CN101880676A - Constructing method of nifA gene-introduced rhizobium japonicum genetic engineering bacterial strain HD-SFH-01 - Google Patents

Abstract

The invention relates to a constructing method of a nifA gene-introduced rhizobium japonicum genetic engineering bacterial strain HD-SFH-01, belonging to a constructing method of a rhizobium japonicum genetic engineering bacterial strain and solving the problems of less increase on soybean nodule quantity and little soybean yield increase after the traditional rhizobium japonicum genetic engineering bacterial strain is inoculated to a soybean seedling. The method comprises the steps of: extracting rhizobium japonicum genome DNA for carrying out PCR amplification to obtain a target gene nifA, purifying and then connecting with a TA clone carrier pMD18-T to obtain a connecting product and then converting into escherichia coli; carrying out multiple enzyme digestion, connection and verification to obtain a recombinant prokaryote expression carrier pTR-Plac-nifA; and converting the recombinant prokaryote expression carrier pTR-Plac-nifA into soybean indigenous rhizobium. The bacterial strain HD-SFH-01 is inoculated into the soybean seedling, the rhizobium quantity is increased, and the soybean yield is increased by 3.80 percent.

Description

The construction process of nifA gene-introduced rhizobium japonicum genetic engineering bacterial strain HD-SFH-01

Technical field

The present invention relates to the construction process of rhizobium japonicum genetic engineering bacterial strain.

Background technology

Nitrogen is the essential substance that constitutes protein and nucleic acid, is requisite bio-feritlizer in the agriculture production.Biological nitrogen fixation is meant that dinitrogen passes through the process of the catalysis formation ammonia of nitrogen-fixing microorganism nitrogenase system.Biology can directly absorb airborne nitrogen as nutriment, and they are converted into amino acid and protein again with molecular nitrogen elder generation reduction ammonification, and this is an important component part of Nitrogen Cycling on the earth.The biological nitrogen fixation of occurring in nature has three kinds of forms, a kind of be bacterium from growing nitrogen-fixing, be meant that microorganism independently lives, independently carry out fixed nitrogen; Another kind is a symbiotic nitrogen fixation, refers to that vinelandii and the host plant in the soil forms the symbiotic nitrogen fixation system, carries out biological nitrogen fixation; The third is an association nitrogen fixation, and it is meant a kind of special symbiotic nitrogen fixation that some nitrogen-fixing bacteria forms at the higher plant rhizosphere.Symbiotic nitrogen fixation has important economic value in agriculture production, wherein the symbiotic nitrogen fixation system that constitutes of leguminous plants and root nodule bacterium most importantly.

At present, rhizobium japonicum genetic engineering bacterial strain is after being inoculated into soybean seedling, and is few to the increase of soybean plant strain dross quantity, little to soybean yields amplification.

Summary of the invention

The objective of the invention is in order to solve existing rhizobium japonicum genetic engineering bacterial strain after being inoculated into soybean seedling, increase to soybean plant strain dross quantity is few, the problem little to soybean yields amplification, and the construction process of nifA gene-introduced rhizobium japonicum genetic engineering bacterial strain HD-SFH-01 is provided.

The construction process of nifA gene-introduced rhizobium japonicum genetic engineering bacterial strain HD-SFH-01 carries out according to the following steps: one, fast living type rihizobium japonicum 15067 is activated and spread cultivation, extract genomic dna then; Two, adopt the homologous sequence cloning, with fast living type rihizobium japonicum 15067 genomic dnas is that template is carried out pcr amplification, obtain goal gene nifA, adopt TaKaRa Agarose GeL DNAPurification Kit to carry out purifying then, again the goal gene nifA behind the purifying is connected with TA cloning vector pMD18-T, obtains to connect product; Three, will connect the screening and the evaluation of product transformed into escherichia coli DH5 α and positive colony, adopt alkaline lysis method of extracting plasmid vector pUC19 then, again goal gene nifA and plasmid vector pUC19 are carried out double digestion through SacI and SalI respectively, reclaim required purpose fragment, connect and structure recombinant vectors pUC19-nifA; Four, be that template is carried out pcr amplification with recombinant vectors pUC19-nifA, obtain P _Lac-nifA fragment (the nifA gene fragment that has the lac promotor) is then with P _Lac-nifA fragment and pET-28a plasmid vector carry out double digestion through NcoI and SacI respectively, reclaim required purpose fragment, connect and structure recombinant vectors pET-28a-P _Lac-nifA; Five, with recombinant vectors pET-28a-P _Lac-nifA is that template is carried out pcr amplification, obtains P _Lac-nifA-T7 fragment (fragment that has the nifA gene and the T7 terminator of lac promotor) is then with P _Lac-nifA-T7 fragment and pTR102 plasmid vector carry out single endonuclease digestion through KpnI respectively, reclaim required purpose fragment, connect and structure reorganization prokaryotic expression carrier pTR-P _Lac-nifA; Six, adopt the triparental mating technology with prokaryotic expression carrier pTR-P _Lac-nifA is transformed in the soybean original inhabitants root nodule bacterium, promptly finishes the structure of nifA gene-introduced rhizobium japonicum genetic engineering bacterial strain HD-SFH-01; Wherein the used upstream primer of pcr amplification is 5 '-CGC in the step 2 GTCGACTTATGACTGAATTATCC-3 ', downstream primer are 5 '-CGC GAGCTCACCGAATTTAGATCTT-3 '; The used upstream primer of pcr amplification is 5 '-CGC in the step 4 CCATGGATTACCGCCTTTGAGT-3 ', downstream primer are 5 '-CGC GCGGCCGCTGCAAGGCGATTAA-3 '; The used upstream primer of pcr amplification is 5 '-CGC in the step 5 GGTACCATTGTGAGCGGATAAC-3 ', downstream primer are 5 '-GCG GGTACCTAGTTCCTCCTTTCAG-3 '.

The nif gene is mainly the gene of coding nitrogenase mixture, and the coded product of nifA is responsible for the nif gene transcription.NifA is the positive regulator gene of transcribing, and the transcription activating albumen of coding is found in klebsiella pneumoniae at first.Therefore the product NifA albumen of nifA gene has atpase activity, is the just adjusting product of transcribing, and its direct catalysis nif gene and fix expression of gene becomes the core factor of biological nitrogen fixation process.In addition, it also participates in the symbiosis process of nodulation of root nodule bacterium and influences color, quantity, distribution, growth and the differentiation etc. of root nodule.

Constructed nifA gene-introduced rhizobium japonicum genetic engineering bacterial strain HD-SFH-01 among the present invention, after being inoculated into soybean seedling, soybean plant strain dross quantity increases, and soybean yields is had a big increase, and average soybean yields increases about 3.80%; The present invention has actual value to improving Heilongjiang Province's soybean yields and improving the ecological environment.

Description of drawings

Fig. 1 is the tree-shaped figure of phylogeny of soybean original inhabitants root nodule bacterium in the embodiment one; Fig. 2 is the bacterium colony luminous detection figure of rhizobium japonicum genetic engineering bacterial strain HD-SFH-01 nifA gene-introduced in the embodiment one; Fig. 3 is the bacterium colony luminous detection figure that adopts the bacterial strain that changes empty carrier in the embodiment one.

Embodiment

Technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.

Embodiment one: the construction process of the rhizobium japonicum genetic engineering bacterial strain HD-SFH-01 that present embodiment is nifA gene-introduced carries out according to the following steps: one, fast living type rihizobium japonicum 15067 is activated and spread cultivation, extract genomic dna then; Two, adopt the homologous sequence cloning, with fast living type rihizobium japonicum 15067 genomic dnas is that template is carried out pcr amplification, obtain goal gene nifA, adopt TaKaRa Agarose GeL DNA Purification Kit to carry out purifying then, again the goal gene nifA behind the purifying is connected with TA cloning vector pMD18-T, obtains to connect product; Three, will connect the screening and the evaluation of product transformed into escherichia coli DH5 α and positive colony, adopt alkaline lysis method of extracting plasmid vector pUC19 then, again goal gene nifA and plasmid vector pUC19 are carried out double digestion through SacI and SalI respectively, reclaim required purpose fragment, connect and structure recombinant vectors pUC19-nifA; Four, be that template is carried out pcr amplification with recombinant vectors pUC19-nifA, obtain P _Lac-nifA fragment (the nifA gene fragment that has the lac promotor) is then with P _Lac-nifA fragment and pET-28a plasmid vector carry out double digestion through NcoI and SacI respectively, reclaim required purpose fragment, connect and structure recombinant vectors pET-28a-P _Lac-nifA; Five, with recombinant vectors pET-28a-P _Lac-nifA is that template is carried out pcr amplification, obtains P _Lac-nifA-T7 fragment (fragment that has the nifA gene and the T7 terminator of lac promotor) is then with P _Lac-nifA-T7 fragment and pTR102 plasmid vector carry out single endonuclease digestion through KpnI respectively, reclaim required purpose fragment, connect and structure reorganization prokaryotic expression carrier pTR-P _Lac-nifA; Six, adopt the triparental mating technology with prokaryotic expression carrier pTR-P _Lac-nifA is transformed in the soybean original inhabitants root nodule bacterium, promptly finishes the structure of nifA gene-introduced rhizobium japonicum genetic engineering bacterial strain HD-SFH-01; Wherein the used upstream primer of pcr amplification is 5 '-CGC in the step 2 GTCGACTTATGACTGAATTATCC-3 ', downstream primer are 5 '-CGC GAGCTCACCGAATTTAGATCTT-3 '; The used upstream primer of pcr amplification is 5 '-CGC in the step 4 CCATGGATTACCGCCTTTGAGT-3 ', downstream primer are 5 '-CGC GCGGCCGCTGCAAGGCGATTAA-3 '; The used upstream primer of pcr amplification is 5 '-CGC in the step 5 GGTACCATTGTGAGCGGATAAC-3 ', downstream primer are 5 '-GCG GGTACCTAGTTCCTCCTTTCAG-3 '.

Fast living type rihizobium japonicum 15067 is taken from DSMZ of Institute of Micro-biology of the Chinese Academy of Sciences in the present embodiment step 1.

Concrete operations in the present embodiment step 1: fast living type rihizobium japonicum 15,067 three rides of 4 ℃ of preservations are inoculated into YMA solid medium (N.F,USP MANNITOL of 10g, the KH of 0.2g ₂PO ₄, 0.2g MgSO ₄7H ₂The CaCl of the NaCl of O, 0.2g, the yeast extract paste of 0.1g, 5g ₂, 18.0～20.0g agar powder add distilled water and be settled to 1000mL), put into constant incubator, cultivate 72h down at 28 ℃; Picking list colony inoculation places on the air shaking table to the YMA liquid nutrient medium, and at 28 ℃, 180r/min shaking culture 72h carries out bacterial strain and spreads cultivation, and extracts fast genomic dna of giving birth to type rihizobium japonicum 15067 with the CTAB method then and also detects with 1% agarose gel electrophoresis.

Concrete operations in the present embodiment step 3: adopt CaCl ₂Legal system is equipped with the bacillus coli DH 5 alpha competent cell; To connect product and bacillus coli DH 5 alpha competent cell mixing under aseptic condition, adopt the then warm suddenly mode of bathing of ice bath to make its conversion, add SOC liquid nutrient medium (the 2M MgCl of 0.25MKCl, the 5mL of NaCl, the 10mL of the peptone of 20g, the yeast extract of 5g, 5g ₂, 20mL 1M glucose add distilled water and be settled to 1000mL), 37 ℃ of shaking table 180r/min shaking culture 1h coat bacterium liquid and to carry out indigo plant on the LB flat board that contains Amp, IPTG, X-Gal and screen in vain by the amount of different gradients; With white colony on the aseptic toothpick picking flat board in 1mL LB liquid nutrient medium, place 37 ℃ of shaking tables, the 180r/min incubated overnight is a template with bacterium liquid then, the PCR that carries out positive colony identifies, gets 5 μ L PCR products after reaction finishes and carries out the detection of 1% agarose gel electrophoresis; The bacterium liquid activation that qualification result is positive is delivered to order-checking company and is checked order.

Soybean original inhabitants root nodule bacterium are to separate from periphery soybean planting zone, Harbin City, Heilongjiang Province to obtain in the present embodiment step 6; The employing universal primer PCR (P9:5 '-AGA GTT TGA TCC TGG CTC AG-3 ' and P10:5 '-AGA GTT TGA TCC TGG CTC AG-3 ') the indigenous root nodule bacterium 16S rDNA of amplification, obtain its 16S rDNA sequence; 16S rDNA sequence in the 16S rDNA sequences of the indigenous root nodule bacterium that obtain and the GenBank database is carried out the homology compare of analysis, and constructing system growth tree (Fig. 1), know this indigenous rhizobium by inference in Chinese rhizobium (Sinorhizobium) according to the homology analysis result.

Rhizobium japonicum genetic engineering bacterial strain HD-SFH-01 nifA gene-introduced in the present embodiment compares with adopting the bacterial strain (TC: the pTR102 empty carrier is transformed indigenous root nodule bacterium) that changes empty carrier:

Cover dropping capraldehyde solution 50mL at cultured single bacterium colony flat board, then flat board is inverted in the darkroom, again film is placed on dull and stereotyped about 1min of going up, being placed on then in the developing solution develops manifests (centre should mobile film so that its contact with developing solution is full and uniform) to observing blackspot, fast film is put into clear water, fully wash developing solution off, be put in photographic fixing 10min in the stop bath again, use flushing with clean water 30min at last, so far the X-ray sheet just completes; The X-ray sheet the results are shown in Figure 2 and 3, rhizobium japonicum genetic engineering bacterial strain HD-SFH-01 nifA gene-introduced in the present embodiment as can be known successfully constructs.

Gained reorganization prokaryotic expression carrier pTR-P in the present embodiment step 5 _LacThe mensuration of the genetic stability of-nifA, and forward to and do further checking in the green plant, concrete steps are as follows:

1, genetic stability detects under the spontaneous condition:

After transconjugant is purified, picking is 5 at random, switching 10 times continuously on the YMA medium of added with antibiotic not, several single bacterium colonies of last picking respectively dibblings at the YMA of added with antibiotic with not on the YMA medium of added with antibiotic, 28 ℃ are cultured to and grow bacterium colony, check the luminous of bacterium colony;

2, genetic stability detects under the symbiosis condition:

Bacterial strain behind the mark is made bacteria suspension, be inoculated in the soybean plant strain root, and with indigenous root nodule bacterium with do not inoculate and compare, illumination cultivation is gathered in the crops to 40d, and the root nodule of gathering in the crops is cut in half, and back-off is in the culture dish bottom, Dropwise 50 μ L capraldehyde solution in culture dish, in the darkroom, promptly can be observed luminously, develop and, calculate the percentage that luminous root nodule accounts for all root nodules thus with the X-ray sheet record that exposes; HD-SFH-01, the conservation rate of the recombinant plasmid of TC after transferring continuously is shown in Table 1;

Table 1

Strains tested	??1	??2	??3	??4	??5	??6	??7	??8
									??HD-SFH-01	??100	??100	??100	??100	??100	??100	??100	??100
??TC	??100	??100	??100	??100	??100	??100	??100	??100

Inoculate rhizobium japonicum genetic engineering bacterial strain HD-SFH-01 and TC under the soybean condition of pot respectively and be cultured to the appearance of soybean plant strain root nodule, gather the root nodule on the soybean plant strain root system, behind surface sterilization, cut root nodule with scalper, back-off is in the culture dish bottom, observe luminescence activity, with the luminescence activity of X-mating plate exposure record root nodule; The result is as shown in table 2, and as seen from the luminous detected result of root nodule, the mark bacterial strain is infecting in the process of dross mutually with plant, and marker gene is stable.

Table 2

Engineering strain	Detect root nodule number (individual)	Luminous root nodule number (individual)	Stability (%)
				??HD-SFH-01	??67	??67	??100
??TC	??57	??57	??100

The green plant experiment:

Select the uniform soybean seeds of the full color and luster of shape, treated, seed is in sterilized water fully behind the imbibition, in 28 ℃ of vernalization 2d; After the vernalization in the sowing sterile soil, the no nitrogen nutrient solution of every day pouring, treat that soybean grows first cotyledon after, inoculation rihizobium japonicum engineering strain, behind the cultivation 45d, picking 30 strain soybean plant strains are equally divided into 3 groups at random; Measure the over-ground part root nodule number of every group 10 strain soybean plant strains; The inoculation different strains sees Table 3 to the influence of soybean plant strain growing state; Significant result analysis in table 4;

Table 3

Project	The root nodule number/	Soybean yields/g
			Do not connect bacterium	??0	??0.381
The original inhabitants rihizobium japonicum	??6.0333	??0.395
			??TC(pTR102)	??6.0666	??0.3943

Project	The root nodule number/	Soybean yields/g
			??TL(pTR-P lac-nifA)	??7.5333	??0.41

Annotate: per 1 data are all taken from the mean value of 10-15 sample, repeat 3 times.

Table 4

Project	The root nodule number	Soybean yields
			Original inhabitants root nodule bacterium/TC (pTR102) vs. does not connect bacterium	??**	??*
Original inhabitants root nodule bacterium vs.TC (pTR102)	??ns	??ns
			HD-SFH-01vs. do not connect bacterium	??**	??**
HD-SFH-01vs. indigenous root nodule bacterium/TC (pTR102)	??**	??**

Annotate: * * represents difference extremely significantly (P＜0.01), and * represents significant difference (P＜0.01), and ns represents difference not remarkable (P＞0.01).

The result shows: nifA gene-introduced rhizobium japonicum genetic engineering bacterial strain HD-SFH-01 is behind the inoculation soybean seedling, the influence of aspects such as root nodule number that soybean plant strain is tied and soybean yields all with the indigenous rihizobium japonicum of inoculation, control group TC (pTR102) compares the difference that all exists in various degree, wherein transgenic engineering bacterial strain HD-SFH-01 does not compare with indigenous rihizobium japonicum/TC (pTR102) with connecing bacterium, and is remarkable at the heteropole that is wanting in of soybean plant strain root nodule number and soybean yields; Compare with the soybean plant strain of the indigenous bacterium of inoculation, the soybean yields of inoculation transgenic engineering bacterial strain HD-SFH-01 increases about 3.80%.

Embodiment two: present embodiment and embodiment one are different is that the reaction system of pcr amplification in the step 2 is 25 μ L reaction systems, is made up of following ingredients:

The composition consumption

400ng/ μ L gives birth to type rihizobium japonicum 15067 genomic dnas 1.0 μ L soon

0.2μM?PnifA1??????????????????????????????1.0μL

(5’-CGC GTCGACTTATGACTGAATTATCC-3’)

0.2μM?PnifA2??????????????????????????????1.0μL

(5’-CGC GAGCTCACCGAATTTAGATCTT-3’)

10×Ex?Taq?Buffer??????????????????????????2.5μL

0.4mMdNTP?Mixture??????????????????????????1.5μL

Taq archaeal dna polymerase 0.5 μ L

Sterilized water 17.5 μ L

The pcr amplification condition is: 94 ℃ of sex change 10min, and 94 ℃ of sex change 1min, 65 ℃ of annealing 30s, 72 ℃ are extended 1min, totally 35 circulations, 72 ℃ are extended 7min, 4 ℃ of insulations again.Other step and parameter are identical with embodiment one.

Embodiment three: present embodiment is different with one of embodiment one to two is that the concrete operations step of TaKaRa Agarose GeL DNA Purification Kit in the step 2 is with reference to specification sheets.Other step and parameter are identical with one of embodiment one to two.

TaKaRa Agarose GeL DNA Purification Kit is precious biological sepharose DNA purification kit in the present embodiment.

Embodiment four: present embodiment is different with one of embodiment one to three is that goal gene nifA is connected with TA cloning vector pMD18-T in the step 2 concrete operations step is with reference to TaKaRa pMD18-T Vector test kit specification sheets.Other step and parameter are identical with one of embodiment one to three.

TaKaRa pMD18-T Vector is precious biological pMD18-T carrier in the present embodiment.

Embodiment five: what present embodiment was different with one of embodiment one to four is that the system of double digestion is as follows in the step 3:

The composition consumption

Goal gene nifA 13.0 μ L

Restriction endonuclease SacI 1.0 μ L; SalI 1.0 μ L

10×T?Buffer??????????????3.75μL

BSA (bovine serum albumin) 0.25 μ L

Sterilized water 6.0 μ L.

Other step and parameter are identical with one of embodiment one to four.

Embodiment six: what present embodiment was different with one of embodiment one to five is that the system of double digestion is as follows in the step 3:

The composition consumption

Plasmid vector pUC19 15.0 μ L

Restriction endonuclease SacI 1.0 μ L; SalI 1.0 μ L

10×T?Buffer????????????????3.75μL

BSA?????????????????????????0.25μL

Sterilized water 4.0 μ L.

Other step and parameter are identical with one of embodiment one to five.

Embodiment seven: present embodiment is different with one of embodiment one to six is that the system that connects in the step 3 is as follows:

The composition consumption

10 * T ₄Dna ligase damping fluid 2.5 μ L

Goal gene 10.0 μ L after enzyme is cut

Plasmid vector pUC19 2.0 μ L after enzyme is cut

T ₄Dna ligase 1.0 μ L

Sterilized water 9.5 μ L.

Other step and parameter are identical with one of embodiment one to six.

Embodiment eight: what present embodiment was different with one of embodiment one to seven is that the system of double digestion is as follows in the step 4:

The composition consumption

P _Lac-nifA fragment 13.0 μ L

Restriction endonuclease NcoI1.0 μ L; SacI 1.0 μ L

10×T?Buffer????????????3.75μL

BSA?????????????????????0.25μL

Sterilized water 6.0 μ L.

Other step and parameter are identical with one of embodiment one to seven.

Embodiment nine: what present embodiment was different with one of embodiment one to eight is that the system of double digestion is as follows in the step 4:

The composition consumption

PET-28a plasmid vector 15.0 μ L

Restriction endonuclease NcoI1.0 μ L; SacI 1.0 μ L

10×T?buffer????????????3.75μL

BSA?????????????????????0.25μL

Sterilized water 4.0 μ L.

Other step and parameter are identical with one of embodiment one to eight.

Embodiment ten: present embodiment is different with one of embodiment one to nine is that the reaction system of pcr amplification in the step 4 is 25 μ L reaction systems, is made up of following ingredients:

The composition consumption

400ng/ μ L recombinant vectors pUC 19-nifA 1.0 μ L

0.2μM?LBnifA1???????????????????????????1.0μL

(5’CGC CCATGGATTACCGCCTTTGAGT-3’)

0.2μM?LBnifA2???????????????????????????1.0μL

(5’-CGC GCGGCCGCTGCAAGGCGATTAA-3’)

10×Ex?Taq?Buffer????????????????????????2.5μL

0.4mMdNTP?Mixture????????????????????????1.5μL

Taq archaeal dna polymerase 0.5 μ L

Sterilized water 17.5 μ L

The pcr amplification condition is: 94 ℃ of sex change 10min, and 94 ℃ of sex change 1min, 65 ℃ of annealing 30s, 72 ℃ are extended 1min, totally 35 circulations, 72 ℃ are extended 7min, 4 ℃ of insulations again.Other step and parameter are identical with one of embodiment one to nine.

Embodiment 11: present embodiment is different with one of embodiment one to ten is that the system that connects in the step 4 is as follows:

The composition consumption

10 * T ₄Dna ligase damping fluid 2.5 μ L

P after enzyme is cut _Lac-nifA sheet 10.0 μ L

PET-28a plasmid vector 2.0 μ L after enzyme is cut

T ₄Dna ligase 1.0 μ L

Sterilized water 9.5 μ L.

Other step and parameter are identical with one of embodiment one to ten.

Embodiment 12: present embodiment is different with one of embodiment one to 11 is that the reaction system of pcr amplification in the step 5 is 25 μ L reaction systems, is made up of following ingredients:

The composition consumption

400ng/ μ L recombinant vectors pET-28a-P _Lac-nifA 1.0 μ L

0.2μM?LBTnifA1???????????????????????????1.0μL

(5’-CGC GGTACCATTGTGAGCGGATAAC-3’)

0.2μM?LBTnifA2???????????????????????????1.0μL

(5’-GCG GGTACCTAGTTCCTCCTTTCAG-3’)

10×Ex?Taq?Buffer?????????????????????????2.5μL

0.4mMdNTP?Mixture?????????????????????????1.5μL

Taq archaeal dna polymerase 0.5 μ L

Sterilized water 17.5 μ L

The pcr amplification condition is: 94 ℃ of sex change 10min, and 94 ℃ of sex change 1min, 65 ℃ of annealing 30s, 72 ℃ are extended 1min, totally 35 circulations, 72 ℃ are extended 7min, 4 ℃ of insulations again.Other step and parameter are identical with one of embodiment one to 11.

Sequence table

＜110〉Heilongjiang University

＜120〉construction process of nifA gene-introduced rhizobium japonicum genetic engineering bacterial strain HD-SFH-01

<160>6

 

<210>1

<211>26

<212>DNA

＜213〉artificial sequence

 

<220>

＜223〉the sequence PnifA1 of PCR upstream primer.

 

<400>1

 

cgcgtcgact?tatgactgaa?ttatcc?26

 

<210>2

<211>25

<212>DNA

＜213〉artificial sequence

 

<220>

＜223〉the sequence PnifA2 of PCR downstream primer.

 

<400>2

cgcgagctca?ccgaatttag?atctt?25

 

<210>3

<211>25

<212>DNA

＜213〉artificial sequence

 

<220>

＜223〉the sequence LBnifA 1 of PCR upstream primer.

 

<400>3

cgcccatgga?ttaccgcctt?tgagt?25

 

<210>4

<211>25

<212>DNA

＜213〉artificial sequence

 

<220>

＜223〉the sequence LBnifA 2 of PCR downstream primer.

 

<400>4

cgcgcggccg?ctgcaaggcg?attaa?25

 

<210>5

<211>25

<212>DNA

＜213〉artificial sequence

 

<220>

＜223〉the sequence LBTnifA1 of PCR upstream primer.

 

<400>5

cgcggtacca?ttgtgagcgg?ataac?25

 

<210>6

<211>25

<212>DNA

＜213〉artificial sequence

 

<220>

＜223〉the sequence LBTnifA2 of PCR downstream primer.

 

<400>6

gcgggtacct?agttcctcct?ttcag?25

Claims (10)

1. the construction process of nifA gene-introduced rhizobium japonicum genetic engineering bacterial strain HD-SFH-01, the construction process that it is characterized in that nifA gene-introduced rhizobium japonicum genetic engineering bacterial strain HD-SFH-01 carries out according to the following steps: one, fast living type rihizobium japonicum 15067 is activated and spread cultivation, extract genomic dna then; Two, adopt the homologous sequence cloning, with fast living type rihizobium japonicum 15067 genomic dnas is that template is carried out pcr amplification, obtain goal gene nifA, adopt TaKaRa Agarose GeL DNAPurification Kit to carry out purifying then, again the goal gene nifA behind the purifying is connected with TA cloning vector pMD18-T, obtains to connect product; Three, will connect the screening and the evaluation of product transformed into escherichia coli DH5 α and positive colony, adopt alkaline lysis method of extracting plasmid vector pUC19 then, again goal gene nifA and plasmid vector pUC19 are carried out double digestion through SacI and SalI respectively, reclaim required purpose fragment, connect and structure recombinant vectors pUC19-nifA; Four, be that template is carried out pcr amplification with recombinant vectors pUC19-nifA, obtain P _Lac-nifA fragment is then with P _Lac-nifA fragment and pET-28a plasmid vector carry out double digestion through NcoI and SacI respectively, reclaim required purpose fragment, connect and structure recombinant vectors pET-28a-P _Lac-nifA; Five, with recombinant vectors pET-28a-P _Lac-nifA is that template is carried out pcr amplification, obtains P _Lac-nifA-T7 fragment is then with P _Lac-nifA-T7 fragment and pTR102 plasmid vector carry out single endonuclease digestion through KpnI respectively, reclaim required purpose fragment, connect and structure reorganization prokaryotic expression carrier pTR-P _Lac-nifA; Six, adopt the triparental mating technology with prokaryotic expression carrier pTR-P _Lac-nifA is transformed in the soybean original inhabitants root nodule bacterium, promptly finishes the structure of nifA gene-introduced rhizobium japonicum genetic engineering bacterial strain HD-SFH-01; Wherein the used upstream primer of pcr amplification is 5 '-CGC in the step 2 GTCGACTTATGACTGAATTATCC-3 ', downstream primer are 5 '-CGC GAGCTCACCGAATTTAGATCTT-3 '; The used upstream primer of pcr amplification is 5 '-CGC in the step 4 CCATGGATTACCGCCTTTGAGT-3 ', downstream primer are 5 '-CGC GCGGCCGCTGCAAGGCGATTAA-3 '; The used upstream primer of pcr amplification is 5 '-CGC in the step 5 GGTACCATTGTGAGCGGATAAC-3 ', downstream primer are 5 '-GCG GGTACCTAGTTCCTCCTTTCAG-3 '.

2. the construction process of nifA gene-introduced rhizobium japonicum genetic engineering bacterial strain HD-SFH-01 according to claim 1, the reaction system that it is characterized in that pcr amplification in the step 2 is 25 μ L reaction systems, is made up of following ingredients:

The composition consumption

400ng/ μ L gives birth to type rihizobium japonicum 15067 genomic dnas 1.0 μ L soon

0.2μM?PnifA1???????????????????????????????????1.0μL

5’-CGC GTCGACTTATGACTGAATTATCC-3’

0.2μM?PnifA2???????????????????????????????????1.0μL

5’-CGC GAGCTCACCGAATTTAGATCTT-3’

10×Ex?Taq?Buffer???????????????????????????????2.5μL

0.4mMdNTP?Mixture???????????????????????????????1.5μL

Taq archaeal dna polymerase 0.5 μ L

Sterilized water 17.5 μ L

The pcr amplification condition is: 94 ℃ of sex change 10min, and 94 ℃ of sex change 1min, 65 ℃ of annealing 30s, 72 ℃ are extended 1min, totally 35 circulations, 72 ℃ are extended 7min, 4 ℃ of insulations again.

3. the construction process of nifA gene-introduced rhizobium japonicum genetic engineering bacterial strain HD-SFH-01 according to claim 1 is characterized in that the system of double digestion is as follows in the step 3:

The composition consumption

Goal gene nifA 13.0 μ L

Restriction endonuclease SacI 1.0 μ L; SalI 1.0 μ L

10×T?Buffer????????????????????????????3.75μL

BSA?????????????????????????????????????0.25μL

Sterilized water 6.0 μ L.

4. the construction process of nifA gene-introduced rhizobium japonicum genetic engineering bacterial strain HD-SFH-01 according to claim 1 is characterized in that the system of double digestion is as follows in the step 3:

The composition consumption

Plasmid vector pUC19 15.0 μ L

Restriction endonuclease SacI 1.0 μ L; SalI 1.0 μ L

10×T?Buffer??????????????????????3.75μL

BSA???????????????????????????????0.25μL

Sterilized water 4.0 μ L.

5. the construction process of nifA gene-introduced rhizobium japonicum genetic engineering bacterial strain HD-SFH-01 according to claim 1 is characterized in that the system that connects in the step 3 is as follows:

The composition consumption

10 * T ₄Dna ligase damping fluid 2.5 μ L

Goal gene 10.0 μ L after enzyme is cut

Plasmid vector pUC19 2.0 μ L after enzyme is cut

T ₄Dna ligase 1.0 μ L

Sterilized water 9.5 μ L.

6. the construction process of nifA gene-introduced rhizobium japonicum genetic engineering bacterial strain HD-SFH-01 according to claim 1 is characterized in that the system of double digestion is as follows in the step 4:

The composition consumption

P _Lac-nifA fragment 13.0 μ L

Restriction endonuclease NcoI1.0 μ L; SacI 1.0 μ L

10×T?Buffer?????????????????????????3.75μL

BSA??????????????????????????????????0.25μL

Sterilized water 6.0 μ L.

7. the construction process of nifA gene-introduced rhizobium japonicum genetic engineering bacterial strain HD-SFH-01 according to claim 1 is characterized in that the system of double digestion is as follows in the step 4:

The composition consumption

PET-28a plasmid vector 15.0 μ L

Restriction endonuclease NcoI1.0 μ L; SacI 1.0 μ L

10×T?buffer??????????????????????3.75μL

BSA???????????????????????????????0.25μL

Sterilized water 4.0 μ L.

8. the construction process of nifA gene-introduced rhizobium japonicum genetic engineering bacterial strain HID-SFH-01 according to claim 1, the reaction system that it is characterized in that pcr amplification in the step 4 is 25 μ L reaction systems, is made up of following ingredients:

The composition consumption

400ng/ μ L recombinant vectors pUC19-nifA 1.0 μ L

0.2μM?LBnifA1??????????????????????????1.0μL

5’-CGC CCATGGATTACCGCCTTTGAGT-3’

0.2μM?LBnifA2??????????????????????????1.0μL

5’-CGC GCGGCCGCTGCAAGGCGATTAA-3’

10×Ex?Taq?Buffer???????????????????????2.5μL

0.4mMdNTP?Mixture???????????????????????1.5μL

Taq archaeal dna polymerase 0.5 μ L

Sterilized water 17.5 μ L

The pcr amplification condition is: 94 ℃ of sex change 10min, and 94 ℃ of sex change 1min, 65 ℃ of annealing 30s, 72 ℃ are extended 1min, totally 35 circulations, 72 ℃ are extended 7min, 4 ℃ of insulations again.

9. the construction process of nifA gene-introduced rhizobium japonicum genetic engineering bacterial strain HD-SFH-01 according to claim 1 is characterized in that the system that connects in the step 4 is as follows:

The composition consumption

10 * T ₄Dna ligase damping fluid 2.5 μ L

P after enzyme is cut _Lac-nifA fragment 10.0 μ L

PET-28a plasmid vector 2.0 μ L after enzyme is cut

T ₄Dna ligase 1.0 μ L

Sterilized water 9.5 μ L.

10. the construction process of nifA gene-introduced rhizobium japonicum genetic engineering bacterial strain HD-SFH-01 according to claim 1, the reaction system that it is characterized in that pcr amplification in the step 5 is 25 μ L reaction systems, is made up of following ingredients:

The composition consumption

400ng/ μ L recombinant vectors pET-28a-P _Lac-nifA 1.0 μ L

0.2μM?LBTnifA???????????????????????????11.0μL

5’-CGC GGTACCATTGTGAGCGGATAAC-3’

0.2μM?LBTnifA2????????????????????1.0μL

5’-GCG GGTACCTAGTTCCTCCTTTCAG-3’

10×Ex?Taq?Buffer??????????????????2.5μL

0.4mMdNTP?Mixture??????????????????1.5μL

Taq DNA polysaccharase 0.5 μ L

Sterilized water 17.5 μ L

The pcr amplification condition is: 94 ℃ of sex change 10min, and 94 ℃ of sex change 1min, 65 ℃ of annealing 30s, 72 ℃ are extended 1min, totally 35 circulations, 72 ℃ are extended 7min, 4 ℃ of insulations again.

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