阅读说明：本技术 一种旱柳1,7-二磷酸景天庚酮糖磷酸酶及其编码基因和应用 (Salix matsudana 1, 7-sedoheptulose diphosphate phosphatase, and coding gene and application thereof ) 是由 陈艳红 张健 江钰娜 余春梅 刘国元 钟非 连博琳 刘昱 朱星兆 于 2020-07-23 设计创作，主要内容包括：本发明提供了一种旱柳1,7-二磷酸景天庚酮糖磷酸酶及其编码基因和应用,涉及基因工程技术领域。基因来源于旱柳,命名为SmSBPase基因,基因的序列如SEQ ID NO:1所示,其编码的蛋白质为1,7-二磷酸景天庚酮糖磷酸酶,其氨基酸序列如SEQ ID NO:2所示。本发明提供的基因及其编码的蛋白质具有1,7-二磷酸景天庚酮糖磷酸酶的功能,得到的含有SmSBPase基因的转基因拟南芥耐盐性显著提高,为植物耐盐性的研究中提供了优异的基因资源和酶资源。(The invention provides salix matsudana 1, 7-diphosphoric acid sedoheptulose phosphatase, a coding gene and application thereof, and relates to the technical field of genetic engineering. The gene is derived from salix matsudana and named as SmSBPase gene, the sequence of the gene is shown as SEQ ID NO. 1, the coded protein is sedoheptulose-1, 7-diphosphate, and the amino acid sequence is shown as SEQ ID NO. 2. The gene and the protein coded by the gene have the functions of 1, 7-sedoheptulose diphosphate phosphatase, the salt tolerance of the obtained transgenic arabidopsis containing the SmSBPase gene is obviously improved, and excellent gene resources and enzyme resources are provided for the research of the salt tolerance of plants.)

1. A gene of sedoheptulose 1, 7-diphosphate phosphatase of salix matsudana, which is characterized in that: the sequence of the gene is shown as SEQID NO. 1.

2. A protein encoded by the gene of claim 1, wherein: the amino acid sequence of the protein is shown as SEQ ID NO. 2.

3. A recombinant T vector comprising the gene of claim 1, wherein: the recombinant T vector is obtained by the following steps:

(1) acquisition of the SmSBPase gene: extracting RNA of the salix matsudana leaves, performing reverse transcription to synthesize cDNA, and performing PCR amplification by using the following primers to obtain a PCR product 1, namely a SmSBPase cDNA sequence, wherein the nucleotide sequence is shown as SEQ ID NO. 3;

primer 1: SmSBPase CDS-F

5’-AACTTGGACATCCAGCTAAAATG-3’；

Primer 2: SmSBPase CDS-R

5’-TTAAGCGGCAGCTCCAACAGTAAC-3’；

(2) Obtaining of recombinant T vector: adding A to the end of the PCR product 1 obtained in the step (1), connecting with a pGEM-T vector, transforming an Escherichia coli DH5 alpha competent cell by the obtained connecting product, culturing overnight in an LB solid culture medium containing 5-bromo-4-chloro-3-indole-D-galactoside, X-gal and 100ug/ml ampicillin, selecting a single colony, culturing overnight in an LB liquid culture medium, extracting a plasmid, and obtaining a recombinant T vector, namely pGEM-T-SmSBPase.

4. A recombinant plant expression vector comprising the gene of claim 1, wherein: the recombinant plant expression vector is obtained by the following steps:

(1) ligation of plant expression vector Pwm 101: taking the pGEM-T-SmSBPase plasmid obtained in the claim 3 as a template, and carrying out PCR amplification by using the following primers to obtain a PCR product 2, wherein the nucleotide sequence of the PCR product is shown as SEQ ID NO. 4;

primer 3: pwm101-SmSBPase-F

TCGAGCTTTCGCGAGCTCGGTACCATGGAGACTGGTGTAGCATGCTG；

Primer 4: pwm101-SmSBPase-R

GCATGCCTGCAGGTCGACTCTAGATTAAGCGGCAGCTCCAACAGTAAC；

(2) Obtaining of recombinant plant expression vector: and (2) performing recombinant connection on the PCR product 2 obtained in the step (1) and a linearized Pwm101 vector, performing escherichia coli DH5 alpha competent cell transformation on the connection product, culturing overnight in an LB solid culture medium containing 50ug/ml kanamycin, selecting a single colony, culturing overnight in an LB liquid culture medium, and extracting a plasmid to obtain a recombinant plant expression vector, namely Pwm 101-SmSBPase.

5. A transgenic Arabidopsis thaliana containing the gene of claim 1, characterized in that: the obtaining of the transgenic arabidopsis thaliana comprises the following steps:

transforming the recombinant plant expression vector Pwm101-SmSBPase obtained in the claim 4 into competent cells of agrobacterium GV3101 to obtain recombinant bacteria GV3101/Pwm101-SmSBPase, transforming the recombinant bacteria GV3101/Pwm101-SmSBPase into Arabidopsis thaliana, screening positive transgenic strains T1 generation on MS culture medium containing 50mg/L hygromycin, culturing and screening T1 generation transgenic strains for two generations to obtain homozygous T3 generation transgenic Arabidopsis thaliana seeds, directly sowing the T3 generation transgenic Arabidopsis thaliana seeds, and normally growing under the long-day condition in the greenhouse to obtain transgenic Arabidopsis thaliana.

6. The use of the gene according to claim 1 in plant salt tolerance.

The technical field is as follows:

the invention relates to the technical field of genetic engineering, in particular to salix matsudana 1, 7-diphosphori sedoheptulose phosphatase, and a coding gene and application thereof.

Background art:

soil salinity is one of the major adverse environmental factors affecting plant organ growth and productivity. About 10 hundred million hectares of land are affected by salt damage and tend to increase year by year, accounting for more than 6% of the total land area in the world. High concentrations of salt can lead to osmotic stress, Na⁺And Cl^-The accumulation of stress and the production of Reactive Oxygen Species (ROS) negatively affect plant metabolism and growth and result in crop losses of $ 273 million per year. In china, it is estimated that about 30% of saline soils can be reclaimed by various strategies to ensure grain safety and improve economic environment. Selection and breeding of salt tolerant plants will be the most efficient and direct strategy in saline soil reclamation. More salt-tolerant plants are obtained through genetic engineering, the key components of a plant salt-tolerant network are firstly solved, the molecular mechanism of the plant salt-tolerant network is disclosed, and the method has important theoretical and practical significance.

Salix matsudana, also called Chinese willow, is one of the important tree species in Salix family and Salix genus, is light-loving and cold-resistant, can grow in wet land and dry land, and is native to northeast China. The salix matsudana is an important tree species for ornamental, greening and timber use. The genome of the salix matsudana is mostly allopetraploid, and the salt tolerance of the salix matsudana is higher than that of diploid closely related species, so that the salix matsudana plays an important ecological role by improving beach soil and relieving salinization when planted in coastal high-salinity beach in China. Less research is currently being conducted on the salt-tolerant molecular mechanism of salix matsudana than on other model plants and crops.

The sedoheptulose-1, 7-diphosphate (SBPase) is a key enzyme in the plant Calvin cycle process and is also one of the main rate-limiting enzymes in the plant photosynthesis pathway, and slight inhibition of SBPase activity can result in the reduction of the regeneration capacity of the Calvin cycle and the reduction of the photosynthetic activity. Genetic engineering research proves that the biological function of SBPase in various plants is obviously improving the photosynthetic fixed CO of the plants₂The efficiency and photosynthetic capacity of the plant growth promoting agent, and the accumulation of saccharides in the plant and the growth of the plant. It has also been shown that SBPase plays an important role in alleviating oxidative and salt stress. At present, the salix matsudana 1, 7-diphosphori sedoheptulose phosphatase (SBPase) and reports thereof on seed germination and seedling salt resistance are not found.

The invention content is as follows:

the invention aims to provide Salix matsudana 1, 7-sedoheptulose diphosphate as well as a coding gene and application thereof, wherein the gene is derived from Salix matsudana (Salix matsudana) and named as SmSBPase gene, and the coded protein is sedoheptulose diphosphate 1, 7-bisphosphate.

On one hand, the invention provides a gene of salix matsudana 1, 7-sedoheptulose diphosphate gene, wherein the sequence of the gene is shown as SEQ ID NO. 1; the amino acid sequence of the protein coded by the gene is shown as SEQ ID NO. 2.

In one aspect, there is provided a recombinant T vector comprising the above gene, the recombinant T vector being obtained by the steps of:

(1) acquisition of the SmSBPase gene: extracting RNA of the salix matsudana leaves, performing reverse transcription to synthesize cDNA, and performing PCR amplification by using the following primers to obtain a PCR product 1, namely a SmSBPase cDNA sequence, wherein the nucleotide sequence is shown as SEQ ID NO. 3;

primer 1: SmSBPase CDS-F

5’-AACTTGGACATCCAGCTAAAATG-3’；

Primer 2: SmSBPase CDS-R

5’-TTAAGCGGCAGCTCCAACAGTAAC-3’；

(2) Obtaining of recombinant T vector: adding A to the end of the PCR product 1 obtained in the step (1), connecting with a pGEM-T vector, transforming an Escherichia coli DH5 alpha competent cell by the obtained connecting product, culturing overnight in an LB solid culture medium containing 5-bromo-4-chloro-3-indole-D-galactoside, X-gal and 100ug/ml ampicillin, selecting a single colony, culturing overnight in an LB liquid culture medium, extracting a plasmid, and obtaining a recombinant T vector, namely pGEM-T-SmSBPase.

In one aspect, a recombinant plant expression vector containing the above gene is provided, and the recombinant plant expression vector is obtained by the following steps:

(1) ligation of plant expression vector Pwm 101: the pGEM-T-SmSBPase plasmid obtained above is taken as a template, and PCR amplification is carried out by using the following primers to obtain a PCR product 2, wherein the nucleotide sequence of the PCR product is shown as SEQ ID NO. 4;

primer 3: pwm101-SmSBPase-F TCGAGCTTTCGCGAGCTCGGTACCATGGAGACTGGTGTAGCATGCTG;

primer 4: pwm101-SmSBPase-R GCATGCCTGCAGGTCGACTCTAGATTAAGCGGCAGCTCCAACAGTAAC;

(2) obtaining of recombinant plant expression vector: and (2) performing recombinant connection on the PCR product 2 obtained in the step (1) and a linearized Pwm101 vector, performing escherichia coli DH5 alpha competent cell transformation on the connection product, culturing overnight in an LB solid culture medium containing 50ug/ml kanamycin, selecting a single colony, culturing overnight in an LB liquid culture medium, and extracting a plasmid to obtain a recombinant plant expression vector, namely Pwm 101-SmSBPase.

In another aspect, a transgenic arabidopsis containing the above gene is provided, the obtaining of the transgenic arabidopsis comprises the following steps:

transforming competent cells of agrobacterium GV3101 by the obtained recombinant plant expression vector Pwm101-SmSBPase to obtain recombinant bacteria GV3101/Pwm101-SmSBPase, transforming the recombinant bacteria GV3101/Pwm101-SmSBPase to Arabidopsis thaliana, screening positive transgenic strains T1 generation on MS culture medium containing 50mg/L hygromycin, obtaining homozygous transgenic strains T3 generation transgenic Arabidopsis thaliana seeds by two generation culture and screening processes of the transgenic strains T1 generation, directly sowing the transgenic Arabidopsis thaliana seeds of T3 generation, and normally growing under the long-day condition in the greenhouse to obtain the transgenic Arabidopsis thaliana.

The invention also provides the application of the gene in plant salt tolerance.

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

the gene and the protein coded by the gene have the functions of 1, 7-diphospho sedoheptulose phosphatase; the transgenic arabidopsis thaliana containing the SmSBPase gene obtained by the invention has obviously improved salt tolerance, and provides excellent gene resources and enzyme resources for the research of plant salt tolerance.

Drawings

FIG. 1 shows the expression levels of the SmSBPase gene in WT, L20 and L22 transgenic lines.

FIG. 2 shows SBPase activity in WT, L20 and L22 transgenic lines.

FIG. 3 is the observation of germination of SmSBPase-transferred Arabidopsis L20 and L22 lines and wild type Arabidopsis seeds for 7 days under different concentrations of NaCl.

FIG. 4 shows the statistics of germination rates of SmSBPase-transferred Arabidopsis L20 and L22 strains and wild type Arabidopsis seeds in 7 days under NaCl treatment at different concentrations.

FIG. 5 is a graph of changes in soluble sugar content of leaves of wild type and transgenic L20 and L22 line plants treated for 24h and 1 week under normal conditions of growth and salt stress.

FIG. 6 is a graph showing the change in leaf sucrose content of plants of wild type and transgenic lines L20 and L22 treated for 24h and 1 week under normal conditions of growth and salt stress.

FIG. 7 is a graph of leaf starch content changes in wild type and transgenic L20 and L22 line plants treated for 24h and 1 week under normal conditions of growth and salt stress.

FIG. 8 is a graph showing the change in chlorophyll content of leaf blades of plants of wild type and transgenic lines L20 and L22 treated for 48h under normal conditions of growth and salt stress.

Detailed Description

In order to make the technical means, the original characteristics, the achieved purposes and the effects of the invention easily understood, the invention is further explained with the following embodiments, but the following embodiments are only the preferred embodiments of the invention, and not all embodiments. Based on the embodiments in the implementation, other embodiments obtained by those skilled in the art without any creative efforts belong to the protection scope of the present invention.

The experimental methods in the following examples are conventional methods unless otherwise specified, and the drugs, materials, reagents and the like used in the following examples are commercially available without otherwise specified.