CN104059925A - Application of Arabidopsis thaliana zinc finger protein gene GIS2 in promoting growth of tobacco tentacles - Google Patents
Application of Arabidopsis thaliana zinc finger protein gene GIS2 in promoting growth of tobacco tentacles Download PDFInfo
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- CN104059925A CN104059925A CN201410255006.3A CN201410255006A CN104059925A CN 104059925 A CN104059925 A CN 104059925A CN 201410255006 A CN201410255006 A CN 201410255006A CN 104059925 A CN104059925 A CN 104059925A
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Abstract
The invention discloses application of an Arabidopsis thaliana zinc finger protein gene GIS2 in promoting growth of tobacco tentacles. The nucleotide sequence of the Arabidopsis thaliana zinc finger protein gene GIS2 is disclosed as SEQ ID NO.1. The application comprises the following steps: connecting the Arabidopsis thaliana zinc finger protein gene GIS2 into a plant expression vector to construct a recombinant expression vector, and transforming tobacco with the recombinant expression vector. By cloning the Arabidopsis thaliana zinc finger protein gene GIS2 and carrying out genetic transformation in the tobacco, the overexpression of the exogenous GIS2 can obviously increase the quantity of the tobacco leaf tentacles and enhance the tentacle growth density without influencing the quantity of protective hair which does not secrete secondary metabolites. The application is beneficial to researching the regulation mechanism of tentacle formation and development and the genetic development of tentacle density, enhancing the aroma quality of the tobacco leaf, and researching the mechanism for controlling the tobacco tentacle and aroma formation.
Description
Technical field
The invention belongs to technical field of bioengineering, relate in particular to a kind of Arabidopis thaliana zinc finger protein gene GIS2 in the application promoting in the growth of tobacco glandular hairs.
Background technology
Tobacco has become one of most important cash crop on 21 century world market, it is not only more than 100 a national tobacco grower revenue source, and whole tobacco industry relates to different production departments and wholesale and retail link, become the Important Economic strength of many countries; Along with the development of tobacco, the place of many countries and the tax revenue of local government have had and have increased substantially.
Tobacco is high benefit crop, and cultivated area and the output of China tobacco all rank first in the world.In all Tobacco Types, tobacco planting is the most extensive.China is the country that worldwide production flue-cured tobacco is maximum, and China's tobacco planting area and ultimate production all occupy first place in the world, and its yield of flue-cured tobacco accounts for 50% left and right of global yield of flue-cured tobacco, and development sound tobacco is no matter to tobacco grower or cigarette industry is all very important.Tobacco is again high taxes and profits commodity, agriculture taxes and profits 30% left and right, and industrial taxes and profits are more than 60%, 1987-2001, the taxes and profits that tobacco industry is realized add up to be in first of national economy every profession and trade for continuous 14 years, for huge contribution has been made in nation-building.Therefore, the good tobacco bred of research and development Comprehensive Traits, improves the production level of tobacco for increasing country and local finance income, solves agriculture, rural areas and farmers problem and new countryside construction tool and is of great significance.
The problem that tobacco breeding emphasis is considered is tobacco leaf fragrance and tobacco leaf security etc., and improving flavouring essence quality is one of core of domestic and international tobacco breeding.Tabacco fragrance deficiency is a principal element that affects quality of tobacco, and the tobacco industry developing direction of low nicotine, low tar, makes the problem of tobacco leaf insufficient fragrance more outstanding especially at present.Tobacco glandular hairs are important component parts of tobacco leaf epidermis system, the gluey secretory product of Tobacco Glandular Trichome and tabacco fragrance material produce closely related, and glandular hairs density and type directly affect the quality and quantity of Trichome exudates, and the amount of secretory product directly affects the flavouring essence quality of blade, glandular hairs density is larger, the glandular hairs proportion of many cells head is larger, blade flavouring essence quality better (tobacco leaf glandular hairs and secretory product progress thereof. Guizhou Agricultural Sciences, 2009,37 (8): 61～64).Therefore, the seed selection of the research of Tobacco Glandular Trichome to tobacco new variety and the enforcement of High Quality Tobacco cultivation technique, and the research of raising tobacco aroma matter and perfume quantity all has important directive significance.Although carried out large quantity research aspect tobacco glandular hairs both at home and abroad at present, but glandular hairs are formed and the regulatory mechanism of growth and the genetic development of glandular hairs density, particularly between glandular hairs and tobacco aroma formation, aspect the research of relation, be also nowhere near, therefore, from the mechanism of molecular level research control tobacco glandular hairs and fragrance formation, not only there is important theory significance, also there is potential using value simultaneously.
Summary of the invention
The invention provides a kind of Arabidopis thaliana zinc finger protein gene GIS2 in the application promoting in the growth of tobacco glandular hairs, in tobacco, heterogenous expression Arabidopis thaliana finger protein gene GIS2 can improve the stand density of tobacco leaf glandular hairs.
Arabidopis thaliana zinc finger protein gene GIS2 is in the application promoting in the growth of tobacco glandular hairs, and the nucleotide sequence of described Arabidopis thaliana zinc finger protein gene GIS2 is as shown in SEQ ID NO.1.
Described application, comprising: described Arabidopis thaliana zinc finger protein gene GIS2 is connected in plant expression vector, builds and obtain recombinant expression vector, then by described recombinant expression vector transformation of tobacco.
The structure of recombinant expression vector can adopt ordinary method, as adopted Gateway system (Invitrogen company) that Arabidopis thaliana GIS2 gene (being Arabidopis thaliana zinc finger protein gene GIS2) is connected in plant expression vector.
Described plant expression vector can be pH2GW7, pK2GW7 etc.
In described recombinant expression vector, the promotor that starts described Arabidopis thaliana GIS2 genetic expression is strong promoter.Strong promoter can start Arabidopis thaliana GIS2 gene overexpression, can improve the stand density of tobacco leaf glandular hairs.Described strong promoter is specifically as follows 35S promoter.
When transformation of tobacco, can adopt the method for agrobacterium mediation converted, described Agrobacterium is specifically as follows Agrobacterium GV3101, EHA105 etc.
The kind of described tobacco can be the western cigarette of coral, but is not limited only to the western cigarette of coral.
The present invention also provides a kind of recombinant expression vector, comprises initial carrier and inserts the goal gene of described initial carrier, and the nucleotide sequence of described goal gene is as shown in SEQ ID NO.1.
In described recombinant expression vector, described initial carrier can be pH2GW7, pK2GW7 etc.
The present invention also provides a kind of recombinant conversion that comprises described plant expression vector.
In described recombinant conversion, Host Strains can be Agrobacterium, is specifically as follows Agrobacterium GV3101, EHA105 etc.
Compared with prior art, beneficial effect of the present invention is:
The present invention is by clone's Arabidopis thaliana GIS2 gene; and in tobacco, carry out genetic transformation; overexpression external source Arabidopis thaliana GIS2 gene can significantly improve tobacco leaf glandular hairs quantity, improves glandular hairs stand density, and the protective hair quantity of not secreting secondary metabolite is not affected.The present invention contributes to study glandular hairs formation and the regulatory mechanism of growing and the genetic development of glandular hairs density, based on the substantial connection of glandular hairs and tobacco aroma, the present invention can improve tobacco leaf flavouring essence quality, contribute to research to control the mechanism of tobacco glandular hairs and fragrance formation, there is important theory significance and potential using value.
Brief description of the drawings
Fig. 1 is wild-type (WT) and transgene tobacco (35S:GIS2) blade table fur quantity statistics in the different length of time.
Embodiment
Below in conjunction with specific embodiment, the present invention is further explained.
Embodiment 1GIS2 gene overexpression vector construction and tobacco genetic transformation
(1) get Arabidopis thaliana inflorescence, liquid nitrogen grinding, adds TRIzol reagent (Invitrogen company), extracts RNA.Getting 0.5 μ g RNA carries out reverse transcription reaction and obtains cDNA.
(2) according to the GIS2 gene order design special primer on Genbank, add Sal I and Not I restriction enzyme site at primer 5 ‵ ends respectively, sequence is as follows:
Primer 1:5 ‵-TCAACTGTCGACAGCCATCCAGAGTCATAACCA-3 ‵;
Primer 2: 5 ‵-AAGATAGCGGCCGCGAATGGAACTAGAGGCGTAGA-3 ‵.
(3) carry out pcr amplification taking full-length cDNA as template.
Adopt high-fidelity enzyme
hS DNA Polymerase (TaKaRa Code:DR010A) system:
5×PrimeSTAR Buffer(Mg
2+plus)10μl，
DNTP Mixture (each 2.5mM) 4 μ l,
Primer 1 (10 μ M) 1 μ l,
Primer 2 (10 μ M) 1 μ l,
Template cDNA1 μ l,
HS DNA Polymerase0.5μl，
Sterile purified water adds to cumulative volume 50 μ l.
PCR cycling condition is: 98 DEG C of denaturations 5 minutes, and 98 DEG C of sex change 10 seconds, 55 DEG C of annealing 15 seconds, 72 DEG C are extended 90 seconds, 30 circulations of increase, last 72 DEG C are extended 10 minutes, finish to react.
(4) will after the recovery of PCR product, cut and be connected to Gateway cloning system (the application adopts the Gateway system of Invitrogen company) entry vector pENTR1A by enzyme, transform bacillus coli DH 5 alpha, check order and analyze, GIS2 gene order is as shown in SEQ ID NO.1.
(5) correct order-checking pENTR1A plasmid and Overexpression vector pH2GW7 containing GIS2 total length CDS are carried out to LR permutoid reaction.
Use Gateway LR Clonase TM Enzyme Mix (Invitrogen company, Cat.No.11791-019), system is as follows: Entry clone (entry vector) 50-150ng, Destination vector (object carrier) 150ng, 5 × LR Clonase Reaction Buffer2 μ l, ddH
2o up to8 μ l, adds 2 μ l LR Clonase enzyme mix, and of short duration vortex mixes for twice, slightly centrifugal, in 25 DEG C of water-bath 8h, then add 1 μ l Proteinase K solution (Proteinase K solution) to mix, place for 37 DEG C and within 10 minutes, finish reaction.
Get 5 μ l reaction product and transform bacillus coli DH 5 alpha, picking PCR checking positive colony extracts plasmid after cultivating and carries out double digestion checking.
(6) from-80 DEG C of refrigerators, take out EHA105 competent cell, utilize electric shocking method to transform Agrobacterium EHA105.
Competent cell is placed on ice and thaws, and the object plasmid of getting 2 μ l left and right is added in competent cell, mixes and is placed on 30min on ice, the more all mixtures in centrifuge tube are added in the electric shock cup of precooling, places on ice.Open Bole's electroporation, program is adjusted to Agr, draw the LB liquid nutritional base of 1ml, electric shock cup salient point is pushed to electroporation inwardly, click plus key, hear buzzer, take out rapidly electric shock cup, and add the LB liquid nutritional base of 1ml, re-suspended cell to electric shock cup cup, be transferred in the centrifuge tube of 1.5ml, be placed on interior 28 DEG C of eppendorf mixed instrument, more than 700rpm shakes 2.5h, coat (Rif (Rifampin) 50mg/L on resistance YEP flat board, Spec (spectinomycin hydrochloride) 100mg/L), be inverted for 28 DEG C and cultivate 2 evenings so that grow single bacterium colony.
(7) the Agrobacterium mono-clonal that picking grows, to containing in corresponding antibiotic 5mL YEP liquid nutrient medium (corresponding microbiotic is Rif50mg/L, spec100mg/L), 28 DEG C of 150rpm shaking culture 48 hours.By the bacterium shaking by volume 1:10 add 28 DEG C of YEP liquid nutrient mediums (containing corresponding microbiotic), be cultured to OD600>1.
(8) bacterium shaking being cooked to tobacco leaf contaminates:
1) MS liquid nutrient medium (PH7.0) is by 10 times (2mL bacterium liquid+18mL MS solution) of bacterium liquid dilution;
2) utilize aseptic tobacco (kind is the western cigarette of coral) tissue cultured seedling.Aseptic tobacco leaf cuts edge and main vein, is cut into 0.5 × 0.5cm
2size;
3) explant cutting soaks 5min in Agrobacterium bacterium liquid;
4) blot explant surface bacterium liquid with aseptic filter paper, proceed to the MS substratum of upper berth one deck filter paper, 25 DEG C of dark cultivations 3 days;
5) after three days, material is forwarded in the division culture medium (MS minimum medium+2mg/L6-BA+0.5mg/LNAA) after suitable selection and carries out illumination cultivation, 25 DEG C ± 2 DEG C of temperature, photoperiod 14hr illumination/8hr illumination;
6), wait growing to 2-3cm when high, cut root induction (basic MS+hpt (Totomycin) 35mg/L+cef (cephamycin) 250mg/L) in the root media that budlet proceeds to suitable screening;
7) root grows to 10cm above (about surrounding), and has some amount and can remove sealed membrane, in group training chamber, tempers 2-3 days, is then transplanted in flowerpot, in room temperature growth 2-3 week, finally transplants to field planting.Grow after about 20d when the tobacco seedling moving on in soil, get tobacco leaf and extract DNA, detect transfer-gen plant by PCR method.
The character observation of embodiment 2 transgene tobaccos
The epidermal hair of tobacco is by having or not gland to be divided into two large classes, and the one, can produce the glandular hairs of secretory product, the 2nd, do not there is the protective hair of secretion capacity, wherein glandular hairs are divided into long handle glandular hairs and short handle glandular hairs according to form again.
The transgene tobacco that embodiment 1 is obtained carries out glandular hairs phenotype statistics; as shown in Figure 1; overexpression Arabidopis thaliana GIS2 gene (sequence as shown in SEQ ID NO.1) in tobacco; compare with wild-type; can extremely significantly promote the 20 day length of time the 3rd and the 4 long glandular hairs of tobacco leaf upper surface (long handle glandular hairs) quantity and short glandular hairs (short bright glandular hairs) quantity (P<0.01), difference is not remarkable not secrete epidermal hair (protective hair) quantity of secondary metabolite.We have also added up the 40 day length of time and the 60 day length of time the 7th and the quantity of 8 leaf upper surface tobacco glandular hairs and protective hair (long epidermal hair and short epidermal hair) simultaneously; result shows that overexpression Arabidopis thaliana GIS2 gene can significantly increase the long glandular hairs quantity of tobacco and short glandular hairs quantity in tobacco, and difference is not remarkable not secrete epidermal hair (long epidermal hair, the short epidermal hair) quantity of secondary metabolite.
Claims (9)
1. Arabidopis thaliana zinc finger protein gene GIS2, in the application promoting in the growth of tobacco glandular hairs, is characterized in that, the nucleotide sequence of described Arabidopis thaliana zinc finger protein gene GIS2 is as shown in SEQ ID NO.1.
2. application as claimed in claim 1, is characterized in that, comprising: described Arabidopis thaliana zinc finger protein gene GIS2 is connected in plant expression vector, builds and obtain recombinant expression vector, then by described recombinant expression vector transformation of tobacco.
3. application as claimed in claim 2, is characterized in that, described plant expression vector is pH2GW7 or pK2GW7.
4. application as claimed in claim 2, is characterized in that, in described recombinant expression vector, the promotor that starts described Arabidopis thaliana zinc finger protein gene GIS2 expression is strong promoter.
5. application as claimed in claim 4, is characterized in that, described strong promoter is 35S promoter.
6. a recombinant expression vector, comprises initial carrier and inserts the goal gene of described initial carrier, it is characterized in that, the nucleotide sequence of described goal gene is as shown in SEQ ID NO.1.
7. recombinant expression vector as claimed in claim 6, is characterized in that, described initial carrier is pH2GW7 or pK2GW7.
8. recombinant conversion that comprises recombinant expression vector as described in claim 6 or 7.
9. recombinant conversion as claimed in claim 8, is characterized in that, Host Strains is Agrobacterium GV3101 or EHA105.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106834306A (en) * | 2017-03-16 | 2017-06-13 | 贵州省烟草科学研究院 | The application of tobacco C2H2 type zinc finger protein genes Nt540 |
| CN108070594A (en) * | 2017-12-22 | 2018-05-25 | 河南农业大学 | Tobacco glandular hairs TTR1 promoters, its expression vector and its application |
| CN113234726A (en) * | 2021-06-21 | 2021-08-10 | 贵州省烟草科学研究院 | Tobacco glandular hair specific promoter pNtTCP9a and application thereof |
-
2014
- 2014-06-10 CN CN201410255006.3A patent/CN104059925A/en active Pending
Non-Patent Citations (2)
| Title |
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| 孟小芳: "拟南芥GIS家族基因在烟草中功能的验证", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
| 蔡刘体等: "烟草叶片表面腺毛形态的扫描电镜观察", 《农业生物技术科学》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106834306A (en) * | 2017-03-16 | 2017-06-13 | 贵州省烟草科学研究院 | The application of tobacco C2H2 type zinc finger protein genes Nt540 |
| CN106834306B (en) * | 2017-03-16 | 2020-10-23 | 贵州省烟草科学研究院 | Application of tobacco C2H2 type zinc finger protein gene Nt540 |
| CN108070594A (en) * | 2017-12-22 | 2018-05-25 | 河南农业大学 | Tobacco glandular hairs TTR1 promoters, its expression vector and its application |
| CN108070594B (en) * | 2017-12-22 | 2020-10-27 | 河南农业大学 | Tobacco glandular hairTTR1Promoter, expression vector and application thereof |
| CN113234726A (en) * | 2021-06-21 | 2021-08-10 | 贵州省烟草科学研究院 | Tobacco glandular hair specific promoter pNtTCP9a and application thereof |
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Application publication date: 20140924 |