by PCR amplification was ligated into the pMD19-T expression vector using the pMD19-T Ligation Kit, and then transformed into competent Escherichia coli DH5α to obtain positive recombinant bacteria (refer to the instructions of the kit for specific operation methods). Finally, the nucleotide sequence was obtained by sequencing.

[0038] The results showed that the amplified specific gene had a reading frame of 2004 bp, encoding 667 amino acids. The gene sequence was translated using an ORF (Organic Reading Frame) from the bioinformatics analysis website (Protparam: http: / / expasy.org / tools) to obtain the protein sequence, the amino acid sequence of which is shown below. Figure 1As shown. Through searching, querying, and comparing nucleic acid and amino acid sequences using the bioinformatics analysis website (NCBI: https: / / www.ncbi.nlm.nih.gov), it was found that this gene and its protein have 100% similarity to the BEL1-LIKE HOMEODOMAIN transcription factor MaBEL1 gene sequence (XM_009422319.2) and protein sequence, and were named MaBEL1.

[0039] Analysis of the gene sequence of the BEL1-LIKE HOMEODOMAIN transcription factor MaBEL1:

[0040] Phylogenetic trees were constructed using MaBEL1, showing the highest homology with MaBEL1-like4 in bananas and SlBEL1-like4 in tomatoes. (See results below.) Figure 2 .

[0041] Bioinformatics analysis of the amino acid sequence of the MaBEL1 protein, performed at https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi, revealed that MaBEL1 contains POX and Homeobox_KN protein domains. (See attached image for details.) Figure 3 .

[0042] The gene sequence of the banana BEL1-LIKE HOMEODOMAIN transcription factor MaBEL1 was cloned into the pBE-GFP vector.

[0043] Transformation of Agrobacterium: Mix 100 μl of competent Agrobacterium GV3101 cells with 200-300 ng of expression vector, add to an electrode cup, select the bacteria A9 program, electroporate Agrobacterium GV3101, add 300-500 μl of antibiotic-free LB liquid medium, incubate at 200 rpm and 28℃ for 2-3 h, take 10-300 μl of bacterial culture and spread it on LB (containing Rif+Kana) solid plates, and incubate upside down at 28℃ for 2-3 days.

[0044] Tobacco infection: Single colonies were picked and cultured in 5 ml of LB liquid medium (containing Rif + kana) at 200 rpm and 28℃ for 24-36 h. Subsequently, 5 μl of bacterial suspension was added to 5 ml of LB liquid medium (containing Rif + kana) and cultured for about 12 h. The OD600 value was measured. Generally, after 12 h of culture, when the OD value is between 0.5 and 0.6 (logarithmic growth phase; if the OD value exceeds 0.8, the competent cell transformation efficiency is greatly reduced), the process was completed in a clean bench. Centrifugation was performed at 3500 rpm for 15 min at room temperature, the supernatant was discarded, 10 ml of infection solution was added, and centrifugation was performed again at 3500 rpm for 10 min at room temperature to precipitate the bacterial cells (at room temperature). The supernatant was discarded, and 5 ml of infection solution was added to bring the final concentration OD value to between 0.5 and 0.6. After standing for 2-3 h, and after 36-72 h of infection with tobacco, observation and photography were performed using a Zeiss Axioskep 2 Plus fluorescence microscope with the spectral detector wavelength set at 488 nm.

[0045] For detailed operating procedures, please refer to the following references:

[0046] Zunyang Song, Xiuhua Lai, Yulin Yao, Jiajia Qin, Xiaochun Ding, QiuliZheng, Xuequn Pang, Weixin Chen, Xueping Li, Xiaoyang Zhu*. F-box protein EBF1 and transcription factor ABI5-like regulate banana fruit chilling-induced ripening disorder. Plant Physiology. 2022, 188(2): 1312-1334.

[0047] Xiuhua Lai 1 Xiaoyang Zhu 1, Hangcong Chen, Xuequn Pang, Weixin Chen,Xueping Li*, Zunyang Song*. The MaC2H2-like zinc finger protein is involved in ripening and ripening disorders caused by chilling stress via the regulation of softening-related genes in ' Fenjiao' banana. PostharvestBiology and Technology. 2022, 186: 111817.

[0048] The upstream and downstream primer sequences (5'-3') used are MaBEL1-pBE-GFP-F and MaBEL1-pBE-GFP-R, respectively. The upstream primer has an Xbal I restriction site added, and the downstream primer has a BamH I restriction site added. The primer sequences are shown in Table 1.

[0049] Table 1

[0050]

[0051] The results showed that MaBEL1 is a nuclear protein located in the cell nucleus. (See attached image for details.) Figure 4 .

[0052] To investigate the expression characteristics of MaBEL1, banana fruits stored at 25℃, 11℃, and 7℃ for 0, 3, 6, 9, and 12 days were used as materials to study the effect of low-temperature stress on MaBEL1 expression levels. Real-time quantitative PCR (RT-qPCR) was used for quantitative analysis. The specific method is as follows: MaACTIN-F and MaACTIN-R were used as internal control primers, and MaBEL1-F and MaBEL1-R were used as quantitative PCR primers. The primer sequences are shown in Appendix Table 1. The RT-qPCR reaction system was as follows: 2×SYBR Green Mix: 10 µL, cDNA template: 2.0 µL, primers: 0.4 µL each, water: 7.2 µL, total volume: 20 µL. The reaction program was: 94℃ for 6 min; 94℃ for 10 s, 56℃ for 20 s, 72℃ for 35 s, for 40 cycles. The CT value was used as the qPCR result. -△CT To calculate the relative expression level of genes.

[0053] The results showed that low temperature stress at 11℃ and 7℃ significantly inhibited... MaBEL1 The expression level at 7°C was significantly lower than that at 11°C, such as... Figure 5As shown.

[0054] As can be seen, this embodiment can successfully obtain the gene sequence of the BEL1-LIKE HOMEODOMAIN transcription factor MaBEL1.

[0055] Example 2

[0056] This embodiment discloses MaBEL1 Acquisition of banana fruit via transient overexpression, including

[0057] (1) Construction of MaBEL1 overexpression vector.

[0058] Based on the obtained MaBEL1 gene sequence, the CDS sequence of MaBEL1 was constructed into the pMDC32 overexpression vector using a gateway method to obtain the MaBEL1-pMDC32 recombinant vector. This MaBEL1-pMDC32 recombinant vector was then transformed into Agrobacterium GV3101. Subsequently, the GV3101 Agrobacterium containing the MaBEL1-pMDC32 recombinant vector was... (The specific method is described in: ZunyangSong, Xiuhua Lai, Yulin Yao, Jiajia Qin, Xiaochun Ding, Qiuli Zheng, XuequnPang, Weixin Chen, Xueping Li, Xiaoyang Zhu*. F-box protein EBF1 and transcription factor ABI5-like regulate banana fruit chilling-induced ripening disorder. Plant Physiology. 2022, 188(2): 1312-1334.)

[0059] (2) Transient overexpression of MaBEL1 in banana fruit.

[0060] Agrobacterium GV3101 containing the MaBEL1-pMDC32 recombinant vector and Agrobacterium PMDC32 overexpressing empty vector were injected into the fruit from the tail end of the banana fruit, with 2-3 ml of bacterial solution. The specific method is as follows: Zunyang Song, Xiuhua Lai, Yulin Yao, Jiajia Qin, Xiaochun Ding, Qiuli Zheng, Xuequn Pang, Weixin Chen, Xueping Li, Xiaoyang Zhu*. F-box protein EBF1 and transcription factor ABI5-like regulate banana fruit chilling-induced ripening disorder. PlantPhysiology. 2022, 188(2): 1312-1334.

[0061] Transiently overexpressed banana fruits were placed at 7℃ for storage, and chilling injury symptoms were observed. Samples were taken at 0, 4, 7, and 10 days after treatment and stored at -80℃. The transient overexpression in banana fruits was validated, and the conductivity, malondialdehyde, and proline content were measured.

[0062] Verification of MaBEL1 transient overexpression in banana fruits. RNA was extracted from banana fruits containing MaBEL1 transient overexpression and those containing the empty vector, reverse transcribed, and analyzed by RT-qPCR. This confirmed that we successfully obtained MaBEL1 transient overexpression in banana fruits. Figure 6 ).

[0063] The effect of transient overexpression of MaBEL1 on the cold resistance of banana fruits was tested. The results showed that, compared with the empty expression vector, transient overexpression of MaBEL1 exhibited milder chilling injury symptoms, significantly induced proline content, significantly reduced electrical conductivity and malondialdehyde content, and had a lower chilling injury index. These results indicate that transient overexpression of MaBEL1 significantly improves the cold resistance of banana fruits. Figure 7 ).

[0064] Effects of MaBEL1 transient overexpression on fatty acid desaturation and flavonoid synthesis genes in banana fruit: The results showed that, compared with the empty expression vector, MaBEL1 transient overexpression significantly induced the expression of genes related to fatty acid desaturation and flavonoid synthesis. Figure 8 ).

[0065] The following shows sequences SEQ ID NO. 1 and SEQ ID NO. 2 respectively:

[0066] SEQ ID NO. 1

[0067]

[0068] SEQ ID NO. 2:

[0069] MATYFHGAPEIQPDGLQTLYLMNPSYVGYTDAAAPANMVFLNSTMNSVNSINLAQTGQQQHFVGIPLQSAAQPHDSHRSQPIHASHDVPAVQGLLPRSHYNLWTTATSSNPVDMASQFGPWRTSTAPSAQQGLSLSLSPHEMVAPAQEITPASAANEVKIAASASM ASGAANGGSGLQSFLMGTKYLKAAQQLLDEVVNVGKGVKDEAAKGAPLKNPADSSNVELKDPGAGTSEGNTSAKRGADLTTAERQELQMKKAKLINMLEEVEQRYRQYHHQMQIVVSSFEAVAGYGSARTYTALALRTISKQFRCLRDAITAQIRETSKSLGEEDSK SGGSRLRFIDHHLRQQRALQQLGMIQQNAWRPQRGLPERSVSILRAWLFEHFLHPYPKDSDKLMLAKQTGLTRSQVSNWFINARVRLWKPMVEEMYLEELKDQEQNNSEENASNGDANGSSTSKSNAQQEGSPTGTAPSESLKIDPNQTLAAVQPSSFAHGQSPAFH DKDAIEQPSFKKAKRHVNSVAHSIGIEVKPDETNNQDILMKLIDGRHRGGEQGYPLIPGNTSHGGSYGAYPIGGELGRFDAEQFAPRFSGNGVSLTLGLQHSESLPLSGAQPSFLSSESMQMGRRVEMNTEAGDYCSLNNNAAVAHSSNAAAFRAERGFLLNYYQNS

[0070] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Application of BEL1-LIKE HOMEODOMAIN transcription factor MaBEL1 in improving the cold resistance of banana fruits, wherein the sequence of MaBEL1 is shown in SEQ ID NO.

1.

2. Application of the protein sequence of BEL1-LIKE HOMEODOMAIN transcription factor MaBEL1 in improving the cold resistance of banana fruit, the protein sequence being shown in SEQ ID NO. 2.

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