CN103146745B - Plant RNA (Ribose Nucleic Acid) interference vector for inhibiting lignin from synthesizing, and construction method and application of plant RNA interference vector - Google Patents

Plant RNA (Ribose Nucleic Acid) interference vector for inhibiting lignin from synthesizing, and construction method and application of plant RNA interference vector Download PDF

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CN103146745B
CN103146745B CN201310079414.3A CN201310079414A CN103146745B CN 103146745 B CN103146745 B CN 103146745B CN 201310079414 A CN201310079414 A CN 201310079414A CN 103146745 B CN103146745 B CN 103146745B
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plant
acid fragment
lignin
interference vector
alfalfa
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CN103146745A (en
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韩阳
王红艳
段亚楠
卢福荣
茹艺
李曹娜
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Liaoning University
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Abstract

The invention relates to a plant RNA (Ribose Nucleic Acid) interference vector for inhibiting lignin from synthesizing, and a construction method and application of the plant RNA interference vector. The plant RNA interference vector is that some nucleotide sequences (serving as forward nucleic acid fragments) of alfalfa CCoAOMT as shown in a sequence table SEQ ID No.1 and some nucleotide sequences (serving as reverse nucleic acid fragments) of the alfalfa CCoAOMT as shown in a sequence table SEQ ID No.2 are inserted into a plant expression vector pFGC5941, thus constructing the plant RNA interference vector pFGC5941-CCoAOMTi. The obtained interference vector is used for transforming agrobacterium tumefaciens EHA105 competent cells, so as to obtain agrobacterium engineering bacteria containing the interference vector. The agrobacterium engineering bacteria containing the interference vector is utilized for transforming the plant, and therefore, the plant with low lignin content can be cultivated. By adopting the method, a transgenic plant with normal growth and low lignin content can be gained, and germplasm resources are provided for cultivating low-lignin grass and papermaking plant and the like.

Description

A kind of suppress lignin synthesis plant RNA interference carrier and construction process and application
Technical field
The invention belongs to biological technical field, be specifically related to a kind ofly suppress the structure of the plant interference carrier of lignin synthesis and cultivating the application in low content of lignin plant.
Background technology
Xylogen is the macromole phenol polymer that in plant materials, a class is important, and its enormous amount, is only second to Mierocrystalline cellulose at the content of occurring in nature, occupies the second of organic polymer, usually accounts for about 10% of plant materials dry weight, even can reach 30% trees.Xylogen plays a part and important in the growing of terrestrial plant.But the deposition that in herbage, xylogen is excessive but affects the absorption of livestock to herbage Middle nutrition composition; Xylogen in industrial plant also increases the cost utilizing vegetable fibre.Therefore, utilize in industry at husbandry sector and vegetable fibre and wish to obtain the low new plant strain of content of lignin always, thus improve the nutritive property of herbage or reduce industrial production cost.
Summary of the invention
The object of this invention is to provide a kind of plant RNA interference carrier suppressing lignin synthesis, this interference carrier contains part alfalfa cCoAOMT(caffeoyl coenzyme A-O-methyltransgerase) gene order.CCoAOMT is one of lignin synthesis key enzyme, and the gene of coding CCoAOMT is cCoAOMTgene.
Another object of the present invention is to provide a kind of preparation method suppressing the plant RNA interference carrier of lignin synthesis.
Another object of the present invention is to provide a kind of plant RNA interference carrier of lignin synthesis that suppresses and applies in the low content of lignin plant of cultivation, provides good material for cultivating low content of lignin herbage etc.
Another object of the present invention is to provide a kind of method that interference carrier by structure proceeds to vegetable cell, utilizes the method, this interference carrier is proceeded to vegetable cell, obtains the normal low content of lignin plant of growth through cultivating.
To achieve these goals, the present invention adopts following technical measures: a kind of plant RNA interference carrier suppressing lignin synthesis, and described plant RNA interference carrier is: inserted in plant expression vector pFGC5941 as the nucleotide sequence shown in forward kernel acid fragment and sequence table SEQ ID NO.2 as inverse kernel acid fragment by the nucleotide sequence shown in sequence table SEQ ID NO.1.Nucleotide sequence shown in sequence table is the alfalfa inserting restriction enzyme site cCoAOMTfragment .
The construction process of the plant RNA interference carrier of above-mentioned suppression lignin synthesis, comprises the following steps:
1) extraction of alfalfa total serum IgE
2) reverse transcription obtains alfalfa cDNA;
3) with corresponding restriction enzyme site, respectively as the alfalfa of forward kernel acid fragment and inverse kernel acid fragment cCoAOMTthe pcr amplification of nucleotide sequence;
4) the forward kernel acid fragment of pcr amplification and inverse kernel acid fragment reclaim through cutting glue, are connected respectively on T carrier;
5) double digestion is connected to forward kernel acid fragment in carrier T and plant expression vector pFGC5941, and electrophoresis reclaims object fragment, and T4 ligase enzyme connects, and obtains intermediate carrier pFGC5941-CCoAOMT1;
6) double digestion is connected to inverse kernel acid fragment in carrier T and pFGC5941-CCoAOMT1, and electrophoresis reclaims object fragment, and T4 ligase enzyme connects, and obtains plant RNA interference carrier pFGC5941-CCoAOMTi.
By plant RNA interference carrier---pFGC5941-CCoAOMTi plasmid proceeds in agrobacterium tumefaciens, obtains restructuring agrobacterium tumefaciens.
Above-mentioned plant RNA interference carrier is cultivating the application in low content of lignin plant, and its method is as follows:
1) transductive process adopts leaf disk method
Get plant (as tobacco or other plant) healthy and strong blade, be cut into 0.5 ㎝ 2the leaf dish of left and right, in division culture medium after preculture 2d, puts into the above-mentioned restructuring agrobacterium tumefaciens containing plant RNA interference carrier pFGC5941-CCoAOMTi and soaks 10min, be inoculated in division culture medium, light culture 2d after taking-up.Proceed to screening division culture medium, carry out the induction of kalamycin resistance screening and indefinite bud.The indefinite bud of induction is cut from base portion, goes in root media and take root.When root system development is complete, during root length 1.5 ~ 2.0cm, transplant regrowth, obtain transfer-gen plant.
2) PCR of transfer-gen plant detects
Extracting transgenic line genomic dna, take genomic dna as template, and 35S promoter adds the forward gene fragment shown in sequence table SEQ ID NO.1 for detecting target, carries out PCR reaction; PCR primer order-checking, comparison.By the method screening positive transgenic plant.
Upstream primer: 5 '-GCACGACACTCTGGTCTACTC-3 '
Downstream primer: 5 '-TTATTTAATGGGCTAAAAATGGTT-3 '.
3) acquisition of normal low content of lignin plant is grown
The mensuration of transfer-gen plant content of lignin: the content being measured sample xylogen by the Klason method of classics.The material that screening content of lignin reduces by more than 10% compared with wild-type continues to cultivate.
The mensuration of transfer-gen plant physiological and biochemical index: the transfer-gen plant that screening content of lignin reduces by more than 10% compared with wild-type continues to cultivate, with outward appearance, plant height, Weight per plant for index, relatively transgenosis and the difference of wild-type material in growth potential is the final normal low content of lignin plant of growth obtained with growth potential and wild-type without the transfer-gen plant of significant difference.
The invention has the beneficial effects as follows: the present invention adopts and derives from alfalfa cCoAOMTthe partial gene sequence of (caffeoyl coenzyme A-O-methyltransgerase), construct a kind of plant RNA interference carrier suppressing lignin synthesis, adopt agrobacterium-mediated transformation that this interference carrier is proceeded to vegetable cell, the normal low content of lignin plant of growth is obtained, for the herbage, papermaking material etc. cultivating low content of lignin provides germ plasm resource through cultivating.
Accompanying drawing explanation
Fig. 1 is double digestion checking interference carrier forward kernel acid fragment;
M1:Marker (2000bp); M2:Marker (15000bp); 1 ~ 6; Recombinant vectors digestion products.
Fig. 2 is double digestion checking interference carrier inverse kernel acid fragment;
M1:Marker (15000bp); M2:Marker (2000bp); 1 ~ 3; Recombinant vectors digestion products.
Fig. 3 is that the PCR of transformed plant (tobacco) detects (2000bp Marker);
M:Marker (2000bp); 1 ~ 5; Having target fragment, is positive plant; 6: driftlessness fragment, be WT lines; 7: recombinant plasmid: 8: water.
Embodiment
embodiment 1: alfalfa cCoAOMTthe clone of gene fragment
1. the extraction of alfalfa total serum IgE
(1) get the tender alfalfa of children and organize 100mg, add liquid nitrogen grinding; In mortar, add 1mL RNAiso Plus, room temperature leaves standstill melts completely to sample, continues to be ground to the transparent shape of lysate;
(2) homogenate is transferred in centrifuge tube; 12000 r/min, 4 DEG C, centrifugal 5min; Getting supernatant moves in new centrifuge tube;
(3) in supernatant liquor, add chloroform (1/5 volume of RNAiso Plus), concussion 15s, after solution is fully emulsified, then room temperature leaves standstill 5min;
(4) 12000 r/min, 4 DEG C, centrifugal 15min; Aspirate supernatant is transferred in another new centrifuge tube;
(5) in supernatant, add isopyknic Virahol, fully after mixing, room temperature leaves standstill 10min;
(6) 12000 r/min, 4 DEG C, centrifugal 10min; Abandon supernatant, slowly add the ethanol 1mL of 75%; 12000 r/min, abandon ethanol after centrifugal 5min by 4 DEG C;
(7) drying at room temperature precipitation 2 ~ 5min, adds appropriate RNase-free water dissolution precipitation, in-80 DEG C of preservations after RNA precipitation is dissolved completely, obtains RNA template.
2. obtain alfalfa cDNA through reverse transcription
(1) synthesis of alfalfa cDNA first chain.By obtain RNA template (3 μ l), Primer Mix(2 μ l), dNTP Mix(4 μ l) and RNase-Free Water(4 μ l) configure reaction system, hatch 10min for 70 DEG C, rapid ice bath 2min.Of short duration centrifugal.Continue in above reaction solution, add 5 × RT Buff(4 μ l), 0.1M DTT(2 μ l), hatch 2min for 42 DEG C; Add 1 μ l HiFi-MMLV(200U/ μ l), hatch 50min for 42 DEG C.Hatch 15min for 70 DEG C.After reaction terminates, of short duration centrifugal, be placed in cooled on ice.
(2) alfalfa cDNA synthesis.Above-mentioned reaction product, as template, carries out PCR reaction, obtains alfalfa cDNA sequence.
3. with the alfalfa of corresponding restriction enzyme site cCoAOMTthe amplification of Gene Partial fragment
With alfalfa cDNA for template, amplification cCoAOMTgene fragment.
Amplification forward kernel acid fragment, upstream primer 5'-GGCGCGCCACGGAGAAGGAAAGCAAA-3,
Downstream primer 5'-ATTTAAATGCGGAGGGGCGCGTCGGG-3'.
Amplification inverse kernel acid fragment, upstream primer 5'-TCCCCCGGGACGGAGAAGGAAAGCAAA-3',
Downstream primer 5'-GCTCTAGAGCGGAGGGGCGCGTCGGG-3'.
Pcr amplification, the forward kernel acid fragment obtained as shown in sequence table SEQ ID NO.1 through checking order and the inverse kernel acid fragment as shown in sequence table SEQ ID NO.2.
embodiment 2: the preparation suppressing the plant RNA interference carrier of lignin synthesis
The forward kernel acid fragment with corresponding restriction enzyme site embodiment 1 obtained and inverse kernel acid fragment are connected respectively on pMD19-T carrier; With asci and swai double digestion is connected to forward kernel acid fragment on pMD19-T carrier and plant expression vector pFGC5941, reclaims object fragment, and T4 ligase enzyme connects, and obtains intermediate carrier pFGC5941-CCoAOMT1; With xbai and smai double digestion contains pMD19-T carrier and the intermediate carrier pFGC5941-CCoAOMT1 of inverse kernel acid fragment, reclaims object fragment, and T4 ligase enzyme connects, and is built into the plant RNA interference carrier pFGC5941-CCoAOMTi suppressing lignin synthesis.
Double digestion carrier pFGC5941-CCoAOMTi identifies, result as depicted in figs. 1 and 2, obtains 2 fragments, and size is consistent with design.
With interference carrier pFGC5941-CCoAOMTi for template, 35S promoter adds forward gene fragment for detecting target, carries out PCR reaction; PCR primer serves Hai Shenggong order-checking, and order-checking is correct.
embodiment 3: plant RNA interference carrier transform Agrobacterium tumefaciens.
1. the preparation of Agrobacterium competent cell
The single colony inoculation of picking agrobacterium tumefaciens is in 3ml LB liquid nutrient medium; 220 r/min, 28 DEG C, shaking culture is to OD 600=0.5; Draw 1.5ml bacterium liquid in centrifuge tube, ice bath 10min; 5000 r/min, centrifugal 30s, abandoning supernatant, precipitation suspends with 1.5 ml 0.5M NaCl, ice bath 20min; 5000 r/min, centrifugal 30s, abandoning supernatant; Often effective 100 μ l 20mM CaCl 2suspend, for transforming.
2. the direct transformation Agrobacterium of plasmid
Plasmid pFGC5941-CCoAOMTi 10ul is added, ice bath 30 min in 50 μ l Agrobacterium competent cells; Put into liquid nitrogen 1min, then put into 37 DEG C of water-bath water-bath 5min immediately; Take out centrifuge tube, add 0.5mlLB, 28 DEG C, 220 r/min shaking culture 4h; Take out bacterium liquid in containing coated plate on the LB flat board of kantlex, be inverted under 28 DEG C of conditions in incubator and cultivate.Within about 2 days, bacterium colony is visible, obtains agrobacterium tumefaciens of recombinating.
3. agrobacterium tumefaciens of recombinating is identified
Extract plasmid DNA in a small amount, PCR identifies, containing plasmid pFGC5941-CCoAOMTi in restructuring agrobacterium tumefaciens.
embodiment 4: plant RNA interference carrier transformed tobacco plant
1. transduce (employing leaf disk method)
Get the healthy and strong blade of tobacco, be cut into 0.5 ㎝ 2the leaf dish of left and right, the leaf outside of belly is placed in downwards on division culture medium, temperature 26 DEG C ± 1 DEG C, illumination 16h/d, cultivates 2d.Blade is put into and is cultured to OD 600(0.5-0.6) soak 10min in restructuring agrobacterium tumefaciens bacterium liquid, suck unnecessary bacterium liquid with aseptic filter paper, the leaf outside of belly is inoculated into downwards in division culture medium, temperature 26 DEG C ± 1 DEG C, light culture 2d.Proceed to the screening division culture medium containing kantlex, carry out the induction of resistance screening and indefinite bud, culture temperature 26 DEG C ± 1 DEG C, illumination 16h/d.Cultivate about 7d, callus occurs blade; Continue to cultivate about 10d, callus differentiates indefinite bud.When indefinite bud grows to 1.5cm height, it is cut from base portion, separate with leaf dish, go to root media, culture temperature 26 DEG C ± 1 DEG C, illumination 16h/d.Cultivate about 10d, adventive root occurs, and when root system development is complete, during the long 1.5 ~ 2.0cm of root, is cultivated by Transplantation of Regenerated Plantlets, Routine Management to the basin that Nutrition Soil is housed.Transgenic tobacco plant is obtained through aforesaid operations.
2. the PCR of transgenic tobacco plant detects
Extract test kit operation by plant genome DNA, extracting transgene tobacco genomic dna, take genomic dna as template, and 35S promoter adds forward gene fragment for clone's target, carries out PCR reaction; PCR primer order-checking, comparison.By the method screening positive transgenic plant.
Upstream primer: 5 '-GCACGACACTCTGGTCTACTC-3 '
Downstream primer: 5 '-TTATTTAATGGGCTAAAAATGGTT-3 '
Positive transgenic plant is screened by PCR.
As shown in Figure 3, after testing, target fragment is contained in regeneration plant.Collect target fragment, deliver the raw work order-checking in Shanghai, sequencing result conforms to original design, is judged as positive plant.
embodiment 5: the acquisition growing normal low content of lignin plant.
1. the mensuration of transgenic tobacco plant content of lignin
Be taken at the transgenosis of cultivating in Nutrition Soil and wild-type tobacco blade is dried to constant weight, get 100mg as sample, measured the content of sample xylogen by the Klason method of classics.After testing, in transgenic tobacco plant content of lignin comparatively wild-type on average reduce by more than 10%.
Klason method: after sample liquid nitrogen grinding, accurately takes sample 100 mg after drying to constant weight, and adds 12.5ml 72 % H 2sO 4, 30 DEG C of digestion 1h, are diluted to 4 % by postdigestive mixed solution sterilized water, and 120 DEG C of the 2nd digestion 1h, by hot solution by drying the sand core funnel (W to constant weight 1) filter, and with hot water injection 3 times, residue puts into baking oven together with funnel, 60 DEG C of (W that dry to constant weight 2), and be calculated as follows content of lignin value.Each sample content of lignin replication 3 times, averages.
Content of lignin (mg/100mg)=W 2– W 1.
2. the mensuration of transfer-gen plant physiological and biochemical index
With outward appearance, plant height, Weight per plant for index, comparing transgenosis and the difference of wild-type material in growth potential, is the normal low content of lignin plant of growth finally obtained with growth potential and wild-type without the transgenic line of significant difference.
<110> Liaoning University
<120> mono-kind suppresses the plant RNA interference carrier of lignin synthesis and construction process thereof and application
<160> 2
 
<210> 1
<211> 684
<212> DNA
<213> alfalfa cCoAOMTnucleic acid fragment (as forward kernel acid fragment)
<400> 1
GCTATTAGTAGGGCATTCGCGCATCCTGCGAAGAATCTTGGGTATACTTCTCCATATTATTTCTTTATAAACAATGATTCCCCCCGGACTCTCTTTCTGTCCGGAGTCTTAAAGAATGAATTCTATTGAGTTGTGGTTGTTGACCAACAAATTATGCATGGGAGAAAGATTCACAAAGGAAAACTAACCTTGATCCAGAAAGAAAGGGGCCCCTTCATCGCATCGGGTTGGGTTGGCCCATCAGAATTTGTGCATTGCATCCTACCTAATTCGTTAGCGCTTTTTCTTTTAAGAATGCATCTGGTTCTATCTTTCTTTCGCCAGTTAGTCCACCTTTTAGTGATTCTAGTAATTCTGGTTTGATAGTGCTTAGAATGTCTCTTTCATATTGAGCAATTTTGTCTAATGGCATTCGATCACAGAATCCATTGACAGCTGCATAAATTACTAGAATTTGTTTTTCAATTGGAAGTGGTGCATATTGTGGTTGTTTTAGTACTTCTGTAAGCCTTGCACCTCTATTGAGTAATGCCTGAGTCGCAGCATCAAGGTCTGAGCCAAATTGAGCAAAGGCGGCCACTTCGCGATATTGTGCCAATTCAAGTTTTAAACTACCGCAGACTTGTTTCATAGCTTTCAACTGAGCGGCAGACCCGACGCGCCTCTCGCATTTTTTATATATAA
 
<210> 2
<211> 686
<212> DNA
<213> alfalfa cCoAOMTnucleic acid fragment (as inverse kernel acid fragment)
<400> 2
CCTTGCCGGCTAGCTGAGCTATGAACAGTCTGCGGTAGTTTAAAACTTGAATTGGCACAATATCGCGAAGTGGCCGCCTTTGCTCAATTTGGCTCAGACCTTGATGCTGCGACTCAGGCATTACTCAATAGAGGTGCAAGGCTTACAGAAGTACTAAAACAACCACAATATGCACCACTTCCAATTGAAAAACAAATTCTAGTAATTTATGCAGCTGTCAATGGATTCTGTGATCGAATGCCATTAGACAAAATTGCTCAATATGAAAGAGACATTCTAAGCACTATCAA ACCAGAATTACTAGAATCACTAAAAGGTGGACTAACTGGCGAAAGAAAGATAGAACCAGATGCATTCTTAAAAGAAAAAGCGCTAACGAATTAGGTAGGATGCAATGCACAAATTCTGATGGGCCAACCCAACCCGATGCGATGAAGGGGCCCCTTTCTTTCTGGATCAAGGTTAGTTTTCCTTTGTGAATCTTTCTCCCATGCATAATTTGTTGGTCAACAACCACAACTCAATAGAATTCATTCTTTAAGACTCCGGACAGAAAGAGAGTCCGGGGGGAATCATTGTTTATAAAGAAATAATATGGAGAAGTATACCCAAGATTTTCTTCGCAGGATGCGCGAATTTGCCCTACTAATTATCCTTTGCTTTCCTTCTCCGCCCCGGGGGGAAGG
 

Claims (2)

1. suppress a construction process for the plant RNA interference carrier of lignin synthesis, it is characterized in that comprising the following steps:
1) extraction of alfalfa total serum IgE;
2) reverse transcription obtains alfalfa cDNA;
3) with corresponding restriction enzyme site, respectively as the alfalfa of forward kernel acid fragment and inverse kernel acid fragment cCoAOMTthe pcr amplification of nucleotide sequence; The alfalfa of described forward kernel acid fragment cCoAOMTnucleotide sequence as shown in SEQ ID NO.1, the alfalfa of described inverse kernel acid fragment cCoAOMTnucleotide sequence is as shown in SEQ ID NO.2;
4) the forward kernel acid fragment of pcr amplification and inverse kernel acid fragment reclaim through cutting glue, are connected respectively on T carrier;
5) double digestion is connected to forward kernel acid fragment in carrier T and plant expression vector pFGC5941, and electrophoresis reclaims object fragment, and T4 ligase enzyme connects, and obtains intermediate carrier pFGC5941-CCoAOMT1;
6) double digestion is connected to inverse kernel acid fragment in carrier T and pFGC5941-CCoAOMT1, and electrophoresis reclaims object fragment, and T4 ligase enzyme connects, and obtains plant RNA interference carrier pFGC5941-CCoAOMTi.
2. the plant RNA interference carrier built in accordance with the method for claim 1 is cultivating the application in low content of lignin plant, it is characterized in that method is as follows:
1) the restructuring Agrobacterium tumefaciens transformation plant leaf of leaf disk method containing plant RNA interference carrier pFGC5941-CCoAOMTi is adopted; Described plant is tobacco;
2) vanes is cultivated and is obtained regeneration plant;
3) Transplantation of Regenerated Plantlets is obtained transfer-gen plant to the basin that Nutrition Soil is housed, in transfer-gen plant, content of lignin reduces by more than 10%.
CN201310079414.3A 2013-03-13 2013-03-13 Plant RNA (Ribose Nucleic Acid) interference vector for inhibiting lignin from synthesizing, and construction method and application of plant RNA interference vector Active CN103146745B (en)

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CN103642827B (en) * 2013-10-17 2017-05-17 吉林省农业科学院 RNA interference vector suitable for herbicide selection marker Bar gene
CN105532251B (en) * 2016-01-26 2018-10-23 西北农林科技大学 A kind of cultural method improving clover digestibility
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