MaDREB1F gene and application thereof

Abstract

The application discloses a MaDREB1F gene, and a nucleotide sequence of the MaDREB1F gene is shown as SEQ ID NO:1. Experimental research results reveal a mechanism of MaDREB1F in mediating non-biological stress resistance of bananas, and determine new targets (MaAOC4, MaACO20 and MaERF11) of MaDREB1F, which are beneficial to design breeding strategies to improve the resistance of crops to cold and drought stress.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant genetic engineering technology, and particularly relates to a MaDREB1F gene and application thereof. BACKGROUND

[0002] The AP2 / ERF superfamily of transcription factors is subdivided into three families, namely ERF, AP2 and RAV. The ERF family contains the ERF and DREB subfamilies, with a single AP2 domain and few introns, and plays a key role in plant responses to abiotic stress. In Arabidopsis, DREBs are divided into six subgroups, of which the A1 subgroup mainly includes CBFs (CBF1-4). CBFs have been characterized in many plant species, and overexpression of CBFs induces cold-responsive (COR) gene expression and increases plant resistance to cold stress in plants such as rice and tomato. Although this indicates the functional conservation of CBFs in cold resistance, other roles have been verified for different CBF members. Extensive overexpression and silencing studies have shown that CBF1 and CBF3 play a core role in cold acclimation. CBF4 plays a role in plant resistance to drought stress, while CBF2 negatively regulates salt and dehydration stress in Arabidopsis. Overexpression of orthologs of Arabidopsis CBFs (OsDREB1A, OsDREB1B, OsDREB1E, OsDREB1F and OsDREB1G) increases the resistance of rice to drought and / or salt stress. Although the role of CBFs in plant stress resistance has been studied to some extent, however, the common mechanism of multiple stress resistance mediated by CBFs / DREB1s is still unclear.

[0003] Accumulating evidence suggests that the key target of CBFs / DREB1s is COR genes. COR genes include COR / LEA, early dehydration-induced (ERD), low-temperature-induced (LTI) and desiccation (RD) genes. The products of COR genes mainly include key enzymes involved in cell wall modifiers, osmolyte biosynthesis, lipid metabolism, carbohydrate metabolism, hormone-responsive proteins and chaperones. CBF can regulate transcriptional changes in many genes related to stress response, transcription factors, kinases, hormone signaling, carbohydrate metabolism and cell wall modification. However, in addition to COR genes, little is known about the target genes directly bound by CBF.

[0004] Banana is one of the world's most nutritious fruits and staple foods. It is widely distributed in tropical and subtropical developing countries, but the incidence of abiotic stress and plant diseases in these regions is high, which seriously reduces the yield and quality of bananas. In addition, banana plants are sensitive to cold, drought and salt stress. It is necessary to study the molecular mechanism of banana response to abiotic stress to enhance its resistance to abiotic stress. Some genes, including MusaPIP2; 6, MusaPIP1; 2, MusaNAC042, MusaWRKY71, MusaSAP1, MusaSNAC1, MaPIP2-7 and MaSIP2-1, have been shown to positively regulate the resistance of banana to abiotic stress. However, there is currently no evidence of the genetic function and molecular mechanism of the AP2 / ERF subfamily in response to banana abiotic stress. SUMMARY

[0005] In the present application, based on the results of previous studies, further in-depth analysis found that MaDREB1F has the function of activating MaAOC4, MaACO20 and MaERF11 promoters.

[0006] The main achievements of this study include the following aspects:

[0007] Firstly, we disclose a MaDREB1F gene, the nucleotide sequence of which is shown as SEQ ID NO: 1. The gene sequence and its function have not been published in other prior art documents.

[0008] On the other hand, we analyzed the function of MaDREB1F gene in detail and clarified its use. Specifically includes:

[0009] The application of MaDREB1F gene in activating MaAOC4 promoter, the nucleotide sequence of MaAOC4 is shown as SEQ ID NO: 2.

[0010] The application of MaDREB1F gene in activating MaACO20 promoter, the nucleotide sequence of MaACO20 is shown as SEQ ID NO: 3.

[0011] The application of MaDREB1F gene in activating MaERF11 promoter, the nucleotide sequence of MaERF11 is shown as SEQ ID NO: 4.

[0012] The beneficial effects achieved by the present application are:

[0013] The present application discloses the sequence of MaDREB1F gene and determines several new targets (MaAOC4, MaACO20 and MaERF11) of MaDREB1F, which is conducive to designing banana breeding strategies to improve crop stress resistance. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 , Figure 2 : Yeast one-hybrid assay results graph. Analysis of the interaction of MaDREB1F and MaAOC4 / MaACO20 promoters. Self-activation of the promoters was tested on SD / -Ura+AbA medium and the interaction of MaDREB1F and MaAOC4 / MaACO20 promoters was tested on SD / -Leu+AbA medium.

[0015] Figures 3-6 : Dual luciferase activity assay results graph. Analysis shows that MaDREB1F activates MaAOC4 / MaACO20 promoter under normal, cold and drought conditions.

[0016] Figure 7 , Figure 8 : Yeast one-hybrid assay results graph. Analysis of the interaction of MaDREB1F and MaERF11 promoters and MaERF11 and MaACO20 promoters. Self-activation of the promoters was tested on SD / -Ura+AbA medium and the interaction between MaDREB1F and MaERF11 promoters and MaERF11 and MaACO20 promoters was tested on SD / -Leu+AbA medium.

[0017] Figure 9 , Figure 10 : Dual luciferase activity assay results graph. Analysis shows that MaDREB1F activates MaERF11 promoter under normal and drought conditions and MaERF11 activates MaACO20 promoter. DETAILED DESCRIPTION

[0018] In order to facilitate the understanding of the technical content of the present application, the present application will be further described in detail below in combination with specific examples.

[0019] 1. Materials and methods

[0020] Plant material and growth conditions

[0021] Banana seedlings (Musa acuminata L. AAA group, cv. Brazil) were grown in a greenhouse (28°C, 70% relative humidity, 16 hours light / 8 hours dark cycle). Banana seedlings were subjected to stress treatment at the five-leaf stage. For cold treatment, banana seedlings were placed in an incubator at 8°C for 47 hours and recovered for 7 days. For osmotic treatment, banana seedlings were treated with 200 mM mannitol for 17 days. For NaCl treatment, banana seedlings were treated with 300 mM NaCl for 24 days.

[0022] qRT-PCR

[0023] Banana leaves, with or without cold, drought and salt treatments, were collected and subjected to qRT-PCR for MaDREB1F expression on Mx3000P instrument (Stratagene, CA, USA). Relative gene expression was determined using the 2-ΔΔCt method. Three biological replicates were performed for each sample.

[0024] Cloning and subcellular localization of MaDREB1F

[0025] The coding sequence of MaDREB1F was cloned from Musa acuminata L. AAA group by RT-PCR. The forward primer sequence was 5'- ATGGAGTTCGAGGAATCGTC-3' and the reverse primer sequence was 5'- TTATTCATCGTTCCACAGTGGC-3'. The PCR product was cloned into pMD19-T vector and sequenced. The full-length cDNA sequence with the stop codon removed was inserted into pCAMBIA1302 vector to generate MaDREB1F:GFP fusion. The pCAMBIA1302-MaDREB1F-GFP was transiently expressed in onion epidermal cells using a biolistic particle delivery system (PDS-1000, BIORAD). After 48 hours of incubation on MS medium, fluorescence was measured using a fluorescence microscope. The excitation / emission wavelength for GFP was 485 / 515 nm.

[0026] Transcriptional activation activity of MaDREB1F protein

[0027] The complete coding sequence, N-terminal and C-terminal of MaDREB1F were amplified by PCR and then cloned into pGBKT7 vector. The construct was transformed into yeast strain AH109, which was subsequently plated on SD / Trp- or SD / His- plates. Growth of yeast cells on medium containing X-gal was used to assess transcriptional activation activity.

[0028] Generation of transgenic plants

[0029] The pCAMBIA1302-MaDREB1F-GFP construct was transferred into Agrobacterium strain GV3101. Overexpression of MaDREB1F in banana plants (Musa acuminata L. AA group, cv. Mas) was performed using the Agrobacterium-mediated method reported in our previous study. Hygromycin-resistant transgenic plants were detected by amplifying the hpt-ll gene from pCAMBIA1302. Then, T2 generation was obtained. Southern blot analysis was used to further verify the integration of MaDREB1F in L1 and L2 genomes.

[0030] Southern blot analysis

[0031] Genomic DNA was digested with EcoRI enzyme and transferred onto a nylon membrane. The cDNA probe of hpt-ll in pCAMBIA1302 was amplified with a primer set (5'-GCTCCTACAAATGCCATCATTGC-3' and 5'GATAGTGGGATTGTGCGTCATCCC-3') and labeled with a random primer labeling system. The hybridization was performed according to the manufacturer's instructions (Roche 11745832910, USA).

[0032] Transcriptome analysis

[0033] RNA extraction kit (EX1882, G-CLONE, Beijing, China) was used. 3 ug of RNA from each sample was converted to cDNA by RevertAid First-Strand cDNA Synthesis Kit (Promega, Madison, WI, USA). Eighteen cDNA libraries were constructed and sequenced using Illumina GAII. After removing the adaptor sequences using the FASTX toolkit, clean reads were used for further analysis. FastQC was used to assess the quality of the sequences. The cufflinks with alignment files were used to assemble the transcriptome. FPKM was used to calculate the gene expression levels. DEGseq was used to identify differentially expressed genes based on three replicate reads per gene (fold change > 2; P value < 0.05).

[0034] Yeast one-hybrid assay (Y1H)

[0035] To characterize the interaction between MaDREB1F and target promoters, each promoter sequence was constructed into pAbAi as a bait vector. The coding sequence of MaDREB1F was constructed into pGADT7 as a prey vector. The prey and bait constructs were co-transformed into Y1H yeast strain and then cultured on SD / -Leu+AbA 200 selective medium at 30°C for 3 days.

[0036] Dual-luciferase activity assay

[0037] Dual-luciferase assay was performed according to previous methods. The promoter and coding sequence of genes were cloned into pGreenII 0800-LUC and pGreenII 62-SK vectors, respectively, and then transferred into Agrobacterium tumefaciens GV3101. The transformed A. tumefaciens was infiltrated into tobacco leaves using the injection method. The dual-luciferase reporter assay system was used to examine the activity of firefly LUC and Renilla luciferase (REN). Three biological replicates were performed for each assay.

[0038] 2Results and analysis

[0039] MaDREB1F encodes a DREB transcription factor

[0040] To investigate the function of MaDREB1F, we cloned the open reading frame (ORF) of MaDREB1F, which is 687 bp in length and encodes 229 amino acids. BLASTX analysis showed that MaDREB1F has relatively high sequence identity to EgDREB1F from Elaeis guineensis (60%), PdDREB1F from Phoenix dactylifera (60%), and AcDREB1F from Ananas comosus (58%). The deduced MaDREB1F protein has the typical sequence characteristics of a conserved AP2 domain, a nuclear localization signal (NLS), and an activation domain. Subcellular localization analysis showed that the fluorescence of 35S::GFP was distributed throughout the cell, while the fluorescence of 35S::MaDREB1F-GFP chimeras was localized in the nucleus. Transcriptional activity analysis showed that yeast cells containing pGBKT7-MaDREB1F and pGBKT7-MaDREB1F-C grew well in SD / His- medium and were blue in SD / His- medium containing X-gal, indicating that MaDREB1F protein and its C-terminal have a transcriptional activity domain. These results indicate that MaDREB1F encodes a DREB transcription factor in banana.

[0041] Generation of transgenic banana plants overexpressing MaDREB1F

[0042] To study the role of MaDREB1F in plants, MaDREB1F was introduced into pCAMBIA1302 vector. Flower tips of Musa acuminata L. AA group, cv. Mas were sliced into thin pieces and then placed on differentiation medium to induce callus growth. Two millimeter sections cut from callus were infected with Agrobacterium tumefaciens EHA105 containing binary vector. Infected callus sections were transferred to differentiation medium to induce shoot growth. Shoots were cultured to 3 cm height, after which the explants were placed in rooting medium. When roots grew to 8 cm, the plantlets were moved to coconut coir medium for 100 days. Finally, we obtained 27 hygromycin resistant lines, of which 6 transgenic lines could be verified by PCR amplification of hpt-ll gene from the binary vector. Then, T2 generation was replicated from these transgenic lines. To provide molecular evidence for these transgenic lines, we carried out detailed molecular characterization. Southern blot analysis showed that MaDREB1F transgene was integrated in multiple copies in transgenic lines L1 and L2. In addition, PCR amplification showed the presence of hpt-ll gene from the vector in L1 and L2. The expression level of MaDREB1F was 6-10 fold higher in L1 and L2 than in WT. These results indicate that MaDREB1F was successfully overexpressed in banana plants.

[0043] Identifying cold and drought response genes affected by MaDREB1F overexpression

[0044] To gain deeper insights into the transcriptional changes caused by MaDREB1F overexpression, comparative transcriptome analysis was performed on WT and overexpressing MaDREB1F plants before and after drought and cold treatment.

[0045] After cold treatment, a total of 8902 and 8811 DEGs (differentially expressed genes) were identified from transgenic lines (TL)_cold / TL and WT_cold / WT, respectively. Of these, 4812 genes were uniquely identified in TL_cold / TL; 4721 genes were found only in WT_cold / WT; 4090 genes were commonly regulated in WT_cold / WT and TL_cold / TL. Among the commonly regulated genes, the expression of 1336 genes changed more in TL_cold / TL than in WT_cold / WT (fold change > 2). A total of 6148 genes were identified as cold response genes affected by MaDREB1F overexpression.

[0046] After drought treatment, a total of 10839 and 2794 DEGs were identified from WT_drought / WT and TL_drought / TL, respectively. Among them, 8994 genes were only found in WT_drought / WT; 949 genes were uniquely identified in TL_drought / TL; 1845 genes were commonly regulated in WT_drought / WT and TL_drought / TL. Among the commonly regulated gene set, the expression changes of 868 genes in TL_drought / TL were greater than that in WT_drought / WT (fold change > 2). A total of 1817 genes were identified as drought-responsive genes affected by MaDREB1F overexpression.

[0047] We further overlapped the cold- and drought-responsive genes affected by MaDREB1F overexpression and found that 720 genes were commonly regulated by MaDREB1F overexpression after cold and drought treatment. These genes belong to 37 categories of GO enrichment analysis. Notably, these GO terms include carbohydrate transport (GO_0008643), sugar transmembrane transporter activity (GO_0051119), response to abscisic acid (GO_0009737), oxidoreductase activity (GO_0016491), oxidoreductase activity acting on C (GO_0016641), oxidation-reduction process (GO_0055114), and response to oxygen-containing compounds (GO_1901700), indicating that carbohydrate metabolism, ABA response, and oxidation-reduction-related processes are commonly affected by MaDREB1F overexpression after cold and drought treatment. These seven GO terms contain 146 genes, whose expression patterns are divided into four groups: (i) (TL_cold / TL)>(WT_cold / WT) and (TL_drought / TL)>(WT_drought / WT) contain 73 genes; (ii) (TL_cold / TL)<(WT_cold / WT) & (TL_drought / TL)<(WT_drought / WT) contain 18 genes; (iii) (TL_cold / TL)>(WT_cold / WT) & (TL_drought / TL)<(WT_drought / WT) contain 30 genes; (iv) (TL_cold / TL)<(WT_cold / WT) & (TL_drought / TL)>(WT_drought / WT) contain 25 genes. This indicates that more genes (73 genes) in these biological processes exhibit common induction, but in MaDREB1F overexpressing plants, fewer genes (18 genes) exhibit common inhibition compared to WT after cold and drought treatment.

[0048] Common regulation of jasmonic acid and ethylene biosynthesis by MaDREB1F overexpression under cold and drought conditions

[0049] Among the commonly regulated genome of MaDREB1F overexpression after cold and drought treatments, we found that the expression of six genes encoding key enzymes in jasmonic acid (JA) biosynthesis were significantly affected. Under cold treatment, phospholipase A1 (PAL1) and two lipoxygenases (LOX) (GSMUA_Achr3G07870_001 and GSMUA_Achr3G11780_001) were repressed in both WT and transgenic plants, but the degree of repression was alleviated in transgenic plants. In addition, allene oxide cyclase (AOC) and acyl-CoA oxidase (ACX) did not show significant changes in WT, but were significantly induced in transgenic plants under cold treatment. Under drought treatment, PAL1, LOX (GSMUA_Achr3G11780_001) and ACX were repressed in WT, but the degree of repression was alleviated in MaDREB1F overexpression plants. In addition, another LOX (GSMUA_Achr3G07870_001) and AOC were repressed in WT plants, but were significantly induced in transgenic plants after drought treatment. These results suggest that MaDREB1F activated or alleviated the repression of genes in the JA biosynthesis pathway under cold and drought conditions.

[0050] The transcripts of five genes encoding enzymes related to ethylene biosynthesis were commonly affected by MaDREB1F overexpression after cold and drought treatments. Ethylene biosynthesis includes three core enzymatic steps: S-adenosyl-l-methionine synthase (SAMS), ACS and ACO. Under cold treatment, SAMS was down-regulated, one ACS (GSMUA_Achr5G08880_001) did not show significant changes in WT, but they were significantly up-regulated in MaDREB1F overexpression plants. One ACS (GSMUA_Achr4G29150_001) and two ACOs were up-regulated in both WT and transgenic plants, with significantly higher induction in transgenic plants than in WT. Under drought treatment, SAMS was repressed in both WT and transgenic plants, but the degree of repression was alleviated in transgenic plants. Two ACSs and one ACO (GSMUA_AchrUn_randomG26050_001) were repressed in WT plants, but were significantly induced in transgenic plants. One ACO (GSMUA_AchrUn_randomG26050_001) was up-regulated in both WT and transgenic plants, but the degree of induction was significantly enhanced in transgenic plants. These results suggest that MaDREB1F activated or alleviated the repression of genes in the ethylene biosynthesis pathway under cold and drought conditions.

[0051] In particular, we observed that the expression of 22 ERFs was generally affected by overexpression of MaDREBlF after cold and drought treatments. Under cold treatment, 15 ERFs were induced or repressed more in transgenic plants than in WT. Under drought treatment, 21 ERFs showed more induction or less repression in transgenic plants than in WT.

[0052] Transcriptional regulation of MaAOC4, MaACO20 and MaERFl 1 by MaDREBlF

[0053] The results showed that the yeast cells containing MaDREBlF-MaAOC4Pro and MaDREBlF-MaACO20Pro survived on the selective medium, indicating the interaction between MaDREBlF and MaAOC4 promoter and between MaDREBlF and MaACO20 promoter Figure 1 、 Figure 2 ).

[0054] To evaluate the effect of MaDREBlF on MaAOC4 / MaACO20 promoters, dual luciferase assays were performed. The results showed that under normal, cold or drought conditions, tobacco leaves containing MaDREBlF and luciferase (LUC) driven by MaAOC4Pro or MaACO20Pro exhibited significantly higher LUC activity than those containing SK vector and LUC driven by MaAOC4Pro or MaACO20Pro Figures 3-6 ). This indicates that MaDREBlF has an effect on activating MaAOC4 and MaACO20 promoters.

[0055] Interestingly, ethylene response factor (MaERFl 1) is present in the commonly used regulatory genes set by MaDREBlF, and the promoter region of MaERFl 1 contains DRE / CRT cis-elements. Therefore, we used yeast one-hybrid assay to study the interaction between MaERFl 1 promoter and MaDREBlF, and found that the yeast cells containing MaDREBlF-MaERFl 1Pro survived on the selective medium, indicating the interaction between MaDREBlF and MaERFl 1 promoter

[0056] MaDREBlF-MaERFl 1Pro survived on the selective medium, indicating the interaction between MaDREBlF and MaERFl 1 promoter Figure 7 ). In addition, the yeast one-hybrid assay also showed the interaction between MaERFl 1 and MaACO20 promoter Figure 8 ). Further dual luciferase assays showed that MaDREBlF has an effect on activating MaERFl 1 promoter, and MaERFl 1 also activated MaACO20 promoter after drought treatment Figure 9 、 Figure 10 ).

[0057] The above merely provides part of the preferred embodiments of the present application, and is not intended to limit the present application. The protection scope of the present application is not limited to the above. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. MaDREB1F Genes are activated MaAOC4 promoter, MaACO20 promoters and MaERF11 The application of promoters is characterized by, The MaAOC4 The nucleotide sequence is shown in SEQ ID NO:

2. MaACO20 The nucleotide sequence is shown in SEQ ID NO:

3. MaERF11 The nucleotide sequence is shown in SEQ ID NO:4; The MaDREB1F The gene was cloned from Musa acuminata L. AAA group using the forward primer sequence 5'-ATGGAGTTCGAGGAATCGTC-3' and the reverse primer sequence 5'-TTATTCATCGTTCCACAGTGGC-3'.

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