CN113817038B

CN113817038B - Protein VaVPAC derived from small beans and application of encoding gene thereof in aspect of enhancing drought resistance of tobacco

Info

Publication number: CN113817038B
Application number: CN202111273342.7A
Authority: CN
Inventors: 沙爱华; 陈银华; 易勇; 向艳涛; 魏正欣; 蒋浩中; 黄林涛
Original assignee: Hainan University
Current assignee: Hainan University
Priority date: 2021-10-29
Filing date: 2021-10-29
Publication date: 2023-09-19
Anticipated expiration: 2041-10-29
Also published as: CN113817038A

Abstract

The invention provides a protein VaVPAC derived from small beans and application of a coding gene thereof in enhancing drought resistance of tobacco. The amino acid sequence of the protein VaVPAC is shown as SEQ ID NO.1, and comprises 376 amino acids. The gene sequence of the coded protein is shown as SEQ ID NO.2, and the nucleotide length is 1131bp. Experiments prove that the VaVPAC protein and the coding gene thereof can obviously improve the drought resistance of tobacco.

Description

Protein VaVPAC derived from small beans and application of encoding gene thereof in aspect of enhancing drought resistance of tobacco

Technical Field

The invention relates to the technical field of plant bioengineering, in particular to a protein VaVPAC derived from small beans and application of a coding gene thereof in enhancing drought resistance of tobacco.

Background

With the continuous aggravation of global climate warming in recent years, drought stress has become one of main abiotic stress causing crop yield reduction, and grain loss caused by drought in China accounts for more than 50% of all natural disasters. Thus, solving the drought problem is an important challenge in achieving sustainable development of agriculture.

The traditional crop drought-resistant breeding has long period, large investment and is limited by factors such as narrow drought-resistant germplasm resources, and the like, so that the current drought-resistant breeding has slow progress. The biotechnological breeding can break the inter-species restriction and provide a new way for efficient drought-resistant breeding, so that the identification of drought-resistant gene resources is a key for obtaining new varieties of drought-resistant transgenic crops. After the drought-resistant germplasm resources of the small beans are identified, the extremely drought-resistant germplasm resources of the small beans are found, the extremely drought-resistant and extremely sensitive germplasm resources of the small beans are used as materials, the gene expression difference between the drought-resistant germplasm resources and the sensitive germplasm resources under the drought stress condition is analyzed by adopting a transcriptome sequencing method, the drought-resistant gene VaVPAC (document: characterization of Drought-Responsive Transcriptome During Seed Germination in Adzuki Bean (Vigna angular L.) by PacBio SMRT and Illumina Sequencing, front. Genet,2020, 11:996.) is identified, and the VaVPAC can be proved to improve the drought resistance of plants through the overexpression of virus expression vectors in tobacco, and can be used as a drought-resistant gene resource for drought-resistant breeding of plants.

Disclosure of Invention

Aiming at the defects existing in the prior art, the invention provides the protein VaVPAC from small beans and the application of the coding gene thereof in enhancing the drought resistance of tobacco.

The scheme of the invention specifically comprises the following aspects:

in one aspect, there is provided the use of the protein VaVPAC for enhancing drought resistance in tobacco. The amino acid sequence of the protein is shown as SEQ ID NO. 1. The gene sequence of the encoding protein is shown as SEQ ID NO. 1.

On the other hand, the invention also provides a method for identifying the drought-resistant function of the gene, which comprises the following steps: the target gene is introduced into PVX-LIC vector, after tobacco leaf is injected, the target gene is over-expressed by PVX virus replication, drought is caused by a complete water control mode, and whether the plant survives under drought condition is observed, so that drought resistance of the target gene is identified. Wherein the introduction is by inserting the target gene between LIC1 and LIC2 sites of PVX-LIC vector.

The invention also provides a preparation method of the protein VaVPAC coding gene, which comprises the following steps:

extracting total RNA by using small bean seeds subjected to mannitol stress treatment with the mass concentration of 9.0% for 36 hours as a material, carrying out reverse transcription to obtain cDNA, carrying out PCR amplification by using the cDNA as a template under the guidance of a primer VaVPAC-F1 and a primer VaVPAC-R1, recovering a PCR product, and purifying to obtain a DNA fragment.

The invention has the following effects:

experiments prove that the tobacco obtained by injecting the recombinant expression vector PVX-LIC-VaVPAC containing the protein VaVPAC coding gene into tobacco for transient over-expression has drought resistance obviously stronger than that of the tobacco expressed by wild and empty vector under drought conditions. The invention has important significance in enhancing drought resistance of plants by utilizing VaVPAC protein.

Drawings

Fig. 1: amplification results of the nucleotide sequence encoded by the cDNA of the VaVPAC gene. Wherein M is D2000Plus Marker, and the sizes of the bands from top to bottom are 5000, 3000, 2000, 1000, 750, 500, 250 and 100bp in sequence.

Fig. 2: agrobacteria PCR identification of the introduced recombinant plasmid PVX-LIC-VaVPAC plasmid. 1-7 are monoclonal numbers, H ₂ O is a blank. "M" is Marker.

Fig. 3: RT-PCR detects VaVPAC expression in tobacco plants. M: DL2000 Plus marker; lanes 1-5 are, respectively, non-injected normal growth tobacco, non-injected drought treated tobacco, transformed PVX-LIC empty vector tobacco, transformed PVX-LIC- -VaVPAC plasmid tobacco.

Fig. 4: overexpression of VaVPAC tobacco phenotype under drought stress. The upper panels are from left to right, respectively, tobacco not injected, PVX-LIC empty vector transformed tobacco, PVX-LIC-VaVPAC plasmid transformed tobacco before drought treatment (0 d). The lower graph shows, from left to right, normal tobacco growth without injection 15d, tobacco drought treatment transformed with PVX-LIC empty vector 15d, tobacco drought treatment transformed with PVX-LIC-VaVPAC plasmid 15d, respectively.

Detailed Description

In order to better understand the technical content of the present invention, the following provides specific examples to further illustrate the present invention.

Example 1 Small Bean protein VaVPAC, its coding Gene and recombinant expression vector acquisition

Extracting total RNA by using bean seeds treated for 36h by 9.0% mannitol as a material, carrying out reverse transcription to obtain cDNA, carrying out amplification by a conventional PCR method under the guidance of a primer VaVPAC-F1 and a primer VaVPAC-R1 by using the cDNA as a template, detecting a PCR amplification product by 1% agarose gel electrophoresis after the reaction is finished, and recovering and purifying a DNA fragment of about 1131bp (figure 1); the gene fragment was ligated to the vector PVX-LIC using LIC reaction (the T-DNA fragment of the vector PVX-LIC contained the lethal gene ccdB and on both sides thereof the recognition sequences of LIC reaction the vector was the same as that of the laboratory, literature: zhao J, liu Q, hu P, et al (2016) An efficient Potato virus X-based microRNA silencing in Nicotiana benthamiana. Sci Rep 6:20573), and recombinant vector PVX-LIC-VaVPAC was obtained, which was confirmed by sequencing to be a 1131bp DNA fragment shown in SEQ ID NO.2 inserted between LIC1 and LIC2 sites of the vector PVX-LIC (FIG. 2). The gene shown in SEQ ID No.2 was designated VaVPAC and encodes a protein consisting of 376 amino acids shown in SEQ ID No. 1.

The sequences of the above primers are as follows:

VaVPAC-F1 5’-CGACGACAAGACCCTATGGCGAGTAGGTACTGGGCAG-3’VaVPAC-R1 5’-GA GGAGAAGAGCCCTCAAGCAAGATTGATAGTGA-3’

example 2 acquisition of recombinant Agrobacterium tumefaciens

The recombinant vector PVX-LIC-VaVPAC obtained in example 1 was subjected to freeze thawing to transform Agrobacterium tumefaciens GV3101 (literature: amanda M Davis, anthony Hall, andrew J Miller, chiarina Darrah and Seth J Davis, protocol: streaming sub-protocols for floral-dip transformation and selection of transformants in Arabidopsis thaliana,2009,5:310.1186/1746-4811-5-3; public available from Changjiang university), and Agrobacterium tumefaciens GV3101 containing the recombinant vector PVX-LIC-VaVPAC was obtained and designated as GV3101/PVX-LIC-VaVPAC;

the agrobacterium tumefaciens GV3101 containing the empty vector PVX-LIC is obtained by transforming the empty vector PVX-LIC into the agrobacterium tumefaciens GV3101 through a freeze thawing method, and the recombinant agrobacterium is named GV3101/PVX-LIC.

Example 3 obtaining and identification of transiently expressed transgenic tobacco

1. Acquisition of transgenic tobacco

An Agrobacterium suspension was prepared using the two recombinant agrobacteria GV3101/PVX-LIC-VaVPAC and GV3101/PVX-LIC obtained in example 2, and the volume ratio of the culture solution to the cells in the suspension was 1:1. The smoke-producing seeds are sowed in a culture medium (turf: vermiculite: perlite is mixed in a volume ratio of 1:3:0.5) and cultured in an artificial greenhouse. When tobacco grows to 4-5 leaves, injection of the topmost fully expanded new leaf begins. 1mL of the bacteria suspension is respectively sucked by a disposable injector, the needle of the injector is removed, the lower part of the leaf is propped against by fingers, the bacteria suspension in the injector is lightly forced to be pumped and permeated into leaf tissues, 2 leaves are injected into each piece of tobacco, and 5 plants are respectively injected into GV3101/PVX-LIC-VaVPAC and GV3101/PVX-LIC. The injected tobacco plants are covered with a plastic film and cultivated for 24 hours in a dark place and then transferred to a greenhouse for cultivation under the photoperiod of 25 ℃ and 16 hours of illumination/8 hours of darkness. Tobacco without agrobacterium was used as wild type control and grown under the same growth conditions.

2. Molecular detection of transgenic tobacco

Taking the positive transgenic plant of VaVPAC obtained in the step one, transferring the empty carrier plant and the wild plant, respectively extracting total RNA, carrying out reverse transcription to obtain cDNA, taking the cDNA as a template, carrying out RT-PCR amplification by using a specific primer VaVPAC-F25' -GTCCACAACTCCGCATCTCAAC-3' and a downstream primer VaVPAC-R2 5' -GCTTCATCCCAAACAAACCTAG-3, and taking tobacco actin as an internal reference and using a primer FC 5'-CCCTCCCACATGCTATTCT-3', RC5'-AGAGCCTCCAATCCAGACA-3'. The results are shown in FIG. 3. The result shows that the target gene VaVPAC is not expressed in the trans-empty vector plant and the wild plant; the expression of the target gene VaVPAC in the transgenic VaVPAC plant shows that the transgenic tobacco strain with the transient expression of VaVPAC is obtained.

3. Drought-resistant phenotype identification of transgenic tobacco

Taking the tobacco strain of the transgenic VaVPAC, the tobacco strain of the empty vector and the wild strain obtained in the step one, carrying out drought stress treatment after injection for 7d, and observing that the tobacco with the injection of the PVX-LIC-VaVPAC4 gene has good drought resistance when the wild strain and the empty vector control plant are not injected seriously wilted when the soil water content is reduced to 7.16% for 15d (see figure 4). Therefore, the VaVPAC gene can obviously improve the drought resistance of tobacco, and the gene can be used for drought resistance breeding of plants or crops.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.

Sequence listing

<110> university of Hainan

<120> protein VaVPAC derived from small bean and application of coding gene thereof in enhancing drought resistance of tobacco

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<170> SIPOSequenceListing 1.0

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<212> PRT

<213> Vigna angularis L.

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Met Ala Ser Arg Tyr Trp Ala Val Ser Leu Pro Val His Asn Ser Ala

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Ser Gln Leu Trp Asn Gln Ile Gln Glu Arg Ile Ser Lys His Ser Phe

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Asp Thr Pro Leu Tyr Arg Phe Asn Ile Pro Asn Leu Arg Val Gly Thr

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Leu Asp Ser Leu Leu Ser Leu Ser Asp Asp Leu Ala Lys Ser Asn Asn

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Glu Arg Val Ser Gly Val Asp Ser Gly Gly Leu Thr Val Asp Gly Val

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Pro Thr Met Ser Pro Leu Lys Glu Ile Val Asp Gly Ile His Ser Gln

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Val Ala Lys Ile Glu Asp Asp Leu Lys Val Arg Val Ser Glu Tyr Asn

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Asn Ile Arg Ser Gln Leu Asn Ala Ile Asn Arg Lys Gln Thr Gly Ser

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Leu Ala Val Arg Asp Leu Ser Asn Leu Val Lys Pro Glu Asp Ile Ile

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Gln Lys Asp Trp Leu Ser Ser Tyr Glu Thr Leu Thr Asn Tyr Val Val

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Arg Glu Lys Val Phe Gln Ile Arg Asp Phe Glu Tyr Ser Gln Glu Thr

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His Glu Asn Arg Lys Gln Glu Leu Asp Lys Leu Val Gln Asp Gln Glu

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Val Phe Ser Ser Trp Met His Phe Cys Ala Val Arg Leu Phe Ala Glu

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aaccaaattc aagaacgaat ctccaaacat tccttcgaca ctcctctcta ccgattcaat 120

atccctaacc tccgcgttgg aaccctagat tctctgctct ctctcagtga tgacctcgcc 180

aagtcgaaca atttcgtgga aggagtgacg cacaagatca ggcgccagat tgaggagctc 240

gagagagttt ctggtgtgga cagtggcggc ctcaccgttg acggtgtccc cgtggattcc 300

tatttgacta ggtttgtttg ggatgaagcc aagtacccca ccatgtcgcc tttgaaggag 360

atcgtggatg gtattcacag tcaagtggca aagattgagg atgatctcaa ggttcgtgtt 420

tctgagtata acaatattcg tagtcagctt aatgctatca accgaaagca aactggaagc 480

ttagcagtcc gtgatctttc caacttggtt aagccagagg acattataac ttcagaaaat 540

ttaactaccc tccttgcaat tgtttccaag tattcacaga aggactggct ttcaagctac 600

gaaacattga caaattatgt ggtccccagg tcttctaaga agttgtacga ggacaacgaa 660

tatgctctgt atactgtaac actctttagt cgagttgcag ataattttag aactagtgca 720

cgggaaaaag tgttccaaat tcgtgacttt gaatacagtc aagaaacaca cgagaaccga 780

aagcaagagt tagataaatt ggtacaagat caggaaagac tgaaggcttc tctgttgcaa 840

tggtgcttta ccagttatgg agaggttttc agttcctgga tgcacttttg tgctgtgcgt 900

ttatttgccg agagcattct gagatatggt ctgccaccat ctttcttggc atgcgttttg 960

gctccgtctg tgaaatctga gaagaaagtg cgctctatcc ttgaaacgtt gaatgatagt 1020

acaaacagtg catactggaa gattgaggat gacgtaggta ctgggatggt tggccttgca 1080

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gaggagaaga gccctcaagc aagattgata gtga 34

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gtccacaact ccgcatctca ac 22

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gcttcatccc aaacaaacct ag 22

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agagcctcca atccagaca 19

Claims

1. The application of the protein VaVPAC in enhancing the drought resistance of tobacco is characterized in that the amino acid sequence of the protein VaVPAC is shown as SEQ ID NO. 1.

2. The use of the protein VaVPAC according to claim 1 for enhancing drought resistance of tobacco, wherein the preparation method of the protein VaVPAC comprises the following steps:

extracting total RNA (ribonucleic acid) by taking bean seeds subjected to 36h of mannitol stress treatment with the mass concentration of 9.0% as a material, carrying out reverse transcription to obtain cDNA (complementary deoxyribonucleic acid), carrying out PCR (polymerase chain reaction) amplification by taking the cDNA as a template under the guidance of a primer VaVPAC-F1 and a primer VaVPAC-R1, recovering a PCR product and purifying to obtain a DNA fragment;

the sequence of the primer VaVPAC-F1 is as follows:

5’-CGACGACAAGACCCTATGGCGAGTAGGTACTGGGCAG-3’；

the sequence of the primer VaVPAC-R1 is as follows:

5’-GAGGAGAAGAGCCCTCAAGCAAGATTGATAGTGA-3’。