CN102115759A

CN102115759A - Transgenic sweet sorghum with altered lignin composition and process of preparation thereof

Info

Publication number: CN102115759A
Application number: CN2010102658614A
Authority: CN
Inventors: 艾斯塔瓦·巴苏; 迈瑞娜·库马尔·买提; 萨塔如帕·嘉; 苏米特拉·库马尔·森; 巴尼布拉塔·潘迪
Original assignee: Nagarjuna Energy Pvt Ltd
Current assignee: Nagarjuna Energy Pvt Ltd
Priority date: 2009-08-21
Filing date: 2010-08-23
Publication date: 2011-07-06
Also published as: AP2010005382A0

Abstract

The present invention provides a sweet sorghum plant characterized by altered lignin content and/or altered lignin composition compared to a wild plant and this is achieved by manipulating the expression of caffeoylCoA-O-methyltransferae (CCoAOMT) in particular and optionally caffeic acid-O-methyltranferase (COMT) in sweet sorghum by incorporation of a construct comprising an isolated DNA sequence represented by SEQ ID NO 2 and optionally SEQUENCE ID NO 1.

Description

Transgenic sweet sorghum and preparation method thereof with lignin component of change

The present invention is 12/545 of submission on August 21st, 2009, the partial continuous application case of No. 699 U. S. applications, this application are the continuation applications according to united states patent law the 120th (35U.S.C. § 120) of the PCT/IB2008/000380 international patent application submitted to the 20 days February in 2008 of the right of priority of the 1481/CHE/2006 Indian patent application submitted in requirement on February 21st, 2007.These disclosed content whole are to be incorporated into this with reference to the mode of quoting.

Technical field

The present invention relates to biological technical field, relate in particular to the exploitation of the plant of lignin component with change.

Background technology

Sweet sorghum (Sweet sorghum), sweet sorghum kind (Sorghum bicolor (L.) Moench) provides the fringe grain of the production that can be used as wine, sugar, syrup, fuel etc. and unique crop of stem stalk.Sweet sorghum is compared with other crop and has the following advantages:

Vegetative period (about 4 months) and water requirement (two strain crops surpass 8000m ³) than vegetative period of sugarcane and water requirement (be respectively 12 to 16 months, 36000m ³) low 4 times.

The cultivation expense of sweet sorghum is low 3 times than sugarcane.

Seminal propagation.

Be suitable for the mechanize crop production.

The method ethanol production that is derived from sweet sorghum is environmentally friendly with respect to the production process from syrup.

The ethanol burning quality is better---be derived from sugarcane and compare and have less sulphur and higher octane value.

Referring to International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) Fact Book, " sweet sorghum: food, raw material, feed and fuel crop " of announcing in 2006.

But the matter of utmost importance of utilizing this plant is to have xylogen, and it influences the leaching process of useful material unfriendly.Except syrup, the stem stalk provides great potential for biological fuel production.Yet,, as other crop plants, influence the separation of available sugar (for example wood sugar, pectinose, glucose etc.) owing to there is xylogen.

Xylogen is complicated phenylpropionic acid base polymer.Plant comprises the xylogen based on about 25-30% of dry weight.The xylogen major sedimentary is in the cell walls of support and conductive tissue (for example fiber and tracheary element).The mechanical rigid of xylogen is strengthened these tissues, so that tracheary element can tolerate the negative pressure that is produced by transpiration under the situation that does not have tissue to subside.

Yet xylogen is disadvantageous in other all respects.Xylogen has reduced the digestibility of animal-feed crop and xylogen must have been removed in smashing slurry and paper-making process, wherein needs to utilize environmentally harmful pharmaceutical chemicals.Xylogen is as also the utilization of plant and trees biomass being had negative impact.

Xylogen is considered to be formed by right-tonquinol (p-coumaryl alcohol), lubanol (coniferyl alcohol) and sinapyl alcohol (sinapyl alcohol) monomer dehydrogenation polymerization.These monomers are synthetic by phenylpropionic acid class synthesis path (phenylpropanoid pathway).Structurally only the 3C at aromatic ring (aromatic ring) is different with the methoxyl group of 5C position for these monomers.Change the type that monomeric ratio is determining xylogen (for example H, G, S xylogen).In the enzyme glycolysis process, the G xylogen provides the resistance higher than S xylogen.Owing to the moleculartie that has greater amt in the G xylogen makes it fine and close more, thereby present higher resistance.

Therefore, preferably develop the sweet sorghum plant of lignin component with change.With comparing of self-sow, described component comprises more S xylogen and less G xylogen.In order to realize this goal, also need the plant of modification must have more biomass content and its biochemical system structure helps downstream processing.

The component of having taked several different methods reduction or change content of lignin is to increase the S/G ratio.Yet, found that it is contradiction, this may be owing to the understanding that lacks the xylogen biosynthetic pathway, and owing to adopt inappropriate decreasing method that is used for the xylogen biosynthetic enzyme activity, the selection that this comprises the structure of transgenosis, the promotor that is adopted, box the most important thing is the screening of transformant.Regulation and control have reduced content of lignin as the initial reaction step of the enzyme of phenylalanine ammonia lyase (phenylalanine ammonia lyase), styracin-4-hydroxylase (cinnamate4-hydroxylase), 4-coumaric acid CoA ligase (4-hydroxycinnamate CoA ligase).Yet, this can cause polyphenic effect, it comprises the leaf shape of change, and the form of the pollination activity that partial fluorescence pathology, plant be short and small, reduce, the flower of change and pigmentation, the benzene soluble L-Ala of reduction level, the S/G of reduction such as compare at (people such as Elkind, 1990; People such as Bate, 1994; People such as Sewalt, 1997).The change of being undertaken by other worker or the S/G of modification than and the similar effect that obtains is to cause phenotype defective plant.The result shows, the activity downward modulation of coffic acid O-methyltransgerase (caffeic acid O-methyltransferase) can cause the biosynthetic violent decline of syringyl lignin (syringyl lignin), and it is little to the influence of guaiacyl xylogen (guaiacyl lignin), this result is unfavorable, because the latter has higher resistance to chemical degradation.Yet, till carrying out that day of any change of lignin component in the sweet sorghum at all by downward modulation COMT gene.

Therefore, the objective of the invention is to develop a kind of sweet sorghum plant that has the lignin component of change and on biochemistry, be suitable for the modification of downstream process.In order to realize this purpose, the contriver attempts to find out the gene that can change the lignin component that changes in the sweet sorghum plant, and the Chinese sorghum plant of the COMT downward modulation by similar gene silencing methods production in described plant and the existing research is compared easier enzymolysis.

Summary of the invention

The present invention relates generally to the peptide sequence that can change lignin component in the sweet sorghum.The downward modulation of polynucleotide described in the sweet sorghum plant causes formation, the higher free sugared content of the S xylogen more with respect to the G xylogen and shows in the plant of modification with the plant of self-sow compares morphological specificity preferably.The computer based analysis that isolating sequence is carried out demonstrates a kind of similarity with the CCoAOMT of vertical homology (arthologous) plant.Yet, do not have data openly to have CCoAOMT and its effect in the lignin component in regulating sweet sorghum up to now.Following polynucleotide sequence is shown in SEQ ID NO 1, and it has class CCoAOMT character, and its sequence is partly discerned.

According to a first aspect of the invention, provide isolating polynucleotide sequence, wherein obtained in the Chinese sorghum of this sequence by coding caffeoyl coenzyme A-O-methyltransgerase (CCoAOMT), shown in SEQ ID NO 1.According to some aspects, DNA can be cDNA.

Another object of the present invention relates to the gene silencing construct (mediated gene silencing construct) that a kind of RNA interferes (RNAi) mediation, comprise: the suitable fragments of Nucleotide shown in the SEQ ID NO 1 (suitable fragment), the described Nucleotide promotor (promoter) that is operably connected, wherein said nucleotide fragments have double-stranded justice-antisense orientation and impel and at least a portion SEQ ID NO 1 produced RNA interferes (RNAi) in the body.Further purpose of the present invention is to provide a kind of carrier (vector) that contains construct, and in purposes more of the present invention, described carrier can be the pUC18 carrier.

Further purpose of the present invention relates to forward and the reverse primer shown in SEQ ID NO 3 and 4.Described primer helps the CCoAOMT that increases.

Be unexpectedly, when finding the SEQ ID NO 1 in reducing sweet sorghum, make these plants that plant is better than obtaining to reduce COMT at biomass (biomass), starch (starch), free sugared content (free sugar content) and phenotypic characteristic aspects such as (phenotypical properties).

Description of drawings

Fig. 1: compare control plant (left side), anti--CCoAOMT class transforms the raising growth of Chinese sorghum T0 plant (right side).

Fig. 2: (in field) has the raising growth of the transgenosis Chinese sorghum (T1 plant) of the downward modulation CCoAOMT genoid shown in the SEQ ID NO1 in the field.

Fig. 3: control plant (left side) and CCoAOMT genoid transform the panicle (panicle) of plant (right side).

Fig. 4: compare control plant (left side), the variation of the seed size of anti--CCoAOMT plant (right side).

Fig. 5: two strands mediates anti--CCoAOMT construct and promotes endogenous (endogenous) CCoAOMT gene is produced the interior RNAi of special body.

Fig. 6: the pcr amplification of the marker gene by COMT and CCoAOMT transition lines is selected plant.

The T1 filial generation of Fig. 7: CCoAOMT and the southern of T0 transformant and control plant thereof analyze (southern analysis) and show the genetically modified inherited character of integrating.

Embodiment

Therefore, the invention provides a kind of sweet sorghum plant, it is characterized in that: compare with wild plant, it has the lignin component of change, and this is to realize by the expression of polynucleotide (polynucleotide) in sweet sorghum shown in the downward modulation SEQ ID NO 1.

One of purpose of the present invention provides the gene silencing of RNA interference (RNAi) mediation of the native gene shown in the SEQ ID NO 1, makes when external source (exogenous) Nucleotide is transcribed the downward modulation of the activity of endogenous SEQ ID NO 1.

In addition, the present invention also provides the production method of a kind of sweet sorghum of improvement, and the sweet sorghum of this improvement has the lignin component of change.Described method comprises: utilize nucleotide sequence transfection shown in the SEQ ID NO 1 to have the vegetable cell of the gene silencing box of double-stranded RNA (dsRNA) mediation, the G-content of lignin with reduction is compared in the promotion of nucleotide sequence shown in the described SEQ ID NO 1 to RNA interference (RNAi) in the native gene generation body and with wild plant.

Develop two kinds of transgenosis lines, a kind of have anti--COMT, another kind has anti--SEQ ID NO1, and compare anti--COMT and transform plant, anti--SEQ ID NO 1 transforms plant and demonstrates required and useful phenotypic characteristic, such as having accelerated growth, have the panicle proterties, increased each paniculiform seed amount, increased size and weight, and therefore provide multi-biomass more to be used for the starting material that use in the downstream and higher amount of starch and free sugared content.In this application, will be called " C-plant " and " T-plant " by the construct plant transformed of anti--COMT and anti--SEQ ID NO 1.

Gene by 1 pair of sweet sorghum of SEQ ID NO improves has profound significance.The Chinese sorghum biomass can be used to zymotechnique.In zymotechnique, the very important point just is to use the biomass with high sugared content and preferred soluble sugar.Here need should be mentioned that based on morphological analysis and the biochemical analysis to improved Chinese sorghum plant, biomass and content of cellulose all have significantly as can be known increases.

Seek the new gene in the sweet sorghum that can change lignin component:

A] overheated phenol processes makes whole RNA and sophisticated Chinese sorghum stem isolated:

Get the fresh Chinese sorghum stem of 1gm, and it is minced into segment, in liquid nitrogen, in mortar, segment Chinese sorghum stem is crushed then, until it is powdered with pestle.(β-ME) adds described powder with a little Polyvinylpyrolidone (PVP) (PVP) and 200 μ l beta-mercaptoethanols.Earlier 5mlRNA is extracted the saturated phenol of damping fluid and 5ml and mix, and heat in 80 ℃ water-bath, the mixed solution after will heating then adds powder and thorough mixing, makes described powder dissolution in mixed solution.Then, sample dissolution is transferred in the centrifuge tube and in 80 ℃ water-bath the heating 20 minutes.Then, described centrifuge tube is kept 5-10 minute at room temperature, and in centrifuge tube, add 5ml trichloromethane (chloroform), make the abundant whirling motion of centrifuge tube (vortexed) subsequently.Then, make centrifuge tube at room temperature with 10, centrifugal 10 minutes of 000rpm gets the upper strata stillness of night (supernatant) to place corex pipe (Glass tubing of ultraviolet printing opacity).With 1/10 ^ThThe sodium acetic acid (Na-acetate) of volume and the frozen ethanol of 2 volumes add the described upper strata stillness of night, and make its precipitation of spending the night in-20 ℃.Second day, make described corex pipe in 4 ℃ with 10, centrifugal 10 minutes of 000rpm.Abandon the upper strata stillness of night and air-dry particulate.Soluble particles and it being distributed in the microfungi pipe in 1ml DEPC treating water.Add the saturated phenol of equal volume, shake mixed solution gently, subsequently with it with 10, centrifugal 5 minutes of 000rpm obtains the upper strata stillness of night subsequently.Repeat above-mentioned phenol step, become limpid up to the described upper strata stillness of night.After the phenol step, add the trichloromethane of equal quantities, subsequently with mixed solution with 10, centrifugal 5 minutes of 000rpm obtains the upper strata stillness of night subsequently.Subsequently with 1/10 ^ThThe sodium acetic acid of volume and the frozen ethanol of 2 volumes add the described upper strata stillness of night, and make it carry out 1 hour precipitation in-20 ℃.Then, with 10, centrifugal 10 minutes of 000rpm uses 70% ethanol (alcohol) to clean particulate subsequently with precipitation solution, subsequently to its thorough drying, makes particle dissolution in methane amide and remain on and be used in-70 ℃ preserving at last.

The component of RNA extraction damping fluid (pH8):

LiCl-100mM

TRIS-100mM

EDTA-10mM

SDS -1％

By oligose (dT) cellulosic matrix purified mRNA:

Oligose (dT) cellulose suspension of 100mg is inserted in the 1ml elution buffer, and make it keep room temperature the whole night.Remove elution buffer by short period of time rotation (short spin).Post bed and the equilibrium of 1ml binding buffer liquid.Remove binding buffer liquid by the short period of time rotation.1mgRNA sample in the methane amide precipitated in preceding a whole night, and the cleaning of use ethanol, and drying also is dissolved in the binding buffer liquid of 1ml fully, 65 ℃ of heating 5 minutes, and cooled off 5 minutes on ice cube fast.The RNA sample is inserted in oligomerization (dT) cellulosic matrix then.Follow gently and shake, make in conjunction with continuing 30 minutes.Make this suspension centrifugation by the short period of time rotation.Use the 1ml cleaning buffer solution to clean the post bed 3 times, each 30 minutes, to remove binding buffer liquid.Use 200 μ l elution buffers, 13, centrifugation is 30 seconds under the 000rpm rotating speed, uses 2 poly (A of 200 μ l elution buffers extraction ⁺) mRNA.Readjust elution pool by the NaCl that correspondingly increases 0.5M.Whole process begins to repeat twice from the step that adds binding buffer liquid wash-out again.Once more in conjunction with, clean once more, the sample that forms of wash-out uses 1/10 of Na-acetate once more ^ThPost bed and alcoholic acid 2.5 ^Th Post bed precipitation 1 hour.Use 70% ethanol cleaning granular substance, dry also the suspension once more inserted the DEPC treated water.

The composition of damping fluid:

A) oligomerization (dT) binding buffer liquid

TRIS-HCL(pH7.5) ——10mM

NaCl ——500mM

EDTA ——1mM

SDS ——0.5％

B) oligomerization (dT) cleaning buffer solution

TRIS-HCL(pH7.5) ——10mM

NaCl ——100mM

EDTA ——1mM

C) oligomerization (dT) elution buffer

TRIS-HCL(pH7.5)?——10mM

EDTA ——1mM

The cDNA's of isolating mRNA is synthetic from sophisticated stem:

Utilize reverse transcription, prepare cDNA from mRNA, this mRNA separates from sweet sorghum.Primer makes it by 5 ' to 3 ' common extension to the mRNA annealing enzyme that can be used to be inverted the freedom 3 ' end of recording the enzyme extension is provided, by forming hybrid molecule with mRNA template paired complementary base guiding, this hybrid molecule by with the based composition of complementary cDNA chain paired template ribonucleic acid chain.After the initial mRNA degraded, use the synthetic complementary DNA chain of archaeal dna polymerase, change strand cDNA into duplex cDNA.

The degenerate primer that application designs from the gene conservative aminoacid sequence from the allos botanical system, by 5 ' and 3 ' RACE (Rapid Amplification of cDNA Ends, rapid amplifying cDNA end), use PCR (polymerase chain reaction) and separate required cDNA completely.With fixed oligomerization (dT) ₁₈Primer and six aggressiveness primers at random use the RT-PCR conversion unit (Standard RT-PCR Reaction kit) of the standard of Roche Holding Ag (Roche) to synthesize cDNA.At the PCR test tube that is arranged in aseptic, nuclease free, thin-walled on ice, the order of listing by according to the form below adds composition, is the prepared in reaction templa-primer mixture of 20 μ l.

The mixing of templa-primer:

Composition	Volume	Ultimate density
			Poly (A ⁺)mRNA	1μl	10ng poly (A ⁺)mRNA
Fixed oligose (dT) ₁₈Primer, 50pmol/ μ l	1μl	?2.5μM

Six aggressiveness primers at random, 600pmol/ μ l	2μl	60μM
			PCR water	9μl

Cumulative volume 13 μ l

In thermal block cycler,,, make the second structure sex change 65 ℃ of following heated die plate primer mixtures 10 minutes with the lid of heating.At once test tube is placed on ice.The order that according to the form below is listed adds the remaining component of RT mixture.

Composition

Volume

Ultimate density

Transcription factor ThermoScript II reaction buffered soln (5 *)	4μl	?1×(8mM?MgCl)
			Protection thing ribonuclease inhibitor, 40U/ μ l	0.5μl	?20U
The deoxynucleoside acid mixture, 10mM each	5μl	?1mM?each
			The transcription factor ThermoScript II, 20U/ μ l	0.5μl	?10U

Final volume 13 μ l

Carefully mix also centrifugation reagent simply, and under 25 ℃, reagent was placed on last 10 minute of thermal block cycler, be 55 ℃ then and placed 30 minutes down.By heating 5 minutes down, make transcription factor ThermoScript II (Transcriptor Reverse Transcriptase) inactivation at 85 ℃.Test tube is placed on ice, and reaction finishes.

The amplification of primer design and synthetic and PCR:

Design 5 ' and 3 ' primer of two degradeds.For the clone of pcr amplified fragment is introduced in BamHI site on the 5 ' primer and the EcoRI site on the 3 ' primer.The transfer amino acid sequence analysis of amplified fragments shows identical with allogenic CCoAOMT.The sequence of the primer of degraded is:

Forward primer: 5 ' gaa ttc gga tcc a (c/t) c a (a/g) g tng gnc a (c/t) a a 3 ' (SEQ ID NO3)

Reverse primer: 5 ' gaa (g/a) tt cca nag ngt (g/a) tt (g/a) tc (g/a) ta 3 ' (SEQ ID NO4)

Part SEQ ID NO 1 segmental clone:

When in primer, introducing BamHI and EcoRI, use Restriction Enzyme BamHI and EcoRI digestion pcr amplified fragment and carrier pUC18 to be used for the clone.The fragment that the carrier of digestion and expansion increase is all by LMP sepharose purifying.The product of purifying and T4DNA ligase enzyme bundle.Change the part of binding mixture into the DB10B competent cell, and be placed on Ampicillin Trihydrate (the 100 μ g/ml) flat board.Select the bacterium colony of conversion.From the clone, separate the plastid DNA of reorganization and plastid DNA is checked order.Use specific primer (SEQ ID NO 5 and 6) clone's fragment further to increase, and use BamHI and Sacl digestion by specific primer, and under the help of any key with BamHI and BgIII site, in justice-antisense orientation combination.Under promotor (promoter), should justice-antisense fragment be arranged in two carriers, to produce dsRNA, it is the CCoAOMT generation carrier of Agrobacterium, is used for the agrobacterium mediation converted of plant.

The sequence of specific primer is:

Forward primer: 5 ' gaa ttc gga tcc cat cag gaa gta ggg cac aa, 3 ' (SEQ ID NO 5)

Reverse primer: 5 ' cc gag ctc gtt cca cag tgt gtt gtc ata, 3 ' (SEQ ID NO 6)

Separate total length SEQ ID NO 1:

Use the s-generation 5 '/3 ' RACE test kit and separate total length SEQ ID NO 1 with 3 ' end from the cDNA amplification 5 ' of Chinese sorghum set by step.Then, cloned sequence and evaluation.Primer generates from the nucleotide sequence of the part SEQ ID NO 1 of evaluation.

Clone SEQ ID NO 1 in the TA carrier:

Fragment behind the pcr amplification is by LMP sepharose purifying, and is cloned in the TA carrier.Then, part connection (ligation) mixture is transformed on DH10B competent cell neutralization of ammonia penicillin G (100ug/ml) flat board.Clone after screening transforms, and by the order-checking evaluation.

Clone's evaluation and based on the clone after the software analysis order-checking:

By the white clone (white clone) of the initial evaluation and screening of isolating plasmid DNA, with BamHI and EcoRI digestion, to identify existing of inserting.Then, for further affirmation, use is analyzed and sequence alignment W analysis as NCBI blast based on the analytical system of software, checks order to being used for each segmental these three of cloning by the big dyeing terminator method (Big Dye Terminator method) that checks order.

From the nucleotide sequence that sequencing result obtained:The sequence of gene finds to have 759 bases.The DNA sequences encoding of gene is found to finish at base 759 places from base 1.Nucleotide sequence hereinafter is called SEQ ID NO 1, and it is following structure:

5’-

ATGGCCGAAAACGGCGAAGAGCAGCAGCAGGCGAACGGCAACGGCGAGCAGAAGACCCGG

CATCAGGAAGTAGGGCACAAGAGCCTGCCTGCTCAAGAGCGACGGACGAGCTCTACCAGTACATCCT

GGACACGAGCGTGTACCCGCGGGAGCCGGAGAGCATGAAGGAGCTCCGCGAGCGAGATCACC

GCCAAGCACCCATGGAACCTGATGACGACCTCCGCCGACGAGGGGCAGTTCCTCAACAT

GCTCATCAAGCTCATCGGCGCCAAGAAGACCATGGAGATCCGCGTCTACACCGGCTACTC

CCTCCTTGCTACTGCCATGGCTCTTCCCGATGATGGCAAGATTCTAGCTATGGATATTAAC

CGGGAAAACTACGAGATTGGTCTTCCAGTGATTGAAAAGGCTGGACTGGCCCACAAGAT

CGACTTCCGCGAGGGCCCCGCGCTCCCCGTCCTCGACGACCTCATCGCCGACGAGAAGA

ACCACGGGTCGTTCGACTTCGTCTTCGTGGACGCCGACAAGGACAACTACCTCAACTACC

ACGACCGGCTGCTCAAGCTGGTGAAGCTGGGGGGCCTCATCGGCTATGACAACACACTG

TGGAACGGGAGCGTCGTGCTGCCCGACGACGCCCCGATGCGGAAGTACATTCGCTTCTA

CCGCGATTTCGTCCTCGTCCTGAACAAGGCGCTCGCGGCGGATGATCGCGTCGAGATCTG

CCAGCTCCCCGTCGGTGACGGTGTGACGCTGTGCCGGCGCGTCAAGTGA-3’(SEQ?ID?NO?1)

For further characterization based on the Computer Analysis sequence:

Then, use expense uncommon (Jellyfish) software translation dna sequence dna in Jerry to obtain aminoacid sequence.Sequence after the translation is:

MAENGEEQQANGNGEQKTRHQEVGHKSLLKSDELYQYILDTSVYPREPESMKELREITAKHP

WNLMTTSADEGQKLNMLIKLIGAKKTMEIRVYTGYSLLATAMALPDDGKILAMDINRENYEI

GLPVIEKAGLAHKIDFREGPALPVLDDLIADEKNHGSFDFVFVDADKDNYLNYHDRLLKLVKLG

GLIGYDNTLWNGSVVLPDDAPMRKYIRFYRDFVLVLNKALAADDRVEICQLPVGDGVTLCRR

VK*(SEQ?ID?NO?2)

Then, using the blastP among the NCBI) aminoacid sequence after this translation carries out the blast analysis, finds that the CCoAOMT of itself and close relative's corn has maximum homology.Therefore, use sequence contrast W software (as described below), further the homology of the CCoAOMT skeuomorph of obtainable corn in analytical sequence and the Genbank database shows that its CCoAOMT skeuomorph 95% with corn is identical.

Corn C CoAOMT1------

MATTATEAAPAQEQQANGNGEQKTRHSEVGHKSLLKSDDLYQYILDTSVYPREP(SEQ?ID?NO?7)

Chinese sorghum CCoAOMT------------

MAENGEEQQANGNGEQKTRHQEVGHKSLLKSDELYQYILDTSVYPREP(SEQ?ID?NO?8)

Corn C CoAOMT2

MATTATEATKTTAPAQEQQANGNGNGEQKTRHSEVGHKSLLKSDDLYQYILDTSVYPREP(SEQ?ID?NO?9)

Paddy rice CCoAOMT----

MAEAASAAAAATTEQANGSSGGEQKTRHSEVGHKSLLKSDDLYQYILETSVYPREH(SEQ?ID?NO?10)

******************：******：*******

Corn C CoAOMT1------

ESMK

ELREVTAKHPWNLMTTSADEGQFLNMLIKLIGAKKTMEIGVYTGYSLLATALALPE(SEQ?ID?NO?11)

Chinese sorghum CCoAOMT

ESMKELREITAKHPWNLMTTSADEGQFLNMLIKLIGAKKTMEIRVYTGYSLLATAMALPD(SEQ?ID?NO?12)

Corn C CoAOMT2

ESMKELREITAKHPWNLMTTSADEGQFLNMLIKLIGAKKTMEIGVYTGYSLLATALALPE(SEQ?ID?NO?13)

Paddy rice CCoAOMT

ECMKELREVTANHPWNLMTTSADEGQFLNLLLKLIGAKKTMEIGVYTGYSLLATALAIPD(SEQ?ID?NO?14)

＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊

Corn C CoAOMT1------

DGTILAMDINRFNYELGLPCIEKAGVAHKIDFRFGPALPVLDDLIAEEKNHGSFDFVFVD(SEQ?ID?NO?15)

Chinese sorghum CCoAOMT

DGKILAMDINRENYEIGLPVIEKAGLAHKIDFREGPALPVLDDLIADEKNHGSFDFVFVD(SEQ?ID?NO?16)

Corn C CoAOMT2

DGTILAMDINRENYELGLPCINKAGVGHKIDFREGPALPVLDDLVADKEQHGSFDFAFVD(SEQ?ID?NO?17)

Paddy rice CCoAOMT

DGTILAMDINRENYELGLPSIEKAGVAHKIDFREGPALPVLDQLVEEEGNHGSFDFVFVD(SEQ?ID?NO?18)

Corn C CoAOMT1

ADKDNYLNYHERLLKLVKLGGLIGYDNTLWNGSVVLPDDAPMRKYIRFYRDFVLVLNKAL(SEQ?ID?NO?19)

Chinese sorghum CCoAOMT

ADKDNYLNYHDRLLKLVKLGGLIGYDNTLWNGSVVLPDDAPMRKYIRFYRDFVLVLNKAL(SEQ?ID?NO?20)

Corn C CoAOMT2

ADKDNYLNYHERLLKLVRPGGLIGYDNTLWNGSVVLPDDAPMRKYIRFYRDFVLALNSAL(SEQ?ID?NO?21)

Paddy rice CCoAOMT

ADKDNYLNYHERLMKLVKPGGLVGYDNTLWNGSVVLPADAPMRKYIRYYRDFVLELNSAL(SEQ?ID?NO?22)

Corn C CoAOMT 1AADDRVEICQLPVGDGVTLCRRVK (SEQ ID NO23)

Chinese sorghum CCoAOMT AADDRVEICQLPVGDGVTLCRRVK (SEQ ID NO 24)

Corn C CoAOMT2 AADDRVEICQLPVGDGVTLCRRVK (SEQ ID NO25)

Paddy rice CCoAOMT AADHRVEICQLPVGDGITLCRRVK (SEQ ID NO26)

The role of assessment SEQ ID NO 1 in sweet sorghum:

Then, be the similarity of obtainable sequence in recognition sequence and the database, sequence stands NCBIblast, finds its protein height with the supposition of Chinese sorghum identical (97% is identical).Although it is similar to the CCoAOMT gene that the protein of this supposition has been considered to, its function does not also have tested.Therefore, we take method to set up the effect of isolating gene, because the nucleotide sequence of gene is more important than its function in the antisense strategy, so the method for taking is that (in-planta) decrement is regulated CCoAOMT in metatarsus.

Comprise the preparation of the construct of SEQ ID NO 1:

Promotor: DNA transcribes mRNA and is regulated by the zone of the DNA that is called promotor.Promoter region comprise transmit signal to RNA polymerase make its in conjunction with DNA, and will be wherein DNA chain start the base sequence of transcribing, form corresponding complementary RNA chain of mRNA as template.Because therefore 5 ' zone and 3 ' regional complementarity of RNA chain will generate double-stranded RNA, this double-stranded RNA is used the mechanisms of degradation that produces short intervening rna (RNAi) subsequently.Promoter sequence comprises TATA box consensus sequence (TATAAT (SEQ ID NO 27)), it typically is 20-30 the base pair (bp) that be positioned at the transcription initiation site upstream (by convention, for from respect to transcripting start point-30 to-20bp).The TATA box is the unique upstream promoter element that has the relative fixed position with respect to starting point.The CAAT box consensus sequence is the center with-75, but works from either direction in the quite unfixed distance from starting point.Another common promoter element is to be positioned at-90 GC box, and it comprises consensus sequence GGGCGG (SEQ ID NO 28).It can occur with a plurality of copies and in either direction.Can also find that at promoter region other gives the sequence of maximum efficiency.In the combination of promotor and structure gene, promotor preferably is arranged in itself and the distance of allos transcripting start point and the identical position of distance at the transcripting start point of its natural background.Promotor can be composing type or induce sexual type.

The corn ubiquitin promoter of using in research of the present invention has been presented in the most tissues and has highly been activated and structurally express.This corn ubiquitin promoter is included in first intron of selective expression's corn ubiquitin gene in the plant.Promotor is cloned in the carrier in the HindIII/BamHI site, has placed introducing SEQ ID NO 1 segmental dsRNA under this site.Under the 2x 35S promoter, comprise the selectable marker gene hygromix phosphotransferase, can screen vegetable cell with required construct.

Just adding the segmental appropriate area generation of required cDNA dsRNA construct by connexon with opposite direction.This construct is imported to the expression vector that is used for the Chinese sorghum Plant Transformation, to find its role in pathways metabolism.Carrier preferably comprises the protokaryon replication orgin with extensive host range.Also should comprise selective marker, make it can screen bacterial cell with required construct.Suitable protokaryon selective marker comprises the antibiotic resistance as kantlex.

The dna sequence dna that in carrier, can also present as everyone knows, other coding additional function.For example, under the situation of Agrobacterium-mediated Transformation, can also comprise and be used for the T-DNA sequence of subsequent transfer to plant chromosome.

In order in plant, to express, adopt two carriers that can import goal gene therein.Recombinant expression cassettes also will comprise plant promoter zone, transcripting start point and Transcription Termination subsequence except comprising needed sequence.Typically comprise the special Restriction Enzyme site of 5 ' and 3 ' end of box, be inserted in the carrier of preexist so that can be easy to.Those skilled in the art also know the sequence of control eukaryotic gene expression.

The generation of transgenic plant:

Two kinds of transgenic lines have been developed.Instead-COMT (anti-COMT) thaumatropy, be called " C-plant " plant hereinafter and have anti--SEQ ID NO 1, be called the plant of " T-plant ".The T-plant demonstrates the seed number of every fringe of growth that satisfactory and useful phenotypic character for example improves, fringe type, increase, the size of increase and weight, and therefore provide more biomass as being used for the raw material that use in the downstream.

The conversion of sweet sorghum (Sorghum bicolor, Chinese sorghum):

With the seed-coat of aseptic distillation water washing with mercury chloride (7 minutes) bacterium of going out of polysorbas20 (5 minutes) and 0.2%.To hatch on the sterilising filtration paper that is soaked with sterile purified water of seed in culture dish then.After hatching 3 days in the dark, cut off the stem apex of generation and use the infection substratum that wherein has agrobacterium suspension to infect 20 minutes.Hatching explant on the substratum altogether, and keeping in the dark 3 days in 25 ℃.Between or with cefotaxime acid (cefotaxime) and distilled water wash explant, preventing bacterial contamination, and it is transferred to have 2 of 2mg/l, on the MS of the sucrose of 4-D, 30gm/l and the cefotaxime acid of 250mg/l, kept 12 days in the dark, to generate callus.Cut off callus part at the otch end of stem apex, and it is transferred to the regeneration culture medium (sucrose with 30g/l with hygromycin selection, the MS of the BAP of 2mg/l and the Totomycin of 2mg/l) on, remains under 2: 1 illumination/dark cycle condition of 28 ℃.After 2 weeks, the callus of green is transferred on the same medium of the selective marker (Totomycin of 4mg/l) that contains greater concn.The bud that obtains is transferred on the same medium of Totomycin (5mg/l) 2 months, the cultivation of regularly going down to posterity in per 2 weeks with greater concn.The blastogenesis of elongation is longer than on the root media to generate root.Exist at last ¹/ ₂Use the hygromycin selection Cheng Miao of 6mg/l on the MS liquid nutrient medium.The seedling that is generated by single callus is called as monosystem.

Agrobacterium mediation converted:

Agriculture bacillus mediated method for transformation of the present invention can be divided into several steps.Basic step comprises the infection step; Be total to culturing step; Selectable sleep step (resting step); The screening step; And regeneration step.

In infecting step, separate cell to be transformed, and make its contact Agrobacterium.If target cell is immature embryo, separates embryo, and make the suspension of its contact Agrobacterium.As mentioned above, Agrobacterium has been transformed into and has comprised goal gene or purpose Nucleotide.This Nucleotide is inserted in the T-DNA zone of carrier.The general molecular engineering of using among the present invention is by the molecular cloning of (eds.) such as for example Sambrook: laboratory manual, 1989, cold spring harbor laboratory's magazine, cold spring port (Cold Spring Harbor Laboratory Press, Cold Spring Harbor N.Y.) provides.

Can influence transformation frequency in infection step and the concentration that is total to the Agrobacterium of using in the culturing step.Similarly, very the Agrobacterium of high density can damage tissue to be transformed, for example immature embryo, and cause the reaction of callus to reduce.Therefore, the concentration of the Agrobacterium of using in the method for the present invention can change along with the genotype of the Chinese sorghum of the tissue of the bacterial strain of the Agrobacterium of using, conversion, conversion etc.In order to optimize the specific Chinese sorghum system or the Transformation Program of tissue, tissue to be transformed (for example immature embryo) can be hatched with the Agrobacterium of various concentration.Similarly, can evaluate and test the concentration of various Agrobacteriums with the degree that marker gene is expressed and transformed.Although the concentration of Agrobacterium can change, the Agrobacterium of using among the present invention, usually OD600nm is 0.7 to 1.0.

In order to optimize transformation efficiency, generally tissue to be transformed is joined in the Agrobacterium and agrobacterium suspension that comprises a certain concentration for liquid contact phase.Contact helps the maximum contact of cell/tissue and agrobacterium suspension to be transformed mutually.The time that cell contacts with agrobacterium suspension is at least about 3 minutes to about 15 minutes, is preferably about 4 minutes to about 10 minutes, more preferably about 5 minutes to about 8 minutes.

The liquid contact of infecting step is to add 68.5g/l sucrose in liquor MS substratum mutually, and 36g/l glucose, 100 μ M Syringylethanones (acetosyringone), pH are adjusted into 5.2 and form.Other substratum of the present invention is: culture medium (adds 20g/l sucrose among the MS altogether, 10g/l glucose, 2mg/l 2,4-D, 100 μ M Syringylethanones and 8.5g/l agar, pH 5.8), microbial culture substratum (YEP substratum-yeast extract-10g/l, peptone-10g/l and sodium-chlor-5g/l), infect substratum and (add 68.5g/l sucrose among the MS, 36g/l glucose and 100 μ M Syringylethanones, pH 5.2), regeneration culture medium (adding 30g/l sucrose among the MS, 2mg/l BAP and 8.5g/l agar) and root media ( ¹/ ₂Add 20g/l sucrose, 0.5mg/l IAA and 0.5mg/l NAA among the MS).

The concentration of Agrobacterium between period of infection

The O.D.-of Agrobacterium is between 0.7-1.0

Follow-up common culturing step, or in the sleep step (if use) is accepted selective pressure to screen those and received and just expressed the cell from the polypeptide of allos Nucleotide that is imported by Agrobacterium with cell transformed.Cell is under the situation of embryo, embryo is transferred on the flat board with solid medium, and this solid medium comprises microbiotic and the selective agent that suppresses the Agrobacterium growth.The selective agent that is used to screen transformant is positioned at select to be used to comprise at least one in super binary vector (super binary vector) and the preferential growth of the explant of the selectable mark insertion that imports by Agrobacterium.

Usually, the known any substratum that is suitable for cultivating Chinese sorghum of affiliated technical field all can be applicable to screen step, for example comprises to be suspended with 30g/l sucrose, and 2mg/l 2, and the N6 salt of 4-D or the substratum of MS salt kept 15 days in the dark.During the screening, culturing embryo forms up to observing callus.Typically, can grow into diameter at the callus of selecting to grow on the substratum and be about 1.5 to the 2cm size.

After callus reaches suitable size, cultured calli several weeks in the dark on regeneration culture medium, be typically about 1 to 3 week, so that the somatic embryo maturation.Preferred regeneration culture medium comprises comprising and is suspended with 30g/l sucrose, the MS substratum of 2mg/l BAP and 8.5g/l agar.Cultured calli on root media is cultivated in illumination/dark cycle then, up to bud and root growth.

Then seedling is transferred to an about week in the pipe that comprises root media, made more of its g and Ds.Then with in the soil mixture in the jar of plant transplanting in the greenhouse.

The supposition of dsRNA inductive transforms the screening of system

Be used for inducing genetically modified supposition T through Totomycin seed selection regenerated dsRNA ₀Transformant is further screened by pcr analysis, to determine existing of hygromycin gene.Thereby, pcr analysis uses genome DNA, and this DNA is from the individuality of conversion and control plant and one group of gene specific primer (5 ' primer: atg aaa aag cct gaa ctc acc gcg acg tct and 3 ' primer: separate gca tca gct cat cga gag cct gcg cga cgg) of non-conversion.In all transformants, observe the amplified production of about 547bp, show to have hygromycin gene.This amplification does not exist in non-conversion jowar plant.Therefore, can expect, can be inserted into the T of supposition when dsDNA inductive COMT and CCoAOMT box gene ₀Change in the sub-genome, and keep being connected with hygromycin gene (hptll).

The Southern of transgenic plant analyzes: the Southern that carries out transgenic plant analyzes to detect the quantity of duplicating of integration mode and integrator gene construct.In order to carry out this analysis DNA isolation from control plant, T0 and T1 plant.The DNA of the 10mg of every group of plant is digested by HindIII and electrophoresis in 1% sepharose.This gel is transferred on the nylon membrane (GE health care company) then, and respectively with COMT with the CCoAOMT gene probe is crosslinked and terminal crossing (Fig. 6 and 7).

The T1 filial generation forms from the seed of pollination certainly of T0 plant.By allowing them to grow in comprising the basic medium of Totomycin, this seed is screened to come out.When having Totomycin, find that eight kinds of seeds germinate.The seedling of resistance is allowed to grow into the stage of maturity then in container.Use two gene specific primers by PCR, and when hygromycin gene exists, analyze plant.At last, carry out T ₁The Southern of transgenic plant analyzes, to detect the quantity of duplicating of integration mode and integrator gene composition.In reverse sequence ID NO 1 transgenic plant, can observe single integral body except T1-1.In addition, under all conditions, find that integration mode is quite similar.Enzymic activity in the T1 plant is compared about the same with the T0 plant.

Transform the endogenous enzyme activity analysis of system:

Enzymic activity in the tissue of the sweet sorghum that transforms is analyzed, to obtain the change that xylogen is formed.The results are shown in Table 1 for it.Table 2 clearly illustrates that the downward modulation shown in the SEQ ID NO 1 has caused the minimizing of content of lignin.The result shows that content of lignin reduces about 14% and 27% respectively in the stem tissue in C-plant and T-plant.Table 3 discloses the S/G ratio in transforming plant and control plant.The result shows that S/G is than increasing in two kind of plant.The change of the S/G ratio in the T-plant is more obvious.Therefore, can expect that the increase of this S/G ratio helps the particularly extractability of the fibrous material in the T-plant of transgenic plant.

The enzymic activity of table 1:C and T axis tissue

The change of the growth parameter(s) of transgenic lines:

Transgenic lines shows the change of grower when contrasting with control plant.The increase of plant height and internode connect each other apart from the increase and the internode of length.Except the vegetalitas growth that increases, to compare with control plant, change also is observed in the seed sizes of transgenic plant.The increase of seed sizes is checked and approved by average seed weight.Through finding that comparison increases by 45% according to seed approximately.

The biochemical analysis of the sweet sorghum plant that changes:

Analyze the content and the component of xylogen in the transgenic plant with chemical process: with regard to G type and S type, lignin component is by reductive cleavage (reductive cleavage, the derivatization that DFRC) causes decision.As standard, the acetyl derivative of monomer lignin degradation product utilization lubanol and sinapyl alcohol is by the GC-FID analysis confirmation.T-plant lignin total content is compared with control plant and has been reduced about 28%.But the C-plant is compared with control plant and has only reduced about 14%.

Table 2 utilizes the content of lignin of the Chinese sorghum stem of acetyl bromide spectrphotometric method for measuring change

The lignin component of table 3C and T plant haulm tissue

The Chinese sorghum plant	S type (%)	G type (%)	The S/G ratio
				Control plant	22.29	77.71	0.29
C-type plant	39.99	60.01	0.66
				T-type plant	63.51	36.49	1.74

The Mierocrystalline cellulose estimation

Collected one section (fresh weight 20mg) of contrast and transgenic plant stem stalk, in liquid nitrogen, refrigerated and grind into fine powder.This powder was handled 30 minutes in the container of containing boiling water with acetic acid/nitron (10: 1) of 3ml, cooled off and precipitate 15 to 20 minutes.The storage supernatant is used to estimate soluble sugar content.Pellet is cleaned and continues 1 hour with 67% vitriolization.At this moment, treated sample is diluted 15 times.Handle with untreated sample in all add the 5ml anthrone reagent and mix.In being housed, the container of boiling water kept 10 minutes, fast cooling and under the 630nm condition, measure 0.D..This result is as the mean value of 3 samples in each example.

Content according to carbohydrate in schematic design estimate seed described in the method and the stem stalk tissue.We have noticed that content of cellulose has increased by 48% in the C-plant, have increased by 36% (table 4) in the T-plant.Although content of cellulose is than higher (table 4) in the C-plant, what soluble sugar increased in the T-plant (table 5) is more, notices that this point is very important.Higher soluble sugar content is useful to fermenting process.

The mensuration of content of cellulose in the table 4 Chinese sorghum stem stalk tissue

Soluble sugar Determination on content in the table 5 Chinese sorghum stem stalk tissue

Starch estimation in the seed

Seed homogenizes in 80% hot ethanol to remove sugar and centrifugal.Pellet cleans and drying repeatedly with 80% ethanolic soln.Pellet was handled 20 minutes with the water of 5ml and the perchloric acid of 6.5ml 52%.Centrifugal extract is also collected supernatant liquor.Diluted 250 times of supernatant liquor.The 5ml anthrone reagent adds in the sample of 1ml, kept 8 minutes in the container of boiling water is housed and measure O.D. under 630nm.This result is as the mean value of 3 samples in each example.

According to contents of starch in schematic design estimate seed described in the method and the stem stalk tissue.Notice that equally in C-plant and T-plant, contents of starch has increased about 20% and 30% (table 3) respectively in the seed.Can expect that the increase of amount of starch is just not saying other component may be that the seed weight increase causes.

The mensuration of starch content in table 6 seed

Take an overall view of the present invention, quoted various patents and publication as proof, the open of these patents and publication incorporated among the present invention as a reference at this as a whole, so that the present situation in field related to the present invention to be described more fully.

Though the present invention has described present advised preferred embodiment, the present invention is not so limited.On the contrary, the present invention attempts to cover the various modifications in the spirit and scope that are included in above-mentioned detailed description and is equal to the change mode.

Claims

1. one kind is separated the oligonucleotide sequence that obtains from the Chinese sorghum of coding caffeoyl coenzyme A-O-methyltransgerase, shown in SEQ ID NO 1.

2. isolating oligonucleotide sequence according to claim 1 is characterized in that DNA is cDNA.

3. the gene silencing box of a double-stranded mediation, it comprises the Nucleotide shown in the SEQ ID NO 1, the described Nucleotide promotor that is operably connected, wherein Nucleotide is positioned at justice-antisense orientation, is convenient at least a portion SEQ ID NO 1 produced that RNA disturbs in the body.

4.SEQ forward primer and the reverse primer shown in the ID NO 5-6.

5. the carrier that comprises the described construct of claim 3.

6. carrier according to claim 5 is characterized in that application carrier is a binary vector.

7. the transgenic plant that comprise the described gene silencing box of claim 3.