阅读说明：本技术 一种采用绿藻pmi作为番茄遗传转化筛选标记基因的方法 (Method for adopting green alga PMI as tomato genetic transformation screening marker gene ) 是由 林渊源 黄俊潮 于 2019-10-24 设计创作，主要内容包括：本发明提供一种采用绿藻PMI作为番茄遗传转化筛选标记基因的方法。该方法包括绿藻PMI的克隆,植物双元表达载体的构建,番茄的转化及转基因番茄的鉴定等步骤。该基因能替代目前广泛使用的抗生素基因、抗除草剂基因及大肠杆菌来源PMI等异源基因作为标记基因应用于番茄的遗传转化,获得更具食品安全性、生物安全性和生态安全性的转基因番茄。(The invention provides a method for selecting a marker gene by using green alga PMI as tomato genetic transformation. The method comprises the steps of cloning green alga PMI, constructing a plant binary expression vector, transforming tomato, identifying transgenic tomato and the like. The gene can replace the currently widely used antibiotic gene, herbicide resistance gene, escherichia coli source PMI and other heterologous genes as marker genes to be applied to genetic transformation of tomatoes, and the transgenic tomatoes with higher food safety, biological safety and ecological safety are obtained.)

1. A method for adopting green alga PMI as a tomato genetic transformation screening marker gene is characterized in that the marker gene is the origin of green alga, and a screening agent is safe and nontoxic mannose, and the method comprises the following steps:

(1) cloning of PMI

Cloning a PMI phosphomannose isomerase gene from Chlorococcum sp by a PCR technology to obtain a safety marker gene, wherein a nucleotide sequence is shown as a sequence table SEQ ID NO. 1;

(2) construction of expression vectors

After enzyme digestion, the marker gene is connected and introduced into a binary vector PBI121 to replace the original kana resistance gene NPT II, the new vector has no NPT II mark and carries a safety screening marker PMI, is named as PMI-pBI121, and is introduced into agrobacterium LBA4404 by adopting an electric shock transformation method;

(3) genetic transformation of tomato

Transforming tomatoes by adopting an agrobacterium-mediated leaf disc method, culturing for 2 days, transferring into screening culture mediums containing mannose with different concentrations for screening culture, transferring into a rooting culture medium after budding, and rooting to obtain transgenic plants;

(4) identification of transgenic tomato

PCR identification is carried out to determine whether the plant is a transgenic plant; chlorophenol red experiments verify whether the plants are transgenic plants.

2. The method of claim 1, wherein the green algae is Chlorococcumsp.

3. The method of claim 1, wherein the expression vector is constructed by deleting an antibiotic marker gene.

4. The method of claim 1, wherein the expression vector is PMI-PBI 121.

5. The method of claim 1, wherein the mannose concentration is 6 g/L.

6. The use of the method of claim 1 for the construction of transgenic tomato using the green alga PMI as a marker gene for genetic transformation in tomato.

7. The use of the method of claim 6 for the construction of transgenic tomato using the PMI from green algae as a marker gene for genetic transformation screening of tomato, characterized in that the transgenic tomato is constructed by the following method:

cloning of PMI: cloning a PMI phosphomannose isomerase gene from Chlorococcum sp by a PCR technology to obtain a safety marker gene, wherein a nucleotide sequence is shown as a sequence table SEQ ID NO. 1;

construction of expression vector: after enzyme digestion, the marker gene is connected and introduced into a binary vector PBI121 to replace the original kana resistance gene NPT II, the new vector has no NPT II mark and carries a safety screening marker PMI, is named as PMI-pBI121, and is introduced into agrobacterium LBA4404 by adopting an electric shock transformation method;

genetic transformation of tomato: transforming tomatoes by adopting an agrobacterium-mediated leaf disc method, culturing for 2 days, transferring into screening culture mediums containing mannose with different concentrations for screening culture, transferring into a rooting culture medium after budding, and rooting to obtain transgenic plants;

identification of transgenic tomato: identifying whether the plant is a transgenic plant by using PCR; chlorophenol red experiments verify whether the plants are transgenic plants.

8. A security marker gene having a nucleotide sequence shown in SEQ ID No.1, which is obtained by cloning a PMI phosphomannose isomerase gene of Chlorococcum sp.

Technical Field

The invention relates to the field of genetic engineering, in particular to screening and application of a safety screening marker gene in transgenic plants.

Background

Tomato, originally produced in central and south america, is an annual or perennial herb of the solanaceae family, also known as tomato and persimmons. The tomato is rich in lycopene, beta-carotene and other various nutritional ingredients, is very beneficial to body health, and is one of the most widely planted vegetables and fruits in the world. Since the first transgenic tomato variety "Flavr-Savr" was approved for marketing in the United states in 1994, transgenic tomatoes have been approved for marketing in various countries and regions of the United states, European Union, Japan, Latin America, and the like. The transgenic tomato is developed vigorously, various storage-resistant, high-nutrition, disease-resistant, salt-resistant, cold-resistant and drought-resistant transgenic tomato varieties are successfully developed in succession, the excellent varieties are the basis of the development of the tomato industry, more and more transgenic tomatoes with excellent characters are planted commercially, and the commercial opportunity of the transgenic tomato industry is unlimited.

With the vigorous development of transgenic biotechnology, commercial transgenic tomatoes are continuously appeared, and people pay more and more attention to the safety problem of transgenic foods. The resistance marker gene derived from pathogenic bacteria contained in the transgenic plant is seriously attacked by people and becomes an important factor for restricting the development of the transgenic organism. Transgenic screening generally uses antibiotics and herbicides as screening markers, and the risks are mainly as follows: whether the resistance markers induce broad-spectrum resistance of microorganisms and plants, and generate 'super bacteria' and 'super weeds' which cannot be killed by the existing antibiotics and herbicides; whether the use of resistance markers affects ecological balance and other potential negative effects. At present, people mainly solve the safety problem of the selection marker in transgenic plants from two directions: firstly, the mark is avoided or eliminated, the process is complex and difficult, and the mark cannot be generally adopted. The second is the development of a biologically safe selection marker. In recent years, scientists also propose to utilize the plant gene and the regulation and control original as selection markers of transgenic crops as much as possible, develop the green selection markers, eliminate the worry of people about the potential safety hazard of transgenic plants, and have good development prospects. For example, mannose-6-phosphate isomerase (PMI) derived from Escherichia coli converts mannose-6-phosphate into fructose-6-phosphate, and the PMI gene can be used as a potential selection marker gene.

The tomato transgenic industry is developed rapidly, people obviously have higher requirements on the safety of a marker gene, but no excellent and safe genetic transformation screening system exists at present, and the development of a safety marker gene derived from green algae for applying to tomato transgenic engineering is an urgent need.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: provides a method and application of using green algae gene as tomato genetic transformation safety screening marker.

In order to achieve the above purpose of the present invention, the present invention provides the following technical solutions:

a method for adopting green alga PMI as a tomato genetic transformation screening marker gene, wherein the marker gene is the origin of green alga, and a screening agent is safe and nontoxic mannose, comprises the following steps:

cloning of PMI

Cloning a PMI phosphomannose isomerase gene from Chlorococcumsp by a PCR technology to obtain a safety marker gene, wherein the nucleotide sequence is shown as SEQ ID No. 1;

construction of expression vectors

After enzyme digestion, the marker gene is connected and introduced into a binary vector PBI121 to replace the original kana resistance gene NPT II, the new vector has no NPT II mark and carries a safety screening marker PMI, is named as PMI-pBI121, and is introduced into agrobacterium LBA4404 by adopting an electric shock transformation method;

genetic transformation of tomato

Transforming tomatoes by adopting an agrobacterium-mediated leaf disc method, culturing for 2 days, transferring into screening culture mediums containing mannose with different concentrations for screening culture, transferring into a rooting culture medium after budding, and rooting to obtain transgenic plants;

identification of transgenic tomato

PCR identification is carried out to determine whether the plant is a transgenic plant; chlorophenol red experiments verify whether the plants are transgenic plants.

According to the method for adopting the green alga PMI as the tomato genetic transformation screening marker gene, the green alga is Chlorococcumpsp.

According to the method for adopting the green alga PMI as the tomato genetic transformation screening marker gene, the antibiotic marker gene is deleted in the construction of the expression vector.

According to the method for adopting the green alga PMI as the tomato genetic transformation screening marker gene, the expression vector is PMI-PBI 121.

According to the method for adopting the green alga PMI as the tomato genetic transformation screening marker gene, the screening concentration of mannose is 6 g/L.

The invention also provides application of the method for adopting the green alga PMI as the tomato genetic transformation screening marker gene in establishment of transgenic tomatoes.

According to the application, the transgenic tomato is established by adopting the following method:

cloning of PMI: cloning a PMI phosphomannose isomerase gene from Chlorococcumsp by a PCR technology to obtain a safety marker gene, wherein the nucleotide sequence is shown as SEQ ID No. 1;

construction of expression vector: after enzyme digestion, the marker gene is connected and introduced into a binary vector PBI121 to replace the original kana resistance gene NPT II, the new vector has no NPT II mark and carries a safety screening marker PMI, is named as PMI-pBI121, and is introduced into agrobacterium LBA4404 by adopting an electric shock transformation method;

genetic transformation of tomato: transforming tomatoes by adopting an agrobacterium-mediated leaf disc method, culturing for 2 days, transferring into screening culture mediums containing mannose with different concentrations for screening culture, transferring into a rooting culture medium after budding, and rooting to obtain transgenic plants;

identification of transgenic tomato: identifying whether the plant is a transgenic plant by using PCR; chlorophenol red experiments verify whether the plants are transgenic plants.

The invention also provides a safety marker gene with a nucleotide sequence shown as SEQ ID NO.1, which is obtained by cloning PMI phosphomannose isomerase gene from Chlorococcums sp.

Compared with the prior art, the invention has the advantages that:

firstly, an ecological environment-friendly tomato genetic transformation safety screening and marking system originated from green algae is established: the PMI gene is amplified from green algae to construct a plant binary expression vector, then tomato is transformed, and a new tomato transformation system is established by taking edible mannose as a screening agent to obtain a transformed plant. Compared with the screening marker gene derived from other microorganisms, the PMI is derived from edible green algae, and the screening agent is safe and nontoxic, so that the PMI has food safety and ecological safety.

And secondly, the antibiotic marker gene is removed from the transformation vector, and the multiple cloning sites are reserved, so that the cloning of the exogenous gene is facilitated, and the transformation vector is a pure natural plant gene expression vector which can be used for genetic engineering.

Thirdly, as the tomato is used as a model plant, the high-efficiency broad-spectrum plant screening method can be applied to screening and transformation of other plants, and has wide prospect.

In a word, the PMI gene is cloned from unicellular chlorenchucumsp of a plant ancestor to be used as a selective marker gene of a new generation of a plant gene transfer technology, a novel screening method for positively screening transformed cells by taking mannose as a screening agent is established, the defects existing in the way that currently widely used antibiotics or herbicide-resistant genes and PMI genes derived from escherichia coli are used as marker genes can be replaced, the safety of food, biology and ecology is better possessed, the public worry about the safety of the marker genes can be relieved, and the development of the transgenic tomato industry is promoted.

Drawings

FIG. 1 is a schematic representation of the intermediate vector PTZ 57-P-T;

FIG. 2 is a schematic diagram of plant binary vector pBI 121;

FIG. 3 is a schematic diagram of the vector PTZ57-P-PMI-T after cloning of PMI into FIG. 1;

FIG. 4 is a schematic diagram of the PMI-pBI121 vector after cloning of PMI into FIG. 2;

FIG. 5 is a graph showing the effect of tomato cotyledons after 6 weeks growth on MS-induced recovery medium with different mannose concentrations: the a-d culture media respectively contain 0, 3, 6 and 9g/L mannose;

FIG. 6 is a schematic diagram of PMI-pBI121 transgenic tomato seedling cultivation: a is a growth diagram of cotyledons in a mannose induced germination MS culture medium of 6 g/L; b is 1-2cm bud; c is rooted positive bud; d is the transgenic tomato seedlings in the soil;

FIG. 7 is a schematic diagram of PCR detection of PMI-pBI121 transgenic tomato seedlings: WT is wild tomato, and 1-10 is transformed tomato;

FIG. 8 is a schematic diagram of chlorophenol red detection of PMI activity in transgenic leaves of PMI-pBI121 tomato.

Detailed Description

The present invention will be further described with reference to the following examples, which are not intended to limit the scope of the present invention.

Unless otherwise specified, the primer synthesis and DNA sequencing in the present invention were carried out by Shanghai Czeri bioengineering, Inc.; restriction enzyme, ligase, high purity plasmid miniprep kit, and DNA fragment recovery kit used in the present invention were purchased from NEB and operated according to the methods described in the specification.