CN110205330A - Tobacco heat shock protein HSP22 and its application - Google Patents
Tobacco heat shock protein HSP22 and its application Download PDFInfo
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Abstract
The invention belongs to transgenic tobacco fields, and in particular to 22 gene of tobacco heat shock protein HSP and its apply patent application.The gene base sequence is as shown in SEQ ID NO.1.Tobacco heat shock protein HSP 22 is made of 192 amino acid residues, wherein the SHSP structural domain that 68-159 amino acids are conservative.The albumen is related to colors content in plant leaf blade, and after reducing the protein expression, colors content is substantially reduced in blade, the colors are as follows: neoxathin, violaxanthin, lutein, chlorophyll a/b, beta carotene.The present invention has found that it is highly relevant with tobacco colors content, after by the gene silencing, colors content is substantially reduced in tobacco by the Primary Study to specific tobacco heat shock protein HSP 22.Based on this characteristic, certain application foundation and reference can be cultivated for new product of tobacco.
Description
Technical field
The invention belongs to transgenic tobacco fields, and in particular to tobacco heat shock protein HSP 22 and its application patent Shen
Please.
Background technique
The Nicotiana Dicotyledoneae Tubiflorae Solanaceae Nicotiana plant that people are usually sucked, Nicotiana
(Nicotiana) have more than 60 kind, and being used to prepare sucked tobacco is mainly two cultivars, respectively Nicotiana tabacum
(also known as safflower tobacco,Nicotiana tabacum) and makhorka (Nicotiana rustica), wherein the former occupies master
Area is wanted, and the cultivated area of the latter is relatively small.
Cultivation tobacco by quality of tobacco feature, biological character and cultivation modulator approach the features such as can be divided into flue-cured tobacco, suncured tabacco,
Six air-curing of tobacco leaves, burley tobaccos, Turkish tobaccos and Nicotiniana rustica types, wherein flue-cured tobacco is to cultivate most wide Nicotiana tabacum.China's tobacco planting
Area and total output rank first in the world.As a kind of leaf with industrial crops, the cultivation technique of flue-cured tobacco is different from other crop fields
Crop does not require nothing more than certain yield of tobacco, and more focuses on quality of tobacco.Quality of tobacco determines the availability of tobacco leaf, directly
Connect influence cigarette commodity color and commodity value, be also related to the economic benefit of tobacco grower, be tobacco business life and
Starting point.To make to establish oneself in an unassailable position in following domestic and international market competition, meet domestic and international cigarette enterprise to sound tobacco
Increasing need, it is necessary to improve quality of tobacco and safety.
Phytochrome is a kind of important compound in tobacco, mainly includes chlorophyll and carotenoid substance.Leaf
Largely degradation disappears green element in tobacco maturation and tobacco leaf modulated process, it is a kind of unfavorable chemical component in dry tobacco leaf,
It is one of the index being tightly controlled in tobacco leaf grading often with blue foreign smell.If chlorophyll does not have during modulation treatment
There is fully degraded that just tobacco leaf is dried, different degrees of " bluish yellow cigarette " will be baked." bluish yellow cigarette " not only exterior quality is bad, combustion
Harmful substance can be also generated when burning, and then influences tobacco grade and quality.
During tobacco fermentation alcoholization, on the one hand chlorophyll porphyrin ring can degrade and generate azoles, to increase
Sleep is fragrant, reduces blue foreign smell；On the other hand, the phytol that chlorophyll hydrolysis generates can further be degraded into neophytadiene, and
It is degraded into plant furans again, and then is converted to the fresh and sweet ingredient of tobacco leaf.Therefore, chlorophyll is important latent aromatic substance, sufficiently
After degradation advantageously to cigarette matter.
Tobacco uranidin is mainly carotenoid, and tobacco leaf carotenoid content and quality of tobacco, which exist to be positively correlated, to close
System, is on the one hand that tobacco leaf exterior quality is directly related with such component content；Another aspect carotenoid be tobacco aroma at
The important precursor divided, perfume quantity and flavouring essence quality to tobacco are positive correlations.Flavor component in tobacco leaf is very big by one
Part is the catabolite of carotenoid, wherein many compounds are aroma components crucial in tobacco, such as irisone, greatly
Horse ketone and isophorone etc..
Based on the important function of pigment in above-mentioned tobacco leaf, tobacco colors controlling gene is furtherd investigate, benefit
New tobacco bred is constructed with genetic engineering, can establish applications well basis to improve tobacco bred.
Summary of the invention
Based on the research of tobacco colors content controlling gene, it is an object of that present invention to provide a kind of tobacco heat shock eggs
White 22 gene of HSP and its application in terms of tobacco pigment content of material adjusting, to establish base for new product of tobacco cultivation
Plinth.
Details are as follows for the technical solution that the application is taken.
The encoding gene of tobacco heat shock protein HSP22HSP22, base sequence is as shown in SEQ ID NO.1, wherein specifically
Property nucleic acid fragment be 202-477 bit base.
The codingHSP22Application of the gene in the regulation of leaf pigment substance content, using gene silent technology or
Person's gene overexpresses method, by adjusting tobacco HSP22 expressing quantity, to adjust colors content feelings in control tobacco leaf
Condition, the colors are as follows: neoxathin, violaxanthin, lutein, chlorophyll a/b, beta carotene.
Tobacco heat shock protein HSP22, amino acid sequence is as shown in SEQ ID NO.2, by 192 amino acid residue groups
At wherein the SHSP structural domain that 68-159 amino acids are conservative.
Application of the tobacco heat shock protein HSP22 in the regulation of leaf pigment substance content, the albumen and leaves of plants
Colors content is related in piece, and after reducing the protein expression, colors content is substantially reduced in blade, the pigment
Substance are as follows: neoxathin, violaxanthin, lutein, chlorophyll a/b, beta carotene.
Utilize the encoding geneHSP22New product of tobacco breeding method, pass through transgenic technology, transient expression technology
Or genome editing technique, building containHSP22Virus induction silent carrier, the RNAi interference vector, overexpression vector of gene
Or genome editor's carrier, transformation of tobacco, screening obtain the new product of tobacco of pigment content variation；
Specifically for example: utilizing the technology of virus induced gene silencing (VIGS), interferenceHSP22The expression of gene makes its silencing,HSP22Colors content is remarkably decreased in gene silencing plant, and then obtains the new variety of plant of pigment content decline.
The present invention has found that it contains with tobacco colors by the Primary Study to specific tobacco heat shock protein HSP 22
Measure highly relevant, after by the gene silencing, colors content is substantially reduced in tobacco.Based on this characteristic,
Certain application foundation and reference can be cultivated for new product of tobacco.
Detailed description of the invention
Fig. 1 is the phenotype comparison diagram of tobacco TRV2-PDS, TRV2-GFP and TRV2-NtHSP22 carrier conversion group of the present invention；
Fig. 2 is the relative expression quantity of the gene in NtHSP22 gene silencing plant compared with adjoining tree；
Fig. 3 is the primary pigments comparision contents in the tobacco leaf and control tobacco leaf of Gene Silencing.
Specific embodiment
Explanation is further explained to the application below with reference to embodiment, before introducing specific embodiment, with regard to following implementations
Specific experiment background information situation is briefly discussed below in example.
Biomaterial:
Ben's tobacco, a kind of common tobacco-containing material, tobacco planting is in Zhengzhou tobacco institute planting base, pot for growing seedlings in following embodiments
Middle nursery is divided into seedlings for two weeks after germinateing, and is planted in polypots (10cm × 10cm), 22 DEG C, under 16h light/8h dark condition into
Daily fertilizer and water management of row etc.；
VIGS carrier employed in following embodiments is a kind of viral vectors (tobacco from Tobacco rattle virus
Rattle virus, TRV), the TRV2 specifically utilized is saved by Zhengzhou tobacco research institute gene center, is screened with that is blocked
Label and 35S promoter, while TRV2 can be used to carry and convert external source with multiple cloning sites such as EcoR I and BamH I
Gene；
Experiment reagent:
LB liquid medium contains in 1L content: 10 g bacto peptones (bacteriological peptone)；10 g chlorine
Change sodium (NaCl)；5 g yeast extracts (yeast extract), autoclave sterilization；
YEB fluid nutrient medium contains in 1L content: 5g beef extract (beef extract)；5 g bacto peptones
(bacteriological peptone)；5 g sucrose (sucrose)；1 g yeast extract (yeast extract)；2 mL
1M magnesium sulfate (MgSO4), autoclave sterilization；
1M 2- (N- morpholine) ethanesulfonic acid (MES) stock solution: ddH2O dissolution, filtration sterilization, -20 DEG C store for future use；
200 mM acetosyringone (Acetosyringone, As) stock solutions: dimethyl sulfoxide (DSMO) dissolution, -20 DEG C of storages
It is spare；
MMA(100 mL): 1 mL(1 M) MgCl2；1 mL(1 M, pH5.6) MES；75 μ L(200 mM) As.
Embodiment 1
The present embodiment is with regard to tobaccoNtHSP22The building process of gene cloning and silent carrier is briefly discussed below.
(1) tobaccoNtHSP22Gene cloning
It according to early period for tobacco gene group and correlation HSP22 gene studies, selects special coded sequence for target fragment, designs
PCR amplification primer sequence is as follows:
NtHSP22-F:5 '-ACCGAATTCGCTCAGACTGGAAGGAAACA- 3 ',
NtHSP22-R:5 '-ACCGGATCCGCTTTAATGTCCTCACCACC- 3 '；
Using the cDNA of tobacco K326 blade as template, PCR amplification acquisition is carried outNtHSP22Gene；
PCR amplification program are as follows: 95 DEG C of 3 min of initial denaturation；95 DEG C of denaturation 15s, 55 DEG C of annealing 15s, 72 DEG C of extension 30s, 34 are followed
After ring, 72 DEG C thoroughly extend 5min；
Agarose gel electrophoresis detection is carried out to pcr amplification product, and it is spare to recycle electrophoresis product.
(2) building recombination TRV2-NtHSP22 carrier
By in step (1) pcr amplification product carry out EcoRI, BamHI double digestion, while to empty carrier TRV2 carry out EcoRI,
BamHI double digestion, is separately recovered digestion products, is attached using T4 DNA ligase；
Connection product is converted into E. coli competent DH5 α, converted product is coated on containing 50mg/L after conversion operation
On the LB solid medium of Kan, culture night is spent at 37 DEG C；
Further progress PCR is identified after selecting positive single colonie amplification, and combines sequence verification, it is ensured that obtains building correctly weight
Group carrier TRV2-NtHSP22.
It should be understood that
TobaccoNtHSP22Gene, including 579 bases, specific base sequence are as follows:
ATGGTGAAAACAACTGTTAGTCTTTTGAGCTTTCTAGTGTTAGCAATGGCTGTAGTCTTATTTCTTCCATCA
CAAACTGAAGCACTAATGCCATACACACGCCCTTTTTGGGACTTAACGTTCCCACCAGAAGATCCTTTCAAGATTC
TTGAACAAATCCCACTAACCATCCCAAAAGGGGTCGATTCAATCGCCTTAGCTCGCTCAGACTGGAAGGAAACAGG
AACAGAGCACGTAATTACCCTCGACATACCAGGGATGAAAAGGGATGACATCAAGATCGAGGTGGAAGAGAACAGG
GTGTTGAGAATCATTGGGGAAAGGAAAATAGAGGAAGAAGTTGAAGGAGAAAAGTGGCACAGAGCTGAGAGGACTT
CTGGAAAATTCTGGAGACAGTTTAGGTTACCTGGGAATGCAGATTTGGAACACATAAAGGCTCATTTGGAAAATGG
TGTCTTGAAGATTACTGTGCCAAAGTTGGCTGAAGAGAAGAAGAAACAGACAAAGGTGATTAATATTGCTGAGGAT
GGTAACTCTGCTGGTGGTGAGGACATTAAAGCTACCAAAGCTGAGATGT；
Tobacco heat shock protein HSP22, including 192 amino acid, specific amino acid sequence are as follows:
MVKTTVSLLSFLVLAMAVVLFLPSQTEALMPYTRPFWDLTFPPEDPFKILEQIPLTIPKGVDSIALARSDWK
ETGTEHVITLDIPGMKRDDIKIEVEENRVLRIIGERKIEEEVEGEKWHRAERTSGKFWRQFRLPGNADLEHIKAHL
ENGVLKITVPKLAEEKKKQTKVINIAEDGNSAGGEDIKATKAEM。
Embodiment 2
On that basis of example 1, using the VIGS technology of mediated by agriculture bacillus, inventor is further by constructed recombination TRV2-
NtHSP22 carrier has converted tobacco plant, and has done verifying analysis, specific experiment process letter with regard to corresponding plants character mutation situation
It is situated between as follows.
(1) Agrobacterium is converted
It should be noted that the operation of reference implementation example 1 and the prior art, inventor are prepared for TRV2-GFP, TRV2-PDS simultaneously
Recombinant vector is as control, specific conversion process are as follows:
By TRV2-GFP(vehicle Control), TRV2-PDS(VIGS efficiency control) and TRV2-NtHSP22 positive colony plasmid,
It is converted respectively by electroporated mode into Agrobacterium GV3101 competent cell, utilizes Kan containing 50mg/L and 50mg/
The YEB plate of L Rif carries out culture screening, after 2d is cultivated in 28 DEG C of inversions, there is the agriculture of purpose gene using bacterium colony PCR screening zone
Bacillus.
(2) transfection bacterium solution is prepared
The positive Agrobacterium colonies of screening gained in step (1) (are contained into 50 mg/L Kan and 50 in the YEB fluid nutrient medium of 5 mL
Mg/L Rif) in, 28 DEG C, overnight incubation under the conditions of 250 rpm；
50uL overnight culture is taken to be seeded in the YEB fluid nutrient medium (containing 50 mg/L Kan) of 50 mL, culture to OD600 =
1.0 ~ 1.5 or so, then 4000g is centrifuged 5 min, collects thallus, then with MMA (1 mL(1 M) MgCl2；1 mL(1 M,
PH5.6) MES；75 μ L(200 mM) As) it is resuspended, adjust OD600 =1.0 or so；
After being finally placed at room temperature for 3 h or so, as transfection bacterium solution.
(3) instantaneous conversion
It, will be prepared in step (2) using 1 mL gauge hypodermic device using the Ben's tobacco leaf of 3 ~ 4 w seedling ages as experimental material
Transfection is injected in tobacco leaf with bacterium solution, and the tobacco after injection continues to cultivate in artificial incubator, observes character mutation.
Tobacco phenotypes situation of change after injecting 3 weeks is as shown in Figure 1.It can be seen that the During Agrobacterium containing TRV2-PDS
Plant Newborn Leaves have bleaching phenomenon, illustrate to infect success；And TRV2-GFP group is then without significant change, corresponding TRV2-
NtHSP22 group tobacco leaf color then has significant change, shows NtHSP22 gene and pigment content height phase in tobacco leaf
It closes.
Further NtHSP22 expression conditions are detected by qRT-PCR, as a result as shown in Fig. 2, can see
Out, TRV2- NtHSP22 infects in plant, and the expression quantity of NtHSP22 significantly reduces.
Further, inventor is to experimental group (TRV2- NtHSP22 disseminates plant) and control group (TRV2-GFP dip dyeing plant
Strain) in phytochrome (neoxathin, violaxanthin, lutein, chlorophyll a/b, beta carotene) content situation detected
(according to People's Republic of China (PRC) tobacco business standard YC/T 382-2010, " measurement of tobacco and tobacco product plamochromic pigment is efficient
Liquid chromatography " is detected), as a result as shown in Fig. 3 and following table.
Table 1, phytochrome content declines percentage in fresh tobacco leaves sample
。
It can be seen that neoxathin in experimental group, violaxanthin, lutein, chlorophyll a, chlorophyll b and β recklessly from upper table result
The various pigment contents such as radish element are decreased significantly compared with the control group, this is further demonstrated that, pass through silencing NtHSP22 base
Cause can regulate and control the content of phytochrome in tobacco leaf, and then can establish certain technical foundation for quality of tobacco regulation.
SEQUENCE LISTING
<110>Zhengzhou Tobacco Research Institute of CNTC
<120>tobacco heat shock protein HSP22 and its application
<130> none
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 577
<212> DNA
<213> Nicotiana tabacum
<400> 1
atggtgaaaa caactgttag tcttttgagc tttctagtgt tagcaatggc tgtagtctta 60
tttcttccat cacaaactga agcactaatg ccatacacac gccctttttg ggacttaacg 120
ttcccaccag aagatccttt caagattctt gaacaaatcc cactaaccat cccaaaaggg 180
gtcgattcaa tcgccttagc tcgctcagac tggaaggaaa caggaacaga gcacgtaatt 240
accctcgaca taccagggat gaaaagggat gacatcaaga tcgaggtgga agagaacagg 300
gtgttgagaa tcattgggga aaggaaaata gaggaagaag ttgaaggaga aaagtggcac 360
agagctgaga ggacttctgg aaaattctgg agacagttta ggttacctgg gaatgcagat 420
ttggaacaca taaaggctca tttggaaaat ggtgtcttga agattactgt gccaaagttg 480
gctgaagaga agaagaaaca gacaaaggtg attaatattg ctgaggatgg taactctgct 540
ggtggtgagg acattaaagc taccaaagct gagatgt 577
<210> 2
<211> 192
<212> PRT
<213> Nicotiana tabacum
<400> 2
Met Val Lys Thr Thr Val Ser Leu Leu Ser Phe Leu Val Leu Ala Met
1 5 10 15
Ala Val Val Leu Phe Leu Pro Ser Gln Thr Glu Ala Leu Met Pro Tyr
20 25 30
Thr Arg Pro Phe Trp Asp Leu Thr Phe Pro Pro Glu Asp Pro Phe Lys
35 40 45
Ile Leu Glu Gln Ile Pro Leu Thr Ile Pro Lys Gly Val Asp Ser Ile
50 55 60
Ala Leu Ala Arg Ser Asp Trp Lys Glu Thr Gly Thr Glu His Val Ile
65 70 75 80
Thr Leu Asp Ile Pro Gly Met Lys Arg Asp Asp Ile Lys Ile Glu Val
85 90 95
Glu Glu Asn Arg Val Leu Arg Ile Ile Gly Glu Arg Lys Ile Glu Glu
100 105 110
Glu Val Glu Gly Glu Lys Trp His Arg Ala Glu Arg Thr Ser Gly Lys
115 120 125
Phe Trp Arg Gln Phe Arg Leu Pro Gly Asn Ala Asp Leu Glu His Ile
130 135 140
Lys Ala His Leu Glu Asn Gly Val Leu Lys Ile Thr Val Pro Lys Leu
145 150 155 160
Ala Glu Glu Lys Lys Lys Gln Thr Lys Val Ile Asn Ile Ala Glu Asp
165 170 175
Gly Asn Ser Ala Gly Gly Glu Asp Ile Lys Ala Thr Lys Ala Glu Met
180 185 190
Claims (8)
1. the encoding gene of tobacco heat shock protein HSP22HSP22, which is characterized in that its base sequence such as SEQ ID NO.1 institute
Show, wherein nucleic acid specific fragment is 202-477 bit base.
2. the coding described in claim 1HSP22Application of the gene in the regulation of leaf pigment substance content, feature exist
In using gene silent technology or gene overexpression method, by adjusting tobacco HSP22 expressing quantity, to adjust control
Colors content situation in tobacco leaf, the colors are as follows: neoxathin, violaxanthin, lutein, chlorophyll a/b, β-Hu
Radish element.
3. encoding gene described in claim 1HSP22Encoded tobacco heat shock protein HSP22, which is characterized in that its amino acid
Sequence is made of as shown in SEQ ID NO.2 192 amino acid residues, wherein the SHSP that 68-159 amino acids are conservative
Structural domain.
4. application of the tobacco heat shock protein HSP22 described in claim 3 in the regulation of leaf pigment substance content, feature exist
In, the albumen is related to colors content in plant leaf blade, after reducing the protein expression, colors content in blade
It is substantially reduced, the colors are as follows: neoxathin, violaxanthin, lutein, chlorophyll a/b, beta carotene.
5. utilizing encoding gene described in claim 1HSP22New product of tobacco breeding method, which is characterized in that by turning base
Because technology, transient expression technology or genome editing technique, building containHSP22Virus induction silent carrier, the RNAi of gene
Interference vector, overexpression vector or genome editor's carrier, transformation of tobacco, screening obtain the tobacco new product of pigment content variation
Kind.
6. new product of tobacco breeding method as claimed in claim 5, which is characterized in that utilize virus induced gene silencing skill
Art, interferenceHSP22The expression of gene makes its silencing,HSP22Colors content is remarkably decreased in gene silencing plant, in turn
Obtain the new variety of plant of pigment content decline.
7.PCR expands encoding gene described in claim 1HSP22Primer sequence, which is characterized in that primer is designed specifically to:
NtHSP22-F:5 '-ACCGAATTCGCTCAGACTGGAAGGAAACA- 3 ',
NtHSP22-F:5 '-ACCGGATCCGCTTTAATGTCCTCACCACC- 3 '.
8. utilizing the encoding gene of primer described in claim 7HSP22PCR amplification method, which is characterized in that with tobacco K326
CDNA be template, using NtHSP22-F, NtHSP22-F as primer, carry out PCR amplification.
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| CN110903371A (en) * | 2019-12-19 | 2020-03-24 | 中国烟草总公司郑州烟草研究院 | Tobacco cellobiose-related protein NtCRP1 and application thereof |
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| CN110903371B (en) * | 2019-12-19 | 2022-06-07 | 中国烟草总公司郑州烟草研究院 | Tobacco cellobiose-related protein NtCRP1 and application thereof |
| CN111499712A (en) * | 2020-06-04 | 2020-08-07 | 中国烟草总公司郑州烟草研究院 | Tobacco NtDREB-1B L2 transcription factor and application thereof |
| CN111533794A (en) * | 2020-06-04 | 2020-08-14 | 中国烟草总公司郑州烟草研究院 | Tobacco NtDREB-1BL1 transcription factor and application thereof |
| CN113151296A (en) * | 2021-03-22 | 2021-07-23 | 云南中烟工业有限责任公司 | Tobacco heat shock protein related gene and application thereof |
| CN113151296B (en) * | 2021-03-22 | 2022-09-13 | 云南中烟工业有限责任公司 | Tobacco heat shock protein related gene and application thereof |
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