CN107641620A

CN107641620A - Natrinema altunense sp high-salt tolerance relevant protein and its application

Info

Publication number: CN107641620A
Application number: CN201710963244.3A
Authority: CN
Inventors: 朱诚; 丁艳菲; 江琼
Original assignee: China Jiliang University
Current assignee: China Jiliang University
Priority date: 2017-10-17
Filing date: 2017-10-17
Publication date: 2018-01-30
Anticipated expiration: 2037-10-17
Also published as: CN107641620B

Abstract

The invention discloses a kind of method for improving Rice Resistance characteristic of disease, and external source nasod genes are specifically transferred into rice, and the nasod genes are the genes for encoding Natrinema altunense sp high-salt tolerance relevant protein.The nasod trans-genetic hybrid rice that turns obtained by the method for the present invention shows the excellent water resistant bacterial blight of rice.

Description

Natrinema altunense sp high-salt tolerance relevant protein and its application

Technical field

The present invention relates to a kind of method for improving Rice Resistance characteristic of disease, and in particular to external source nasod genes are transferred into rice, The nasod genes are the genes for encoding Natrinema altunense sp high-salt tolerance relevant protein.

Background technology

Halophilic archaea is a kind of archeobacteria lived in hypersaline environment.Bacteria rhodopsin albumen (bacteriorhodopsin.BR) it is protein on its cell membrane, it has typical seven transmembrane structures and CD-ROM drive kinoplaszm Sub- pumping function, there is higher development prospect and application value.Natrinema altunense sp is used as a kind of common ancient bacterium, its molecular level Research is carried out relatively fewer, therefore largely obtains its genetic resources, seems to study the mechanism of their adaptation extreme environment It is particularly important.Natrinema altunense sp belongs to ancient bacterium, is the biotype entirely different with eucaryote, bacterium, is also far from by people Recognize, there is wide researching value and application prospect itself as a kind of brand-new living resources.It is resistant to extreme environment Molecule mechanism is once cracked, and certainly will may apply to medicine and agriculture field, greatly improve their production and research level. The current research about Natrinema altunense sp is mainly limited to the separation discriminating of bacterium, general histochemistry, biochemical composition and substantially Physical signs.

Genetically modified plants are will to be obtained using the means of genetic engineering from animal, plant and microorganism or artificial synthesized Exogenous origin gene integrator makes stable heredity and the plant with new economical character correctly expressed and obtained into Plant Genome Thing.With the development of agricultural biotechnologies, oneself is widely used in agricultural production transgenic technology, is educated disease-resistant, pest-resistant, degeneration-resistant Kind etc. plays an important role.After gene transformation plant, we also need to use plant of the multiple means to acquisition Strain is screened and identified, obtains the transfer-gen plant of the positive.First, it would be desirable to it is thin to determine whether foreign gene is transferred to plant Whether born of the same parents, then detection are incorporated into the genome W of plant and its mode of integration into the foreign gene of plant cell, also finally Need detection be incorporated into foreign gene in Plant Genome whether transcript and expression.

The content of the invention

The present invention will encode the gene of Natrinema altunense sp (Natrinema altunense sp.) high-salt tolerance relevant protein (hereinafter referred to as nasod genes), is transferred in rice, and the original intention of inventor is by being transferred to nasod genes in rice, improving The high-salt tolerance ability of rice, is but surprisingly found that, turning nasod trans-genetic hybrid rice has very strong resistance against diseases, especially water resistant The bacterial blight of rice, so as to complete the present invention.

Therefore, one embodiment of the present invention there is provided a kind of method for turning nasod gene disease resisting rices, described Nasod genes have base sequence shown in SEQ ID No.1.

The present invention is that protein expression differences of the Natrinema altunense sp AJ2 under different salinity is started with, by SDS-Page, HPLC, amino acid sequencing, gene cloning etc. are studied, and obtain Natrinema altunense sp AJ2 high-salt tolerance relevant protein NASOD and phase Corresponding nasod genes.And nasod genes are transferred to Bacillus coli expression, it have studied NASOD fusion proteins and improve Escherichia coli The influence of salt resistance ability.

Specifically, the application is：The gene for encoding Natrinema altunense sp high-salt tolerance relevant protein is transferred to Host Strains, made Host Strains express Natrinema altunense sp high-salt tolerance relevant protein, obtain the recombinant bacterial strain with resistance to high salt ability.Then it is heavy from this NASOD albumen is identified in group bacterial strain.

Method by nasod genes by conventional transgenic, is transferred to rice, turns nasod trans-genetic hybrid rice so as to obtain, and Identify the performance of the bacterial blight of rice of transgenic paddy rice T1 strains.

Brief description of the drawings

Fig. 1：PFGC5941 plasmid maps.

Fig. 2：The amplification of nasod genes.

Fig. 3：The digestion verification of pFGC5941-nasod plasmids.

Fig. 4：T0 detects for the bar gene PCRs of rice.

Fig. 5：T0 detects for the nasod gene PCRs of rice.

Fig. 6：Scab length column diagram after rice leaf inoculation leaf spot bacteria.

Embodiment

With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in This：

Embodiment 1：The the isolating and purifying of NASOD albumen, amino acid sequencing, gene cloning

1) Natrinema altunense sp AJ2 (is presented) culture and existed by A. by Life Science College Institute of Micro-biology of Zhejiang University professor Wu Min Under 1.7M and 3.0M NaCl concentrations, sampled when growing into middle exponential growth.Carry out SDS-Page analyses and HPLC analyses. With reference to two analysis results, differential protein is obtained, and is purified with HPLC and SDS-Page to obtain this albumen.

B. amino acid sequencing.Obtain NASOD amino acid sequences 25 residues of N-terminal.

2) a. is compared by blast, and NASOD should belong to Natrinema altunense sp SOD families.According to the Halophiles reported SOD sequences, in conservative region design degenerate primer (SOD-PF2：CTNCCVTACGACTACGAYGC, SOD-PR2： GTAGTAKGARTG YT C CCAGACG), PCR amplifications, sequencing obtain one section of gene order among nasod ORF.

B. using the gene order about 500bp obtained in step a as masterplate, DNA probe (DIG labelling methods) is prepared, to AJ2's The XhoI digestion products of genomic DNA, southern hybridization is done, obtain a hybridization signal.

C. the result hybridized according to step b southern, the XhoI digestions of the genomic DNA of hybridization signal position are produced Thing reclaims, and using Taq enzyme filling-in end, is connected to carrier T.Convert escherichia coli DH5a.By transformed bacteria bed board.

D. step b DNA probe is used, for the plate of step c pavings, does colony hybridization.The single bacterium colony for choosing hybridization signal is surveyed Sequence.Obtain nasod ORF complete sequences, and its 5 ' upstream and 3 ' downstream sequences.

3) result of the 2nd step and the 1st step is compared, the difference of only one amino acid sequence, it is believed that be that amino acid is surveyed The error of sequence.

The final ORF total lengths 603bp (sequence is shown in SEQ ID No.1) for obtaining nasod, 5 ' upstream sequences have 286bp altogether, and 3 ' Downstream sequence has 670bp altogether.

It encodes 200 amino acid, molecular weight about 22.4kDa.In the amino acid sequence that it is deduced, have known extremely thermophilic 35 in salt bacterium SOD 37 conservative amino acid residues.

The homology of NASOD and known Natrinema altunense sp SOD on amino acid shows this albumen category 70%~83% In the member of Natrinema altunense sp SOD families.NASOD is as SOD albumen, with known Halophiles and the SOD albumen of other species There should be superoxide dismutase function.

Embodiment 2：Turn the preparation of nasod trans-genetic hybrid rice

Nipponbare (Nipponbare) and Anhui round-grained rice 97 are that laboratory is routinely reserved seed for planting.

Plasmid pFGC5941, coli strain DH5 α, Xanthomonas campestris (Xanthomonas oryzae Pv.oryzae, Xoo), agrobacterium strains EHA105, pMD18-T carrier, be laboratory conventional material, or directly buy Commercially available product.It is described to use reagent and enzyme, all it is general commercially available product, test apparatus is laboratory routine test instrument.

Primer synthesizes entrusts Hangzhou Rui Pu Gene Tech. Company Limited to complete with gene sequencing.

Test method and flow：

1. prepare Escherichia coli DH-5 α competence

2. the digestion and connection of plasmid

3. thermal shock method converts E. coli competent

4. the extraction of plasmid

5. prepare Agrobacterium tumefaciems EHA105 competence

6. freeze-thaw method converts Agrobacterium

7. the genetic transformation of rice

8. the PCR detections of transgenic paddy rice

9.Southern blot are detected

10. the Total RNAs extraction of transgenic paddy rice blade

11. fluorescence quantitative PCR method analyzes the expression quantity of transgenic paddy rice

Above test method refers to (An improved protocol for efficient transformation And regeneration of diverse indica rice cultivars, Khirod K Sahoo, Amit K Tripathi, Ashwani Pareek, Sudhir K Sopory and Sneh L Singla-Pareek, Plant Methods2011)。

The design of primers of table 1

PFGC5941 is chosen as rice transformation expression vector.Reacted by PCR and add NcoI at the both ends of primer With two restriction enzyme sites of BamHI.The plasmid pGEX-sod containing nasod genes preserved with laboratory is (reference can be made to application number 200810163748.8 Chinese patent) it is that masterplate obtains PCR primer, pMD-18 carriers are connected, after being sequenced correctly, while enzyme PFGC5941 carriers and pMD18-nasod are cut, finally connects purpose fragment and digestion carrier.PFGC5941 plasmid maps See Fig. 1, nasod genetic fragments are shown in Fig. 2 (M is 1Kb Ladder), and digestion verification is as shown in Figure 3 (M is 1Kb Ladder).

Embodiment 3：Turn acquisition and the Molecular Detection of nasod trans-genetic hybrid rice

1. the PCR checkings of transgenic paddy rice strain

Using rice material Nipponbare and the mature embryo of Anhui round-grained rice 97 as explant, Agrobacterium-mediated genetic transformation, warp are carried out Callus induction, the screening of kanamycin-resistant callus tissue, the differentiation culture of kanamycin-resistant callus tissue are crossed, obtains Basta resistances T0 for plant, its transfer 37 plants of nasod gene Nipponbare plant (T0), turn 97 41 plants of nasod genes Anhui round-grained rice.

Choose using transgenosis water of the Nipponbare as 8 plants of the transgenic paddy rice of rice material and with Anhui round-grained rice 97 for rice material 7 plants of extraction genomic DNAs of rice, with SOD-NcoI-F, SOD-BamHI-R is that primer (being shown in Table 1) enters performing PCR identification, after testing all For the positive, PCR testing results as shown in Figure 4, Figure 5, wherein, M is 100bp Ladder, 1-15 be T0 for DNA, 16 be control. T0 is positioned over greenhouse-grown for rice plant, T1 is for transgenic paddy rice seed for harvest.

2. the Southern blot detections of transgenic paddy rice strain

For further identification positive plant and T-DNA integration, mark is used as by the use of bar genetic fragments (about 311bp) Remember probe, Southern blot detections are carried out for plant to T1, extract transformed plant and non transformed plants genomic DNA, limitation Property restriction endonuclease EcoRI complete degestions, carry out hybridization verification afterwards.Genomic DNA is after single endonuclease digestion, by hybridizing display with probe Hybrid belt can be shown that the copy number situation that exogenous DNA is integrated in rice genome.Testing result：TSOD1-1、TSOD1-6 It is 1 copy with TSOD1-8, TSOD1-7 and TSOD1-9 are 2 copies, and TSOD1-11 is 3 copies, and TSOD1-17 is 4 Copy, single number of copies is at most (Southern Blot results are not shown).

Embodiment 4：The anti-disease enzyme of transgenic paddy rice

The determination of rice leaf spot bacteria inoculation condition

1) according to the preparation method of inoculation bacterium solution, bacterium solution OD600 values are adjusted to 1.0 by gradient with sterilized water, 0.8, 0.5、0.3、0.1。

2) each concentration chooses 3 plants similar of tillering stage Nipponbare of growth conditions, is inoculated with sword-like leave, finding out can cause rice to send out The minimum bacterial leaf-blight bacteria concentration of disease.

3) through experiment, OD=0.5 bacterial leaf spot bacterial concentration is the suitable concn that concentration is relatively low and Nipponbare can be made to fall ill.

4) each 5 plants of the rice of 5 different strains is inoculated with the bacterium solution of OD=0.5 concentration, as a result find strain TSOD1-9 and TSOD1-17 has preferable disease resisting effect, and the two strains are formally mainly chosen during experiment and repeat to test.

The inoculation and identification of bacterial blight of rice

(1) step：

1) 10 plants similar of rice of upgrowth situation, numbering mark are selected in each strain.

2) sword-like leave of rice selected by the OD=0.5 prepared bacterial leaf spot bacterium solution inoculation is used.

3) each leaf incidence after observing 3 days, 5 days, 10 days, scab length is recorded.

(2) disease-resistant result：

Table 2 is inoculated with transgenic paddy rice leaf spot lesion length average value (unit after leaf spot bacteria：mm)

	3 days	5 days	10 days
				TSOD1-9	0.00	1.26	17.21
TSOD1-17	0.27*	2.25*	34.12*
				Control	1.74*	10.41*	64.20*

The average value (table 2) of the lower 10 groups of scab length of each strain different onset number of days is calculated respectively, finds each morbidity day Number.TSOD1-9 is respectively less than control group, significant difference with TSOD1-17 scab length, and TSOD1-9 scabs length is most short, refers to Fig. 6, rice leaf are inoculated with scab length column diagram after leaf spot bacteria.TSOD1-9, TSOD1-17 represent transgenic line；CK To compare Nipponbare strain；* represent that transgenic line scab length is notable in the level differences of P ＜ 0.05 compared with the control.

Sequence table

<110>Metering university of China

<120>Natrinema altunense sp high-salt tolerance relevant protein and its application

<130>

<160>7

<170>PatentIn version 3.4

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<211>603

<212>DNA

<213>Natrinema altunense sp.

<400>1

atgactgatc acgaacttcc accactcccg tacgattacg acgcgctcga accggcactg 60

tccgaacagg tactgacctg gcatcacgat acgcaccacc agggctacgt caacggcctc 120

aacgccgccg aggagaccct cgcggagaac cgcgaggagg gcgacttcgg ctcgacgccc 180

ggtgccctca aaaacgttac tcacaacggc tgtggtcact atctccacac gctgttctgg 240

gagaacatgt cccccaacgg cggcggcgag ccggacggcg acctcgccga ccgcatcgag 300

gaggacttcg gatcctacga gggctggaaa ggcgagttcg aggccgctgc cggtgccgcc 360

ggtggctggg cactgctggt gtacgatccg gttgcgaagc aacttcgcaa cgtcgcggtc 420

gacaagcacg accagggcgc gctctggggc gcacatccag tgctcgcgct ggacgtctgg 480

gagcactcct actactacga ctacggtccg gaccgcggag acttcatcga cgccttcttc 540

gacgtcgtca actgggagaa ggccgaagag gagtaccaga cctgcctcga ccacttcgag 600

taa 603

Claims

A kind of 1. method for improving Rice Resistance characteristic of disease, it is characterised in that external source nasod genes, the nasod are transferred into rice Gene is the gene for encoding Natrinema altunense sp high-salt tolerance relevant protein.
2. the method for claim 1, wherein external source nasod genes have the sequence that SEQ ID No.1 are represented.
3. method as claimed in claim 1 or 2, wherein, the disease resistance is the water resistant bacterial blight of rice.
4. method as claimed in claim 1 or 2, wherein, the external source nasod genes have 2 or 4 copies.
5. method as claimed in claim 4, wherein, the external source nasod genes have 2 copies.
6. the gene for encoding Natrinema altunense sp high-salt tolerance relevant protein is cultivating the application among disease resistance rice varieties.
7. application as claimed in claim 6, the gene have the sequence that SEQ ID No.1 are represented.