阅读说明：本技术 菊叶薯蓣转基因毛状根诱导方法 (Dioscorea composita transgenic hairy root induction method ) 是由 谢君 周建婵 钟春梅 于 2020-05-20 设计创作，主要内容包括：本发明提供了一种菊叶薯蓣转基因毛状根诱导方法,包括组培苗培养,转基因侵染菌液的制备,侵染与共培养,毛状根的诱导,毛状根的除菌培养,毛状根的液体扩大培养。本发明提供了针对菊叶薯蓣转基因毛状根的诱导方法,以菊叶薯蓣去根组培苗作为外植体,发根农杆菌C58C1作为诱导菌株,在特定浓度的乙酰丁香酮条件下可高效诱导产毛状根,诱导率可达50％～85％,同时本发明可根据需求诱导产生含不同目的基因的转基因毛状根。本发明对菊叶薯蓣功能基因的鉴定、薯蓣皂苷合成等生物代谢途径的研究、分子育种、次生代谢物产业化生产等具有重要意义。(The invention provides a dioscorea composita transgenic hairy root induction method, which comprises the steps of tissue culture seedling culture, preparation of transgenic infection bacterial liquid, infection and co-culture, induction of a hairy root, degerming culture of the hairy root and liquid expansion culture of the hairy root. The invention provides an induction method for dioscorea composita transgenic hairy roots, which takes dioscorea composita root-removed tissue culture seedlings as explants and agrobacterium rhizogenes C58C1 as induction strains, can efficiently induce and produce hairy roots under the condition of acetosyringone with specific concentration, and the induction rate can reach 50-85 percent. The invention has important significance for the identification of dioscorea composita functional genes, the research of biological metabolic pathways such as dioscin synthesis and the like, molecular breeding, the industrial production of secondary metabolites and the like.)

1. A dioscorea composita transgenic hairy root induction method is characterized by comprising the following steps:

s1, culturing tissue culture seedlings

Removing roots of the rooted dioscorea composita tissue culture seedlings, and then transferring the rooted dioscorea composita tissue culture seedlings to a subculture medium for pre-culturing for 10-20 days to obtain explants;

s2, preparation of transgenic engineering bacteria

Preparing competent cells by carrying out amplification culture on the activated agrobacterium rhizogenes C58C1, then transferring the competent cells into an expression vector containing a target gene, and screening and identifying to obtain transgenic agrobacterium rhizogenes C58C1 engineering bacteria;

s3, preparation of transgenic infected bacterial liquid

Carrying out amplification culture on the transgenic agrobacterium rhizogenes C58C1 engineering bacteria of the step S2 to obtain OD₆₀₀Centrifuging at 0.5-0.6 deg.C to collect strain, and suspending with 1/2MS liquid culture medium containing acetosyringone to OD₆₀₀Obtaining an infection bacterial liquid at 0.5-0.6;

s4, infection and co-culture

Infecting the explant obtained in the step S1 in the infecting bacterium solution obtained in the step S3 for 15-30 min, removing the explant and removing liquid on the surface of the explant after infection is finished, then transferring the explant to an MS solid culture medium containing acetosyringone, and carrying out dark co-culture for 2-3 days at 23-27 ℃;

s5, induction of hairy roots

Taking out the explant from the co-culture medium, soaking the explant in sterile water containing cefotaxime sodium antibiotic for 8-10 minutes, and then washing the explant for 2-4 times by using pure sterile water; removing water on the surface of the explant after the explant is cleaned, and then transferring the explant to an MS solid culture medium containing cefotaxime sodium for culture;

s6, degerming culture of hairy roots

Removing stems and leaves of the explants 12-16 days after rooting of the explants, transferring the remaining hairy roots to a new MS solid culture medium containing cefotaxime sodium, transferring every two weeks, and continuously reducing the working concentration of the cefotaxime sodium until the working concentration is zero;

s7, liquid amplification culture of hairy roots

The hairy roots after the sterilization culture are separated into one strip from the explant, and then inoculated into 1/2MS liquid culture medium for shake culture.

2. The method for inducing transgenic hairy roots of Dioscorea composita according to claim 1, further comprising PCR detection of transgenic hairy roots: extracting hairy root DNA, and performing PCR identification to identify genes including rol B and rol C and transferred target genes.

3. The method for inducing transgenic hairy roots of Dioscorea composita according to claim 1 or 2, wherein the subculture medium is MS + 0.05-0.3 mg/L NAA + 1.2-1.7 mg/L6-BA.

4. The method for inducing the transgenic hairy roots of Dioscorea composita according to claim 1 or 2, wherein the pre-culture in step S1 is carried out at 23-27 ℃, the illumination intensity is 1500-2500 lx, the illumination time is 14-18 h per day, and the culture is carried out for 10-20 days.

5. The method for inducing the transgenic hairy roots of Dioscorea composita according to claim 1 or 2, wherein the infection in step S4 is carried out at 26-30 ℃ and 130-160 r/min by shaking for 15-30 min.

6. The method for inducing transgenic hairy roots of Dioscorea composita according to claim 1 or 2, wherein the concentration of acetosyringone in 1/2MS liquid medium containing acetosyringone in step S3 and MS solid medium containing acetosyringone in step S4 is 100-600 μmol/L.

7. The method for inducing transgenic hairy roots of Dioscorea composita according to claim 1 or 2, wherein the concentration of cefotaxime sodium in sterile water containing cefotaxime sodium antibiotic and MS solid culture containing cefotaxime sodium in step S5 is 200-300 mg/L.

8. The method for inducing the transgenic hairy roots of Dioscorea composita according to claim 1 or 2, wherein the cultivation in step S5 is carried out at 23-27 ℃, with an illumination intensity of 1500-2500 lx, for 14-18 h per day, for 3-5 days.

9. The method for inducing transgenic hairy roots of Dioscorea composita according to claim 1 or 2, wherein the working concentration of cefotaxime sodium in the MS culture medium of the first transfer in step S6 is 250mg/L, the working concentration of cefotaxime sodium of the second transfer is 100mg/L, and finally the material is transferred to MS solid culture medium without Cef.

10. The method for inducing transgenic hairy roots of Dioscorea composita according to claim 1 or 2, wherein the gene of interest is GFP.

Technical Field

The invention relates to the technical field of plant genetic engineering, in particular to a dioscorea composita transgenic hairy root induction method.

Background

Dioscorea composita Hemsl is a perennial wound herbaceous plant of Dioscoreaceae, is native to Mexico, and is one of the main cultivars for producing diosgenin, a hormone medicine raw material, in Mexico. China successfully introduced in a Xishuangbanna tropical vegetable garden for the first time in 1978, is characterized by high yield of dioscorea tuber and high content of diosgenin and starch, and is a preferred raw material for producing diosgenin and fuel ethanol by fermentation. However, as wild yam resources are excessively exploited, the regeneration speed of the wild yam resources cannot meet the market demand, the yield of diosgenin cannot meet the production demand of steroid pharmaceutical industry, and the contradiction between supply and demand is more prominent. Therefore, by optimizing good yam resources and researching how to improve the yield of roots or tubers and the content of the diosgenin, the growth rule of the diosgenin in the plant body is mastered, the content of the diosgenin is stabilized, and the like, the potential huge economic benefits are realized.

Agrobacterium rhizogenes (Agrobacterium rhizogenes) is a gram-negative Agrobacterium of the genus Agrobacterium of the family Rhizobiaceae, which is highly invasive, can infect most dicotyledons and few monocotyledons as well as gymnosperms, and has wide applications in the field of plant genetic engineering. The Ri plasmid carried by the agrobacterium rhizogenes is independent double-stranded circular DNA located outside the agrobacterium rhizogenes chromosome, and comprises a Vir region and a T-DNA region. When the agrobacterium rhizogenes infects plants, small molecular phenolic compounds are generated at the wound of the plants after the plants are wounded, so that a Vir region gene group on an Ri plasmid is induced and activated, a T-DNA region is regulated and controlled, and rol genes (rol B, rol C and the like) which are genetic loci for inducing hairy roots are transferred into plant host cell genes, thereby causing the change of plant morphology and metabolism and leading to the generation of hairy roots. Hairy roots generated by agrobacterium rhizogenes induced plants have the characteristics of rapid growth, hormone autotrophy, simple growth conditions, high and stable secondary metabolite content, high differentiation degree, difficult variation and the like, and are commonly used as models of a plurality of important researches.

Dioscorea belongs to monocotyledons, and monocotyledons do not synthesize phenols such as acetosyringone, so that hairy roots are difficult to induce. According to the related research reports of hairy root induction, there are few related research reports about the successful induction of hairy roots by monocotyledons, and only preliminary research reports about the induction of hairy roots of dioscorea zingiberensis (Chengyongjiu, Shenjunhao, Pangjun. the induction of hairy roots of dioscorea zingiberensis and the production of diosgenin [ J ]. Hubei university (Nature science edition), 2009 01: 79-83.), which applies Agrobacterium rhizogenes such as R1601, A4 and the like to perform hairy root induction by taking stem segments, leaves and callus of dioscorea zingiberensis as explants, finds that only the callus can be induced to generate hairy roots, and has low induction efficiency of 34.5%, while related technologies about the induction of hairy roots of dioscorea zingiberensis have not been reported at present.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide a dioscorea composita transgenic hairy root induction method.

The above object of the present invention is achieved by the following technical solutions:

a dioscorea composita transgenic hairy root induction method comprises the following steps:

s1, culturing tissue culture seedlings

Removing roots of the rooted dioscorea composita tissue culture seedlings, and then transferring the rooted dioscorea composita tissue culture seedlings to a subculture medium for pre-culturing for 10-20 days to obtain explants;

s2, preparation of transgenic engineering bacteria

Preparing competent cells by carrying out amplification culture on the activated agrobacterium rhizogenes C58C1, then transferring the competent cells into an expression vector containing a target gene, and screening and identifying to obtain transgenic agrobacterium rhizogenes C58C1 engineering bacteria;

s3, preparation of transgenic infected bacterial liquid

Carrying out amplification culture on the transgenic agrobacterium rhizogenes C58C1 engineering bacteria of the step S2 to obtain OD₆₀₀Centrifuging at 0.5-0.6 deg.C to collect strain, and suspending with 1/2MS liquid culture medium containing acetosyringone to OD₆₀₀Obtaining an infection bacterial liquid at 0.5-0.6;

s4, infection and co-culture

Infecting the explant obtained in the step S1 in the infecting bacterium solution obtained in the step S3 for 15-30 min, removing the explant and removing liquid on the surface of the explant after infection is finished, then transferring the explant to an MS solid culture medium containing acetosyringone, and carrying out dark co-culture for 2-3 days at 23-27 ℃;

s5, induction of hairy roots

Taking out the explant from the co-culture medium, soaking the explant in sterile water containing cefotaxime sodium antibiotic for 8-10 minutes, and then washing the explant for 2-4 times by using pure sterile water; removing water on the surface of the explant after the explant is cleaned, and then transferring the explant to an MS solid culture medium containing cefotaxime sodium for culture;

s6, degerming culture of hairy roots

Removing stems and leaves of the explants 12-16 days after rooting of the explants, transferring the remaining hairy roots to a new MS solid culture medium containing cefotaxime sodium, transferring every two weeks, and continuously reducing the working concentration of the cefotaxime sodium until the working concentration is zero;

s7, liquid amplification culture of hairy roots

The hairy roots after the sterilization culture are separated into one strip from the explant, and then inoculated into 1/2MS liquid culture medium for shake culture.

Preferably, the PCR detection step of the transgenic hairy roots is also included: extracting hairy root DNA, and performing PCR identification to identify genes including rol B and rol C and transferred target genes.

Preferably, the subculture medium is MS + 0.05-0.3 mg/L NAA + 1.2-1.7 mg/L6-BA.

Further preferably, the subculture medium is MS +0.1mg/L NAA +1.5 mg/L6-BA.

Preferably, the pre-culture in the step S1 is carried out at 23-27 ℃, the illumination intensity is 1500-2500 lx, the illumination time is 14-18 h per day, and the culture lasts 10-20 days.

Further preferably, the pre-culture of step S1 is performed at 25 deg.C, illumination intensity of 2000lx, illumination time of 16h per day, and culture period of about two weeks.

Preferably, step S2 is activating agrobacterium rhizogenes C58C1 and making competent cells, then transforming expression vector containing target gene into competent cells of the strain, recovering strain, centrifugally enriching strain, coating on YEB solid medium containing rifampicin antibiotic, after single strain grows out, picking single colony for culture, and performing PCR identification and enzyme digestion identification, positive transgenic engineering strain C58C 1.

Further preferably, the freeze-thaw method is used for transforming the expression vector containing the target gene into agrobacterium rhizogenes; the strain recovery condition is 28 ℃, and shaking culture is carried out for 3h at 180 r/min; the working concentration of rifampicin antibiotic used was 50 mg/L.

Preferably, the infection in the step S4 is carried out at 26-30 ℃ and 130-160 r/min by shaking for 15-30 min.

Further preferably, the infection in the step S4 is carried out at 28 ℃ and 150r/min for 20min with shaking.

Preferably, the concentration of acetosyringone in the 1/2MS liquid culture medium containing acetosyringone in the step S3 and the MS solid culture medium containing acetosyringone in the step S4 is 100-600 μmol/L.

Further preferably, the concentration of the acetosyringone is 400-600 mu mol/L.

Preferably, the concentration of cefotaxime sodium in the sterile water containing cefotaxime sodium antibiotic and the MS solid culture containing cefotaxime sodium in the step S5 is 200-300 mg/L.

Further preferably, the concentration of cefotaxime sodium is 250 mg/L.

Preferably, in the step S5, the cultivation is carried out at 23-27 ℃, the illumination intensity is 1500-2500 lx, the illumination time is 14-18 h per day, and the cultivation lasts for 3-5 days.

Further preferably, in the step S5, the cultivation is performed at a temperature of 25 ℃, a light intensity of 2000lx, a light time of 16h per day, and 3-5 days.

Preferably, the working concentration of cefotaxime sodium in the MS culture medium of the first transfer of step S6 is 250mg/L, the working concentration of cefotaxime sodium of the second transfer is 100mg/L, and finally the material is transferred into the MS solid culture medium without Cef.

Preferably, the gene of interest is GFP. But not limited to the gene, expression vectors containing different target genes can be constructed according to different research requirements, the expression vectors are transferred into agrobacterium rhizogenes C58C1 competence to prepare transgenic engineering bacteria, and then the transgenic hairy roots expressing the target genes are induced.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides an induction method for dioscorea composita transgenic hairy roots, which takes dioscorea composita root-removed tissue culture seedlings as explants and agrobacterium rhizogenes C58C1 as induction strains, can efficiently induce and produce hairy roots under the condition of acetosyringone with specific concentration, and the induction rate can reach 50-85 percent. The invention has important significance for the identification of dioscorea composita functional genes, the research of biological metabolic pathways such as dioscin synthesis and the like, molecular breeding, the industrial production of secondary metabolites and the like.

Drawings

FIG. 1 shows the induction and culture of transgenic hairy root of Dioscorea composita.

FIG. 2 shows the colony PCR identification of Agrobacterium rhizogenes C58C1+ pCang-eGFP transgenic engineering bacteria. M: DNA Marker; 1-4: 4 pseudotransformants of C58C1+ pCang-eGFP.

FIG. 3 shows PCR detection of transgenic hairy roots of Smallanthus sonchifolius. 1 and 2: root DNA of wild Dioscorea composita seedlings; 3: plasmid DNA of Agrobacterium rhizogenes C58C 1; 4: plasmid DNA of transgenic Agrobacterium rhizogenes C58C1+ PcanaG-GFP; 5 and 6: transgenic Agrobacterium rhizogenes C58C1+ PcanaG-GFP induced hairy root DNA from explants.

FIG. 4 shows the explant optimization results induced by hairy roots. The right side is the switching state of the leaf experimental group, and the left side is the switching state of the rooting-free tissue culture seedling experimental group.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

Agrobacterium rhizogenes C58C1, R1601, K599, A4 were all purchased from Shanghai Bionetwork Rachu Biotech Ltd.