CN103014029A - Nicotiana tabacum isoflavone reductase-like (NtIRL) gene and application thereof - Google Patents

Abstract

The invention discloses a new member NtIRL2 of a Nicotiana tabacum isoflavone reductase-like (NtIRL) gene family. The sequence of an open reading frame of the NtIRL2 is shown as 79<th> nucleotide to 1011<th> nucleotide in SEQ ID NO.10. The invention performs systemic bioinformatic analysis on the members NtIRL1 and NtIRL2 of the NtIRL gene family, sets forth the expression features of the NtIRL1 and NtIRL2 genes in each tissue and organ of Nicotiana tabacum, constructs a plus sense expression vector and an antisense expression vector, and verifies that the NtIRL1 and NtIRL2 genes are coded with activated nicotine synthesis correlated enzymes through genetic transformation. The NtIRL1 and NtIRL2 genes play vital roles in the biosynthesis and metabolic pathway of tobacco nicotine, participate in the fungal disease defense of tobacco plants, can be used for regulating the nicotine content of Nicotiana tabacum and improving the disease resistance, and is beneficial to the gene engineering breeding of high-quality tobacco having low nicotine content and high fungal disease resistance.

Description

Common tobacco isoflavones reductase gene and application thereof

Technical field

The invention belongs to gene engineering technology field, relate to a kind of tobacco gene, also relate to the application of this gene.

Background technology

Tobacco is Dicotyledoneae Tubiflorae Solanaceae Nicotiana plant.Nicotiana (Nicotiana) has more than 60 to plant, and common tobacco in 2 cultivars (claiming again safflower tobacco, Nicotiana tabacum) occupies main area, and the area of Folium Nicotianae rusticae (Nicotiana rustica) is very little.The cultivation tobacco can be divided into flue-cured tobacco, suncured tabacco, air-curing of tobacco leaves, burley tobaccos, Turkish tobaccos and six types of rustica by quality of tobacco characteristics, biological character and cultivation modulator approach etc., and wherein flue-cured tobacco is to cultivate the widest common tobacco.China's tobacco planting area and ultimate production all rank first in the world.Use cash crop as a kind of leaf, the cultivation technique of flue-cured tobacco is different from other field crop, and not only requiring has certain yield of tobacco, and more pays attention to quality of tobacco.Quality of tobacco determines the operability of tobacco leaf, directly affects color and the commodity value of cigarette commodity, also is related to tobacco grower's economic benefit, is life and the starting point of tobacco industry.Stand on the invincible position for making in the competition of the domestic and international market in future, satisfy domestic and international cigarette enterprise to the ever-increasing demand of sound tobacco, must improve quality of tobacco and security.

(claim again Nicotine, nicotine) be one of main factor that affects quality of tobacco and security to nicotine.The fragrance that an amount of nicotine will be become reconciled with suitable physiological strength to the smoker and jealous causes the excitement of nervus centralis appropriateness and lax, make the people in high spirits, happy, be emotionally stable; Excessive nicotine then pungency is excessively strong, and health is harmful to health.In addition, nicotine content also will keep eurythmy with other chemical composition content in the tobacco, could produce good overall quality, and for example the ratio of water-soluble sugar and nicotine is commonly used to estimate strength and the level of comfort of tobacco.Therefore, suitable nicotine content is the target that the low evil of high-quality leaf tobacco production is pursued, and also is the urgent task of pendulum in face of the tobacco scientific worker.

The low evil of the high-quality of generally acknowledging at present cured tobacco leaf chemical ingredients is: total reducing sugar 18%-22%, reducing sugar 16%-18%, reducing sugar/total reducing sugar ratio 〉=0.9, total nitrogen 1.5%-3.5%, protein 7%-9%, nicotine 1.5%-3.5%, reducing sugar/nicotine is than 8-12, and the nitrogen base ratio is 1 or is slightly less than 1, potassium 〉=2%, chlorine≤1%, potassium/chlorine ratio＞4.The visual appearance of China's cured tobacco leaf at present oneself through substantially reaching or near international sound tobacco standard, but the Harmony of tobacco leaf chemical composition is inadequate, especially middle and upper part tobacco leaf, outstanding behaviours is that nicotine content is higher, and the leading indicator (reducing sugar/nicotine ratio, nitrogen base ratio) of weighing tobacco leaf chemical composition Harmony does not meet the standard of sound tobacco.Therefore, reduce the nicotine content of middle and upper part tobacco leaf, improve the Harmony of tobacco leaf chemical composition, to increase the industrial applicability of tobacco leaf, the security that improves tobacco leaf is the emphasis of cigarette quality improvement.

Fall under the limited condition of alkali effect in the routine techniques measure, the gene that will encode the synthetic rate-limiting enzyme of nicotine or have an enzyme of mediation function with engineered means imports in the tobacco, cultivates low nicotine High Quality Tobacco kind, is a very promising new way.Know that at present putrescine-N-Methyl transferase (PMT) and quinolate phosphoribosyl transferase (QPT) are the maximum rate-limiting enzymes in 2 intermediate biosynthesizing of nicotine, other substrates or enzyme have regulating effect in some cases, but its regulatory mechanism shortage is understood in depth.The synthetic of nicotine is combined with pyrrole ring and finished by nicotinic acid, and this process not yet can be separated with qualitative this enzyme at present by the catalysis of nicotine synthetic enzyme institute, therefore about nicotine finally synthetic mechanism and regulation mechanism it be unclear that.In addition, tobacco is many at present to be used for genetically engineered research as mode crop rather than purpose plant, although the engineered achievement in research of resistance plant can directly apply to the resistance breeding of tobacco, breeding has little significance but the engineered achievement in research of quality-improving is mostly to cigarette quality, this be because, on the one hand the molecular mechanism of tobacco self physiological metabolism lacked deep research, tobacco is different from conventional farm crop on the other hand, and its quality depends primarily on the comprehensive action of the materials such as nicotine, sugar, acid, alcohol, fat, pigment, inorganics in the tobacco.Therefore, be necessary that the clone and the transformation of tobacco that carry out the tobacco smoke alkaloid biosynthesis related genes carry out functional verification, the biosynthetic molecular mechanism of tobacco smoke alkaloid is carried out deep research, for the improvement of the genetically engineered of cigarette quality provides new strategy, advance the molecular breeding of low nicotine High Quality Tobacco.

In addition, through national tobacco infectious disease investigation, the tobacco fungal disease has been found 28 kinds in China, accounts for 47.5% of global known tobacco infectious disease, the financial loss that causes accounts for 37.8% of total losses due to the tobacco infectious disease, occupies first of all kinds of diseases.And the tobacco fungal disease has the trend of increasing the weight of in recent years, has a strong impact on the tobacco yield and quality and plants the cigarette income.On current cured tobacco production, the control of disease and pest still depends on chemical agent to a great extent, applications of pesticide amount is excessive, application times is on the high side, and be main (particularly sterilant) mainly with riskiest pesticide, the pesticide abuse phenomenon is serious, this has brought no small side effect to cured tobacco production, is mainly manifested in the following aspects: increase production cost; The variation of pathogenic bacteria generation physiological strain or insect are developed immunity to drugs, cause the large generation of disease; Destroy the eubiosis, kill natural enemy, reduce the Natural control action of natural enemy; Increase pesticide residue, reduce the tobacco leaf security.Therefore, excavate the function Analysis of Defence Genes Involved relevant with the Resistance In Tobacco fungal disease, carry out the Tobacco resistance improvement by the genetic engineering modification, strengthen tobacco to the protection capability of fungal disease, to reduce applications of pesticide amount, improve the security of tobacco leaf, have important theory and realistic meaning for the Sustainable Healthy Development of tobacco industry.

Summary of the invention

In view of this, the object of the invention is to common tobacco isoflavones reductase enzyme (NtIRL) gene family is cloned and transformation of tobacco carries out functional analysis, for the genetic engineering breeding of High Quality Tobacco provides new strategy and instrument.

For achieving the above object, the present invention adopts the RACE technology, take the total RNA of root of common tobacco high-nicotine kind Longli safflower cigarette as material, full-length cDNA and the genome sequence of NtIRL gene family have been cloned, to its bioinformatic analysis that has carried out system, disclosed the number of members of NtIRL gene family in the common tobacco, illustrated the expression characteristic of these members in each histoorgan of common tobacco, make up justice and antisense plant expression vector, and finished functional analysis by genetic transformation.

Result of study shows, common tobacco NtIRL gene family has NtIRL1 and NtIRL2 totally 2 members, the genome sequence of NtIRL1 and NtIRL2 is respectively 1946bp and 2089bp, full length cDNA sequence is respectively 1257bp and 1261bp, the open reading frame (ORF) that a 933bp is all arranged at the 79-1011bp place of cDNA, all protein of 310 amino acid compositions of coding.The consistence of the genome sequence of NtIRL1 and NtIRL2, mRNA sequence, coding region sequence is respectively 84.9%, 95.1%, 96.9%, and the coding region consistence is higher than non-coding region far away, has proved the conservative property of coding region; The consistence of coding protein sequence and similarity are respectively 95.5% and 97.4%, illustrate also to have high conservative on amino acid levels.NCBI Blastn homology the analysis showed that, the mRNA of NtIRL1 is corresponding to the NtA622mRNA that has reported (Genbank accession number D28505), genome sequence is corresponding to the NsA622 gene of having reported (Genbank accession number AB071165), and NtIRL2 then is new clone of the present invention.They with have significant homology from isoflavones reductase enzyme (IFR) genes of the plants such as soybean, with phenyl coumarane benzylic ether reductase enzyme (PCBER) gene of other plant certain homology is arranged, also have the homology of core section with the reductase enzyme of other type.Although the homology of NtIRL1 and NtIRL2 is very high, obvious differentiation has occured at the space-time characterisation of expressing in the two.NtIRL1 expression amount in root is the highest, and suitable expression is arranged in the stem, and weak expression is arranged in the flower bud, does not express fully in 20 days the organs such as seed at leaf, flower, capsule and after blooming; NtIRL2 has stronger tissue specificity, expresses the byest force in root, and extremely weak expression is arranged in stem, does not all express in 20 days the organs such as seed at flower bud, leaf, flower, capsule and after blooming; The transcriptional level of NtIRL2 in root is apparently higher than NtIRL1.

The present invention adopts SacI/BamHI double digestion directed cloning mode, respectively NtIRL1 and NtIRL2 genome sequence forward are inserted among the plant expression vector pCambia2301G, by the CaMV35S promoters driven, 2 just plant expression vector pNtIRL1 and pNtIRL2 have been made up; Common conservative fragments NtIRLA in NtIRL1 and the NtIRL2 full-length cDNA oppositely is inserted among the plant expression vector pCambia2301G, by the CaMV35S promoters driven, made up the antisense plant expression vector pNtIRLA of 1 NtIRL gene family member co-suppression.Above-mentioned justice and antisense plant expression vector are transformed respectively Agrobacterium, obtained to carry the Agrobacterium engineering strain of corresponding plant expression vector, further utilize agriculture bacillus mediated leaf dish method to transform common tobacco.The result shows, obvious external form variation does not all appear in the transgene tobacco that the NtIRL gene is expressed in overexpression and inhibition, have normal plant forms and growth characteristics, tobacco leaf color is compared also without significant difference with transgenosis cigarette strain not, but, the nicotine content of tobacco leaves overwhelming majority of the transgenic line of overexpression NtIRL1 or NtIRL2 more not transgenosis cigarette strain has in various degree raising, suppress to express the NtIRL gene transgenic line nicotine content of tobacco leaves all more not the strain of transgenosis cigarette in various degree decline is arranged, and transgene tobacco blade nicotine content changing conditions becomes positive correlation with the gene expression abundance of NtIRL gene in root system, NtIRL1 and the NtIRL2 gene synthetic relevant enzyme of activated nicotine of can both encoding is described, important role in the biosynthetic metabolism approach of tobacco smoke alkaloid.In addition, turning the change of NtIRL gene plant by nicotine accumulation has also affected its disease-resistant performance, overexpression NtIRL1 or NtIRL2 gene, and nicotine content increases, and the anti-balck shank ability of cigarette strain strengthens; Suppress the expression of NtIRL gene, nicotine content reduces, and the anti-damping-off ability of cigarette strain weakens; Illustrate that the nicotine in the tobacco secondary metabolite also is the plant protecting chemical class material with defense function, can strengthen the ability of cigarette strain opposing fungal disease.

According to above-mentioned result of study, the present invention proposes following technical scheme:

1. the open reading frame sequence of common tobacco isoflavones reductase gene NtIRL2 is shown in 79-1011 position Nucleotide among the SEQ ID No.10.

2. the full length cDNA sequence of common tobacco isoflavones reductase gene NtIRL2 is shown in SEQ ID No.10.

3. the genome sequence of common tobacco isoflavones reductase gene NtIRL2 is shown in SEQ ID No.12.

4. the just plant expression vector that contains NtIRL2 gene open reading frame sequence or genome sequence.

Further, described just plant expression vector is between the BamHI and SacI multiple clone site with the NtIRL2 genome sequence forward insertion vector pCambia2301G shown in the SEQ ID No.12, replaces gus gene wherein and gets; Described carrier pCambia2301G downcuts the gus expression casette with HindIII and EcoRI double digestion from the pBI121 plasmid, is connected on the pCambia2301 of same double digestion carrier again and gets.

5. contain the altogether antisense plant expression vector of conservative fragments of NtIRL gene family member, described NtIRL gene family member is NtIRL1 and NtIRL2; The full length cDNA sequence of described NtIRL1 gene is shown in SEQ ID No.9; The full length cDNA sequence of described NtIRL2 gene is shown in SEQ ID No.10.

Further, described antisense plant expression vector is with NtIRL gene family member altogether between the BamHI and SacI multiple clone site of the reverse insertion vector pCambia2301G of conservative fragments NtIRLA, replaces gus gene wherein and gets; Described NtIRL gene family member is total to the nucleotide sequence of conservative fragments NtIRLA shown in 1-1179 position Nucleotide among the SEQ ID No.9; Described carrier pCambia2301G downcuts the gus expression casette with HindIII and EcoRI double digestion from the pBI121 plasmid, is connected on the pCambia2301 of same double digestion carrier again and gets.

6. the microbial transformant that contains above-mentioned just plant expression vector.

7. the microbial transformant that contains above-mentioned antisense plant expression vector.

8. the application in the molecular breeding of common tobacco isoflavones reductase gene NtIRL2 aspect common Nicotine Content in Tobacco Leaf and resistance to fungal disease.

Further, described fungal disease is balck shank and damping-off.

9. the application in the molecular breeding of common tobacco isoflavones reductase gene NtIRL1 aspect common Nicotine Content in Tobacco Leaf and resistance to fungal disease, the full length cDNA sequence of described NtIRL1 gene is shown in SEQ ID No.9, and genome sequence is shown in SEQ ID No.11.

Further, described fungal disease is balck shank and damping-off.

Beneficial effect of the present invention is: the present invention clones the NtIRL gene family, the clear and definite member of NtIRL gene family, found new gene NtIRL2, NtIRL gene family member NtIRL1 and NtIRL2 have been carried out the bioinformatic analysis of system, illustrated the expression characteristic of these members in each histoorgan of common tobacco, and structure justice and antisense plant expression vector, confirmed NtIRL gene family member NtIRL1 and the NtIRL2 synthetic relevant enzyme of activated nicotine of all encoding by the genetic transformation tobacco, important role in the biosynthetic metabolism approach of tobacco smoke alkaloid, and the resistance to fungal disease that participates in plant is defendd, thereby for genetic engineering modification and the molecular breeding of common Nicotine Content in Tobacco Leaf and resistance to fungal disease aspect provides important target gene, the molecular regulation that carries out the NtIRL gene by the transgenosis means helps tobacco smoke alkaloid to modify and the disease resistance improvement.

Description of drawings

In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is described in further detail below in conjunction with accompanying drawing, wherein:

Fig. 1 is the genomic dna of Longli safflower cigarette, and wherein 1 swimming lane is DNAMarker, and 2,3 swimming lanes are genomic dna.

Fig. 2 is the total RNA of each organ of Longli safflower cigarette, and wherein 1 swimming lane is DNA Marker, and the 2-8 swimming lane is followed successively by root, stem, leaf, flower, flower bud, capsule and blooms total RNA of rear 20 days seeds.

Fig. 3 is total cDNA of Longli safflower cigarette, and wherein 1 swimming lane is DNAMarker, the total cDNA of 2 swimming lanes for obtaining.

Fig. 4 is 3 ' cDNA RLM-RACE of NtIRL gene family, and wherein 1 swimming lane is DNA Marker, the nest that 2,3 swimming lanes the are respectively 3 ' cDNA end thing of expanding production.

Fig. 5 is that the terminal monoclonal PCR of NtIRL gene family 3 ' cDNA detects, and wherein the M swimming lane is DNAMarker, and the 1-23 swimming lane is respectively the terminal mono-clonal of 3 ' cDNA.

Fig. 6 is 5 ' cDNA RLM-RACE of NtIRL gene family, and wherein 1 swimming lane is DNA Marker, and 2,3 swimming lanes are respectively one of 5 ' cDNA end thing of expanding production.

Fig. 7 is that the terminal monoclonal PCR of NtIRL gene family 5 ' cDNA detects, and wherein 1 swimming lane is DNA Marker, and the 2-7 swimming lane is respectively the terminal mono-clonal of 5 ' cDNA.

Fig. 8 is the full-length cDNA amplification of NtIRL gene family, and wherein 1 swimming lane is DNAMarker, and 2,3 swimming lanes are respectively pcr amplification product.

Fig. 9 is the full-length gene group sequence amplification of NtIRL gene family, and wherein 1 swimming lane is DNA Marker, and 2,3 swimming lanes are respectively the genome sequence pcr amplification product.

Figure 10 is the nucleotide sequence of NtIRL1 gene and the aminoacid sequence of derivation thereof, wherein initiator codon ATG and terminator codon TAG underline mark with runic, intron gray background mark, alternative transcription initiation site and variable tailing site underline mark with oblique runic, the tailing signal wavy line mark of inferring, NADPH is in conjunction with conservative territory GGTGYIG frame mark, the general conservative amino acid residues K of Binding Capacity ₁₃₅Add the gray background mark with frame, the NmrA of prediction guards territory I9～T239 underscore mark.

Figure 11 is the nucleotide sequence of NtIRL2 gene and the aminoacid sequence of derivation thereof, wherein initiator codon ATG and terminator codon TAG underline mark with runic, intron gray background mark, alternative transcription initiation site and variable tailing site underline mark with oblique runic, the tailing signal wavy line mark of inferring, NADPH is in conjunction with conservative territory GGTGYIG frame mark, the general conservative amino acid residues K of Binding Capacity ₁₃₅Add the gray background mark with frame, the NmrA of prediction guards territory I9～T239 underscore mark.

Figure 12 is the sequence multiple ratio pair of NtIRL family protein and other plant PIP albumen, " GxxGxxG " of wherein being combined with NADPH is conservative in conjunction with territory square frame mark, the special insertion point of Activities of Some Plants IFR albumen and PLR albumen is with lower wavy line mark, infer in the consensus sequence that the amino-acid residue of being combined with cofactor adds the square frame mark with shade, deduction underlines mark with the amino-acid residue of Binding Capacity with runic.

Figure 13 is the phylogenetic tree of NtIRL family protein and other plant PIP albumen.

Figure 14 is the secondary structure of NtIRL1 and NtIRL2 albumen, wherein long, in long and short and the shortest vertical line represent respectively α spiral, extended chain, β-bend and curling at random.

Figure 15 is the tertiary structure of NtIRL1 and NtIRL2 albumen.

Figure 16 is the evaluation of NtIRL gene family number of members, and wherein scheming A is the probe mark result, and figure B is that enzyme is cut the result, and figure C is Southern blot results of hybridization.

Figure 17 is that the RT-PCR that NtIRL1 and NtIRL2 gene are expressed in each organ of common tobacco detects, and wherein Ro is root, and St is stem, and Le is leaf, and Fl is flower, and Bu is flower bud, and Ca is capsule, and 20D is the rear 20 days seed of blooming.

Figure 18 is the T-DNA part drawing of NtIRL1 gene family member justice plant expression vector pNtIRL1 and pNtIRL2.

Figure 19 is that the BamHI/SacI double digestion of NtIRL1 gene family member justice plant expression vector pNtIRL1 and pNtIRL2 is identified.

Figure 20 is the T-DNA part drawing of NtIRL1 gene family member antisense plant expression vector pNtIRLA.

Figure 21 is that the PCR of NtIRL1 gene family member antisense plant expression vector pNtIRLA identifies.

Figure 22 is that the SacI/BamHI double digestion of NtIRL1 gene family member antisense plant expression vector pNtIRLA is identified.

Figure 23 is the bacterium liquid PCR detection after NtIRL1 gene family member justice and antisense plant expression vector transform Agrobacterium.

Figure 24 is for adopting agriculture bacillus mediated leaf dish method that NtIRL gene family member is transformed common tobacco, and wherein A is the growth of resistant buds, and B is the resistant buds root culture, and C is the water planting hardening, and D is for transplanting transgenic seedling.

Figure 25 is that the GUS active mass chemical staining of transgenic tobacco plant detects, and wherein A is the anti-Kan individual plant of the large gold dollar overexpression of safflower NtIRL1 gene root system GUS dyeing, and CK is unconverted tobacco, and all the other are the large gold dollar transfer-gen plant of safflower; B is the anti-Kan individual plant of the large gold dollar overexpression of safflower NtIRL2 gene root system GUS dyeing, and CK is unconverted tobacco, and all the other are the large gold dollar transfer-gen plant of safflower; C is the anti-Kan individual plant of Da Fu cigarette Antisense Suppression root system GUS dyeing, and CK is unconverted tobacco, and all the other are Da Fu cigarette transfer-gen plant; D is the anti-Kan individual plant of Longli safflower cigarette Antisense Suppression root system GUS dyeing, and CK is unconverted tobacco, and all the other are Longli safflower cigarette transfer-gen plant.

Figure 26 is that the PCR of the anti-Kan seedling of the large gold dollar overexpression of safflower NtIRL1 gene detects, and wherein M is DNA standard molecular weight DL2000, CK ⁺Positive contrast (Agrobacterium engineering strain LBA4404-pNtIRL1 bacterium liquid), CK ^-The strain of negative contrast cigarette, 1～15 is the large gold dollar transfer-gen plant of safflower of random choose.

Figure 27 is that the PCR of the anti-Kan seedling of the large gold dollar overexpression of safflower NtIRL2 gene detects, and wherein M is DNA standard molecular weight DL2000, CK ⁺Positive contrast (Agrobacterium engineering strain LBA4404-pNtIRL2 bacterium liquid), CK ^-The strain of negative contrast cigarette, 1～18 is the large gold dollar transfer-gen plant of safflower of random choose.

Figure 28 is that the PCR of the anti-Kan seedling of Da Fu cigarette Antisense Suppression detects, and wherein M is DNA standard molecular weight DL2000, CK ⁺Positive contrast (Agrobacterium engineering strain LBA4404-pNtIRLA bacterium liquid), CK ^-The strain of negative contrast cigarette, 1～22 is the Da Fu cigarette transfer-gen plant of random choose.

Figure 29 is that the PCR of the anti-Kan seedling of Longli safflower cigarette Antisense Suppression detects, and wherein M is DNA standard molecular weight DL2000, CK ⁺Positive contrast (Agrobacterium engineering strain LBA4404-pNtIRLA bacterium liquid), CK ^-The strain of negative contrast cigarette, 1～22 is the Longli safflower cigarette transfer-gen plant of random choose.

Figure 30 is that the large gold dollar of safflower turns the RT-PCR analysis that NtIRL1 expresses in the NtIRL1 gene plant, wherein M is DNA standard molecular weight DL2000, the strain of the negative contrast cigarette of CK, 8 and the 22 transgenosis cigarette strains for GUS dyeing and PCR detection jack to jack adapter, 29 for GUS dyeing feminine gender but PCR detects positive transgenosis cigarette strain, and 2,3,7,11,18,50,51,59,61 and 62 detect the transgenosis cigarette strains of two positives for GUS dyeing and PCR.

Figure 31 is that the large gold dollar of safflower turns the RT-PCR analysis that NtIRL2 expresses in the NtIRL2 gene plant, wherein M is DNA standard molecular weight DL2000, the strain of the negative contrast cigarette of CK, 40 and the 76 transgenosis cigarette strains for GUS dyeing and PCR detection jack to jack adapter, 75 for GUS dyeing feminine gender but PCR detects positive transgenosis cigarette strain, and 28,34,42,44,49,51,60,62,70 and 79 detect the transgenosis cigarette strains of two positives for GUS dyeing and PCR.

Figure 32 is that the RT-PCR of NtIRL1 genetic expression in the Da Fu cigarette Antisense Suppression transfer-gen plant analyzes, wherein M is DNA standard molecular weight DL2000, the strain of the negative contrast cigarette of CK, 20 and the 37 transgenosis cigarette strains for GUS dyeing and PCR detection jack to jack adapter, 86 and 93 for GUS dyeing feminine gender but PCR detects positive transgenosis cigarette strain, and 1,8,13,40,70,74,78 and 80 detect the transgenosis cigarette strains of two positives for GUS dyeing and PCR.

Figure 33 is that the RT-PCR of NtIRL2 genetic expression in the Da Fu cigarette Antisense Suppression transfer-gen plant analyzes, wherein M is DNA standard molecular weight DL2000, the strain of the negative contrast cigarette of CK, 20 and the 37 transgenosis cigarette strains for GUS dyeing and PCR detection jack to jack adapter, 86 and 93 for GUS dyeing feminine gender but PCR detects positive transgenosis cigarette strain, and 1,8,13,40,70,74,78 and 80 detect the transgenosis cigarette strains of two positives for GUS dyeing and PCR.

Figure 34 is the Southern blot detection that the large gold dollar of safflower turns the NtIRL1 gene plant, the wherein negative contrast cigarette of CK strain, the 8 transgenosis cigarette strains for GUS dyeing and PCR detection jack to jack adapter, 2,3,11,12,18,24,47,56 and 62 is that GUS dyeing and PCR detect two positive transgenosis cigarette strains.

Figure 35 is that the Southern Blot of Da Fu cigarette Antisense Suppression transfer-gen plant detects, the wherein negative contrast cigarette of CK strain, the 37 transgenosis cigarette strains for GUS dyeing and PCR detection jack to jack adapter, 1,6,7,8,13,19,21,30 and 40 is that GUS dyeing and PCR detect two positive transgenosis cigarette strains.

Figure 36 is that the large gold dollar of safflower turns NtIRL1 gene plant nicotine content of tobacco leaves mutation analysis, the wherein negative contrast of CK, the 8 transgenosis cigarette strains for GUS dyeing and PCR detection jack to jack adapter; The transgenosis cigarette strains of two positives are detected in 2～62 (except 8) for GUS dyeing and PCR.

Figure 37 is that the large gold dollar of safflower turns NtIRL2 gene plant nicotine content of tobacco leaves mutation analysis, the wherein negative contrast of CK, the 40 transgenosis cigarette strains for GUS dyeing and PCR detection jack to jack adapter; The transgenosis cigarette strains of two positives are detected in 1～82 (except 40) for GUS dyeing and PCR.

Figure 38 is Da Fu cigarette Antisense Suppression transfer-gen plant nicotine content of tobacco leaves mutation analysis, the wherein negative contrast of CK, the 37 transgenosis cigarette strains for GUS dyeing and PCR detection jack to jack adapter; The transgenosis cigarette strains of two positives are detected in 1～95 (except 37) for GUS dyeing and PCR.

Figure 39 is Longli safflower cigarette Antisense Suppression transfer-gen plant nicotine content of tobacco leaves mutation analysis, the wherein negative contrast of CK, and 1～30 is GUS dyeing and the two positive transgenosis cigarette strains of PCR detection.

Figure 40 is the large gold dollar overexpression of safflower NtIRL1 gene plant (left side) and the not phenotype comparison of transfer-gen plant (right side).

Figure 41 is the large gold dollar overexpression of safflower NtIRL2 gene plant (left side) and the not phenotype comparison of transfer-gen plant (right side).

Figure 42 is Da Fu cigarette Antisense Suppression transfer-gen plant (left side) and the not phenotype comparison of transfer-gen plant (right side).

Figure 43 is Longli safflower cigarette Antisense Suppression transfer-gen plant (right side) and the not phenotype comparison of transfer-gen plant (left side).

Figure 44 is that the disease resistance of the large gold dollar overexpression of safflower NtIRL1 transfer-gen plant changes, and wherein 1～6 is the large gold dollar overexpression of safflower NtIRL1 gene plant, 7～11 negative adjoining trees.

Figure 45 is that the disease resistance of the large gold dollar overexpression of safflower NtIRL2 transfer-gen plant changes, and wherein 1～4 is the large gold dollar overexpression of safflower NtIRL2 gene plant, 5～7 negative adjoining trees.

Figure 46 is that the disease resistance of Da Fu cigarette Antisense Suppression NtIRL gene family member transfer-gen plant changes, the negative adjoining tree of CK1, CK2 and CK3 wherein, and 1～5 is Da Fu cigarette Antisense Suppression transfer-gen plant.

Embodiment

Hereinafter with reference to accompanying drawing, specific embodiments of the invention are described in detail.The experimental technique of unreceipted actual conditions among the embodiment, usually according to normal condition, the condition described in the molecular cloning experiment guide (third edition, J. Pehanorm Brooker etc. work) for example, or the condition of advising according to manufacturer.

The large gold dollar of common Performance Liquid Chromatography Analysis for Nicotine in Tobacco kind safflower that uses among the embodiment, common tobacco high-nicotine kind Da Fu cigarette and Longli safflower cigarette are provided the field general planting by the Guizhou Province Tabacco Science and Technology Institute.Plant tissue RNA extraction agent box, a small amount of glue recovery test kit are Shanghai China Shun biotechnology company limited product in a small amount.GeneRacer Kit is Invitrogen company product.LA Taq archaeal dna polymerase, pMD18-T carrier are precious biotechnology (Dalian) company limited product.PCR DIG Probe Synthesis Kit is Roche company product.RNA PCR Kit Ver.3.0 is TaKaRa company product.Examining order is entrusted Shanghai English to complete biotechnology Services Co., Ltd and is finished.Carrier pCambia2301G by Chai Yourong make up (Genes For Plant Tolerance verticillium dahliae acceptor proteinoid gene and MBL Molecular cloning and expression of gene. Agricultural University Of Southwest, 2003), to downcut its gus expression casette (3032bp) with HindIII and EcoRI double digestion from the pBI121 plasmid, be connected to after the recovery on the pCambia2301 of same double digestion carrier and get final product, it contains a NPTII gene expression frame and two expression of plants frames that CaMV35S starts, express gus gene for one, can realize the double-tagging screening of Kan and GUS activity, gus gene in another expression cassette can be used to replace to goal gene, realizes the expression of foreign gene.

One, NtIRL gene family clone and characterization of molecules

1, the extraction of genome DNA and total RNA

Get Longli safflower cigarette plant tender leaf, extract genome DNA with the CTAB method.Gained DNA carries out 1.0% agarose gel electrophoresis and detects (Fig. 1), and adopts metric measurement OD after diluting 200 times ₂₆₀, OD ₂₈₀, OD ₂₃₀Equivalent.The result shows that gained genome DNA integrity is good, and RNA digestion is more complete, and purity is higher, can be used for pcr amplification and Southern hybridization.

Get respectively root, stem, leaf, flower bud, flower, the capsule of Longli safflower cigarette plant and the rear 20 days seed of blooming, extract the total RNA of each organ with a small amount of plant tissue RNA extraction agent box, and remove the DNA that wherein contains with DNase I.The total RNA of each organ of gained detects (Fig. 2) through 1.0% agarose gel electrophoresis, and quality is good, and the feature band is clear, without obviously RNA degraded, pollutes without DNA; Through spectrophotometry, purity is better, can satisfy the requirement that RACE operation and sxemiquantitative RT-PCR detect.

2, the acquisition of total cDNA

Get the total RNA of root of Longli safflower cigarette, with GeneRacer Kit dephosphorylation with after removing the mRNA cap sequence, be connected with oligonucleotide RNA Oligo, carry out reverse transcription with GeneRacer Oligo dT primer, obtain the first chain cDNA; Then, take gained the first chain cDNA as template, use GeneRacer5 ' primer and GeneRacer3 ' primer, carry out pcr amplification with LA Taq archaeal dna polymerase, obtain total cDNA.The PCR program is: 94 ℃ of denaturation 4min; Then 94 ℃ of sex change 1min anneal for 68 ℃ and also extend 6min, totally 28 circulations; Last 72 ℃ are extended 10min.The total cDNA of gained detects (Fig. 3) through 1.0% agarose gel electrophoresis, present the traction of size between 200bp～10kb, the center of gravity zone is between 500bp～4kb, and nucleus is about 1.5kb, illustrate that reverse transcription is complete, obtained high-quality total cDNA.

3, the clone of NtIRL gene family 3 ' cDNA end

Take the Longli total cDNA of safflower cigarette as template, carry out one of NtIRL gene family 3 ' RACE end with gene-specific primer NtIRL-31 (SEQ ID No.1) and GeneRacer3 ' primer (SEQ ID No.2) and expand reaction, the PCR program is: 94 ℃ of denaturation 4min; Then 94 ℃ of sex change 1min, 52 ℃ of annealing 1min, 72 ℃ are extended 1min, totally 28 circulations; Last 72 ℃ are extended 10min.Then, expand production thing as template take one, carry out the nido reaction of NtIRL gene family 3 ' RACE end with gene-specific primer NtIRL-32 (SEQ ID No.3) and GeneRacer3 ' nested primer (SEQID No.4), PCR program and expands reacting phase together.The nest thing of expanding production detects (Fig. 4) through 1.0% agarose gel electrophoresis, expands the band that an about 500bp of size, conforms to the expection length of purpose fragment.Reclaim test kit with a small amount of glue and cut glue recovery purpose fragment, be connected with the pMD18-T carrier, transform the bacillus coli DH 5 alpha competent cell, carry out the screening of amicillin resistance and blue hickie.Get a plurality of hickie mono-clonals, the PCR that carries out Insert Fragment detects (Fig. 5), and the result shows that there is certain length polymorphism in these monoclonal Insert Fragments.Selecting a collection of representative mono-clonal with length polymorphism from the PCR positive monoclonal checks order, carry out the Blast analysis to recording sequence, the result shows, the homology of these Insert Fragments and woods tobacco NsA622 gene (GenBank accession number AB071165) is the highest, 2 difference 3 ' cDNA that represented the NtIRL gene family are terminal, length is respectively 480bp and 484bp (disregarding poly dA), and there is two or more mutability tailing sites in each gene.

4, the clone of NtIRL gene family 5 ' cDNA end

Take the Longli total cDNA of safflower cigarette as template, use GeneRacer5 ' primer (SEQ ID No.5) and gene-specific primer NtIRL-51 (SEQ ID No.6) to carry out one of NtIRL gene family 5 ' RACE end and expand reaction, one of PCR program and 3 ' RACE end expands reacting phase together.One thing of expanding production detects (Fig. 6) through 1.0% agarose gel electrophoresis, expands the band that an about 500bp of size, conforms to the expection length of purpose fragment, therefore need not to carry out nest and expands and react.Reclaim test kit with a small amount of glue and cut glue recovery purpose fragment, be connected with the pMD18-T carrier, transform the bacillus coli DH 5 alpha competent cell, carry out the screening of amicillin resistance and blue hickie.Get a plurality of hickie mono-clonals, the PCR that carries out Insert Fragment detects (Fig. 7), and the result shows that these monoclonal Insert Fragment length are consistent, does not have polymorphism.Choosing the PCR positive monoclonal checks order, to record and carry out the Blast analysis after sequence is removed 5 ' terminal manual splice sequence, the result shows, these Insert Fragments and woods tobacco NsA622 gene homology are the highest, 2 difference 5 ' cDNA that represented the NtIRL gene family are terminal, and length is 417bp (disregarding 5 ' terminal manual splice sequence).

5, the clone of NtIRL gene family full-length cDNA

According to the sequence information of above-mentioned NtIRL gene family 5 ' cDNA end and 3 ' cDNA end, design forward primer FNtIRL (SEQ ID No.7) and reverse primer RNtIRL (SEQ ID No.8).Take Longli safflower cigarette the first chain cDNA as template, with FNtIRL primer and RNtIRL primer, pcr amplification NtIRL gene family full-length cDNA.The PCR program is: 94 ℃ of denaturation 4min; Then 94 ℃ of sex change 1min, 55 ℃ of annealing 1min, 72 ℃ are extended 2min, totally 35 circulations; Last 72 ℃ are extended 10min.Gained PCR product detects (Fig. 8) through 1.0% agarose gel electrophoresis, expands the band that the about 1.2kb that conforms to the expection size.Reclaim test kit with a small amount of glue and cut glue recovery purpose fragment, be connected with the pMD18-T carrier, transform the bacillus coli DH 5 alpha competent cell, carry out the screening of amicillin resistance and blue hickie.Getting a plurality of hickie mono-clonals checks order, to record sequence and 5 ' cDNA end sequence and 3 ' cDNA end sequence and carry out multiple ratio pair, found that 2 different full length cDNA sequences, length is respectively 1257bp and 1261bp (disregarding poly dA), respectively called after NtIRL1 (SEQ ID No.9) and NtIRL2 (SEQ ID No.10).

6, the clone of NtIRL gene family full-length gene group sequence

Take the genome DNA of Longli safflower cigarette as template, with FNtIRL primer (SEQ ID No.7) and RNtIRL primer (SEQ IDNo.8) pcr amplification NtIRL gene family full-length gene group sequence, the PCR program increases identical with full-length cDNA.Gained PCR product detects (Fig. 9) through 1.0% agarose gel electrophoresis, expands the band that the about 1.9kb that conforms to the expection size.Reclaim test kit with a small amount of glue and cut glue recovery purpose fragment, be connected with the pMD18-T carrier, transform the bacillus coli DH 5 alpha competent cell, carry out the screening of amicillin resistance and blue hickie.Getting a plurality of hickie mono-clonals checks order, analysis records sequence, found that 2 different full-length gene group sequences, length is respectively 1946bp and 2089bp (disregarding poly dA), respectively called after NtIRL1 gene (SEQ IDNo.11) and NtIRL2 gene (SEQ ID No.12).Sequence alignment shows that they are inconsistent at exon 1 and full length cDNA sequence.

7, the protein molecular signature analysis of NtIRL gene family and derivation thereof

Searching with translation, molecular weight calculating etc. of ORF carried out at Vector NTI Advance 9.0.The CDD search of Genbank BLAST and protein sequence is carried out at NCBI (www.ncbi.nlm.nih.gov), and the structure prediction of the NtIRL albumen of derivation carries out at the software platform that the networks such as www.expasy.org, www.softberry.com provide.

(1) nucleotide sequence analysis of NtIRLI and NtIRL2 gene

The nucleotide sequence of NtIRL1 gene and the aminoacid sequence of derivation thereof are as shown in figure 10.NtIRL1 genome total length is 1946bp, is comprised of 4 introns and 5 exons, and except the cut mode of the 2nd intron is the GC/AG, other intron is all followed the AG/GT of standard ... the AG/AT cut mode.The NtIRL1cDNA total length is 1257bp, and the ORF of a 933bp is arranged at the 79-1011bp place, and (5 ' UTR) is 78bp to 5 ' non-coding region, and (3 ' UTR) is 246bp to 3 ' non-coding region.The dA of genome sequence, dG, dT, dC content are respectively 32.53%, 16.96%, 32.43%, 18.09%; The dA of cDNA sequence, dG, dT, dC content are respectively 30.87%, 19.97%, 29.44%, 19.73%, meet the characteristic feature of low dG+dC content.The dG+dC content of ORF is 41.37%, the dG+dC content of 5 ' UTR and 3 ' UTR is respectively 39.74% and 33.33%, introne 1,2,3,4 dG+dC content are respectively 25.11%, 24.55%, 33.01%, 27.41%, and the non-coding region particularly dG+dC content of 3 ' UTR and 4 introns is starkly lower than the coding region.This gene does not have typical poly (A) tailing signal AATAAA in 3 ' UTR, but A ₁₇₇₀ATAAT ₁₇₇₅And A ₁₉₂₀ATGAA ₁₉₂₅It may be atypical tailing signal.

The nucleotide sequence of NtIRL2 gene and the aminoacid sequence of derivation thereof are as shown in figure 11.NtIRL2 genome total length is 2089bp, is comprised of 4 introns and 5 exons, and except the cut mode of the 2nd intron is the GC/AG, other intron is all followed the AG/GT of standard ... the AG/AT cut mode.The NtIRL2cDNA total length is 1261bp, and the ORF of a 933bp is arranged at the 79-1011bp place, and 5 ' UTR is 78bp, and 3 ' UTR is 250bp.The dA of genome sequence, dG, dT, dC content are respectively 32.93%, 16.61%, 33.08%, 17.38%, the dA of cDNA sequence, dG, dT, dC content are respectively 32.08%, 19.53%, 29.25%, 19.14%, meet the characteristic feature of low dG+dC content.The dG+dC content of ORF is 41.16%, the dG+dC content of 5 ' UTR and 3 ' UTR is respectively 38.46% and 31.60%, introne 1,2,3,4 dG+dC content are respectively 22.46%, 26.54%, 30.15%, 27.35%, and the non-coding region particularly dG+dC content of 3 ' UTR and 4 introns is starkly lower than the coding region.This gene does not have typical poly (A) tailing signal AATAAA in 3 ' UTR, but A ₁₉₁₀ATAAT ₁₉₁₅And A ₂₀₆₃ATGA A ₂₀₆₈It may be atypical tailing signal.

2 members of NtIRL gene family are carried out sequence analysis, the result shows, the genome sequence consistence of NtIRL1 and NtIRL2 is 84.9%, the mRNA sequence identity is 95.1%, the coding region consistence is 96.9%, the coding region consistence is higher than non-coding region far away, has proved the conservative property of coding region.

Genome sequence and full length cDNA sequence to NtIRL1 and NtIRL2 carry out the analysis of Blastn homology.The results are shown in Table 1, they and Nicotiana A622 gene have the highest homology, and the mRNA of NtIRL1 is corresponding to the NtA622mRNA that has reported, its genome sequence is corresponding to the NsA622 gene of having reported, NtIRL2 then is new clone of the present invention; They with have significant homology from the IFR gene of soybean, white birch, European pear, clover, garbanzo, Arabidopis thaliana, barley, lotus flower, potato, pea, also has higher homology with the PCBER gene from black poplar, Chinese hemlock spruce, torch pine, the capsule of weeping forsythia, white pine, belong to SDR gene family PIP subfamily member, probably the secondary metabolism of tobacco there is material impact, and the defensive raction of involved in plant.

Table 1NtIRL gene and other plant PIP family member's nucleotide sequence homology is analyzed

(2) the Argine Monohydrochloride sequential analysis of NtIRL1 and NtIRL2 gene derivation

NtIRL1 and NtIRL2 gene be the protein of 310 amino acid compositions of coding all, and theoretical molecular (Mw) is respectively 34.652kDa and 34.650kDa, and iso-electric point (pI) is respectively 5.58 and 5.78.During the amino acid of NtIRL1 albumen formed, the content of Isoleucine the highest (9.03%) secondly was leucine (8.71%), and what content was minimum is halfcystine and tryptophane (being 0.65%).The amino acid of NtIRL2 albumen also is the content the highest (9.03%) of Isoleucine in forming, and secondly is leucine and Methionin (being 8.39%), and what content was minimum is halfcystine and tryptophane (being 0.65%).The number of acidic amino acid in two NtIRL albumen (account for respectively amino acid sum 12.90% and 12.58%) all is slightly more than basic aminoacids (account for respectively amino acid sum 11.29% and 10.97%), all is slightly acidic albumen.

Find that by sequence alignment NtIRL1 and NtIRL2 albumen have very high homology at amino acid levels, consistence and similarity illustrate that respectively up to 95.5% and 97.4% there is high conservative in protein level.NCBI BLASTp analyzes (table 2) and shows that the homology of the aminoacid sequence of NtIRL1 and NtIRL2 albumen and Nicotiana A622 albumen is the highest, and consistence is all more than 95%; With the IFR albumen from European pear, soybean, white birch, Arabidopis thaliana, potato, barley, lotus flower, clover, garbanzo, pea widely homology is arranged, consistence is respectively 50%～66% and 50%～67%, and similarity is respectively 68%～80% and 69%～81%; With the PCBER albumen from the capsule of weeping forsythia, black poplar, Chinese hemlock spruce, white pine, hemp, torch pine certain homology is arranged, consistence is respectively 58%～63% and 59%～64%, and similarity is respectively 75%～79% and 77%～79%; With the PLR albumen from western red cedar lower homology is arranged, consistence is respectively 47% and 50%, and similarity is respectively 66% and 67%; NtIRL in evolutionary system is nearer than PCBER and PLR with the relation of IFR in explanation.

The amino acid sequence homologous analysis of table 2NtIRL albumen and other plant PIP albumen

The aminoacid sequence of NtIRL1, NtIRL2 albumen and IFR, PCBER, PLR albumen is carried out multiple ratio pair.Result (Figure 12) shows, NtIRL1, NtIRL2 albumen and IFR, PCBER, PLR albumen are the same, all there is one section special, conserved sequence " GGTGYIG " in N-terminal, supposition may be and the coenzyme NADP 11 calmodulin binding domain CaM that wherein amino-acid residue Y and I supposition is combined with the phosphate group of NADPH.Variation has occured in above-mentioned conserved sequence in Activities of Some Plants, as having become GPTGAIG in the garbanzo, become GPTGAIG in the clover, become GATGAIG in the pea, become GPTGAIG in the lotus flower, become GATGYIG in the white pine, become GATGYIG in the torch pine, supposition may have different catalytic substrates relevant from enzyme.Other conservative region of NtIRL1 and NtIRL2 albumen is also consistent with the type sequence of PIP albumen, may guard site R with all aminoacid residue of cofactor and Binding Capacity as known ₃₈, K ₄₇, K ₁₃₅, R ₁₃₉, D ₂₇₆Deng all generation variations in NtIRL1 and NtIRL2 albumen, just change has occured in the position, space on protein molecular.Infer that thus NtIRL1 and NtIRL2 albumen should have and other plant PIP albumen especially the same typical catalytic activity of leguminous plants IFR albumen.It can also be seen that from Figure 12 NtIRL1, NtIRL2 albumen and some PCBER, PLR albumen have higher homology in the subregion, show that the encoding gene of these enzymes has original common ancestor.

In above-mentioned sequence multiple ratio cluster analysis is carried out on the basis.Result (Figure 13) shows, the IFR albumen of 10 kind of plant has been divided into 5 evolution branches, soybean and barley become separately a subgroup separately, and garbanzo, clover, pea and lotus flower are poly-to be a subgroup, and Arabidopis thaliana, white birch, European pear and potato are poly-to be a subgroup; The PCBER albumen of 6 kind of plant has been divided into 3 evolution branches, and torch pine, white pine and Chinese hemlock spruce are poly-to be a subgroup, and black poplar and the capsule of weeping forsythia are poly-to be a subgroup, and hemp becomes separately a subgroup; NtIRL1, NtIRL2, NtA622 and NsA622 albumen form a closely subgroup, and with the close together of leguminous plants IFR albumen, supposition NtIRL albumen has the defense function with leguminous plants IFR protein similar.

NCBI is conservative, and the domain search result shows the K of NtIRL1 and NtIRL2 albumen ₈～G ₈₈And I ₉～T ₂₃₉The zone all exists AdoHycase (c109931) and two conservative property structural domains of NmrA (pfam05368).The hydrolysis of AdoHycase catalysis AdoHcy generates adenosine and homocysteine, plays keying action in the activity of regulating various methylferases.NmrA is a negative transcription regulaton factor relevant with the posttranslational modification of AreA transcription factor, is the integral part that nitrogen metabolism suppresses system in the control fungi.In the conservative territory of long 211 amino acid whose AdoHycase, the aminoacid sequence of NtIRL1 and NtIRL2 albumen is compared with it, and score value is respectively 54.52bits and 51.82bits, and the E value is respectively 3e-08 and 1e-07; In the conservative territory of long 232 amino acid whose NmrA, the aminoacid sequence of NtIRL1 and NtIRL2 albumen is compared with it, and score value is respectively 271.33bits and 270.56bits, and the E value is respectively 1e-73 and 3e-73.

The Protscal analytical results shows, hydrophobic maximum value is 2.200 (the 269th amino acids) in the NtIRL1 albumen, minimum value is-2.378 (the 173rd amino acids), having 7 hydrophobic regions, is respectively the 5th～16,57～65,75～87,126～135,148～166,221～229,262～272 amino acids (average hydrophobic value is respectively 0.825,0.918,0.866,0.991,0.654,0.592,0.858).Hydrophobic maximum value is 2.200 (the 269th amino acids) in the NtIRL2 albumen, minimum value is-2.378 (the 173rd amino acids), having 8 hydrophobic regions, is respectively 5-16 position, 32-40 position, 57～65,75～87,125～136,148～166,221～229,261～272 amino acids (average hydrophobic value is respectively 0.825,0.524,0.917,0.866,0.871,0.654,0.592,1.042).From average hydrophobic value and the hydrophobic chain length of NtIRL1 and NtIRL2 albumen hydrophobic domain, these hydrophobic domains only have slight hydrophobicity.

Membrane spaning domain to NtIRL albumen predicts that the result shows that there are 2 possible membrane spaning domains in NtIRL1 albumen, and the 1st is Y ₁₄₉～L ₁₆₈20 amino-acid residues, by entering in the film outside the film; The 2nd is Y ₁₅₁～L ₁₆₈18 amino-acid residues, entered in the film outward by film.There are 3 possible membrane spaning domains in NtIRL2 albumen, and the 1st is Y ₁₄₉～L ₁₆₈20 amino-acid residues, the 2nd is P ₂₆₀～A ₂₇₈19 amino-acid residues, both by entering in the film outside the film; The 3rd is Y ₁₅₁～L ₁₆₈18 amino-acid residues, entered in the film outward by film.

Maximum reaction velocity and the Michaelis-Menton constant of the phosphorylation enzyme of protein kinase C.The Substratspezifitaet of the phosphorylation enzyme of casein kinase i I.Protein tyrosine kinase plays an important role in differentiation, growth and the activation of regulating cell.The N-myristoylation of protein affects the specificity of enzyme, myristoylation protein Growth of Cells, Development And Differentiation, signal transduction, tumour occur and the processes such as the copying of virus, assembling in play an important role.The glycosylation of protein not only affects the active of enzyme but also affects the space conformation of protein.In view of the importance in these chemically modified sites of enzyme, the present invention predicts the chemically modified site of NtIRL albumen.SignalP3.0 and TargetP1.1 prediction think that there are not signal peptide in NtIRL1 and NtIRL2 albumen, are a nonsecreting type albumen.The NetNGlyc1.0 prediction is thought, all there is the significant N-glycosylation site of prediction on the 211st asparagine residue of NtIRL1 and NtIRL2 albumen, prediction probability is respectively 0.7421 and 0.7467, the goodness of fit all is 9/9, because the protein of no signal peptide can not have N-glycosylation mechanism, even so NtIRL1 and NtIRL2 albumen have the significant glycosylation site of prediction, may really not carry out in vivo glycosylation yet.The NetPhos2.0 prediction thinks that NtIRL1 albumen has 11 significant phosphorylation sites, and wherein Serine is 4,4 of Threonines, 3 in tyrosine; NtIRL2 has 15 significant phosphorylation sites, and wherein Serine is 6,6 of Threonines, and 3 in tyrosine, abundant phosphorylation site explanation phosphorylation modification may be the prerequisite of NtIRL family protein performance function.PROSITE predictive display (table 3), NtIRL1 and NtIRL2 albumen respectively have 4 protein kinase C phosphorylation sites, and wherein the position in 2 sites is different, and this may mean that NtIRL1 and NtIRL2 albumen certain differentiation occurred at biological function; NtIRL1 and NtIRL2 albumen respectively have 7 casein kinase i I phosphorylation sites, 2 Tyrosylprotein kinase phosphorylation sites and 4 N-myristoylation sites; the position is all quite fixing, this means that the phosphorylation of phosphorylation, Tyrosylprotein kinase of casein kinase i I and N-myristoylation may bring into play its biological function to the NtIRL family protein and have vital role.PROSITE is in full accord with NetNGlyc1.0 to predicting the outcome of NtIRL family protein N-glycosylation site.

The chemically modified site of table 3PROSITE prediction NtIRL family protein

Softberry-ProtComp6.1, SubLoc v1.0, PSORT and CELLO v.2.5 wait the Subcellular Localization instrument all with NtIRL1 and NtIRL2 protein localization in tenuigenin, think that thus the NtIRL family protein is the plant PIP albumen of standard type, infer that its biological function is close with other plant IFR protein function.

SOPMA has carried out predicting (Figure 14) to NtIRL family protein precursor secondary structure, and the secondary structure of NtIRL family protein is mainly by α spiral, extended chain with curlingly at random consist of, and the β-bend proportion seldom.NtIRL1 albumen is curling proportion the highest (38.39%) at random, secondly is alpha-helix (33.55%), secondly is extended chain (21.29%) again, is β-corner (6.77%) at last; NtIRL2 albumen alpha-helix proportion the highest (40.32%) secondly is at random curling (36.13%), secondly is extended chain (19.03%) again, is β-corner (4.52%) at last.Alpha-helix mainly is positioned at two ends in the NtIRL1 albumen, has 1 larger alpha-helix in their middle part, and extended chain, curling and β-corner interts therein at random, is close to uniform mode and distributes with a kind of; Alpha-helix mainly is positioned at two ends in the NtIRL2 albumen, has 1 larger alpha-helix in their middle part, and extended chain and β-corner mainly are distributed in N end and middle part, at random curling staggered interting therein.

Adopt First Approach mode that NtIRL1 and NtIRL2 albumen have been carried out tertiary structure prediction (Figure 15) in http://swissmodel.expasy.org/ website, find that they have the typical tertiary structure feature of enzyme.According to 2gasA (the X ray diffractive crystal structure of clover IFR) tertiary structure of NtIRL1 albumen has been carried out the homology modeling, the two consistence is 53.9%.According to 1qycB (the X ray diffractive crystal structure of torch pine PCBER) tertiary structure of NtIRL2 albumen has been carried out the homology modeling, the two consistence is 60.13%.The tertiary structure of inferring is comprised of N-stub area and C-stub area, in the middle of these two zones a very large breach is arranged, and in order to bound substrates and generation catalyzed reaction, is the important structure characteristics of enzyme.As can be seen from the figure, NADPH binding site and substrate specificity binding site are all on the breach surface, the NADPH binding site is the confession H site of NtIRL, and the substrate specificity binding site determines the specificity of reduction reaction especially, also further specifies catalyzed reaction and occurs in dehiscence furrow.Can find out that by the 3D structure of inferring NtIRL1 albumen is the most similar to clover IFR structure, NtIRL2 albumen is the most similar to torch pine PCBER structure, has the exemplary functions structure of PIP enzyme.

8, the evaluation of NtIRL gene family number of members

Take the plasmid of the sub-NTA622G-2 of Longli safflower cigarette full-length gene group sequence clone as template, with NtIRL-31 primer (SEQ IDNo.1) and RNtIRL primer (SEQ ID No.8), 62 ℃ of annealing, amplification obtains the fragment (not containing the restriction enzyme site in the reverse primer) of 662bp, the 4th exon afterbody, the 4th intron and the 5th exon corresponding to the NtIRL gene family are most of, (G+C) %=37.0%, calculating Tm with Roche digoxin test kit is 63.2 ℃.With the template of this fragment as hybridization probe, adopt PCR DIG ProbeSynthesis Kit that probe is carried out digoxin-dUTP mark, the electrophoretic band of probe obviously lags behind (Figure 16 A) than unlabelled contrast behind the witness marking, and the probe mark success is described.Get Longli safflower cigarette genome DNA, carry out single endonuclease digestion with EcoRI, the HindIII, SacI and the XbaI that do not have recognition site in the probe sequence respectively, enzyme is cut product and is carried out Southern gel electrophoresis (Figure 16 B), as seen 4 kinds of enzymes are cut substantially and are put in place, it is best that wherein the enzyme of EcoRI is cut effect, HindIII is also more satisfactory, SacI and XbaI substantially put in place but do not reach perfect condition, the distribution of traction is not very even, lay particular stress on the macromole district, but do not have obvious band, do not affect crossbreeding effect.The complete rear transferring film of electrophoresis, fix, wash film, with digoxin-dUTP label probe in 43 ℃ of hybridization, the development, observe the quality band on the nylon membrane, as seen 4 kinds of enzyme butt formulas have all only produced 2 specific bands, illustrate that only there are 2 members (Figure 16 C) in the NtIRL gene family in common tobacco gene group.

9, the expression specificity of NtIRL gene family member between common tobacco organ

Get respectively root, stem, leaf, flower bud, flower, the capsule of Longli safflower cigarette and rear 20 days seeds total RNA of totally 7 organs of blooming, with the synthetic total cDNA of Oligo (dT) Adaptor primer reverse transcription of RNA PCR Kit Ver.3.0.Sequence according to Arabidopis thaliana 26S rRNA, design primers F 26S (SEQ ID No.13) and R26S (SEQ ID No.14), be used for the increasing conservative fragments of common tobacco house-keeping gene Nt26S534bp, this fragment be as interior mark, for detection of with the cDNA concentration of regulating reverse transcription.By the multiple compare of analysis to NtIRL gene family member's full length cDNA sequence and genome sequence, design NtIRL1 gene specific primer FNtIRL-1S (SEQ ID No.15) and RNtIRL-1S (SEQ ID No.16), and NtIRL2 gene specific primer FNtIRL-2S (SEQ ID No.17) and RNtIRL-2S (SEQ ID No.18), be respectively applied to the specifically expressing situation that RT-PCR detects NtIRL1 and NtIRL2 gene.Take Longli safflower cigarette genome DNA as template, adopt respectively above-mentioned FNtIRL-1S+RNtIRL-1S, FNtIRL-2S+RNtIRL-2S combination of primers to carry out 55～68 ℃ grads PCR, determine that the highest annealing temperature that above-mentioned 2 pairs of primers can effectively increase is respectively 66 ℃ and 68 ℃.The pMD18-T plasmid that NtIRL1 or NtIRL2 full-length cDNA is arranged take the clone respectively under above-mentioned annealing temperature, adopts above-mentioned 2 pairs of combination of primers to increase as template simultaneously, proves almost to intersect amplification between NtIRL1 and the NtIRL2 gene.

According to interior mark amplification, regulate the initial cDNA template of each organ amount to consistent, then respectively take total cDNA of root, stem, leaf, flower bud, flower, capsule and the rear 20 days seeds of blooming as template, detect NtIRL1 and the expression of NtIRL2 gene in these organs with FNtIRL-1S+RNtIRL-1S, FNtIRL-2S+RNtIRL-2S combination of primers respectively.The results are shown in Figure 17, the interior mark amplified band size of each sample room is basically identical with concentration, shows that the quality of the initial RNA of each sample, the total cDNA of reverse transcription and quantity have consistence and comparability; The expression amount of NtIRL1 gene in root is the highest, and suitable expression is arranged in stem, and weak expression is arranged in the flower bud, does not express fully in 20 days the seed at leaf, flower, capsule and after blooming; The NtIRL2 gene has stronger tissue specificity, expresses the byest force in root, and extremely weak expression is arranged in stem, does not all express in 20 days the seed at flower bud, leaf, flower, capsule and after blooming; The transcriptional level of NtIRL2 in root is apparently higher than NtIRL1.Generally speaking, NtIRL gene family member is predominant expression in root system mainly, infers that it has vital role in the secondary metabolism process of tobacco root, and wherein the NtIRL2 gene is occupied an leading position.

Two, the structure of NtIRL gene family member justice and antisense plant expression vector

1, the structure of NtIRL gene family member justice plant expression vector

Respectively NtIRL1 and NtIRL2 full-length gene group sequence forward are inserted between the BamHI and SacI multiple clone site of carrier pCambia2301G, replace gus gene wherein, made up by CaMV35S promoters driven, NOS terminator stop transcribing, chain NtIRL1 and NtIRL2 justice plant expression vector pNtIRL1 and the pNtIRL2 (Figure 18) that reporter gene gus and selection markers gene nptII are arranged respectively in the T-DNA border simultaneously.The BamHI/SacI double digestion qualification result (Figure 19) of recombinant plasmid shows, from pNtIRL11 and pNtIRL2, downcut respectively the fragment of an about 1900bp of size and 2000bp, consistent with NtIRL1 and NtIRL2 full-length gene group sequence size, show NtIRL1 and NtIRL2 full-length gene group sequence directed cloning in carrier pCambia2301G.

2, the structure of NtIRL gene family member antisense plant expression vector

NtIRL gene family member is total to conservative fragments NtIRLA oppositely to be inserted between the BamHI and SacI multiple clone site of carrier pCambia2301G, replace gus gene wherein, made up by CaMV35S promoters driven, NOS terminator stop transcribing, the chain NtIRL gene family antisense plant expression vector pNtIRLA (Figure 20) that reporter gene gus and selection markers gene nptII are arranged respectively in the T-DNA border simultaneously.Concrete construction step is as follows: take the total cDNA of root of Longli safflower cigarette as template, with primers F NtIRLA (SEQ IDNo.19) and RNtIRLA (SEQ ID No.20), with the common conservative fragments NtIRLA of the PfuDNA polysaccharase pcr amplification NtIRL gene family member of high-fidelity.The PCR program is: 94 ℃ of denaturation 2min; Then 94 ℃ of sex change 1min, 55 ℃ of annealing 1min, 72 ℃ are extended 2min, totally 35 circulations; Last 72 ℃ are continued to extend 10min.The PCR product is after agarose gel electrophoresis is identified, recovery purpose fragment is carried out end and is added A, then subclone enters the pMD18-T plasmid, carry out the Insert Fragment order-checking, the sequencing result demonstration has obtained the NtIRLA fragment of sequence shown in 1-1179 position Nucleotide among the SEQ IDNo.9, the sub-plasmid of extracting positive colony, carry out SacI and BamHI double digestion, reclaim the NtIRLA fragment, the carrier pCambia2301G with the same double digestion of warp is connected again, namely gets recombinant plasmid pNtIRLA.With this recombinant plasmid transformed bacillus coli DH 5 alpha, with primers F NtIRLA and RNtIRLA the resistance bacterium colony mono-clonal bacterium liquid through kantlex (Kan)+Streptomycin sulphate (Str)+Rifampin (Rif) screening being carried out PCR identifies, the result shows, the size of recombinant plasmid and prediction in the same size (Figure 21); This recombinant plasmid is carried out double digestion with SacI and BamHI identify, the result shows, has downcut the fragment of an about 1200bp of size from recombinant plasmid, consistent with the NtIRLA clip size (Figure 22).Show purpose fragment NtIRLA directed cloning in carrier pCambia2301G.

Three, the structure of NtIRL gene family member Agrobacterium engineering strain

Adopt liquid nitrogen cold shock method to transform the agrobacterium tumefaciens lba4404 competent cell just plant expression vector pNtIRL1, pNtIRL2 and antisense plant expression vector pNtIRLA respectively, coat on the LB flat board that contains 100mg/L Kan, 40mg/L Rif and 20mg/L Str, be inverted for 28 ℃ and cultivated 2 days, the single bacterium colony of picking resistance, be inoculated in the LB liquid nutrient medium that 800 μ l contain 100mg/L Kan, 40mg/L Rif and 20mg/L Str, 28 ℃, 250rpm shaking culture are to OD ₆₀₀=0.6～0.8, to get bacterium liquid and carry out PCR method detection (Figure 23), positive monoclonal is Agrobacterium engineering strain LBA4404-pNtIRL1, LBA4404-pNtIRL2 and LBA4404-pNtIRLA.

Four, NtIRL gene family member Agrobacterium engineering strain transforms common tobacco

1, Transformation of tobacco-agriculture bacillus mediated Ye Panfa

Respectively Agrobacterium engineering strain LBA4404-pNtIRL1, LBA4404-pNtIRL2 are transformed the large gold dollar of common Performance Liquid Chromatography Analysis for Nicotine in Tobacco kind safflower.Agrobacterium engineering strain LBA4404-pNtIRLA is transformed respectively common tobacco high-nicotine kind Da Fu cigarette and Longli safflower cigarette.Concrete steps are as follows: frozen Agrobacterium engineering strain is thawed is cultured to logarithmic phase after the activation, and centrifugal 5 minutes of 5000rpm collects thalline, uses MS ₀(MS+30g/L sucrose pH5.8) is regulated bacterial concentration to OD to liquid nutrient medium ₆₀₀Be 0.2～0.5, for contaminating.With 1% chlorine bleach liquor, the 7～8min that sterilizes, rinsed with sterile water 6～8 times is seeded to MS with tobacco seed ₀(MS+8g/L agar+30g/L sucrose pH5.8), is that 25 ℃, photoperiod are to cultivate under the condition of 16h/d in temperature to solid medium, after about one month, gets the aseptic seedling of robust growth as transforming explant.Get young tender, the healthy and strong blade of aseptic seedling, remove master pulse, be cut into the leaf dish of about 0.8cm * 0.8cm.About 100 leaf dishes are put Agrobacterium engineering strain MS ₀(carry out blank conversion with the agrobacterium tumefaciens lba4404 bacterium liquid that does not contain expression vector simultaneously) among the resuspended liquid 200mL, 28 ℃, 100rpm are contaminated 10min, pull the leaf dish out, suck unnecessary bacterium liquid with sterilization thieving paper.The leaf dish that blots is lain in the common culture medium MS that adds lid layer filter paper ₁(MS ₀+ 2.0mg/L6-BA+0.1mg/L NAA) on, seals culture dish with sealed membrane, cultivate altogether about 48h in 23 ℃-25 ℃, dark place, grow light white bacterial plaque to the explant edge.Then with leaf dish adaxial and its surface upwards, lie in screening division culture medium MS ₂(MS ₁+ 500mg/L cephamycin (Cef)+150mg/L Kan) on, seals culture dish with sealed membrane, under 27 ℃-29 ℃, the condition of photoperiod 14h/d, cultivate, after about 3 weeks, downcut the leaf margin with bud, insert screening division culture medium MS ₂Middle continuation was cultivated more than 1 month, and 2 all subcultures once.When treating that Multiple Buds generally grows to 2cm above (Figure 24 A), it is cut from base portion and leaf dish, with not with the budlet insertion root media MS of callus ₃(MS ₀+ 300mg/L Cef+150mg/L Kan) root induction in is to cultivate under the condition of 14h/d in 27 ℃-29 ℃, photoperiod, and 2 all subcultures once screening and culturing 2-3 generation, are eliminated Albino Seedling and lopsided seedling.Treat that most of resistance seedling has grown root in great numbers and root growth to 2～3cm (Figure 24 B), plant height reaches 10cm when above, select normal, the eugonic resistance seedling of form, clean the agar of root, water planting hardening 2～3d (Figure 24 C), be transplanted to again in the nutrition pot growth (Figure 24 D) under natural condition.Anti-Kan seedling 89 strains of final acquisition safflower large gold dollar overexpression NtIRL1 gene, anti-Kan seedling 104 strains of the large gold dollar overexpression of safflower NtIRL2 gene, anti-Kan seedling 129 strains of Da Fu cigarette Antisense Suppression, anti-Kan seedling 152 strains of Longli safflower cigarette Antisense Suppression, obtain simultaneously with after the agrobacterium tumefaciens lba4404 blank conversion at the regrowth that does not contain substratum that Kan screening presses and grow, with it as the strain of negative control cigarette.

2, the GUS active mass chemical staining of transgenic tobacco plant detects

Before resistance individual plant system and the negative control water planting hardening, edulcoration agar is got a bit of root system and is carried out GUS tissue activity staining examine, and the root system of the positive individual plant of GUS can produce blue reaction.The result shows, the GUS staining examine positive has 58 strains (Figure 25 A) in the anti-Kan seedling of the large gold dollar overexpression of 89 strain safflowers NtIRL1 gene; The GUS staining examine positive has 60 strains (Figure 25 B) in the anti-Kan seedling of the large gold dollar overexpression of 104 strain safflowers NtIRL2 gene; The GUS staining examine positive has 77 strains (Figure 25 C) in the anti-Kan seedling of 129 strain Da Fu cigarette Antisense Suppression; The GUS staining examine positive has 93 strains (Figure 25 D) in the anti-Kan seedling of 152 strain Longli safflower cigarette Antisense Suppression.

3, the PCR of transgenic tobacco plant detects

Take the DNA of individual plant extracting as template, adopt 35S promoter primers F 35S3N and RNtIRL (SEQ ID No.8), overexpression resistance individual plant system is carried out PCR detect, further identify whether changed the goal gene sequence in the transfer-gen plant over to.The result shows have 73 strain PCR results to be positive in the anti-Kan seedling of the large gold dollar overexpression of 89 strain safflowers NtIRL1 gene, occurs and expection 2.0kb feature band of the same size (Figure 26); There are 79 strain PCR results to be positive in the anti-Kan seedling of the large gold dollar overexpression of 104 strain safflowers NtIRL2 gene, occur and expection 2.1kb feature band of the same size (Figure 27).Reclaim at random respectively the as a result purpose fragment of the conversion individual plant system of the positive of 3 PCR, TA is cloned on the pMD18-T carrier, carry out sequence verification, the sequence similarity of sequencing result and NtIRL1 and NtIRL2 gene justice fragment all reaches 100%, illustrates by the NtIRL1 of CaMV35S promotor startup and the just fragment of NtIRL2 gene to be incorporated in the tobacco chromogene group.

Take the DNA of individual plant extracting as template, adopt 35S promoter primers F 35S3N and FNtIRLA (SEQ ID No.21), carry out PCR and detect suppressing to express resistance individual plant system, further identify whether changed the goal gene sequence in the transfer-gen plant over to.The result shows have 98 strain PCR results to be positive in the anti-Kan seedling of 129 strain Da Fu cigarette Antisense Suppression, occurs and expection 1.2kb feature band of the same size (Figure 28); There are 116 strain PCR results to be positive in the anti-Kan seedling of 152 strain Longli safflower cigarette Antisense Suppression, occur and expection 1.2kb feature band of the same size (Figure 29).Reclaim at random respectively the as a result purpose fragment of the conversion individual plant system of the positive of 3 PCR, TA is cloned on the pMD18-T carrier, carry out sequence verification, the sequence similarity of sequencing result and NtIRLA antisense fragment reaches 100%, illustrates that the NtIRLA antisense fragment that is started by the CaMV35S promotor has been incorporated in the tobacco chromogene group.

4, the RT-PCR of transgenic tobacco plant detects

24h after the large gold dollar of safflower turns NtIRL1 or NtIRL2 gene plant and pinches, choose consistent negative control cigarette strain root system 1 strain of growth, GUS dyeing and goal gene PCR detect transgenosis cigarette strain root system 2 strains of jack to jack adapter, GUS dyeing feminine gender but goal gene PCR detects positive transgenosis cigarette strain root system 1 strain, GUS dyeing and goal gene PCR detect two positive transgenosis cigarette strain root system 10 strains, extract respectively total RNA, Oligo (dT) Adaptor primer and AMV ThermoScript II with RNA PCR Kit are carried out reverse transcription, the total cDNA that obtains take reverse transcription again is as template, carry out pcr amplification with NtIRL1 gene specific primer FNtIRL-1S (SEQ ID No.15) and RNtIRL-1S (SEQ ID No.16) and NtIRL2 gene specific primer FNtIRL-2S (SEQ ID No.17) and RNtIRL-2S (SEQ ID No.18), take the conservative fragments of house-keeping gene Nt26S as interior mark, detect the expression of goal gene in the transgenic tobacco plant.The large gold dollar of safflower turns the RT-PCR analysis that NtIRL1 expresses in the NtIRL1 gene plant and sees Figure 30, GUS dyeing and goal gene PCR detect the transgenosis cigarette strain (8 and 22) of jack to jack adapter and GUS dyeing feminine gender but goal gene PCR detects the NtIRL1 gene expression amount of the transgenosis cigarette strain (29) of the positive compares no significant difference with the strain of negative control cigarette, and the NtIRL1 gene expression amount of the transgenosis cigarette strain of GUS dyeing and the two positives of goal gene PCR detection all is higher than negative control, 3 plant (3 wherein, 18 and 62) expression amount is extremely strong, 7 plant (2,7,11,50,51,59 and 61) expression amount is stronger, the difference of expression amount may be that different different institutes with the insertion copy number cause T-DNA in the position that genome inserts, illustrate that the strain of transgenosis cigarette not only integrated the just fragment of NtIRL1 gene, and can be transcribed into mRNA, the NtIRL1 gene has obtained overexpression in the strain of transgenosis cigarette.The large gold dollar of safflower turns the RT-PCR analysis that NtIRL1 expresses in the NtIRL2 gene plant and sees Figure 31, as seen GUS dyeing and goal gene PCR detect the transgenosis cigarette strain (40 and 76) of jack to jack adapter and GUS dyeing feminine gender but goal gene PCR detects the NtIRL1 gene expression amount of the transgenosis cigarette strain (75) of the positive compares no significant difference with the strain of negative control cigarette, and the NtIRL1 gene expression amount of the transgenosis cigarette strain of GUS dyeing and the two positives of goal gene PCR detection all is higher than negative control, wherein the expression amount of 2 plant (34 and 79) is extremely strong, 5 plant (28,44,51,70 and 62) expression amount is stronger, 3 plant (42,49 and 60) expression amount is a little more than negative control, illustrate that the strain of transgenosis cigarette not only integrated the just fragment of NtIRL2 gene, and can be transcribed into mRNA, the NtIRL2 gene has obtained overexpression in the strain of transgenosis cigarette.

According to above-mentioned same procedure, 24h after Da Fu cigarette Antisense Suppression transfer-gen plant is pinched, choose consistent negative control cigarette strain root system 1 strain of growth, GUS dyeing and goal gene PCR detect transgenosis cigarette strain root system 2 strains of jack to jack adapter, GUS dyeing feminine gender but goal gene PCR detects positive transgenosis cigarette strain root system 2 strains, GUS dyeing and goal gene PCR detect two positive transgenosis cigarette strain root system 8 strains, extract respectively total RNA, adopt sxemiquantitative RT-PCR method to detect the expression of goal gene in the transgenic tobacco plant.The RT-PCR of NtIRL1 and NtIRL2 genetic expression analyzes and sees Figure 32-33 in the Da Fu cigarette Antisense Suppression transfer-gen plant, as seen GUS dyeing and goal gene PCR detect the transgenosis cigarette strain (20 and 37) of jack to jack adapter and GUS dyeing feminine gender but goal gene PCR detects the NtIRL1 of positive transgenosis cigarette strain (86 and 93) does not compare not reduction with the strain of negative control cigarette with the NtIRL2 gene expression amount, the difference of expression amount may be that the spatial distribution position difference of cigarette strain sampling root system causes, and NtIRL1 and the NtIRL2 gene expression amount of the transgenosis cigarette strain of GUS dyeing and the two positives of goal gene PCR detection all are lower than negative control, wherein the NtIRL1 expression amount of 2 plant (8 and 78) extremely a little less than, 5 plant (1,40,70,74 and 80) NtIRL1 expression amount a little less than, the NtIRL1 expression amount of 1 plant (13) is a little less than negative control, 3 plant (1,8 and 78) do not detect the expression of NtIRL2 gene, 3 plant (70,74 and 80) NtIRL2 expression amount a little less than, the NtIRL2 expression amount of 2 plant (13 and 40) is a little less than negative control, the difference of expression amount may be that different different institutes with the insertion copy number cause T-DNA in the position that genome inserts, illustrate that the strain of transgenosis cigarette not only integrated NtIRLA antisense fragment, and have transcriptional activity, NtIRL1 and NtIRL2 gene have obtained to suppress to express in the strain of transgenosis cigarette.

5, the Southern blot of transgenic tobacco plant detects

Turning the NtIRL1 gene plant at the large gold dollar of safflower transplanted rear one month, choose respectively consistent negative control cigarette strain 1 strain of growth, GUS dyeing and goal gene PCR detect transfer-gen plant 1 strain of jack to jack adapter, GUS dyeing and goal gene PCR detect two positive transfer-gen plant 9 strains, adopt the genomic dna of CTAB method extracting same area blade, detect with carrying out Southern blot behind the HindIII complete degestion.The results are shown in Figure 34, the hybrid belt that only has 2 native genes (NtIRL1 and NtIRL2) in the DNA sample of the transgenosis cigarette strain (8) of the strain of negative control cigarette and GUS dyeing and goal gene PCR detection jack to jack adapter, and GUS dyeing and goal gene PCR detect two positive transgenosis cigarette strains (2,3,11,12,18,24,47,56 and 62) in the DNA sample except above-mentioned 2 hybrid belts, the hybrid belt that 1-4 bar transgenosis integration event causes has also appearred in addition, wherein the just fragment of 1 plant (3) chromosomal integration is 4 copies, the just fragment of 2 plant (11 and 12) chromosomal integration is 3 copies, the just fragment of 1 plant (47) chromosomal integration is two copies, confirms that further the strain of transgenosis cigarette successfully integrated the just fragment of NtIRL1 gene.

The Southern blot that carries out Da Fu cigarette Antisense Suppression plant according to above-mentioned same procedure detects.The results are shown in Figure 35, the hybrid belt that only has 2 native genes (NtIRL1 and NtIRL2) in the DNA sample of the transgenosis cigarette strain (37) of the strain of negative control cigarette and GUS dyeing and goal gene PCR detection jack to jack adapter, and GUS dyeing and goal gene PCR detect two positive transgenosis cigarette strains (1,6,7,8,13,19,21,30,40) in the DNA sample except above-mentioned 2 hybrid belts, the hybrid belt that 1-2 bar transgenosis integration event causes has also appearred in addition, wherein the antisense fragment of 2 plant (1 and 13) chromosomal integration is two copies, confirms that further the strain of transgenosis cigarette successfully integrated NtIRLA antisense fragment.

6, the nicotine content of transgenic tobacco plant and correlated character analysis of variance

For the phenotypic variation of research transgene tobacco, choose the consistent regrowth of growth conditions, transplant simultaneously, carry out parallel management and character observation.Comprise: what the large gold dollar GUS of safflower dyeing and goal gene PCR detected two positives turns 32 strains of NtIRL1 gene regrowth, GUS dyeing feminine gender but goal gene PCR detect positive NtIRL1 gene regrowth 8 strains that turn, GUS dyeing and goal gene PCR detection jack to jack adapter turn 8 strains of NtIRL1 gene regrowth, 8 strains of negative control regrowth; What the large gold dollar GUS of safflower dyeing and goal gene PCR detected two positives turns 40 strains of NtIRL2 gene regrowth, GUS dyeing feminine gender but goal gene PCR detect positive NtIRL2 gene regrowth 10 strains that turn, GUS dyeing and goal gene PCR detection jack to jack adapter turn 10 strains of NtIRL2 gene regrowth, 10 strains of negative control regrowth; Da Fu cigarette GUS dyeing and goal gene PCR detect two positive regenerated transgenic seedling 50 strains, GUS dyeing feminine gender but goal gene PCR detects positive regenerated transgenic seedling 12 strains, GUS dyeing and goal gene PCR detect regenerated transgenic seedling 10 strains of jack to jack adapter, 10 strains of negative control regrowth; Longli safflower cigarette GUS dyeing and goal gene PCR detect two positive regenerated transgenic seedling 60 strains, GUS dyeing feminine gender but goal gene PCR detects positive regenerated transgenic seedling 10 strains, GUS dyeing and goal gene PCR detect regenerated transgenic seedling 10 strains of jack to jack adapter, 10 strains of negative control regrowth.In addition, the flowers are in blossom at the center puts disposable pinching, and the large gold dollar individual plant of safflower stays 18 on leaf, and Da Fu cigarette individual plant stays 16 on leaf, and Longli safflower cigarette individual plant stays 16 on leaf, pinches and gets 4-6 reciprocal position leaf in rear 30 days, and 105 ℃ completed 15 minutes, measured nicotine content after 70 ℃ of oven dry.

(1) nicotine content of transgenic tobacco plant detects

Turn NtIRL1 gene T from the large gold dollar of safflower ₁In the strain, the strain of selecting the similar 26 strain GUS dyeing of appearance growing way and PCR detection pair positive strain and 1 strain GUS dyeing and PCR to detect jack to jack adapter is carried out the detection of blade nicotine content.The result shows (Figure 36), blade nicotine content and the negative control of jack to jack adapter strain (8) are suitable, the blade nicotine content of the two positive strains of 24 strains has raising in various degree and has obvious concord with the expression amount of NtIRL1 gene than negative control, wherein there are 6 strain nicotine contents to exceed negative control more than 50%, be up to 1.75 times of negative control, there are 10 strain nicotine contents to be higher than 0.3～0.5 times of negative control, illustrate that the NtIRL1 gene has all obtained overexpression in these strains, promoted the biosynthesizing of tobacco leaf nicotine; The blade nicotine content of the two positive strains of 2 strains is lower than negative control, may co-suppression occur with endogenous genes involved.

Turn NtIRL2 gene T from the large gold dollar of safflower ₁In the strain, the strain of selecting the similar 31 strain GUS dyeing of appearance growing way and PCR detection pair positive strain and 1 strain GUS dyeing and PCR to detect jack to jack adapter is carried out the detection of blade nicotine content.The result shows (Figure 37), blade nicotine content and the negative control of jack to jack adapter strain (40) are basically identical, the blade nicotine content of the two positive strains of 29 strains has rising in various degree and has obvious concord with the expression amount of NtIRL2 gene than negative control, wherein there are 7 strain nicotine contents to exceed negative control more than 50%, be up to 1.85 times of negative control, there are 13 strain nicotine contents to be higher than 0.3～0.5 times of negative control, illustrate that the NtIRL2 gene has obtained overexpression in these strains, promoted effective accumulation of nicotine in the tobacco leaf; The blade nicotine content of the two positive strains of 2 strains is lower than negative control, may have the co-suppression effect of endogenous genes involved.

From Da Fu cigarette transgenosis T ₁In the strain, select 33 similar GUS dyeing of appearance growing way and PCR detects two positive strains and the strain of 1 strain GUS dyeing and PCR detection jack to jack adapter is carried out the detection of blade nicotine content.The result shows (Figure 38), blade nicotine content and the negative control of jack to jack adapter strain (37) are suitable, the blade nicotine content of the two positive strains of 33 strains has decline in various degree and has obvious concord with the expression amount of NtIRL gene than negative control, 3 strain nicotine contents are wherein arranged not as good as 50% of negative control, there are 21 strain nicotine contents to be lower than negative control 30%～50%, illustrate that NtIRL gene family member has all obtained establishment in these strains, hindered the synthetic of tobacco leaf nicotine.

From Longli safflower cigarette transgenosis T ₁In the strain, 30 similar GUS of selection appearance growing way dye and the two positive strains of PCR detection are carried out the nicotine content of tobacco leaves detection.The result shows (Figure 39), the blade nicotine content of the two positive strains of 30 strains has in various degree decline than negative control, wherein have 1 strain nicotine content only negative contrast 17%, 9 strain nicotine contents are arranged not as good as 40% of negative control, the nicotine content range of decrease of all the other strains is all more than 30%, illustrate that NtIRL gene family member has obtained establishment in these strains, reduced effective accumulation of nicotine content in tobacco leaf.

Comprehensive the above results, the expression amount of NtIRL gene family on the nicotine content of tobacco leaves impact are obviously inferred the NtIRL gene family synthetic relevant enzyme of activated nicotine of encoding, and play an important role in the biosynthetic metabolism approach of tobacco smoke alkaloid.

(2) Phenotypic Observation of transgenic tobacco plant

Phenotypic Observation is found (Figure 40～43), obvious phenotype does not appear in overexpression and the transgene tobacco that suppresses to express to be changed, have normal plant forms and growth characteristics, tobacco leaf color and contrast infer that also without significant difference NtIRL gene family member may have vital role in the tobacco secondary metabolism.

(3) disease resistance of transgenic tobacco plant changes

The large gold dollars of safflower of choosing GUS dyeing and the two positives of PCR detection turn 10 strains of NtIRL1 gene plant and 10 strains of negative control plant, and random alignment is potted plant in little soil with black shank bacterium, parallel management.The result shows (Figure 44), and the balck shank sickness rate of negative control plant is 50%, does not fall ill and the large gold dollar of safflower turns the NtIRL1 gene plant.

The large gold dollars of safflower of choosing GUS dyeing and the two positives of PCR detection turn 8 strains of NtIRL2 gene plant and 8 strains of negative control plant, and random alignment is potted plant in little soil with black shank bacterium, parallel management.The result shows (Figure 45), and the balck shank sickness rate of negative control plant is 75%, does not fall ill and the large gold dollar of safflower turns the NtIRL2 gene plant.

Choose GUS dyeing and PCR and detect two positive 8 strains of Da Fu cigarette transfer-gen plant and 8 strains of negative control plant, in the potted plant soil in omiting with rhizoctonia solani of random alignment, parallel management.The result shows (Figure 46), and the damping-off sickness rate of Da Fu cigarette transfer-gen plant is 62.5%, and the negative control plant does not fall ill.

Can find out that from the above results NtIRL gene family member has affected the disease-resistant performance of transfer-gen plant by the nicotine accumulation that changes the acceptor plant, overexpression NtIRL gene, nicotine content increases, and the anti-balck shank ability of cigarette strain strengthens; Suppress the expression of NtIRL gene, nicotine content reduces, and the anti-damping-off ability of cigarette strain weakens; Thereby illustrate that the nicotine in the tobacco secondary metabolite also is the plant protecting chemical class material with defense function, can strengthen the ability of cigarette strain opposing fungal disease.Carry out the molecular regulation of NtIRL gene by the transgenosis means, will be conducive to the genetic engineering breeding of low nicotine, high resistance to fungal disease High Quality Tobacco.

Explanation is at last, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although by invention has been described with reference to the preferred embodiments of the present invention, but those of ordinary skill in the art is to be understood that, can make various changes to it in form and on the details, and not depart from the spirit and scope of the present invention that appended claims limits.

Claims (10)

1. the open reading frame sequence of common tobacco isoflavones reductase gene NtIRL2 is shown in 79-1011 position Nucleotide among the SEQ ID No.10.

2. the full length cDNA sequence of common tobacco isoflavones reductase gene NtIRL2 is shown in SEQ ID No.10.

3. the genome sequence of common tobacco isoflavones reductase gene NtIRL2 is shown in SEQ ID No.12.

4. the just plant expression vector that contains claim 1 or 3 described sequences.

5. just plant expression vector according to claim 4, it is characterized in that: described just plant expression vector is between the BamHI and SacI multiple clone site with the genome sequence forward insertion vector pCambia2301G of common tobacco isoflavones reductase gene NtIRL2, replaces gus gene wherein and gets; Described carrier pCambia2301G downcuts the gus expression casette with HindIII and EcoRI double digestion from the pBI121 plasmid, is connected on the pCambia2301 of same double digestion carrier again and gets.

6. contain the altogether antisense plant expression vector of conservative fragments of common tobacco isoflavones reductase gene family member, described common tobacco isoflavones reductase gene family member is NtIRL1 and NtIRL2; The full length cDNA sequence of described NtIRL1 gene is shown in SEQ IDNo.9; The full length cDNA sequence of described NtIRL2 gene is shown in SEQ ID No.10.

7. antisense plant expression vector according to claim 6, it is characterized in that: described antisense plant expression vector is with common tobacco isoflavones reductase gene family member altogether between the BamHI and SacI multiple clone site of the reverse insertion vector pCambia2301G of conservative fragments NtIRLA, replaces gus gene wherein and gets; Described common tobacco isoflavones reductase gene family member is total to the nucleotide sequence of conservative fragments NtIRLA shown in 1-1179 position Nucleotide among the SEQ ID No.9; Described carrier pCambia2301G downcuts the gus expression casette with HindIII and EcoRI double digestion from the pBI121 plasmid, is connected on the pCambia2301 of same double digestion carrier again and gets.

8. the microbial transformant that contains claim 4 or 5 described just plant expression vectors or the described antisense plant expression vector of claim 6.

9. the application in the molecular breeding of each described sequence of claims 1 to 3 aspect common Nicotine Content in Tobacco Leaf and resistance to fungal disease.

10. the application in the molecular breeding of common tobacco isoflavones reductase gene NtIRL1 aspect common Nicotine Content in Tobacco Leaf and resistance to fungal disease, the full length cDNA sequence of described NtIRL1 gene is shown in SEQ ID No.9, and genome sequence is shown in SEQ ID No.11.

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