CN117417946A

CN117417946A - Gene NtBRL3 for improving drought resistance of tobacco, protein and application of gene in tobacco

Info

Publication number: CN117417946A
Application number: CN202311556712.7A
Authority: CN
Inventors: 杨竞; 刘洋; 仆田秀南; 王麟麒; 万克; 罗徐; 石远帅
Original assignee: China Tobacco Corp Guizhou Provincial Co
Current assignee: China Tobacco Corp Guizhou Provincial Co
Priority date: 2023-11-21
Filing date: 2023-11-21
Publication date: 2024-01-19

Abstract

The invention discloses a gene NtBRL3 for improving drought resistance of tobacco, protein and application thereof in tobacco. The nucleotide sequence of the gene NtBRL3 for improving the drought resistance of the tobacco is shown as SEQ ID NO. 1. The gene NtBRL3 for improving the drought resistance of the tobacco can be combined with brassinosteroids and used for starting transmission of brassinosteroids signals, activating expression of a gene downstream of the brassinosteroids signals and further improving the drought resistance of the tobacco. Through verification, the overexpression of the NtBRL3 gene of the tobacco has a positive correlation with the drought resistance of the tobacco. The genetic transformation technology is adopted to over-express the NtBRL3 gene, and the drought resistance of the tobacco is obviously improved under drought stress conditions.

Description

Gene NtBRL3 for improving drought resistance of tobacco, protein and application of gene in tobacco

Technical Field

The invention relates to the technical field of biology, in particular to a gene NtBRL3 for improving drought resistance of tobacco, a protein and application thereof in tobacco.

Background

Tobacco (Nicotiana tabacum l.) is a good commercial crop and plays a very important role in national economy. The quality of tobacco determines the sales amount to a certain extent, so that the quality of tobacco is imperative to be improved.

Drought becomes one of the most prominent natural disasters worldwide, and drought stress can adversely affect tobacco yield and quality. Especially in recent years, due to global warming, water resources in various places are lacking from the past years and drought occurs. Drought has serious influence on the growth and development of tobacco, and causes the reduction of different degrees of production quality, thus becoming an important influencing factor in tobacco production.

Disclosure of Invention

The invention mainly aims to provide a gene for drought resistance of tobacco and application of the gene in tobacco so as to reduce the technical problem of influence of drought on growth and development of tobacco.

In order to achieve the aim, the application provides a gene NtBRL3 for improving the drought resistance of tobacco, and the nucleotide sequence of the gene NtBRL3 for improving the drought resistance of the tobacco is shown as SEQ ID NO. 1.

The application provides a protein for improving drought resistance of tobacco, which is formed by encoding a gene NtBRL3 as set forth in claim 1, and the amino acid sequence of the protein is shown as SEQ ID NO. 2.

The application also provides application of the gene NtBRL3 for improving the drought resistance of the tobacco in the tobacco.

According to an embodiment of the present application, the method comprises the steps of:

the recombinant vector was obtained by ligating the vector pSH737 and the gene NtBRL3 by double digestion with XbaI and KpnI.

And (3) introducing the recombinant vector into competent cells of the agrobacterium tumefaciens to obtain the recombinant agrobacterium tumefaciens.

And (3) transforming the aseptic seedling leaves of the tobacco through the recombinant agrobacterium tumefaciens medium guide vane disk to obtain the resistant tobacco.

According to an embodiment of the present application, the vector pSH737 comprises a promoter, a resistance screening gene, a reporter gene, the promoter being 35S, the resistance screening gene being NPTII, the reporter gene being GUS.

According to an embodiment of the present application, the step of transforming the sterile seedling leaf of tobacco through the recombinant agrobacterium tumefaciens mediated leaf disc comprises:

culturing the recombinant agrobacterium tumefaciens until the OD600 of the bacterial liquid reaches 0.5, centrifuging the bacterial liquid, and re-suspending to obtain a re-suspension.

Cutting tobacco aseptic seedling leaves into leaf blocks, immersing the leaf blocks in the heavy suspension for 8min, and culturing the immersed leaf blocks into the resistant tobacco.

According to an embodiment of the present application, the step of culturing the submerged leaf mass into the resistant tobacco comprises:

and (3) after the leaf blocks are dried by the bacterial liquid, inoculating the leaf blocks into a symbiotic culture medium, culturing the leaf blocks in a screening culture medium after dark culture for 2 days, culturing the leaf blocks for 2 weeks for a secondary time, cutting out the leaf blocks and transferring the leaf blocks into a rooting culture medium when the resistant buds grow to about 2cm, and obtaining the resistant tobacco.

According to an embodiment of the present application, the symbiotic medium comprises: 4.432g/L MS, 1.00 mg/L6-BA, 0.1mg/L NAA, 30g/L sucrose and 7.5g/L agar powder.

According to an embodiment of the present application, the screening medium comprises: 4.432g/L MS, 1.00 mg/L6-BA, 0.1mg/L NAA, 30g/L sucrose, 7.5g/L agar powder, 100mg/L timentin and 100mg/L kanamycin.

According to an embodiment of the present application, the rooting medium comprises: 2.216g/L MS, 20g/L sucrose, 7.5g/L agar powder, 100mg/L telangiectasia, and 100mg/L kanamycin.

The beneficial effects of the invention at least comprise:

the gene NtBRL3 for improving the drought resistance of the tobacco can be combined with brassinosteroids and starts the transmission of brassinosteroids signals, and activates the expression of a gene at the downstream of the brassinosteroids signals, so that the drought resistance of the tobacco is improved. Through verification, the overexpression of the NtBRL3 gene of the tobacco has a positive correlation with the drought resistance of the tobacco. The genetic transformation technology is adopted to over-express the NtBRL3 gene, and the drought resistance of the tobacco is obviously improved under drought stress conditions. The NtBRL3 can promote drought resistance of tobacco and application of candidate genes related to drought obtained through transcriptome analysis, is used as important candidate genes for developing genetic engineering and improving breeding of tobacco, and provides a technical reserve for future research on the action mechanism of BRL3 genes and application of improving drought resistance quality of tobacco.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to the structures shown in these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a genetic transformation of tobacco; wherein, (A) is symbiotic culture of tobacco infected by agrobacterium tumefaciens; (B) growing callus for transfer to a screening medium; (C) cutting off about 2cm long resistant buds to induce rooting; (D) PCR was performed on tobacco used in drought experiments; (E) Photographs of transgenic tobacco before transplanting (transgenic upper part, wild lower part); (F) tobacco transplantation and survival;

FIG. 2 is a phenotypic identification of transgenic tobacco and wild type tobacco under drought treatment; (a) a pre-drought stress phenotype; (B) post drought treatment phenotypic conditions; ntBRL3ox: transgenic tobacco; WT: wild type;

FIG. 3 shows the results of the determination of the GST, POD, CAT, SOD, MDA content, the proline content and the soluble sugar content of transgenic tobacco and wild-type tobacco after drought treatment;

FIG. 4 is a graph showing the relative expression levels of candidate genes in wild type and transgenic.

The achievement of the object, functional features and advantages of the present invention will be further described with reference to the drawings in connection with the embodiments.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, are intended to fall within the scope of the present invention.

Moreover, the technical solutions of the embodiments of the present invention may be combined with each other, but it is necessary to be based on the fact that those skilled in the art can implement the embodiments, and when the technical solutions are contradictory or cannot be implemented, it should be considered that the combination of the technical solutions does not exist, and is not within the scope of protection claimed by the present invention.

The application provides a gene NtBRL3 for improving drought resistance of tobacco, wherein the nucleotide sequence of the gene NtBRL3 for improving drought resistance of the tobacco is shown as SEQ ID NO. 1.

Obtaining the target gene: sequence number of the Arabidopsis BRL3 gene on TAIR (https:// www.arabidopsis.org/i ndex. Jsp): AT3G13380.1 the CDS sequence of the Arabidopsis thaliana is found out through the sequence number of the BRL3 gene, the obtained CDS sequence of the Arabidopsis thaliana BRL3 gene is converted into the protein sequence of the Arabidopsis thaliana BRL3 gene through TBtools software, and the NtBRL3 gene which belongs to tobacco and has close likelihood is screened and identified by using a maximum likelihood Method (ML). Blast analysis was performed on the CDS sequence of the obtained tobacco NtBRL3 gene through NCBI to determine that the corresponding gene was the sought target gene (XM_ 016615644.1).

The nucleotide sequence of the target gene is shown as SEQ ID NO. 1. The method comprises the following steps:

CCCCACCACACCCAAAAACCTATTATCACTCGCAAAACACCTGCAGCAGCAGCTTCTATATGTATGTACTGTATCTGTCAATTGTCATTCCAAATAGCAGCAAAATCTCCCCTTTCTTAACAAACTCAAAGACTGTGACTTGAATGAACAACAAGAATCAGGTTTTACAAGATTGTAAGATGGAGGGGCCGTATTTCCTTAAGTAGGGAAGGAAAGAATTTTTTTTTTTTCAGCAAAGATGAGCTACAAACTATTATCACCAAGTGGGGTGATGAAAGATGTTGTCTTTCTCATA

ATGTTTCTGAGTTGCTGTTTTGTGGTGGTATCAAATGCAAGAAAATTGGC

TGCTAATGATCAAGTAGGTAGCTTAATTGCCTTCAAGAAATCTTCTGTTG

AATTTGATCCAAATGGGTTTTTGAATGACTGGAGTTTTTCTTCTTCTTCAA

CTCCTTGCACTTGGAATGGGATTTCATGCTCAAATGGTCAAGTTGTTGAG

CTCAATCTCTCTACTGCAGACCTTAGTGGGCCCCTTCATCTCTCTCATCTC

ATGGCTTTGCCTACTCTGCTTCGCCTTCACTTCACTGGCAACAATTTCTAT

GGCAATCTTTCCTCAACAGCTGATTCTTGTAGCTTTGAGTTTCTTGATTTA

TCAGCTAACAATTTCTCTGAGACTCTTGTTTTAGAGCCTTTATTGCAGTCT

TGTGATAGAATTAAGTACCTTAATGTCTCTGGGAATTCAATTCATGGAGT

AGTTGGTCTCAAGTTTGGACCTTCACTGTTGCAGCTTGACTTATCAAGTA

ACACAATATCAGATGTAGGCATTTTAAGTTATGCACTGTCCAATTGCCAA

AATTTGAATGTGCTTAATATCTCCTCAAATAAGCTATCTGGCAAACTAAA

AAGCTCCCTCTCTTCTTGCAAGAGCCTTTCTGTTCTTGATCTCTCGCATAA

TAACTTTACTGGAGAACTCAATGGTCTTGATTTTGGGACTTGTCAAAATC

TTACTGTCCTCAATTTGTCTTTCAACAACCTTACTTCAACTGAGTTCCCTC

CCACCTTGGCAAATTGCTTGAGCCTCCATACACTGGACGTTGGCCATAAT

TCGATCCAAACGAAGATTCCTGGCGAATTATTGGTGAAGCTAAAGAGTT

TGAAGCGATTGGTGCTAGCCCACAATCACTTTTTTGAAGAAATTCCGTCA

GAATTGGGGCAGACTTGCAGCACACTTGAGGAGCTTGATCTTTCTGGAA

ACCAGCTGACCGGTGAACTTCCTTCAACTTTTAAATTGTGCTCCTCACTA

TTCAGTCTCAACCTTGGTAATAATGAGCTATCGGGCGATTTCTTGAATAC

TGTTATCAGTTCACTCACAAGTGTTAGGTATCTTTACTTACCATTCAACA

ACATAACTGGTCATGTACCAAGGTCTTTGGCAAACTGTACAAAACTTGA

GGTGCTAGACCTCAGTTCAAATGTGTTAACTGGGAATGTACCTTTTGAGT

TTTGTTTAGCAGCCTCAGCCTCAGGGTTTCCTCTGGAGAAGATGCTGTTA

GCCGGTAATTATCTTACTGGAACAGTGCCAGCACAGCTCGGACTTTGTAG

GAACCTCAGGAAGATTGATCTCAGCTTCAATAAACTGACGGGTTCAATC

CCATTGGAGATTTGGACCCTTCCAAATCTTTCGGAATTGATAATGTGGGC

TAATAATCTCACAGGTGAAATTCCTCAAGGCATTTGCATCAGTGGAGGA

AACCTTCAGACCCTCATTCTCAATAACAATTTCCTCACTGGGGAACTTCC

ACAGTCCATTACCAATTGTACCAACTTGGTTTGGGTTTCGTTATCCAGCA

ACCGACTTTCTGGAGAGATACCACATGGAATTGGGAATCTTGCAAATCTT

GCTATCCTTCAGTTGGGTAATAACTCACTTACTGGACCAATCCCTCAGGG

ATTAGGTACGTGCAGGAACTTGATATGGCTAGATTTGAATAGCAATGCTC

TAACCGGCTCAATCCCTCCCGAGCTTGCTGATCAAGCCGGCCTCGTTAAT

CCTGGAATTGTTTCTGGGAAGCAGTTTGCTTTTGTGAGAAATGAGGGTGG

GACTGAATGCAGAGGTGCTGGTGGATTGGTTGAGTTTGAGGGTATCCGT

GAAAAGAGGCTTGCGATTTTTCCTATGGTTCACTCCTGCCCATCAACTAG

GATCTATTCTGGGACGACAGTGTACACCTTCACGAGCAACGGTAGCATG

ATTTACCTTGATCTCTCCTACAATGCTTTATCAGGAACTATTCCTGAGAAT

TTAGGTTCAATGAGCTTTCTTCAAGTTCTGAATTTGGGACACAACAATTT

CACTGGAACCATTCCGTTCAACTTTGGAGGTCTGAAGATAGTTGGAGTTC

TCGATCTGTCACATAACAGCCTTCAGGGATTCATTCCACCCTCGTTAGGA

GGCCTTTCGTTTCTTAGTGATCTTGACGTCTCAAACAATAACCTATCTGG

AGCGATTCCTTCTGGCGGACAGCTGACTACTTTTCCAGCTTCAAGATATG

AAAACAATTCAGGCCTTTGTGGGGTTCCCCTTCCTCCTTGTGGCTCTGGC

AAGGGACACCGTTCATCAGGCATTTACAATCATAAGAATAAAAAGCCTA

CAACTATAGGAATGGTGGTTGGAATTATGGTCTCTCTTGTTTGTATTGTC

CTACTTATCGTCGCCCTTTATAGGATTAAAAAGACTCAGAAGGAGGAGG

AGAAGAGAGATAAATACATTGAGAGCCTTCCAACTTCTGGCAGTAGCAG

CTGGAAGCTATCAAGCGTTCCCGAGCCACTTAGCATTAATGTGGCAACAT

TTGAAAAACCATTGAGGAAACTCACCTTTGGACATCTTCTAGAAGCAACT

AATGGGTTCAGCTCAGAAAGCTTGATTGGGTCTGGAGGTTTTGGTGAAGT

CTACAAGGCTCAGTTGAGAGATGGAAGTACGGTCGCAATCAAGAAGCTT

GTACATGCAACAAGTCAAGGAGACAGAGAATTCATGGCTGAAATGGAG

ACTATTGGAAAAATCAAACATAGAAACCTTGTCCCTTTGTTGGGATACTG

CAAAATTGGAGAGGAAAGGCTTCTTGTATACGAGTACATGAAATGGGGA

AGTCTCGAATCTGTTCTTCACGAAGGGGAGAAAGGAGGGATGATTCTTG

ATTGGGCAGTTAGGAAGAAGATTGCAATAGGATCAGCAAGAGGATTAGC

ATTTCTTCACCACAGCTGCATACCCCACATAATTCATCGCGATATGAAGT

CCAGTAATGTACTTTTAGACGAAAACTTTGAGGCACGGGTTTCTGACTTT

GGAATGGCAAGGCTGGTAAATGCACTGGATACTCATCTGAGTGTGAGTA

CACTTGCTGGGACTCCAGGTTATGTTCCTCCAGAATACTACCAAAGTTTT

CGGTGCACAGCAAAAGGGGATGTGTACAGTTATGGTGTCATATTGTTGG

AGCTTCTTTCCGGTAAGAGACCAATAGATCCTCGCGAATTTGGTGAGGAT

AACAATCTAGTTGGATGGGCAAAGCAGCTTCATAATGCGAAAAGAAGTC

ATGAGATCCTGGATTCTGAGCTAATAACAAATCTCTCTGGTGATGCTGAA

CTATATCATTATTTAAAGGTTGCATTTGAATGCCTTGACGAGAAACCTTA

CAAAAGGCCAACAATGATTCAAGTGATGACCAAGTTTAAAGAACTACAA

GCTGATTCAGAAAGCGATATTCTTGATGGCATTTCACTGAAAGGTTCTAT

ACTAGAGGAATCGCAAGAAAGAGAACCTTAATCAATGATATTTGATCTT

TTCTCTTCTTTTTGATTGCCTTATGGATAGTTAGAAACTTTTATGTTAATG

GTTCTTACTTGTGAGATTCATAGGAGTTTATACTTCAGATTTTGCCTACTG

TAAATATTAGAACATGATGAAGATGTGAAATTGGTTTAGATTAATGCGC

GAATGAATTTTCCTATTTCTTGTCCCA

the gene NtBRL3 for improving the drought resistance of the tobacco can be combined with brassinosteroids and used for starting transmission of brassinosteroids signals, activating expression of a gene downstream of the brassinosteroids signals and further improving the drought resistance of the tobacco. Through verification, the overexpression of the NtBRL3 gene of the tobacco has a positive correlation with the drought resistance of the tobacco. The genetic transformation technology is adopted to over-express the NtBRL3 gene, and the drought resistance of the tobacco is obviously improved under drought stress conditions. The NtBRL3 can promote drought resistance of tobacco and application of candidate genes related to drought obtained through transcriptome analysis, is used as important candidate genes for developing genetic engineering and improving breeding of tobacco, and provides a technical reserve for future research on the action mechanism of BRL3 genes and application of improving drought resistance quality of tobacco.

The method is specifically as follows:

MSYKLLSPSGVMKDVVFLIMFLSCCFVVVSNARKLAANDQVGSLIAFKKSSVEFDPNGFLNDWSFSSSSTPCTWNGISCSNGQVVELNLSTADLSGPLHLSHLMALPTLLRLHFTGNNFYGNLSSTADSCSFEFLDLSANNFSETLVLEPLLQS CDRIKYLNVSGNSIHGVVGLKFGPSLLQLDLSSNTISDVGILSYALSNCQNLNVLNISSNKLSGKLKSSLSSCKSLSVLDLSHNNFTGELNGLDFGTCQNLTVLNLSFNNLTSTEFPPTLANCLSLHTLDVGHNSIQTKIPGELLVKLKSLKRLVLAHNHFFEEIPSELGQTCSTLEELDLSGNQLTGELPSTFKLCSSLFSLNLGNNELSGDFLNTVISSLTSVRYLYLPFNNITGHVPRSLANCTKLEVLDLSSNVLTGNVPFEFCLAASASGFPLEKMLLAGNYLTGTVPAQLGLCRNLRKIDLSFNKLTGSIPLEIWTLPNLSELIMWANNLTGEIPQGICISGGNLQTLILNNNFLTGELPQSITNCTNLVWVSLSSNRLSGEIPHGIGNLANLAILQLGNNSLTGPIPQGLGTCRNLIWLDLNSNALTGSIPPELADQAGLVNPGIVSGKQFAFVRNEGGTECRGAGGLVEFEGIREKRLAIFPMVHSCPSTRIYSGTTVYTFTSNGSMIYLDLSYNALSGTIPENLGSMSFLQVLNLGHNNFTGTIPFNFGGLKIVGVLDLSHNSLQGFIPPSLGGLSFLSDLDVSNNNLSGAIPSGGQLTTFPASRYENNSGLCGVPLPPCGSGKGHRSSGIYNHKNKKPTTIGMVVGIMVSLVCIVLLIVALYRIKKTQKEEEKRDKYIESLPTSGSSSWKLSSVPEPLSINVATFEKPLRKLTFGHLLEATNGFSSESLIGSGGFGEVYKAQLRDGSTVAIKKLVHATSQGDREFMAEMETIGKIKHRNLVPLLGYCKIGEERLLVYEYMKWGSLESVLHEGEKGGMILDWAVRKKIAIGSARGLAFLHHSCIPHIIHRDMKSSNVLLDENFEARVSDFGMARLVNALDTHLSVSTLAGTPGYVPPEYYQSFRCTAKGDVYSYGVILLELLSGKRPIDPREFGEDNNLVGWAKQLHNAKRSHEILDSELITNLSGDAELYHYLKVAFECLDEKPYKRPTMIQVMTKFKELQADSESDILDGISLKGSILEESQEREP

According to the invention, an agrobacterium-mediated method is utilized to transform the NtBRL3 key gene into tobacco through agrobacterium tumefaciens, and a transgenic plant is obtained through tissue culture, so that the T1 generation is obtained. The drought resistance is stronger in tobacco, and the drought resistance can be further promoted to be applied to genetic breeding of tobacco. Compared with the traditional breeding technology, the method has the advantage that the breeding period is long, and the breeding time is greatly shortened.

According to an embodiment of the present application, the symbiotic medium comprises: 4.432g/L MS (MS medium), 1.00 mg/L6-BA (6-benzylaminopurine), 0.1mg/L NAA (naphthylacetic acid), 30g/L sucrose and 7.5g/L agar powder.

The technical scheme of the present application will be described below with reference to specific embodiments.

The relevant media, reagents and materials will first be described.

(1) YEP solid medium: 10g/L peptone+10 g/L yeast extract+5 g/L sodium chloride+7.5 g/L agar powder

(2) YEP liquid medium: 10g/L yeast extract+10 g/L peptone+5 g/L sodium chloride

(3) Symbiotic medium: 4.432g/L MS+1.00 mg/L6-BA+0.1 mg/L NAA+30g/L sucrose+7.5 g/L agar powder

(4) Screening the culture medium: 4.432g/L MS+1.00 mg/L6-BA+0.1 mg/L NAA+30g/L sucrose+7.5 g/L agar powder+100 mg/L timentin+100 mg/L kanamycin

(5) Rooting medium: 2.216g/L MS, 20g/L sucrose, 7.5g/L agar powder, 100mg/L telangiectan and 100mg/L kanamycin

The tobacco variety used was tobacco (Nicotiana tabacum l.), tobacco K326 aseptic seedling.

In the whole, the application of the gene NtBRL3 for improving the drought resistance of the tobacco in the tobacco comprises the following steps of;

(1) Acquisition of the Gene of interest

(2) Construction of NtBRL 3-purpose Gene vector

(3) Preparation of competent cells of Agrobacterium tumefaciens

(4) Vector plasmid introduction into Agrobacterium tumefaciens

(5) The genetic transformation of tobacco is carried out,

(6) Identification of resistant tobacco plants

(7) Transgenic tobacco T1 generation drought treatment

(8) Detection of candidate Gene expression levels by fluorescent quantitative qRT-PCR

Wherein the NtBRL3 gene expression element is synthesized by Shanghai crown biotechnology Co., ltd, the BRL3 expression vector is obtained by double digestion of vector pSH737 and BRL3 fragments by XbaI and KpnI, and then ligation and transformation, and the vector contains 35S promoter and NtBRL3 gene expression element, and screening marker gene and reporter gene. Phs737 was used as vector, 35S as promoter, NPTII as resistance selection gene, and GUS as reporter gene.

In the step 5, the dark culture and the light culture are performed, the light culture is performed at 25 ℃ in a tissue culture room with a 16-hour light/8-hour dark period for 2 d.

The primers identified by PCR in step 6:

Forward primer(SEQ ID NO.3)：ATCACTCGCAAAACACCTGC；

Reverse primer(SEQ ID NO.4)：AGTGAAGTGAAGGCGAAGCA；

and 7, performing drought experiments to obtain tobacco seedlings growing to a period of five leaves and one heart. The transgenic tobacco and wild-type tobacco were each provided with 3 biological replicates. Repeatedly irrigating with 20% PEG6000 solution for 7 days, wherein each plant is irrigated with 50mL of the solution per pot;

in the step 7, candidate genes are obtained by screening according to the standard that the p value is < 0.005 and log2FoldChange > 1.

Specifically, the application of the gene NtBRL3 for improving the drought resistance of the tobacco in the tobacco comprises the following steps:

1. obtaining the target gene: sequence number of the Arabidopsis BRL3 gene on TAIR (https:// www.arabidopsis.or g/index. Jsp): AT3G13380.1 the CDS sequence of the Arabidopsis thaliana is found out through the sequence number of the BRL3 gene, the obtained CDS sequence of the Arabidopsis thaliana BRL3 gene is converted into the protein sequence of the Arabidopsis thaliana BRL3 gene through TBtools software, and the NtBRL3 gene which belongs to tobacco and has close likelihood is screened and identified by using a maximum likelihood Method (ML). The CDS sequence of the obtained tobacco NtBRL3 gene was subjected to Bleast analysis by NCBI to determine that the corresponding gene was the target gene (XM_ 016615644.1).

Construction of NtBRL 3-purpose Gene vector:

the NtBRL3 gene expression element is synthesized by Shanghai Xueguan Biotechnology Co., ltd, and the BRL3 expression vector is obtained by double digestion of the vector pSH737 and BRL3 fragments by XbaI and KpnI and then ligation and transformation, and the vector contains the 35S promoter and the NtBRL3 gene expression element as well as the screening marker gene and the reporter gene.

The plasmid containing the expression vector pSH737-35S-BRL3 is transformed into the agrobacterium tumefaciens strain LBA4404 by a freeze thawing method, the agrobacterium tumefaciens positive strain is screened by Kan (kanamycin) of 100mg/L, rif (rifampicin) of 20mg/L, colony PCR detection is carried out on the positive strain by using a primer pair (SEQ ID NO.3 and SEQ ID NO. 4), and the agrobacterium tumefaciens strain with positive PCR result is cultivated at minus 80 ℃ in a enlarging mode for seed preservation. The specific steps are that LBA4404 competent cells stored at-80 ℃ are placed in ice for thawing for 10min; adding 5 mu L of plasmid DNA into each competent cell, gently flicking and uniformly mixing, and then carrying out ice bath for 30min; after quick freezing for 5min by liquid nitrogen, immediately carrying out water bath for 2min at 37 ℃; adding 900 mu L of preheated YEP liquid culture medium at 37 ℃,28 ℃ and carrying out shaking culture at 200rpm for 3 hours; centrifuging at room temperature at 4000 Xg for 1min, and discarding the supernatant; adding 100 mu L of YEP liquid culture medium into the thalli, and sucking and beating by a gun head to mix uniformly; and (3) a proper amount of bacterial liquid is coated on a YEP flat plate culture medium containing 100mg/L Kan and 20mg/L Rif, and the flat plate culture medium is placed in a constant temperature incubator at 28 ℃ for 2d, and the bacterial liquid is preserved at-80 ℃.

3. Preparation of Agrobacterium tumefaciens competent cells:

preparation of Agrobacterium tumefaciens competent cells: taking out Agrobacterium tumefaciens strain stored in-80deg.C refrigerator, dipping the strain solution with inoculating needle, and adding strain solution containing Rif (20mg.L ^-1 ) Streaking the YEP plate medium of the antibiotics to enable the antibiotics to pick out single colonies; the YEP solid medium with the inoculated strain is inverted and placed in a constant temperature incubator at 28 ℃ to culture, single colony is picked up from the plate medium, inoculated in 5mL of strain containing Rif (20 mg.L) ^-1 ) Shake culturing in YEP liquid medium of antibiotics at 28deg.C and 200 rpm; 1mL of the bacterial liquid was taken from the YEP medium, and inoculated into 50mL of a medium containing Rif (20 mg. L) ^-1 ) In the YEP liquid culture medium, shake culturing is carried out at 28 ℃ and 200rpm until the OD600 is 0.5-0.6; taking 1.5ml centrifuge tube, adding 18 μl glycerol (15% concentration) into each tube, pre-cooling the centrifuge tube on ice, and sterilizing CaCl ₂ (0.1mol·L ^-1 ) Pre-cooling the solution on ice; subpackaging bacterial liquid with an OD600 value of 0.5-0.6 into a 10mL centrifuge tube, centrifuging at 4 ℃ and 5000rpm for 10min, and pouring out supernatant; 200. Mu.L of pre-chilled CaCl was added to a 10ml centrifuge tube ₂ The solution is lightly blown by a pipetting gun to make the thalli re-suspended evenly; taking 100 mu L of heavy suspension from a 10mL centrifuge tube, adding the heavy suspension into a 1.5mL centrifuge tube pre-cooled in advance, uniformly mixing the heavy suspension with glycerol, and then quickly freezing the heavy suspension with liquid nitrogen; the prepared competent cells of the agrobacterium tumefaciens are preserved in an ultralow temperature refrigerator at the temperature of minus 80 ℃.

Plasmid extraction used the root kit.

The operation steps are as follows:

1. column equilibration step, adding 500. Mu.L of equilibration liquid BL into the adsorption column CP3 (the adsorption column is placed in the collecting tube), centrifuging at 12,000rpm (13,400 Xg) for 1min, pouring out the waste liquid in the collecting tube, and putting the adsorption column back into the collecting tube.

2. 1-5ml of the overnight cultured bacterial liquid was taken and added to a centrifuge tube, and the supernatant was centrifuged at 12,000rpm (13,400 Xg) for 1min using a conventional bench centrifuge, and the supernatant was removed as much as possible.

3. To the centrifuge tube with the bacterial pellet left, 250. Mu.L of solution P1 (please check whether RNaseA had been added) was added and bacterial pellet was thoroughly suspended using a pipette or vortex shaker.

4. 250 μl of solution P2 was added to the centrifuge tube, and the tube was gently turned upside down 6-8 times to allow the cells to be fully lysed.

5. 350. Mu.L of solution P3 was added to the centrifuge tube, immediately gently turned upside down 6-8 times, and thoroughly mixed, at which time a white flocculent precipitate was formed. Centrifuge at 12,000rpm (-13,400 Xg) for 10min.

6. The supernatant collected in the previous step is transferred to the adsorption column CP3 by a pipette. The waste liquid in the collection tube was poured off by centrifugation at 12,000rpm (13,400 Xg) for 30-60sec, and the adsorption column CP3 was placed in the collection tube.

7. 600. Mu.L of the rinse PW was added to the in-suction CP3, centrifuged at 12,000rpm (13,400 Xg) for 30-60sec, the waste liquid in the collection tube was poured out, and the adsorption column CP3 was placed in the collection tube.

8. And repeating the operation step 7.

9. The column CP3 was placed in a collection tube and centrifuged at 12,000rpm (13,400 Xg) for 2min in order to remove the residual rinse solution from the column.

10. The adsorption column CP3 was placed in a clean centrifuge tube, 50-100. Mu.L of elution buffer EB was added dropwise to the middle portion of the adsorption membrane, and the solution was placed at room temperature for 2min, centrifuged at 12,000rpm (13,400 Xg) for 2min, and the plasmid solution was collected in the centrifuge tube.

4. Vector plasmid introduction into Agrobacterium tumefaciens

Taking out the competent cells of the agrobacterium tumefaciens stored in the ultralow temperature refrigerator at the temperature of minus 80 ℃ and putting the competent cells on ice for 10min, and thawing the competent cells; weighing 5 mu L of DNA plasmid containing NtBRL3, adding into competent cells of agrobacterium tumefaciens, slightly flicking, mixing, placing on ice for 30min, and then quick-freezing with liquid nitrogen for 5min; placing the centrifuge tube in a water bath at 37deg.C for 2min to obtain a solutionDissolving to obtain recombinant agrobacterium tumefaciens; into a centrifuge tube, 900. Mu.L of a solution containing Rif (20 mg. Multidot.L) ^-1 ) YEP liquid medium of antibiotics, 28 ℃,200rpm, shake culture for 4h; centrifuging at 5000 Xg for 1min at room temperature, and pouring out supernatant; adding 100u LYEP liquid culture medium into the centrifuge tube, and gently mixing; a proper amount of bacterial liquid is coated on the substrate containing Rif (20 mg.L) ^-1 ) And Kan (20 mg.L) ^-1 ) On the YEP solid culture medium, the YEP solid culture medium is inversely placed in a constant temperature incubator at 28 ℃ for 2 days, and single colony is selected for seed preservation for standby.

5. Genetic transformation of tobacco

Shan Junla agrobacterium containing the NtBRL3 expression vector selected in the step are subjected to shaking culture on a shaking table, when the OD600 of the bacterial liquid reaches 0.5, the bacterial liquid is centrifuged, and then the bacterial liquid is resuspended by a resuspension for later use. Tobacco is genetically transformed by agrobacterium tumefaciens-mediated leaf disc transformation using tobacco aseptic seedling lamina as an explant. Cutting tobacco aseptic seedling leaf into 1cm×1cm small leaf pieces, immersing in the resuspended bacteria liquid, and keeping the stopwatch for 8min, and continuously shaking the bacteria liquid to make full contact with the leaf. The leaves were nipped onto sterile paper with forceps to blot the bacterial solution, see FIG. 1 (A), into symbiotic medium. After 2d of dark culture, see FIG. 1 (B), the cells were inoculated into a selection medium and cultured for 2 weeks for a further time. Referring to FIG. 1 (C), when the resistant buds grow to about 2cm, cutting out the resistant buds, transferring the resistant buds into rooting culture medium, and when the tobacco roots are thicker, hardening off and transplanting. The tobacco is resistant tobacco.

6. Identification of resistant tobacco plants

Referring to FIG. 1 (E), when tobacco plants are transplanted to survive and grow to a certain size, tobacco leaves are taken to extract DNA, and plants transformed with the over-expression vector are amplified by the primers of tobacco NtBRL3 (primers shown as SEQ ID NO.3 and SEQ ID NO. 4). The PCR was performed using tobacco, and the target band was obtained at 200 bp. Referring to FIG. 1 (D), 20. Mu.L of the system comprised 2. Mu.L of tobacco genomic DNA, 0.5. Mu.L of tobacco BRL3-Forward, 0.5. Mu.L of BRL3-Reverse, 10. Mu.L of Taq DNA polymerase and 7. Mu.L of ddH ₂ O. The amplification procedure was: 94 ℃ for 5min;94 ℃,30sec,55 ℃,30sec,72 ℃,60sec,30 cycles; extending at 72℃for 10min.Tobacco NtBRL3 was used as positive control, water was used as blank control, and wild-type tobacco genomic DNA was used as negative control.

7. Transgenic tobacco T1 generation drought treatment

Referring to fig. 1 (F), wild-type tobacco with consistent growth vigor and tobacco seedlings over-expressing NtBRL3 were selected for drought stress treatment. Firstly, after 3 times of washing with distilled water, transferring the tobacco seedlings to a pot plant, and when the tobacco seedlings survive stably to 5 leaves and one heart, simulating drought stress by using 20% of PEG-6000 for 7 days. Physiological and biochemical analysis was performed on the T1 generation. See fig. 2 (a) and 2 (B), (a) is a pre-drought stress phenotype; (B) In order to achieve the phenotype after drought treatment, the growth and development of wild plants are severely inhibited, leaves wither to a certain extent, the withered leaves turn yellow, and the influence of NtBRL3ox plants is smaller, so that the result shows that the drought resistance of transgenic plants is improved by over-expressing the NtBRL3 genes. Wherein, ntBRL3ox: transgenic tobacco; WT: wild type.

Under drought stress, glutathione S-transferase (GST) activity was increased in both groups. However, the GST activity in transgenic tobacco was significantly increased, and the GST activity of transgenic tobacco was 1.51-fold higher than that of wild type. The Peroxidase (POD) activity of transgenic tobacco was 1.26 times that of wild type. Referring to fig. 3, fig. 3 is a graph showing the results of the measurement of GST, POD, CAT, SOD, MDA content, proline content and soluble sugar content of transgenic tobacco and wild-type tobacco after drought treatment. The results show that under drought stress, catalase (CAT) in both transgenic tobacco and wild type increased significantly, but the transgenic tobacco increased more than the wild type by 1.55 times. Typically, the superoxide dismutase (SOD) activity in transgenic tobacco is slightly higher than wild type. Under drought stress, the SOD activity of N transgenic tobacco is 1.09 times that of wild type.

Malondialdehyde (MDA) content increases for both genotypes under drought stress. The MDA content of the transgenic tobacco and the MDA content of the wild type are obviously increased compared with the control, and the transgenic tobacco content is increased by 1.50 times compared with the wild type. Likewise, the proline (Pro) content and soluble sugar content of transgenic tobacco were also significantly higher than that of wild type, 2.07-fold and 1.97-fold, respectively. The above results indicate that overexpression of NtBRL3 can improve the removal capacity of ROS in transgenic plants by improving the enzymatic activities of GST, POD, SOD and CAT, so that the accumulation of ROS is reduced, and the drought resistance of the transgenic plants is improved.

8. Detection of candidate Gene expression levels by fluorescent quantitative qRT-PCR

Genes related to drought tolerance are obtained through transcriptome analysis, and are involved in plant response to drought stress or drought induction genes are involved in plant response to drought stress or are induced by drought, GST, PLA, KCS, LOX, RD, MAH1, HCT, WSD1 and WAK.

Total RNA from samples was extracted using TRNzol-A+ (Tiangen Biochemical Co., ltd., beijing) kit and was reverse transcribed to cDNA using PrimeScript II 1ststrandcDNASynthesis kit (Soy Co., ltd., beijing), and the cDNA was diluted 10-fold for fluorescent quantitative PCR detection to determine the expression of POR1 gene, and specific primers for the above genes were designed using PrimerPremier5.0 software, with L25 gene as an internal reference. qRT-PCR assay was performed on a BioRadCTXConnectTM real-time quantitative PCR apparatus (Bio-Rad Co.). The qRT-PCR is a general high-sensitivity dye method quantitative PCR detection kit for the Nanjinouzan, and the qRT-PCR reaction system is configured according to the use instruction on the kit. The conditions and system of qRT-PCR were as follows, one denaturation cycle (94 ℃,5 min), then 40 amplification cycles (94 ℃,30 seconds, 50 ℃,45 seconds), and signal acquisition (72 ℃,113 seconds), qRT-PCR selected 50. Mu.L of system: nuclear-free Water 20. Mu.L, biorun Pfu PCR Mix. Mu. L, template 1. Mu. L, primer (+) (100. Mu.M) 2. Mu. L, primer (-) (100. Mu.M) 2. Mu.L. Referring to FIG. 4, FIG. 4 is a graph showing the relative expression amounts of candidate genes in wild type and transgenic types. The relative expression levels of the junction genes were analyzed with 2- Δct. The data were analyzed by variance using SPSS software and error bars represent ± standard deviation of the data from three independent experiments. Different letters indicate significant P <0.05 differences and very significant P <0.01 differences. The drought gene expression amount of the transgenic plant is increased, which indicates that the over-expression of NtBRL3 can improve the expression of related drought resistance genes.

In the above technical solution of the present invention, the above is only a preferred embodiment of the present invention, and therefore, the patent scope of the present invention is not limited thereto, and all the equivalent structural changes made by the description of the present invention and the content of the accompanying drawings or the direct/indirect application in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. The gene NtBRL3 for improving the drought resistance of the tobacco is characterized in that the nucleotide sequence of the gene NtBRL3 for improving the drought resistance of the tobacco is shown as SEQ ID NO. 1.

2. A protein for improving drought resistance of tobacco is characterized in that the protein is formed by encoding a gene NtBRL3 as set forth in claim 1, and the amino acid sequence of the protein is shown as SEQ ID NO. 2.

3. Use of the gene NtBRL3 for improving drought resistance of tobacco according to claim 1 in tobacco.

4. The use according to claim 3, characterized by the following steps:

the recombinant vector is obtained by connecting an XbaI and KpnI double-restriction enzyme cutting vector pSH737 and the gene NtBRL 3;

introducing the recombinant vector into competent cells of agrobacterium tumefaciens to obtain recombinant agrobacterium tumefaciens;

5. The use according to claim 4, wherein the vector pSH737 comprises a promoter, a resistance screening gene, a reporter gene, wherein the promoter is 35S, the resistance screening gene is NPTII, and the reporter gene is GUS.

6. The use according to claim 4, wherein said step of transforming sterile seedling leaves of tobacco through said recombinant agrobacterium tumefaciens mediated disc comprises:

culturing the recombinant agrobacterium tumefaciens until the OD600 of the bacterial liquid reaches 0.5, centrifuging the bacterial liquid, and re-suspending to obtain a re-suspension;

7. The use according to claim 4, wherein the step of culturing the submerged leaf mass into the resistant tobacco comprises:

8. The use of claim 7, wherein the symbiotic medium comprises: 4.432g/L MS, 1.00 mg/L6-BA, 0.1mg/L NAA, 30g/L sucrose and 7.5g/L agar powder.

9. The use according to claim 7, wherein the screening medium comprises: 4.432g/L MS, 1.00 mg/L6-BA, 0.1mg/L NAA, 30g/L sucrose, 7.5g/L agar powder, 100mg/L timentin and 100mg/L kanamycin.

10. The use according to claim 7, wherein the rooting medium comprises: 2.216g/L MS, 20g/L sucrose, 7.5g/L agar powder, 100mg/L telangiectasia, and 100mg/L kanamycin.