KR20120009130A - Protein and its Gene HTR2 of laver, giving High-Temperature-Resistance - Google Patents

Abstract

PURPOSE: A laver-derived high temperature resistant protein in bacteria, algae, and plants and a gene encoding the protein are provided to enhance gene expression and laver production. CONSTITUTION: A protein which is isolated from laver and has high temperature resistance contains an amino acid sequence of sequence number 1. The protein is a 15kDa protein with 144 amino acids. A gene encoding the protein is a cDNA having a sequence of sequence number 2. A recombinant vector contains the gene, HTR2. A transformant is prepared by introducing the recombinant vector to the host such as bacteria, sea algae, or plants.

Description

Protein and its Gene ΗＴＲ２ of laver, giving High-Temperature-Resistance}

The present invention, which is isolated from laver, relates to a protein that provides high temperature resistance to bacteria, seaweeds and plants, and a gene encoding the same, a recombinant vector comprising the gene and a transformant by the recombinant vector.

Marine life accounts for about 80% of the world's species, and seaweeds are not only used for food, fertilizers, and health foods, but also for the production of useful substances, for the discovery of new materials, and for biomonitoring or bioremediation. It is an important biological resource and genetic resource used. In particular, seaweed is a representative seaweed produced in Korea along with seaweed and kelp.

Since most useful seaweeds inhabit the intertidal zones (seaweed, shellfish, blue sea, etc.) that are submerged at high tide and over water at low tide, and are always submerged in the sea, but not so deep (seaweed, auditory, maternity, etc.) It is made in intertidal or azo zones on the coast.

In particular, algae farmed in intertidal zones are frequently exposed to high temperature direct sunlight, causing many to die or delay growth.

On the other hand, the temperature of seawater tends to increase gradually with global warming. Increasing the temperature of fish farms causes a sharp drop in the production of algae.

An object of the present invention is to provide proteins, genes, and the like that give high temperature resistance to algae, such as laver and chlamidomonas.

In addition, an object of the present invention is to provide proteins and genes that impart high temperature resistance to bacteria and plants as well as seaweeds.

The present invention for achieving the above object relates to a protein of SEQ ID NO: 1, which provides high temperature resistance to an individual as isolated from Porphyra seriata .

<SEQ ID NO 1>

MAASKTLAAAFAVALLALLTLTAAAPAADTAAADAATAAYDALHVEGISGAAEAPALDGLTDRSGYYRCRSYFVCGSKKVKVPVKHSHPCYYKEAPSILKSSRSSGLCRPDPYSCKPATCIGWRKCTCDACKTVIFTHNIWCEK

The protein according to the present invention is a ˜15 kDa protein (pI, 8.42) consisting of 144 amino acids.

The present invention also relates to a gene encoding the protein, preferably cDNA comprising SEQ ID NO: 2 below.

<SEQ ID NO 2>

ATG GCG GCT TCC AAG ACC CTC GCC GCC GCG TTT GCC GTT GCG CTC CTG GCA CTC CTG ACC CTG ACC GCC GCC GCC CCC GCA GCG GAC ACT GCC GCC GCC GAC GCC GCA ACC GCC GCG TAC GAC GC GG GG GC GC TCGG GG GCG GCG GAG GCA CCC GCC CTC GAC GGC CTC ACC GAC CGG TCG GGC TAC TAC CGC TGC CGC TCC TAC TTT GTG TGC GGC AGC AAG AAG GTG AAA GTG CCT GTC AAG CAC TCG CAC CCG TGC TAC AAC ATC GAG ATC TCG TCC CGC AGC AGC GGC CTG TGC CGG CCG GAC CCG TAC AGC TGC AAG CCC GCC ACG TGC ATC GGC TGG CGC AAG TGC ACG TGC GAT GCG TGC AAG ACG GAT ATC TTC ACC CAT AAC ATC TGG TGC GAG

In the present invention, the proteins and genes can be modified, such as substitution, deletion, insertion, etc. as appropriate within the range of maintaining the characteristics (high temperature characteristics), and the modified proteins and genes are also within the scope of the present invention. It is natural for me.

The present invention also relates to a recombinant vector and a transformant obtained by introducing the recombinant vector into any one host selected from the group consisting of bacteria, algae and plants.

As described above, the proteins and genes according to the present invention can be used as useful genes because they impart high temperature resistance to algae and the like.

In other words, according to the present invention, when the temperature rises, the production of the seaweed disease or physiological disorders tend to decrease the production of the seaweed, which increases the expression of the gene according to the present invention, thereby giving high temperature resistance to increase the productivity of the seaweed. .

In addition, by applying the gene of the present invention to various seaweeds for bioenergy production, it is possible to increase the production of biomass of seaweeds.

1 is a table showing the protein sequence of SEQ ID NO: 1, the cDNA of SEQ ID NO: 2 and the base sequence before and after.
Figure 2 is a photograph of the RT-PCR results showing that a large amount of HTR2 gene is expressed at high temperatures according to the present invention.
3 is shown a conceptual diagram of a Pseudomonas expression vectors Cloud pCR102_ HTR2 for containing HTR2 gene according to the present invention.
4 is a photograph showing that the HTR2 gene according to the present invention exhibits high temperature resistance even in chlamidomonas .

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings. It should be understood, however, that the appended drawings illustrate only the contents and scope of technology of the present invention, and the technical scope of the present invention is not limited thereto. It will be apparent to those skilled in the art that various changes and modifications can be made within the scope of the technical idea of the present invention based on these examples.

In particular, in the following examples, the gene according to the present invention was inserted into the plasmid pCR102 to produce and use an expression vector. However, it will be apparent to those skilled in the art that various kinds of plasmids suitable for the characteristics of the host may be utilized.

The present inventors grew various types of laver fronds at normal temperature growth conditions and high temperature treatment conditions, and then selected ESTs with increased expression at high temperature conditions by comparing the ESTs produced by them. Subsequently, a gene showing the best high temperature resistance among the selected ESTs was isolated and named as HTR2 (High Temperature Response gene2).

The gene HTR2 according to the present invention, whose expression is increased by high temperature, is derived from Porphyraseriata seriata and encodes a basic protein (pI = 8.4) of about 15.2 kDa consisting of 144 amino acids. Is considered to be a new gene with no similarity.

Meanwhile, as a result of transforming the high temperature sensitive bacteria and the model alga Chlamydomonas with the HTR2 gene according to the present invention, it was confirmed that these transformants obtained resistance to high temperature.

On the other hand, the HTR2 gene according to the present invention can be isolated by using a conventional PCR using the primers of SEQ ID NOs: 3 and 4 as exemplarily shown in the following examples as a template of total DNA or RNA of the hairpinning process. Can be. Therefore, the deposit of the gene is omitted.

The present invention will be described in detail through the following examples.

Example 1 Isolation of High Temperature Tolerant Gene from Seaweed

(1) Preparation of living body and sample

Porphyra seriata was obtained from the Seaweed Bio Research Center.

The laver filaments were harvested from the medium for the production of cDNA library and immediately frozen using liquid nitrogen. The laver fronds were transferred to 25 ^° C incubator for 6 hours and then incubated for 6 hours and 12 hours before harvesting.

(2) RNA isolation and cDNA library construction

Total RNA was extracted from the prepared samples according to the method suggested by the manufacturer using TRI REAGENT ^™ -RNA ISOLATION REAGENT.

The extracted RNA was confirmed by formaldehyde agarose gel electrophoresis after treatment with RNase free DNA. mRNA was isolated from extracted total RNA using Absolute mRNA purification kit (Stratagene), and cDNA was synthesized from about 5 ug of mRNA using ZAP Express cDNA Synthesis Kit "(Stratagene, San Diego, Calif.). Among them, only about 500bp or more of cDNA was selected and cloned into ZAP expression vector, and then packaged using "Gigapack Gold III Packaging Extract" (Stratagene) to prepare cDNA library. One cDNA library was prepared from the fronds, and cDNA library was prepared from the hairy fronds that were matured at 37 ° C. for 6 hours and 12 hours, respectively.

(3) ESTs generation and isolation of high temperature resistant genes

The prepared primary lambda phage library was converted to phagemid and then cultured in agar medium containing antibiotics. In order to generate ESTs (Expressed Sequence Tags), clones grown in the medium were randomly selected and plasmid DNA was extracted. Then, nucleotide sequences were determined in one direction only from the 5'-end of the cDNA using a T3 primer. After sequencing, the vector sequence was cleaved and the low quality sequence or the sequence whose length was less than 100bp was removed.

To find genes that induce or increase expression in high temperature conditions, ESTs generated from the patterned cDNA library under normal culture conditions and the high-temperature treated patterned cDNA library were compared. Through comparison of ESTs, only those transcripts with increased expression more than 10-fold at high temperature were selected and named these genes as high temperature responsive (HTR) genes.

Among the selected HTR genes, HTR2 was the same as SEQ ID NO: 2 having 435 bases, and the protein was confirmed to be a protein (pI, 8.42) of SEQ ID NO: 1 of about 15 kDa composed of 144 amino acids. 1 shows the protein sequence of SEQ ID NO: 1, the HTR2 cDNA of SEQ ID NO: 2, and the base sequence before and after it.

(4) Reconfirmation of increase in expression level at high temperature

The isolated HTR2 cDNA was reconfirmed as follows to increase the expression level during the high temperature culture of seaweed.

Incubate at 24 hours or more at normal culture temperature (about 12 ° C), and transfer the sample to 25 ° C. The samples were collected at predetermined time intervals and total RNA was extracted.

Using the extracted RNA as a template, RT-PCR was performed as follows. 1 μl of the extracted RNA (1 μg), oligo (dT) primer and 1 μl of 10mM dNTP Mix were added, and the total volume was 13 μl, followed by 5 min at 65 ° C. and 1 min on ice. Then add 4µl of 5 ㅧ First-strand Buffer, 1µl of 0.1mM DTT, 1µl of RNaseOUT ^TM Recombinant RNase Inhibitor, and 1µl of SuperScript ^TM III RT (Invitrogen) .The total reaction volume is 20µl for 30 ~ 60 minutes at 50 ℃. And incubated at 70 ° C. for 15 minutes.

Primer for RT-PCR was prepared and used as shown in Table 1 with reference to the cDNA of SEQ ID NO: 2 shown in Figure 1 and the base sequence before and after.

The RT-PCR results electrophoresed on 1% agarose gel are shown in FIG. 2.

As shown, it was confirmed that the cDNA expression is gradually increased from the initial high temperature culture of laver after 12 hours.

Example 2 Confirmation of High Temperature Resistance in Chlamydomonas

It was examined whether HTR2 according to the present invention imparts high temperature resistance to Chlamydomonas, a model seaweed.

After all the total RNA of a stem Seaweed, using the primers of Table 2 amplifies only the ORF sequence of HTR2 gene PCR, and inserting it into a Chlamydomonas expression NcoI vector in pCR102 and EcoRV place in a usual manner HTR2 gene to prepare a vector pCR102_ HTR2 for expression (see Fig. 3).

Conventional method for wild type Chlamydomonas reinhardtii cc-125 [The vector has hygromycin resistance gene, so it is transformed by glass bead transformation method and cultured for 2 to 3 weeks, and then selected in medium containing 15 mg / L hygromycin] by introducing the pCR102_ HTR2 to obtain a Chlamydomonas transformants.

pCR102_ the body HTR2 introduced transformant 10 ⁵ ~ 10 ³ / ㎖ ⁵ days at high temperatures of the inoculated solution was inoculated about 37 ℃ 5㎕l on solid medium diluted to a concentration of the culture and then transferred to culture in 25 ℃ 7 The growth of the cells after 1 and 14 days was observed and the photograph is shown in 4. In the figure, W / T refers to the wild type strain, and V-form refers to the blank strain in which the pCR102 vector is introduced into the wild type strain. The control represents the case of incubation at 25 ° C. without high temperature treatment.

As shown, in the case of W / T or V-form finally killed by high temperature treatment for 5 days, the transgenic Chlamydomonas introduced HTR2 according to the present invention was inhibited to some extent by the high temperature treatment It can be seen that the growth is recovered by returning to normal temperature.

Therefore, it was confirmed that HTR2 according to the present invention derived from laver can impart high temperature resistance to other species.

<110> Republic of korea <120> Protein and its Gene HTR2 of laver, giving          High-Temperature-Resistance <130> P0710-009 <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 144 <212> PRT <213> Porphyra sp. <400> 1 Met Ala Ala Ser Lys Thr Leu Ala Ala Ala Phe Ala Val Ala Leu Leu   1 5 10 15 Ala Leu Leu Thr Leu Thr Ala Ala Ala Pro Ala Ala Asp Thr Ala Ala              20 25 30 Ala Asp Ala Ala Thr Ala Ala Tyr Asp Ala Leu His Val Glu Gly Ile          35 40 45 Ser Gly Ala Ala Glu Ala Pro Ala Leu Asp Gly Leu Thr Asp Arg Ser      50 55 60 Gly Tyr Tyr Arg Cys Arg Ser Tyr Phe Val Cys Gly Ser Lys Lys Val  65 70 75 80 Lys Val Pro Val Lys His Ser His Pro Cys Tyr Tyr Lys Glu Ala Pro                  85 90 95 Ser Ile Leu Lys Ser Ser Arg Ser Ser Gly Leu Cys Arg Pro Asp Pro             100 105 110 Tyr Ser Cys Lys Pro Ala Thr Cys Ile Gly Trp Arg Lys Cys Thr Cys         115 120 125 Asp Ala Cys Lys Thr Val Ile Phe Thr His Asn Ile Trp Cys Glu Lys     130 135 140 <210> 2 <211> 435 <212> DNA <213> Porphyra sp. <400> 2 atggcggctt ccaagaccct cgccgccgcg tttgccgttg cgctcctggc actcctgacc 60 ctgaccgccg ccgcccccgc agcggacact gccgccgccg acgccgcaac cgccgcgtac 120 gacgcgctgc acgttgaggg catctcgggc gcggcggagg cacccgccct cgacggcctc 180 accgaccggt cgggctacta ccgctgccgc tcctactttg tgtgcggcag caagaaggtg 240 aaagtgcctg tcaagcactc gcacccgtgc tactacaagg aggcgccgtc gatcctcaag 300 tcgtcccgca gcagcggcct gtgccggccg gacccgtaca gctgcaagcc cgccacgtgc 360 atcggctggc gcaagtgcac gtgcgatgcg tgcaagacgg tgatcttcac ccataacatc 420 tggtgcgaga agtga 435 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 atggcggctt ccaagaccct c 21 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 tcacttctcg caccagatgt ta 22 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 caccatggcg gcttccaaga c 21 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 cgatatcact tctcgcacca gatgtta 27

Claims (5)

A protein of SEQ ID NO: 1 conferring high temperature resistance, isolated from laver.
The gene HTR2 encoding the protein according to claim 1.
The method of claim 2,
Gene characterized in that the cDNA comprising SEQ ID NO: 2.
A recombinant vector comprising the gene according to claim 2.
A transformant obtained by introducing the recombinant vector according to claim 4 into any one host selected from the group consisting of bacteria, seaweeds and plants.

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