CN110092822A - Clerodendron trichotomum NAC gene C tNAC1 and its application - Google Patents
Clerodendron trichotomum NAC gene C tNAC1 and its application Download PDFInfo
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- CN110092822A CN110092822A CN201910530119.2A CN201910530119A CN110092822A CN 110092822 A CN110092822 A CN 110092822A CN 201910530119 A CN201910530119 A CN 201910530119A CN 110092822 A CN110092822 A CN 110092822A
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- clerodendron trichotomum
- gene
- tnac1
- trichotomum
- clerodendron
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Abstract
The invention discloses clerodendron trichotomum NAC gene C tNAC1 and its applications.The present invention chooses the clerodendron trichotomum of two provenances, clone obtains a new NAC gene from clerodendron trichotomum tissue, it is named as CtNAC1, its nucleotide sequence is as shown in SEQ ID NO.1, the amino acid sequence of albumen is expressed as shown in SEQ ID NO.2, and CtNAC1 gene expression amount is measured analysis by qRT-PCR technology to CtNAC1 gene expression amount in clerodendron trichotomum different tissues and under the clerodendron trichotomum salt stress different disposal time in blade, the results showed that NAC gene C tNAC1 expresses higher in the stem of clerodendron trichotomum.Under salt stress processing, gene expression amount highest when reacting 12h participates in the growth and development of clerodendron trichotomum stem and the response of salt stress, can be used for improveing in the adversity gene engineering of plant, have a extensive future.
Description
Technical field
The invention belongs to gene engineering technology fields, and in particular to clerodendron trichotomum NAC gene C tNAC1 and its application.
Background technique
NAC gene family is distributed widely in each species of plant kingdom, be maximum transcription factor family specific to plant it
One (Zhou Honghui etc., 2017), NAC gene family have five subprovinces A, B, C, D, E, are originally found in petunia, Zhi Hou
It has also been found that NAC gene family (Duan Aoqi etc., 2018), has been demonstrated that NAC gene family wide participation plant sends out in other plant
Educate, plant biological and abiotic stress response, and can enhance plant to the defence of stress and tolerance (Tak H et al.,
2018), as in white birch BpNAC012 salt stress and osmotic stress tolerance (Hu et al., 2018) can be enhanced.
Clerodendron trichotomum (Clerodendrum trichotomum Thunb.) also known as clerodendron trichotomum are Verenaceae
(Verbenaceae) paulownia category (Clerodendrum L.) shrub or dungarunga are remitted, flowers and fruits are beautiful, are a kind of summer to see flower, winter
The excellent ornamencal flower and tree (Yang Xiulian etc., 2019) of Ji Guanguo.Furthermore clerodendron trichotomum has medicinal function, and leaf, limb and root are equal
Can be used as medicine, have the effects that anti-hypertension, arthritis, rheumatism and it is anti-inflammatory (Hu Haijun etc., 2014；Et al., 2017).
The coastal soil salinization is always a worldwide problem, and suitable coastal Soiline-alkali plants is selected to restore coastal ecology
Key measure (He et al., 2014；Li et al., 2010).Clerodendron trichotomum has bad abiotic stress good anti-
Property possesses salt tolerant (Yue Yuanzheng etc., 2018), resistance to water logging (Zeng De waits quietly, 2013), high temperature resistant (Chen Yan etc., 2015) and drought-resistant
Merits such as (Wei Juan etc., 2009), especially there is excellent resistance to salt stress, are that one kind can be used for restoring coastal salt-soda soil
The salt-tolerant plant (Li Ya etc., 2017) of ecology.Clerodendron trichotomum divides under salt stress about it at present as excellent salt-tolerant plant
The research of sub- response mechanism is still relatively lacking.Molecules in response mechanism of the clerodendron trichotomum under salt stress is verified, for clerodendron trichotomum
It is had a very important significance on ecological recovery.
Summary of the invention
Goal of the invention: for the deficiency of technology, the object of the present invention is to provide clerodendron trichotomum NAC gene C tNAC1, with reality
Further research of the existing NAC gene in clerodendron trichotomum salt stress response mechanism.It is a further object of the present invention to provide Haizhou
The expression albumen of Changshan NAC gene C tNAC1.Another object of the present invention is to provide said gene and is improving the clerodendron trichotomum salt side of body
Compel the application in tolerance, expression of the gene in the stem of clerodendron trichotomum is higher.Under salt stress processing, when reacting 12h
Gene expression amount highest participates in the growth and development of clerodendron trichotomum stem and root and the response of salt stress, can be used in the degeneration-resistant of plant
Genetic engineering improvement, has a extensive future.
Technical solution: in order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention are as follows:
Clerodendron trichotomum NAC gene C tNAC1, nucleotide sequence is as shown in SEQ ID NO.1.
The expression albumen of clerodendron trichotomum NAC gene C tNAC1, amino acid sequence is as shown in SEQ ID NO.2.
The clerodendron trichotomum NAC gene C tNAC1 is improving the application in clerodendron trichotomum Salt Stress Tolerance.
The expression vector of the clerodendron trichotomum NAC gene C tNAC1.
The host strain of the clerodendron trichotomum NAC gene C tNAC1.
The clerodendron trichotomum NAC gene C tNAC1 cloning primer are as follows: upstream primer: 5 '-
CACTAACAAATCCCCAACTCAT-3′；Downstream primer: 5 '-CTCAAGCTCAAAGGAAAAGAAC-3 '.
The utility model has the advantages that compared with prior art, the present invention chooses the clerodendron trichotomum of two provenances, from clerodendron trichotomum tissue
Clone obtains a new NAC gene, is named as CtNAC1, and by qRT-PCR technology in clerodendron trichotomum different tissues
CtNAC1 gene expression amount and CtNAC1 gene expression amount is measured in blade under the clerodendron trichotomum salt stress different disposal time
Analysis, the results showed that NAC gene C tNAC1 expresses higher in the stem of clerodendron trichotomum.Under salt stress processing, base when reacting 12h
Because of expression quantity highest, the growth and development of clerodendron trichotomum stem and the response of salt stress are participated in, can be used for the adversity gene work in plant
Cheng Gailiang has a extensive future.
Detailed description of the invention
Fig. 1 is CtNAC1 gene expression spirogram in clerodendron trichotomum different tissues；
Fig. 2 is CtNAC1 gene expression spirogram in blade under the clerodendron trichotomum salt stress different disposal time.
Specific embodiment
The present invention is further illustrated combined with specific embodiments below, but these examples are not intended to limit the invention.
Main material to be tested used in following embodiment are as follows: use and be planted in Nanjing Forestry University's Baima teaching research base
Two clerodendron trichotomum provenances in Tai'an, Yancheng in ground.
Embodiment 1
The selection of 1 clerodendron trichotomum histoorgan
Sampling time is on September 13rd, 2018.Under weather condition ideal conditions, clerodendron trichotomum tissue sample is acquired.Choosing
It selects well-grown, height and more consistent 3 plant of growing way (repeating as 3 biology) to be sampled, every plant is adopted respectively
Collect the tissue such as root, stem, leaf, flower, fruit, is put into after acquisition in the centrifuge tube of sterile no enzyme, and take back experiment with liquid nitrogen flash freezer rapidly
Room is set -80 DEG C of ultra low temperature freezers and is saved backup.
The preparation of 2 Stress treatment materials
When acquiring clerodendron trichotomum tissue sample, while the cuttings of two provenances is acquired, cuttings cuttage is (precious in mixed-matrix
Zhu Yan: vermiculite: peat: husky=1: 1: 1: 1:2) in, it is placed in culturing room's culture (25 DEG C, 60%RH), periodically pours maintenance, is guaranteed
Normal growth uses after cuttings grows 4 pairs with blade.All Stress treatments are completed in artificial climate incubator
Clerodendron trichotomum cuttage seeding is put into every bottle of culture medium and is by (Ningbo southeast instrument plant RDN-1000-3), the last week to be processed
It in the 1/4MS culture medium of 150mL, moves into artificial climate incubator and adapts to one week, setting light application time is 14h, diurnal temperature
It is 25/21 DEG C, intensity of illumination 180mmol/m2/ s, relative humidity 60%.Cuttings is placed in the culture medium containing 1.0%NaCl
In (every bottle is 150mL culture medium, is determined used in the present embodiment with reference to clerodendron trichotomum salt-tolerant threshold 0.8%NaCl concentration
NaCl concentration), in stress 0h, 2h, 6h, 12h, for 24 hours, 48h and 72h acquire cuttings blade.
3 clerodendron trichotomum CtNAC1 clone
Clerodendron trichotomum Total RNAs extraction is all made of the EASY spin Plus kit of Beijing Ai Delai biology Co., Ltd,
The RNA of extraction first passes through the integrality that RNA is verified with 1.5% agarose gel electrophoresis, all OD260/OD2801.8~2.0
Between, OD260/OD230> 2.0, RNA at least 2 clear bands in all samples, 18S/28S band substantially 2:1 can just be adopted
With.The detection of nucleic acids instrument UV5NANO detection RNA mass and concentration produced with Mettler company.Use Beijing Quan Shi King Company
EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix reverse transcription reagent box reagent
Box synthesizes first chain of cDNA.
A clerodendron trichotomum NAC full length gene is obtained according to existing transcript profile sequencing data storehouse, uses primer
5.0 design primer of premier, NAC transcription factor full length gene clone's forward primer 5 ' to 3 ', which is held, is
CACTAACAAATCCCCAACTCAT, reverse primer 5 ' to 3 ' are held as CTCAAGCTCAAAGGAAAAGAAC, design of primers principle,
Method (Wang Xi etc., 2018, http://kns.cnki.net/kcms/ of PCR reaction system and response procedures referring to Wang Xi et al.
Detail/detail.aspx? filename=NNXB201804021&dbcode=CJFD&dbname=cJFD2018&v
=), by gel extraction, product is connected and is converted, selected 3 positive colonies and send to the survey of Nanjing Jin Sirui biology Co., Ltd
Sequence.
It is 1253bp that length is obtained after sequencing, as shown in SEQ ID NO.1, is CtNAC1 by this sequence designations, encodes egg
White is the sequence of 333 amino acid composition, as shown in SEQ ID NO.2.
4 clerodendron trichotomum CtNAC1 expression analysis
QRT-PCR is carried out using the Ex TaqTM dyestuff of TaKaRa company, fluorescent quantitation instrument model Sai Mofei company
plied Biosystems StepOne PCR System.Use 5.0 design primer of primer premier, forward primer 5 '
It holds to 3 ' as CCGCCATTTCTGGTGTTCA, reverse primer 5 ' to 3 ' is held as GGCAGGTTGTCCGAGTTCC, internal reference selection class
Be applicable under the clerodendron trichotomum tissue and salt stress that screen before topic group reference gene (UBCE2 (primer composition sequence:
F:GCAAAGGCTGATTGATGAGATTC R:CCTCAACATTGTCTTGGGTGG) and RPL (primer composition sequence
F:AGTCAATGGTGGCGATGTAGC
R:CCCTTGGTCACTCCGATAATGT)), fluorescence real-time quantitative design of primers principle, PCR reaction system and program
Referring to mother Hong Na et al. method (Mu et al., 2017, https: //link.springer.com/article/
10.1007%2Fs12041-017-0769-8).
Choose the two provenance clerodendri,folium, flower, fruit, root and stems as tissue sample and choose two provenance Haizhou it is normal
Blade under the cuttage seeding salt stress of mountain uses UBCE2 and RPL as reference gene, carries out glimmering in real time as Stress treatment sample
Fluorescent Quantitative PCR.As a result as shown in Figure 1, in the tissue, CtNAC1 gene is expressed at most in stem, the gene expression amount in spending
At least, gene expression amount of provenance CtNAC1 in Tai'an in stem is 13.27 times in spending, base of Yancheng provenance CtNAC1 in stem
Because expression quantity be spend in 55.56 times.Tai'an provenance and Yancheng provenance CtNAC1 gene expression amount are at identical group on the whole
It knits under position, the gene expression amount of Tai'an provenance is higher than the gene expression amount of Yancheng provenance.
As a result as shown in Fig. 2, CtNAC1 gene expression amount preceding 12h in Yancheng provenance and Tai'an provenance is up-regulation, it
The trend that gene expression amount is lowered is shown afterwards.Tai'an provenance CtNAC1 is in 46.86 times that the expression quantity of 12h is 0h, Yancheng provenance
It is then 18.71 times, down regulation trend is presented in gene expression amount after 12h.
Sequence table
<110>Nanjing Forestry University
<120>clerodendron trichotomum NAC gene C tNAC1 and its application
<130> 1
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1253
<212> DNA
<213>clerodendron trichotomum (Clerodendrum trichotomum Thunb.)
<400> 1
caatttttct caagctcaaa ggaaaagaac tgttttcatt ttcgtctgcc ggatcagaaa 60
aatgggagtc cgacaaaccg accagctttc acaattgagt ttgcctccag gatttcggtt 120
ctacccgact gatgaggagc ttttggtgca gtacctctgc agaaaagttg ctggacagca 180
cttctctcta cagattattg cagatattga tttgtacaaa tttgatcctt gggaccttcc 240
cagcaaagct ttgtttggag aaaaagaatg gtattttttc accccaagag acagaaagta 300
cccaaatgga tcacggccca acagagtagc aggctctgga tattggaaag ccacagggac 360
tgataaggtt attacgactg aagggagaaa agtgggtatt aagaaagccc ttgtttttta 420
cgttggaaaa gcacctaaag gaaccaaaac caactggatt atgcatgaat acaggctttc 480
tgattctccc agaaaaaatg tcagcgctag gttggatgat tgggttcttt gtcggattta 540
caagaagaac tcaagcgcac aaaagcccgc catttctggt gttcagagca aagagtacag 600
tagtctggga tcgtcatcat cctcttcatc tcaatacgac gacatgctgg agtcgctgac 660
agaaattgat gaccgtttcc tctctttacc taaaatgaaa tctcagcagg aagactataa 720
gctaaatctc cagcacttgg gctctggaaa ttttgattgg gccacactag ctgggctcaa 780
cctactgccg gaactcggac aacctgccca gcagacacaa atgattagta gtagcatgaa 840
ttgtcagaat gacatatatg ccccttcttt gcatcacctt tgtgggaatg cggatgtggt 900
ggatgaagaa gtccagagtg ggctcaaagc acagcgggtg ggccgatctg aattttatcc 960
acaaaatgtc ggcgtgttgg gtcaggggct gtcaaacttg gttgacccgt tttctgttcg 1020
gcacccgacc cagtctggtg ggttgggttt taggaagtga atactaaatg agttggggat 1080
ttgttagtga taggtcaata gctagggaga ttctgtaaat acatagaatt ctttgggcta 1140
tcgtcttccg ggccataaaa tatagaatac cggctttgcc cccattcaaa aaaatttata 1200
ttcacgatca catttaaaat gtattttgtc gcaggggatg agtgtgaaat ttg 1253
<210> 2
<211> 333
<212> PRT
<213>clerodendron trichotomum (Clerodendrum trichotomum Thunb.)
<400> 2
Lys Met Gly Val Arg Gln Thr Asp Gln Leu Ser Gln Leu Ser Leu Pro
1 5 10 15
Pro Gly Phe Arg Phe Tyr Pro Thr Asp Glu Glu Leu Leu Val Gln Tyr
20 25 30
Leu Cys Arg Lys Val Ala Gly Gln His Phe Ser Leu Gln Ile Ile Ala
35 40 45
Asp Ile Asp Leu Tyr Lys Phe Asp Pro Trp Asp Leu Pro Ser Lys Ala
50 55 60
Leu Phe Gly Glu Lys Glu Trp Tyr Phe Phe Thr Pro Arg Asp Arg Lys
65 70 75 80
Tyr Pro Asn Gly Ser Arg Pro Asn Arg Val Ala Gly Ser Gly Tyr Trp
85 90 95
Lys Ala Thr Gly Thr Asp Lys Val Ile Thr Thr Glu Gly Arg Lys Val
100 105 110
Gly Ile Lys Lys Ala Leu Val Phe Tyr Val Gly Lys Ala Pro Lys Gly
115 120 125
Thr Lys Thr Asn Trp Ile Met His Glu Tyr Arg Leu Ser Asp Ser Pro
130 135 140
Arg Lys Asn Val Ser Ala Arg Leu Asp Asp Trp Val Leu Cys Arg Ile
145 150 155 160
Tyr Lys Lys Asn Ser Ser Ala Gln Lys Pro Ala Ile Ser Gly Val Gln
165 170 175
Ser Lys Glu Tyr Ser Ser Leu Gly Ser Ser Ser Ser Ser Ser Ser Gln
180 185 190
Tyr Asp Asp Met Leu Glu Ser Leu Thr Glu Ile Asp Asp Arg Phe Leu
195 200 205
Ser Leu Pro Lys Met Lys Ser Gln Gln Glu Asp Tyr Lys Leu Asn Leu
210 215 220
Gln His Leu Gly Ser Gly Asn Phe Asp Trp Ala Thr Leu Ala Gly Leu
225 230 235 240
Asn Leu Leu Pro Glu Leu Gly Gln Pro Ala Gln Gln Thr Gln Met Ile
245 250 255
Ser Ser Ser Met Asn Cys Gln Asn Asp Ile Tyr Ala Pro Ser Leu His
260 265 270
His Leu Cys Gly Asn Ala Asp Val Val Asp Glu Glu Val Gln Ser Gly
275 280 285
Leu Lys Ala Gln Arg Val Gly Arg Ser Glu Phe Tyr Pro Gln Asn Val
290 295 300
Gly Val Leu Gly Gln Gly Leu Ser Asn Leu Val Asp Pro Phe Ser Val
305 310 315 320
Arg His Pro Thr Gln Ser Gly Gly Leu Gly Phe Arg Lys
325 330
Claims (6)
1. clerodendron trichotomum NAC gene C tNAC1, nucleotide sequence is as shown in SEQ ID NO.1.
2. the expression albumen of clerodendron trichotomum NAC gene C tNAC1 described in claim 1, amino acid sequence such as SEQ ID
Shown in NO.2.
3. clerodendron trichotomum NAC gene C tNAC1 described in claim 1 is improving the application in clerodendron trichotomum Salt Stress Tolerance.
4. containing the expression vector of clerodendron trichotomum NAC gene C tNAC1 described in claim 1.
5. containing the host strain of clerodendron trichotomum NAC gene C tNAC1 described in claim 1.
6. clerodendron trichotomum NAC gene C tNAC1 according to claim 1, which is characterized in that the NAC gene C tNAC1's
Cloning primer are as follows: upstream primer: 5 '-CACTAACAAATCCCCAACTCAT-3 '；Downstream primer: 5 '-
CTCAAGCTCAAAGGAAAAGAAC-3′。
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