CN106148393A

CN106148393A - The open country chrysanthemum flame early blossoming strain of safety label is obtained by transgenic method

Info

Publication number: CN106148393A
Application number: CN201510252695.7A
Authority: CN
Inventors: 张旸; 解莉楠; 赵月明; 丁兵; 王江; 李玉花
Original assignee: Northeast Forestry University
Current assignee: Northeast Forestry University
Priority date: 2015-05-18
Filing date: 2015-05-18
Publication date: 2016-11-23

Abstract

Obtain open country chrysanthemum ' flame ' early blossoming strain by turning AtLEAFY gene, belong to gene engineering technology field.The present invention utilizes agrobcterium-mediated transformation, by perfect genetic conversion system, and utilize the non-antibiotic marker PMI to bio-safety, screening obtains the early blossoming strain of the open country chrysanthemum ' flame ' with safety label, not only robust plant, flowering time Zao 9-11 days than wild type, full-bloom stage is in left and right at the beginning of by the end of September 10 months, meet the demand of flower in red-letter day, there is the highest afforestation and ornamental value.

Description

The open country chrysanthemum flame early blossoming strain of safety label is obtained by transgenic method

Technical field

The invention belongs to gene engineering technology field, relate to the early blossoming new lines obtaining a kind of open country chrysanthemum ' flame '.

Background technology

Open country chrysanthemum (Chrysanthemum morifolium); for Compositae Chrysanthemum, herbaceos perennial; it is one of China tradition outdoor cropping ornamental plant, there is the features such as pattern is abundant, florescence length, plant type are neat, be widely used in scale afforestation.At the cold district of high latitude, open country chrysanthemum can normal growth, but it was relied on by the photoperiod, and flowering time has focused largely on JIUYUE to October, and full-bloom stage is just met the frost phase, and open country chrysanthemum pattern after the Frost's Descent starts to take off change, caused that its sight declines, viewing period significantly shortens.But the rise of technique for gene engineering, may be oriented breeding and do not change other character, i.e. use the Protocols in Molecular Biology of advanced person, genes of interest or DNA fragmentation by carrier or are introduced directly into recipient cell, make hereditary material reconfigure.Owing to the safety issue of transgenic technology receives much concern, the present invention is to utilize non-antibiotic marker gene 6-Phophomannose isomerase gene (PMI) to bio-safety, with mannose for screening medium, only proceed to the outer planting somatic cell energy metabolism 6-phosphomannose of PMI gene, thus providing energy for the differentiation of cell normal growth, the cell not proceeding to PMI gene then can not continued growth in screening culture medium.

Flos Chrysanthemi genetic transforming method mainly has agrobacterium-mediated transformation and direct guiding method, and agrobacterium-mediated transformation, with advantages such as its easily operation, big, the good stabilities of low expense, high efficiency, insertion DNA fragmentation, becomes the prefered method in transgenic technology.Transgenic flower can substantially overcome the blindness of current conventional breeding, shortens breeding cycle, can create with high content of technology, market competition advantage obvious Flower New Variety kind.Flowers have high ornamental value, the tremendous economic interests simultaneously also contained, and the development of flower gene engineering is very rapid, and since Meyer in 1987 etc. obtain transgenic petunia, flower gene engineering has become the new trend of flower breeding.It is by suppression endogenous gene or imports exogenous gene thus directional transformation flowers, shortens the time of breeding of new variety, and can break through incompatible restriction between kind, introduce the gene of other species, improves flower characteristic.

Summary of the invention

The invention aims to obtain open country chrysanthemum ' flame ' new lines of Blooming, by technique for gene engineering, from arabidopsis, clone LEAFY gene.Utilize Gateway technique construction plant over-express vector, the carrier pCAMBIA1301-PMI-AtLFY electricity of structure is proceeded in Agrobacterium EHA105.Infect outer implant open country chrysanthemum ' flame ' blade by Agrobacterium and carry out genetic transformation, through preculture, co-culture, early stage screening and culturing, later stage screening and culturing, take root the stages such as screening and culturing, utilizing bio-safety labelling phosphomannose isomerase (PMI) gene on carrier to screen in the culture medium containing mannose, final acquisition turns resistant plant 218 strain of AtLFY gene.Then these resistant plants utilize the methods such as PCR, GUS dyeing, Western blot carry out molecular Biological Detection.Have detected PMI gene and the specific gene AtLFY of resistant plant during PCR detection respectively, detection obtains positive plant 13 strain turning AtLFY gene.This 13 strain transfer-gen plant and wild type are proceeded to field simultaneously, observe transfer-gen plant budding the time, begin to take time, contain and take time and compare with WT lines, screening obtains open country chrysanthemum ' flame ' early blossoming strain, make its full-bloom stage early than the phase in the Frost's Descent, extending and view and admire the time, therefore the present invention carries out the florescence control to open country chrysanthemum ' flame ' is the most necessary.

Accompanying drawing explanation

Fig. 1 is pCAMIB1301 carrier figure；Fig. 2 is that resistant plant PCR detects electrophoretogram；Fig. 3 is the GUS dyeing testing result figure of transfer-gen plant；The phenotypic evaluation (squaring period) of Fig. 4 transfer-gen plant；The phenotypic evaluation (revealing the red phase) of Fig. 5 transfer-gen plant；The phenotypic evaluation (just opening the phase) of Fig. 6 transfer-gen plant；The phenotypic evaluation (full-bloom stage) of Fig. 7 transfer-gen plant.

Detailed description of the invention

Technical solution of the present invention is as follows:

(1) clone and the over-express vector of target gene AtLEAFY builds

First with the method for RT-PCR, from arabidopsis, clone obtains gene LEAFY (Sequence ID:ref | NM_125579.1, Length:1263), utilizes Gateway technique construction over-express vector, and the primer of construction of expression vector is:

attB-LFY–F 5′-AAAAAGCAGGCTATGGATCCTGAAGGTTTCACG-3′

attB-LFY–R 5′-AGAAAGCTGGGTCTAGAAACGCAAGTCGTCGC-3′

attB-adapter-F 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3′

attB-adapter-R 5′-GGGGACCACTTTGTACAAGAAAGCTGGGT-3′

Then utilize Agrobacterium multigelation method, by the process LAN built in body proceeds to Agrobacterium competent cell, Agrobacterium bacterium solution is all applied on a LB solid medium flat board, 20 DEG C of quiescent culture 24hrs.The single bacterium colony of picking is (Kana and rif resistance) in LB fluid medium, shakes at 28 DEG C and uses attB-adapter-F/R primer to carry out PCR detection after bacterium is cultivated, PCR primer is sent order-checking and to preserve strain standby.

(2) genetic transformation and the transfer-gen plant of open country chrysanthemum ' flame ' is identified

Carrying out open country chrysanthemum ' flame ' genetic transformation first with agriculture bacillus mediated method, concrete grammar is as follows:

(1) preculture: with open country chrysanthemum ' flame ' aseptic seedling young leaflet tablet for examination material, blade is cut into 1cm × 1cm fritter, is inoculated on pre-culture and carries out preculture 1 day.

(2) bacterium is shaken: 3mL LB liquid+3 μ L Kana+3 μ L rif adds 5 μ L Agrobacterium bacterium solution, 28 DEG C of 160rpm 12hrs.

(3) infect and co-culture: the bacterium solution overnight shaken being taken 1ml and adds in 50ml LB liquid and add 50 μ L Kana, 28 DEG C of 160rpm about two hours, survey its OD₆₀₀Value so that it is reach 0.5-0.6.Putting in bacterium solution by pre-incubated blade and adding acetosyringone concentration is 500mg/L, infects 10min, bacterium solution to be shaken gently for when infecting, and makes each piece of outer implant can be fully contacted with bacterium solution.Suck the unnecessary bacterium solution of blade surface attachment with sterilizing filter paper, after blade surface is without obvious moisture, is then accessed and co-culture in culture medium, light culture 2 days.

(4) screening and culturing: use the mode selecting pressure to be incremented by screen, the blade co-cultured is transferred in superclean bench in the culture medium of early stage screening, when on outer implant blade it can be seen that proceeded to after small callus later stage screening and culturing culture medium is further screened, within every 15 days, change a subculture.

(5) root culture: when a length of 1cm to the 2cm of bud of differentiation when, resistance Seedling is proceeded in root media, hestening rooting, within every 15 days, changed a subculture by 20 days, it is thus achieved that resistant plant.

Then with obtain open country chrysanthemum ' flame ' resistant plant STb gene as template, with LR reaction plasmid as positive control, with wild type Flos Chrysanthemi as negative control, carry out PCR detection with PMI gene and AtLFY gene specific primer respectively.

(6) culture medium prescription

Precultivation medium: MS+BA 0.5mg/L+NAA 1.5mg/L+sucrose 30g/L

Co-culture culture medium: 1/2MS+BA 0.5mg/L+NAA 1.5mg/L+sucrose 30g/L

Delay culture medium: MS+BA 0.5mg/L+NAA 1.5mg/L+sucrose 30g/L

Early stage screening culture medium: MS+BA 0.5mg/L+NAA 1.5mg/L+sucrose 22g/L+mannose 8g/L+cef 300mg/L

Later stage screening culture medium: MS+BA 0.5mg/L+NAA 1.5mg/L+sucrose 20g/L+mannose 10g/L+cef 300mg/L

Root culture culture medium: 1/2MS+sucrose 20g/L+mannose 10g/L+cef 100mg/L

Then transgenic open country chrysanthemum ' flame ' identified preliminary PCR, takes young leaflet tablet, is dipped in GUS dye liquor, with vacuum filter, it is carried out vacuum infiltration, after being repeated several times, puts it into insulation 20-22hrs in 37 DEG C of incubators.Proceed to blade the ethanol of 75% carries out decolouring 3 to 5 days, period constantly renew 75% ethanol, present yellow-white to negative control material.Observe under naked eyes or microscope, the blue fleck i.e. GUS expression sites in white background.

Finally transfer-gen plant is carried out phenotypic evaluation, the positive plant 13 strain transfer-gen plant and the wild type that detection are obtained proceed to field simultaneously, observe transfer-gen plant budding the time, begin to take time, contain and take time and WT lines compares, screening obtains open country chrysanthemum ' flame ' the early blossoming strain 9 of advance flowering period 7-11 days.

Claims

1. it is outer implant with open country chrysanthemum ' flame ' blade, by agrobcterium-mediated transformation, utilizes and contain on pCAMIB1301 carrier There is non-antibiotic marker gene 6-Phophomannose isomerase gene (PMI) to bio-safety, and with mannose for screening medium, it is thus achieved that turn AtLEAFY gene has open country chrysanthemum ' flame ' the Blooming strain of safety label, and flowering time is than the early 7-11 of not genetically modified open country chrysanthemum ' flame ' My god.