Tobacco cold-tolerance gene NtCIP and its application

Abstract

The invention discloses a method for regulating the low temperature resistance of tobacco NtCIP Gene and its application. The gene knockout mutant of tobacco was obtained by gene editing technology. The results of low temperature tolerance phenotype identification showed that compared with wild-type tobacco, the knockout strain wilted more severely under low temperature stress and its low temperature tolerance was significantly reduced, indicating that NtCIP Positively regulates tobacco's low-temperature tolerance. The functional identification of this gene provides important genetic resources and theoretical basis for tobacco breeding for low-temperature tolerance.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a tobacco low-temperature-resistant gene NtCIP and an application thereof. Background Art

[0002] Tobacco (Nicotiana tabacum L.), originating in warm South America, is a thermophilic, cold-tolerant leaf crop. Its optimum growth temperature is 25°C to 28°C. However, tobacco is sensitive to cold stress, with temperatures as low as -2°C to -3°C resulting in its death. Cold stress is particularly acute during the seedling stage. At the six- to seven-leaf stage, exposure to temperatures as low as 12°C for approximately two weeks can cause premature flowering, reducing leaf yield and quality, and ultimately, farmers' income. In tobacco-growing areas of southern China, tobacco plants are often subject to "late spring cold snaps" after transplanting, exposing them to cold stress. Currently, timely transplanting is the primary method of combating these "late spring cold snaps." While this approach can mitigate losses caused by cold temperatures to some extent, it does not fundamentally address the problem. Further research is needed to identify and characterize cold-tolerant genes in tobacco plants and to develop more tolerant varieties.

[0003] CBF (C-repeat binding factor) is an early-discovered transcription factor involved in plant response to cold stress. CBF transcription factors belong to the DREB subfamily of the AP2 / ERF (Apetala2 / Ethylene responsive factor) transcription factor family, and therefore all CBF transcription factors possess a highly conserved AP2 / ERF domain. Currently, the most well-studied cold signaling pathway is the ICE-CBF-COR (Inducer of CBF expression-C-repeat binding factor-Cold-responsive genes) pathway. In this pathway, ICE is rapidly expressed upon cold-induced expression, promoting CBF gene expression. CBF then binds to the CRT / DRE (C-repeat / dehydration-responsive element) cis-acting element in the COR gene promoter, activating COR gene expression and thereby enhancing plant resistance to cold.

[0004] Currently, the identification of low-temperature tolerance genes in tobacco is very limited. Summary of the Invention

[0005] In the early stages of this study, a gene, NtCIP, that interacts with the key transcription factor NtCBF for low-temperature tolerance was identified through a yeast two-hybrid screening library. This gene was then functionally characterized, providing a genetic resource and theoretical basis for molecular breeding of tobacco varieties with low-temperature tolerance.

[0006] The present invention provides an application of an NtCIP protein or an NtCIP protein encoding gene in cultivating plants or plant varieties with enhanced or reduced low-temperature tolerance.

[0007] Specifically, the plant is dicotyledonous or monocotyledonous, preferably the plant is tobacco.

[0008] In a specific embodiment, the enhanced low temperature tolerance is achieved by overexpressing the NtCIP protein encoding gene in the plant.

[0009] More specifically, the overexpression is achieved by overexpressing the NtCIP protein encoding gene in a transgenic plant through genetic transformation, for example, the genetic transformation is mediated by Agrobacterium or by a biolistic method.

[0010] Specifically, the reduction of low temperature tolerance is achieved by knocking out the endogenous NtCIP protein encoding gene in the plant or weakening its expression.

[0011] Preferably, the knockout is achieved by homologous recombination or gene editing; the attenuation can be achieved by RNA interference; more preferably, the target used is ATACGGGTTTGAATCCGACCCGG or TATCCGACCCGACAGAAACCGGG.

[0012] Specifically, the amino acid sequence of the NtCIP protein is shown in SEQ ID No: 2, or a homologous NtCIP protein from tobacco with the same function. More specifically, the CDS amino acid sequence of the gene encoding the NtCIP protein is shown in SEQ ID No: 1.

[0013] In addition, the method further includes the step of identifying the low temperature tolerance of the transgenic plant (for example, by treating the plant at 4°C for 6-12 hours and observing the appearance of the plant), and optionally further planting to obtain pure varieties.

[0014] Using gene editing technology, tobacco knockout mutants of the NtCIP gene were generated. Phenotypic characterization of cold tolerance revealed that, compared to wild-type tobacco, the two knockout lines exhibited more severe wilting under low-temperature stress and significantly reduced cold tolerance, indicating that NtCIP positively regulates cold tolerance in tobacco. This suggests that overexpressing this gene in plants can also improve cold tolerance. The functional characterization of this gene provides an important genetic resource and theoretical foundation for cold tolerance breeding in tobacco. For example, plants sensitive to cold tolerance can be cultivated as control materials or breeding materials for specialized conditions, while plants with enhanced cold tolerance can be cultivated to minimize the impact of low temperatures on plant growth, thereby providing superior cold-tolerant varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the phylogenetic tree analysis diagram of NtCIP (XP_016484498.1).

[0016] Figure 2 This is a homology comparison analysis diagram of the NtCIP protein sequence.

[0017] Figure 3 The peak map near the cip-a2 target site (left) and the DNA sequence alignment map (right).

[0018] Figure 4 The peak map near the cip-a3 target site (left) and the DNA sequence alignment map (right).

[0019] Figure 5 This is a sequence alignment of NtCIP proteins in knockout strains cip-a2 (top) and cip-a3 (bottom) and WT.

[0020] Figure 6 The phenotypes of the wild type and cip mutant strains after being treated with low temperature (4°C) for 8 hours. DETAILED DESCRIPTION

[0021] 1 Materials and Methods

[0022] 1.1 Gene sequence acquisition and feature analysis

[0023] Primers were designed using the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov) NtCIP mRNA sequence (accession number: XM_016629012.1):

[0024] NtCIP-CDS-F:TCTCTCCTCATCCCTTCACC

[0025] NtCIP-CDS-R:AGATGAGCGAATTGGGGCAA

[0026] PCR product size: 749 bp.

[0027] The gene was amplified from the cDNA of the leaves of the flue-cured tobacco variety Xiangyan No. 7. The PCR reaction system and procedure are as follows (Table 1):

[0028] Table 1 PCR reaction system and procedure

[0029]

[0030] The obtained PCR products were sequenced to determine the CDS sequence and protein sequence of the gene.

[0031] According to the NtCIP protein sequence (accession number: XP_016484498.1), MEGA7.0 software was used to analyze gene homology and the neighbor-joining method was used to construct a phylogenetic tree. DNA, CDS and protein sequences were aligned using DNAMAN software.

[0032] 1.2 Functional identification of NtCIP in tobacco cold tolerance

[0033] 1.2.1 Construction of gene editing vector

[0034] (1) Target design

[0035] Using the online analysis tool http: / / crispor.gi.ucsc.edu / , we designed target sites within gene exons. The following two specific targets were identified: Target1, ATACGGGTTTGAATCCGACCCGG, and Target2, TATCCGACCCGACAGAAACCGGG. The first base and the final NGG residue were removed from both target sites, leaving only 19 nt. The 19-nt target sequence of Target1 (in italics) was inserted into primers DT1-F0 and DT1-BsF. The inverted complementary sequence of the 19-nt target of Target2 (in italics) was inserted into primers DT2-R0 and DT2-BsR.

[0036] Then, primers for constructing the CRSIPR vector were designed, and their sequence information is as follows:

[0037] DT1-BSF:ATATATGGTCTCGATTGTACGGGTTTGAATCCGACCGTT

[0038] DT1-F0:TGTACGGGTTTGAATCCGACCGTTTTAGAGCTAGAAATAGC

[0039] DT2-R0:ACGGTTTCTGTCGGGTCGGATCAATCTCTTAGTCGACTCTAC

[0040] DT2-BSR:ATTATTGGTCTCGAAACGGTTTCTGTCGGGTCGGATCAA

[0041] (2) PCR amplification

[0042] Four-primer PCR amplification was performed using the pCBC-DT1T2 vector (chloramphenicol resistance) as a template. DT1-BsF / DT2-BsR is the normal primer concentration (100 μM); DT1-F0 / primer DT2-R0 is the diluted concentration (10 μM). 4 μL of each of the four primers was taken and added to 4 μL of sterile water to prepare a 20 μL primer stock solution. High-fidelity enzyme For Max DNA Polymerase amplification, add 2 μL of primer master mix to 50 μL of the system. The PCR product size is 626 bp. PCR amplification was performed using the following protocol: denaturation at 98°C for 10 s, annealing at 55°C for 5 s, and extension at 72°C for 20 s for 30 cycles.

[0043] The target fragment (626 bp) was cut out under ultraviolet light by 1.5% agarose gel electrophoresis at 5 v / cm for 20 min, and the PCR product was recovered according to the operating instructions of a common agarose gel DNA recovery kit.

[0044] (3) Ligation of knockout vector

[0045] The connection system and reaction conditions are as follows (Table 2):

[0046] Table 2 Connection system

[0047]

[0048] (4) Transformation and identification

[0049] 10 μL of the ligation product was transformed into competent E. coli (following the competent E. coli transformation method in the kit).

[0050] Transform the cells onto kanamycin-resistant plates, incubate at 37°C for 12 h, and perform colony PCR using primers U626-IDF and U629-IDR. Sequencing was performed using primers U626-IDF and U629-IDF.

[0051] Pick 10 single colonies for PCR identification. The colony PCR and sequencing primers are as follows:

[0052] U626-IDF:TGTCCCAGGATTAGAATGATTAGGC

[0053] U629-IDF:TTAATCCAAACTACTGCAGCCTGAC

[0054] U629-IDR:AGCCCTCTTTCTTTCGATCCATCAAC

[0055] Size: 726bp.

[0056] The PCR reaction system and procedure are as follows (Table 3):

[0057] Table 3 Colony PCR reaction system and procedure

[0058]

[0059] Take 100 μL of the bacterial solution corresponding to the three positive bands of about 726 bp for sequencing, and inoculate the remaining 400 μL of bacterial solution into 10 ml of LB containing kanamycin resistance. Shake the tube and wait for the sequencing results to come out. Extract the plasmid from the bacterial solution with correct sequencing and store it in a -20℃ refrigerator.

[0060] 1.2.2 Tobacco genetic transformation

[0061] (1) Preparation of Agrobacterium

[0062] Add 1 μL of plasmid to 50 μL of GV3101 competent Agrobacterium cells (following the Agrobacterium transformation protocol in the kit). Transform into kanamycin-resistant plates and incubate at 28°C for 48 hours before colony PCR analysis. The PCR reaction system and procedure are shown in Table 3. PCR products were detected by gel electrophoresis. If the bacterial sample and positive control show clear, correctly sized electrophoretic bands, and the negative control shows no bands, the bacterial sample is suitable for tobacco transformation.

[0063] (2) Tobacco genetic transformation

[0064] Tobacco seeds (Xiangyan No. 7) were sterilized in 75% alcohol for 30 seconds, rinsed with sterile water for 1 minute, and then disinfected with 84 disinfectant for 3-5 minutes. Three rinses were performed with sterile water, each for 1 minute. The sterilized tobacco seeds were sown on germination medium at 23°C with a photoperiod of 16 hours light / 8 hours dark. The seeds were incubated for 4-5 weeks. Sterile tobacco leaves were cut into small pieces with a scalpel and inoculated onto the pre-incubation medium. Agrobacterium was selected and placed in the inoculation solution. An Agrobacterium suspension was prepared to an OD600 of 0.2. Tobacco leaves pre-incubated for 2-3 days were inoculated with the Agrobacterium suspension for 10-15 minutes. The inoculated leaves were then inoculated onto filter paper, the liquid was aspirated, and the leaves were inoculated onto co-cultivation medium for 48-72 hours. The leaves were then transferred to induction medium for callus induction, and callus tissue was grown for approximately 10 days. Select calli that meet the criteria and inoculate them onto a screening medium for the appropriate resistance. Incubate at 23°C with a photoperiod of 16 hours light / 8 hours dark for 15-30 days. Inoculate actively growing positive calli onto differentiation medium, 4-5 calli per dish. Incubate at the same temperature, photoperiod, and duration as for the screening medium. If seedlings form from the calli during differentiation, inoculate them onto seedling-strengthening medium for growth.

[0065] 1.2.3 Sequencing analysis of positive seedlings

[0066] Use Nanjing Novozymes Biotech Co., Ltd. Genomic DNA was extracted from leaves of positive seedlings using the Plant DNA Isolation MiniKit (refer to the manufacturer's instructions for detailed methods). PCR amplification of knockout lines was performed using the high-fidelity enzyme PrimeSTAR Max DNA Polymerase.

[0067] A pair of primers was designed to perform PCR detection on target sites Target1 and Target2 simultaneously.

[0068] The primer sequence information is as follows:

[0069] NtCIP-F1:GAGAAGAAGAATCCCAAGAAATACAAG

[0070] NtCIP-R1: CAAACGGAAAATCACCACCATAT

[0071] The target fragment length is approximately 384 bp.

[0072] The PCR amplification system and procedure are shown in Table 4.

[0073] Table 4 Target site detection PCR reaction system and procedure

[0074]

[0075] The resulting PCR products were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. Sequencing peak plots and sequence alignment were analyzed using SnapGeneViewer and DNAMAN, respectively.

[0076] 1.2.4 Identification of low temperature tolerance of knockout strains

[0077] Seeds from transgenic pure lines and wild-type plants with uniformly plump and large grains were selected, sterilized, and placed in a 4°C low-temperature treatment for 48 hours. They were then sown in 9-cm-diameter pots and covered with transparent plastic lids to maintain heat and moisture. When the seeds reached four leaves and a single heart, the lids were removed. The incubation temperature was set at 23°C, and the photoperiod was set at 16 hours of light and 8 hours of darkness. Thirty days after sowing (at the six-leaf, one-heart stage), seedlings of uniform growth were selected and placed in a 4°C incubator for low-temperature treatment. The phenotypes were observed and photographed.

[0078] 2 Results Analysis

[0079] 2.1 Analysis of NtCIP Sequence Characteristics

[0080] The CDS sequence and protein sequence of NtCIP were obtained by PCR amplification and sequencing.

[0081] (1) NtCIP CDS sequence (SEQ ID NO: 1):

[0082] ATGGCTGAGTCGACTCAACCAATTAAACGTTCAAGAGAAGAAGAATCCCAAGAAATACAAGAAAATTTATGTGAAGGTAA

[0083] TAATACAAAGCGCCATAAATCATCTTATAATGATATCCTCTCCATTCTTGAAGAGGAAGAATACGGGTTTGAATCCGACC

[0084] CGGTTTCTGGGTCCGACCTATTCGCCACCCTTCAACAAGAACTTCTCCCAAATGACGGGTTTGAGGCCGACCCGGTTTCT

[0085] GTCGGGTCGGATATTTCTTCTAAAGAAGATGAAGTAGAAGATGACAGAAGTAGTGTTATTAGACACCTTCTAGAAGCTTC

[0086] TGATGATGAACTAGGTATTCCCAGTGGAGATGGAACCAACGGTGTAGATTTAACGTTTGCTGAAGAAAATGAAACATATG

[0087] GTGGTGATTTTCCGTTTGCTATATCTGATGGTTTATTATGGGAGTTTGAAGATGAAGCTGCTAACTATTATTCTTTGTTA

[0088] CAGTCCGAACTTTTTATGTAG

[0089] (2) NtCIP protein sequence (SEQ ID NO: 2):

[0090] MAESTQPIKRSREEESQEIQENLCEGNNTKRHKSSYNDILSILEEEEYGFESDPVSGSDLFATLQQELLPNDGFEADPVS

[0091] VGSDISSKEDEVEDDRSSVIRHLLEASDDELGIPSGDGTNGVDLTFAEENETYGGDFPFAISDGLLWEFEDEAANYYSLL

[0092] QSELFM

[0093] Phylogenetic tree analysis revealed that NtCIP was most closely related to NsCIP (XP_009778551.1) of Nicotiana tabacum. Figure 1 Protein sequence comparison analysis found that the NtCIP protein sequence has a high homology with the CIP gene of Solanaceae crops ( Figure 2 ).

[0094] 2.2 Screening of cip homozygous knockout strains

[0095] By sequencing and analyzing the target sites of transgenic positive seedlings, two successfully edited lines, cip-a2 and cip-a3, were screened. The mutation site of cip-a2 line showed a clear single peak ( Figure 3 The left figure in the middle shows that the strain is a homozygous mutation. Sequence comparison revealed that a base A was inserted near the first target site and two bases TT were deleted near the second target site ( Figure 3 The mutation site of cip-a3 strain showed a clear single peak ( Figure 4 The left figure in the middle shows that the strains are all homozygous mutations. Sequence comparison revealed a 41-base deletion near the second target site ( Figure 4 Protein sequence comparison revealed that NtCIP protein translation terminated prematurely in cip-a2 and cip-a3 strains ( Figure 5 ), indicating that the NtCIP gene function was disrupted in these two knockout lines.

[0096] 2.3 Functional identification of the NtCIP gene in tobacco low temperature tolerance

[0097] like Figure 6 As shown, under normal temperature conditions, the NtCIP knockout lines cip-a2 and cip-a3 showed no significant phenotypic differences from Xiangyan 7 (WT). After 8 hours of low-temperature treatment at 4°C, the two knockout lines cip-a2 and cip-a3 experienced whole-plant wilting, while the WT leaves only showed slight drooping. These results indicate that the chilling tolerance of the cip-a2 and cip-a3 lines is significantly reduced. These results suggest that NtCIP positively regulates chilling tolerance in tobacco.

Claims

1. A use of an NtCIP protein or a gene encoding an NtCIP protein in cultivating plants or plant varieties with reduced low-temperature tolerance; the reduced low-temperature tolerance is achieved by knocking out the endogenous NtCIP protein encoding gene in the plant or weakening its expression; the plant is tobacco; The amino acid sequence of the NtCIP protein is shown in SEQ ID No:

2.

2. The use according to claim 1, characterized in that The knockout is achieved by homologous gene recombination or gene editing; the attenuation is achieved by RNA interference.

3. The use according to claim 2, characterized in that The targets used in the knockout were ATACGGGTTTGAATCCGACCCGG or TATCCGACCCGACAGAAACCGGG.

4. The use according to claim 1, wherein The CDS sequence of the NtCIP protein encoding gene is shown in SEQ ID No:

1.

5. The use according to any one of claims 1 to 4, characterized in that The step of identifying the low temperature tolerance of the resulting plant or plant variety is also included.

6. The use according to claim 5, characterized in that The low temperature tolerance of the plant or plant variety is identified by treating the plant at 4°C for 6 to 12 hours and observing the appearance of the plant.

7. The use according to claim 6, characterized in that It also includes the step of further cultivation to obtain purebreds.

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