CN111635903A

CN111635903A - Method for enhancing plant viability

Info

Publication number: CN111635903A
Application number: CN202010512039.7A
Authority: CN
Inventors: 郭东林; 董晓雨; 周思莹; 石卓; 李洪
Original assignee: Harbin Normal University
Current assignee: Harbin Normal University
Priority date: 2020-06-08
Filing date: 2020-06-08
Publication date: 2020-09-08

Abstract

The invention provides a method for enhancing plant viability, which comprises the steps of constructing alfalfa YSL6 gene as a plasmid of a plant gene expression vector, transforming agrobacterium into the plasmid, and culturing the agrobacterium and a plant seedling together to obtain a transgenic plant seedling, so that the iron content and the iron storage capacity in a plant are improved, and the plant viability is enhanced. The transgenic plant obtained by the method for enhancing the plant viability has large leaves, greener color, dark seed color and black-like seeds, and the seeds are fuller than the original seeds of the plant.

Description

Method for enhancing plant viability

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a method for enhancing plant viability.

Background

Alfalfa (Medicago sativa L.) is the earliest and most widely cultivated high-quality perennial leguminous forage grass planted in the world at present, and has the advantages of high grass yield, strong regeneration, long utilization period, strong palatability, rich nutrition and the like. Alfalfa has been planted for over two thousand years in China, and is the largest pasture in China.

The threat of soil salinization to the production, ecological environment and sustainable development of agriculture and animal husbandry is a worldwide problem, and the global salinization soil area reaches 9.5 × 108hm²The total area of salinized land in China is about 5 hundred million mu, 3000 more than ten thousand mu of saline-alkali land is available in Heilongjiang province, and the area of severe saline-alkali land is about 5 hundred million mu. The great difficulty of the utilization of the alkaline soil is that iron exists in a slightly soluble form and is difficult to be absorbed by plants, so that the plants are lack of iron. The alfalfa grows in saline-alkali soil with extremely low content of available iron and shows the resistance to saline-alkali and the tolerance to iron deficiency. This suggests a unique iron metabolism mechanism in alfalfa bodies. The capability of alfalfa to resist abiotic stress has important significance for the cultivation and breeding research of alfalfa, and the solution of the capability of resisting abiotic stress is an important content for improving the productivity. The OPT gene plays an important role in the process of transferring substances of plants, and the plants use amino acid obtained by hydrolyzing oligopeptide in cells by the OPT gene as an important source of carbon and nitrogen, so that nitrogen and carbon are provided for growth and development, and the germination of seeds, the development of embryos and the accumulation of biomass are influenced. The alfalfa can grow in saline-alkali soil with extremely low effective iron content, and shows saline-alkali resistance and tolerance to iron deficiency. Recent studies show that the YS/YSL subfamily of the oligopeptide transporter family plays an important role in chelated metal transport, particularly iron transport, has different expression modes and has a wide range of transport substrates.

In conclusion, the YSL gene of alfalfa has high application value.

Disclosure of Invention

Based on the content, the invention provides a method for enhancing plant viability, which comprises the steps of constructing alfalfa YSL6 gene as a plasmid of a plant gene expression vector, transforming agrobacterium into the plasmid, and culturing the agrobacterium tumefaciens and a plant seedling together to obtain a transgenic plant seedling, so that the iron content and the iron storage capacity in the plant are improved, and the plant viability is enhanced;

further, the method specifically comprises the following steps:

s1: cloning YSL6 gene of alfalfa from alfalfa by PCR technology;

s2: constructing a gene expression vector Pbi121-Ms YSL6 plasmid of a plant;

s3: transforming the Pbi121-Ms YSL6 plasmid into Agrobacterium;

s4: co-culturing the plantlet and the Pbi121-Ms YSL6 plasmid, and performing bud induction, rooting induction and transplantation domestication after culture to finally obtain a transgenic plantlet with strong survival ability;

further, the S1 specifically includes:

s11: extracting alfalfa RNA;

s12, extracting alfalfa RNA as a template, and obtaining cDNA by a reverse transcription PCR method;

s13: gel recovery of target gene bands: recovering a target gene fragment Ms YSL6 obtained by PCR amplification by adopting a DNA gel recovery kit;

s14: construction of cloning vector pMD18T-MsYSL 6: connecting Ms YSL6 target gene segment to a cloning vector pMD18T, and transferring the connection product into escherichia coli competence by a heat shock method;

s15: sequencing the Ms YSL6 gene and analyzing by bioinformatics;

further, the S11 specifically includes:

s111: putting 0.1g of the whole alfalfa plant into a mortar precooled by liquid nitrogen, and grinding the whole alfalfa plant into fine powder;

s112: the powder was transferred to a 1.5mL sterile centrifuge tube and 450. mu.L RL was added;

s113: transferring all the liquid to a filter column, centrifuging at 12000rpm for 5min, and carefully sucking the supernatant into a new RNase-Free centrifuge tube;

s114: slowly adding 0.5 times of absolute ethyl alcohol in volume of the supernatant, uniformly mixing, transferring the obtained solution and the precipitate into an adsorption column, centrifuging at 12000rpm for 60s, pouring off waste liquid in the collecting tube, and putting the adsorption column back into the collecting tube;

s115: add 350. mu.L of deproteinized solution to the adsorption column, centrifuge at 12000rpm for 60s, discard the filtrate, and place the adsorption column back into the collection tube.

S116: preparing DNase working solution: adding 70 mu L of RDD solution into 10 mu L of DNase I reaction solution, and gently and uniformly mixing;

s117: adding 80 μ LDNase working solution onto adsorption column filter membrane, and standing for 15 min;

s118: adding 350 μ L deproteinized solution into centrifugal adsorption column, centrifuging at 12000rpm for 60s, discarding filtrate, and placing adsorption column back into collection tube;

s119: adding 300 μ L of rinsing solution into the centrifugal adsorption column, standing at room temperature for 2min, centrifuging at 12000rpm for 60s, removing filtrate, and placing the adsorption column back into the collecting tube;

s1110: step S119 is repeated.

S1111: centrifuging at 12000rpm for 2min, removing filtrate, standing the adsorption column at room temperature for 5min to completely air-dry the residual rinsing solution in the adsorption column;

s1112: transferring the centrifugal adsorption column into an RNase-free collection tube, adding 30 mu L of RNA eluent, standing for 1-2min, centrifuging at 12000rpm for 2min to obtain an RNA sample in the collection tube, and storing at-80 ℃;

further, the S14 specifically includes:

s141: taking out the Escherichia coli competence from a refrigerator at minus 80 ℃, quickly putting the Escherichia coli competence on ice, adding 20ul of the ligation product after the Escherichia coli competence is dissolved, and carrying out ice bath for 30 min;

s142: taking out, rapidly placing in 42 deg.C water bath for heat shock for 1min 15s, rapidly inserting into ice, and ice-cooling for 2 min. Adding 800ul LB liquid culture medium, placing on a 200rpm shaking table, shaking at 37 deg.C for 1 h;

s143: taking out the centrifuge tube, centrifuging at 4000rpm for 5min, taking out 800ul of supernatant, re-suspending the residual thallus, and smearing the residual 200ul of bacterial liquid on two LB solid culture media containing ampicillin resistance in a ratio of 3: 1;

s144: carrying out inverted culture in an incubator at 37 ℃ overnight; picking single colony on the culture medium for identification;

further, the S15 specifically includes:

s151: predicting the open reading frame of MsYSL6 gene by using the software ORF Finder;

s152: comparing the predicted amino acid sequence of MsYSL6 by using NCBI protein BlAST to hopefully obtain the structure domain of MsYSL65 protein, comparing the structure domain with the structure domain of the alfalfa OPT family oligopeptide transporter, and finding out the difference site of the amino acid in the corresponding structure domain by using software ClustalX2.1;

s153, analyzing the theoretical isoelectric point of the MsYSL6 protein by using ProtParam software, and analyzing the transmembrane domain of the MsYSL6 protein by using TMHMM 2.0;

s154: secondary domains of MsYSL6 protein were analyzed using the SOPMA software prediction.

S155: analyzing the subcellular localization of the MsYSL6 protein by using ngLOC software;

s156: protein sequences of YSL and OPT transport protein families of plants are obtained from NCBI, and are compared by using software ClustalX2 and MEGA6, and a phylogenetic evolutionary tree of the plants is constructed.

The method of claim 5, wherein the reaction system of S14 is: mu.l of pMD18-Tvector, 3. mu.l of the target gene fragment solution, and 1. mu.l of dd H₂O, 5 μ l of Solution I, and the reaction conditions are as follows: the reaction temperature is 16 ℃, the reaction time is more than 12h, and the reaction is carried out overnight;

furthermore, the S3 also includes identification of transformed Agrobacterium, wherein the identification method comprises selecting a single colony with uniform growth, performing colony PCR amplification with gene-specific primers, and the reaction system comprises Template DNA colony, 1 μ l10 × Buffer, 0.8 μ l d NTP Mix, 0.4 μ l MsYSL6-F, 0.4 μ l MsYSL6-R, 0.05 μ l R Taq, and 7.35 μ l dd H₂O, the reaction conditions are ① 94 ℃, 5min, ② 94 ℃, 30s, ③ 55 ℃, 30s, ④ 72 ℃, 2min, 30s, ⑤ 72 ℃, 10min, ⑥ 4 ℃, 1h and ① - ⑥ for 30 times of circulation;

further, the S4 specifically includes:

s41: carrying out aseptic culture on the plant plantlets, and using leaves after the plantlets grow into big seedlings for infection experiments;

s42: cutting tobacco leaves into squares of about 1cm multiplied by 1cm, and pre-culturing for 48 h;

s43: activating the agrobacterium cultured in the S3, and infecting the tobacco in the S42 with the bacterial liquid;

s44: placing the leaf in S41 on sterile filter paper, sucking off bacterial liquid attached to the tobacco in S43, placing the back of the leaf upwards on a culture medium of MS1, and culturing in dark for 2 d;

s45: transferring the leaves subjected to dark culture for 2 days in S44 to an MS2 culture medium for bud induction;

s46: when the buds induced in the S45 are differentiated, cutting off the buds, transferring the buds to an MS3 culture medium, and carrying out rooting culture;

s47: transferring the rooted tobacco seedlings to soil for culture for subsequent functional verification and physiological analysis;

further, the S43 specifically includes: coating pBI121-MsYSL6 positive bacteria on a YEB solid culture medium containing Km, Str and Rif in an ultraclean workbench, performing inverted culture for 48h at 28 ℃, then suspending the bacteria by 1/2MS liquid culture medium to obtain bacterial liquid, diluting the bacterial liquid by 1/2MS until the OD600 is about 0.4-0.6, and transferring the bacterial liquid to a 50mL conical flask for infecting tobacco;

the invention has the following beneficial effects:

1. according to the method for enhancing the plant viability, the obtained transgenic plant has large leaves and greener color, the color of seeds is dark and tends to be black, and the seeds are fuller than the original seeds of the plant;

2. the method for enhancing the plant viability improves the iron content in the plant and the iron storage capacity, thereby increasing the iron and chlorophyll content in the leaves, enhancing the photosynthesis, further improving the iron nutrition status of the plant and enhancing the low iron resistance of the plant.

Drawings

FIG. 1 is a diagram showing the acquisition of a tobacco resistant plant transformed with MsYSL6 gene in example 1 of the present invention;

FIG. 2 is a diagram showing the results of partial PCR identification in example 1 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. On the contrary, the invention is intended to cover alternatives, modifications, equivalents and alternatives which may be included within the spirit and scope of the invention as defined by the appended claims. Furthermore, in the following detailed description of the present invention, certain specific details are set forth in order to provide a better understanding of the present invention. It will be apparent to one skilled in the art that the present invention may be practiced without these specific details.

The invention is further described with reference to the following figures and specific examples, which are not intended to be limiting. The following are preferred examples of the present invention:

the invention provides a method for enhancing the viability of plants, which improves the content of iron in the plants and the capacity of storing the iron, and enables the plants to grow better in adverse circumstances in the development process of organisms due to the increase of the content of the iron. Thus, plant viability is enhanced. And because of the rise of the iron content, the iron and chlorophyll content in the leaves is increased, the photosynthesis is enhanced, the iron nutrition condition of the plants is further improved, the low iron resistance of the plants in the saline-alkali soil is enhanced, and the plants grow in the saline-alkali soil with extremely low effective iron content, so that the soil is improved.

The invention is realized by the following technical scheme:

a method of enhancing the viability of a plant, said method comprising the steps of:

s1, cloning YSL6 gene of alfalfa from alfalfa by adopting PCR technology;

s2, constructing a plant gene expression vector Pbi121-Ms YSL6 plasmid;

s3, transforming Agrobacterium with the Pbi121-Ms YSL6 plasmid;

and S4, co-culturing the plantlet and the Pbi121-Ms YSL6 plasmid, and performing bud induction, rooting induction and transplantation domestication after culture to finally obtain the transgenic plant with strong survival ability.

The specific content of the S1 includes:

s11, extracting alfalfa RNA:

(1) 0.1g of the whole alfalfa plant is put into a mortar precooled by liquid nitrogen and ground into fine powder.

(2) The powder was transferred to a 1.5mL sterile centrifuge tube and 450. mu.L RL (confirming the addition of β -mercaptoethanol) was added.

(3) All the liquid was transferred to a filtration column, centrifuged at 12000rpm for 5min and the supernatant carefully pipetted into a new RNase-Free centrifuge tube.

(4) Slowly adding 0.5 times of the volume of the supernatant of absolute ethyl alcohol, mixing uniformly, transferring the obtained solution and the precipitate into an adsorption column, centrifuging at 12000rpm for 60s, pouring off the waste liquid in the collection tube, and putting the adsorption column back into the collection tube.

(5) Add 350. mu.L of deproteinized solution to the adsorption column, centrifuge at 12000rpm for 60s, discard the filtrate, and place the adsorption column back into the collection tube.

(6) Preparing DNase working solution: add 70. mu.L of RDD solution into 10. mu.L of DNase I reaction solution, and mix gently.

(7) Adding 80 μ LDNase working solution onto the filter membrane of the adsorption column, and standing for 15 min.

(8) Add 350. mu.L deproteinized solution to the centrifugation and adsorption column, centrifuge at 12000rpm for 60s, discard the filtrate, and place the adsorption column back into the collection tube.

(9) Adding 300 μ L of rinsing solution into the centrifugal adsorption column, standing at room temperature for 2min, centrifuging at 12000rpm for 60s, discarding the filtrate, and placing the adsorption column back into the collection tube.

(10) Step 9 is repeated.

(11) Centrifuging at 12000rpm for 2min, discarding the filtrate, and standing the adsorption column at room temperature for 5min to completely air-dry the residual rinse solution in the adsorption column.

(12) Transferring the centrifugal adsorption column into RNase-free collection tube, adding 30 μ L RNA eluent, standing for 1-2min, centrifuging at 12000rpm for 2min to obtain RNA sample in the collection tube, and storing at-80 deg.C.

S12: synthesis of cDNA

Using the extracted RNA as a template, obtaining cDNA by a reverse transcription PCR method, wherein the reaction process comprises the following steps:

(1) preparing a reaction solution I:

Oligo(dT)201μl

RNA 5μl

RNase Free H2O6μl

Total 12μl

(2) and mixing the reaction solution I, placing the mixture in a water bath at 65 ℃ for 5min, and quickly placing the mixture in ice after the water bath.

(3) Preparing a reaction solution II:

reaction solution I12. mu.l

5×RT Buffer 4μl

dNTPs Mixture 2μl

RNase Inhibitor 1μl

Reverse Transcriptase 1μl

Total 20μl

Reaction procedure:

30℃10min

42℃20min

99℃5min

5min S1.2 at 4 ℃, amplifying target genes:

s121, primer sequence: MsYSL6F: 5'-ATGGGTACAGAAACA-3'

MsYSL6R:5’-TCAGCTGCTTGCGGAA-3’

S122, PCR amplification procedure: 94 ℃ for 5min, 30 cycles of 94 ℃ for 30s, 55 ℃ for 30s, 72 ℃ for 2min for 30s, and finally 72 ℃ for 10 min; detecting by 1% agarose gel electrophoresis;

s13, recovering a target gene band by glue:

recovering a target gene fragment Ms YSL6 (alfalfa YSL6) obtained by PCR amplification by adopting a DNA gel recovery kit;

s14, constructing a cloning vector pMD18T-MsYSL 6: connecting Ms YSL6 target gene segment to a cloning vector pMD18T, and transferring the connection product into escherichia coli competence by a heat shock method; the concrete content is as follows:

s141, taking out the escherichia coli competence from a refrigerator at minus 80 ℃, quickly putting the escherichia coli competence on ice, adding 20ul of the ligation product after the escherichia coli competence is dissolved, and carrying out ice bath for 30 min;

and S142, taking out, quickly placing in a water bath at 42 ℃ for heat shock for 1min 15S, quickly inserting into ice, and carrying out ice bath for 2 min. Adding 800ul LB liquid culture medium, placing on a 200rpm shaking table, shaking at 37 deg.C for 1 h;

s143, taking out the centrifuge tube, centrifuging at 4000rpm for 5min, taking out 800ul of supernatant, re-suspending the residual thallus, and smearing the residual 200ul of bacterial liquid on two LB solid culture media containing ampicillin resistance according to the ratio of 3: 1;

s144, carrying out inverted culture in an incubator at 37 ℃ overnight; picking single colony on the culture medium for identification;

s15, sequencing of Ms YSL6 gene and bioinformatics analysis:

s151, predicting the open reading frame of MsYSL6 gene by using the software ORF Finder.

S152, the predicted amino acid sequence of MsYSL6 is compared by using NCBI protein BLAST to hopefully obtain the structure domain of MsYSL65 protein, compared with the structure domain of the alfalfa OPT family oligonucleotide transporter, and the difference site of the amino acid in the corresponding structure domain is found by using software ClustalX2.1.

S153, analyzing the theoretical isoelectric point of the MsYSL6 protein by using ProtParam software. The transmembrane domain of the MsYSL6 protein was analyzed using TMHMM 2.0.

S154, predicting and analyzing the secondary structure domain of the MsYSL6 protein by using SOPMA software.

S155, using ngLOC (http:// genome. unmac. edu/ngLOC/index. html) software to analyze the subcellular localization of MsYSL6 protein.

S156, obtaining protein sequences of other species YSL and OPT transport protein families such as Medicago truncatula (Medicago truncatula), soybean (Glycine max), Arabidopsis thaliana (Arabidopsis thaliana) and rice (Oryza sativa) from NCBI, comparing by using software ClustalX2 and MEGA6, and constructing phylogenetic evolutionary tree thereof.

The reaction system of S1.4 is as follows: mu.l of pMD18-T vector, 3. mu.l of the target gene fragment solution, and 1. mu.l of dd H₂O, 5 μ l of Solution I.

The reaction conditions of S1.4 are as follows: the reaction temperature is 16 ℃, and the reaction time is more than 12h (overnight).

The specific content of the S2 includes:

s21, extracting plasmids of pMD18T-Ms YSL6 and pBI 121: extracting plasmids of pMD18T-MsYSL6 and pBI121 by AXYGEN kit;

s22, digesting plasmids of pMD18T-Ms YSL6 and pBI 121:

s221, Xba I and Sma I are adopted to carry out double digestion on plasmid DNA of pBI121, and the reaction system of double digestion is as follows: 50 μ l of pBI121 plasmid, 10 μ l of 10 XM Buffer, 2 μ l of Xba I, 2 μ l of Sma I and 10 μ l of BSA;

s222, plasmid DNA of pMD18T-Ms YSL6 was double digested with Xba I and Sma I in the reaction system of 50. mu.l of PCR product, 10. mu.l of 10 × M Buffer, 2.5. mu.l of Xba I, 2.5. mu.l of Sma I, 5. mu.l of BSA and 30. mu.l of ddH₂O；

S23, recovering the restriction enzyme products of plasmid DNA of pMD18T-Ms YSL6 and pBI121 by adopting a DNA gel recovery kit;

s24, connecting the expression vector pBI121 and the target gene fragment Ms YSL6, wherein the reaction system during connection is as follows: 5. mu.l of plasmid DNA digest of pBI121, 3. mu.l of digest of Ms YSL6, 1. mu.l of 10 XT 4DNA Ligase Buffer and 1. mu.l of T4DNA Ligase; the reaction conditions are as follows: the reaction temperature is 16 ℃, and the reaction time is more than 12h (overnight);

s25, ligation product: pBI121-Ms YSL6 transformed Escherichia coli competence;

s26, identification of recombinant plasmid:

(1) colony PCR identification is carried out, MsYSL6 gene primers containing Xba I and Sma I enzyme cutting sites are used for PCR identification, and the reaction system is as follows:

ddH₂O7.35μl、10×PCR Buffer1μl

、dNTP Mix(2.5mmol/L)0.8μl、MsYSL6-Xba I(10μmol/L)

0.4μl、MsYSL6-Sma I(10μmol/L)0.4μl、rTaq DNA polymerase(5U/μl)0.05μl、Template DNA

single colony reaction procedure: (30 cycles)

(2) The plasmid DNA of the recombinant pBI121-Ms YSL6 was extracted for enzyme digestion verification, and the reaction system for the enzyme digestion verification was 5. mu.l of the pBI121-MsYSL6 recombinant plasmid, 1. mu.l of 10 × T Buffer, 1. mu.l of BSA, 0.5. mu.l of Xba I, 0.5. mu.l of Sma I, and 2. mu.l of ddH₂O；

The specific content of the S3 includes:

(1) the recombinant plasmid pBI121-MsYSL6 is transformed by a freeze-thaw method, which comprises the following steps:

firstly, taking the competent cells stored at the temperature of-80 ℃ and placing the competent cells in ice for thawing.

② adding 10 mul of pBI121-MsYSL6 recombinant plasmid into 100 mul of Agrobacterium tumefaciens competent cell GV3101, and stirring uniformly by hand to the bottom of the tube.

And thirdly, standing on ice for 5min, using liquid nitrogen for 5min, using water bath at 37 ℃ for 5min, and carrying out ice bath for 5 min.

Fourthly, 700 mul of YEB liquid culture medium without antibiotics is added, and shaking culture is carried out for 2-3h at the temperature of 28 ℃.

Fifthly, carrying out centrifugation at 6000rmp for 1min to collect bacteria, leaving 100 mul of supernatant to resuspend bacteria blocks, coating the bacteria liquid on YEB solid culture medium containing Km (50mg/mL) + Str (50mg/mL) + Rif (50mg/mL), and carrying out dark culture in an incubator at 28 ℃ for 2-3d by inverting.

The S3 also comprises identifying the transformed Agrobacterium, wherein the identification method comprises selecting a single colony with uniform growth, and performing colony PCR amplification with gene specific primers, and the reaction system comprises Template DNA colony, 1 μ l10 × Buffer, 0.8 μ l d NTP Mix, 0.4 μ l MsYSL6-F, 0.4 μ l MsYSL6-R, 0.05 μ l R Taq, and 7.35 μ l dd H₂O, the reaction conditions are ① 94 ℃, 5min, ② 94 ℃, 30s, ③ 55 ℃, 30s, ④ 72 ℃, 2min, 30s, ⑤ 72 ℃, 10min, ⑥ 4 ℃, 1h and ① - ⑥ for 30 times of circulation;

the specific content of the S4 includes:

s41, sterile culture of plant seedlings:

(1) placing wild type tobacco seed in sterilized centrifuge tube, sterilizing with 75% alcohol for 3min, and washing with sterilized water for 6-8 times.

(2) Then treated with 1mL of 10% sodium hypochlorite for 5min, and washed with sterilized water for 6-8 times.

(3) And uniformly spreading the treated seeds on an MS solid culture medium, and culturing to grow seedlings.

(4) And transferring the plantlets into a bottle filled with an MS solid culture medium, continuously culturing to grow into large plantlets, and using the leaves for infection experiments.

S42, cutting tobacco leaves into squares of about 1cm × 1cm, and pre-culturing for 48 h.

S43, activating the agrobacterium:

coating pBI121-MsYSL6 positive bacteria on YEB solid culture medium containing Km, Str and Rif in a clean bench, culturing for 48h at 28 ℃, then suspending the bacteria by 1/2MS liquid culture medium to obtain bacterial liquid, and then diluting the bacterial liquid by 1/2MS until OD600 is about 0.4-0.6. The bacterial solution used above was transferred to a 50mL Erlenmeyer flask for infestation of tobacco.

S44, taking out the pre-cultured leaf blades, infecting in the bacterial liquid for 6min, continuously reversing the process to ensure that the leaf blades are fully contacted with the bacterial liquid, taking out the leaf blades, placing the leaf blades on sterile filter paper, sucking off the attached bacterial liquid, placing the back of the leaf blades upwards on a culture medium of MS1, and culturing in the dark for 2 d.

S45, shoot induction:

the leaves were transferred to MS2 medium for shoot induction, and the medium was changed every 3 days and every 2 weeks after 3 consecutive times.

S46, rooting induction:

when the differentiated buds grow to about 2cm, the differentiated buds are cut off and transferred to an MS3 culture medium for rooting culture.

S47, transplanting and domesticating:

and transferring the rooted tobacco seedlings to soil for culture for subsequent functional verification and physiological analysis.

The tobacco plant obtained after the transformation is the tobacco resistant plant of the transgenic MsYSL6 gene.

Example 1:

as shown in FIG. 1, pBI121-MsYSL6 positive bacteria were spread on YEB solid medium containing Km, Str and Rif, and bacterial colonies were resuspended in 1/2MS to obtain bacterial suspension, which was then diluted with 1/2MS to an OD600 of about 0.4 to 0.6. The MsYSL6 gene is genetically transformed to tobacco by an agrobacterium-mediated leaf disc method, and a resistant plant of the MsYSL6 gene can be obtained. The infected leaves are first cultured on MS1 in the dark for 2-3d, then transferred to MS2 for screening, callus can grow on the periphery of the successfully infected leaves, and then buds are differentiated, while callus and buds cannot grow on the periphery of the unsuccessfully infected leaves. And after cutting the resistant buds, transferring the cut resistant buds to an MS3 culture medium for rooting culture to obtain 25 kanamycin resistant plants in total, and obtaining 13 positive plants after PCR identification. Transplanting the grown resistant plants to a soil-containing: vermiculite is 2: 1 in the flowerpot.

And carrying out PCR identification on Km resistant plants. The plasmid pBI121-MsYSL6 is used as a positive control, wherein 13 strains can amplify fragments about 600bp and are consistent with the positive control fragments, which can preliminarily prove that MsYSL6 is transferred into tobacco, and partial PCR identification results are shown in figure 2.

The agrobacterium-mediated leaf disc method used in the invention is processed and improved, compared with the prior experimental method, the method has the advantages of convenience, rapidness and short consumed time, and avoids the defect that the concentration is difficult to control in the prior bacteria shaking process. In the present study, it was found that the efficiency of callus induction when a tobacco plant with a bright green color was used as an explant was significantly higher than that of young, yellow tobacco plants.

The above-described embodiment is only one of the preferred embodiments of the present invention, and general changes and substitutions by those skilled in the art within the technical scope of the present invention are included in the protection scope of the present invention.

Claims

1. A method for enhancing plant viability is characterized in that alfalfa YSL6 gene is constructed to be used as a plasmid of a plant gene expression vector, agrobacterium is transformed by the plasmid and is cultured together with plant seedlings to obtain transgenic plant seedlings, and then the iron content and the iron storage capacity of the plant are improved, and the plant viability is enhanced.

2. The method according to claim 1, characterized in that it comprises in particular the steps of:

s1: cloning YSL6 gene of alfalfa from alfalfa by PCR technology;

s2: constructing a gene expression vector Pbi121-Ms YSL6 plasmid of a plant;

s3: transforming the Pbi121-Ms YSL6 plasmid into Agrobacterium;

s4: co-culturing the plantlet and the Pbi121-Ms YSL6 plasmid, and performing bud induction, rooting induction and transplantation domestication after culture to finally obtain the transgenic plantlet with strong survival ability.

3. The method according to claim 2, wherein the S1 specifically includes:

s11: extracting alfalfa RNA;

s15: the Ms YSL6 gene was sequenced and analyzed using bioinformatics.

4. The method according to claim 3, wherein the S11 specifically comprises:

s112: transferring the powder to a 1.5mL sterile centrifuge tube, and adding 450. mu.L RL;

s115: adding 350 μ L deproteinized solution into adsorption column, centrifuging at 12000rpm for 60s, discarding filtrate, and placing adsorption column back into collection tube;

s1110: repeating step S119;

s1112: transferring the centrifugal adsorption column into RNase-free collection tube, adding 30 μ L RNA eluent, standing for 1-2min, centrifuging at 12000rpm for 2min to obtain RNA sample in the collection tube, and storing at-80 deg.C.

5. The method according to claim 3, wherein the S14 specifically comprises:

s142: taking out, rapidly placing in 42 deg.C water bath, thermally shocking for 1min 15s, rapidly inserting into ice, ice-cooling for 2min, adding 800ul LB liquid culture medium, placing in 200rpm shaking table, shaking at 37 deg.C for 1 h;

s144: carrying out inverted culture in an incubator at 37 ℃ overnight; single colonies on the medium were picked for identification.

6. The method according to claim 3, wherein the S15 specifically comprises:

s154: predictive analysis of the secondary domain of MsYSL6 protein using SOPMA software;

7. The method of claim 5, wherein the reaction system of S14 is: mu.l of pMD18-Tvector, 3. mu.l of the target gene fragment solution, and 1. mu.l of dd H₂O, 5 μ l of Solution I, and the reaction conditions are as follows: the reaction temperature is 16 ℃, the reaction time is over 12h, and the reaction is carried out overnight.

8. The method of claim 2, wherein S3 further comprises identifying the transformed Agrobacterium by selecting a single colony with uniform growth and performing colony PCR amplification using gene-specific primers, wherein the reaction system comprises Template DNA colony, 1 μ l of 10 × Buffer, and 0.8 μ l of dNTP Mix, 0.4. mu.l of MsYSL6-F, 0.4. mu.l of MsYSL6-R, 0.05. mu.l of rTaq and 7.35. mu.l of dd H₂O, the reaction conditions are ① 94 ℃, 5min, ② 94 ℃, 30s, ③ 55 ℃, 30s, ④ 72 ℃, 2min, 30s, ⑤ 72 ℃, 10min, ⑥ 4 ℃, 1h and ① - ⑥ for 30 times of circulation.

9. The method according to claim 2, wherein the S4 specifically includes:

s47: and transferring the rooted tobacco seedlings to soil for culture for subsequent functional verification and physiological analysis.

10. The method according to claim 9, wherein S43 is specifically: coating pBI121-MsYSL6 positive bacteria on YEB solid culture medium containing Km, Str and Rif in a clean bench, culturing for 48h at 28 ℃ in an inverted mode, suspending the bacteria by 1/2MS liquid culture medium to obtain bacterial liquid, diluting the bacterial liquid by 1/2MS until OD600 is about 0.4-0.6, and transferring the bacterial liquid to a 50mL conical flask for infecting tobacco.