CN107267550B

CN107267550B - Anti-freeze lily and method for transforming lily by using anti-freeze protein gene

Info

Publication number: CN107267550B
Application number: CN201710565356.3A
Authority: CN
Inventors: 颜笑洒; 张业胜; 李艳霞; 王文; 董扬
Original assignee: Yunnan Nabo Biotechnology Co ltd
Current assignee: Yunnan Shixiete Biotechnology Co ltd
Priority date: 2017-07-12
Filing date: 2017-07-12
Publication date: 2020-10-30
Anticipated expiration: 2037-07-12
Also published as: CN107267550A

Abstract

The invention discloses an anti-freeze lily and a method for transforming the lily by using an anti-freeze protein gene. The anti-freezing lily is obtained by converting lily with an antifreeze protein gene. The Antifreeze protein gene is AF1(Antifreeze protein gene). The AF1 gene is inserted into a Pcambia1390 skeleton vector and is started by adopting a PCISV promoter, so that an AF1 gene plant expression vector P1390-PCISV-AF1 is constructed. The antifreeze protein AF1 gene is successfully transferred into lily, and a positive transgenic plant has good antifreeze effect, which is very beneficial to the cultivation of lily in autumn and winter in the north of China.

Description

Anti-freeze lily and method for transforming lily by using anti-freeze protein gene

Technical Field

The invention belongs to the technical field of transgenosis, and particularly relates to an anti-freezing lily and a method for transforming lily by using an anti-freezing protein gene.

Background

Lily (Lilium brownii var. viridulum Baker) is one of the flowers with the widest planting area after four cut flowers. China is the main origin of lily, and among more than one hundred lily varieties found in the world, more than fifty varieties originally produced in China exist. With the rapid development of the world economy, the flower industry develops rapidly all over the world, and lily grows up, particularly since the nineties of the last century, the lily enters a large development period, and the demand and the yield are greatly improved.

The lily grows at a proper temperature of 20-25 ℃ in the daytime, 10-15 ℃ at night, the growth of below 5 ℃ or above 28 ℃ is influenced, the growth of below 0 ℃ is usually greatly influenced, leaves are frostbitten and vitrified, bulbs are frostbitten, and tweezers are lightly clamped to form paste. The freeze resistance detection of four different lily varieties seed balls imported from Holland in 2007, such as Weanxihui, shows that the activity of seed ball scales is greatly reduced after low-temperature freezing treatment below 0 ℃; after low-temperature freezing treatment at the temperature below-2 ℃, the activity of partial seedball terminal buds is greatly reduced. The temperature difference in winter and summer is very large in north China, and the lily is easily influenced by cold flow in growth in autumn and winter, so that the yield is reduced, and the anti-freezing research of the lily has great necessity.

The AFPs gene is firstly found in fish in the last 60 th century, and has little influence on the melting point due to the special anti-freezing property, namely, the freezing point of a solution is lowered, so that the AFPs gene arouses the interest of wide scientists once found, and if the scientists transfer the AFPs into drosophila to improve the anti-freezing property or transfer the AFPs into protoplasts of tobacco, tomatoes, corns and the like. At present, no one has transferred the antifreeze gene into lily to improve the antifreeze capacity.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides an anti-freeze lily and a method for transforming the lily by using an anti-freeze protein gene. The invention transfers the antifreeze protein gene into lily by means of transgenosis, so that the lily generates antifreeze protein after transcription and translation, which is beneficial to expanding the lily planting.

In a first aspect of the invention, an antifreeze lily is provided, which is obtained by transforming lily with an antifreeze protein gene.

The Antifreeze protein gene is AF1(Antifreeze protein gene), the gene is derived from polar frozen fish (antifichoris minor), and the gene coding sequence is shown in SEQ ID NO. 3.

The AF1 gene is inserted into a Pcambia1390 skeleton vector and is started by adopting a PCISV promoter, so that an AF1 gene plant expression vector P1390-PCISV-AF1 is constructed.

The AF1 gene is obtained by optimizing plant codons of the polar frozen fish gene AFPIII, the coding sequence of the polar frozen fish gene AFPIII is shown as SEQ ID No.4, and the optimized gene coding sequence is shown as SEQ ID No. 3.

Because the antifreeze protein AFPIII gene is derived from polar frozen fish, codon optimization is carried out on the antifreeze protein AFPIII gene, the AF1 gene obtained after optimization has the bias of plants on the premise of not changing the amino acid sequence, and is easier to express in the plants.

In a second aspect of the present invention, there is also provided a method for transforming lily with an antifreeze protein gene, comprising the steps of:

inserting an AF1 gene into a Pcambia1390 skeleton vector by an enzyme digestion connection method, and constructing an AF1 gene plant expression vector P1390-PCISV-AF1 by adopting a PCISV promoter for starting;

step (2) transforming the expression vector P1390-PCISV-AF1 into an agrobacterium strain by electric shock, and transforming the vector P1390-PCISV-AF1 into the agrobacterium strain after detecting a colony by PCR;

and (3) transforming the AF1 gene into receptor lily callus by an agrobacterium-mediated genetic transformation method, determining that the AF1 gene is integrated into a lily genome through PCR identification, performing growth recovery culture on the callus on a non-resistance culture medium, and transferring the callus to a differentiation culture medium for culture to obtain a lily positive plant.

Further, the Pcambia1390 in the step (1) comprises an EcoR I/Spe I enzyme cutting site, a primer pair P1390-PCISV-F/R is used, a PCISV promoter is used as a template, PCR reaction is carried out, enzyme cutting and connecting reaction of the Pcambia1390 vector and the PCISV promoter is carried out, and enzyme cutting and connecting reaction is carried out by temperature change circulation; and then thermally shocking the ligation product to transform E.coliDH5 alpha competent cells, screening resistant plasmids by using an LB plate, extracting plasmids to obtain an intermediate expression vector P1390-PCISV, and carrying out sequencing verification on the PCISV-F/R by using a primer specific to the PCISV promoter.

Furthermore, the PCISV promoter needs to be modified before being connected with a vector Pcambia1390, so that the PCISV promoter has an EcoR1Kpn I-Pml I-Sac I-Xba I-Spe I enzyme cutting site.

Further, the downstream of the modified PCISV promoter carries an enzyme cutting site and the sequence is Kpn I-PmL I-SacI-Xba I-Spe I, Kpn I and Spe I are selected as candidate sites, the enzyme cutting site of the synthesized AF1 gene is modified to be provided with Kpn I and Spe I enzyme cutting sites, a primer pair is used for carrying out PCR experiments on P1390-AF1-F/R and an AF1 gene is used as a template, then endonuclease Kpn I and Spe I are selected to complete the connection of the P1390-PCISV and the AF1 gene, a connection product is thermally shocked to transform E.coli DH5 alpha competent cells, an LB plate is used for screening resistant plasmids, a small amount of plasmids are extracted to obtain an AF1 gene plant expression vector, and AF1-F/R is sequenced and verified by using AF1 specific primers.

Further, adopting an electric shock method to transform the expression vector P1390-PCISV-AF1 into an agrobacterium strain in the step (2), carrying out shake culture on the bacterium solution after electric transformation at 28-30 ℃ for 1.5-2.5 h, coating a plate on a YM solid culture medium, and culturing for 1d (day) in a constant temperature incubator at 28-30 ℃; picking a single colony in a YM liquid culture medium, carrying out shake culture at 28-30 ℃ for 1.5-2.5 h until the OD value is 0.3-0.6, and carrying out colony PCR amplification on the hygromycin gene by using a primer pair HYG-f/r.

Further, the scale of the seed ball of the lily aseptic seedling is used in the step (3), the middle part and the lower part of the scale are taken, and the scale is induced to slice to grow callus; adding YM liquid culture medium added with antibiotics into a sterilized tissue culture bottle, adding the bacterial liquid of the agrobacterium strain obtained in the step (2) into the YM culture medium, culturing until OD of the bacterial liquid is between 0.2 and 0.6, and activating the strain; centrifuging at low temperature, removing supernatant, adding half of suspension culture medium, mixing, and adding the rest half of suspension culture medium; pouring the bacterial liquid into a triangular flask, adding the lily callus blocks into the triangular flask, sealing, vacuumizing to 0.3-0.5 kPa in a vacuum pump, and continuing for 10-20 min to infect; and transferring the callus blocks to a screening culture medium after culturing for 3-5 days on the co-culture medium, inoculating the callus blocks which are not dead after screening to a new screening culture medium again after culturing for 10-20 days, and performing secondary screening culture for 20-40 days until resistant seedlings are harvested.

Preferably, the infection conditions are that the OD value of the culture solution is 0.2, the infection time is 15min, the AS concentration in the co-culture medium is 100umol/L, and the co-culture time is 3 days.

The invention is characterized in that the antifreeze gene is adopted for transformation, the antifreeze protein expressed by the antifreeze gene is a protein directly interacting with ice crystals, and the antifreeze protein has the characteristics of preventing the formation of the ice crystals, regulating and controlling the growth of extracellular ice crystals, inhibiting the recrystallization of the ice, maintaining the non-freezing state of body fluid and the like, and can reduce the freezing point of aqueous solution in a non-colligative form, thereby enabling species to have the antifreeze capacity. However, as the antifreeze protein AFPIII gene is derived from polar frozen fish, if the gene is directly transformed into lily, the AFPIII gene can not express the antifreeze protein in plants smoothly, so the invention optimizes the codon of the antifreeze protein, ensures that the expression of the optimized AF1 gene has the bias of plants on the premise of not changing the amino acid sequence, and is easier to express in lily, thereby successfully obtaining the antifreeze lily.

The invention is characterized in that Pcambia1390 is used as a skeleton vector, a promoter PCISV is selected to start expression, and the invention modifies the promoter PCISV in order to connect the virus promoter PCISV with the skeleton vector, so that the PCISV promoter has an EcoR1Kpn I-Pml I-Sac I-Xba I-Spe I enzyme cutting site.

The invention is characterized in that the positive seedlings are difficult to be successfully obtained by adopting the existing infection conditions and culture conditions, so the experimental conditions are screened, and the OD values of the bacteria liquid for a transgenic system are 0.2, 0.4, 0.6, 0.8 and 1.0; infection time: 5min, 10min, 15min and 20 min; co-culture time: 1d, 2d, 3d, 4d, 5d, 6 d; acetosyringone (AS) concentration (umol/L): 10, 20, 30, 40, 50, 60, 70, 80, 100; and finally, according to the group with the best transient expression result, determining that the key infection condition is that the OD value is 0.2, the AS concentration is 100umol/L, the infection time is 15min, and the co-culture time is 3d, and finally obtaining the resistant lily seedlings.

Compared with the prior art, the invention has the following beneficial effects: the antifreeze protein AF1 gene is successfully transferred into lily, and a positive transgenic plant has good antifreeze effect, which is very beneficial to the cultivation of lily in autumn and winter in the north of China. From the results of the anti-freezing experiment, it can be seen that the AFPIII gene from the source and the polar anti-freezing fish successfully lowers the freezing point of the intracellular water environment by more than 2 ℃, which is consistent with the findings of scientists so far.

Drawings

FIG. 1 is a flow chart of expression vector construction;

FIG. 2 shows the PCR detection results of Agrobacterium colonies, where M: DL2000 marker; 1-8 represent PCR results for different monoclonal colonies;

FIG. 3 shows the resistant seedlings obtained; in the figure, 1 is lily callus infected by agrobacterium; 2, vacuumizing by a vacuum pump for continuous infection; 3, primary screening after co-culture; 4, secondary screening;

FIG. 4 is a diagram showing the results of PCR detection of transgenic plants, in which 1 is a control group and 2-7 are experimental groups M.DL2000marker;

FIG. 5 is a photograph of an anti-freezing experiment at-2 ℃ A/C: the left side of the picture before freezing treatment is a positive seedling, and the right side is a wild type; B/D: the picture of the day 13 after freezing treatment is positive seedlings on the left side and wild type on the right side;

FIG. 6 is a photograph of an anti-freezing experiment at-4 ℃; A/C: the left side of the picture before freezing treatment is a positive seedling, and the right side is a wild type; B/D: the picture of the day 10 of the freezing treatment is that the left side is a positive seedling and the right side is a wild type;

FIG. 7 is a schematic diagram of the optimization of the AFPIII codon sequence.

Detailed Description

The technical solution of the present invention will be described in further detail with reference to specific examples, but the present invention is not limited to the following technical solutions.

The lily adopted in the embodiment is one of the varieties which are favored by consumers, also called ' double jia of absolute generations ' and English name Conca D ' or, and is easy to plant, golden in color, robust in leaves and dark green.

Bacterial strains and plasmids

The antifreeze protein gene AF1 optimized by a codon in the laboratory is synthesized by a company; coli strain DH5 alpha, Agrobacterium strain EHA105, Pcambia1390 backbone vectors were all stored in this laboratory.

Enzyme and biochemical reagent

T4-DNA ligase, Taq DNA polymerase, dNTP mix, DNA Marker DL2000 and the like are all TAKARA company reagents;

a common DNA product purification kit (centrifugal column type), an agarose gel DNA recovery kit (common centrifugal column type);

plasmid mini-extraction kits (centrifugal column type) are all kits of Tiangen company;

EcoR I, Spe I, Kpn I, etc. are NEB company reagents;

the viral promoter PCISV and the antifreeze gene AF1 were synthesized by Shanghai Czeri bioengineering, Inc.

Plant material and culture medium composition

The receptor material is Lily bulb scale of Lily bulb variety (Lily Conca D' or).

The basic culture medium is MS culture medium. The culture medium formula of each tissue culture stage is as follows:

induction medium: MS +2 mg/L2, 4-D +1 mg/L6-BA + 0.8% agar;

suspension culture medium: MS +0.25mg/L6-BA +0.25 mg/L2, 4-D + 2% sucrose + 1% glucose, pH 5.20;

co-culture medium: MS +0.25mg/L6-BA +0.25 mg/L2, 4-D + 2% sucrose + 1% glucose + 0.8% agar +100umol/L AS

Screening a culture medium: MS +0.25mg/L6-BA +0.25 mg/L2, 4-D + 3% sucrose + 0.8% agar + carbenicillin 250mg/L + hygromycin 20mg/L, pH 5.80;

rooting culture medium: MS +0.1mg/L NAA + 3% sucrose + 0.8% agar, pH 5.80.

YM solid medium: 3g/L yeast powder, 5g/L tryptone, 5g/L maltose, 5g/L D-glucose and 16g/L agar

YM liquid medium: 3g/L yeast powder, 5g/L tryptone, 5g/L maltose and 5g/L D-glucose

LB solid culture plate: 5g/L yeast powder, 10g/L tryptone, 5g/L sodium chloride and 16g/L agar

The primers involved in the following examples are shown in Table 1.

TABLE 1

The framework vector Pcambia1390 sequence is shown as SEQ ID NO.1, the promoter PCISV sequence is shown as SEQ ID NO.2, the AF1 gene coding sequence is shown as SEQ ID NO.3, and the polar frozen fish gene AFPIII coding sequence is shown as SEQ ID NO. 4.

Example 1

Construction of intermediate expression vector P1390-PCISV

Pcambia1390 does not contain the viral promoter PCISV, the promoter PCISV is synthesized by the entrusted Gene Synthesis, and Pcambia1390 contains an EcoR I/Spe I cleavage site, so the synthesized PCISV promoter is modified to have the EcoR1Kpn I-Pml I-Sac I-Xba I-Spe I cleavage site.

Carrying out PCR by using a primer pair P1390-PCISV-F/P1390-PCISV-R and a PCISV promoter as a template, carrying out enzyme digestion and connection reaction on a Pcambia1390 vector and the PCISV promoter according to the table 2, and carrying out enzyme digestion and connection by using a variable temperature cycle, wherein the cycle is about 10-15 and the temperature is 37 ℃ for 5 min; 5min at 10 ℃ and 5min at 20 ℃; finally 5min at 37 ℃. And thermally shocking the ligation product to transform E.coli DH5 alpha competent cells, screening resistant plasmids by LB solid culture medium containing 50mg/L, extracting plasmids in small amount to obtain an intermediate expression vector P1390-PCISV (see figure 1), and carrying out sequencing verification on the PCISV-F/R by using primers specific to the PCISV promoter.

Table 2: connection reaction system of P1390 vector and PCISV promoter

Reagent	Addition amount (μ L)	Final concentration
			10×CutSmart Buffer	1.5	1×
10mM ATP	1.5	1mM
			Pcambia1390 plasmid	60-80ng	4-6ng/μL
PCR product of PCISV promoter	1ul
			EcoRI SpeI	10U	0.1-0.2U/μL
T4DNA ligase	35U	2-3U/μL
			ddH₂O	To 15μL

Construction and identification of AF1 gene plant expression vector

The codon optimization process of the AF1 gene is shown in FIG. 7.

The downstream of the modified PCISV promoter carries an enzyme cutting site and a sequence Kpn I-PmL I-Sac I-Xba I-speI, Kpn I and Spe I are selected as candidate sites, carrying out enzyme cutting site modification on the synthesized AF1 gene, carrying out enzyme cutting site modification on the synthesized AF1 gene to enable the synthesized AF1 gene to have Kpn I and Spe I enzyme cutting sites, carrying out PCR experiment by using a primer pair P1390-AF1-F/P1390-AF1-R and taking the AF1 gene as a template, then, the connection of the P1390-PCISV and the AF1 gene is completed by selecting endonucleases Kpn I and Spe I according to the enzyme cutting connection method shown in the table 1, and further thermally shocking the ligation product to transform E.coli DH5 alpha competent cells, screening resistant plasmids by using LB plates containing 50mg/L, extracting plasmids in small quantity to obtain AF1 gene plant expression vectors (shown in figure 1), and carrying out sequencing verification on AF1-F/R by using AF1 specific primers.

Expression vector transformation of agrobacterium

Transforming the expression vector into an agrobacterium strain EHA105 by adopting an electric shock method, carrying out shake culture on the bacterium solution after electric transformation at 28 ℃ for 2h at 200r/min in a YM liquid culture medium, coating a flat plate on the YM solid culture medium (adding 100mg/L Kan and 60mg/L Rif), and culturing for 1d in a constant-temperature incubator at 28 ℃; single colonies were picked up in 10mL of liquid YM medium (100 mg/L Kan, 60mg/LRif added), shake-cultured at 28 ℃ and 200r/min until OD was 0.3-0.6, and colony PCR amplification was performed on hygromycin gene using primer pair HYG-f/r.

Obtaining of transgenic lily with AF1 gene

Using the bulb scales of the lily aseptic seedlings, taking the middle parts and the lower parts of the scales, and inducing the scale slices to grow callus on an induction culture medium; adding about 50mL YM liquid culture medium added with antibiotics into a sterilized tissue culture bottle, taking 500-; centrifuging at low temperature for 10min, discarding supernatant, adding half of suspension culture medium, mixing, and adding the rest half of suspension culture medium; pouring the bacterial liquid into a triangular flask, adding the callus pieces, sealing, vacuumizing to 0.4kPa in a vacuum pump, and continuously infecting for 15 min; and culturing for 3 days on the co-culture medium, transferring to a screening medium, culturing for 15 days, inoculating the callus blocks which are not dead after screening to a new screening medium again, screening for 30 days for the second time until resistant seedlings are harvested, and inoculating the resistant seedlings into a rooting medium for culturing.

PCR detection of transgenic plants

Taking the leaves of the resistant plant and the wild control plant, and extracting the plant genome DNA by adopting a CTAB method. The extracted DNA was used as a template, and AF1-F/R was amplified using AF1 gene-specific primers. Samples were taken at 2ul and checked electrophoretically on a 1% agarose gel.

Antifreeze test for transgenic positive seedlings

Plant freezing resistance refers to the ability of a plant to tolerate low temperatures below freezing. The overwintering capability of the plant can be detected by field tests, but the freezing resistance is different from the overwintering capability, and the freezing treatment and activity measurement are the reliable method for determining the freezing resistance. In 1930, Dexter et al first proposed the measurement of frost resistance of plants by electrical conduction, and since then, the techniques for measuring frost resistance of plants have been developed in a long time, they are roughly classified into three types, i.e., growth recovery test, cell membrane property change, and cell metabolism change due to change in membrane structure function. In the experiment, a growth recovery test is adopted, namely the ability of the plant or organ to recover growth or form callus is observed at a proper temperature (a greenhouse or a growth box) after freezing treatment, the method is reliable and intuitive, but sometimes is influenced by tissue dormancy, most cells of the plant are in a living state, but do not grow; sometimes it is difficult to distinguish where the injury occurs and this method tends to take a long time.

The present example uses a biochemical incubator as a temperature control incubator with a temperature control error of ± 0.1 ℃. 7 temperature gradients of 6 ℃,4 ℃, 2 ℃, 0 ℃, minus 1 ℃, minus 2 ℃ and minus 3 ℃ are designed for testing the critical temperature of the growth of the negative seedlings. Each gradient was set for 6 experimental replicates and observations and photographs were taken for a period of 13 days; after the photographing is finished, the seedlings are placed at room temperature (24 ℃) for a period of time again, and whether the seedlings can normally recover growth is observed.

And (3) carrying out an anti-freezing experiment on the positive transgenic seedling on the basis of the detected critical growth temperature: placing positive seedlings and negative seedlings in a biochemical incubator at the critical temperature, setting 6 experiments to be repeated, and observing and photographing for a period of time; and (3) continuously reducing the temperature at the critical temperature, simultaneously putting the positive seedlings and the negative seedlings in a biochemical incubator, setting 6 experiments to repeat, and culturing and observing for a period of time. After the photographing is finished, the seedlings are placed at room temperature (24 ℃) for a period of time again, and whether the seedlings can normally recover growth is observed.

Results and analysis

Identification of agrobacterium transformed by expression vector

The constructed plant expression vector is transformed into agrobacterium by an electric shock transformation method, screened on additional Kan and Rif culture media, and selected to be resistant clone for colony PCR identification, and a target band is amplified, and the result shows that the plant expression vector is successfully introduced into agrobacterium strain EHA105 (figure 2).

Acquisition of resistant seedlings

Infecting lily callus for 15min, co-culturing for 3d, transferring the co-cultured explant to selective culture medium, browning and killing most of the explant, only normally differentiating resistant buds, inoculating the callus blocks which are not killed by screening to new screening culture medium, and screening for 30d for the second time until resistant seedlings are harvested.

PCR detection of transgenic plants

PCR detection is carried out on the genome DNA of the resistant plant by adopting a target gene (AF1 gene) primer, a specific strip of about 250bp is amplified, the size of the specific strip is matched with the size (267bp) of a DNA fragment defined by the target gene primer, and a target strip does not appear in a wild type control plant, so that the fact that the target gene is transferred into the resistant plant is proved.

Antifreeze detection of transgenic positive seedlings

After continuous observation and photographing for 13 days, the following results were obtained:

(1) 6 negative seedlings in total are frozen to death at the temperature of minus 3 ℃ for 5 days;

(2) 6 negative seedlings in total are frozen to death at the temperature of minus 2 ℃ for 7 days;

(3) 6 negative seedlings in total grow normally at-1 ℃ for 13 days;

(4) 6 negative seedlings in total grow normally at the temperature of 0-6 ℃ for 13 days;

selecting-2 ℃ as the critical temperature of lily growth, wherein the leaf of the non-transgenic lily is frostbitten and is vitrified when the critical temperature is lower than the critical temperature; the bulb is frostbitten, and the forceps are lightly clamped to form paste; after transfer to room temperature, the frozen seedlings (seedlings at-2 ℃ and-3 ℃) failed to recover growth, and the seedlings recovered normal growth under other temperature gradients.

Therefore, the subsequent anti-freezing experiment of the positive seedlings is carried out at the temperature of-2 ℃ and-4 ℃. Through experiments and observation for 13 days, the following results are found:

(1) at-4 ℃ for 13 days, 6 negative seedlings are completely frozen, 6 positive seedlings are all normally grown, and the seedlings can be observed to grow obviously; after the temperature is shifted to room temperature, the positive seedlings can still grow normally.

(2) At-2 ℃ for 13 days, 6 negative seedlings are completely frozen, 6 positive seedlings are all normally grown, and the seedlings can be observed to grow obviously; after the temperature is shifted to room temperature, the positive seedlings can still grow normally.

FIG. 5 shows photographs and details of seedlings at-2 ℃ and FIG. 6 shows photographs and details of seedlings at-4 ℃.

In conclusion, although China is the main origin of lily, due to the characteristics of cold winter and hot summer in north, large annual temperature difference and daily temperature difference, the growth of lily is greatly influenced in autumn and winter, so that the anti-freezing experiment of lily has great necessity, and the anti-freezing experiment has guiding significance in lily planting and also has good reference effect in breeding of other crops. The antifreeze protein is a protein which directly interacts with ice crystals, has the characteristics of preventing the formation of the ice crystals, regulating and controlling the growth of extracellular ice crystals, inhibiting the recrystallization of the ice, maintaining the non-freezing state of body fluid and the like, can reduce the freezing point of an aqueous solution in a non-colligative form, but has little influence on the melting point, so that the difference between the melting point and the freezing point of the aqueous solution is caused, and the difference is called thermal hysteresis activity. Cold inducible proteins are also present in plants, which have properties much like AFP of fish and insects and are therefore considered to be antifreeze proteins, but plant antifreeze proteins have a lower thermal lag value than fish and insects and are therefore selected for fish antifreeze proteins.

In Agrobacterium-mediated gene transformation, T-DNA is typically transferred into the host genome from the right border to the left border in sequence, so that sequences near the right border enter the plant genome before the left border sequence. In this vector, the hygromycin gene is near the left border and the gene of interest is near the right border, thus indicating that the gene of interest must have integrated into the plant genome when the resistance gene is detected.

For a transgenic system, the embodiment also carries out system exploration and optimization, and the OD values of bacterial liquid are 0.2, 0.4, 0.6, 0.8 and 1.0; infection time: 5min, 10min, 15min and 20 min; co-culture time: 1d, 2d, 3d, 4d, 5d, 6 d; acetosyringone (AS) concentration (umol/L): 10, 20, 30, 40, 50, 60, 70, 80, 100; and finally, according to the group with the best transient expression result, determining that the key infection condition is that the OD value is 0.2, the AS concentration is 100umol/L, the infection time is 15min, and the co-culture time is 3d, and finally obtaining the resistant seedlings.

The antifreeze experiment perfectly indicates that the AF1 gene is successfully transferred into the lily, and the positive transgenic plant has good antifreeze effect, which is very beneficial to the cultivation of the lily in autumn and winter in the north of China. From the results of the anti-freezing experiment, it can be seen that the AFPIII gene derived from polar anti-freezing fish successfully lowers the freezing point of the intracellular water environment by more than 2 ℃, which is consistent with the findings of scientists so far.

Sequence listing

<110> Yunan Nabo Biotech Co., Ltd

<120> an antifreeze lily and a method for transforming lily by using antifreeze protein gene

<141>2017-07-12

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cgaggccagg cggcgcggtg ccttccgtga ggacgcattg accgaggccg acgccctggc 1320

ggccgccgag aatgaacgcc aagaggaaca agcatgaaac cgcaccagga cggccaggac 1380

gaaccgtttt tcattaccga agagatcgag gcggagatga tcgcggccgg gtacgtgttc 1440

gagccgcccg cgcacgtctc aaccgtgcgg ctgcatgaaa tcctggccgg tttgtctgat 1500

gccaagctgg cggcctggcc ggccagcttg gccgctgaag aaaccgagcg ccgccgtcta 1560

aaaaggtgat gtgtatttga gtaaaacagc ttgcgtcatg cggtcgctgc gtatatgatg 1620

cgatgagtaa ataaacaaat acgcaagggg aacgcatgaa ggttatcgct gtacttaacc 1680

agaaaggcgg gtcaggcaag acgaccatcg caacccatct agcccgcgcc ctgcaactcg 1740

ccggggccga tgttctgtta gtcgattccg atccccaggg cagtgcccgc gattgggcgg 1800

ccgtgcggga agatcaaccg ctaaccgttg tcggcatcga ccgcccgacg attgaccgcg 1860

acgtgaaggc catcggccgg cgcgacttcg tagtgatcga cggagcgccc caggcggcgg 1920

acttggctgt gtccgcgatc aaggcagccg acttcgtgct gattccggtg cagccaagcc 1980

cttacgacat atgggccacc gccgacctgg tggagctggt taagcagcgc attgaggtca 2040

cggatggaag gctacaagcg gcctttgtcg tgtcgcgggc gatcaaaggc acgcgcatcg 2100

gcggtgaggt tgccgaggcg ctggccgggt acgagctgcc cattcttgag tcccgtatca 2160

cgcagcgcgt gagctaccca ggcactgccg ccgccggcac aaccgttctt gaatcagaac 2220

ccgagggcga cgctgcccgc gaggtccagg cgctggccgc tgaaattaaa tcaaaactca 2280

tttgagttaa tgaggtaaag agaaaatgag caaaagcaca aacacgctaa gtgccggccg 2340

tccgagcgca cgcagcagca aggctgcaac gttggccagc ctggcagaca cgccagccat 2400

gaagcgggtc aactttcagt tgccggcgga ggatcacacc aagctgaaga tgtacgcggt 2460

acgccaaggc aagaccatta ccgagctgct atctgaatac atcgcgcagc taccagagta 2520

aatgagcaaa tgaataaatg agtagatgaa ttttagcggc taaaggaggc ggcatggaaa 2580

atcaagaaca accaggcacc gacgccgtgg aatgccccat gtgtggagga acgggcggtt 2640

ggccaggcgt aagcggctgg gttgtctgcc ggccctgcaa tggcactgga acccccaagc 2700

ccgaggaatc ggcgtgacgg tcgcaaacca tccggcccgg tacaaatcgg cgcggcgctg 2760

ggtgatgacc tggtggagaa gttgaaggcc gcgcaggccg cccagcggca acgcatcgag 2820

gcagaagcac gccccggtga atcgtggcaa gcggccgctg atcgaatccg caaagaatcc 2880

cggcaaccgc cggcagccgg tgcgccgtcg attaggaagc cgcccaaggg cgacgagcaa 2940

ccagattttt tcgttccgat gctctatgac gtgggcaccc gcgatagtcg cagcatcatg 3000

gacgtggccg ttttccgtct gtcgaagcgt gaccgacgag ctggcgaggt gatccgctac 3060

gagcttccag acgggcacgt agaggtttcc gcagggccgg ccggcatggc cagtgtgtgg 3120

gattacgacc tggtactgat ggcggtttcc catctaaccg aatccatgaa ccgataccgg 3180

gaagggaagg gagacaagcc cggccgcgtg ttccgtccac acgttgcgga cgtactcaag 3240

ttctgccggc gagccgatgg cggaaagcag aaagacgacc tggtagaaac ctgcattcgg 3300

ttaaacacca cgcacgttgc catgcagcgt acgaagaagg ccaagaacgg ccgcctggtg 3360

acggtatccg agggtgaagc cttgattagc cgctacaaga tcgtaaagag cgaaaccggg 3420

cggccggagt acatcgagat cgagctagct gattggatgt accgcgagatcacagaaggc 3480

aagaacccgg acgtgctgac ggttcacccc gattactttt tgatcgatcc cggcatcggc 3540

cgttttctct accgcctggc acgccgcgcc gcaggcaagg cagaagccag atggttgttc 3600

aagacgatct acgaacgcag tggcagcgcc ggagagttca agaagttctg tttcaccgtg 3660

cgcaagctga tcgggtcaaa tgacctgccg gagtacgatt tgaaggagga ggcggggcag 3720

gctggcccga tcctagtcat gcgctaccgc aacctgatcg agggcgaagc atccgccggt 3780

tcctaatgta cggagcagat gctagggcaa attgccctag caggggaaaa aggtcgaaaa 3840

ggtctctttc ctgtggatag cacgtacatt gggaacccaa agccgtacat tgggaaccgg 3900

aacccgtaca ttgggaaccc aaagccgtac attgggaacc ggtcacacat gtaagtgact 3960

gatataaaag agaaaaaagg cgatttttcc gcctaaaact ctttaaaact tattaaaact 4020

cttaaaaccc gcctggcctg tgcataactg tctggccagc gcacagccga agagctgcaa 4080

aaagcgccta cccttcggtc gctgcgctcc ctacgccccg ccgcttcgcg tcggcctatc 4140

gcggccgctg gccgctcaaa aatggctggc ctacggccag gcaatctacc agggcgcgga 4200

caagccgcgc cgtcgccact cgaccgccgg cgcccacatc aaggcaccct gcctcgcgcg 4260

tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg 4320

tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg 4380

gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata ctggcttaac 4440

tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac 4500

agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg cttcctcgct cactgactcg 4560

ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg 4620

ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag 4680

gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac 4740

gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga 4800

taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt 4860

accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc 4920

tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc 4980

cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta 5040

agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat 5100

gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca 5160

gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct 5220

tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt 5280

acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct 5340

cagtggaacg aaaactcacg ttaagggatt ttggtcatgc attctaggta ctaaaacaat 5400

tcatccagta aaatataata ttttattttc tcccaatcag gcttgatccc cagtaagtca 5460

aaaaatagct cgacatactg ttcttccccg atatcctccc tgatcgaccg gacgcagaag 5520

gcaatgtcat accacttgtc cgccctgccg cttctcccaa gatcaataaa gccacttact 5580

ttgccatctt tcacaaagat gttgctgtct cccaggtcgc cgtgggaaaa gacaagttcc 5640

tcttcgggct tttccgtctt taaaaaatca tacagctcgc gcggatcttt aaatggagtg 5700

tcttcttccc agttttcgca atccacatcg gccagatcgt tattcagtaa gtaatccaat 5760

tcggctaagc ggctgtctaa gctattcgta tagggacaat ccgatatgtc gatggagtga 5820

aagagcctga tgcactccgc atacagctcg ataatctttt cagggctttg ttcatcttca 5880

tactcttccg agcaaaggac gccatcggcc tcactcatga gcagattgct ccagccatca 5940

tgccgttcaa agtgcaggac ctttggaaca ggcagctttc cttccagcca tagcatcatg 6000

tccttttccc gttccacatc ataggtggtc cctttatacc ggctgtccgt catttttaaa 6060

tataggtttt cattttctcc caccagctta tataccttag caggagacat tccttccgta 6120

tcttttacgc agcggtattt ttcgatcagt tttttcaatt ccggtgatat tctcatttta 6180

gccatttatt atttccttcc tcttttctac agtatttaaa gataccccaa gaagctaatt 6240

ataacaagac gaactccaat tcactgttcc ttgcattcta aaaccttaaa taccagaaaa 6300

cagctttttc aaagttgttt tcaaagttgg cgtataacat agtatcgacg gagccgattt 6360

tgaaaccgcg gtgatcacag gcagcaacgc tctgtcatcg ttacaatcaa catgctaccc 6420

tccgcgagat catccgtgtt tcaaacccgg cagcttagtt gccgttcttc cgaatagcat 6480

cggtaacatg agcaaagtct gccgccttac aacggctctc ccgctgacgc cgtcccggac 6540

tgatgggctg cctgtatcga gtggtgattt tgtgccgagc tgccggtcgg ggagctgttg 6600

gctggctggt ggcaggatat attgtggtgt aaacaaattg acgcttagac aacttaataa 6660

cacattgcgg acgtttttaa tgtactgaat taacgccgaa ttaattcggg ggatctggat 6720

tttagtactg gattttggtt ttaggaatta gaaattttat tgatagaagt attttacaaa 6780

tacaaataca tactaagggt ttcttatatg ctcaacacat gagcgaaacc ctataggaac 6840

cctaattccc ttatctggga actactcaca cattattatg gagaaactcg agcttgtcga 6900

tcgacagatc cggtcggcat ctactctatt tctttgccct cggacgagtg ctggggcgtc 6960

ggtttccact atcggcgagt acttctacac agccatcggt ccagacggcc gcgcttctgc 7020

gggcgatttg tgtacgcccg acagtcccgg ctccggatcg gacgattgcg tcgcatcgac 7080

cctgcgccca agctgcatca tcgaaattgc cgtcaaccaa gctctgatag agttggtcaa 7140

gaccaatgcg gagcatatac gcccggagtc gtggcgatcc tgcaagctcc ggatgcctcc 7200

gctcgaagta gcgcgtctgc tgctccatac aagccaacca cggcctccag aagaagatgt 7260

tggcgacctc gtattgggaa tccccgaaca tcgcctcgct ccagtcaatg accgctgtta 7320

tgcggccatt gtccgtcagg acattgttgg agccgaaatc cgcgtgcacg aggtgccgga 7380

cttcggggca gtcctcggcc caaagcatca gctcatcgag agcctgcgcg acggacgcac 7440

tgacggtgtc gtccatcaca gtttgccagt gatacacatg gggatcagca atcgcgcata 7500

tgaaatcacg ccatgtagtg tattgaccga ttccttgcgg tccgaatggg ccgaacccgc 7560

tcgtctggct aagatcggcc gcagcgatcg catccatagc ctccgcgacc ggttgtagaa 7620

cagcgggcag ttcggtttca ggcaggtctt gcaacgtgac accctgtgca cggcgggaga 7680

tgcaataggt caggctctcg ctaaactccc caatgtcaag cacttccgga atcgggagcg 7740

cggccgatgc aaagtgccga taaacataac gatctttgta gaaaccatcg gcgcagctat 7800

ttacccgcag gacatatcca cgccctccta catcgaagct gaaagcacga gattcttcgc 7860

cctccgagag ctgcatcagg tcggagacgc tgtcgaactt ttcgatcaga aacttctcga 7920

cagacgtcgc ggtgagttca ggctttttca tatctcattg ccccccggga tctgcgaaag 7980

ctcgagagag atagatttgt agagagagac tggtgatttc agcgtgtcct ctccaaatga 8040

aatgaacttc cttatataga ggaaggtctt gcgaaggata gtgggattgt gcgtcatccc 8100

ttacgtcagt ggagatatca catcaatcca cttgctttga agacgtggtt ggaacgtctt 8160

ctttttccac gatgctcctc gtgggtgggg gtccatcttt gggaccactg tcggcagagg 8220

catcttgaac gatagccttt cctttatcgc aatgatggca tttgtaggtg ccaccttcct 8280

tttctactgt ccttttgatg aagtgacaga tagctgggca atggaatccg aggaggtttc 8340

ccgatattac cctttgttga aaagtctcaa tagccctttg gtcttctgag actgtatctt 8400

tgatattctt ggagtagacg agagtgtcgt gctccaccat gttatcacat caatccactt 8460

gctttgaaga cgtggttgga acgtcttctt tttccacgat gctcctcgtg ggtgggggtc 8520

catctttggg accactgtcg gcagaggcat cttgaacgat agcctttcct ttatcgcaat 8580

gatggcattt gtaggtgcca ccttcctttt ctactgtcct tttgatgaag tgacagatag 8640

ctgggcaatg gaatccgagg aggtttcccg atattaccct ttgttgaaaa gtctcaatag 8700

ccctttggtc ttctgagact gtatctttga tattcttgga gtagacgaga gtgtcgtgct 8760

ccaccatgtt gggcccggcg cgccaagctt gaattcggat ccggtaccct gcaggagctc 8820

gtcgactcta gaccatggta gatctgacta gtgttaacg 8859

<210>2

<211>352

<212>DNA

<213> Artificial Synthesis

<400>2

acagagggat ttctctgaag atcatgtttg ccagctatgc gaacaatcat cgggagatct 60

tgagccaatc aaagaggagt gatgtagacc taaagcaata atggagccat gacgtaaggg 120

cttacgccat tacgaaataa ttaaaggctg atgtgacctg tcggtctctc agaaccttta 180

ctttttatat ttggcgtgta tttttaaatt tccacggcaa tgacgatgtg acctgtgcat 240

ccgctttgcc tataaataag ttttagtttg tattgatcga cacgatcgag aagacacggc 300

catttggacg atcatttgag agtctaaaag aacgagtctt gtaatatgtt tt 352

<210>3

<211>267

<212>DNA

<213> Artificial Synthesis

<400>3

atgaagtctg ctattttgac tggattgttg tttgttttgt tgtgtgttga tcatatgtct 60

tctgctcatc aagcttctat tgttgctaat caattgattc ctattaatac tgctttgact 120

cctattatga ctaaggctca agttgttact cctttgggaa ttcctgctga agatattcct 180

agaattattg gaatgcaagt taatagagct gttgctttgg gaactacttt gatgcctgat 240

atggttaagg gatatcctcc taattaa 267

<210>4

<211>310

<212>DNA

<213>Anarhichas minor

<400>4

ttaattaatt actaattaat taagtgtcag ccacagccat gaagtcagct attttaactg 60

gtttgctttt cgtcctcctt tgtgtcgacc acatgagttc agcccaccag gcgtccattg 120

tggccaacca gctgatcccc ataaatactg ccctgactcc gataatgacg aaggcgcagg 180

tggtcacccc attgggcatc cctgccgagg acattccccg aataatcgga atgcaagtga 240

acagggcagt ggcgttgggc acaaccctca tgccagacat ggtgaaaggg taccccccga 300

attagaagtt 310

Claims

1. An antifreeze protein gene is characterized in that the antifreeze protein gene is AF1 gene, and the gene sequence is shown in SEQ ID NO. 3.

2. The antifreeze protein gene of claim 1, wherein the AF1 gene is obtained by plant codon optimization of polar frozen fish gene AFPIII, the sequence of the polar frozen fish gene AFPIII is shown as SEQ ID No.4, and the optimized gene sequence is shown as SEQ ID No. 3.

3. A method for transforming lily with the antifreeze protein gene of claim 1, comprising the steps of:

transforming the AF1 gene into receptor lily callus by an agrobacterium-mediated genetic transformation method, and determining that the AF1 gene is integrated into a lily genome through PCR identification; and (3) performing growth recovery culture on the callus on a non-resistant culture medium, and then transferring the callus to a differential culture medium for culture, wherein the infection conditions are that the OD value of a culture bacterial liquid is 0.2-1.0, the infection time is 5-15min, and the AS concentration in a co-culture medium is 10-100 umol/L, so AS to obtain the lily positive plant.

4. The method of claim 3, wherein in step (1), Pcambia1390 comprises an EcoR I/Spe I cleavage site, a PCR reaction is performed using a primer pair P1390-PCISV-F/R using a PCISV promoter as a template, an enzymatic ligation reaction of the Pcambia1390 vector and the PCISV promoter is performed, and an enzymatic ligation reaction is performed using a temperature-variable cycle; then thermally shocking the ligation product to transform E, coli DH5 alpha competent cells, screening resistant plasmids by using an LB plate, extracting plasmids to obtain an intermediate expression vector P1390-PCISV, and carrying out sequencing verification on the PCISV-F/R by using specific primers of a PCISV promoter;

the PCISV promoter is required to be modified before being connected with a vector Pcambia1390, so that the PCISV promoter has EcoRI, Kpn I, Pml I, Sac I, Xba I and Spe I enzyme cutting sites.

5. The method of claim 4, wherein the modified PCISV promoter carries a cleavage site downstream of the promoter and Kpn I, PmL I, Sac I, Xba I, and Spe I in the order named, Kpn I and Spe I are selected as candidate sites, the synthesized AF1 gene is modified to have Kpn I and Spe I cleavage sites, PCR experiments are performed using the primer pair P1390-AF1-F/R and AF1 gene as a template, then the ligation of P1390-PCISV and AF1 gene is completed using the endonucleases Kpn I and Spe I, and the ligation product is heat shock transformed into E. coli DH5 alpha competent cells, resistant plasmids are screened using LB plate, and AF1 gene plant expression vectors are obtained, and AF1-F/R is sequence verified using AF1 specific primers.

6. The method as claimed in claim 3, wherein the expression vector P1390-PCISV-AF1 is transformed into Agrobacterium strain in step (2) by electric shock method, the bacterial liquid after electric transformation is cultured for 1.5-2.5 h at 28-30 ℃ under shaking, and the bacterial liquid is spread on YM solid culture medium and cultured for 1d in 28-30 ℃ constant temperature incubator; picking a single colony in a YM liquid culture medium, carrying out shake culture at 28-30 ℃ for 1.5-2.5 h until the OD value is 0.3-0.6, and carrying out colony PCR amplification on the hygromycin gene by using a primer pair HYG-f/r.

7. The method of claim 3, wherein the step (3) uses scales of seed bulbs of the aseptic lily seedlings to induce the scale slices to grow callus; adding YM liquid culture medium containing antibiotics into a sterilized tissue culture bottle, adding the bacterial liquid of the agrobacterium strain obtained in the step (2) into the YM liquid culture medium, culturing until OD of the bacterial liquid is between 0.2 and 0.6, and activating the strain; centrifuging at low temperature, removing supernatant, adding suspension culture medium, and mixing; pouring the bacterial liquid into a triangular flask, adding the lily callus blocks into the triangular flask, sealing, vacuumizing to 0.3-0.5 kPa in a vacuum pump, and continuing for 10-20 min to infect; and culturing on the co-culture medium for 3-5 days, transferring to a screening culture medium, culturing for 10-20 days, inoculating the callus blocks which are not dead after screening to a new screening culture medium again, performing secondary screening culture for 20-40 days until resistant seedlings are harvested, and inoculating the resistant seedlings into a rooting culture medium for culturing.

8. The method of claim 3, wherein the infection conditions are that the bacterial liquid is cultured until the OD value is 0.2, the infection time is 15min, the AS concentration in the co-culture medium is 100umol/L, and the co-culture time is 3 days.