CN112522293B

CN112522293B - Heidelano Yang Linzhi acyl inositol specific phospholipase C encoding gene PsnPI-PLC and application thereof

Info

Publication number: CN112522293B
Application number: CN202011573064.2A
Authority: CN
Inventors: 孙尧; 王雷; 李瑶; 黄国庆; 吴琼; 曹涤非; 薛佳莹; 孙鑫
Original assignee: Institute of Advanced Technology of Heilongjiang Academy of Sciences
Current assignee: Institute of Advanced Technology of Heilongjiang Academy of Sciences
Priority date: 2020-12-24
Filing date: 2020-12-24
Publication date: 2023-06-27
Anticipated expiration: 2040-12-24
Also published as: CN112522293A

Abstract

A small black Yang Linzhi acyl-inositol specific phospholipase C encoding gene PsnPI-PLC and application thereof relate to a small black Yang Linzhi acyl-inositol specific phospholipase C encoding gene PsnPI-PLC and application thereof. The aim is to provide a small black Yang Linzhi acyl inositol specific phospholipase C encoding gene PsnPI-PLC and application thereof. The nucleotide sequence of the coding gene PsnPI-PLC of the small black Yang Linzhi acyl-inositol specific phospholipase C is shown as a sequence table Seq ID No:1, the amino acid sequence is shown as a sequence table Seq ID No:2, the gene is applied to improving the salt tolerance of plants. The salt tolerance of the PsnPI-PLC over-expressed tobacco plant is superior to that of a non-transgenic plant, the gene over-expression can improve the salt tolerance of tobacco, and the PsnPI-PLC over-expressed tobacco plant has higher application value in the field of tree salt tolerance breeding.

Description

Heidelano Yang Linzhi acyl inositol specific phospholipase C encoding gene PsnPI-PLC and application thereof

Technical Field

The invention relates to a gene, a coded protein and application thereof, in particular to a small black Yang Linzhi acyl inositol specific phospholipase C coded gene PsnPI-PLC.

Background

Lipid signaling is an important component of plant signal transduction, and organisms promote signal cascade and transmission through changes in lipid concentration, thereby regulating vital activities of cells and individuals. Phospholipase C (PLC) is a member of the phospholipase family, and can hydrolyze phospholipids to generate a head containing a phosphate group and Diacylglycerol (DAG), and can be classified into non-specific phospholipase C (NPC) and phosphatidylinositol-specific phospholipase C (PI-PLC) according to their substrates of action.

Most PLCs in animals and plants are PI-PLCs (hereinafter referred to as PLCs), which mainly consist of PH domain (N terminal), EF hand domain, X domain, Y domain and C2 domain (C terminal). Plants only exist for one subtype of PLC, structurally close to the plcδ subfamily, which lacks a substrate that is PH domain, which is not catalytically active but can help the PLC anchor to the plasma membrane. In the case of PH domain deletions, the PLC binds to PIP2 via the junction sequence between X-Y domains. The amino acid sequences of X and Y domains are most conserved, and the two domains are formed by alternating alpha helices and beta sheets and are catalytic centers of enzymes; EF chiral domain has 1 typical available Ca ²⁺ The combined helix-turn-helix structure, most plant PLCs only contain 2 EF chiral domains (4 in animals), whether the absence of EF chiral domains affects the binding of metal ions has not been clear so far; in some plants, C2 domain regulates PLC binding to phospholipids, a process that is subject to Ca ²⁺ Regulation can be accomplished when the EF chiral domain is absent, which has an auxiliary effect on maintaining catalytic activity.

Poplar has extremely high economic and ecological values, but most poplar varieties have weaker drought resistance and salt and alkali resistance. Populus simonixP. Nigra is a hybrid of Populus simonixP and Populus PopuLus, and has the advantages of rapid growth, strong adaptability, cold resistance, drought resistance, barren resistance, and slight saline-alkali resistance, and is an important greening and afforestation tree species. However, in areas with water shortage and severe salinization, the growth and development of poplar are affected. For cultivating Yang Shukang stress-resistant new varieties, a large number of stress-resistant genes are applied to poplar molecular breeding research to obtain efficient and stable stress-resistant poplar plants, and a relevant gene action mechanism and complete upstream and downstream signal paths are required to be clear, so that the breeding process is improved pertinently on the basis, a foundation is laid for the research of the salt tolerance mechanism of poplar, and the method has important significance.

Disclosure of Invention

The invention aims to provide a small black Yang Linzhi acyl inositol specific phospholipase C coding gene PsnPI-PLC and application thereof.

The nucleotide sequence of the coding gene PsnPI-PLC of the small black Yang Linzhi acyl-inositol specific phospholipase C is shown as a sequence table Seq ID No: 1.

The small black Yang Linzhi phosphatidylinositol specific phospholipase C coding gene PsnPI-PLC is applied to improving the salt tolerance of plants.

The small black Yang Linzhi phosphatidylinositol specific phospholipase C coding gene PsnPI-PLC is applied to breeding of salt-tolerant transgenic plants.

The beneficial effects of the invention are as follows:

the coding gene of the small black Yang Linzhi acyl inositol specific phospholipase C, namely the poplar salt tolerance related gene PsnPI-PLC, provided by the invention can be used for improving the salt tolerance of plants or used for breeding salt tolerance transgenic plants. The transgenic tobacco is obtained by constructing a PsnPI-PLC over-expression vector and carrying out leaf disk transfection, the transgenic strain grows for 30 days in an MS culture medium with 150mM NaCl, the strain height is higher than that of a non-transgenic strain, the POD, the SOD and the proline are all obviously higher than that of the non-transgenic strain, and the MDA is obviously lower than that of the non-transgenic strain. The result shows that the salt tolerance of the PsnPI-PLC over-expressed tobacco plant is superior to that of a non-transgenic plant, the gene over-expression can improve the salt tolerance of tobacco, and the gene has higher application value in the field of forest salt tolerance breeding.

Drawings

FIG. 1 is a prediction diagram of a PsnPI-PLC conserved region;

FIG. 2 is a prediction diagram of PsnPI-PLC domain elements;

FIG. 3 is a diagram of a multiple sequence alignment of PsnPI-PLC and other 10 plant PI-PLC proteins;

FIG. 4 is a graph showing the comparison of strain height changes of PsnPI-PLC transgenic and non-transgenic tobacco after NaCl stress; wherein a is CK, b is P10, c is P20, d is P21;

FIG. 5 is a graph showing comparison of POD content measurements of PsnPI-PLC transgenic and non-transgenic tobacco after NaCl stress; wherein a is CK, b is P10, c is P20, d is P21;

FIG. 6 is a graph showing the analysis and determination of SOD content in transgenic tobacco under NaCl stress; wherein a is CK, b is P10, c is P20, d is P21;

FIG. 7 is a graph showing the MDA content analysis and determination of transgenic tobacco under NaCl stress; wherein a is CK, b is P10, c is P20, d is P21;

FIG. 8 is a graph comparing proline content measurements of PsnPI-PLC transgenic and non-transgenic tobacco after salt stress; wherein a is CK, b is P10, c is P20, d is P21.

Detailed Description

The first embodiment is as follows: the nucleotide sequence of the encoding gene PsnPI-PLC of the small black Yang Linzhi acyl-inositol specific phospholipase C of the embodiment is shown as a sequence table Seq ID No: 1.

The coding gene of the small black Yang Linzhi phosphatidylinositol specific phospholipase C, namely the poplar salt tolerance related gene PsnPI-PLC, provided by the embodiment can be used for improving the salt tolerance of plants or used for breeding salt tolerance transgenic plants. The transgenic tobacco is obtained by constructing a PsnPI-PLC over-expression vector and carrying out leaf disk transfection, the transgenic strain grows for 30 days in an MS culture medium with 150mM NaCl, the strain height is higher than that of a non-transgenic strain, the POD, the SOD and the proline are all obviously higher than that of the non-transgenic strain, and the MDA is obviously lower than that of the non-transgenic strain. The result shows that the salt tolerance of the PsnPI-PLC over-expressed tobacco plant is superior to that of a non-transgenic plant, the gene over-expression can improve the salt tolerance of tobacco, and the gene has higher application value in the field of forest salt tolerance breeding.

The second embodiment is as follows: the first difference between this embodiment and the specific embodiment is that: the amino acid sequence of the protein coded by the gene is shown as a sequence table Seq ID No: 2. The other is the same as in the first embodiment.

And a third specific embodiment: the small black Yang Linzhi acyl inositol-specific phospholipase C encoding gene PsnPI-PLC of the embodiment is applied to improving the salt tolerance of plants.

The specific embodiment IV is as follows: the small black Yang Linzhi acyl inositol-specific phospholipase C encoding gene PsnPI-PLC of the embodiment is applied to breeding of salt-tolerant transgenic plants.

The embodiment is applied to salt-tolerant tree breeding.

The effect of the invention is verified by the following experiments:

example 1:

1. cloning of poplar PI-PLC Gene

Using the Populus deltoides cDNA sequence as a template, a sequence such as Seq ID No was designed using Primer5 based on Populus deltoides PI-PLCORF sequence information in the JGI database (https:// phytozome. JGI. Doe. Gov/pz/portal. Html): 3 and Seq ID No:4 (orf 7-1F and orf 7-1R), and amplifying the PsnPI-PLC gene fragment.

The PCR system is as follows:

ddH ₂ o makes up a volume of 10. Mu.L.

The reaction procedure is 94 ℃ pre-denaturation for 5min; denaturation at 94℃for 30s, renaturation at 52℃for 30s, extension at 72℃for 2min,30 cycles; the target fragment was recovered by using a gel recovery kit after extension at 72℃for 10 min.

The recovered gene fragment was ligated to pMD19-T vector, the ligation product was transformed into E.coli competent cells, and monoclonal was selected using Seq ID No:5 and Seq ID No: the pMD19-T universal primers M13+ and M13-shown in FIG. 6 were subjected to PCR as follows:

ddH ₂ o makes up a volume of 10. Mu.L.

The reaction procedure is 94 ℃ pre-denaturation for 5min; denaturation at 94℃for 30s, renaturation at 52.1℃for 30s, extension at 72℃for 2min,30 cycles; extending at 72 ℃ for 10min, and carrying out sequencing verification on the obtained specific fragment to finally obtain the sequence represented by Seq ID No:1, and a poplar PI-PLCcDNA sequence shown in the specification.

2. Poplar PI-PLC gene structural domain verification and sequence analysis

The full length 1764bp of the poplar PI-PLCcDNA sequence obtained in the embodiment codes a sequence with the sequence table of Seq ID No:2, and a protein having an amino acid sequence of the polypeptide. The protein-related parameters were analyzed using ProtParam software (https:// web. Expasy. Org/protParam /), and the results indicated that the protein had a molecular weight of 66.6kDa and a theoretical isoelectric point of 5.82. The protein conservation region was analyzed by NCBI's CD-search (https:// www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb. Cgi) function, and the analysis result is shown in FIG. 1, the poplar PI-PLC gene obtained in this example contains a conserved PLC domain and belongs to phosphatidylinositol-specific phospholipase C encoding gene; analysis results of InterPro (http:// www.ebi.ac.uk/Interpro /) show that the poplar PI-PLC gene encoded protein obtained in the embodiment comprises EF chiral domain, X domain, Y domain and C2 domain (figure 2); these characteristics are consistent with the basic characteristics of the phosphatidylinositol-specific phospholipase C family, and a new member of the phosphatidylinositol-specific phospholipase C gene family, named PsnPI-PLC, was initially determined.

The NCBI Blast program (https:// Blast. NCBI. Nlm. Nih. Gov/Blast. Cgi) was used to search for homologous sequences, and 11 other plant PI-PLC amino acid sequences were selected for multiple sequence alignment by mega software analysis.

The selected sequence is as follows:

AtPLC1 NP_568881；AtPLC2 NP_001030660；AtPLC3 NP_195565；AtPLC8 NP_001327564；AtPLC9 NP_190306；ZmPLC1 AAS45137；SlPLC4 NP_001234181；GmPLC12 XP_006601953；OsPLC1 XP_015646464；OsPLC2 XP_015628700；OsPLC3 XP_015618103。

the PLC protein sequences are respectively from plants such as arabidopsis thaliana, rice and the like, the multi-sequence comparison result is shown in figure 3, the homology of PsnPI-PLC and each plant PLC is not high, but the selected genes all have the conserved structural characteristics of PI-PLC. At the same time, the differences between different PI-PLCs within the same species are evident. Although OsPLC1 and OsPLC3 are reported to participate in plant salt tolerance regulation, the fact that EF chiral domain is absent between the two is confirmed through conservative domain analysis, so that compared with Psn PI-PLC, the structure of the PSn PI-PLC is greatly different, and researches on proteins similar to Psn PI-PLC in Blast results in the plant salt tolerance field are not reported at present.

Example 2: construction of poplar PsnPI-PLC gene overexpression vector

Design such as Seq ID No:7 and Seq ID No:8, the specific primers 7-1F-SmaI and 7-1R-SacI are cloned by taking poplar cDNA as a template, and the PsnPI-PLC gene is cloned by the following reflecting system:

ddH ₂ o makes up a volume of 10. Mu.L.

The reaction procedure is 94 ℃ pre-denaturation for 5min; denaturation at 94℃for 30s, renaturation at 60.5℃for 30s, extension at 72℃for 2min,30 cycles; extending at 72 ℃ for 10min, recovering amplified fragments by using a gel recovery kit, carrying out Sma I and Sac I double digestion on the PCR product and pROKII vector, respectively recovering the products and connecting by using T4 ligase, and transforming competent cells of the escherichia coli by using the method such as Seq ID No:7 and Seq ID No:8, carrying out PCR verification on specific primers 7-1F-SmaI and 7-1R-SacI, submitting to sequencing, wherein the sequencing primers are universal primers 35S and M13F, the positive clone is named pROKII-PsnPLC, extracting plasmid and transforming the agrobacterium strain EHA105, and carrying out cryopreservation on the pROKII-PsnPLC over-expressed agrobacterium strain at the temperature of minus 80 ℃ for later use.

Example 3: obtaining of PsnPI-PLC gene-transferred tobacco strain and analysis of plant salt tolerance

The pROKAI-PsnPLC overexpression vector in example 2 was used to transform tobacco by Agrobacterium-mediated leaf disc method, T2 seeds of transgenic and non-transgenic tobacco were sterilized and then sown in MS medium containing 0 and 150mM NaCl, transgenic and non-transgenic tobacco seedlings with the same size in 3-5d post-germination growth period were transferred into secondary medium containing 0 and 150mM NaCl for cultivation, and leaf DNA thereof was used as template, using as the seed of Seq ID No:9 and Seq ID No:10, and positive plants are determined by resistance screening and molecular detection and after 30d culture in a secondary culture medium, one wild type (ck) and three groups of transgenic lines (named p10, p20 and p21 respectively, 6 biological repeats per group) are selected for measuring physiological and biochemical indexes.

The measuring of the physiological index includes: plant height, POD content, SOD content, MDA content and proline content. Wherein POD content measurement uses POD measurement kit produced by Nanjing built bioengineering research institute; the Pro content measurement adopts a proline content kit produced by Suzhou Ming biotechnology Co., ltd; and the determination of the SOD content and the MDA content adopts an MDA detection kit and a total SOD activity detection kit (NBT method) produced by Biyun Tian biotechnology.

FIG. 4 shows the results of the measurement of transgenic (p 10, p20, p 21) and non-transgenic (ck) tobacco plant heights under normal growth and 150mM NaCl conditions, the transgenic plant heights under normal growth conditions were not significantly different from the ck plant height, while each plant height under salt stress conditions was significantly lower than the normal growth conditions, while the transgenic plant heights were slightly higher than the ck, wherein the p20 plant height was 2 times (p < 0.01) and the p21 plant height was 1.66 times (p < 0.05) of the ck, whereby it was seen that each tobacco plant growth was affected by salt stress, but the transgenic tobacco was significantly less affected than the ck, compared to the normal conditions.

Salt stress results in the production of intracellular active oxygen, while Peroxidases (POD) and superoxide dismutase (SOD) scavenge free radicals, reducing damage to cells from salt stress. FIGS. 5 and 6 show the results of the determination of the transgene and the amounts of ckPOD and SOD, respectively, wherein the POD has no obvious difference in each strain under normal conditions, and each transgenic strain is significantly higher than ck after salt stress treatment; the SOD detection method is slightly changed, each reagent of the reaction system is halved, and when the inhibition percentage in xanthine oxidase coupling reaction is 50%, the activity of the SOD in the reaction system is defined as 1 activity unit. After salt stress treatment, the SOD content of each transgenic line was higher than ck. Therefore, the free radical scavenging capacity of the transgenic strain is higher than that of ck, and the transgenic strain has better salt tolerance.

The Malondialdehyde (MDA) content reflects the extent of damage to cell membrane lipid peroxidation, and FIG. 7 shows the results of the determination of the MDA content of the transgene and the MDA content of ck at 150mM NaCl, wherein the MDA content of each transgenic line is lower than ck, p10 is 0.41 times (p < 0.01) of ck, p20 is 0.51 times (p < 0.01) of ck, and p21 is 0.47 times (p < 0.01) of ck. This shows that the damage condition of the transgenic strain is better than ck, and the transgenic strain has better stress resistance.

The proline (Pro) and soluble sugar and other small molecular substances can be used as osmotic adjusting substances to improve the salt tolerance of plants, and FIG. 8 shows the results of the normal growth and the determination of the transgene and ck Pro content under the condition of 150mM NaCl, the transgene strain has no obvious difference from ck under the normal condition, and the Pro content of the transgene strain is higher than that of ck after salt stress treatment.

In conclusion, through the analysis of the physiological indexes of transgenic tobacco, the salt tolerance of plants can be obviously improved after the PI-PLC gene of the populus euphratica is determined to be over-expressed, and the method has a good application prospect in the field of forest salt tolerance breeding.

Sequence listing

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<120> small black Yang Linzhi acyl inositol specific phospholipase C encoding gene PsnPI-PLC and application thereof

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Val Pro Ala Leu Ala Leu Leu Arg Ile Glu Val His Glu Tyr Asp

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Claims

1. Heidelano Yang Linzhi acyl inositol specific phospholipase C encoding genePsnPI-PLCThe application of the gene in improving the salt tolerance of plants is characterized in that the nucleotide sequence of the gene is shown as a sequence table Seq ID No:1 is shown in the specification; the plant is populus euphratica or tobacco.

2. Heidelano Yang Linzhi acyl inositol specific phospholipase C encoding genePsnPI-PLCThe application of the gene in breeding salt-tolerant transgenic plants is characterized in that the nucleotide sequence of the gene is shown as a sequence table Seq ID No:1 is shown in the specification; the plant is populus euphratica or tobacco.